Effects of testosterone metabolites on copulation, medial preoptic dopamine, and NOS-immunoreactivity in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. In a previous study, we identified 17beta-estradiol (E(2)) as the metabolite of testosterone (T) that maintains MPOA basal extracellular DA levels. However, the presence of dihydrotestosterone (DHT), an androgenic metabolite of T, is required for the female-induced increase in MPOA DA observed during copulation. Recently, we reported that assays of MPOA tissue DA content showed that castrates actually had more stored DA than did gonadally intact males. Therefore, the reduction in extracellular levels in castrates was not due to decreased availability of DA; most likely it was due to decreased release. Furthermore, T upregulates neuronal nitric oxide synthase (nNOS) in the MPOA. NO has been implicated in the regulation of DA release in the MPOA. It is not known, however, which metabolite(s) of T regulate(s) tissue stores of DA and/or nNOS in the MPOA of male rats. The present experiments were designed to test the following: (1) whether E(2), DHT, or the combination of the two influences MPOA DA tissue levels, an indication of stored DA, in male rat castrates; and (2) whether E(2), DHT, or the combination of the two influences NOS-ir in the MPOA of castrated male rats. The results indicate that E(2) up-regulates nNOS-ir in the MPOA and maintains tissue content of DA at levels similar to those in T-treated rats. DHT did not influence nNOS-ir, while attenuating the effect of castration on tissue DA content.     

Effects of testosterone metabolites on copulation and medial preoptic dopamine release in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. The recent presence of testosterone (T) may be necessary for this precopulatory increase in release. Previously, the postcastration loss of copulatory ability mirrored the loss of the DA response to an estrous female, and the restoration of copulation with exogenous T was concurrent with the reemergence of this DA response. The present study investigated the effectiveness of the two major metabolites of T in maintaining copulation and basal and female-stimulated DA levels. Adult male rats were castrated and received daily injections of estradiol benzoate (EB), dihydrotestosterone benzoate (DHTB), EB + DHTB, testosterone propionate (TP), or oil vehicle for 3 weeks. Microdialysis samples were collected from the MPOA during baseline conditions, exposure to an estrous female behind a barrier, and copulation testing. EB + DHTB- and TP-treated animals had normal basal DA levels and showed a precopulatory DA response, and most copulated normally. EB-treated castrates had high basal DA levels, but failed to show a female-stimulated increase; most intromitted, but none ejaculated. DHTB- and oil-treated groups had low basal levels of extracellular DA that did not increase during copulation testing; most failed to mount and none ejaculated. These results suggest that E maintains normal basal levels of extracellular DA in the MPOA, which are sufficient for suboptimal copulation, but that androgen is required for the female-stimulated increase in DA release and for facilitation of ejaculation.     

Testosterone restoration of copulatory behavior correlates with medial preoptic dopamine release in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. We have reported an increase in dopamine (DA) release in the MPOA of male rats shortly before and during copulation. Postcastration loss of copulatory ability mirrored the loss of the precopulatory DA response to an estrous female. The present study investigated the time courses of restoration, rather than loss, of the MPOA DA response to a receptive female and of copulation in long-term castrates. Male rats were castrated and tested for loss of copulatory ability 21 days later. They then received 2, 5, or 10 daily subcutaneous injections of testosterone propionate (TP, 500 microg) or oil. Microdialysate samples were collected from the MPOA during baseline, exposure to a female behind a barrier, and copulation. Extracellular DA was measured using HPLC-EC. None of the six 2-day-TP-treated animals copulated, nor did they show elevated DA release in the MPOA in the presence of a receptive female. Five of the nine 5-day-TP-treated animals ejaculated; three intromitted without ejaculating; and one failed to copulate, with all but the noncopulating animal showing elevated DA release. All of the six 10-day-TP-treated animals copulated and also demonstrated an increase in MPOA DA. None of the oil controls copulated or showed an increase in DA release. Therefore, a consistent relationship between MPOA DA release during exposure to a receptive female and the subsequent ability of the male to copulate was observed.     

