Involvement of cyclic guanosine monophosphosphate (cGMP) and cytosolic guanylate cyclase in the regulation of synaptic ribbon numbers in rat pineal gland

In the rat pineal gland N-acetyltransferase (NAT) activity and synaptic ribbon (SR) numbers display a circadian rhythm. It is well-known that NAT activity is regulated by adrenergic mechanisms involving cyclic adenosine monophosphate (cAMP) as a second messenger. However, the mechanism involved in the regulation of SR numbers has not been established so far. In the present in vitro study, we have investigated the effects of 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), a cyclic guanosine monophosphate (cGMP) analog, and stimulation of guanylate cyclase on SR numbers. Incubation with 8-bromo-cGMP increased SR numbers in a dose- and time-dependent manner. Further, stimulation of the cytosolic guanylate cyclase also resulted in increased SR numbers. Adrenergic agonists stimulated cGMP but did not alter SR numbers. These findings suggest that cGMP is involved as a second messenger in the regulation of SR numbers. Since the adrenergically stimulated increase in cGMP did not influence SR numbers, a non-adrenergic cGMP metabolic pathway seems to be involved in the regulation of SR numbers in the rat pineal gland.     

银杏叶提取物抗血小板聚集机制研究

目的 通过体外观察银杏叶提取物(EGB)对血小板磷酸二酯酶(PDE)、核苷酸环化酶[腺苷酸环化酶(AC)、鸟苷酸环化酶(GC)]活性和环核苷酸[环腺苷酸(cAMP)、环鸟苷酸(cGMP)]水平的影响,探讨EGB抗血小板聚集的可能机制.方法 采集2周内未服任何药物的3例健康志愿者肘静脉血(柠檬酸钠抗凝),血小板分离洗涤后分别观察不同浓度EGB对血小板cAMP、cGMP水平及经超声匀浆后分离的PDE、AC、GC活性的影响,并以加同等体积药物溶剂为空白对照.结果 随着EGB浓度的增加,血小板cAMP水平明显增高,PDE3活性明显抑制,变化呈剂量依赖性;大剂量EGB(80 mg/L)对PDE5有轻度抑制作用;不同浓度EGB对cGMP水平和PDE2、AC、GC活性均无明显影响.结论 EGB通过抑制血小板PDE3活性,提升cAMP水平从而起到抗血小板聚集作用.

Abstract：

Objective To investigate the effect of Ginkgo biloba extract (EGB) on changes of activities of platelet phosphodiesterase (PDE) and nucleotide cyclase (adenylate cyclase [AC] and guanylate cyclase [GC]), and levels of cyclic nucleotide (cylic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]), and investigate the mechanism of platelet anti-aggregation with EGB. Methods Blood samples from 3 healthy volunteers, not taken any drugs within 2 weeks of the experiments, were anticoagulated with 3.8% sodium citrate. After isolating and washing, platelet was given various concentrations of EGB, and then applied for measurement of the levels cAMP and cGMP and the activities of PDE, AC and GC isolated from platelet sonic homogenates. Controls were given the same volume of bulk drug solvent. Results EGB dose-dependence was noted: the higher the level of EGB, the higher the cAMP level and the more active the PDE3. EGB at high-dose could slightly inhibit the PDE5 activity. EGB in any dosages could not affect the cGMP level, and activities of PDE2, AC and GC. Conclusion EGB has platelet anti-aggregation effect through inhibiting the PDE3 activity and increasing the cAMP level.     

