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Mechanisms underlying arteriovenous malformations (AVMs) are poorly understood. Using mice with endothelial cell (EC) expression of constitutively active Notch4 (Notch4*EC), we show decreased arteriolar tone in vivo during brain AVM initiation. Reduced vascular tone is a primary effect of Notch4*EC, as isolated pial arteries from asymptomatic mice exhibited reduced pressure-induced arterial tone ex vivo. The nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-l-arginine (L-NNA) corrected vascular tone defects in both assays. L-NNA treatment or endothelial NOS (eNOS) gene deletion, either globally or specifically in ECs, attenuated AVM initiation, assessed by decreased AVM diameter and delayed time to moribund. Administering nitroxide antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl also attenuated AVM initiation. Increased NOS-dependent production of hydrogen peroxide, but not NO, superoxide, or peroxynitrite was detected in isolated Notch4*EC brain vessels during AVM initiation. Our data suggest that eNOS is involved in Notch4*EC-mediated AVM formation by up-regulating hydrogen peroxide and reducing vascular tone, thereby permitting AVM initiation and progression.

Here we describe mechanistically distinct enzymes (a kinase, a guanosine triphosphatase, and a ubiquitin protein hydrolase) that function in disparate biochemical pathways and can also act in concert to mediate a series of redox reactions. Each enzyme manifests a second, noncanonical function-transnitrosylation-that triggers a pathological biochemical cascade in mouse models and in humans with Alzheimer's disease (AD). The resulting series of transnitrosylation reactions contributes to synapse loss, the major pathological correlate to cognitive decline in AD. We conclude that enzymes with distinct primary reaction mechanisms can form a completely separate network for aberrant transnitrosylation. This network operates in the postreproductive period, so natural selection against such abnormal activity may be decreased.

Tramadol, an analgesic classified as an "atypical opioid", exhibits both opioid and non-opioid mechanisms of action. This study aimed to explore these mechanisms, specifically the opioid-, cannabinoid-, nitric oxide-, and potassium channel-based mechanisms, which contribute to the peripheral antinociception effect of tramadol, in an experimental rat model. The nociceptive threshold was determined using paw pressure withdrawal. To examine the mechanisms of action, several substances were administered intraplantarly: naloxone, a non-selective opioid antagonist (50 µg/paw); AM251 (80 µg/paw) and AM630 (100 µg/paw) as the selective antagonists for types 1 and 2 cannabinoid receptors, respectively; nitric oxide synthase inhibitors L-NOArg, L-NIO, L-NPA, and L-NIL (24 µg/paw); and the enzyme inhibitors of guanylatocyclase and phosphodiesterase of cGMP, ODQ, and zaprinast. Additionally, potassium channel blockers glibenclamide, tetraethylammonium, dequalinium, and paxillin were used. The results showed that opioid and cannabinoid receptor antagonists did not reverse tramadol's effects. L-NOarg, L-NIO, and L-NPA partially reversed antinociception, while ODQ completely reversed, and zaprinast enhanced tramadol's antinociception effect. Notably, glibenclamide blocked tramadol's antinociception in a dose-dependent manner. These findings suggest that tramadol's peripheral antinociception effect is likely mediated by the nitrergic pathway and sensitive ATP potassium channels, rather than the opioid and cannabinoid pathways., Competing Interests: 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.

Graphical abstract Abstract The objective of this study is to design a new family of biodegradable synthetic polymeric biomaterials for providing a tunable inhibition of macrophage's nitric oxide synthase (NOS) pathway. l -Arginine (Arg) is the common substrate for NOS and arginase. Both two metabolic pathways participate in the wound healing process. An impaired wound healing, such as diabetic or other chronic wounds is usually associated with an overproduction of NO by macrophages via the NOS pathway. In this study, a new family of l -nitroarginine (NOArg) based polyester amide (NOArg-PEA) and NOArg-Arg PEA copolymers (co-PEA) were designed and synthesized with different composition ratios. The NOArg-PEA and NOArg-Arg co-PEAs are biodegradable (more than 50% degradation in vitro in 4 days at 37 °C), biocompatible and did not activate the resting macrophage immune response per se. When classically activated or alternatively activated macrophages (CAM/AAM) were incubated with NOArg-PEA and NOArg-Arg co-PEAs, the treatments decreased the NO production of CAM, increased the arginase activity in both CAM and AAM, increased TGF-β1 production of CAM to various degrees and had no significant effect on TNF-α production. Diabetic rat models were used to evaluate the efficacy of NOArg-PEA and NOArg-Arg co-PEAs on wound healing. Diabetic rats treated with 2-NOArg-4 PEA, 2-NOArg-4-Arg-4 20/80, and 2-NOArg-4-Arg-4 50/50 biomaterials achieved 40%–80% faster-wound healing when compared with the control on day 7. The data from the histological and immunohistochemical analysis showed that the 2-NOArg-4-Arg-4 20/80 and 2-NOArg-4-Arg-4 50/50 treatments led to more AAM phenotypes (CD206) and arginase I production in wound tissue than the control during the first 7 days, i.e., suggesting pro-healing wound microenvironment with improved re-epithelialization of wound healing. A similar trend was retained until day 14. The 2-NOArg-4-Arg-4 20/80 and 2-NOArg-4-Arg-4 50/50 treatments also increased the collagen deposition and angiogenesis in the healing wound between day 7 and day 14. Both in vitro and in vivo data of this study showed that this new family of NOArg-Arg co-PEA biomaterials have the potential as viable alternatives for treating impaired wound healing, such as diabetic or other types of chronic wounds. Statement of Significance Diabetic or other chronic wounds is usually associated with an overproduction of NO and pro-inflammatory signals by macrophages. Arginine supplement or NOS inhibitors administration failed to achieve an expected improved wound healing because of the dynamic complexity of arginine catabolism, the difficulty in transition from pro-inflammatory to pro-healing, and the short-term efficacy. We designed and synthesized a new family of water-soluble and degradable nitroarginine-arginine polyester amides to rebalance NOS/arginase metabolism pathways of macrophages. They showed tunable immunomodulating properties in vitro. The in vivo studies were performed to evaluate their efficacy in accelerating the healing. These new biomaterials have the potential as viable alternatives for treating impaired wound healing. The general audience of Acta Biomaterialia should be interested in these findings. [ABSTRACT FROM AUTHOR]

The effect of various selective glutamate agonists upon the rate of generation of reactive oxygen species (ROS), was examined in an isolated synaptoneurosomal (microsac) fraction derived from rat cerebral cortex. The rates of ROS generation were determined by a fluorescent probe. Agonists specific for each of the three major glutamate inotropic receptor sites (NMDA, kainic acid, alpha-amino-3-hydroxy-5-methyl-4-isoxalolpropionic acid, AMPA), were able to enhance rates of ROS generation. The metabotropic glutamate agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid, (ACPD), was inactive in this regard. Stimulation of ROS was most pronounced in the case of kainate. Such results could not be replicated by use of ion-channel active agents, veratridine and A23817. Pretreatment with the kainate antagonist, 6-cyano-7-quinoxaline-2,3-dione (CNQX), was not able to block the kainate-induced elevation of ROS. Domoic acid, a kainate agonist, also enhanced microsac ROS generation. Neurological damage may result from generation of excess free radicals, and this may be effected by glutamate agonists acting by means independent of their ionotropic properties.

The casts of Pompeii bear witness to the people who died during the Vesuvius 79 AD eruption. However, studies on the cause of death of these victims have not been conclusive. A previous important step is the understanding of the post-depositional processes and the impact of the plaster in bones, two issues that have not been previously evaluated. Here we report on the anthropological and the first chemical data obtained from the study of six casts from Porta Nola area and one from Terme Suburbane. A non-invasive chemical analysis by portable X-ray fluorescence was employed for the first time on these casts of Pompeii to determine the elemental composition of the bones and the plaster. Elemental profiles were determined providing important data that cross-referenced with anthropological and stratigraphic results, are clearly helpful in the reconstruction of the perimortem and post-mortem events concerning the history of these individuals. The comparative analyses carried out on the bone casts and other collections from burned bones of the necropolis of Porta Nola in Pompeii and Rome Sepolcreto Ostiense, and buried bones from Valencia (Spain), reveal the extent of high temperature alteration and post-depositional plaster contamination. These factors make bioarchaeological analyses difficult but still allow us to support asphyxia as the likely cause of death., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Alapont et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Abstract: Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N G-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5–200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70–90% and the tissue cGMP level by 60–80%. By contrast, chronic application of l-NNA between postnatal days 4–21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10–50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum. [Copyright &y& Elsevier]

In the present study, we investigated whether magnesium sulphate activates the L-arginine/NO/cGMP pathway and elicits peripheral antinociception. The male Swiss mice paw pressure test was performed with hyperalgesia induced by intraplantar injection of prostaglandin E2. All drugs were administered locally into the right hind paw of animals. Magnesium sulphate (20, 40, 80 and 160 μg/paw) induced an antinociceptive effect. The dose of 80 μg/paw elicited a local antinociceptive effect that was antagonized by the non-selective NOS inhibitor, L-NOArg, and by the selective neuronal NOS inhibitor, L-NPA. The inhibitors, L-NIO and L-NIL, selectively inhibited endothelial and inducible NOS, respectively, but were ineffective regarding peripheral magnesium sulphate injection. The soluble guanylyl cyclase inhibitor, ODQ, blocked the action of magnesium sulphate, and the cGMP-phosphodiesterase inhibitor, zaprinast, enhanced the antinociceptive effects of intermediate dose of magnesium sulphate. Our results suggest that magnesium sulphate stimulates the NO/cGMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.