Alterations in nitric oxide-cGMP pathway in ventricular myocytes from obese leptin-deficient mice

Leptin is a regulator of body weight and affects nitric oxide (NO) production. This study was designed to determine whether the myocardial NO-cGMP signal transduction system was altered in leptin-deficient obese mice. Contractile function, guanylyl cyclase activity, and cGMP-dependent protein phosphorylation were assessed in ventricular myocytes isolated from genetically obese (B6.V-Lepob) and age-matched lean (C57BL/6J) mice. There were no differences in baseline contraction between the lean and obese groups. After stimulation with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10-6 and 10-5 M) or a membrane-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10(-6) and 10(-5) M), cell contractility was depressed. However, 8-Br-cGMP had significantly greater effects in obese mice than in lean controls with percent shortening reduced by 47 vs. 39% and maximal rate of shortening decreased by 46 vs. 36%. The negative effects of SNAP were similar between the two groups. Soluble guanylyl cyclase activity was not attenuated. This suggests that the activity of the cGMP-independent NO pathway may be enhanced in obesity. The phosphorylated protein profile of cGMP-dependent protein kinase showed that four proteins were more intensively phosphorylated in obese mice, which suggests an explanation for the enhanced effect of cGMP. These results indicate that the NO-cGMP signaling pathway was significantly altered in ventricular myocytes from the leptin-deficient obese mouse model.     

Oxytocin in the medial preoptic area facilitates male sexual behavior in the rat

Oxytocin (OT) is a versatile neuropeptide that is involved in a variety of mammalian behaviors, and its role in reproductive function and behavior has been well established. The majority of pharmacological studies of the effects of OT on male sexual behavior have focused on the paraventricular nucleus (PVN), ventral tegmental area (VTA), hippocampus, and amygdala. Less attention has been given to the medial preoptic area (MPOA), a major integrative site for male sexual behavior. The present study investigated the effects of intra-MPOA administration of OT and (d(CH2)51, Tyr(Me)2, Thr4, Orn8, Tyr-NH29)-vasotocin, an OT antagonist (OTA), on copulation in the male rat. The relationship between OT receptor (OTR) binding levels in the MPOA and sexual efficiency was also explored. Microinjection of OT into the MPOA facilitated copulation in sexually experienced male rats, whereas similar injections of an OTA inhibited certain aspects of copulation but had no significant effect on locomotor activity in an open field. Contrary to expectation, sexually efficient males had lower levels of OTR binding in the rostral MPOA compared to inefficient animals. The present data suggest that OT activity in the MPOA is not necessary for the expression of male sexual behavior but is sufficient to facilitate copulatory behaviors and improve sexual efficiency in sexually experienced male rats. These data also suggest that OTR activity in the MPOA stimulates anogenital investigation, facilitates the initiation of copulation, and plays a role in the sensitization effect of the first ejaculation on subsequent ejaculations.     

Dissociated functional pathways for appetitive and consummatory reproductive behaviors in male Syrian hamsters

In many species, including Syrian hamsters, the generation of male reproductive behavior depends critically on the perception of female odor cues from conspecifics in the environment. The behavioral response to these odors is mediated by a network of steroid-sensitive ventral forebrain nuclei including the medial amygdala (MA), posterior bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA). Previous studies have demonstrated that each of these three nuclei is required for appropriate sexual behavior and that MA preferentially sends female odor information directly to BNST and MPOA. It is unknown, however, how the functional connections between MA and BNST and/or MPOA are organized to generate different aspects of reproductive behavior. Therefore, the following experiments used the asymmetrical pathway lesion technique to test the role of the functional connections between MA and BNST and/or MPOA in odor preference and copulatory behaviors. Lesions that functionally disconnected MA from MPOA eliminated copulatory behavior but did not affect odor preference. In contrast, lesions that functionally disconnected MA from BNST eliminated preference for volatile female odors but did not affect preference for directly contacted odors or copulatory behavior. These results therefore demonstrate a double dissociation in the functional connections required for attraction to volatile sexual odors and copulation and, more broadly, suggest that appetitive and consummatory reproductive behaviors are mediated by distinct neural pathways.     

