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Background: This work aimed to prepare niosomal formulations of an anticancer agent [mefenamic acid (MEF)] to enhance its cancer targeting. 131 I was utilized as a radiolabeling isotope to study the radio-kinetics of MEF niosomes., Methods: niosomal formulations were prepared by the ether injection method and assessed for entrapment efficiency (EE%), zeta potential (ZP), polydispersity index (PDI) and particle size (PS). MEF was labeled with 131 I by direct electrophilic substitution reaction through optimization of radiolabeling-related parameters. In the radio-kinetic study, the optimal 131 I-MEF niosomal formula was administered intravenously (I.V.) to solid tumor-bearing mice and compared to I.V. 131 I-MEF solution as a control., Results: the average PS and ZP values of the optimal formulation were 247.23 ± 2.32 nm and - 28.3 ± 1.21, respectively. The highest 131 I-MEF labeling yield was 98.7 ± 0.8%. The biodistribution study revealed that the highest tumor uptake of 131 I-MEF niosomal formula and 131 I-MEF solution at 60 min post-injection were 2.73 and 1.94% ID/g, respectively., Conclusion: MEF-loaded niosomes could be a hopeful candidate in cancer treatment due to their potent tumor uptake. Such high targeting was attributed to passive targeting of the nanosized niosomes and confirmed by radiokinetic evaluation., (© 2023. The Author(s).)

Background: 5-azacitidine is a very potent chemotherapeutic agent that suffers from certain disadvantages., Objective: This study aims to prepare gold nanoparticles as a new nano-formula of 5-azacitidine that can improve its bioavailability and decrease its side effects., Methods: 5-azacytidine-loaded GA-AuNPs were prepared and characterized by UV-Vis spectroscopy, infrared (IR), and electronic transmission microscope (TEM). This new platform was characterized in vitro by measuring its zeta potential, particle size, and drug loading efficacy, and the anti-proliferative effect on the MCF-7 cell line was evaluated. In vivo biodistribution studies of 99m Tc-5-aza solution and 99m Tc-5-aza-gold nano formula were conducted in tumor-bearing mice by different routes of administration (intravenous and intra-tumor)., Results: 5-Aza-GA-AuNPs formula was successfully prepared with an optimum particle size of ≈34.66 nm, the zeta potential of -14.4 mV, and high entrapment efficiency. 99m Tc-5-Aza-GA-AuNPs were successfully radiosynthesized with a labeling yield of 95.4%. Biodistribution studies showed high selective accumulation in tumor and low uptake in non-target organs in the case of the 5-Aza-GA-AuNPs formula than the 99m Tc-5-azacitidine solution., Conclusion: 99m Tc-5-Aza-GA-AuNPs improved the selectivity and uptake of 5-azacitidine in cancer. Moreover, 99m Tc-5-Aza-GA-AuNPs could be used as hopeful theranostic radiopharmaceutical preparation for cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A niosomal formula of acemetacin was developed to improve its tumor targeting and radio-kinetic evaluation was performed using 131 I. Niosomes were prepared by ether injection method and characterized for particle size (PS), polydispersity index (PDI), zeta potential (ZP), entrapment efficiency (EE%) and in vitro drug release. Factors affecting radiolabeling with 131 I were studied and optimized. Radio-kinetic evaluation was done for 131 I-ACM optimum niosomal formula by intravenous (I.V) administration to solid tumor bearing mice and compared to I.V 131 I-ACM solution as a control. The average droplet size, zeta potential and in vitro release after 24 h for the optimum formula were 315.23 ± 5.37 nm, -9.16 ± 2.91 and 76 %, respectively. The greatest labeling yield of 131 I-ACM was 93.1 ± 1.1 %. Radio-kinetic evaluation showed a maximum tumor uptake of 5.431 %ID/g for 131 I-ACM niosomal formula and 2.601 %ID/g for 131 I-ACM solution at 60 min post I.V. injection. As a conclusion, niosomal formula increased tumor uptake of ACM by passive targeting of the nanosized niosomes. In addition, chemotherapeutic effect of ACM and radiotherapeutic effect of 131 I were successfully combined in one treatment regimen using 131 I-ACM niosomes which could be used as a hopeful dual anticancer therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mucormycosis is a fatal invasive illness most frequently seen in immunocompromised hosts with uncontrolled diabetes, hematological malignancies, organ transplantation, or long-term steroid treatment. It has a poorer outcome than other fungal diseases due to its rapid spread and resistance to antifungal agents. We report a rare case of disseminated mucormycosis including the bone marrow, peritoneum, lung, and lymph nodes in an apparently immunocompetent 58-year-old gentleman who presented with two months of ascites and weight loss. After a thorough analysis, we found aseptate fungal hyphae in the bone marrow and ascitic fluid. In addition, a cottony white, woolly growth indicative of mucor species was seen in the ascitic fluid culture. CT scans of the chest and abdomen indicate characteristics consistent with mucor invasion. We began the patient on tablet posaconazole, but he died on the fifth day. The atypical presentation in an apparently immunocompetent patient and broad dissemination with rare bone marrow involvement emphasizes the disease's invasiveness., Competing Interests: We declare no known conflict of interest., (© 2022 The Authors.)

