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Drug repurposing is considered a valid approach to accelerate therapeutic solutions for rare diseases. However, it is not as widely applied as it could be, due to several barriers that discourage both industry and academic institutions from pursuing this path. Herein we present the case of an academic multicentre study that considered the repurposing of the old drug guanabenz as a therapeutic strategy in amyotrophic lateral sclerosis. The difficulties encountered are discussed as an example of the barriers that academics involved in this type of study may face. Although further development of the drug for this target population was hampered for several reasons, the study was successful in many ways. Firstly, because the hypothesis tested was confirmed in a sub-population, leading to alternative innovative solutions that are now under clinical investigation. In addition, the study was informative and provided new insights into the disease, which are now giving new impetus to laboratory research. The message from this example is that even a repurposing study with an old product has the potential to generate innovation and interest from industry partners, provided it is based on a sound rationale, the study design is adequate to ensure meaningful results, and the investigators keep the full clinical development picture in mind.

The circadian clock regulates a variety of biological processes that are normally synchronized with the solar day. Disruption of circadian rhythms is associated with health problems. Understanding the signaling mechanisms that couple cell physiology and metabolism to circadian timekeeping will help to develop novel therapeutic strategies. The integrated stress response (ISR) is activated by the cellular stressors to maintain physiological homeostasis by orchestrating mRNA translation. Aberrant ISR has been found in a number of neurological diseases that exhibit disrupted circadian rhythms and sleep. Recent work has started to uncover a critical role for the ISR in regulating the physiology of the circadian clock. Guanabenz (2,6-dichlorobenzylidene aminoguanidine acetate) is an orally bioavailable α2-adrenergic receptor agonist that has been used as an antihypertensive for decades. Recent studies demonstrated that guanabenz can regulate the ISR. Here, we assessed the effects of guanabenz on cellular and behavioral circadian rhythms using a multidisciplinary approach. We found that guanabenz can induce the ISR by increasing eIF2α phosphorylation in cultured fibroblasts as well as in the mouse brain. The hyperphosphorylation of eIF2α by guanabenz is associated with the shortened circadian period in cells and animals and the disruption of behavioral circadian rhythms in mice. Guanabenz administration disrupted circadian oscillations of the clock protein Per1 and Per2 in the mouse suprachiasmatic nucleus, the master pacemaker. These results uncover a significant yet previously unidentified role of guanabenz in regulating circadian rhythms and indicate that exacerbated ISR activation can impair the functions of the brain’s circadian clock by disrupting clock gene expression.

The circadian clock regulates a variety of biological processes that are normally synchronized with the solar day. Disruption of circadian rhythms is associated with health problems. Understanding the signaling mechanisms that couple cell physiology and metabolism to circadian timekeeping will help to develop novel therapeutic strategies. The integrated stress response (ISR) is activated by the cellular stressors to maintain physiological homeostasis by orchestrating mRNA translation. Aberrant ISR has been found in a number of neurological diseases that exhibit disrupted circadian rhythms and sleep. Recent work has started to uncover a critical role for the ISR in regulating the physiology of the circadian clock. Guanabenz (2,6-dichlorobenzylidene aminoguanidine acetate) is an orally bioavailable α2-adrenergic receptor agonist that has been used as an antihypertensive for decades. Recent studies demonstrated that guanabenz can regulate the ISR. Here, we assessed the effects of guanabenz on cellular and behavioral circadian rhythms using a multidisciplinary approach. We found that guanabenz can induce the ISR by increasing eIF2α phosphorylation in cultured fibroblasts as well as in the mouse brain. The hyperphosphorylation of eIF2α by guanabenz is associated with the shortened circadian period in cells and animals and the disruption of behavioral circadian rhythms in mice. Guanabenz administration disrupted circadian oscillations of the clock protein Per1 and Per2 in the mouse suprachiasmatic nucleus, the master pacemaker. These results uncover a significant yet previously unidentified role of guanabenz in regulating circadian rhythms and indicate that exacerbated ISR activation can impair the functions of the brain's circadian clock by disrupting clock gene expression. [ABSTRACT FROM AUTHOR]

Abstract Background We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.

Background: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes. [ABSTRACT FROM AUTHOR]

In veterinary medicine, a number of alpha-2 receptor agonists are marketed as sedatives/hypnotics and analgesics, with their principal use being the chemical restraint of large and small animals. Guanabenz (Wytensin®) is an alpha-2 adrenergic receptor agonist marketed for use in humans as an anti-hypertensive agent. Recent reports indicate that guanabenz has been administered to horses in small doses (0.04 mg/kg) for its anti-hypertensive effects. While this offers both benefits of sedation of the horse as well as amelioration of pulmonary hypertension during running exercise and consequent Exercise-Induced Pulmonary Hemorrhage (EIPH), guanabenz is currently proscribed in most racing jurisdictions and its administration to a racing horse can lead to penalties. The Association of Racing Commissioners International (ARCI) lists guanabenz as an ARCI Class 3 agent; Class 3 agents include bronchodilators, anabolic steroids and other drugs with primary effects on the autonomic nervous system, procaine, antihistamines with sedative properties and diuretics and includes amitraz, clonidine, xylazine, detomidine, medetomidine, and romifidine. Guanabenz is unique among alpha-2 agonists in that it differentiates into E- and Z-forms (Fig. 1), with the Z-form lacking hypotensive properties, yet with both E- and Z-forms able to afford relief to cellular stresses related to inflammation or degenerative diseases. The objective of the study was a preliminary description of the pharmacological properties of guanabenz in comparison with clonidine and a number of other alpha-2 agonists. The goal was clinical evaluation of their sedative, analgesic and related activities with the goal of increasing our understanding of the clinical use of such medications and also as a possible prophylaxis for Exercise- Induced Pulmonary Hemorrhage. The clinical study of guanabenz and clonidine was performed in a complete crossover strategy using quantitative markers of sedation, antinociception, heart rate, blood and urine glucose following administration of each compound in five horses. Amitraz, detomidine, medetomidine, romifidine, and xylazine were studied in one horse each. The sedation was quantified by measuring head droop and locomotor activity, while antinociception was measured by Hoof Withdrawal Reflex Latency. Heart rates, urine glucose, urine production and urine specific gravities were also determined by standard clinical chemistry techniques. Guanabenz serum levels and related urinary guanabenz glucuronide levels were determined by established Liquid Chromatography-tandem Mass Spectrometric (LC-MS) methods. In result the clinically effective doses (0.2 mg/kg) of guanabenz produced a rapid and intense sedative effect, with sagging of the lower lip, sunken eyelids, and marked head droop corresponding to plasma guanabenz concentrations that peaked at 120 ng/mL at 2.5 min post-injection (Fig. 2). The initial head height above the ground is considered 100 %, and head heights fell to values ranging 18–40 % with guanabenz, all of which are greater than a 50 % reduction in head height, considered a full clinically useful sedative effect. Despite the intensity of the sedation, all horses remained standing and were able to walk, and the sedation and head droop responses were rapidly reversed by intravenous administration of the alpha-2 receptor antagonist yohimbine, reversals occurring within 10 min of administration. As a pilot investigation this study was extended to six other members of the alpha-2 agonist group, clonidine administered to five horses, and amitraz, detomidine, medetomidine, romifidine, and xylazine to one horse each. Hoof Withdrawal Reflex Latency evaluation demonstrated the considerable analgesic properties of guanabenz, greater than the corresponding potencies among clonidine, detomidine, romifidine, medetomidine and xylazine. Heart rate monitoring showed guanabenz as possessing capacity for prolonged bradycardia, with effects of a single dose lasting for up to 3.5 hr, in contrast with clonidine (1 hr), amitraz (2 hr), detomidine (<1 hr), medetomidine (1 hr), romifidine (2 hr), and xylazine (<1 hr). Peak urine production following guanabenz administration occurred between 1.5 and 3.0 hr after administration (Fig. 6), as indicated by the steeper decline of the urine volume curve during that period. Urine specific gravity dropped to a low of about 1.006 at 2.0 hr after administration and remained at this level for ~1.0 hr. Urine pH remained at 8, and urine protein was negative throughout testing. The other alpha-2 agonists evaluated also caused an increased urine production with a concomitant decrease in specific gravity. The effect of guanabenz had the longest duration on increased urine volume, lasting about 3.0 hr. Xylazine had the shortest diuretic effect, persisting for only about 1.0 hr. Guanabenz along with romifidine and detomidine induced glucosuria whereas other alpha-2 agonists did not. Hyperglycemia and the corresponding glucosuria resulted in a significant diuresis, as shown by the cumulative urine volume. Guanabenz along with amitraz, detomidine and xylazine also produced measurable sedation presenting as reduced locomotor activity (Table 1). While all alpha-2 agonists showed qualitatively similar pharmacological responses, only guanabenz produced an intense and relatively prolonged antinociceptive response. The study is limited by the number of horses examined (five each for guanabenz and clonidine, five for repeat studies that included yohimbine antagonism, and one each for the other agonists). Study design was focused on clinical evaluation of agonist similarities and differences and thus did not specifically generate data for detailed statistical evaluation. In conclusion these studies show that a 100 mg IV dose of guanabenz rapidly induces clinically useful sedation, analgesia and antinociception effects that are more intense and considerably longer-lasting than those produced by other alpha-2 receptor agonists evaluated. Guanabenz also remains detectable in serum up to 8-hours following administration at doses as low as 0.04 mg/kg. In the work reported here, guanabenz administered at 0.2 mg/kg IV showed peak concentrations in serum of 120 ng/ mL at 2.5 min and was detectable for up to 4 hr with its glucuronide metabolite peaking at 120 min post-administration. Although we did not investigate the combination of guanabenz with opioid drugs such as butorphanol for pain management, guanabenz may well be a drug of choice among the other alpha-2 agonists evaluated in this study for administration with opioids for pain management based on maintaining maximum levels of analgesia for longer periods of time. These experiments suggest considerable clinical potential for guanabenz as a sedative and a relatively long-lasting analgesic in equine medicine. Based on these pharmacological properties, guanabenz and related alpha-2 agonists also have considerable potential for clinical use in equine medicine. [ABSTRACT FROM AUTHOR]

Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of periodontitis. In this study, we investigated whether α2-adrenergic receptor (α2-AR) agonist guanabenz which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympathetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in periodontium were examined using micro-computed tomography and hematoxylin-eosin staining, respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-α, IL1-β, and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity. Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity. TH, TNF-α, IL-1β, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks, were lower than those in control rats having high sympathetic activity. This study demonstrated administration of α2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of sympathetic activity in rats.

The aim of this study was to determine, in the diet-induced obesity model in rats, the potential of Guanabenz to reduce body weight and ameliorate some metabolic disturbances. Obesity was induced in rats by a high-fat diet. After 10 weeks, rats were treated intraperitoneally with Guanabenz at the two doses: 2 or 5 mg/kg b.w./day, once daily for 25 days. The spontaneous activity of rats was measured for 24 h on the 1st and 24th day of the Guanabenz treatment with a special radio-frequency identification system. Gastric emptying was measured in intragastric phenol red-treated mice by measuring the color of the stomach homogenate 30 min after phenol red administration. Intraperitoneal administration of Guanabenz for 25 days to obese rats resulted in a significant decrease in body weight compared to the baseline values (about 11% at a dose of 5 mg/kg). Both body weight and the amount of adipose tissue in the groups receiving Guanabenz decreased to the levels observed in the control rats fed only standard feed. The anorectic effect occurred in parallel with a reduction in plasma triglyceride levels. We also confirmed the beneficial effect of Guanabenz on plasma glucose level. The present study demonstrates that the administration of Guanabenz strongly inhibits gastric emptying (about 80% at a dose of 5 mg/kg). Guanabenz can successfully and simultaneously attenuate all the disorders and risk factors of metabolic syndrome: hypertension, hyperglycemia, obesity, and dyslipidemia. However, the exact cellular mechanisms of its action require further research. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) pathology is characterized by cognitive impairment, increased acetylcholinesterase (AChE) activity, and impaired neuronal communication. Clinically, AChE inhibitors are being used to treat AD patients; however, these remain unable to prevent the disease progression. Therefore, further development of new therapeutic molecules is required having broad spectrum effects on AD-related various neurodegenerative events. Since repurposing is a quick mode to search the therapeutic molecules; henceforth, this study was conducted to evaluate the anti-Alzheimer activity of drug guanabenz which is already in use for the management of high blood pressure in clinics. The study was performed employing both cellular and rat models of AD along with donepezil as reference drug. Guanabenz treatment in both the experimental models showed significant protection against AD-specific behavioral and pathological indicators like AChE activity, tau phosphorylation, amyloid precursor protein, and memory retention. In conjunction, guanabenz also attenuated the AD-related oxidative stress, impaired mitochondrial functionality (MMP, cytochrome-c translocation, ATP level, and mitochondrial complex I activity), endoplasmic reticulum stress (GRP78, GADD153, cleaved caspase-12), neuronal apoptosis (Bcl-2, Bax, cleaved caspase-3), and DNA fragmentation. In conclusion, findings suggested the panoptic protective effect of guanabenz on disease-related multiple degenerative markers and signaling. Furthermore, clinical trial may shed light and expedite the availability of new therapeutic anti-Alzheimer's molecule for the wellbeing of AD patients. [ABSTRACT FROM AUTHOR]

Strong evidence suggests that endoplasmic reticulum stress plays a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through altered regulation of proteostasis. Robust preclinical findings demonstrated that guanabenz selectively inhibits endoplasmic reticulum stress-induced eIF2α-phosphatase, allowing misfolded protein clearance, reduces neuronal death and prolongs survival in in vitro and in vivo models. However, its safety and efficacy in patients with ALS are unknown. To address these issues, we conducted a multicentre, randomized, double-blind trial with a futility design. Patients with ALS who had displayed an onset of symptoms within the previous 18 months were randomly assigned in a 1:1:1:1 ratio to receive 64 mg, 32 mg or 16 mg of guanabenz or placebo daily for 6 months as an add-on therapy to riluzole. The purpose of the placebo group blinding was to determine safety but not efficacy. The primary outcome was the proportion of patients progressing to higher stages of disease within 6 months as measured using the ALS Milano-Torino staging system, compared with a historical cohort of 200 patients with ALS. The secondary outcomes were the rate of decline in the total revised ALS functional rating scale score, slow vital capacity change, time to death, tracheotomy or permanent ventilation and serum light neurofilament level at 6 months. The primary assessment of efficacy was performed using intention-to-treat analysis. The treatment arms using 64 mg and 32 mg guanabenz, both alone and combined, reached the primary hypothesis of non-futility, with the proportions of patients who progressed to higher stages of disease at 6 months being significantly lower than that expected under the hypothesis of non-futility and a significantly lower difference in the median rate of change in the total revised ALS functional rating scale score. This effect was driven by patients with bulbar onset, none of whom (0/18) progressed to a higher stage of disease at 6 months compared with those on 16 mg guanabenz (4/8; 50%), the historical cohort alone (21/49; 43%; P = 0.001) or plus placebo (25/60; 42%; P = 0.001). The proportion of patients who experienced at least one adverse event was higher in any guanabenz arm than in the placebo arm, with higher dosing arms having a significantly higher proportion of drug-related side effects and the 64 mg arm a significantly higher drop-out rate. The number of serious adverse events did not significantly differ between the guanabenz arms and the placebo. Our findings indicate that a larger trial with a molecule targeting the unfolded protein response pathway without the alpha-2 adrenergic related side-effect profile of guanabenz is warranted. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it’s urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules.