The role of cGMP in compensatory renal growth (CRG) is uncertain, since inconsistent changes in renal cGMP have been reported following uninephrectomy (UNx) in the rat. The aim of this study was to reassess the change in cGMP following UNx in the conscious rat by sequential measurements of cGMP excretion and to determine the contribution of nitric oxide, an activator of guanylate cyclase, using L-NAME an inhibitor of nitric oxide synthase. In the conscious rat a sustained increase in the urinary excretion of cGMP was produced in the 7-day period following UNx. L-NAME (20 mg/kg per day) prevented the increase in cGMP excretion following UNx, but not compensatory renal growth. Total kidney DNA, however, was reduced by L-NAME. These observations dissociate the increase in cGMP after UNx from the process of renal hypertrophy [ABSTRACT FROM PUBLISHER]

Injection of large doses of ammonium salts leads to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We reported that injecting ammonium acetate (7 mmol/kg) to rats increases the production of superoxide and reduces the activities of some antioxidant enzymes in rat liver and brain. We proposed that these effects induced by ammonia intoxication would be mediated by formation of nitric oxide. To test this possibility we tested whether injection of nitroarginine, an inhibitor of nitric oxide synthase, prevents the effects of ammonia intoxication on antioxidant enzymes and superoxide formation. Following injection of ammonia, glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver by 42%, 54% and 44%, respectively. In brain these activities were reduced by 35%, 46% and 65%, respectively. Glutathione reductase remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both reduction of activity of antioxidant enzymes and increased superoxide radical production were prevented by previous injection of 45 mg/kg of nitroarginine, indicating that ammonia induces increased formation of nitric oxide, which in turn reduces the activity of antioxidant enzymes, leading to increased formation of superoxide. [ABSTRACT FROM AUTHOR]

We have proposed that acute ammonia toxicity is mediated by activation of the N-methyl-D-aspartate type of glutamate receptors. MK-801, a selective antagonist of these receptors, prevents death of animals induced by acute ammonia intoxication as well as ammonia-induced depletion of ATP. It seems therefore that, following activation of the N-methyl-D-aspartate receptors, the subsequent events in ammonia toxicity should be similar to those involved in glutamate neurotoxicity. As it has been shown that inhibitors of nitric oxide synthetase such as nitroargnine prevent glutamate toxicity, we have tested whether nitroarginine prevents ammonia toxicity and ammonia-induced alterations in brain energy and ammonia metabolites. It is shown that nitroarginine prevents partially (≈50%), but significantly death of mice induced by acute ammonia intoxication. Nitroarginine also prevents partially ammonia-induced depletion of brain ATP. It also prevents completely the rise in glucose and pyruvate and partially that in lactate. Injection of nitroarginine alone, in the absence of ammonia, induces a remarkable accumulation of glutamine and a decrease in glutamate. The results reported indicate that nitroarginine attenuates acute ammonia toxicity and ammonia-induced alterations in brain energy metabolites. The effects of MK-801 and of nitroarginine are different, suggesting that ammonia can induce nitric oxide synthetase by mechanisms other than activation of N-methyl-D-aspartate receptors. [ABSTRACT FROM AUTHOR]

1. Previous results suggest that glutamine synthesis in brain could be modulated by nitrix oxide. The aim of this work was to assess this possibility. 2. As glutamine synthetase in brain is located mainly in astrocytes, we used primary cultures of astrocytes to assess the effects of increasing or decreasing nitrix oxide levels on glutamine synthesis in intact astrocytes. 3. Nitric oxide levels were decreased by adding nitroarginine, an inhibitor of nitric oxide synthase. To increase nitric oxide we used S-nitroso- N-acetylpenicillamine, a nitric oxide generating agent. 4. It is shown that S-nitroso- N-acetylpenicillamine decreases glutamine synthesis in intact astrocytes by ≈40–50%. Nitroarginine increases glutamine synthesis slightly in intact astrocytes. 5. These results indicate that brain glutamine synthesis may be modulated in vivo by nitric oxide. [ABSTRACT FROM AUTHOR]

Objective: To investigate the efficacy of the Danlou Fang (DL) Traditional Chinese Medicine formula on microvascular obstruction (no-reflow) through the endothelial/inducible nitric oxide synthase (eNOS/iNOS) pathway in a rat model., Methods: Sprague-Dawley rats were subjected to 60 min of coronary artery occlusion (or sham procedure) followed by 2 h of reperfusion and were then divided into treatment groups: sham, model, DL (500 mg/kg), DL (500 mg/kg) + eNOS inhibitor L-nitroarginine (L-NNA; 7.5 mg/kg), and sodium nitroprusside (SNP; 0.5 mg/kg). There were 16 per group. Areas of no-reflow were determined by thioflavin S staining of heart tissue. Cardiac function was assessed by echocardiography. Myocardial enzymes and antioxidants in serum were measured and analyzed. The relative protein expression levels of eNOS and iNOS were determined by western blotting., Results: DL had a myocardial protective effect on myocardial reperfusion and reduced the area of no-reflow. The serum levels of creatine kinase (CK), myocardial CK isoenzyme CK-MB, and lactate dehydrogenase were significantly lower in the DL group than in the model (P < 0.05). DL treatment also decreased the serum content of malondialdehyde and reactive oxygen species (ROS), increased the activity of superoxide dismutase and nitric oxide, and promoted eNOS expression (P < 0.05) while lowering iNOS expression., Conclusion: DL reduced the area of no-reflow and had a myocardial protective effect that may be associated with the eNOS/iNOS pathway.