High glucose rapidly activates the nitric oxide/cyclic nucleotide pathway in human platelets via an osmotic mechanism

The aim was to evaluate whether high glucose influences the nitric oxide (NO)/cyclic nucleotide pathway in human platelets via osmotic stress and to clarify the role of protein kinase C (PKC) in this phenomenon. The study was carried out on 33 healthy lean male volunteers, aged 28.3+/-1.3 years. NO synthesis was detected as L-citrulline production after L-arginine incubation in platelets incubated for 6 min with 22.0 mM D-glucose and iso-osmolar concentrations of mannitol, L-glucose and fructose. To evaluate the influence of PKC, experiments with D-glucose and mannitol were repeated in the presence of the PKC-beta selective inhibitor LY379196, and NO synthesis was detected after a 6-min incubation with phorbol 12-myristate 13-acetate (PMA), a non-selective PKC activator. Platelet content of guanosine-3',5'-cyclic monophosphate (cGMP) and adenosine-3',5'-cyclic monophosphate (cAMP) was measured by radioimmunoassay in platelets incubated with iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose. NO-dependence of cyclic nucleotide enhancements was evaluated by inhibiting NO synthase and guanylate cyclase. Platelet aggregation to ADP and collagen was evaluated in Platelet-Rich Plasma (PRP) in the presence of a 6-min incubation with D-glucose and mannitol, both without and with LY379196 and the guanylate cyclase inhibitor (H-[1,2,4]Oxadiazolo [4,3-a]quinoxaline-1-one)(ODQ). Iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose, and PMA increased NO production (p=0.0001). Effects of D-glucose and mannitol were blunted by LY379196. D-glucose and mannitol enhanced platelet cGMP and cAMP (p=0.0001) with a mechanism blunted by NO synthase and guanylate-cyclase inhibition, but did not modify platelet aggregation. In conclusion, glucose activates the NO/cyclic nucleotide pathway in human platelets with an osmotic mechanism mediated by PKC-beta.     

Adenosine increases human platelet levels of cGMP through nitric oxide: possible role in its antiaggregating effect

Adenosine is an endogenous antiaggregating substance that influences the platelet responses through specific A-type receptors that activate adenylate cyclase increasing the levels of 3',5'-cyclic adenosine monophosphate (cAMP). In this study, we investigated whether adenosine can also influence the levels of 3',5'-cyclic guanosine monophosphate (cGMP) and decrease the aggregating response of human platelets to adenosine-5-diphosphate (ADP) through this nucleotide. In platelet samples from healthy volunteers, we evaluated the effect of adenosine on ADP-induced aggregation and cyclic nucleotide synthesis. Some experiments were repeated in the presence of dipyridamole (inhibitor of adenosine uptake and phosphodiesterase activity), N(G)-monomethyl-L-arginine (L-NMMA, nitric synthase inhibitor), ionomycin (calcium ionophore), and ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine, inhibitor of nitric oxide (NO)-dependent activation of guanylate cyclase). Adenosine decreased the response to ADP in a concentration-dependent way (analysis of variance, ANOVA: P<.0001): cAMP levels increased from 30.0 +/- 2.0 (control) to 46.0 +/- 3.0 pmol/10(9) platelets (in the presence of 15 mumol/l adenosine) and cGMP levels increased from 5.6 +/- 1.0 (control) to 10.9 +/- 2.0 pmol/10(9) platelets (in the presence of 15 mumol/l adenosine). Also, nucleotide levels measured at the end of aggregation were higher in platelet samples exposed to adenosine than in controls. Dipyridamole at 40 mumol/l slightly increased adenosine's effects on both nucleotides. L-NMMA blunted the effect of adenosine on cGMP both in unstimulated samples and in aggregated platelets without any effect on cAMP synthesis. Platelet exposure to L-NMMA and ambroxol partially prevented adenosine's effect on ADP-induced aggregation. In conclusion, adenosine, which enhances intraplatelet cAMP levels, was determined to also cause an increase in cGMP concentrations through a mechanism that involves NO synthesis. This effect plays a direct role in the adenosine-induced antiaggregation.     

CSF cyclic nucleotides and somatostatin in Parkinson's disease

Concentrations of cyclic adenosine 3',5' monophosphate (cAMP) were significantly lower in parkinsonian patients than in controls, but concentrations of guanosine 3',5' monophosphate (cGMP) were not altered. Both cAMP and cGMP levels were lower in patients with more severe symptoms on the left side of the body. Somatostatin-like immunoreactivity (SLI) was similar in parkinsonian patients and controls. Both cAMP and SLI were significantly related to acetylcholinesterase activity.     