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it's urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules. [ABSTRACT FROM AUTHOR]

Limited therapeutic efficacy of temozolomide (TMZ) against glioblastomas highlights the importance of exploring new drugs for clinical therapy. Sunitinib, a multitargeted receptor tyrosine kinase inhibitor, is currently being tested as therapy for glioblastomas. Unfortunately, sunitinib still has insufficient activity to cure glioblastomas. Our aim was to determine the molecular mechanisms counteracting sunitinib drug sensitivity and find potential adjuvant drugs for glioblastoma therapy. Through in vitro experiments, transcriptome screening by RNA sequencing, and in silico analyses, we found that sunitinib induced glioma apoptotic death, and downregulated genes were enriched in oncogenic genes of glioblastoma. Meanwhile, sunitinib-upregulated genes were highly associated with the protective autophagy process. Blockade of autophagy significantly enhanced sunitinib's cytotoxicity. Growth arrest and DNA damage-inducible protein (GADD) 34 was identified as a candidate involved in sunitinib-promoted autophagy through activating p38-mitogen-activated protein kinase (MAPK) signaling. Higher GADD34 levels predicted poor survival of glioblastoma patients and induced autophagy formation in desensitizing sunitinib cytotoxicity. Guanabenz, an alpha2-selective adrenergic agonist and GADD34 functional inhibitor, was identified to enhance the efficacy of sunitinib by targeting GADD34-induced protective autophagy in glioblastoma cells, TMZ-resistant cells, hypoxic cultured cells, sphere-forming cells, and colony formation abilities. A better combined treatment effect with sunitinib and guanabenz was also observed by using xenograft mice. Taken together, the sunitinib therapy combined with guanabenz in the inhibition of GADD34-enhanced protective autophagy may provide a new therapeutic strategy for glioblastoma. [ABSTRACT FROM AUTHOR]

Polyglutamine expansion diseases are a group of hereditary neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders, such as Huntington's disease, dentatorubral–pallidoluysian atrophy, spinobulbar muscular atrophy and multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited SCA is the type 3 (SCA3), also known as Machado–Joseph disease (MJD), which is an autosomal dominant, progressive neurological disorder. The gene causatively associated with MJD is ATXN3. Recent studies have shown that this gene modulates endoplasmic reticulum (ER) stress. We generated transgenic Caenorhabditis elegans strains expressing human ATXN3 genes in motoneurons, and animals expressing mutant ATXN3-CAG89 alleles showed decreased lifespan, impaired movement, and rates of neurodegeneration greater than wild-type ATXN3-CAG10 controls. We tested three neuroprotective compounds (Methylene Blue, guanabenz and salubrinal) believed to modulate ER stress and observed that these molecules rescued ATXN3-CAG89 phenotypes. Furthermore, these compounds required specific branches of the ER unfolded protein response (UPRER), reduced global ER and oxidative stress, and polyglutamine aggregation. We introduce new C. elegans models for MJD based on the expression of full-length ATXN3 in a limited number of neurons. Using these models, we discovered that chemical modulation of the UPRER reduced neurodegeneration and warrants investigation in mammalian models of MJD.

ABSTRACT Toxoplasma gondii is an intracellular parasite that has infected one-third of humans. The infection is permanent because the replicative form (tachyzoite) converts into a latent tissue cyst form (bradyzoite) that evades host immunity and is impervious to current drugs. The continued presence of these parasitic cysts hinders treatment and leads to chronic infection that has been linked to behavioral changes in rodents and neurological disease in humans. How these behavioral changes occur, and whether they are due to parasite manipulation or the host response to infection, remains an outstanding question. We previously showed that guanabenz possesses antiparasitic activity; here, we show that guanabenz reproducibly lowers brain cyst burden up to 80% in chronically infected male and female BALB/cJ mice when given intraperitoneally but not when administered by gavage or in food. Regardless of the administration route, guanabenz reverses Toxoplasma-induced hyperactivity in latently infected mice. In contrast, guanabenz increases cyst burden when given to chronically infected C57BL/6J mice yet still reverses Toxoplasma-induced hyperactivity. Examination of the brains from chronically infected BALB/cJ and C57BL/6J mice shows that guanabenz decreases inflammation and perivascular cuffing in each strain. Our study establishes a robust model for cyst reduction in BALB/cJ mice and shows for the first time that it is possible to reverse a key behavioral change associated with latent toxoplasmosis. The rescue from parasite-induced hyperactivity correlates with a decrease in neuroinflammation rather than reduced cyst counts, suggesting that some behavioral changes arise from host responses to infection. IMPORTANCE Toxoplasma gondii is a common parasite of animals, including up to one-third of humans. The single-celled parasite persists within hosts for the duration of their life as tissue cysts, giving rise to chronic infection. Latent toxoplasmosis is correlated with neurological dysfunction in humans and results in dramatic behavioral changes in rodents. When infected, mice and rats adapt behaviors that make them more likely to be devoured by cats, the only host that supports the sexual stage of the parasite. In this study, we establish a new mouse model of tissue cyst depletion using a drug called guanabenz and show that it is possible to reverse a key behavior change back to normal in infected animals. We also show that the mechanism appears to have nothing to do with parasite cyst burden but rather the degree of neuroinflammation produced by chronic infection.

Abstract—The body's response to stress is mediated by the interaction of catecholamines with adrenergic α- and β-receptors. It has been established that, in contrast to α1- and β-adrenoceptors, α2-adrenoceptors are involved in feedback loops of the simpatoadrenal system, which controls the release of catecholamines (norepinephrine and epinephrine) to save energy resources and the function of peripheral organs under an overload of catecholamines. Violations of the feedback mechanisms lead to various pathologies, including hypertrophic heart remodeling, followed by the development of heart failure. We previously found the expression of α2-adrenoreceptors in the plasma membrane of cardiomyocytes, where they participate in the regulation of the intracellular level of Ca2+. We suggested that these receptors are able to locally control the response of the heart muscle to stress in addition to the traditional mechanism. We have shown in this study that sarcolemmal α2-adrenoreceptors attenuate the positive inotropic effect of adrenergic stimulation in papillary muscles by controlling the level of free Ca2+ in cytosol. The ability of α2-adrenoreceptor agonists to suppress intracellular spontaneous Ca2+ oscillations increases the efficiency of systolic function of papillary muscles and reduces the likelihood of dangerous ventricular arrhythmia. Thus, α2-adrenoreceptors in the sarcolemmal membrane of cardiomyocytes have their own protective potential, which can prevent pathological remodeling of the heart muscle under chronic stress. [ABSTRACT FROM AUTHOR]

Alpha 2 (α 2 -) adrenoceptor agonists, such as clonidine or dexmedetomidine, have been found to inhibit hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels, not only by reducing intracellular cyclic AMP levels but also by directly blocking HCN channels. In this study, we examined the inhibitory effect of guanabenz, a centrally acting α 2 -adrenoceptor agonist with high specificity for α 2A -subtype, on HCN channels in mesencephalic trigeminal nucleus (MTN) neurons which robustly express HCN channels and have been suggested to coexpress α 2A -adrenoceptors. By performing whole-cell patch-clamp recording on MTN neurons in brainstem slices, hyperpolarization-activated inward current (I h) was examined during guanabenz treatment. Guanabenz inhibited I h in a dose-dependent manner, which was likely to be ZD7288-sensitive HCN current as it did not affect barium-sensitive inward rectifying potassium current. Guanabenz not only inhibited I h but also shifted the voltage-dependent activation curve to hyperpolarizing potentials. Interestingly, I h inhibition by guanabenz was not reversed by α 2 -adrenoceptor antagonist atipamezole treatment or by intracellular cyclic AMP perfusion, suggesting that the inhibition may not result from α 2A -adrenoceptor signalling pathway but from direct inhibition of HCN channels. Coherent to our electrophysiological results, single-cell RT-PCR revealed that most MTN neurons lack α 2A -adrenoceptor mRNA. Our study demonstrates that guanabenz can directly inhibit HCN channels in addition to its primary role of activating α 2A -adrenoceptors. [ABSTRACT FROM AUTHOR]