Sodium thiosulfate, a reversible oxidation product of hydrogen sulfide, has vasodilating and anti-oxidative properties, making it an attractive agent to alleviate damaging effects of hypertension. In experimental settings, inhibition of nitric oxide synthase causes hypertension, renal dysfunction and damage. We hypothesized that thiosulfate would attenuate renal injury and improve renal function, hemodynamics and the efficiency of oxygen utilization for sodium reabsorption in hypertensive renal disease. Additionally, thiosulfate co-administration would further improve these variables when compared to inhibiting the renin-angiotensin system alone. Nitric oxide synthase was inhibited in Sprague Dawley rats by administering N-ω-nitro-L-arginine (L-NNA) in the food for three weeks. After one week, rats were split into two groups; without and with thiosulfate in the drinking water. In a parallel study, rats given N-ω-nitro-L-arginine and the angiotensin converting enzyme inhibitor lisinopril at a relatively low dose in their food were divided into two groups; without and with thiosulfate in the drinking water. Treatment with thiosulfate alleviated hypertension (mean 190 vs. 229 mmHg), lowered plasma urea (mean 11.3 vs. 20.0 mmol/L) and improved the terminal glomerular filtration rate (mean 503 vs. 260 μl/min/100 gbw), effective renal plasma flow (mean 919 vs. 514 μl/min/100 gbw) and oxygen utilization for sodium reabsorption (mean 14.3 vs. 8.6 μmol/μmol). Combining thiosulfate with lisinopril further lowered renal vascular resistance (mean 43 vs. 63 mmHg/ml/min/100 gbw) and prevented glomerulosclerosis. Thus, our results suggest that thiosulfate has therapeutic potential in hypertensive renal disease and might be of value when added to standard antihypertensive therapies., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Ethanol (EtOH) exposure causes alterations of motor coordination, balance, behavior, speech, and certain cognitive functions are considered to be caused partly by impairment of cerebellar circuits function and modulation of synaptic transmission. The cerebellar cortical molecular layer interneuron-Purkinje cell (MLI-PC) synapses are critical for various information integration and transmission, which are sensitive to acute and chronic EtOH exposure. The aim of this study is to investigate the effect of chronic ethanol exposure on the facial stimulation-evoked MLI-PC synaptic transmission in urethane-anesthetized mice, by electrophysiological recording and pharmacological methods. Under current-clamp recording conditions, air-puff stimulation of ipsilateral whisker pad evoked MLI-PC synaptic transmission, which expressed an inhibitory component (P1) followed by a pause of simple spike (SS) firing in cerebellar PCs. Chronic ethanol exposure did not change the latency of the facial stimulation-evoked responses in cerebellar PCs, but induced significant enhancement of the stimulation-evoked MLI-PC synaptic transmission, which expressed increases in amplitude of P1 and pause of SS firing. The amplitude of P1 and pause of SS in ethanol exposure group were significant higher than that in control group. Cerebellar surface application of nitric oxide synthesis (NOS) inhibitor, L-NNA (5 mM) significantly decreased the amplitude of P1 and the pause of SS firing in EtOH exposure group, but did no effect on control group. In contrast, cerebellar surface application of NO donor, SNAP (100 μM) significantly increased the amplitude of P1 and the pause of SS firing in control group, but not in EtOH exposure group. These results indicated that chronic EtOH exposure significantly facilitated the sensory-evoked MLI-PC synaptic transmission via NO signaling pathway in mouse cerebellar cortex., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alkaloids from Ba lotus seeds (ABLS) are a kind of important functional compounds in lotus seeds. The present study was designed to determine its hypertension prophylactic effects in the L-NNA-induced mouse hypertension model. The mice were treated with ABLS, the serum and tissues levels of NO, MDA, ET-1, VEGF, and CGRP were determined using the experimental kits, the mRNA levels of various genes in the heart muscle and blood vessel tissues were further determined by RT-PCR assay. ABLS could reduce the systolic blood pressure (SBP), mean blood pressure (MBP), and diastolic blood pressure (DBP), compared to that of the model control group. After ABLS treatment, the NO (nitric oxide) contents in serum, heart, liver, kidney and stomach of the mice were higher than that of the control mice, but the MDA (malonaldehyde) contents were lower than that of the control mice. The serum levels of ET-1 (endothelin-1), VEGF (vascular endothelial growth factor) were decreased after ABLS treatment, but CGRP (calcium gene related peptide) level was increased. The ABLS treated mice had higher mRNA expressions of HO-1, nNOS, and eNOS and lower expressions of ADM, RAMP2, IL-1β, TNF-α, and iNOS than the control mice. Higher concentration of ABLS had greater prophylactic effects, which were close to that of the hypertension drug captopril. These results indicated the hypertension prophylactic effects of ABLS could be further explored as novel medicine or functional food in the future., (Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Objective: To observe the effect of β₃adrenoceptors (β₃-AR) activation on rat thoracic aorta smooth muscle contractility and the possible related mechanism., Methods: The endothelium removed thoracic aorta was pre-contracted with 30 mmol/L KCl physiological saline solution (PSS). Then the tension of the thoracic aorta was recorded in presence of BRL37344 (BRL) to determine the action of β₃-AR. The tension of the thoracic aorta was also recorded in the presence of Propranolol (PRA), SR59230A (SR), L-NNA, H-89 and Iberiotoxin (IBTX) respectively to reveal the underling mechanism of β₃-AR activation on rat vascular smooth muscle. Immunohistochemistry was adopted to confirm the existence and the distribution of β₃-AR in rat thoracic aorta., Results: The results showed that: (1) The thoracic aorta was relaxed by β₃-AR activation, with a relaxation percentage of (10.59 ± 0.79). (2) β₃-AR was expressed in both endothelial and smooth muscle layer in thoracic aorta sections of rats. (3) PRA did not block the effect of BRL on the thoracic aorta. The relaxation actions of BRL could be antagonized by pre-incubating the thoracic aorta with SR. (4) L-NNA (a NOS inhibitor) and H-89 (a PKA inhibitor) reversed the relaxation effect of BRL on vascular smooth muscle. (5) The effect of BRL was decreased after application of Ibriotoxin (IBTX), a large conductance calcium dependent potassium channel blocker., Conclusion: The results confirmed that activation of β₃-AR led to relaxation of thoracic aorta smooth muscle. The relaxation action of β₃-AR on smooth muscle of rat thoracic aorta was related to activation of NOS and PKA signaling pathway. Large conductance Ca²⁺-K⁺ channels were involved in the relaxation action of β₃-AR activation on rat thoracic aorta smooth muscle.

Endothelial dysfunction is closely associated with hypertension. Protection of vascular endothelial cell is the key to prevention and treatment of hypertension. Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid, isolated from traditional Chinese medicine Uncaria rbyncbopbylla and Semen Raphani respectively, exhibit properties of anti-hypertension and protection of blood vessels. In the present study, we observed the protective effect of the combined use of Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid to the vascular endothelial cell in N'-nitro-L-arginine-induced hypertensive rats and investigate the preliminary mechanism. Blood pressure was detected by non-invasive rats tail method to observe the anti-hypertension effect of drugs. Scanning electron microscopy was used to observe the integrity or shedding state of vascular endothelial cell. The amount of circulating endothelial cells and CD54 and CD62P expression on circulating endothelial cells were tested to evaluate the endothelium function. In this study, we found that the Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid compatibility can effectively lower the blood pressure, improve the structural integrity of vascular endothelium, and significantly reduce the number of circulating endothelial cells. Furthermore, the mean fluorescence intensity of CD54 and CD62P expressed showed decrease after the intervention of Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid compatibility. In conclusion, the combination of effective components of the Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid demonstrated good antihypertension effect and vascular endothelium protective effect. The preliminary mechanism of the protective effect may attribute to relieve the overall low-grade inflammation.

Ethnopharmacological Relevance: The leaves of Taxus chinensis var. mairei (Taxaceae) is used traditionally to fill pillows in some rural areas of China. Its volatile substances have been speculated to be capable of improving sleep quality, making blood pressure stable, and having diuretic capacity as recorded in Ancient Chinese Materia Medica. Using animal models and new technologies, we confirmed the hypotensive potential of volatile components from leaves of Taxus chinensis var. mairei (VCLT)., Materials and Methods: VCLT was obtained by supercritical CO(2) extraction equipment from Taxus chinensis var. mairei fresh leaves. Hypertensive rats were pre-induced by intraperitoneal (i,p.) injection of Nω-Nitro-l-Ariginine (l-NNA) for 15 days (15mg/kg, twice a day), then divided into 5 groups and subjected to the following treatments. l-NNA group (group 1) receiving l-NNA alone (15mg/kg, i.p., twice per day for 6 weeks); in addition to receiving l-NNA same as group 1, Hydrochlorothiazide (HDZ) group (group 2) receiving HDZ (orally administration, 5mg/kg, once per day for 6 weeks); VCLT groups (groups 3-5), including VCLT1, VCLT2, VCLT3. The VCLT rats were housed in an enclosed cage (2 rats/0.064m(3)). VCLT was mixed well and sprayed on fresh leaves surface of Taxus chinensis var. mairei (100ml/kg) with three dosages: 167g/kg (VCLT1), 233g/kg (VCLT2) and 333g/kg (VCLT3), respectively. Systolic Blood Pressure (SBP), plasma nitric oxide (NO), plasma angiotensin II, postprandial blood glucose, fasting blood glucose and blood lipids were determined., Results: VCLT prevented the increase of SBP and plasma angiotensin II in l-NNA treated rats. Although VCLT does not significantly reduce blood triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C), it decreases total cholesterol (TC) while increasing plasma NO levels in a dose-dependent manner., Conclusion: VCLT can be used as a natural and supplementary reagents for the treatment of hypertension., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

We studied the role of nitric oxide (NO) and extra-cellular Ca(2+) on the melanophores in Indian snakehead teleost, Channa punctatus. Increase of Ca(2+) level in the external medium causes pigment aggregation in melanophores. This pigment-aggregating effect was found to be inhibited when the external medium contained spontaneous NO donor, sodium nitro prusside (SNP) at all the levels of concentration tested. Furthermore, it has been observed that SNP keeps the pigment in dispersed state even after increasing the amount of Ca(2+). In order to test whether NO donor SNP causes dispersion of pigments or not is checked by adding the inhibitor of nitric oxide synthase, N-omega-Nitro-L-arginine (L-NNA) in the medium. It has been noted that the inhibitor L-NNA blocked the effect of NO donor SNP causing aggregation of pigments. In that way NO is inhibiting the effect of extracellular Ca(2+), keeping the pigment dispersed.

