Cyclic GMP-mediated Mechanism Research Articles

SP-333, A Guanylate Cyclase-C Agonist, Ameliorates DSS-Colitis in Mice Via a Novel Cyclic GMP-Mediated Mechanism

SP-333, A Guanylate Cyclase-C Agonist, Ameliorates DSS-Colitis in Mice Via a Novel Cyclic GMP-Mediated Mechanism

Release of monoamines and nitric oxide is involved in the modulation of hyperpolarization-activated inward current during acute thalamic hypoxia

Using slices of the dorsal lateral geniculate nucleus, it has been shown that, in the presence of excitatory and inhibitory amino acid antagonists, brief periods of hypoxia (3–4 min of 95% N 2/5% CO 2) induce in thalamocortical neurons an increase in instantaneous input conductance (G N) accompanied by an inward shift in baseline holding current (I BH). These effects have been suggested to be mediated, at least in part, by a positive shift in the voltage-dependence of the hyperpolarization-activated, mixed Na +/K + current (I h) and a change in its activation kinetics which transforms it into an almost instantaneously activated current. In this study, using the whole-cell patch-clamp technique, the contribution of an increased Ca 2+-dependent transmitter release to the hypoxic response of thalamocortical neurons was further investigated using (i) blockers of calcineurin, a Ca 2+/calmodulin-activated phosphatase that selectively regulates Ca 2+-dependent release, and (ii) antagonists of neurotransmitters that are known to modulate I h. Thalamocortical neurons ( n=23) recorded with electrodes filled with calcineurin autoinhibitory fragment (30–250 μM), a membrane impermeable blocker of calcinuerin, showed no difference either in resting, or in the hypoxia-induced changes in, G N, I BH and I h, when compared to thalamocortical cells patched with electrodes that did not contain calcineurin autoinhibitory fragment. In contrast, in 18 of these neurons recorded with calcineurin autoinhibitory fragment-filled electrodes, bath application either of cyclosporin-A (20 μM) or tacrolimus (50–100 μM), two membrane permeable blockers of calcineurin, abolished the effects of hypoxia on G N, I BH, and I h. Separate application of noradrenaline, serotonin, histamine and nitric oxide antagonists produced only a small depression of the hypoxic response, while concomitant bath application of these antagonists decreased the hypoxia-induced changes in G N and I BH by 55 and 42%, respectively ( n=12). Concomitant bath application of 8-bromo-adenosine-3′5′-cyclicmonophosphate and 8-bromo-guanosine-3′5′-cyclicmonophosphate (both 1 mM), which are known to mediate the action of these transmitters on I h, increased G N (40%), decreased I h time-constant of activation (30%) and significantly occluded (50%) the hypoxia-induced effect on G N and I BH. Thalamocortical neurons ( n=6) patched with electrodes filled with 8-bromo-adenosine-3′5′-cyclicmonophosphate and 8-bromo-guanosine-3′5′-cyclicmonophosphate (both 1 mM) showed a larger G N than the one recorded with the standard internal solution, and a significant depression of the hypoxia-induced changes in G N and I BH. These results indicate that during acute thalamic hypoxia an increased release of noradrenaline, serotonin, histamine and nitric oxide is responsible for transforming I h into an instantaneously activating current via cyclic AMP- and cyclic GMP-mediated mechanisms.

Alpha-adrenoceptor-mediated penile erection in dogs: in vivo and in vitro observations.

We have evaluated the role of adrenergic components in the pelvic splanchnic nerve on the erectile function in the dog. Electrical stimulation of pelvic splanchnic nerves increased blood flow in the internal pudendal artery and also elevated the cavernous pressure. These increases were blocked in part by phentolamine or methylene blue, but not by propranolol or atropine. The effects of cholinergic and adrenergic agonists and antagonists on mechanical responses were also examined in muscle strips obtained from various arteries in the intrapelvic region including the internal pudendal artery. Norepinephrine induced contraction in the iliac artery and relaxation in the internal pudendal artery, and both the contraction and relaxation responses were blocked by phentolamine but not by propranolol. These findings suggest that in the dog, alpha-adrenergic components projected through the pelvic splanchnic nerve may contribute to penile erection, together with cyclic GMP-mediated mechanisms.

Secreted form of amyloid precursor protein enhances basal glucose and glutamate transport and protects against oxidative impairment of glucose and glutamate transport in synaptosomes by a cyclic GMP-mediated mechanism.