α2-Adrenoceptors (α2-AR) found in the cardiomyocyte's sarcolemma represent a very important negative feedback for control of myocardial contractility by endogenous catecholamines. Earlier, we showed that the endogenous neurotransmitter agmatine in micromolar concentrations via α2-AR activates the nitric oxide (NO) synthesis, enhancing the Ca2+ pumping into sarcoplasmic reticulum (SR). In the millimolar doses it inhibits Ca2+ sequestration by SR Ca2+ ATPase (SERCA), acting through the first type of imidazoline receptors. Here, we study the functional activity of agmatine, as well as a specific α2-agonist, guanabenz, in respect to spontaneous Ca2+-transients in SHR cardiomyocytes of the early age (2–2.5 months), and adulthood animals (8–9 months). α2-mediated cardioprotective effect was almost twofold decreased in SHR cardiac cells compared to normotensive rats of the corresponding age, despite the fact that both α2A- and α2B-AR protein levels were significantly increased in SHR cardiomyocytes. NO-mediated facilitation of SERCA activity is substantially reduced in SHR cardiomyocytes vs. normotensive rats. These data suggest that the SHR phenotype starting from early age shows signs of the impaired sarcolemmal α2-AR signaling, which can aggravate the development of this cardiovascular pathology. • α2AR signaling is impaired in SHR cardiomyocytes starting from 2 months old. • α2AR-mediated NO synthesis is substantially decreased in SHR vs. normotensive rats. • NO-mediated facilitation of SERCA is practically abrogated in SHR cardiomyocytes. • Protein levels of α2A-AR and α2B-AR subtypes are heightened in SHR cardiomyocytes. • Impaired α2AR-signaling may relative to the deterioration of myocardial functions in SHR. [ABSTRACT FROM AUTHOR]

Aim: Vanishing White Matter (VWM) is a devastating leucoencephalopathy without effective treatment options. Patients have mutations in the EIF2B1-5 genes, encoding the five subunits of eIF2B, a guanine exchange factor that is an important regulator of protein translation. We recently developed mouse models for VWM that replicate the human disease. To study disease improvement after treatment in these mice, it is essential to have sensitive biomarkers related to disease stage. The Bergmann glia of the cerebellum, an astrocytic subpopulation, translocate into the molecular layer in symptomatic VWM mice and patients. This study looked at the prospects of using Bergmann glia pathology as an objective disease marker for VWM. Methods: We defined a new quantitative measurement of Bergmann glia pathology in the cerebellum of VWM mice and patients. To test the sensitivity of this new marker for improvement, VWM mutant mice received long-term treatment with Guanabenz, an FDAapproved anti-hypertensive agent affecting eIF2B activity. Results: Bergmann glia translocation was significantly higher in symptomatic VWM mice and VWM patients than in controls and worsened over the disease course. Both Bergmann glia pathology and cerebellar myelin pathology improved with Guanabenz treatment in mice, showing that Bergmann glia translocation is a sensitive measurement for improvement. Conclusions: Bergmann glia translocation can be used to objectively assess effects of treatment in VWM mice. Future treatment strategies involving compounds regulating eIF2 phosphorylation might benefit VWM patients. [ABSTRACT FROM AUTHOR]

Reduced function of parkin appears to be a central pathogenic event in Parkinson disease (PD). Increasing parkin levels enhances survival in models of PD-related neuronal death and is a promising therapeutic objective. Previously, we demonstrated that the transcription factor ATF4 promotes survival in response to PD-mimetic stressors by maintaining parkin levels. ATF4 translation is up-regulated by phosphorylation of the translation initiation factor eIF2α. The small molecule guanabenz enhances eIF2α phosphorylation by blocking the function of GADD34, a regulatory protein that promotes eIF2α dephosphorylation. We tested the hypothesis that guanabenz, by inhibiting GADD34 and consequently increasing eIF2α phosphorylation and elevating ATF4, would improve survival in models of PD by up-regulating parkin. We found that GADD34 is strongly induced by 6-OHDA, and that GADD34 localization is dramatically altered in dopaminergic substantia nigra neurons in PD cases. We further demonstrated that guanabenz attenuates 6-hydroxydopamine (6-OHDA) induced cell death of differentiated PC12 cells and primary ventral midbrain dopaminergic neurons in culture, and of dopaminergic neurons in the substantia nigra of mice. In culture models, guanabenz also increases eIF2α phosphorylation and ATF4 and parkin levels in response to 6-OHDA. Furthermore, if either ATF4 or parkin is silenced, then the protective effect of guanabenz is lost. We also found similar results in a distinct model of neuronal death: primary cultures of cortical neurons treated with the topoisomerase I inhibitor camptothecin, in which guanabenz limited camptothecin-induced neuronal death in an ATF4- and parkin-dependent manner. In summary, our data suggest that guanabenz and other GADD34 inhibitors could be used as therapeutic agents to boost parkin levels and thereby slow neurodegeneration in PD and other neurodegenerative conditions. [ABSTRACT FROM AUTHOR]

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure.

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure. [ABSTRACT FROM AUTHOR]

Chronic toxoplasmosis which is positively correlated with many neuropsychiatric problems has no curative treatment till now; due to the resistant tissue cysts especially in the brain. In search of an effective treatment, guanabenz-loaded polyethylene glycol poly lactic-co-glycolic acid (PEG-PLGA) nanoparticles was evaluated against chronic experimental toxoplasmosis. For this purpose, each mouse was infected with 10 cysts of Toxoplasma gondii (ME 49 strain). Treated mice received either guanabenz alone (5 mg/kg/day) in subgroup IIa or guanabenz-loaded nanoparticles by full dose in subgroup IIb or guanabenz-loaded nanoparticles by the half dose (2.5 mg/kg/day) in subgroup IIc. Subgroup Ie was treated by pyrimethamine and sulfadiazine. The treatment started on day 25 post-infection for 19 successive days. Then Parasitological, histopathological, immunohistochemical, immunological and ultrastructural morphological studies were performed. The results showed that: subgroup IIb showed the highest statistically significant reduction in the neuroinflammation and brain tissue cysts (77%) with a significant higher efficacy in comparison with pyrimethamine and sulfadiazine and showed the highest level of IFN-γ, while the lowest level was in subgroup IIa. All group II mice showed similar changes of depression and compression of the wall of the cyst. This is marked in subgroup IIb with release of crescent shaped bradyzoite outside the cyst. PEG-PLGA nanoparticles had no toxic effect on the liver or the kidney of the mice. It could be concluded that guanabenz-loaded PEG-PLGA nanoparticles could be promising and safe for treatment of chronic toxoplasmosis. Chronic toxoplasmosis which is positively correlated with many neuropsychiatric problems has no curative treatment till now; due to the resistant tissue cysts. Guanabenzloaded polyethylene glycol poly lacticco-glycolic acid (PEG-PLGA) nanoparticles caused significant brain tissue cysts reduction (77%) in comparison with pyrimethamine and sulfadiazine combination therapy. [Display omitted] • Guanabenz repurposing as an anti- Toxoplasma drug with higher efficacy when incorporated with PEG-PLGA nanoparticles. • Guanabenz-loaded nanoparticles achieved highly significant Toxoplasma brain cyst reduction with reduced neuroinflammation. • Guanabenz-loaded nanoparticles caused marked compression of the cyst wall with release of crescent shaped bradyzoite. • Guanabenz-loaded nanoparticles had an immune stimulant effect by increasing mean interferon gamma level. • PEG-PLGA nanoparticles had no toxic effect on the liver or the kidney. [ABSTRACT FROM AUTHOR]

Salubrinal and guanabenz are two known inhibitors of dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), and they suppress osteoclastogenesis through downregulating nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master molecule of osteoclastogenesis. The mechanism of NFATc1 suppression is not well understood. Using genome-wide microarray analysis, we investigated molecular regulators of osteoclastogenesis, in particular, in response to salubrinal and guanabenz. We identified two sets of genes: a set of genes that were upregulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and downregulated by salubrinal and guanabenz; and the other set of genes that were downregulated by RANKL and upregulated by salubrinal and guanabenz. The microarray and qPCR result revealed that a zinc finger protein, FYVE domain containing 21 (Zfyve21), as well as DNA-damage-inducible transcript 4 (Ddit4), were suppressed by RANKL and upregulated by salubrinal and guanabenz. A partial silencing of Zfyve21 or Ddit4 attenuated salubrinal- and guanabenz-driven suppression of NFATc1. Collectively, this study demonstrates that Zfyve21 and Ddit4 are two inhibitors of osteoclastogenesis. We expect that they may potentially serve as novel targets for preventing bone loss from skeletal diseases such as osteoporosis. [ABSTRACT FROM AUTHOR]