Whole brain spheroid cultures as a model to study the development of nitric oxide synthase-guanylate cyclase signal transduction

Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.     

Cyclic GMP alterations in fetal rat cerebrum after global intrauterine ischemia: Role of guanylate cyclase phosphorylation

Changes in the levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) have been measured in brains of 20-day-old rat fetuses exposed to global intrauterine ischemia. Ischemia of different duration (0.5–30 minutes) did not alter the level of cAMP. In contrast, cGMP levels increased as a result of ischemia. This increase was seen even after a short period of ischemia (less than 5 minutes) and was maximal after 5 minutes, where a threefold increase could be observed. This stimulation was transient: after 30 min of ischemia, cGMP returned to the control level. Accumulation of cGMP can be related to the activation of guanylate cyclase, the activity of which is doubled after 15 minutes of ischemia. Immunoprecipitation of guanylate cyclase after in vivo labeling of the fetal brain with³²P_i revealed a threefold increase in the phosphorylation of the enzyme after 15 minutes of ischemia. The possible role of these modifications in cGMP metabolism during the course of ischemia is discussed. This work was supported in part by the Revson Foundation of the Israel Academy of Sciences and Humanitics, Jerusalem.     

Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content

In this study, we explored the ability of sodium nitroprusside to inhibit the aggregation of human platelets in platelet-rich plasma (PRP) and whole blood and its effects on intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). The experiments investigated dose-dependent effects of nitroprusside starting from concentrations in the range of circulating levels achievable in vivo during drug administration in humans. Furthermore, we investigated the time-course of both antiaggregating action and the influence on cyclic nucleotide synthesis. Results showed that sodium nitroprusside inhibited the aggregation induced by adenosine 5-diphosphate (ADP) and collagen starting from concentration as low as 2 micromol/l. The IC(50) value for ADP-induced aggregation in PRP was 18.7+/-2.4 micromol/l. The inhibition of platelet aggregation showed a time-dependent behaviour and was not reversible within 90 min. The accumulation of intraplatelet cGMP in the presence of sodium nitroprusside exhibited a comparable time-course characterized by an early increase, a steady state and a late further increase. The time-course of cAMP synthesis was very similar to that of cGMP. Our data evidenced a long-lasting inhibition of platelet responses by sodium nitroprusside and excluded a desensitization of platelet guanylyl cyclase after 3-h exposure to nitric oxide (NO). Furthermore, they indicated a role of cAMP accumulation in the antiaggregating effects of nitroso donor: the simultaneous increase of intracellular content of cAMP and cGMP can synergize in the reduction of the platelet responses.     

A functional parameter to study heterogeneity of glial cells in rat brain slices: cyclic guanosine monophosphate production in atrial natriuretic factor (ANF)-responsive cells

Stimulation of guanylate cyclase in vitro by atrial natriuretic factor (ANF) or sodium nitroprusside was studied in rat brain tissue slices biochemically as well as by means of cyclic guanosine monophosphate (cGMP) immunocytochemistry. The ANF-responsive, cGMP-producing cells were studied in the olfactory bulb, the septal area, the hippocampus, the medial amygdala, and the medial preoptic area. These cells, having the ANF-stimulated particulate guanylate cyclase, were characterized as astroglial cells on the basis of their glial fibrillary acidic protein (GFAP) immunostaining, although not all astroglial cells in these areas could be identified as cGMP-immunoreactive cells. Sodium nitroprusside-stimulated soluble guanylate cyclase activity was demonstrated in neuronal cell bodies and varicose fibers and was associated with blood vessel walls. Upon maturation, a significant decrease in cGMP production was found after stimulation by 100 nM ANF-(103-126) in the olfactory bulb, the medial amygdala, and the hippocampus, but not in the septal area; no change was found in these areas in cGMP content after stimulation of cGMP production by 10 microM sodium nitroprusside. Via cGMP immunocytochemistry, no qualitative differences were seen in the ANF-responsive, cGMP-producing cells upon maturation.     