Synaptic dysfunction and degeneration are believed to underlie the cognitive deficits that characterize Alzheimer's disease, and overactivation of glutamate receptors under conditions of increased oxidative stress and metabolic compromise may contribute to the neurodegenerative process in many different disorders. The secreted form of amyloid precursor protein (sAPPalpha), which is released from neurons in an activity-dependent manner, can modulate neurite outgrowth, synaptic plasticity, and neuron survival. We now report that sAPPalpha can enhance glucose and glutamate transport in synaptic compartments. Treatment of cortical synaptosomes with nanomolar concentrations of sAPPalpha resulted in an attenuation of impairment of glutamate and glucose transport induced by exposure to amyloid beta-peptide and Fe2+. The protective effect of sAPPalpha was mimicked by treatment with 8-bromo-cyclic GMP and blocked by a cyclic GMP-dependent protein kinase inhibitor, suggesting that protective action of sAPPalpha is mediated by cyclic GMP. Our data suggest that glucose and glutamate transport can be regulated locally at the level of the synapse and further suggest important roles for sAPPalpha and cyclic GMP in modulating synaptic physiology under normal and pathophysiological conditions.

Propofol-induced relaxation of rat mesenteric arteries: evidence for a cyclic GMP-mediated mechanism.

Experiments were designed to evaluate whether guanosine 3',5'-cyclic monophosphate (cGMP)-mediated mechanisms contribute to vasodilation via propofol in rat mesenteric resistance arteries. Ring segments were suspended in the myograph system for isometric tension recording, and responses to propofol were tested in the presence and absence of methylene blue (MB), an inhibitor of guanylate cyclase. At concentrations > or = 1 microM, propofol caused concentration-dependent relaxation of vessel rings precontracted with U46619 (a thromboxane analog). The effect was not affected by N-monomethyl-L-arginine (L-NMMA; 50 microM). MB (5 microM) reversed propofol-induced vasodilation by 30% (p < 0.001). In contrast, MB has no effect on nifedipine-inhibited vasocontraction. The propofol-induced relaxation was further tested in rings incubated in Ca2+-free solution. U46619-induced contractions were significantly reduced by propofol (40 microM) but not by nifedipine (1 microM). Propofol reduced to a similar degree the contractions obtained to exogenously added calcium chloride in the absence and the presence of MB. Furthermore, propofol (10-100 microM) increased cGMP content in cultured bovine vascular smooth-muscle cells. Soluble guanylate cyclase inhibitors, such as MB and LY83583, attenuated this effect. This investigation suggests that propofol-induced relaxations in small arteries, in addition to inhibition of calcium influx, are mediated by increases of cGMP in the smooth muscle cells.

The negative functional and metabolic effects of muscarinic stimulation are enhanced by beta-adrenergic activation in control and hypertrophic dog hearts in vivo.

The aim of the current study was to determine if the effects of muscarinic stimulation on left ventricular function and metabolism are greater during beta-adrenergic activation, whether a cyclic GMP-mediated mechanism is responsible, and if this is altered by left ventricular hypertrophy (LVH) induced by aortic valve stenosis. Acetylcholine (Ach) (5 micrograms/kg/min) and/or isoproterenol (Iso) (0.1 micrograms/kg/min) was infused into a branch of the left anterior descending (LAD) artery in 8 control and 8 LVH open-chest anesthetized dogs. LVH increased heart weight, heart-to-body weight ratio and systolic left ventricular pressure. LVH reduced muscarinic receptor density (fmol/mg protein) (control: 149.2+/-18.6; LVH: 77.8+/-8.6), but not affinity. Alone, Ach had no effect on regional force, work or metabolism. Iso increased peak force (g) (control: baseline-7.4+/-0.4; Iso-12.4+/-2.2; LVH: baseline-6.7+/-0.8; Iso-16.3+/-2.7, regional work (g mm/min)) (control: baseline-1250+/-186; Iso-1813+/-409; LVH: baseline-927+/-235; Iso-1244+/-222), and O2 consumption (ml O2/min/100 g) (control: baseline-3.3+/-0.2; Iso-8.1+/-2.0; LVH: baseline-4.8+/-1.0; Iso-8.3+/-1.1). During Iso, Ach reduced segment shortening (control: Iso-14.5+/-1.2; Iso+Ach-10.5+/-1.8; LVH: Iso-10.4+/-1.5; Iso+Ach-7.6+/-1.3) and peak force (control: Iso+Ach-7.7+/-1.0; LVH: Iso+Ach-10.5+/-1.4). Ach also reduced work (control: Iso+Ach-875+/-217; LVH: Iso+Ach-776+/-180) and O2 consumption (control: Iso+Ach-3.4+/-0.7; LVH: Iso+Ach-3.6+/-0.6) in the presence of Iso. Cyclic GMP was higher in the LVH animals during all treatments and was elevated from baseline by Ach in both groups. Neither Iso nor Iso+Ach had a significant effect on cyclic GMP. Thus, the negative functional and metabolic effects of muscarinic stimulation are enhanced during beta-adrenergic activation. This does not, however, appear to be dependent on a cyclic GMP-mediated mechanism. Despite reduced number of muscarinic receptors, this response was not altered by pressure-induced cardiac hypertrophy.

