Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities suggests colocalization and segregation of the two transporters in synaptic vesicles.

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug-induced hyperlocomotion and anxiety-related behaviors.

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3-/- ) display anxiety-associated phenotype, increased spontaneous and cocaine-induced locomotor activity and decreased haloperidol-induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward-guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous- and cocaine-induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.

Moderate decline in select synaptic markers in the prefrontal cortex (BA9) of patients with Alzheimer's disease at various cognitive stages.

Synaptic loss, plaques and neurofibrillary tangles are viewed as hallmarks of Alzheimer's disease (AD). This study investigated synaptic markers in neocortical Brodmann area 9 (BA9) samples from 171 subjects with and without AD at different levels of cognitive impairment. The expression levels of vesicular glutamate transporters (VGLUT1&2), glutamate uptake site (EAAT2), post-synaptic density protein of 95 kD (PSD95), vesicular GABA/glycine transporter (VIAAT), somatostatin (som), synaptophysin and choline acetyl transferase (ChAT) were evaluated. VGLUT2 and EAAT2 were unaffected by dementia. The VGLUT1, PSD95, VIAAT, som, ChAT and synaptophysin expression levels significantly decreased as dementia progressed. The maximal decrease varied between 12% (synaptophysin) and 42% (som). VGLUT1 was more strongly correlated with dementia than all of the other markers (polyserial correlation = -0.41). Principal component analysis using these markers was unable to differentiate the CDR groups from one another. Therefore, the status of the major synaptic markers in BA9 does not seem to be linked to the cognitive status of AD patients. The findings of this study suggest that the loss of synaptic markers in BA9 is a late event that is only weakly related to AD dementia.

Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles.

The atypical vesicular glutamate transporter type 3 (VGLUT3) is expressed by subpopulations of neurons using acetylcholine, GABA, or serotonin as neurotransmitters. In addition, VGLUT3 is expressed in the inner hair cells of the auditory system. A mutation (p.A211V) in the gene that encodes VGLUT3 is responsible for progressive deafness in two unrelated families. In this study, we investigated the consequences of the p.A211V mutation in cell cultures and in the CNS of a mutant mouse. The mutation substantially decreased VGLUT3 expression (-70%). We measured VGLUT3-p.A211V activity by vesicular uptake in BON cells, electrophysiological recording of isolated neurons, and its ability to stimulate serotonergic accumulation in cortical synaptic vesicles. Despite a marked loss of expression, the activity of the mutated isoform was only minimally altered. Furthermore, mutant mice displayed none of the behavioral alterations that have previously been reported in VGLUT3 knock-out mice. Finally, we used stimulated emission depletion microscopy to analyze how the mutation altered VGLUT3 distribution within the terminals of mice expressing the mutated isoform. The mutation appeared to reduce the expression of the VGLUT3 transporter by simultaneously decreasing the number of VGLUT3-positive synaptic vesicles and the amount of VGLUT3 per synapses. These observations suggested that VGLUT3 global activity is not linearly correlated with VGLUT3 expression. Furthermore, our data unraveled a nonuniform distribution of VGLUT3 in synaptic vesicles. Identifying the mechanisms responsible for this complex vesicular sorting will be critical to understand VGLUT's involvement in normal and pathological conditions.SIGNIFICANCE STATEMENT VGLUT3 is an atypical member of the vesicular glutamate transporter family. A point mutation of VGLUT3 (VGLUT3-p.A211V) responsible for a progressive loss of hearing has been identified in humans. We observed that this mutation dramatically reduces VGLUT3 expression in terminals (∼70%) without altering its function. Furthermore, using stimulated emission depletion microscopy, we found that reducing the expression levels of VGLUT3 diminished the number of VGLUT3-positive vesicles at synapses. These unexpected findings challenge the vision of a uniform distribution of synaptic vesicles at synapses. Therefore, the overall activity of VGLUT3 is not proportional to the level of VGLUT3 expression. These data will be key in interpreting the role of VGLUTs in human pathologies.

Distribution of vesicular glutamate transporters in the human brain.

Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3) are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe) while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains.

Glutamate receptors of the delta family are widely expressed in the adult brain.