Objective: Clonazepam (CP) is a potent long-acting nitrobenzodiazepine derivative that could be used for targeting peripheral benzodiazepine receptors. Phospholipid magnesome is a new vesicular nanosystem recently developed for brain targeting. Improving the uptake of 131 I-CP to the brain might be effective for the diagnosis and/or radiotherapy of certain brain diseases and/or tumors., Methods: CP was radiolabeled with 131 I using direct electrophilic substitution reaction. Quality control of 131 I-CP was performed using different techniques. Different formulas of 131 I-CP were prepared and characterized according to particle size and polydispersity index. The structural features of the optimized formula were then interpreted using transmission electron microscopy and scanning electron microscopy, whereas pharmacokinetic and in vivo behaviors were estimated using the intravenous and intranasal delivery routes., Results: The heart and blood demonstrated lower uptake of 131 I-CP, which inevitably decreased the nontarget effects of radioiodine. Intranasally administered 131 I-CP-loaded magnesomes (INMg) had noticeably higher brain uptake (7.1 ± 0.09%ID/g) with rapid onset of action within 5 min and effective pharmacokinetic behavior. INMg had a drug targeting efficiency and nose-to-brain direct transport percentage of 121.1% and 94.6%, respectively as well as a relative bioavailability of 441.04 ± 75.5%., Conclusion: The present study showed that 131 I-CP-loaded magnesomes can be a beneficial brain-targeting approach for improving the diagnosis and/or radiotherapy of certain brain diseases., (Copyright © 2021. Published by Elsevier B.V.)

Nanoparticles are frequently used as targeting delivery systems for therapeutic and diagnostic radiopharmaceuticals. Polyethylene oxide-polyacrylic acid (PEO-PAAc) nanogel was prepared via γ-radiation-induced polymerization. Variable factors affecting nanoparticles size were investigated. The nanogel was radiolabeled with the imaging radioisotope 99m Tc and finally conjugated with folic acid to target folate receptor actively. PEO-PAAc-folic acid gel was characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). Biodistribution was studied in normal mice and solid tumor-bearing mice via intravenous and intratumor injections of the radiolabeled PEO-PAAc-folic acid nanogel. Results of biodistribution showed high selective uptake of the prepared complex in tumor muscle compared with normal muscle for both intravenous and intratumor injections. The T/NT ratio was found to be 6.186 and 294.5 for intravenous and intratumor injections, respectively. Consequently, 99m Tc-PEO-PAAc-folic acid complex could be a promising agent for cancer diagnostic imaging., (© 2021 John Wiley & Sons, Ltd.)

Preterm infants frequently present with respiratory insufficiency requiring respiratory assistance. Invasive mechanical ventilation has been associated with several short and long term complications. Therefore, the practice of early use of non-invasive ventilation has been adopted. Nasal CPAP proved efficacy as an initial therapy for preterm infants. Non-invasive positive pressure ventilation is an alternative used to mitigate CPAP failure in infants with apnea or increased work of breathing. High flow nasal cannula gained popularity primarily due to the ease of its use, despite multiple prominent trials that demonstrated its inferiority. Bi-level positive airway pressure and neurally adjusted non-invasive ventilatory are used in infants with apnea and increased work of breathing. The effectiveness of non invasive ventilation tools can be augmented by having a proper protocol for initiation, weaning, skin care, positioning, and developmental care during their application., (© 2020 Publishing services provided by Elsevier B.V. on behalf of King Faisal Specialist Hospital & Research Centre (General Organization), Saudi Arabia.)