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N -(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection ( Fraser et al., 2014b ). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R–like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic. [ABSTRACT FROM AUTHOR]

Integrated stress responses (ISR) may lead to cell death and tissue degeneration via eukaryotic translation initiation factor 2 α (eIF2α)-mediated signaling. Alleviating ISR by modulating eIF2α phosphorylation can reduce the symptoms associated with various diseases. Guanabenz is known to elevate the phosphorylation level of eIF2α and reduce pro-inflammatory responses. However, the mechanism of its action is not well understood. In this study, we investigated the signaling pathway through which guanabenz induces anti-inflammatory effects in immune cells, in particular macrophages. Genome-wide mRNA profiling followed by principal component analysis predicted that colony stimulating factor 2 (Csf2, or GM-CSF as granulocyte macrophage colony stimulating factor) is involved in the responses to guanabenz. A partial silencing of Csf2 or eIF2α by RNA interference revealed that Interleukin-6 (IL6), Csf2, and Cyclooxygenase-2 (Cox2) are downregulated by guanabenz-driven phosphorylation of eIF2α. Although expression of IL1β and Tumor Necrosis Factor-α (TNFα) was suppressed by guanabenz, their downregulation was not directly mediated by eIF2α signaling. Collectively, the result herein indicates that anti-inflammatory effects by guanabenz are mediated by not only eIF2α-dependent but also eIF2α-independent signaling. [ABSTRACT FROM AUTHOR]

Supraphysiological concentrations (SPCs) of triiodo-L-thyronine (T3) have been used in the treatment of a number of nonviral diseases. However, the signaling mechanisms that regulate the function of T3 at these concentrations and their role in modulating cellular stress pathways and antiviral responses are unknown. Here, we have investigated the effects of SPCs of T3 on integrated stress response (ISR) signaling pathways and the replication of vesicular stomatitis virus (VSV). T3 amplified Poly IC-induced activation of RNA-dependent protein kinase, induced phosphorylation of eIF2α, stress granule (SG) formation, IRE1α phosphorylation, XBP1 splicing, and the expression of stress markers. T3 inhibited VSV replication by modulating SG formation and the expression of stress response markers. ISR activator guanabenz also inhibited VSV replication and amplified T3-induced anti-VSV response. To summarize, we have uncovered novel functions of T3 at SPCs as an activator of ISR signaling pathways and an inhibitor of VSV replication. This study offers a proof of principle of the concept that ISR activating agents like SPC of T3 and guanabenz can be potential antiviral agents. [ABSTRACT FROM AUTHOR]

Non-adrenergic ligands that bind to imidazoline receptors (I-R), a selective ligand that binds to alpha2-adrenoceptors (alpha2-AR) and mixed ligands that bind to both receptors were tested for their action on water intake behavior of 24-h water-deprived rats. All drugs were injected into the third cerebral ventricle. Except for agmatine (80 nmol), mixed ligands binding to I-R/alpha2-AR such as guanabenz (40 nmol) and UK 14304 (20 nmol) inhibited water intake by 65% and up to 95%, respectively. The selective non-imidazoline alpha2-AR agonist, alpha-methylnoradrenaline, produced inhibition of water intake similar to that obtained with guanabenz, but at higher doses (80 nmol). The non-adrenergic I-R ligands histamine (160 nmol, mixed histaminergic and imidazoline ligand) and imidazole-4-acetic acid (80 nmol, imidazoline ligand) did not alter water intake. The results show that selective, non-imidazoline alpha2-AR activation suppresses water intake, and suggest that the action on imidazoline sites by non-adrenergic ligands is not sufficient to inhibit water intake.

Inactivating bone-resorbing osteoclasts is a prime therapeutic strategy for the prevention of bone loss in patients with osteopenia and osteoporosis. Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disease characterized by the loss of motor neurons in the motor cortex, brain stem and spinal cord. Currently, there is no cure for this lethal disease. Although the mechanism underlying neuronal cell death in ALS remains elusive, growing evidence supports a crucial role of endoplasmic reticulum (ER) stress in the pathogenesis of ALS. Recent reports show that guanabenz, a novel inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, possesses anti-prion properties, attenuates ER stress and reduces paralysis and neurodegeneration in mTDP-43 Caenorhabditis elegans and Danio rerio models of ALS. However, the therapeutic potential of guanabenz for the treatment of ALS has not yet been assessed in a mouse model of ALS. In the present study, guanabenz was administered to a widely used mouse model of ALS expressing copper zinc superoxide dismutase-1 (SOD1) with a glycine to alanine mutation at position 93 (G93A). The results showed that the administration of guanabenz significantly extended the lifespan, delayed the onset of disease symptoms, improved motor performance and attenuated motor neuron loss in female SOD1 G93A mice. Moreover, western blotting results revealed that guanabenz dramatically increased the levels of phosphorylated-eIF2α (P-eIF2α) protein, without affecting total eIF2α protein levels. The results also revealed a significant decrease in the levels of the ER chaperone glucose-regulated protein 78 (BiP/Grp78) and markers of another two ER stress pathways, activating transcription factor 6α (ATF6α) and inositol-requiring enzyme 1 (IRE1). In addition, guanabenz increased the protein levels of anti-apoptotic B cell lymphoma/lewkmia-2 (Bcl-2), and down-regulated the pro-apoptotic protein levels of C/EBP homologous protein (CHOP), Bcl-2-associated X protein (BAX) and cytochrome C in SOD1 G93A mice. Our findings indicate that guanabenz may represent a novel therapeutic candidate for the treatment of ALS, a lethal human disease with an underlying mechanism involving the attenuation of ER stress and mitochondrial stress via prolonging eIF2α phosphorylation. [ABSTRACT FROM AUTHOR]