Sexual behavior in male rodents

The hormonal factors and neural circuitry that control copulation are similar across rodent species, although there are differences in specific behavior patterns. Both estradiol (E) and dihydrotestosterone (DHT) contribute to the activation of mating, although E is more important for copulation and DHT for genital reflexes. Hormonal activation of the medial preoptic area (MPOA) is most effective, although implants in the medial amygdala (MeA) can also stimulate mounting in castrates. Chemosensory inputs from the main and accessory olfactory systems are the most important stimuli for mating in rodents, especially in hamsters, although genitosensory input also contributes. Dopamine agonists facilitate sexual behavior, and serotonin (5-HT) is generally inhibitory, though certain 5-HT receptor subtypes facilitate erection or ejaculation. Norepinephrine agonists and opiates have dose-dependent effects, with low doses facilitating and high doses inhibiting behavior.     

Photoperiodic influences on sexual behavior in male Syrian hamsters

The effect of photoperiodic conditions on sexual behavior was investigated in male Syrian hamsters that were either gonadally intact, or castrated and treated with low doses of testosterone throughout the experiment. Hamsters were exposed to long (LD 16:8) or short (LD 8:16) days for 7 weeks; for the next 8 weeks, either they were exposed to an intermediate daylength (LD 12:12), or daylength conditions remained unchanged. Sexual behavior was affected by photoperiod conditions in both gonadally intact animals and testosterone-treated castrates, but to different degrees. Intact males exposed to short days for 15 weeks exhibited gonadal regression, and their copulatory performance was impaired. The percentage of animals that intromitted or ejaculated was significantly reduced. Additional measures of sexual performance among the copulating males were also affected. In contrast, among the castrates with testosterone clamped at low but stable levels, the proportion of males that mounted, intromitted, or ejaculated was not affected by photoperiod. However, among the males that continued to copulate, sexual performance changes were present in the short-day castrates that resembled those displayed by the intact males. We infer that these behavioral effects in both hormonal conditions reflect primarily a difficulty in the attainment of intromission. Gonadal regression alone cannot easily account for the behavioral deficits of the intact males, because circulating testosterone levels at the end of the experiment were not significantly different between the gonadally intact hamsters and the castrated, testosterone-treated hamsters exposed continuously to short days. Males transferred from either long or short days to the intermediate-daylength condition responded behaviorally to this photoperiod as if it were a short day, that is, their ejaculatory frequency declined. We conclude that male hamsters exposed to photoinhibitory daylengths exhibit deficits in their sexual behavior, not only because endogenous levels of testosterone decrease, but also because the substrates on which this hormone acts become less responsive. We hypothesize that under physiological conditions, the episodic secretion of testosterone imposes constraints on the maintenance or restoration of copulation, and that the potent behavioral effects achieved by constant-release implants of testosterone may mask the presence of photoperiodically induced alterations in the hamster's sensitivity to this gonadal hormone.     

Cardiac cholinergic NO-cGMP signaling following acute myocardial infarction and nNOS gene transfer

Myocardial infarction (MI) is associated with oxidative stress, which may cause cardiac autonomic impairment. We tested the hypothesis that acute MI disrupts cardiac cholinergic signaling by impairing nitric oxide (NO)-cGMP modulation of acetylcholine (ACh) release and whether the restoration of this pathway following cardiac neuronal NO synthase (nNOS) gene transfer had any bearing on the neural phenotype. Guinea pigs underwent four ligature coronary artery surgery (n = 50) under general anesthesia to induce MI or sham surgery (n = 32). In a separate group, at the time of MI surgery, adenovirus encoding nNOS (n = 29) or enhanced green fluorescent protein (eGFP; n = 30) was injected directly into the right atria, where the postganglionic cholinergic neurons reside. In vitro-evoked right atrial [3H]ACh release, right atrial NOS activity, and cGMP levels were measured at 3 days. Post-MI 24% of guinea pigs died compared with 9% in the sham-operated group. Evoked right atrial [3H]ACh release was significantly (P < 0.05) decreased in the MI group as was NOS activity and cGMP levels. Tetrahydrobiopterin levels were not significantly different between the sham and MI groups. Infarct sizes between gene-transferred groups were not significantly different. The nNOS transduced group had significantly increased right atrial [3H]ACh release, right atrial NOS activity, cGMP levels, and decreased cAMP levels. Fourteen percent of the nNOS transduced animals died compared with 31% mortality in the MI + eGFP group at 3 days. In conclusion, cardiac nNOS gene transfer partially restores the defective NO-cGMP cholinergic pathway post-MI, which was associated with a trend of improved survival at 3 days.     