In this study, water-soluble chitosan lactate (CL) was reacted with lactobionic acid (LA), a disaccharide with remarkable affinity to hepatic asialoglycoprotein (ASGP) receptors, to form dual liver-targeting LA-modified-CL polymer for site-specific drug delivery to the liver. The synthesized polymer was used to encapsulate baicalin (BA), a promising bioactive flavonoid with pH-dependent solubility, into ultrahigh drug-loaded nanoparticles (NPs) via the ionic gelation method. The successful chemical conjugation of LA with CL was tested and the formulated drug-loaded LA-modified-CL-NPs were assessed in terms of particle size (PS), encapsulation efficiency (EE) and zeta potential (ZP) using full factorial design. The in vivo biodistribution and pharmacokinetics of the designed NPs were assessed using 99m Tc-radiolabeled BA following oral administration to mice and results were compared to 99m Tc-BA-loaded-LA-free-NPs and 99m Tc-BA solution as controls. Results showed that the chemical modification of CL with LA was successfully achieved and the method of preparation of the optimized NPs was very efficient in encapsulating BA into nearly spherical particles with an extremely high EE exceeding 90%. The optimized BA-loaded-LA-modified-CL-NPs showed an average PS of 490 nm, EE of 93.7% and ZP of 48.1 mV. Oral administration of 99m Tc-BA-loaded-LA-modified-CL-NPs showed a remarkable increase in BA delivery to the liver over 99m Tc-BA-loaded-LA-free-CL-NPs and 99m Tc-BA oral solution. The mean area under the curve (AUC 0-24 ) estimates from liver data were determined to be 11-fold and 26-fold higher from 99m Tc-BA-loaded-LA-modified-CL-NPs relative to 99m Tc-BA-loaded-LA-free-CL-NPs and 99m Tc-BA solution respectively. In conclusion, the outcome of this study highlights the great potential of using LA-modified-CL-NPs for the ultrahigh encapsulation of therapeutic molecules with pH-dependent/poor water-solubility and for targeting the liver., Competing Interests: The authors declare no conflicts of interest.

Bisphosphonates are widely used for treatment of osteoporosis. Recently, they have been reported to be effective anticancer agents. In this work, we designed some substituted phenyl (azanediyl) bis (methylene phosphonic acid) to be tested for their anticancer effect. Both molecular docking and dynamics studies were used to select the top ranked highly scored compounds. The selected hits showed potential in vitro anticancer effect against some cell lines. Biodistribution pattern and gamma scintigraphy were conducted to the most effective derivative (BMBP) after radiolabeling with 99m Tc. Results of biodistribution and scintigraphic imaging of 99m Tc-BMBP in tumor bearing mice showed a notable tumor affinity, and confirmed the targeting affinity of BMBP to the tumor tissues. As a conclusion, BMBP could act as potential anticancer agent and imaging probe., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The purpose of our study was to improve the delivery of a direct-acting antiviral drug, daclatasvir, to the site of action, liver tissues, using physically and biologically stable cationic bile-based vesicles. Accordingly, cationic bile-based vesicles were prepared as pro-bile-based vesicles and diethylaminoethyl dextran (DEAE-Dx)-stabilized bile-based vesicles to increase their stability without negatively affecting their hepatic affinity. The prepared bile-based vesicles were characterized for particle size, polydispersity index, ζ-potential, in vitro daclatasvir release, and ex vivo permeation using non-everted gut sac intestine. The in vivo biodistribution was experimented after oral administration utilizing the radiolabeling assay, where the liver showed the highest accumulation of the DEAE-Dx-stabilized bile-based vesicles after 4 h, reaching a value of 4.6% ID/g of the total oral administered dose of the labeled drug compared to drug solution, pro-bile-based vesicles, and cationic bile-based vesicles where the accumulation was 0.19, 1.3, and 0.31% ID/g, respectively. DEAE-Dx-stabilized bile-based vesicles increased the drug deposition into the liver about 42-fold compared to oral solution. The high physical stability and the high resistance to opsonization and clearance show that DEAE-Dx-stabilized bile-based vesicles could be efficiently applied for enhancing daclatasvir delivery to the liver after oral administration.

Selective receptors imaging using gamma emitting radiopharmaceuticals allows accurate diagnosis and follow up of many brain related disorders. Levetiracetam, a selective SV2A receptor antiepileptic, was successfully radiolabeled using 99m Tc. Different conditions affecting the labelling process were studied and optimum radiochemical yield of 89.8% was obtained. 99m Tc-levetiracetam was effectively formulated and characterized as microemulsion with particle size of 16.34 ± 5.58 nm and polydispersity index of 0.382 ± 0.05. Parallel biodistribution studies were performed comparing brain targeting efficiency of I.V 99m Tc-levetiracetam solution, I.N 99m Tc-levetiracetam solution and I.N 99m Tc-levetiracetam microemulsion. Brain radioactivity uptake and brain/blood uptake ratio for I.N 99m Tc-levetiracetam microemulsion were higher than the other two routes at all time intervals. Such results present intranasal 99m Tc-levetiracetam microemulsion as the first SPECT tracer for imaging SV2A receptor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The current work presents the synthesis and biological evaluation of new series of coumarin hydrazide-hydrazone derivatives that showed in vitro broad spectrum antitumor activities against resistant pancreatic carcinoma (Panc-1), hepatocellular carcinoma (HepG2) and leukemia (CCRF) cell lines using doxorubicin as reference standard. Bromocoumarin hydrazide-hydrazone derivative (BCHHD) 11b showed excellent anticancer activity against all tested cancer cell lines. Enzyme assays showed that BCHHD 11b induced apoptosis due to activation of caspases 3/7. Moreover, 11b inhibited GST and CYP3A4 in a dose dependent manner and the induced cell death could be attributed to metabolic inhibition. Moreover, 11b microarray analysis showed significant up- and down-regulation of many genes in the treated cells related to apoptosis, cell cycle, tumor growth and suppressor genes. All of the above presents BCHHD 11b as a potent anticancer agent able to overcome drug resistance. In addition, compound 11b was able to serve as a chemical carrier for 99m Tc and the in vivo biodistribution study of 99m Tc-11b complex revealed a remarkable targeting ability of 99m Tc into solid tumor showing that 99m Tc-11b might be used as a promising radiopharmaceutical imaging agent for cancer., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