In response to various stresses including viral infection, nutrient deprivation, and stress to the endoplasmic reticulum, eukaryotic translation initiation factor 2 alpha (eIF2α) is phosphorylated to cope with stress induced apoptosis. Although bone cells are sensitive to environmental stresses that alter the phosphorylation level of eIF2α, little is known about the role of eIF2α mediated signaling during the development of bone-resorbing osteoclasts. Using two chemical agents (salubrinal and guanabenz) that selectively inhibit de-phosphorylation of eIF2α, we evaluated the effects of phosphorylation of eIF2α on osteoclastogenesis of RAW264.7 pre-osteoclasts as well as development of MC3T3 E1 osteoblast-like cells. The result showed that salubrinal and guanabenz stimulated matrix deposition of osteoblasts through upregulation of activating transcription factor 4 (ATF4). The result also revealed that these agents reduced expression of the nuclear factor of activated T cells c1 (NFATc1) and inhibited differentiation of RAW264.7 cells to multi-nucleated osteoclasts. Partial silencing of eIF2α with RNA interference reduced suppression of salubrinal/guanabenz-driven downregulation of NFATc1. Collectively, we demonstrated that the elevated phosphorylation level of eIF2α not only stimulates osteoblastogenesis but also inhibit osteoclastogenesis through regulation of ATF4 and NFATc1. The results suggest that eIF2α-mediated signaling might provide a novel therapeutic target for preventing bone loss in osteoporosis. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Activation of brain α2-adrenoceptors in conscious rodents decreases heart rate (HR) and mean arterial blood pressure (MAP) and increases urine output and urinary sodium excretion. In vitro, α2-adrenoceptor stimulation activates Gαi(1-3), Gαo and Gαs-subunit protein-gated signal transduction pathways. Here we have investigated whether these same Gα-subunit protein-gated pathways mediate the cardiovascular and renal excretory responses to central α2-adrenoceptor activation in conscious Sprague-Dawley rats. EXPERIMENTAL APPROACH Rats were pre-treated by intracerebroventricular injection (i.c.v.) with an oligodeoxynucleotide (ODN) targeted to a Gαi1, Gαi2, Gαi3, Gαo, Gαs or a scrambled (SCR) ODN sequence (25 µg, 24 h). On the day of study, the α2-adrenoceptor agonist guanabenz (50 µg) or saline vehicle, was injected i.c.v. into ODN-pre-treated conscious rats. MAP and HR were recorded, and urine was collected for 150 min. KEY RESULTS In vehicle- and SCR ODN-pre-treated rats, i.c.v. guanabenz decreased MAP and HR, and produced marked diuretic and natriuretic responses. Selective ODN-mediated down-regulation of brain Gαi2-subunit proteins abolished the central guanabenz-induced hypotension and natriuresis. In contrast, following selective Gαs down-regulation, the characteristic hypotensive response to i.c.v. guanabenz was converted to an immediate increase in MAP. The bradycardic and diuretic responses to i.c.v. guanabenz were not blocked by pre-treatment with any ODN. CONCLUSIONS AND IMPLICATIONS There was functional selectivity of Gαi2 and Gαs subunit protein-gated signal transduction pathways in mediating the hypotensive and natriuretic, but not bradycardic or diuretic, responses evoked by central α2-adrenoceptor activation in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Increased central sympathetic outflow secondary to afferent sympathetic excitation has been implicated in the hypertensive effect of the immunosuppressant drug cyclosporine (CSA). The present study investigated the roles of central α2-adrenoceptors and I1-imidazoline receptors in modulating the hypertensive action of CSA. The blood pressure (BP) response to CSA in conscious rats was assessed in the absence and presence of peripherally or centrally acting sympatholytic drugs. Also, the effect of selective pharmacologic blockade of α2 or I1 receptors by yohimbine and efaroxan, respectively, on the pressor response to CSA was evaluated. CSA (20 mg/kg i.v.) produced a rapid increase in BP that peaked (25±4 mm Hg) after approximately 4 min and continued for the 45 min study duration. Ganglionic (hexamethonium 20 mg/kg) or α1-adrenoceptor (prazosin 1 mg/kg) blockade reduced the pressor effect of CSA. Pressor responses to phenylephrine (α1-adrenoceptor agonist) were not affected by CSA, thereby eliminating a possible role for alterations of vascular α1-adrenoceptor responsiveness in CSA hypertension. CSA hypertension was attenuated in rats pretreated intravenously with drugs that reduce central sympathetic tone including clonidine (mixed α2/I1-receptor agonist, 30 μg/kg) or moxonidine (selective I1-receptor agonist, 100 μg/kg) in contrast to no effect for guanabenz (selective α2-receptor agonist, 30 μg/kg). Intracisternal (i.c.) administration of moxonidine also reduced CSA hypertension. Selective blockade of central I1 (efaroxan, 0.15 μg/rat, i.c.) but not α2 (yohimbine, 25 μg/5 μl/rat, i.c.) receptors abolished the hypertensive response to CSA. Together, these findings highlight that CSA elicits its hypertensive effect via disruption of central sympathoinhibitory pathways which include I1-imidazoline receptors. [Copyright &y& Elsevier]

Abstract: In cystic fibrosis respiratory epithelial cells, the absence or dysfunction of the chloride channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator) results in reduced chloride ion transport. In contrast, Ca2+-stimulated Cl− secretion is intact in cystic fibrosis airway epithelia. One possible target for drug discovery aiming at treating cystic fibrosis is to correct the ionic imbalance through stimulation of alternative ionic pathways that may compensate the failure of epithelial Cl− conductance. Here, using a simple high-throughput screening assay to search for Cl− channels modulators in the cystic fibrosis nasal epithelial cell line JME-CF15, the compound guanabenz (Wytensin®), an α2-selective adrenergic agonist was found positive. Using iodide effluxes and electrophysiological recordings, we showed that guanabenz-activated (EC50 =831 nM) a DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) sensitive and Ca2+ dependent Cl− channel (CaCC). Guanabenz activated a linear Cl− channel with unitary single-channel conductance of 8 pS. Recording calcium signals in CF15 cells showed that guanabenz increased the intracellular Ca2+ concentration stimulating an influx of Ca2+. In the absence of extracellular Ca2+, the guanabenz effects on Ca2+ influx and activation of CaCC were both abolished. These data demonstrate that guanabenz activates Ca2+-dependent Cl− channels via a Ca2+ influx in human cystic fibrosis airway epithelial cells. [Copyright &y& Elsevier]

Brain stem noradrenergic cell groups mediating autonomic responses to stress project to airway-related vagal preganglionic neurons (AVPNs). In ferrets, their activation produces withdrawal of cholinergic outflow to the airways via release of norepinephrine and activation of α2A-adrenergic receptors (α2A-AR) expressed by AVPNs. In these studies, we examined the effects of allergen exposure of the airway (AE) with ovalbumin on noradrenergic transmission regulating the activity of AVPNs and, consequently, airway smooth muscle tone. Experiments were performed in vehicle control (Con) and AE ferrets. Microperfusion of an α2A-AR agonist (guanabenz) in close proximity to AVPNs elicited more pronounced effects in Con than AE ferrets, including a decrease in unit activity and reflexly evoked responses of putative AVPN neurons with a corresponding decrease in cholinergic outflow to the airways. Although no differences were found in the extent of noradrenergic innervation of the AVPNs, RT-PCR and Western blot studies demonstrated that AE and repeated exposure to antigen significantly reduced expression of α2A-ARs at message and protein levels. These findings indicate that, in an animal model of allergic asthma, sensitization and repeated challenges with a specific allergen diminish central inhibitory noradrenergic modulation of AVPNs, possibly via downregulation of α2A-AR expression by these neurons. [ABSTRACT FROM AUTHOR]

Agonists active at I1-imidazoline receptors (I1R) not only lower blood pressure but also ameliorate glucose intolerance, insulin resistance, and hyperlipidemia with long-term treatment. We sought to determine the possible mechanism for the lipid-lowering actions of imidazolines in a model of metabolic Syndrome X, the spontaneously-hypertensive obese (SHROB) rat. The acute actions of moxonidine and rilmenidine, selective I1R agonists, were compared to a specific α2-adrenergic receptor agonist, guanabenz, with and without selective receptor blockers. Moxonidine and rilmenidine rapidly reduced plasma triglyceride (20±4% and 21±5%, respectively) and cholesterol (29±9% and 27±9%). In contrast, the specific α2-adrenergic receptor agonist guanabenz failed to reduce plasma lipids. Blocking experiments showed that moxonidine’s actions were mediated by I1R and not α2-adrenergic receptors. To evaluate a hepatic site of action, radioligand binding studies with liver plasma membranes confirmed the presence of I1R. Intraportal moxonidine reduced plasma triglycerides by 23±3% within 10 min. Moxonidine inhibited hepatic triglyceride secretion by 75% compared to vehicle treatment. Tracer studies with 2H2O suggested that moxonidine inhibits de novo fatty acid synthesis. Thus, activation of I1R lowers plasma lipids, with the main site of action probably within the liver to reduce synthesis and secretion of triglycerides. More selective I1R agonists might provide monotherapy for hyperlipidemic hypertension. [ABSTRACT FROM AUTHOR]