Chronic hypoxia alters the function of NOS nerves in cerebral arteries of near-term fetal and adult sheep.

In addition to adrenergic innervation, cerebral arteries also contain neuronal nitric oxide synthase (nNOS)-expressing nerves that augment adrenergic nerve function. We examined the impact of development and chronic high-altitude hypoxia (3,820 m) on nNOS nerve function in near-term fetal and adult sheep middle cerebral arteries (MCA). Electrical stimulation-evoked release of norepinephrine (NE) was measured with HPLC and electrochemical detection, whereas nitric oxide (NO) release was measured by chemiluminescence. An inhibitor of NO synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), significantly inhibited stimulation-evoked NE release in MCA from normoxic fetal and adult sheep with no effect in MCA from hypoxic animals. Addition of the NO donor S-nitroso-N-acetyl-dl-penicillamine fully reversed the effect of l-NAME in MCA from normoxic animals with no effect in MCA from hypoxic animals. Electrical stimulation caused a significant increase in NO release in MCA from normoxic animals, an effect that was blocked by the neurotoxin tetrodotoxin, whereas there was no increase in NO release in MCA from hypoxic animals. Relative abundance of nNOS as measured by Western blot analysis was similar in normoxic fetal and adult MCA. However, after hypoxic acclimitization, nNOS levels dramatically declined in both fetal and adult MCA. These data suggest that the function of nNOS nerves declines during chronic high-altitude hypoxia, a functional change that may be related to a decline in nNOS protein levels.     

Nitric oxide (NO) serves as a retrograde messenger to activate neuronal NO synthase in the spinal cord via NMDA receptors.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.     

βig-h3 regulates store-operated Ca2+ entry and promotes the invasion of human hepatocellular carcinoma cells

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.     

The effect of chronic antipsychotic drug on hypothalamic expression of neural nitric oxide synthase and dopamine D2 receptor in the male rat

Antipsychotic-induced sexual dysfunction is a common and serious clinical side effect. It has been demonstrated that both neuronal nitric oxide (nNOS) and dopamine D2 receptor (DRD2) in the medial preoptic area (MPOA) and the paraventricular nucleus (PVN) of the hypothalamus have important roles in the regulation of sexual behaviour. We investigated the influences of 21 days' antipsychotic drug administration on expression of nNOS and DRD2 in the rat hypothalamus. Haloperidol (0.5 mg/kg/day i.p.) significantly decreased nNOS integrated optical density in a sub-nucleus of the MPOA, medial preoptic nucleus (MPN), and decreased the nNOS integrated optical density and cell density in another sub-nucleus of the MPOA, anterodorsal preoptic nucleus (ADP). Risperidone (0.25 mg/kg) inhibited the nNOS integrated optical density in the ADP. nNOS mRNA and protein in the MPOA but not the PVN was also significantly decreased by haloperidol. Haloperidol and risperidone increased DRD2 mRNA and protein expression in both the MPOA and the PVN. Quetiapine (20 mg/kg/day i.p.) did not influence the expression of nNOS and DRD2 in either the MPOA or the PVN. These findings indicate that hypothalamic nNOS and DRD2 are affected to different extents by chronic administration of risperidone and haloperidol, but are unaffected by quetiapine. These central effects might play a role in sexual dysfunction induced by certain antipsychotic drugs.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

The effect of size,age, and recent egg laying on copulatory choice of the hermaphroditic mollusc Aplysia juliana