Recent reports point to critical roles of glutamate receptor subunit delta2 (GluD2) at excitatory synapses and link GluD1 gene alteration to schizophrenia but the expression patterns of these subunits in the brain remain almost uncharacterized. We examined the distribution of GluD1-2 mRNAs and proteins in the adult rodent brain, focusing mainly on GluD1. In situ hybridization revealed widespread neuronal expression of the GluD1 mRNA, with higher levels occurring in several forebrain regions and lower levels in cerebellum. Quantitative RT-PCR assessed differential GluD1 expression in cortex and cerebellum, and revealed GluD2 expression in cortex, albeit at markedly lower level than in cerebellum. Likewise, a high GluD1/GluD2 mRNA ratio was observed in cortex and a low ratio in cerebellum. GluD1 and GluD2 mRNAs were co-expressed in single cortical and hippocampal neurons, with a large predominance of GluD1. Western blots using GluD1- and GluD2-specific antibodies showed expression of both subunits in various brain structures, but not in non-nervous tissues examined. Both delta subunits were upregulated during postnatal development. Widespread neuronal expression of the GluD1 protein was confirmed using immunohistochemistry. Examination at the electron microscopic level in the hippocampus revealed that GluD1 was mainly localized at postsynaptic density of excitatory synapses on pyramidal cells. Control experiments performed using mice carrying deletion of the GluD1- or the GluD2-encoding gene confirmed the specificity of the present mRNA and protein analyses. Our results support a role for the delta family of glutamate receptors at excitatory synapses in neuronal networks throughout the adult brain.

Design, synthesis and biological evaluation of small-azo-dyes as potent Vesicular Glutamate Transporters inhibitors.

Vesicular Glutamate Transporters (VGLUTs) allow the loading of presynapic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. VGLUTs have proved to be involved in several major neuropathologies and directly correlated to clinical dementia in Alzheimer and Parkinson's disease. Accordingly VGLUT represent a key biological target or biomarker for neuropathology treatment or diagnostic. Yet, despite the pivotal role of VGLUTs, their pharmacology appears quite limited. Known competitive inhibitors are restricted to some dyes as Trypan Blue (TB) and glutamate mimics. This lack of pharmacological tools has heavily hampered VGLUT investigations. Here we report a rapid access to small molecules that combine benefits of TB and dicarboxylic quinolines (DCQs). Their ability to block vesicular glutamate uptake was evaluated. Several compounds displayed low micromolar inhibitory potency when size related compounds are thirty to forty times less potent (i.e. DCQ). We then confirmed the VGLUT selectivity by measuring the effect of the series on vesicular monoamine transport and on metabotropic glutamate receptor activity. These inhibitors are synthesized in only two steps and count among the best pharmacological tools for VGLUTs studies.

The vesicular glutamate transporter VGLUT3 contributes to protection against neonatal hypoxic stress.

Neonates respond to hypoxia initially by increasing ventilation, and then by markedly decreasing both ventilation (hypoxic ventilatory decline) and oxygen consumption (hypoxic hypometabolism). This latter process, which vanishes with age, reflects a tight coupling between ventilatory and thermogenic responses to hypoxia. The neurological substrate of hypoxic hypometabolism is unclear, but it is known to be centrally mediated, with a strong involvement of the 5-hydroxytryptamine (5-HT, serotonin) system. To clarify this issue, we investigated the possible role of VGLUT3, the third subtype of vesicular glutamate transporter. VGLUT3 contributes to glutamate signalling by 5-HT neurons, facilitates 5-HT transmission and is expressed in strategic regions for respiratory and thermogenic control. We therefore assumed that VGLUT3 might significantly contribute to the response to hypoxia. To test this possibility, we analysed this response in newborn mice lacking VGLUT3 using anatomical, biochemical, electrophysiological and integrative physiology approaches. We found that the lack of VGLUT3 did not affect the histological organization of brainstem respiratory networks or respiratory activity under basal conditions. However, it impaired respiratory responses to 5-HT and anoxia, showing a marked alteration of central respiratory control. These impairments were associated with altered 5-HT turnover at the brainstem level. Furthermore, under cold conditions, the lack of VGLUT3 disrupted the metabolic rate, body temperature, baseline breathing and the ventilatory response to hypoxia. We conclude that VGLUT3 expression is dispensable under basal conditions but is required for optimal response to hypoxic stress in neonates.

VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety.

Three different subtypes of H(+)-dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT(1A)-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone.

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Loss of VGLUT1 and VGLUT2 in the prefrontal cortex is correlated with cognitive decline in Alzheimer disease.

Several lines of evidence suggest that the glutamatergic system is severely impaired in Alzheimer disease (AD). Here, we assessed the status of glutamatergic terminals in AD using the first available specific markers, the vesicular glutamate transporters VGLUT1 and VGLUT2. We quantified VGLUT1 and VGLUT2 in the prefrontal dorsolateral cortex (Brodmann area 9) of controls and AD patients using specific antiserums. A dramatic decrease in VGLUT1 and VGLUT2 was observed in AD using Western blot. Similar decreases were observed in an independent group of subjects using immunoautoradiography. The VGLUT1 reduction was highly correlated with the degree of cognitive impairment, assessed with the clinical dementia rating (CDR) score. A significant albeit weaker correlation was also observed with VGLUT2. These findings provide evidence indicating that glutamatergic systems are severely impaired in the A9 region of AD patients and that this impairment is strongly correlated with the progression of cognitive decline. Our results suggest that VGLUT1 expression in the prefrontal cortex could be used as a valuable neurochemical marker of dementia in AD.