The activation of α2-adrenoceptors has been reported to impair memory functions in both rats and humans. The α2-adrenoceptor subtype responsible for this detrimental effect is still unknown. The effect of the α2-agonists clonidine and guanabenz on memory processes, in dependence to the time of administration, was evaluated in the mouse passive avoidance test. Clonidine (0.02–0.2 mg kg-1 i.p.) and guanabenz (0.1–0.3 mg kg-1 i.p.) induced amnesia in a dose-dependent manner. From time–course experiments emerged that the impairment of memory function was detectable only when clonidine and guanabenz were administered 60 min before or immediately after the training test, respectively. This detrimental effect was prevented by pretreatment with the α2-antagonist yohimbine (1–3 mg kg-1 i.p.) and by the α2A-antagonist BRL-44408 (0.3–1 mg kg-1 i.p.). By contrast, the α2B,C antagonists ARC-239 (10 mg kg-1 i.p.) and prazosin (1 mg kg-1 i.p.) did not revert the amnesia induced by both clonidine and guanabenz. At the highest effective doses, clonidine and guanabenz were devoid of behavioral side-effects as well as maintained unaltered the motor coordination, as revealed by the rota-rod test. Furthermore, none of the compounds used modified the spontaneous motility as indicated by the Animex apparatus. These results indicate that clonidine and guanabenz impaired memory processes in a mouse passive avoidance paradigm through the selective activation of the α2A-adrenoceptor subtype. [Copyright &y& Elsevier]

The post-receptorial mechanism of the amnesic action of the α2-agonists clonidine and guanabenz was investigated in the mouse passive avoidance test. Animals were i.c.v. injected with pertussis toxin (PTX) or with antisense oligonucleotides, complementary to the sequence of the α-subunit mRNA of Gi1, Gi2, Gi3, Go1 and Go2 proteins. The administration of PTX (0.25 μg per mouse i.c.v.) reversed the amnesia induced by both α2-agonists. Similarly, anti-Giα1 (6.25–12.5 μg per mouse i.c.v.), anti-Giα3 (3.12–12.5 μg per mouse i.c.v.), anti-Goα1 (12.5–25 μg per mouse i.c.v.) antagonised the detrimental effect induced by clonidine and guanabenz. By contrast, pretreatment with anti-Giα2 (3.12–25 μg per mouse i.c.v.) and anti-Goα2 (12.5–25 μg per mouse i.c.v.) never modified the impairment of memory processes induced by the α2-agonists. At the highest effective doses, none of the compounds used impaired motor coordination (rota rod test), nor modified spontaneous motility and inspection activity, (hole board test). These results indicate the involvement of Gi1, Gi3, and Go1, but not Gi2 and Go2, protein subtypes in the transduction mechanism responsible for the induction of amnesia by clonidine and guanabenz. [Copyright &y& Elsevier]

Procyclidine and ethopropazine, widely used as anti-parkinsonian agents because of their anti-cholinergic action, are also known to have NMDA antagonist properties. Unlike other NMDA antagonists, these agents—because of their anti-cholinergic action—are devoid of neurotoxic side effects. In the present study, we used a sciatic nerve ligation model that produces a hyperalgesic (neuropathic pain) state in adult rats to evaluate the ability of procyclidine or ethopropazine, either alone or in combination with an α2 adrenergic agonist, to ameliorate neuropathic pain. We found that both procyclidine and ethopropazine alleviated thermal hyperalgesia in a dose dependent manner; when a marginally effective dose of these agents was combined with an ineffective dose of an α2 adrenergic agonist (clonidine or guanabenz), the combination therapy provided effective and long-lasting relief from neuropathic pain. In addition, the combination therapy was free from neurotoxic or behavioral side effects, and hyperactivity, a side effect associated with procyclidine monotherapy, was counteracted by clonidine. [Copyright &y& Elsevier]

Moxonidine is a centrally-active imidazoline compound with preferential affinity for imidazoline receptors (IR) over α2-adrenoceptors (α2AR). Clinically, moxonidine has proven advantageous for treating hypertension over pure α2-adrenergic agonists (i.e., guanabenz) due to its lowered incidence of sedative side effects. The present experiments reveal divergent behavioral effects of low doses of moxonidine and guanabenz in C57Bl/6 mice in an exploratory arena. Low-dose moxonidine (0.05 mg kg−1 i.p.) elicited an increase in novel object contacts (+36%) and more movement into central space (+56%; P<0.01) compared to saline-injected controls; whereas guanabenz induced only dose-responsive sedative-like behaviors in the same paradigm. Yet, the two agonists were indistinguishable in terms of blood pressure changes over a similar dose range (0.025–0.1 mg kg−1 i.p.) in consciously free-moving mice (Δ mean±S.E.M.=−12.3±3.2 mm Hg for moxonidine versus −13.5±1.9 mm Hg for guanabenz). As expected of α2AR involvement, the sedative-like effects of guanabenz were completely blocked by pretreatment with the non-imidazoline α2AR-antagonist, SKF86466 (0.5 or 1.0 mg kg−1 i.p.). However, the pro-exploratory effects of low doses of moxonidine (0.05 or 0.1 mg kg−1) were not antagonized by SKF86466. These results suggest that moxonidine acts preferentially through a non-adrenergic mechanism, possibly IR-mediated, to elicit pro-exploratory behavior. [Copyright &y& Elsevier]

Oxidative stress and mitochondrial dysfunction are important aspects of pathogenesis, particularly in the brain, which is highly dependent on oxygen, and the protection of astrocytes is essential for neuroprotection. In this context, imidazoline drugs have been reported to be neuroprotective. Our recent study showed that imidazoline drugs, including guanabenz, inhibit the naphthazarin-induced oxidative cytotoxicity associated with lysosomal destabilization. We now report on a study into the protective effects of rilmenidine and AGN 192403, which have affinity for imidazoline-1 receptors, on the cytotoxicity induced by naphthazarin and inhibitors of mitochondrial respiration in astrocytes. Cytotoxicity was measured grossly by LDH release and by measuring changes in lysosomal membrane stability and features of mitochondrial membrane permeabilization. Naphthazarin-induced cytotoxicity was evidenced by the ordered development of lysosomal acridine orange relocation, decrease in mitochondrial potential, cytochrome c release, and caspase-9 activation, and was inhibited by guanabenz, rilmenidine, and AGN 192403. Antimycin A and rotenone induced mitochondrial dysfunction primarily, and their cytotoxicities were inhibited only by AGN 192403. Rilmenidine and guanabenz may have a lysosomal stabilizing effect, which underlies their protective effects. AGN 192403 might affect the mitochondrial cell death cascades, and had a novel protective effect on the cytotoxicity associated with mitochondrial dysfunction. [Copyright &y& Elsevier]

Oxidative stress is a primary pathogenesis in the brain, which is particularly vulnerable to oxidative stress. Maintenance of astrocyte functions under oxidative stress is essential to prevent neuronal injuries and to recover neuronal functions in various pathologic conditions. Imidazoline drugs have affinities for imidazoline receptors, which are highly distributed in the brain, and have been shown to be neuroprotective. This study presented the protective effects of several imidazoline drugs against oxidative cytotoxicity, in primary cultures of astrocytes. Imidazoline drugs, such as idazoxan, guanabenz, guanfacine, BU224, and RS-45041-190, showed protective effects against naphthazarin-induced oxidative cytotoxicity, as evidenced by LDH release and Hoechst 33342/propidium iodide staining. The imidazoline drugs stabilized lysosomes and inhibited naphthazarin-induced lysosomal destabilization, as evidenced by acridine orange relocation. Guanabenz inhibited, the leakage of lysosomal cathepsin D to cytosol, the decreased mitochondrial potential, and the release of mitochondrial cytochrome c, which were induced by naphthazarin. The lysosomal destabilization by oxidative stress and other apoptotic signals and subsequent cathepsin D leakage to the cytosol can induce apoptotic changes of mitochondria and eventually cell death. Therefore, lysosomal stabilization by imidazoline drugs may be ascribed to their protective effects against oxidative cytotoxicity. [Copyright &y& Elsevier]