Summary

Aplysia juliana is a cross-fertilizing, simultaneous hermaphrodite. During copulation, an individual may act as either a sperm donor or a sperm recipient, or both, when a pair copulates reciprocally. Experiments were conducted with A. juliana to determine if an animal's size, age, or recent egg-laying activity influenced its choice of copulatory role. Animals were isolated except when paired during daily, half-hour trials. In the first experiment, mature animals of different size (weight) but similar age were randomly paired. Animal size had no effect on the initial copulatory role chosen. In the second experiment, young, maturing A. juliana were paired with older animals. Young animals showed no preference in initial copulatory role either as a group or individually. Older A. juliana showed no copulatory preference as a group, but over half of the individuals demonstrated a consistent choice of one role. Some individuals acted almost exclusively as sperm donors, others as sperm recipients, suggesting that as an A. juliana ages, it is likely to develop a preference for a single copulatory role. A record of daily egg mass production was kept for all animals in the second experiment. Production of an egg mass since the last copulation did not affect the copulatory role chosen in the subsequent copulation.     

NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5' proximal promoter fragment (- 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF-deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above-mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF-deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF-deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant-negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO-cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up-regulation of galanin, and for DRG neuron survival and regeneration after axotomy.     

Distribution of NO-induced cGMP-like immunoreactive neurones in the abdominal nervous system of the crayfish,Procambarus clarkii

Nitric oxide (NO) acts as a signalling molecule by activating soluble guanylate cyclase and causing accumulation of the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) in target cells. In order to detect the presence of NO-cGMP signalling pathway in the crayfish abdominal nervous system, accumulation of NO-induced cGMP was investigated by anti-cGMP immunochemistry. Some preparations were incubated in a high-K(+) saline containing an inhibitor of cGMP-degrading phosphodiesterase, 3-isobutyl-1-methyxanthine (IBMX), to activate NO generating neurones, which could release NO in the ganglion, and then immunohistochemistry using an anti-cGMP antibody was performed. The other preparations were incubated in NO donor, sodium nitroprusside (SNP) saline containing IBMX before anti-cGMP immunohistochemistry was performed. The distribution of cGMP-like immunoreactive neurones in high-K(+) treated preparations was similar to that of cGMP-like immunoreactive neurones in NO donor treated preparations. About 70-80 cell bodies and many neuronal branches in the neuropilar area of the ganglion were stained, although no neurones showed immunoreactivity unless preparations were activated by either high-K(+) or the NO donor. Some of them were identical neurones, and they were intersegmental ascending interneurones and motor neurones. Sensory afferents that innervates hind gut showed strong cGMP-like immunoreactivity, although no mechanosensory afferents showed any immunoreactivity. These results strongly suggest the presence of an NO-cGMP signalling pathway that regulates neuronal events in the abdominal nervous system of the crayfish.     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Asymmetric Dimethylarginine and Hepatic Encephalopathy: Cause,Effect or Association?

The methylated derivative of l-arginine, asymmetric dimethylarginine (ADMA) is synthesized in different mammalian tissues including the brain. ADMA acts as an endogenous, nonselective, competitive inhibitor of all three isoforms of nitric oxide synthase (NOS) and may limit l-arginine supply from the plasma to the enzyme via reducing its transport by cationic amino acid transporters. Hepatic encephalopathy (HE) is a relatively frequently diagnosed complex neuropsychiatric syndrome associated with acute or chronic liver failure, characterized by symptoms linked with impaired brain function leading to neurological disabilities. The l-arginine—nitric oxide (NO) pathway is crucially involved in the pathomechanism of HE via modulating important cerebral processes that are thought to contribute to the major HE symptoms. Specifically, activation of this pathway in acute HE leads to an increase in NO production and free radical formation, thus, contributing to astrocytic swelling and cerebral edema. Moreover, the NO-cGMP pathway seems to be involved in cerebral blood flow (CBF) regulation, altered in HE. For this reason, depressed NO-cGMP signaling accompanying chronic HE and ensuing cGMP deficit contributes to the cognitive and motor failure. However, it should be remembered that ADMA, a relatively little known element limiting NO synthesis in HE, may also influence the NO-cGMP pathway regulation. In this review, we will discuss the contribution of ADMA to the regulation of the NO-cGMP pathway in the brain, correlation of ADMA level with CBF and cognitive alterations observed during HE progression in patients and/or animal models of HE.