l-Phosphinothricin (glufosinate or 2-amino-4-((hydroxy(methyl) phosphinyl) butyric acid ammonium salt (AHPB)), which is a structural analog of glutamate, is a recognized herbicide that acts on weeds through inhibition of glutamine synthetase. Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this study focuses on investigating the possibility of repurposing phosphinothricin as a bisphosphonate analogue, particularly in two medicine-related activities: image probing and as an anti-cancer drug. As BP is a competitive inhibitor of human farnesyl pyrophosphate synthase (HFPPS), in silico molecular docking and dynamic simulations studies were established to evaluate the binding and stability of phosphinothricin with HFPPS, while the results showed good binding and stability in the active site of the enzyme in relation to alendronate. For the purpose of inspecting bone-tissue accumulation of phosphinothricin, a technetium ( 99m Tc)-phosphinothricin complex was developed and its stability and tissue distribution were scrutinized. The radioactive complex showed rapid, high and sustained uptake into bone tissues. Finally, the cytotoxic activity of phosphinothricin was tested against breast and lung cancer cells, with the results indicating cytotoxic activity in relation to alendronate. All the above results provide support for the use of phosphinothricin as a potential anti-cancer drug and of its technetium complex as an imaging probe., Competing Interests: The authors declare no conflict of interest.

Lung imaging radiopharmaceuticals are helpful agents for measuring pulmonary blood flow and allow detection of pulmonary embolism and lung cancer. The goal of this study was to develop a novel potential radiopharmaceutical for lung imaging. Zolmitriptan (a selective serotonin receptor agonist) was successfully labeled with 99m Tc via direct labeling method under reductive conditions studying different factors affecting the labeling efficiency. 99m Tc-zolmitriptan was obtained with a maximum labeling yield of 92.5 ± 0.61 % and in vitro stability up to 24 h. Molecular modeling was done to predict the structure of 99m Tc-zolmitriptan and ensure that radiolabeling did not affect binding ability of zolmitriptan to its receptor. Biodistribution studies showed that maximum lung uptake of 99m Tc-zolmitriptan was 23.89 ± 1.2 % injected dose/g tissue at 15 min post-injection and retention in lungs remained high up to 1 h, whereas the clearance from mice appeared to proceed mainly via the renal pathway. Scintigraphic images confirmed the biodistribution results showing a high resolution lung image with low accumulation of radioactivity in other organs except kidneys and urinary bladder. 99m Tc-zolmitriptan is not a blood product and so it is more safe than the currently available 99m Tc-MAA, and its lung uptake is higher than that of the recently discovered 123 I-IPMPD, 99m Tc(CO) 5 I and 99m Tc-DHPM. So, 99m Tc-zolmitriptan could be used as a hopeful radiopharmaceutical for lung scintigraphic imaging.

Clonazepam (CZ) is an anti-epileptic drug used mainly in status epilepticus (SE). The drug belongs to Class II according to BCS classification with very limited solubility and high permeability and it suffers from extensive first-pass metabolism. The aim of the present study was to develop CZ-loaded polymeric micelles (PM) for direct brain delivery allowing immediate control of SE. PM were prepared via thin film hydration (TFH) technique adopting a central composite face-centered design (CCFD). The seventeen developed formulae were evaluated in terms of entrapment efficiency (EE), particle size (PS), polydispersity index (PDI), zeta potential (ZP), and in vitro release. For evaluating the in vivo behavior of the optimized formula, both biodistrbution using 99m Tc-radiolabeled CZ and pharmacodynamics studies were done in addition to ex vivo cytotoxicty. At a drug:Pluronic® P123:Pluronic® L121 ratio of 1:20:20 (PM7), a high EE, ZP, Q8h, and a low PDI was achieved. The biodistribution studies revealed that the optimized formula had significantly higher drug targeting efficiency (DTE = 242.3%), drug targeting index (DTI = 144.25), and nose-to-brain direct transport percentage (DTP = 99.30%) and a significant prolongation of protection from seizures in comparison to the intranasally administered solution with minor histopathological changes. The declared results reveal the ability of the developed PM to be a strong potential candidate for the emergency treatment of SE.