Background and Aim: Hyperin, a flavonol compound extracted from the Chinese herb Abelmoschus manihot L. Medic, is reported to exert protective actions in cerebral ischemic injury. The specific aim of the present study was to study the relaxation of Hyperin in rat isolated basilar artery and identify the underlying cellular mechanisms., Methods: Rat isolated basilar artery segments were cannulated and perfused while being superfused with PSS solution. Vessel images were recorded by video microscopy and diameters measured. Membrane potential was recorded using glass microelectrodes to evaluate the basilar artery smooth muscle cell hyperpolarization., Results: Perfusion of Hyperin (1~100 μM) elicited a concentration-dependent relaxation of basilar artery segments preconstricted with 0.1 μM U46619. The response was significantly inhibited by the removal of the endothelium. Hyperin also elicited marked and concentration-dependent hyperpolarization of smooth muscle cells. 30 μM nitro-L-arginine (an inhibitor of nitric oxide synthase) and indomethacin (an inhibitor of cyclooxygenase), partially inhibited Hyperin-induced relaxation and hyperpolarization leaving an attenuated, but significant, endothelium-dependent relaxation and hyperpolarization. This remaining effect was almost completely blocked by 1mM tetraethylammonium (an inhibitor of Ca(2+)-activated K(+) channels), or by 100 μM DL-propargylglycine, an inhibitor of cystathionine-γ-lyase (a synthase of the endogenous H(2)S)., Conclusion: These findings show that Hyperin produces significant hyperpolarization in rat basilar artery smooth muscle cells and relaxation through both endothelium-dependent and endothelium-independent mechanisms. The underlying mechanisms appeared to be multi-factorial involving nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Our data further suggest that endogenous H(2)S is a component of the EDHF-mediated hyperpolarization and relaxation to Hyperin., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

We recently developed a rat model of cardiorenal failure that is characterized by severe left ventricular systolic dysfunction (LVSD) and low nitric oxide (NO) production that persisted after temporary low-dose NO synthase inhibition. We hypothesized that LVSD was due to continued low NO availability and might be reversed by supplementing NO. Rats underwent a subtotal nephrectomy and were treated with low-dose NO synthase inhibition with N(ω)-nitro-l-arginine up to week 8. After 3 wk of washout, rats were treated orally with either the long-acting, tolerance-free NO donor molsidomine (Mols) or vehicle (Veh). Cardiac and renal function were measured on weeks 11, 13, and 15. On week 16, LV hemodynamics and pressure-volume relationships were measured invasively, and rats were killed to quantify histological damage. On week 15, blood pressure was mildly reduced and creatinine clearance was increased by Mols (both P < 0.05). Mols treatment improved ejection fraction (53 ± 3% vs. 37 ± 2% in Veh-treated rats, P < 0.001) and stroke volume (324 ± 33 vs. 255 ± 15 μl in Veh-treated rats, P < 0.05). Rats with Mols treatment had lower end-diastolic pressures (8.5 ± 1.1 mmHg) than Veh-treated rats (16.3 ± 3.5 mmHg, P < 0.05) and reduced time constants of relaxation (21.9 ± 1.8 vs. 30.9 ± 3.3 ms, respectively, P < 0.05). The LV end-systolic pressure-volume relationship was shifted to the left in Mols compared with Veh treatment. In summary, in a model of cardiorenal failure with low NO availability, supplementing NO significantly improves cardiac systolic and diastolic function without a major effect on afterload.

We have previously demonstrated the vasorelaxant activity of 1,3,4-oxadiazole derivative (NOX-1) through L-type Ca2+ channel blockage. In the present study, we investigated whether the correction of endothelial dysfunction is dependent on the normalization of high blood pressure levels by 1,3,4-oxadiazole derivative (NOX-1) in deoxycorticosterone acetate (DOCA-salt) and N(G)-nitro-l-arginine (L-NNA) hypertensive rats. In DOCA-salt and L-NNA hypertensive rats, the mean systolic blood pressure (MSBB) was 185.3+/-4.7 and 170.2+/-4.1 mmHg, whereas after administration of NOX-1 to hypertensive rats, MSBB was 127.8+/-4.5 and 120.2+/-5.1 mmHg, respectively. To study the endothelial dysfunction, concentration-response curves of norepinephrine (NE) and acetylcholine (Ach) were constructed in rat aortic rings isolated from normotensive, hypertensive (DOCA and L-NNA) and NOX-1 treated rats. NE-induced contractions and Ach-induced relaxations were significantly (p<0.05) decreased and increased, respectively in the aorta of NOX-1 treated rats. Vasorelaxant activity of NOX-1 was not abolished by pretreatment of aortic rings with L-NNA, 1H-[1,2,4] oxadiazolo [4,3-A] quinoxalin-1-one (ODQ), indomethacin or glibenclamide. The results suggest that the endothelial dysfunction can be corrected by the L-type Ca2+ channel blocker with endothelium-independent action and that is dependent on the normalization of high blood pressure levels. The antihypertensive and vasorelaxant effects of NOX-1 are mainly endothelial-independent and it can be used to treat hypertension, a state associated with endothelial dysfunction., (2009 Elsevier Ireland Ltd. All rights reserved.)

Background: In a pilot study we could show that hydroxyethyl starch (HES) induced a significant reduction of endothelium-dependent relaxation (EDR) and the endothelium-derived hyperpolarizing factor (EDHF). In this follow-up study we investigated whether this effect of HES was dose-dependent and whether it could be replicated with other colloids like dextran (DX) and gelatin (GL)., Methods: Rings of fresh porcine coronary arteries were consecutively tested with or without HES, DX or GL (5, 10, or 20 mg/ml). Indomethacin was added in all measurements to eliminate prostacyclin effects. Prostaglandin F₂α was used for contraction and bradykinin (BK, 10⁻¹⁰ to 10⁻⁵ M) for inducing EDR. After blocking nitric oxide (NO) by N-nitro-L-arginine (L-NNA), the experiments were repeated to assess the EDHF-mediated relaxation response to BK., Results: HES induced a reduction in EDR for the BK concentrations of 10⁻⁸ and 10⁻⁷ M (n = 10; p < 0.05). After NO blockage with L-NNA, the relaxation response was reduced especially for the BK concentrations of 10⁻⁶ and 10⁻⁵ M (p < 0.05). GL showed a reduction in EDR with or without NO blockage with L-NNA especially for the BK concentrations of 10⁻⁶ and 10⁻⁵ M (n = 14; p < 0.05). DX induced a significant reduction in EDR for the BK concentrations of 10⁻⁷ and 10⁻⁶ M (n = 12; p < 0.05). After NO blockage with L-NNA, the relaxation response was reduced especially for the BK concentrations of 10⁻⁶ and 10⁻⁵ M (p < 0.05)., Conclusion: For clinically relevant concentrations of HES, DX and GL a significant reduction in both NO-induced and NO-/prostacyclin-independent EDR can be found in epicardial coronary arteries of the pig., (Copyright © 2010 S. Karger AG, Basel.)

N-methyl-D-aspartate (NMDA) receptor and nitricoxide syntheses are the emerging target sites for development of novel drug molecules because their modulation affects the long term potentiation (LTP) process. NMDA receptor antagonists and nitric oxide synthase inhibitors induce amnesia in animals and therefore have been employed for evaluation of efficacy of several novel antiamnesic agents.Bacopa monniera Linn (syn. Brahmi) is commonly used in the ancient Indian medical system for improvement of memory deficit.We have earlier described the involvement of GABAergic and cholinergic system to account for the antiamnesic effects of B. monniera on diazepam- and scopolamine-induced amnesia.In extension to our previous study this study was designed to investigate the downstream mechanism of B. monniera by evaluation of its effect on MK-801 (an NMDA receptor antagonist) and N(w)-nitro-L-arginine (L-NNA) (a nitric oxide inhibitor)induced memory deficit. We used a Morris water maze scale and compared the degree of reversal of amnesia induced by the two agents. Male Swiss albino mice were subjected to a Rotarod muscle incoordination test followed by water maze tasks.Our data revealed that L-NNA and MK-801 produced anterograde and retrograde amnesia and B. monniera significantly attenuated the L-NNA-induced anterograde amnesia, partially reversing L-NNA-induced retrograde amnesia. On the other hand, B. monniera neither attenuated the MK-801-induced anterograde amnesia nor improved retrograde amnesia caused by it.