Distribution and different activation of adenylate cyclase by naf and of guanylate cyclase by NaN3 in neuronal and glial cells separated from rat cerebral cortex

Distribution of adenylate cyclase and guanylate cyclase activities in neuronal perikarya and glial cells separated from rat brain, and cellular differences in activation between of adenylate cyclase by NaF and of guanylate cyclase by NaN3 have been studied. Adenylate cyclase activity was higher in the glial cells than in the neuronal fraction, while guanylate cyclase activity was equally detected in both cell fractions. Adenylate cyclase was mainly derived from the particulate fraction of both brain cell homogenates, whereas the major portion of guanylate cyclase activity was found in their soluble rather than in the particulate fractions. Although bulk-separated neurons and glial cells almost failed to change intracellular cyclic nucleotide levels in response to some putative neurotransmitters, activation of adenylate cyclase by NaF was found to be greater in neuronal than in glial cell fractions, and was observed more clearly in the soluble than in the particulate fractions. Sodium azide greatly increased guanylate cyclase in the particulate fraction, but did not affect it considerably in the soluble one. Addition of catalase to the reaction mixture together with NaN3 further stimulated guanylate cyclase both int he soluble and the particulate fractions. These results suggest that adenylate cyclase and guanylate cyclase without intimate coupling to the transmitter-receptor system, but with activation by NaF or NaN3, may be distributed ubiquitously in the cells separated from rat cerebral cortex.     

Endogenous activation of guanylate cyclase in synaptosomal soluble fraction from rat brain

Guanylate cyclase in crude mitochrondrial (P2) soluble fraction prepared from rat brain, obtained by a hypo-osmotic treatment of P2, showed extremely higher activity than that in the same fraction from other organs. In addition the soluble fraction obtained from synaptosomes (P2 - B) contained the highest enzyme activity among other subfractions of the cerebral P2 examined. Guanylate cyclase activity in the synaptosomal soluble fraction was, however, markedly suppressed by various compounds reacting with free radicals. These results suggest that guanylate cyclase in the synaptosomal soluble fraction may be activated endogenously be a free radical and involved in the regulatory mechanisms for cyclic guanosine monophosphate (cyclic GMP) at presynaptic terminals.     

Inhibitory effect of cyclic guanosine 3',5'-monophosphate (cGMP) on the afferent resting activity in the cephalopod statocyst

The effects of exo- and endogenous cGMP on the resting activity (RA) of afferent crista fibers were studied in isolated preparations of the statocysts of the cuttlefish Sepia officinalis and the squid Sepioteuthis lessoniana. Bath application of the membrane-permeable cGMP analogs 8-bromo-cGMP (B-cGMP) and N(2),2'-o-dibutyryl 3', 5'-cyclic guanosine monophosphate (dB-cGMP), and of the selective inhibitor of cGMP-phosphodiesterase zaprinast (ZAP), caused an inhibition of RA. The inhibitory effects of B-cGMP and dB-cGMP remained when the preparation was pre-treated with: (i) the guanylate cyclase inhibitors 1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one (ODQ) or cystamine (CYS); (ii) the adenylate cyclase inhibitors nicotinic acid (NIC-A), 2',3'dideoxyadenosine (DDA), or MDL-12330A (MDL); (iii) the guanylate cyclase inhibitor methylene blue (M-BLU) and the adenylate cyclase inhibitor MDL combined; or (iv) the nitric oxide (NO) synthase inhibitors N(G)-nitric-L-arginine methyl ester HCl (L-NAME) or N(G)-nitro-L-arginine (L-NOARG). These data indicate that cGMP, as an intracellular messenger, has a tonic inhibitory effect on the RA of afferent crista fibers in cephalopod statocysts.     