Alcohol increases tumor necrosis factor alpha and decreases nuclear factor-kappab to activate hepatic apoptosis in genetically obese mice.

Both obesity and alcohol can cause oxidative stress, cytokine induction, and steatohepatitis. To determine the consequences of their combination, we compared the hepatic effects of moderate ethanol binges in lean and obese ob/ob mice. Mice received water or ethanol (2.5 g/kg) by gastric intubation daily for 4 days, and were killed 2 hours after the last administration. Some obese mice also received pentoxifylline, an inhibitor of tumor necrosis factor-alpha (TNF-alpha) production, before each ethanol administration. In lean mice, these moderate ethanol doses did not increase plasma TNF-alpha and hepatic caspase-3 activity, but triggered some apoptotic hepatocytes. Naive ob/ob mice had a few necrotic and apoptotic hepatocytes, but exhibited little oxidative stress, possibly because of adaptive increases in manganese superoxide dismutase, heat shock protein 70 (Hsp70), mitochondrial cytochrome c, and mitochondrial DNA. Alcohol administration to ob/ob mice did not increase oxidative stress despite increased CYP2E1, but increased plasma TNF-alpha, further increased Hsp70, and profoundly decreased p65 nuclear factor kappaB (NF-kappaB) protein and DNA-binding activity in nuclear extracts. Caspase-3 was activated, and more apoptotic hepatocytes were found in intoxicated obese mice than naive obese mice. In intoxicated obese mice, pentoxifylline fully prevented the increase in plasma TNF-alpha the decrease in nuclear NF-kappaB activity, and the increase in hepatic caspase-3, and it also decreased hepatic triglycerides. In conclusion, obese mice develop adaptations that may limit oxidative stress. Moderate ethanol intoxication does not increase oxidative stress in obese mice, but increases TNF-alpha and also decreases nuclear NF-kappaB activity, thus unleashing the apoptotic effects of TNF-alpha.

Norfloxacin reduces aortic NO synthases and proinflammatory cytokine up-regulation in cirrhotic rats: role of Akt signaling.

BACKGROUND & AIMS: Arterial vasodilation plays a role in the pathogenesis of the complications of cirrhosis. This vasodilation is caused by the overproduction of arterial nitric oxide (NO). Bacterial translocation may be involved in NO synthase (NOS) up-regulation by activating both endothelial NOS (eNOS) and inducible NOS (iNOS). The prevention of intestinal gram-negative translocation by norfloxacin administration corrects systemic circulatory changes by decreasing NO production in cirrhosis. However, the signaling mechanisms for NO overproduction from bacterial translocation are unknown. In this study, we investigated the signal transduction pathway of bacterial translocation-induced aortic NOS up-regulation in cirrhotic rats. METHODS: Proinflammatory cytokine levels, Akt and NOS activities, eNOS phosphorylation, and NOS expressions were assessed in aorta from norfloxacin-treated and untreated cirrhotic rats. Norfloxacin was administered to reduce intestinal bacterial translocation. RESULTS: Aortic eNOS and iNOS protein expressions, Akt activity, and eNOS phosphorylation by Akt at serine 1177 were up-regulated in cirrhotic rats. Norfloxacin administration significantly decreased the incidence of gram-negative translocation and proinflammatory cytokine (tumor necrosis factor-alpha, interferon-gamma, and interleukin-6) levels; norfloxacin also decreased aortic Akt activity, eNOS phosphorylation, and NOS expressions and activities. The decrease in aortic Akt activity and NOS expressions also was obtained after colistin or anti-tumor necrosis factor-alpha antibody administration to cirrhotic rats. CONCLUSIONS: This study identifies a signaling pathway in which bacterial translocation induces aortic NOS up-regulation and thus NO overproduction in cirrhotic rats. These results strongly suggest that bacterial translocation and proinflammatory cytokines play a role in systemic NO overproduction in cirrhosis by the Akt pathway.

Cyclic AMP-phosphodiesterases inhibitor improves sodium excretion in rats with cirrhosis and ascites.

BACKGROUND: The mechanisms responsible for renal dysfunction and sodium retention in cirrhosis remain unclear. Cyclic AMP (cAMP) regulates sodium reabsorption in the proximal nephron. This study investigates the role of cAMP metabolism in renal dysfunction in cirrhosis. METHODS: Renal function was studied by the clearance technique in anesthetized control and cirrhotic rats with or without ascites. cAMP phosphodiesterase (PDE) activity was measured in the renal cortex in vitro. Moroever, the effects on renal function of the intravenous administration of cAMP and rolipram, a powerful and specific cAMP-PDE4 inhibitor, were evaluated. RESULTS: In control and in non-ascitic cirrhotic rats, cAMP administration significantly increased sodium and phosphate excretions, but did not change these excretions in cirrhotic rats with ascites. cAMP-PDE activity was higher in ascitic than in control rats (P < 0.05). Rolipram infusion significantly increased sodium and phosphate excretion only in cirrhotic rats with ascites. CONCLUSION: These results suggest that increased renal cAMP-PDE activity is responsible for resistance to the natriuretic effects of cAMP in cirrhosis and plays a role in the development of ascites.

Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis.

In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock. LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors. In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS. Thus, the aim of this study was to investigate the effects of terlipressin administration (a vasopressin analog) on in vivo LPS-induced aortic iNOS in rats with cirrhosis. LPS (1 mg/kg, intravenously) was administered followed by the intravenous administration of terlipressin (0.05 mg/kg, intravenously) or placebo 1 hour later. Arterial pressure was measured, and contractions to phenylephrine (an alpha(1)-adrenoceptor agonist), iNOS activity, and iNOS expressions (mRNA and protein) were investigated in isolated aortas. LPS-induced arterial hypotension and aortic hyporeactivity to phenylephrine were abolished in rats that received terlipressin. LPS-induced aortic iNOS activity and expression were suppressed in terlipressin-treated rats. In conclusion, in LPS-challenged rats with cirrhosis, terlipressin administration inhibits in vivo LPS-induced aortic iNOS expression. Terlipressin administration may be a novel approach for the treatment of arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors in patients with cirrhosis and septic shock.

Role of shear stress in aortic eNOS up-regulation in rats with biliary cirrhosis.

BACKGROUND & AIMS: In rats with portal vein stenosis, the initial cause of aortic nitric oxide (NO) overproduction might be overactivation of endothelial NO synthase (eNOS) related to increased shear stress. Cardiac output is higher in cirrhosis than in extrahepatic portal hypertension. The aims of this study were to evaluate the role of shear stress, vascular endothelial growth factor (VEGF), and cytokines in aortic eNOS up-regulation in rats with biliary cirrhosis and to compare these results with those in rats with portal vein stenosis. METHODS: NOS activities, NOS protein, heat shock protein (Hsp) 90, and VEGF expressions were studied in rat aortas. Propranolol was administered to rats with cirrhosis to reduce cardiac output and thus shear stress. RESULTS: In cirrhotic rats, the aortic eNOS protein was 3.0 and 1.7 times higher than in control and portal vein-stenosed rats, respectively. In cirrhotic rats, the Hsp90 content was 3.2 and 2.2 times higher than in control and portal vein-stenosed rats, respectively. Propranolol decreased NOS activity by 47% and eNOS and Hsp90 expression by 75% and 72%, respectively. Aortic VEGF expression was decreased in cirrhotic rats. VEGF-induced stimulation of NOS activity was greater in aortas from control rats than in aortas from portal vein-stenosed or cirrhotic rat aortas. eNOS expression was up-regulated after VEGF incubation. After lipopolysaccharide administration, eNOS expression did not change in portal vein-stenosed or cirrhotic rats. CONCLUSIONS: This study shows that in aortas from rats with biliary cirrhosis, systemic vasodilation depends mainly on eNOS up-regulation related to shear stress.

Relationship between protein kinase C alterations and nitric oxide overproduction in cirrhotic rat aortas.

BACKGROUND: Although nitric oxide (NO) overproduction and protein kinase C (PKC) alterations may play a role in systemic haemodynamic changes in cirrhotic rat aortas, the relationship between NO synthase (NOS) hyperactivation and PKC hypoactivation is unknown. Therefore, the relationships between NOS and PKC activities were studied in cirrhotic rat aortas. METHODS: The effects of NOS inhibition by Nw-nitro-L-arginine (LNNA) on the contractile response to phorbol myristate acetate (PMA), a PKC activator, were studied. The effects of NOS inhibition and those of S-nitroso-N acetyl-DL-penicillamine (SNAP), an NO donor, on PKC activity were also evaluated. The effects of PKC activation and inhibition on total NOS and inducible NOS (iNOS) activities were measured. Nitric oxide synthase inhibition caused an increase in PMA-induced contraction and an increase in PKC activity in cirrhotic rat aortas. S-nitroso-N acetyl-DL-penicillamine induced downregulation of PKC activity. Total basal aortic NOS activity was significantly higher in cirrhotic rats than in control rats and activation of PKC by PMA induced a decrease in total aortic NOS activity. Protein kinase C downregulation caused an increase in both total aortic NOS and iNOS activities only in control rats, whereas only iNOS activity increased in cirrhotic rats. CONCLUSION: In cirrhotic rat aortas, NO overproduction plays a role in the decreased PKC activation that leads to reduced aortic contraction. Overactivation of aortic NOS in cirrhotic rats may be because of, in part, the reduced PKC activity.