Arterial hyper-responsiveness to 5-hydroxytryptamine (5-HT) is a hallmark of hypertension, and plasma levels of free 5-HT are elevated in hypertension. We hypothesized that chronic administration of 5-HT would cause blood pressure to 1) rise in normotensive rats and 2) rise significantly more in hypertensive rats. The deoxycorticosterone acetate (DOCA)-salt hypertensive and sham normotensive rat were used. Animals were implanted with minipumps that delivered 5-HT (or vehicle) at a rate of 25 microg/kg/min for 7 days. Free plasma 5-HT was elevated significantly by this protocol. Within 48 h, mean arterial blood pressure measured telemetrically decreased in sham (106 +/- 2 to 83 +/- 2 mm Hg) and in DOCA-salt hypertensive (166 +/- 9 to 112 +/- 3 mm Hg) rats; vehicle did not change blood pressure in either group. Ganglionic blockade (hexamethonium) reduced blood pressure to a greater magnitude in DOCA vehicle-administered rats (peak fall arterial pressure, 91 +/- 14 mm Hg) compared with DOCA 5-HT-administered rats (40 +/- 6 mm Hg). Maximal acetylcholine-induced (NO-dependent) relaxation in phenylephrine-contracted aortic strips was greater in 5-HT-administered (69.2 +/- 9.1% relaxation) versus vehicle-administered (39.7 +/- 14.2%) DOCA rats; aortic endothelial cell nitric oxide synthase expression was higher in the 5-HT- versus vehicle-administered DOCA-salt rats. In normotensive and DOCA-salt hypertensive rats, the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine (0.5 g/l in water) prevented the fall in blood pressure to 5-HT. We conclude that chronic exogenous 5-HT reduces blood pressure in normotensive and hypertensive rats through mechanisms critically dependent on NOS.

Objective: Although, there are some evidences on the contribution of increased sympathoadrenergic activity on long-term nitric oxide synthase (NOS) inhibition induced hypertension, the contribution of sympathetic activity to the development of this model of hypertension are not sufficiently studied. The aim of the present study is to investigate the effects of clonidine on blood pressure and vascular alpha-adrenergic receptors in the long-term N (omega)-nitro-L-arginine (L-NNA) treated rats., Methods: Sixty two Wistar rats were randomly divided into 8 groups. All groups were administrated L-NNA and/or clonidine in two different concentrations for ten days. L-NNA was administrated in concentrations of 15 and 45 mg/100ml to L-NNA15 and L-NNA45 groups, respectively. Clonidine was also administrated in concentrations of 150 and 225 microg/100ml to KLO150 and KLO225 groups, respectively. Blood pressure and heart rates were measured with tail-cuff method and plasma NOx levels with spectrophotometer. The a-adrenoreceptors responses were evaluated in thoracic aorta rings in "in vitro" conditions., Results: Clonidine prevented the L-NNA induced hypertension dose-dependently, but did not effect the heart rates decreased by L-NNA. The heart rates and blood pressure of normotensive rats were not changed by clonidine alone. Plasma NOx levels increased in L-NNA15 group (0.62+/-0.11 micromol/L, p=0.003) but did not change in other groups. The sensitivity of aorta to phenylephrine (-7.33+/-0.11 micromol/L, p=0.001) and clonidine (-7.60+/-0.27 micromol/L, p=0.003) in L-NNA45 group and phenylephrine (-6.94+/-0.13 micromol/L, p=0.002) in L-NNA15 group increased. The sensitivity of aorta to phenylephrine (7.93+/-0.16 micromol/L, p=0.001) in KLO225 group and to clonidine (-7.20+/-0.10 micromol/L, p=0.009) in KLO150 group increased., Conclusion: This study supports the idea suggesting that symphathetic nervous system activation is partly responsible for the development of the long-term NOS inhibition induced hypertension. In conclusion, it was shown for the first time that clonidine prevents the development of long-term NOS inhibition induced hypertension dose-dependently.

Objective: Hydroxyethyl starch (HES) solutions are widely used for fluid resuscitation. We studied the effects of HES on endothelium-dependent relaxation (EDR), especially on the endothelium-derived hyperpolarizing factor (EDHF)., Methods: Four-millimeter-long rings of fresh porcine coronary arteries from the local slaughterhouse were consecutively tested with or without HES (6 mg/ml). Indomethacin (10 micromol/l) was added in all measurements to eliminate prostacyclin effects. Prostaglandin F2alpha (10 micromol/l) was used for contraction and bradykinin (10(-10) to 10(-5) mol/l) for inducing EDR, which was calculated in percentage of the precontraction. After blocking all nitric oxide formation by N-nitro-L-arginine (300 micromol/l), the experiments were repeated to assess the EDHF-mediated relaxation response to bradykinin., Results: HES 6 mg/ml induced a significant (p < 0.01) reduction in EDR (n = 8). After incubation with HES and nitric oxide blockage with N-nitro-L-arginine, the relaxation response was reduced especially for the bradykinin concentrations of 10(-6) mol/l (p < 0.05) and 10(-5) mol/l (p < 0.01)., Conclusion: For the clinically relevant concentration of 6 mg/ml HES, a significant reduction in EDR and the EDHF can be found in epicardial coronary arteries of the pig., (Copyright 2008 S. Karger AG, Basel.)

Background: The importance of nitric oxide (NO) in the pathogenesis of portal hypertension (PHT) has been extensively studied, but whether or not prostacyclin (PGI(2)) plays a role in formation and development of hyperdynamic circutatory state in PHT has not been verified. The present study was undertaken to investigate the possible interaction between prostacyclin (PGI(2)) and nitric oxide (NO) in the hyperdynamic circulatory state of rats with chronic portal hypertension (PHT), by measuring the hemodynamic changes and expression of cyclooxygenase (COX) mRNA in vessels and small intestine after administration of N(omega)-nitro-L-arginine (L-NNA) or indomethacin (INDO) either in the short-term (7 days) or long-term (15 days)., Methods: Ninety-seven male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) induced by injection of CCl(4), prehepatic portal hypertension (PHPH) induced by partial stenosis of the portal vein, and sham-operated controls (SO). Animals of each group received L-NNA or INDO either for 7 or 15 days, with saline as control. Splanchnic hemodynamics was measured by the radioactive microsphere technique. The concentration of NO in serum was determined as the nitrate; nitrite ratio (NO(2)(-)/NO(3)(-), micromol/L) by a colorometric method, and that of PGI(2) was measured by specific radioimmunoassay for its stable hydrolysis product 6-keto-PGF(1alpha) (pg/ml). The reverse transcription-polymerase chain reaction measured the levels of COX-1 mRNA in the superior mesenteric artery, thoracic aorta, and small intestine of these rats., Results: Compared with SO rats, COX-1 mRNA expression and the concentrations of plasma 6-keto-PGF(1alpha) and serum NO(2)(-)/NO(3)(-) were enhanced in both IHPH and PHPH rats; splanchnic vascular resistance (SVR) decreased, but portal venous inflow (PVI) markedly increased (P<0.05). Seven or 15 days of L-NNA treatment reduced COX-1 mRNA expression in these vessels and the small intestine, concomitant with a significant decrease in the concentration of plasma PGI(2) and serum NO in IHPH and PHPH rats (P<0.05). At the same time, PVI decreased but SVR increased significantly (P<0.05). In both IHPH and PHPH rats, the COX-1 mRNA expression and the concentration of plasma PGI(2) after No synthase (NOS) blockade for 15 days were higher than those for 7 days, whereas the hyperdynamic circulatory state was improved after NOS blockade for 15 days compared with 7 days. The concentration of PGI(2) treated by INDO for 15 days was not significantly different from that after 7-day COX blockade, and hemodynamics restored hyperdynamic circulatory state., Conclusions: The hyperdynamic circulatory state in rats with PHT is correlated with the concentration of serum NO. There is a possible interaction between PGI(2) and NO in the hyperhemodynamics of PHT. PGI(2) is probably not the mediator in the formation and development of the hyperdynamic circulatory state in rats with chronic PHT.

We reported upregulation of the 5-hydroxytryptamine (HT) transporter (5-HTT) protein in peripheral arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We hypothesized that upregulated 5-HTT may be generally elevated in hypertensive models and, as a consequence, a higher basal concentration of 5-HT, the 5-HT metabolite 5-hydroxyindoleacetic acid, and an increased 5-HT uptake would occur in peripheral arteries of hypertensive rats compared with normotensive rats. We examined 3 hypertension models: DOCA-salt rats, Nomega-nitro-L-arginine (LNNA) rats, and spontaneously hypertensive rats (SHRs) in our study (systolic blood pressure [mm Hg]: DOCA (D)=197+/-6, SHAM(D)=112+/-4, LNNA (L)=228+/-9, SHAM(L)=128+/-2, SHR=172+/-7, and Wistar-Kyoto [WKY]= 121+/-3). High-pressure liquid chromatography measurements showed lower basal 5-HT concentrations in aorta from DOCA-salt and LNNA rats compared with their SHAM rats but not in SHR compared with WKY. In all of the 5-HT-uptake studies, we used arteries isolated from rats treated with the monoamine oxidase-A inhibitor pargyline to minimize 5-HT metabolism. Exogenous 5-HT was taken up by aorta, and this was inhibited by the 5-HTT inhibitor fluoxetine (1 micromol/L) or fluvoxamine (1 micromol/L). Total 5-HT uptake and 5-HTT-dependent active 5-HT uptake were decreased in aorta from DOCA-salt and LNNA rats compared with SHAM rats, but this was not observed in SHRs compared with WKYs. Western analysis revealed similar expression of 5-HTT in aorta from WKYs and SHRs as opposed to an upregulated 5-HTT in aorta from DOCA-salt and LNNA-hypertensive rats. Our study suggested that an altered serotonergic system by impaired 5-HTT function might play a role in blood pressure regulation in DOCA-salt and LNNA-hypertensive rats.