Nimodipine (NM) is the only FDA-approved drug for treating subarachnoid hemorrhage induced vasospasm. NM has poor oral bioavailability (5-13%) due to its low aqueous solubility, and extensive first pass metabolism. The objective of this study is to develop radiolabeled NM-loaded LPM and to test its ability prolong its circulation time, reduce its frequency of administration and eventually target it to the brain tissue. NM was radiolabeled with 99m Tc by direct labeling method using sodium dithionite. Different reaction conditions that affect the radiolabeling yield were studied. The in vivo pharmacokinetic behavior of the optimum NM-loaded LPM formulation in blood, heart, and brain tissue was compared with NM solution, after intravenous and intranasal administration. Results show that the radioactivity percentage (%ID/g) in the heart of mice following administration of 99m Tc-NM loaded LPM were lower compared with that following administration of 99m Tc-NM solution, which is greatly beneficial to minimize the cardiovascular side effects. Results also show that the %ID/g in the blood, and brain following intravenous administration of 99m Tc-NM-loaded LPM were higher at all sampling intervals compared with that following intravenous administration of 99m Tc-NM solution. This would be greatly beneficial for the treatment of neurovascular diseases. The drug-targeting efficiency of NM to the brain after intranasal administration was calculated to be 1872.82%. The significant increase in drug solubility, enhanced drug absorption and the long circulation time of the NM-loaded LPM could be promising to improve nasal and parenteral delivery of NM.

Migraine attack is a troublesome physiological condition associated with throbbing, intense headache, in one half of the head. Zolmitriptan is a potent second-generation triptan, prescribed for patients with migraine attacks, with or without an aura, and cluster headaches. The absolute bioavailability of zolmitriptan is about 40% for oral administration; due to hepatic first metabolism. Nasal administration would circumvent the pre-systemic metabolism thus increasing the bioavailability of zolmitriptan. In addition, due to the presence of microvilli and high vasculature, the absorption is expected to be faster compared to oral route. However, the bioavailability of nasal administered drugs is particularly restricted by poor membrane penetration. Thus, the aim of this work is to explore the potential of novel nanovesicular fatty acid enriched structures (novasomes) for effective and enhanced nasal delivery of zolmitriptan and investigate their nose to brain targeting potential. Novasomes were prepared using nonionic surfactant, cholesterol in addition to a free fatty acid. A 2 3 full factorial design was adopted to study the influence of the type of surfactant, type of free fatty acid and ratio between the free fatty acid and the surfactant on novasomes properties. The particle size, entrapment efficiency, polydispersity index, zeta potential and % zolmitriptan released after 2 h were selected as dependent variables. Novasomes were further optimized using Design Expert® software (version 7; Stat-Ease Inc., Minneapolis, MN), and an optimized formulation composed of Span® 80:Cholesterol:stearic acid (in the ratio 1:1:1) was selected. This formulation showed zolmitriptan entrapment of 92.94%, particle size of 149.9 nm, zeta potential of -55.57 mV, and released 48.43% zolmitriptan after 2 h. The optimized formulation was further examined using transmission electron microscope, which revealed non-aggregating multi-lamellar nanovesicles with narrow size distribution. DSC, XRD examination of the optimized formulation confirmed that the drug have been homogeneously dispersed throughout the novasomes in an amorphous state. In-vivo bio-distribution studies of 99m Tc radio-labeled intranasal zolmitriptan loaded novasomes were done on mice, the pharmacokinetic parameters were compared with those following administration of intravenous 99m Tc-zolmitriptan solution. Results revealed the great enhancement in zolmitriptan targeting to the brain, with drug targeting potential of about 99% following intranasal administration of novasomes compared with the intravenous drug solution. Zolmitriptan loaded novasomes administered via the nasal route may therefore constitute an advance in the management of acute migraine attacks.

The modulation of cardiovascular responses to the cold pressor test (CPT) as produced by exercise was studied in 13 volunteers. The reproducibility of the measurements selected for the study, i.e. heart rate (HR), blood pressure (BP), blood flow (BF) and skin temperature (ST), was investigated through repeat experiments in the fall of 1994 and the winter of 1995. HR was monitored before, during and after a 10-min period of bicycling at 70% of reserve HR. BP, cutaneous BF and ST were measured before and after exercise. Two CPTs (hand into ice-cold water for 1 min) were performed: one preceding exercise and another at 3 min after exercise. The results obtained allow us to conclude that in non-hypertensive volunteers (1) the pronounced cardiovascular responses (ST, BF and BP) induced by CPT are reproducible (p > 0.2) when compared to basal level values and (2) cardiovascular responses to cold stress are significantly attenuated by exercise (p < 0.03). Our study, therefore, supports and validates the use of our coupled exercise-CPT method in ongoing epidemiological studies attempting to identify individuals at risk for the development of hypertension as well as those most likely to benefit from preventative exercise programs.