Relationships between seizure activity and cyclic nucleotide levels in brain

The effects of pentylenetetrazol on behavior, EEG activity and regional CNS levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) in mice and guinea pigs were studied. Pentylenetetrazol increased cGMP levels in all regions of brain examined (cerebral cortex, hippocampus, striatum and cerebellum) and increased cAMP levels in all regions except striatum. cGMP levels were increased by both sub-convulsant and convulsant doses of pentylenetetrazol. In contrast, cAMP levels were elevated only by concentrations of pentylenetetrazol that produced clinically evident seizures or epileptiform EEG activity. These data indicate that increases in CNS cGMP levels produced by epileptogenic stimuli can occur independently of seizure discharges, whereas accumulation of cAMP requires and is secondary to seizure activity. In conjunction with results of other studies, these data support the hypothesis that cGMP may have a role in seizure genesis and/or propagation, whereas cAMP may be involved in processes that attenuate or terminate seizures.     

Antigenic conservation of brain guanylate cyclase during evolution

A comparative study of brain guanylate cyclase from different animal species (including man, bird, fish and amphibian) has been performed using a specific antibody directed against soluble rat brain guanylate cyclase. Analyses were performed on supernatant fractions by the double-immunodiffusion test, by the protein blotting technique after SDS-polyacrylamide gel electrophoresis and by analytical isoelectric focusing on agarose allowing specific immunodetection of isoelectric patterns. Membrane-bound guanylate cyclase from rat brain and soluble guanylate cyclase from several rat tissues cross-reacted with the antibody. All the brain enzymes tested were found to be identical by double-immunodiffusion. The electrophoretic and isoelectrophoretic profiles of the different brain guanylate cyclases were found to exhibit many common features with some differences between mammalian and non-mammalian enzymes. In human brain, guanylate cyclase has been localized in glial and neuronal cells by immunohistochemistry. The results demonstrate that guanylate cyclase has been well conserved during the course of evolution and are consistent with the involvement of guanylate cyclase and cyclic GMP in basic cellular function.     

Natriuretic Peptides Stimulate Cyclic GMP Production in an Immortalized LHRH Neuronal Cell Line

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro . Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors—which represent GCs—we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.     

Basal, cAMP- and cGMP-dependent protein kinases in human brain tumors

Since the effects of cyclic nucleotides are mediated via protein kinases activation, we have studied the properties and regulation of these enzymes in cytosol and particulate fraction of normal cerebral tissues and of some human brain tumors. We found that distribution and activity of cyclic nucleotide-dependent protein kinases are regulated differently among various brain tumors and in comparison to normal gray and white matter. Pathological tissues show an higher cGMP-dependent protein kinase and this biochemical pattern is particularly evident in tumors with more pronounced malignancy. These data further confirm the hypothesis of a correlation between the increase of cGMP function and cellular growth and malignancy.     

Dysregulation of Cerebellar Adrenomedullin Signaling During Hypertension

Adrenomedullin (AM) is a peptide involved in blood pressure regulation. AM activates three different receptors, the AM type 1 (AM1), type 2 (AM2), and calcitonin gene-related peptide 1 (CGRP1) receptors. AM triggers several signaling pathways such as adenylyl cyclase (AC), guanylyl cyclase (GC), and extracellular signal-regulated kinases (ERK) and modulates reactive oxygen species (ROS) metabolism. Cerebellar AM, AM-binding sites, and its receptor components are altered during hypertension, although it is unknown if these alterations are associated with changes in AM signaling. Thus, we assessed AM signaling pathways in cerebellar vermis of 16-week-old Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Animals were sacrificed by decapitation, and cerebellar vermis was microdissected under stereomicroscopic control. Tissue was stimulated in vitro with AM. Then the production of cyclic guanosine monophosphate (cGMP), nitric oxide (NO) and cyclic adenosine monophosphate (cAMP) were assessed along with ERK1/2 activation and three antioxidant enzymes’ activity: glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). Our findings demonstrate that in the cerebellar vermis of normotensive rats, AM increases cGMP, NO, cAMP production, and ERK1/2 phosphorylation, while decreases basal antioxidant enzyme activity. In addition, AM antagonizes angiotensin II (ANG II)-induced increment of antioxidant enzyme activity. Hypertension blunts AM-induced cGMP and NO production and AM-induced decrease of antioxidant enzyme activity. Meanwhile, AM-induced effects on cAMP production, ERK1/2 activation, and AM-ANG II antagonism were not altered in SHR rats. Our results support a dysregulation of several AM signaling pathways during hypertension in cerebellar vermis.     