The aim of this investigation was to assess the role that NO plays in the antinociceptive activity of tramadol using a rat model of neuropathic pain. Thirty male Wistar rats weighing 200-250 g were randomly divided into five equal groups. The neuropathic pain model used for the study was chronic constrictive injury (CCI) model. Three weeks after the surgical procedure, each rat was tested to assess mechanical threshold in grams using an electronic algometer. After CCI was induced, tramadol hydrochloride was administered by intraperitoneal (i.p.) injection in all groups, and Nomega-nitro-L-arginine (L-NA) and L-arginine were administered i.p. or intrathecally (i.t.) depending on the group. Tramadol was administered in 10 mg/kg doses i.p., L-NA was given in 10 mg/kg doses i.p. and in 30 microg/kg doses i.t.. L-arginine was given in 10 mg/kg doses i. p. and in 50 microg/kg doses i.t.. The multiple agents were given 30 minutes apart from each administration. Intraperitoneal administration of tramadol (Group 1) only increased mechanical threshold in the rats' left hind paw, whereas in i.p. L-NA group (10 mg/kg) (Group 2) produced a significant reduction of the mean mechanical antinociceptive threshold (p<0.05). Like this, in i.t. L-NA group (30 microg/kg) (Group 4) a significant reduction of the mean mechanical antinociceptive threshold (p<0.05) was also observed. The mean threshold values in Group 2 (i.p. tramadol+i.p. L-NA) and Group 4 (i.p. tramadol+i.t. L-NA) were not significantly different. The mean threshold values in Groups 3 (i.p. tramadol+i.p. L-NA+i.p. L-arginine) and 5 (i.p. tramadol+i.t. L-NA+i.t. L-arginine) were also similar. The mean mechanical antinociceptive threshold was significantly increased in Group 3 (i.p. L-NA+L-arginine) and Group 5 (i.t. L-NA+L-arginine) when compared to Group 1 (i.p. tramadol only) (p<0.05 for both). The results of this study support the involvement of the L-arginine/nitric oxide pathway in the antinociceptive effect of tramadol in a rat model of neuropathic pain.

Smooth muscle membrane potential is determined, in part, by K(+) channels. In the companion paper to this article, we demonstrated that superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-l-arginine (l-NNA) are depolarized and express less K(+) channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that l-NNA-induced hypertension reduces the functional expression of K(+) channels in smooth muscle. In whole cell experiments using a Ca(2+)-free pipette solution, current at 0 mV, largely due to voltage-dependent K(+) (K(V)) channels, was reduced approximately 60% by hypertension (2.7 +/- 0.4 vs. 1.1 +/- 0.2 pA/pF). Current at +100 mV with 300 nM free Ca(2+), largely due to large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, was reduced approximately 40% by hypertension (181 +/- 24 vs. 101 +/- 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many K(V) channel types, was reduced approximately 50% by hypertension (1.0 +/- 0.4 vs. 0.5 +/- 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BK(Ca) channels, was reduced approximately 40% by hypertension (86 +/- 14 vs. 53 +/- 19 pA/pF). Differences in BK(Ca) current magnitude are not attributable to changes in single-channel conductance or Ca(2+)/voltage sensitivity. The data support the hypothesis that l-NNA-induced hypertension reduces K(+) current in vascular smooth muscle. Reduced molecular and functional expression of K(+) channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from l-NNA-treated rats.

Smooth muscle membrane potential (E(m)) depends on K(+) channels, and arteries from rats made hypertensive with N(omega)-nitro-l-arginine (LHR) are depolarized compared with control. We hypothesized that decreased K(+) channel function, due to decreased K(+) channel protein expression, underlies E(m) depolarization. Furthermore, K(+) channel blockers should move control E(m) (-46 +/- 1 mV) toward that in LHR (-37 +/- 2 mV) and normalize contraction. The E(m) vs. K(+) relationship was less steep in LHR (23 +/- 2 vs. 28 +/- 1 mV/log K(+) concentration), and contractile sensitivity to K(+) was increased (EC(50) = 37 +/- 1 vs. 23 +/- 1 mM). Iberiotoxin (10 nM), an inhibitor of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, depolarized control and LHR E(m) to -35 +/- 1 and -30 +/- 2 mV, respectively; however, effects on K(+) sensitivity were more profound in LHR (EC(50) = 25 +/- 2 vs. 15 +/- 3 mM). The voltage-dependent K(+) (K(V)) channel blocker 4-aminopyridine (3 mM) depolarized control E(m) to the level of LHR (-28 +/- 1 vs. -28 +/- 1 mV); however, effects on K(+) sensitivity were greater in LHR (EC(50) = 17 +/- 4 vs. 4 +/- 4 mM). Western blots revealed reduced BK(Ca) and K(V)1.5 channel expression in LHR arteries. The findings suggest that diminished expression of K(+) channels contributes to depolarization and enhanced contractile sensitivity. These conclusions are supported by direct electrophysiological assessment of BK(Ca) and K(V) channel function in control and LHR smooth muscle cells.

The aim of this study was to evaluate the impact of poisoning with cadmium in hypertensive doses (50 or 200 ppm in drinking water for three months) on the basal and stimulated release NO effect in the isolated and perfused rat mesenteric bed. Mesenteric artery preparation preconstricted by norepinephrine (0.5 microg/mL) was used to determine changes in its vascular resistance induced by e-NOS synthase blocker, N-omega-nitro-L-arginine (L-NOARG) injected in increasing doses from 1.0 to 200.0 microg or acetylcholine (ACh) administered in doses from 0.05 x 10(-10) to 5.0 x 10(-10) mol before and during L-NOARG infusion (1.0 microg/mL). Vascular reactivity was measured as an increase or decrease in perfusion pressure in the constant flow system. Rats poisoned with 50 or 200 ppm of cadmium demonstrated a significant decrease (P <0.05) in vascular response to L-NOARG used in doses of 50 or 100 microg. The dose-response curve obtained for L-NOARG was shifted to the right and ED50 value was greater in the group of rats given cadmium in a dose of 200 ppm than in the controls (70.3 +/- 10.7 versus 25.7 +/- 4.8 microg, P <0.01). These rats reacted with lower expressed vasodilatation to ACh in doses to 0.2 x 10(-10) mol. In all poisoned rats, L-NOARG enhanced the effect of ACh used in doses from 0.05 to 0.5 x 10(-10) mol, whereas in the control group this effect was only achieved at 0.1 x 10(-10) mol. The serum nitric oxide concentration was decreased (P <0.05) in both groups of cadmium-treated rats. These results suggest that cadmium in hypertensive doses modifies the vascular effect of NO in basal conditions and after stimulation by ACh.

Previous studies have shown that chronic hyperhomocysteinemia is associated with an adverse cardiac remodeling and heart failure. This study, which utilized coronary-perfused hearts and superfused papillary muscle, was designed to determine whether homocysteine acts acutely to alter cardiac contractile function. Left ventricular developed pressure was used as a measure of systolic function in the Langendorff-perfused heart, whereas isometric developed tension was used in papillary muscle. All preparations were bathed in physiological buffer and paced electrically. Initial results showed that homocysteine elicits a relatively rapid onset (maximum effect observed within 5 min), concentration-dependent (10-300 microM), and moderate negative inotropic action (maximum decrease in tension was approximately 15% of control values) in Langendorff-perfused hearts but not in papillary muscle. In contrast, effluent from homocysteine-treated hearts decreased contractility in papillary muscle, and all inotropic actions were largely eliminated when brief Triton X-100 treatment was utilized to inactivate the coronary endothelium in the intact heart. The homocysteine-induced decrease in contractile function was not antagonized by N(omega)-nitro-l-arginine, a nitric oxide synthase inhibitor, or the cyclooxygenase inhibitor indomethacin. Thus data suggest that pathophysiological concentrations of homocysteine elicit an acute negative inotropic effect on ventricular myocardium that is mediated by a coronary endothelium-derived agent other than nitric oxide or products of cyclooxygenase. Future studies are required to elucidate the mechanism by which homocysteine acts to elicit the release of the proposed endothelial mediator, the identity of the proposed paracrine agent, and the mechanism of its negative inotropic action.