Studies presented in this report were designed to investigate the effects of transforming growth factor-beta 1 (TGF-beta 1) on epidermal growth factor (EGF)-mediated stimulation of cAMP accumulation in cardiac myocytes and elucidate the mechanism(s) involved in this modulation. TGF-beta 1 (20 pM) treatment of cardiac myocytes, in a time-dependent manner, decreased the ability of EGF (100 nM) to increase cAMP accumulation. Significant attenuation of EGF-elicited cAMP accumulation was observed 2 h after exposure to TGF-beta 1 and 18 h after addition of TGF-beta 1, the ability of EGF to increase cAMP accumulation was completely obliterated. TGF-beta 1 neither decreased immunoprecipitable EGF receptors in membranes from cardiomyocytes nor altered the specific binding of [125I]EGF to cardiomyocyte membranes. However, TGF-beta 1 decreased the ability of EGF to phosphorylate membrane proteins on tyrosine residues. TGF-beta 1 treatment of cardiomyocytes also decreased the ability of forskolin to augment cAMP accumulation in intact cells and stimulate adenylyl cyclase activity. Similarly, in membranes of TGF-beta 1-treated cells, neither isoproterenol nor EGF stimulated adenylyl cyclase activity. Interestingly, as assessed by the ability of A1F4- to stimulate adenylyl cyclase, TGF-beta 1 did not alter the coupling between Gs and catalytic subunits. Likewise, TGF-beta 1 did not alter the functional activity of the inhibitory regulatory element of the system, Gi. Western analysis of cellular proteins revealed that TGF-beta 1 did not alter the amounts of Ga alpha, Gi alpha 2, and Gi alpha 3. We conclude that TGF-beta 1 attenuates EGF-elicited cAMP accumulation in cardiomyocytes, in part, by decreasing the EGF receptor kinase function and that TGF-beta 1-mediated alterations in the activity of adenylyl cyclase catalytic subunit also contribute toward the regulation of adenylyl cyclase by various agonists.

Previously, we demonstrated that, 48 hours after partial hepatectomy, in the regenerating liver the number of both atrial natriuretic hormone (ANF) receptor subtypes, the guanylyl cyclase-linked and ANF-C receptors, is increased twofold. Subsequently, we demonstrated that activation of ANF-C receptors inhibits growth of hepatocytes. Therefore, studies were performed to determine whether, during hepatic regeneration, the increase in ANF receptor subtypes is accompanied by an increase in their respective transcripts. Our data demonstrate that in the normal and regenerating rat liver, the predominant guanylyl cyclase-linked ANF receptor is of the ANF-A subtype. Moreover, messenger RNA (mRNA) encoding the ANF-A and ANF-C receptors are transiently increased after surgery; the levels of mRNA encoding both receptor subtypes remain unchanged in livers of sham-operated animals. ANF-A receptor mRNA is maximally increased 12 hours after partial hepatectomy, whereas the maximal increase in ANF-C receptor mRNA is observed between 0.5 hour and 4 hours after hepatectomy. The increase in ANF-C receptor transcript is accompanied by increased expression of protein, 4 hours after hepatectomy. However, the ANF-C receptor protein is also elevated 48 hours after partial hepatectomy when ANF-C receptor mRNA levels are not different from controls. Likewise, although ANF-A receptors are increased when hepatic levels of mRNA encoding the protein are maximally elevated, the maximal increase in ANF-A receptor protein occurs at times when transcript levels are low and similar to those in sham-operated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate whether atrial natriuretic factor regulates the growth of hepatocytes and to determine the receptor subtype involved in such modulation, we studied the effect of atrial natriuretic factor 103-126 and clearance receptor binding analogs of atrial natriuretic factor, (des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126) on growth of human hepatoblastoma cells. Atrial natriuretic factor 103-126 and des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 inhibited thymidine incorporation into human hepatoblastoma cells cultured in the presence of bovine serum albumin and epidermal growth factor but not in cells cultured in bovine serum albumin alone. Moreover, atrial natriuretic factor 103-126, des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126, in a concentration-dependent manner, inhibited thymidine incorporation and cell proliferation. As monitored by the ability of des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 to displace 125I-labeled atrial natriuretic factor, epidermal growth factor increased the expression of cell surface clearance receptors. Epidermal growth factor also transiently increased the cellular content of atrial natriuretic factor clearance receptor messenger RNA without altering the levels of guanylyl cyclase-linked atrial natriuretic factor receptor messenger RNA levels. Maximal increase in atrial natriuretic factor clearance receptor messenger RNA coincided with the maximal increase in des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121-displaceable 125I-atrial natriuretic factor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Previously we have shown that epidermal growth factor (EGF) stimulates cardiac adenylyl cyclase and increases cAMP accumulation in the rat heart (Nair et al., Biochem. J. 264, 563-571, 1989). Moreover, we have shown that the stimulation of adenylyl cyclase by EGF in heart is mediated via activation of the stimulatory GTP binding regulatory protein Gs alpha (Nair et al., J. Biol. Chem. 265, 21317-21322, 1990). Since cAMP increases the beating rate of hearts, studies were performed to investigate the effects of EGF on mechanical function of the heart and the role of cAMP in mediating the cardiac effects of EGF. In isolated perfused rat hearts EGF (15 nM) decreased perfusion pressure, increased ventricular contractility and heart rate in a manner similar to that observed with the beta-adrenergic receptor agonist isoproterenol (10 nM). In the presence of the adenosine A1 receptor agonist (-)-N6-(R-phenylisopropyl)-adenosine (PIA, 100 nM) which via activation of the inhibitory GTP binding protein Gi inhibits adenylyl cyclase, the effects of EGF on cAMP accumulation in the heart were markedly attenuated. PIA also decreased the ability of EGF and isoproterenol to alter cardiac contractility and beating rate. However, PIA did not attenuate the increase in heart rate and contractility induced by the alpha-adrenergic agonist phenylephrine which does not stimulate cAMP accumulation in the heart. These data suggest that EGF alters cardiac function by increasing cellular cAMP accumulation.