Influence of lesions of the noradrenergic locus coeruleus system on the cerebral metabolic response to bicuculline-induced seizures

The objective of the present study was to explore if lesions of the ascending noradrenergic pathways, originating in the locus coeruleus, modulate the cerebral metabolic response to bicuculline-induced seizures in rats. Bilateral noradrenergic lesions were performed by 6-hydroxydopamine injections in the caudal mesencephalon, 12–22 days before seizures were induced in animals ventilated on N₂O:O₂ (75:25). After 5 min of seizures the brain was frozen in situ and cerebral cortex and hippocampus were sampled for analysis. Labile phosphates, glycolytic metabolites, cyclic nucleotides, and free fatty acids were measured. In another series, lesioned animals were used for measurements of cerebral oxygen consumption.The noradrenergic lesions neither modified the electroencephalographically recorded seizure discharge, nor did they alter cerebral oxygen consumption or cerebral energy state. However, when compared to sham-operated animals, those with noradrenergic lesions had significantly higher (115% and 68%) glycogen concentrations and lower (50% and 52%) cyclic AMP concentrations in cerebral cortex and hippocampus, respectively, demonstrating the marked influence of noradrenergic activity on adenylate cyclase activity and glycogenolysis. The lesions failed to modulate the rise in free fatty acids in the cerebral cortex, or the cyclic GMP concentrations in the cerebral cortex and hippocampus. Thus, increased noradrenergic activity during status epilepticus does not seem responsible for lipolysis or for activation of guanylate cyclase.     

Endothelium-dependent dilation of feline cerebral arteries: role of membrane potential and cyclic nucleotides

The objective of this study was to characterize the role of membrane potential and cyclic nucleotides in endothelium-dependent dilation of cerebral arteries. Middle cerebral arteries isolated from cats were depolarized and constricted in response to serotonin or when subjected to transmural pressures greater than 50 mm Hg. Acetylcholine (ACh) and ADP caused vasodilation and a sustained, dose-dependent hyperpolarization of up to 20 mV in this artery. The membrane potential change preceded the vasodilation by approximately 6 s. Hyperpolarizations and dilations to ACh and ADP did not occur in preparations without endothelium. The hyperpolarizations were abolished by ouabain (10(-5) M), which also blocked the dilator response to ACh. However, dilations to ADP were unaffected by ouabain. Methylene blue (5 x 10(-5) M), a guanylate cyclase inhibitor, had no effect on the responses to ACh or ADP in the presence or absence of ouabain. Cyclic guanosine monophosphate (cGMP) levels were not altered in cerebral arteries exposed to ACh or ADP. However, ADP did increase cyclic adenosine monophosphate levels in these blood vessels. We conclude that although membrane hyperpolarizations may be adequate to cause vasodilation, at least one other pathway of endothelium-dependent vasodilation also is present in feline cerebral arteries. Cyclic GMP does not appear to be involved in this alternate pathway of dilation.     

Cyclic GMP modulators and excitotoxic injury in cerebral cortical cultures

N-Methyl-D-aspartate (NMDA) receptor activation generates nitric oxide (NO) and cyclic GMP (cGMP) and produces 'excitotoxic' neuronal injury. To examine the possible role of cGMP in excitotoxicity, we evaluated the effects of agents that stimulate or inhibit cGMP activity on the release of lactate dehydrogenase from neuron-enriched cortical cultures. cGMP analogs exhibited no toxicity, and inhibitors of guanylate cyclase or of cGMP-dependent enzymes failed to protect cultures from the toxic effects of NMDA or the NO donor sodium nitroprusside. These findings argue against a role for cGMP in the pathogenesis of excitotoxic neuronal injury.