We assessed the effect of nitric oxide (NO) synthase inhibition on plasma atrial natriuretic peptide (ANP) concentration and content in some brain structures [neurohypophysis (NH), adenohypophysis (AH), medial basal hypothalamus (MHB) and olfactory bulb (OB)] in rats before and after blood volume expansion (BVE). Male Wistar rats were injected i.p. with N(pi)-nitro-L-arginine (L-NNA), 25 mg/kg of body weight, 40 min before the experiment (acute treatment) or L-NNA at a dose of 25 mg/kg body weight, twice a day, for 4 days (chronic treatment). The acute treatment caused an increase in the blood pressure and plasma ANP concentration in rats under basal conditions and after BVE. A decrease in ANP content was observed in the OB and NH, whereas no significant changes were found in the AH or MBH. In chronically treated rats, we also found an increase in blood pressure and in plasma ANP concentration under basal conditions and after BVE. The ANP content increased in the OB, NH and AH. These results indicate that systemic NO synthase inhibition increases ANP concentration in plasma and in areas of the central nervous system. We hypothesize that ANP participates in the hypertension-induced by NO synthesis blockade acting by baroreceptors input to the brain to stimulate ANP release and synthesis that reduces NO prival hypertension.

This study investigated the role of nitric oxide (NO) in the control of right coronary (RC) blood flow at rest and during acute pulmonary hypertension. Experiments were performed in seven chronically instrumented, conscious dogs. NO synthesis was inhibited by systemic administration of N(omega)-nitro-L-arginine (LNA, 35 mg/kg). Inflation of a balloon in the main pulmonary artery raised right ventricular (RV) peak systolic pressure from 34 +/- 2 to 47 +/- 3 mmHg before LNA and from 37 +/- 2 to 47 +/- 3 mmHg after LNA, but did not affect mean systemic arterial pressure. RV O(2) consumption (MVO(2)) increased from 4.4 +/- 0.7 to 6.1 +/- 0.7 ml/min/100 g. 82 % of the elevated RV MVO(2) was provided by RC blood flow, which increased from 46 +/- 7 to 61 +/- 8 ml/min/100 g. After LNA, resting RV MVO(2) and RC flow fell. RC venous PO(2) fell, but RV lactate uptake was not altered. During pulmonary hypertension, the increase in RC blood flow was blunted by LNA, so that only 66 % of the elevated RV MVO(2) was supplied by increased RC flow. Analysis of O(2) supply variables as functions of RV MVO(2) further demonstrated a significant role of NO in regulating RC flow at rest and during moderate pulmonary hypertension. Conclusions NO is required for the RC hyperemic response to acute pulmonary hypertension as well as for normal resting RC blood flow. After blockade of NO synthesis, RV O(2) supply at rest and during pulmonary hypertension was sustained by increased RV O(2) extraction.

Arteries from deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184+/-5 mm Hg] compared with sham rats (SBP=111+/-2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85alpha, p110alpha, p110beta, and p110delta but not p110gamma PI3-kinase subunits; p110delta protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110beta protein density, but neither L-NNA nor DOCA-salt had differences in p85alpha and p110alpha. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 micromol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110delta density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.

Nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-L-arginine (L-NNA) produces L-NNA hypertensive rats (LHR), which exhibit increased sensitivity to voltage-dependent Ca(2+) channel-mediated vasoconstriction. We hypothesized that enhanced contractile responsiveness after NOS inhibition is mediated by depolarization of membrane potential (E(m)) through attenuated K(+) channel conductance. E(m) measurements demonstrated that LHR vascular smooth muscle cells (VSMCs) are depolarized in open, nonpressurized (-44.5 +/- 1.0 mV in control vs. -36.8 +/- 0.8 mV in LHR) and pressurized mesenteric artery segments (-41.8 +/- 1.0 mV in control vs. -32.6 +/- 1.4 mV in LHR). Endothelium removal or exogenous L-NNA depolarized control VSMCs but not LHR VSMCs. Superfused L-arginine hyperpolarized VSMCs from both the control and LHR groups and reversed L-NNA-induced depolarization (-44.5 +/- 1.0 vs. -45.8 +/- 2.1 mV). A Ca(2+)-activated K(+) channel agonist, NS-1619 (10 microM), hyperpolarized both groups of arteries to a similar extent (from -50.8 +/- 1.0 to -62.5 +/- 1.2 mV in control and from -43.7 +/- 1.1 to -55.6 +/- 1.2 mV in LHR), although E(m) was still different in the presence of NS-1619. In addition, superfused iberiotoxin (50 nM) depolarized both groups similarly. Increasing the extracellular K(+) concentration from 1.2 to 45 mM depolarized E(m), as predicted by the Goldman-Hodgkin-Katz equation. These data support the hypothesis that loss of NO activation of K(+) channels contributes to VSMC depolarization in L-NNA-induced hypertension without a change in the number of functional large conductance Ca(2+)-activated K(+) channels.

Background: Vascular endothelial growth factor, a specific endothelial mitogen, plays an important role in myocardial angiogenesis. Previous work has demonstrated increased expression of vascular endothelial growth factor and its receptors in a rat myocardial infarction model, as well as in a pig model of chronic ischemia. The expression of vascular endothelial growth factor and other growth factors after acute myocardial ischemia in patients has not been examined. In this study we examined the expression of vascular endothelial growth factor and its receptors and the responsiveness of human atrial microvessels to vascular endothelial growth factor before and after acute ischemia., Methods: Paired specimens of human atrial tissue were harvested before and after atrial devascularization (ligation) in 16 patients undergoing coronary bypass operations., Results: The messenger RNA (reverse transcriptase-polymerase chain reaction) level of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 were increased by 22.2% +/- 4.2% and 30.7% +/- 7.6%, respectively (P <.05), in the ischemic specimens as compared with the control specimens. Protein expression (Western blotting) of vascular endothelial growth factor and that of vascular endothelial growth factor receptor 1 also were increased significantly by 71.7% +/- 27.8% and 68.2% +/- 27.6%, respectively (P <.05). However, both RNA and protein expressions of another vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2, and fibroblast growth factor and fibroblast growth factor receptor 1 were unchanged. Reactivity of precontracted atrial vessels was examined with video microscopy. Vascular endothelial growth factor-induced (33.9% +/- 2.4% vs 18.3% +/- 2.8% in control and ischemic vessels, respectively; P <.05), fibroblast growth factor-induced (31.6% +/- 3.2% vs 15.8% +/- 4.1%, P <.05), and substance P-induced (84.5% +/- 3.7% vs 54.3% +/- 9.0%, P <.05) microvascular relaxations were decreased in ischemic samples and in the presence of N (G)nitro-L -arginine, whereas responses to sodium nitroprusside were unchanged (90.9% +/- 2.2% vs 91.2% +/- 2.0%)., Conclusions: This study suggests that acute myocardial ischemia in patients results in increased expression of vascular endothelial growth factor but not fibroblast growth factor and that the functional activity of vascular endothelial growth factor receptors and that of other growth factors may be impaired.

Tetrahydrobiopterin (BH4) acts as an important co-factor for endothelial nitric oxide synthase (eNOS). Glucocorticoids have been shown to inhibit expression of the rate-limiting enzyme for tetrahydrobiopterin synthesis, GTP cyclohydrolase, in other cell types. We hypothesized that endothelium-dependent vasodilator responses would be blunted in rats made hypertensive with dexamethasone. Further, we hypothesized that treatment of rat vascular segments with dexamethasone would result in attenuation of endothelial function accompanied by decreased GTP cyclohydrolase expression. We report that endothelium-dependent relaxation responses to the calcium ionophore A23187 are reduced in aortic rings from dexamethasone-hypertensive rats compared with sham values. Dexamethasone incubation abolishes contraction to Nomega-nitro-L-arginine (L-NNA, 10(-5) M) in endothelium-intact aortic rings, and inhibits expression of GTP cyclohydrolase. We conclude that inhibition of BH4 synthesis by glucocorticoid regulation of GTP cyclohydrolase expression may contribute to reduced endothelium-dependent vasodilation characteristic of glucocorticoid-induced hypertension.