Recently we reported the presence of both the guanylyl cyclase-linked (116 kDa) and the ANF-C (66 kDa) atrial natriuretic peptide receptors in the rat liver. Since ANF 103-125 (atriopeptin II) stimulates cGMP production in livers and because cGMP has previously been shown to mimic the actions of cAMP in regulating hepatic carbohydrate metabolism, studies were performed to investigate the effects of atriopeptin II on hepatic glycolysis and gluconeogenesis. Additionally, employing analogs of atrial natriuretic hormone [des-(Q116, S117, G118, L119, G120) ANF 102-121 (C-ANF) and des-(C105,121) ANF 104-126 (analog I)] which bind only the ANF-C receptors, the role of the ANF-C receptors in the hepatic actions of atriopeptin II was evaluated. In perfused livers of fed rats atriopeptin II, but not C-ANF and analog I, inhibited hepatic glycolysis and stimulated glucose production. Moreover, analog I did not alter the ability of atriopeptin II to inhibit hepatic glycolysis. Atriopeptin II, but not C-ANF and analog I, also stimulated cGMP production in perfused rat livers. Furthermore, while atriopeptin II inhibited the activity ratio of pyruvate kinase by 30%, C-ANF did not alter hepatic pyruvate kinase activity. Finally, in rat hepatocytes, atriopeptin II stimulated the synthesis of [14C]glucose from [2-14C]pyruvate by 50% and this effect of atriopeptin II was mimicked by the exogenously supplied cGMP analog, 8-bromo cGMP. Thus atriopeptin II increases hepatic gluconeogenesis and inhibits glycolysis, in part by inhibiting pyruvate kinase activity, and the effects of atriopeptin II are mediated via activation of guanylyl cyclase-linked ANF receptors which elevate cGMP production.

Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.

In isolated perfused rat livers, infusion of the sulfonylureas, glyburide (2.5 microM) and tolbutamide (0.5 mM), stimulated by 2-fold the rate of biliary glutathione secretion. This increase was mainly the result of an apparent increase in the rate of reduced glutathione release by the liver since oxidized glutathione levels in the bile remained unchanged. Sulfonylurea infusion into perfused livers did not alter the rate of glutathione release in the perfusate, indicating that sinusoidal release was not perturbed. N-Benzylimidazole (0.2 mM), an inhibitor of cytochrome P-450, blocked the tolbutamide-mediated increase in biliary release of glutathione. However, the cytochrome P-450 inhibitor did not alter the glyburide-induced increase in biliary glutathione secretion. Glyburide infusion into perfused livers also decreased tissue oxidized glutathione content without altering the total tissue levels of glutathione. The stimulation of biliary glutathione release by sulfonylureas is probably the result of excretion of labile conjugates of glutathione and sulfonylurea metabolites. Although the precise identity of these metabolites is presently unknown, formyltolbutamide and hydroxyglyburide formed during metabolism of tolbutamide and glyburide, respectively, may be the prime candidates for forming labile glutathione conjugates.

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.

The metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in perfused livers was monitored by measuring the rate of 14CO2 production from [1-14C]alpha-ketoglutarate. The rates of 14CO2 production and glucose production from [1-14C]alpha-ketoglutarate were increased with increasing perfusate alpha-ketoglutarate concentrations. Vasopressin, angiotensin II, and the alpha 1-adrenergic agonist phenylephrine stimulated transiently by 2.5-fold the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in the presence and absence of Ca2+ in the perfusion medium. High concentrations of glucagon (1 x 10(-8) M) and 8-p-chlorophenylthio-cAMP (100 microM) (data not shown) also stimulated transiently the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction. However, lower glucagon concentrations (1 x 10(-9) M) stimulated the rate of 14CO2 production from [1-14C]alpha-ketoglutarate only under conditions optimized to fix the cellular oxidation-reduction state at an intermediate level, when glucagon (1 x 10(-9) M)-mediated elevation of cAMP content was greater than that observed under highly oxidizing and reducing conditions. These data indicate that agonists which increase cytosolic free Ca2+ levels stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase complex. Furthermore, the data presented here demonstrate for the first time that physiological glucagon concentrations stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction only under conditions known to be optimal for glucagon-mediated Ca2+ mobilization in the isolated perfused rat liver.