Self-monitoring values of blood pressure may better reflect the average long-term blood pressure value than sporadic measurements in the physician's office and be more useful for blood pressure control. In the present study, we compared the results of self-monitoring of blood pressure values, especially in the morning, with office blood pressure, and related these to progression of chronic renal insufficiency and left ventricular hypertrophy (LVH). Thirty-four patients were selected from 316 subjects with chronic renal insufficiency (average serum creatinine 1.72 ± 0.15 mg/dl, mean age 52.6 ± 3.5 yrs) in accordance with the following criteria (1) office blood pressure was less than 140/90 mmHg, (2) blood pressure was controlled with amlodipine (5-20 mg/day) combined with benazepril (2.5 mg/day), (3) morning blood pressure was greater than 150/90 mmHg at 69 AM and (4) LVH had been determined by echocardiography (posterior wall thickness; PWT ≥ 12 mm). The patients were assigned to 2 groups at random and were given: (1) guanabenz (GB; 2-8 mg at 11 PM, n = 17) or (2) placebo (n = 17). Two years later, the average blood pressure of both groups as measured in the office was not significantly different: however, BP in the morning was significantly reduced from 158 ± 6 to 134 ± 4 mmHg in GB treated group (P < 0.001). In 14 of 17 patients in GB treated group, LVH resolved and there was only mild progression of nephropathy (serum creatinine: 1.69 ± 0.18 to 1.81 ± 0.19 mg/dl). In 12 of 14 patients in placebo group, whose morning blood pressure remained at greater than 150/90 mmHg, LVH was retained and there was moderate progression of nephropathy (serum creatinine: 1.73 ± 0.14 to 2.62 ± 0.50 mg/dl). From these results, it is suggested that antihypertensive treatment with combination therapy based on self-monitoring BP is cardiorenoprotective in patients with chronic renal insufficiency and LVH. [ABSTRACT FROM AUTHOR]

• We present further evidence that guanabenz significantly reduces the number of latent Toxoplasma gondii cysts in BALB/c mice. • Brain cyst counts rebound after cessation of guanabenz treatment without causing symptoms of acute toxoplasmosis. • We tested whether the re-expansion of cysts could be impeded by pyrimethamine during or after guanabenz treatment. Toxoplasma gondii is a protozoan parasite that causes opportunistic infection in immunocompromised individuals. The parasite forms latent tissue cysts that are refractory to current treatments and give rise to life-threatening reactivated infection following immune suppression. Previously, we showed that guanabenz sharply reduces brain cyst count in BALB/c mice harboring latent toxoplasmosis; however, whether cyst count would change once drug treatment stopped was not addressed. In the present study, we observed a rebound in brain cysts following the discontinuation of guanabenz or a guanabenz-pyrimethamine combination therapy. The re-expansion of brain cysts was not accompanied by symptoms of acute toxoplasmosis. We also tested whether the rebound in cyst counts could be ameliorated by administering pyrimethamine during or after guanabenz treatment. [ABSTRACT FROM AUTHOR]

Guanabenz (Wytensin Wyeth-Ayerst Laboratories, Philadelphia, PA) is a centrally acting alpha2 adrenergic agonist. Guanabenz shares structural and pharmacologic properties with the more commonly known sympatholytic agent clonidine. Little is known about guanabenz in overdose. Eleven cases of guanabenz poisoning have been reported in the literature and to the manufacturer to date and followed a clinical course comparable to clonidine poisoning. We report this case of guanabenz poisoning in an elderly woman who sustained protracted hypothermia following overdose. Hypothermia has been commonly associated with clonidine toxicity but has not been previously reported with guanabenz. Postulated mechanisms of clonidine induced hypothermia are discussed. [ABSTRACT FROM AUTHOR]

Guanabenz induced a pressor effect in pithed rats through postsynaptic α-adrenoceptors whereas clonidine activated both vascular α and α-adrenoceptors. Previous treatment with prazosin, and α-antagonist, or depletion of the noradrenergic stores by reserpine produced supersensitivity to the pressor response to clondine only, probably through postsynaptic α-adrenoceptors. The hypotension and bradycardia developed in normotensive rats after intravenous guanabenz administration were abolished by prazosin, whereas the central effects of clonidine were antagonized by both prazosin and yohimbine. Selective destruction of central noradrenergic neurons by [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] (DSP 4) or reserpine plus blockade of catecholamine synthesis by α-methyl-p-tyrosine abolished the hypotension and bradycardia produced by guanabenz but merely reduced the bradycardia from clonidine. The present results suggest that, in rats, guanabenz is a selective stimulant of central α-autoadrenoceptors antagonized by prazosin whereas at a vascular level guanabenz preferentially activates α-adrenoceptors antagonized by yohimbine. The differences observed between the mechanisms by which guanabenz and clonidine produce their central cardiovascular responses might be attributed to their acting on different nuclei. [ABSTRACT FROM AUTHOR]

The effects of two α-agonists (guanfacine and guanabenz) on both the submaxillary and parotid gland of the rat were studied. Whereas guanfacine in doses ranging between 1,000 and 30,000 μg/kg i.v. produced an immediate and persistent secretion of saliva from the submaxillary gland, guanabenz in doses as high as 40,000 μg/kg did not induce measurable secretion either from the parotid or the submaxillary gland. Secretion clicited by guanfacine was not modified by yohimbine (300 μg/kg) but was abolished by prazosin (100 μg/kg). In both glands, low doses of either guanabenz (10 μg/kg) or guanfacine (100 μg/kg) markedly inhibited the secretory responses induced by noradrenaline, methacholine and substance P, but not that induced by isoprenaline. The inhibition caused by the α-agonists was greater for noradrenaline than for either methacholine or substance P. Blockade of α-adrenoceptors with yohimbine (300 μg/kg) did not modify the response to noradrenaline, methacholine or substance P in either gland. However, the same dose of yohimbine injected 5 min before the α-agonists prevented the inhibitory effects of guanfacine and guanabenz on the response induced by either one of the three sialagogic agents. Guanabenz (10 μg/kg) did not modify the increase in mean blood pressure observed after the different doses of noradrenaline employed to induce salivary secretion. Guanabenz (10 μg/kg) and guanfacine (100 μg/kg) did not change the time course of the secretion elicited by either noradrenaline, methacholine or substance P, since the degree of inhibition was of similar magnitude at all the periods of time analyzed. The results obtained give further support to the hypothesis that activation of α-adrenoceptors in the submaxillary as well as parotid gland of the rat inhibits secretory responses which are mediated by either muscarine, substance P and α-receptors and not those elicited by β-adrenoceptors. [ABSTRACT FROM AUTHOR]

The responses of the isolated canine pulmonary vein to guanabenz and to potassium chloride (KCl) were examined by means of the cannula-inserting method. The vessel was perfused by Krebs-Ringer bicarbonate solution at a constant flow rate at 37°C. In the early stage (within 2-3 hours of the perfusion period), guanabenz induced a dose-dependent vasoconstriction, whereas high doses of KCl caused, at most, a slight vasoconstriction. At the late stage (within 8-11 hours of the perfusion period), the dose-response curve for guanabenz was shifted to the left and the maximum response became approximately 3-fold larger than that in the early stage. Vasoconstriction in response to KCl was observed in 8 out of 11 preparations (73%) in the late stage. In both early and late stages, guanabenz-induced responses were similarly antagonized by DG-5128 (a selective α-adrenoceptor antagonist) but not by bunazosin (a selective α-adrenoceptor antagonist). Diltiazem caused an inhibition of guanabenz-induced constrictions, particularly in the late stage. KCl-induced vasoconstriction was not affected by bunazosin or DG-5128, but was markedly suppressed by diltiazem. In preparations in which the endothelium was removed by intraluminal treatment with saponin, the changes in guanabenz- and KCl-induced responses, which were dependent on the perfusion time, were not modified in either stage. Our results suggest that α-adrenoceptor-mediated vasoconstriction is enhanced in a perfusion-time-dependent manner in the isolated and perfused canine pulmonary vein. These enhanced responses mediated by latent α-adrenoceptors are dependent on the influx of extracellular calcium ions. Because the removal of endothelial cells by saponin did not affect the time-dependent enhancement of the response to guanabenz, the removal of endothelial cells by prolonged perfusion may play little part in the time-dependent enhancement of the α-adrenoceptor-mediated response. [ABSTRACT FROM AUTHOR]

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting. [ABSTRACT FROM AUTHOR]