Aim: To examine the role of endothelium in the vascular responses to flavonoids, baicalein, baicalin, cardamonin, alpinetin, and to purified jasmine green tea (-)epicatechin in the isolated rate mesenteric artery rings., Methods: The isometric contraction was measured by Grass force-displacement transducers., Results: Both baicalein and baicalin enhanced the phenylephrine-induced contractile response in the endothelium-intact rings. This enhancement was abolished by pretreatment with the nitric oxide inhibitor NG-nitro-L-arginine or in the absence of the endothelium. Both flavonoids also inhibited the acetylcholine-induced endothelial nitric oxide-dependent relaxation. In contrast, cardamonin, alpinetin or (-)epicatechin induced both endothelium-dependent and -independent relaxation. NG-nitro-L-arginine meyhyl ester or endothelium denudation attenuated the endothelium-dependent relaxation to the same extent., Conclusion: Baicalein and baicalin enhanced the phenylephrine-induced contraction most likely through inhibiting production or/and release of endothelial nitric oxide. Whilst, cardamonin-, alpinetin- or (-)epicatechin-induced endothelium-dependent relaxation is primarily mediated through endothelial nitric oxide.

Men are at greater risk for renal injury than women. We studied whether male rats are more sensitive to the hypertensive and proteinuric effects of chronic nitric oxide synthase (NOS) inhibition than female rats. In addition, we studied whether androgens or estrogens are responsible for differences in sensitivity to proteinuria induced by chronic NOS inhibition. Females and males were treated with 10, 20, 30, and 100 mg/l N(omega)-nitro-L-arginine (L-NNA) during 24 wk. Systolic blood pressure (SBP) and proteinuria were measured regularly and compared with time-control measurements in control females and males. In females and males treatment with 10 mg/l L-NNA had no effect on SBP or proteinuria. Treatment with 20, 30, and 100 mg/l L-NNA resulted in a dose-dependent increase in SBP that was similar in males and females. However, females treated with 20 and 30 mg/l L-NNA were resistant to the development of proteinuria: maximum values were 16 +/- 7 and 46 +/- 21, respectively, vs. 16 +/- 3 mg/day in controls, whereas males treated with those doses showed an increase in proteinuria [139 +/- 35 (P < 0.05) and 318 +/- 82 (P < 0.01), respectively, vs. 55 +/- 11 mg/day in controls]. Treatment with 100 mg/l L-NNA increased proteinuria similarly in both females and males. To study the role of sex hormones in differences in sensitivity to proteinuria induced by mild chronic NOS inhibition, treatment with 20 mg/l L-NNA was repeated in ovariectomized (Ovx) and orchidectomized rats. Ovariectomy did not affect the increase in SBP caused by 20 mg/l L-NNA, but, in contrast to intact females, this dose of L-NNA did cause Ovx rats to develop proteinuria (51 +/- 16 vs. 16 +/- 7 mg/day in control Ovx rats; P < 0.05). Orchidectomy completely prevented the increased SBP as well as proteinuria induced by 20 mg/l L-NNA in male rats. In conclusion, male rats are more sensitive than female rats to develop proteinuria induced by mild chronic NOS inhibition. Estrogens provide some protection in females, whereas androgens are responsible for the increased sensitivity of male rats to proteinuria induced by mild chronic NOS inhibition. Risk factors associated with a compromised nitric oxide system may be more detrimental to the kidney in men than in women.

Nitric oxide (NO) and carbon monoxide (CO) seem to be neurotransmitters in the brain. The colocalization of their respective biosynthetic enzymes, neuronal NO synthase (nNOS) and heme oxygenase-2 (HO2), in enteric neurons and altered intestinal function in mice with genomic deletion of the enzymes (nNOS(Delta/Delta) and HO2(Delta/Delta)) suggest neurotransmitter roles for NO and CO in the enteric nervous system. We now establish that NO and CO are both neurotransmitters that interact as cotransmitters. Small intestinal smooth muscle cells from nNOS(Delta/Delta) and HO2(Delta/Delta) mice are depolarized, with apparent additive effects in the double knockouts (HO2(Delta/Delta)/nNOS(Delta/Delta)). Muscle relaxation and inhibitory neurotransmission are reduced in the mutant mice. In HO2(Delta/Delta) preparations, responses to electrical field stimulation are nearly abolished despite persistent nNOS expression, whereas exogenous CO restores normal responses, indicating that the NO system does not function in the absence of CO generation.

Autoradiography with the nitric oxide synthase (NOS) inhibitor ((3)H)nitro-L-arginine ([(3)H]L-NNA) was used to quantify NOS in cervical and lumbar spinal cord in normal and dystrophic mice. A single homogeneous population of binding sites was seen in all subregions of the gray matter in normal mice and in the superficial dorsal horn in dystrophic mice. However, in dystrophic mice, two populations were revealed in the deeper dorsal, intermediate, and ventral subregions. Pronounced immunoreactivity for neuronal NOS (nNOS), and weak immunoreactivity for endothelial NOS (eNOS), were revealed in all subregions in normal and dystrophic mice. Inducible NOS (iNOS) immunoreactivity was negligible in normal mice but intense in the deeper dorsal, intermediate, and ventral subregions in dystrophic mice. The higher affinity ((3)H)L-NNA binding site colocalized with nNOS and the lower affinity site with iNOS. It is suggested that expression of iNOS is associated with the pathological changes occurring in congenital muscular dystrophy., (Copyright 2000 John Wiley & Sons, Inc.)

Purpose: To investigate the effect of diabetes mellitus (DM) on the density and distribution of nitric oxide synthase (NOS) and the smooth muscle responses to non-adrenergic, non-cholinergic (NANC) nerve stimulation and exogenous nitric oxide (NO) in the rabbit lower urinary tract., Materials and Methods: Transverse sections of detrusor, bladder neck and urethra, from control and six months alloxan-induced DM New Zealand White rabbits were incubated with a radioligand for NOS ([3H]-L-N(G)-nitroarginine). Densitometric analysis was performed on the autoradiographs. NADPH diaphorase histochemistry was also used as a marker for NOS activity. Responses to NANC nerve stimulation (5 to 20 Hz) and to NO (10(-6) to 3x10(-4) M.) on smooth muscle strips from detrusor, bladder neck and urethra were measured in organ baths., Results: NOS binding sites were significantly (p<0.03) more dense in the bladder neck than in the detrusor in both DM and control groups. In DM bladder neck, NOS binding sites were significantly (p<0.04) increased compared with the controls. NADPH diaphorase activity appeared markedly increased in the detrusor, bladder neck and urethra of DM animals compared with controls. The mean IC50 for exogenous NO in control versus DM were not statistically different in the bladder neck (1.03x10(-4) M versus 9.8x10(-5) M) and urethra (8.1x10(-5) M versus 8.8x10(-5) M), but the relaxations to 5x10(-6) M of NO were significantly impaired (p<0.04) in the DM urethral smooth muscle. NANC nerve-mediated relaxations were significantly impaired (p<0.001) in the DM urethral smooth muscle., Conclusions: Alterations of both the NOS binding sites and functional responses to NANC nerve stimulation suggest that NO may have a pathophysiological role in the urinary bladder dysfunction associated with DM.

The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (n=8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O2 content, free fatty acids (FFAs), and lactate and glucose concentrations. Cardiac production of NO metabolites or consumption of O2 or utilization of substrates was calculated as coronary sinus-arterial difference times coronary flow. In end-stage failure, occurring at 29+/-1.6 days, left ventricular end-diastolic pressure was 25+/-1 mm Hg, left ventricular systolic pressure was 92+/-3 mm Hg, mean arterial pressure was 75+/-2.5 mm Hg, and dP/dtmax was 1219+/-73 mm Hg/s (all P<0.05). These changes in hemodynamics were associated with a fall of cardiac NO metabolite production from 0.37+/-0.16 to -0.28+/-0.13 nmol/beat (P<0.05). O2 consumption and lactate uptake did not change significantly from control, while FFA uptake decreased from 0.16+/-0.03 to 0.05+/-0.01 microEq/beat and glucose uptake increased from -2.3+/-7.0 to 41+/-10 microgram/beat (P<0.05). The cardiac respiratory quotient also increased significantly by 28%. In 14 normal dogs the same measurements were performed at control and 1 hour after we injected 30 mg/kg of nitro-L-arginine, a competitive inhibitor of NO synthase .O2 consumption increased from 0.05+/-0.002 mL/beat at control to 0.071+/-0.003 mL/beat after nitro-L-arginine, while FFA uptake decreased from 0.1+/-0.01 to 0.06+/-0.01 microEq/beat, lactate uptake increased from 0.15+/-0.04 to 0.31+/-0.03 micromol/beat, glucose uptake increased from 8.2+/-5.0 to 35.4+/-9.5 microgram/beat, and RQ increased by 23% (all P<0.05). Our results indicate that basal cardiac production of NO falls below normal levels during cardiac decompensation and that there are shifts in substrate utilization. This switch in myocardial substrate utilization also occurs after acute pharmacological blockade of NO production in normal dogs.