The effect of veratridine-mediated depolarization on rat brain synaptosomal respiration in the presence and absence of calcium was investigated. Studies on respiration were performed employing three different pretreatments of the synaptosomes which attempted to deplete endogenous substrates. First, synaptosomes were preincubated for 10 min in the absence of any substrates in medium either containing or devoid of calcium. Second, synaptosomes were preincubated for either 15 or 60-min periods in the presence and absence of calcium, and the incubation medium was changed by centrifugation and resuspension of synaptosomes in their respective media. Irrespective of the prior treatment, maximal stimulation of respiration (400-600%) during veratridine (100 microM) elicited depolarization was observed only when calcium was present in the incubation media. In incubations performed in the absence of calcium, veratridine addition either modestly stimulated (10- and 15-min preincubated synaptosomes) or did not affect (60-min preincubated synaptosomes) the rate of respiration. However, when calcium was added back to these incubations the rate of respiration in the presence of veratridine was stimulated by five- to six-fold. Similarly, the rates of 14CO2 production from [1-14C]- and [2-14C]pyruvate were increased by veratridine only when synaptosomes were incubated in calcium-replete medium. These data indicate that calcium plays an obligatory role in depolarization-elicited stimulation of synaptosomal oxidative processes.

In the absence of any exogenous substrates, glucagon (1 X 10(-9) M) stimulated 45Ca2+ efflux from perfused livers derived from fed rats but not in livers of 24-h-fasted animals. In livers of 24-h-fasted animals perfused under conditions which would decrease cellular NAD(P)H/NAD(P)+ ratio (pyruvate (2.0 mM) or acetoacetate (10.0 mM], glucagon (1 X 10(-9) M) did not stimulate 45Ca2+ efflux. Similarly, in livers of 24-h-fasted animals perfused with substrates which increase cellular NAD(P)H content (lactate (2.0 mM) or beta-hydroxybutyrate (10.0 mM], glucagon (1 X 10(-9) M) did not increase 45Ca2+ efflux. Glucagon (1 X 10(-9) M) elicited an increase in 45Ca2+ efflux from livers of 24-h-fasted animals, only when the livers were perfused with [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate] ratios similar to those reported for livers of fed rats. Stimulation of 45Ca2+ efflux elicited by either 8-CPT-cAMP, a cAMP analog, or high glucagon concentrations (1 X 10(-8) M) was not affected whether livers were perfused with pyruvate (2.0 mM) or lactate (2.0 mM). Administration of isobutylmethylxanthine (50 microM) alone, or glucagon (1 X 10(-9) M) in the presence of isobutylmethylxanthine (50 microM) stimulated 45Ca2+ efflux from livers of 24-h-fasted animals perfused with pyruvate (2.0 mM) but not from livers perfused with lactate (2.0 mM). The ability of glucagon (1 X 10(-9) M) to elevate tissue cAMP levels was also regulated by the oxidation-reduction state of the livers. The data indicate that glucagon-stimulated 45Ca2+ efflux from perfused livers is mediated via cAMP and is dependent on the oxidation-reduction state of the livers.

The effect of oral hypoglycemic sulfonylureas, tolbutamide and glyburide, on metabolic flux through the pyruvate carboxylase reaction was evaluated in liver mitochondria isolated from 24-hr fasted rats. Both these sulfonylureas inhibited the metabolic flux through the pyruvate carboxylase reaction in a concentration dependent manner. Half-maximal inhibition was achieved at tolbutamide and glyburide concentrations of 0.85 mM and 63.3 microM, respectively. Neither sulfonylurea altered the activity of pyruvate carboxylase or the Km of the enzyme for ATP and pyruvate. However, glyburide and tolbutamide decreased mitochondrial ATP content and elevated mitochondrial ADP and AMP levels. The decrease in mitochondrial ATP was greater with 400 microM glyburide compared with 2.0 mM tolbutamide. Glyburide also decreased mitochondrial acetyl-coenzyme A/CoASH ratio. Additionally, glyburide and tolbutamide stimulated pyruvate (5 mM) supported mitochondrial respiration in the absence of ADP. These data indicate that these sulfonylureas inhibit the metabolic flux through the pyruvate carboxylase reaction by decreasing mitochondrial ATP/ADP and acetyl-coenzyme A/CoASH ratios. Decreased mitochondrial nucleotide content and increased mitochondrial respiration caused by sulfonylureas suggest that these compounds may uncouple oxidative phosphorylation.