Nitric oxide reduces depolarization-induced calcium influx in PC12 cells by a cyclic GMP-mediated mechanism

The present study was undertaken to determine whether nitric oxide (NO) alters voltage-dependent changes in intracellular calcium levels ([Ca2+]i) using PC12 cells as a neuronal model. The addition to PC12 cells of sodium nitroprusside (SNP), which spontaneously releases NO in aqueous solution, significantly inhibited the KCl-stimulated increase in [Ca2+]i. The inhibitory action of SNP was concentration-dependent and was mimicked by hydroxylamine which also generates NO. Both L-type (nifedipine sensitive) and N-type (omega-conotoxin sensitive) voltage-dependent Ca2+ channels are present in PC12 cells and may be affected by NO-generating agents. In contrast, SNP did not alter [Ca2+]i in response to purinergic receptor stimulation. Preincubation of PC12 cells with 8-bromo-cyclic GMP also inhibited the KCl-stimulated increase in [Ca2+]i. In addition, inclusion of the guanylyl cyclase inhibitor, LY83583, blocked the inhibitory action of SNP on the voltage-sensitive changes in [Ca2+]i. The results suggest that NO selectively inhibits voltage-dependent calcium influx in neuronal cells through a cyclic GMP-dependent mechanism.

Modulation of neutrophil activity by nitric oxide during acute myocardial ischaemia and reperfusion.

Nitric oxide (NO) exerts an inhibitory effect on polymorphonuclear neutrophil (PMN) function, via a cyclic GMP-mediated mechanism, while PMNs are known to play an important role in myocardial ischaemia-reperfusion injury (MI-R). Since the major source of NO, vascular endothelium, becomes functionally impaired during MI-R, it is attractive to hypothesize that it is this loss of endothelial nitric oxide production that allows PMN adherence and activation. The studies reviewed here add substance to this hypothesis. Authentic NO, administered during MI-R both reduces myocardial necrosis and PMN accumulation, while basal NO release, as estimated by coronary artery ring responses to L-NAME, an NO synthase inhibitor, declines during reperfusion with a time-course mirrored by PMN adherence in the same preparation. Reduction in infarct size and decreased PMN accumulation can also be demonstrated with L-arginine and NO donors. Since endothelial dysfunction leads to PMN adherence and PMNs have been shown to contribute to endothelial dysfunction, it seems probable that a positive feedback loop is generated during MI-R, leading to the amplification of PMN activity and subsequent myocardial damage.

Isoflurane does not vasodilate rat thoracic aortic rings by endothelium-derived relaxing factor or other cyclic GMP-mediated mechanisms.

Endothelium-derived relaxing factor (EDRF) is a potent endogenous vasodilator that has been indirectly suggested to play a role in isoflurane-mediated vasodilation. To examine directly the possible role of EDRF in isoflurane-mediated vasodilation, isolated rat thoracic aortic rings were suspended for isometric tension measurements, equilibrated to a resting tension of 2 g, and constricted with a 50% maximal concentration (EC50) dose of phenylephrine or KCl. Three groups of rings were studied: endothelium-intact, endothelium-denuded, and endothelium-intact rings treated with nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of EDRF synthase. Isoflurane was then added at 1, 2, and 3% in a cumulative manner, allowing 10 min for each concentration to equilibrate. Indomethacin was present in all experiments to prevent the formation of vasoactive prostanoid metabolites. Since EDRF causes vascular relaxation by stimulating soluble guanylyl cyclase and increasing cyclic GMP, the effect of isoflurane on vascular ring cyclic GMP content was determined as an additional indicator of EDRF-mediated dilation. Rings with intact and denuded endothelium were isolated as described above, constricted with phenylephrine, and challenged with methacholine (positive control) or 1, 2, or 3% isoflurane. After 8 min exposure, the rings were flash-frozen in dry-ice-cooled acetone and homogenized in 1 N HCl for subsequent analysis of cyclic GMP content by radioimmunoassay. Isoflurane caused dose-dependent vasodilation of both KCl- and phenylephrine-constricted rings. In the phenylephrine group, at 2% and 3% isoflurane, endothelium-denuded and L-NAME-treated rings relaxed to a greater extent than endothelium-intact rings (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Activated oxygen metabolites as regulators of vascular tone

Oxygen metabolites have been reported to produce vasoconstriction and/or vasodilation in a variety of in vitro or in vivo vascular preparations. Certain basic mechanisms appear to contribute to these responses. Hydrogen peroxide can produce either vasodilation or constriction via stimulation of prostaglandins. The soluble form of guanylate cyclase in vascular smooth muscle, an enzyme which produces the intracellular mediator of relaxation cyclic GMP, is also a site of action of vasoactive O2 metabolites. Guanylate cyclase is directly activated by nanomolar concentrations of nitric oxide (produced by endothelial cells or nitrovasodilator drugs) or H2O2 (via its metabolism by catalase). These cyclic GMP-mediated mechanisms of relaxation are inhibited by superoxide anion, produced from endogenous sources after inhibition of superoxide dismutase or produced by pharmacological agents that undergo redox cycling. In addition, O2 metabolites may modulate vascular tone via the chemical destruction of physiological contractile agents (e.g. norepinephrine) and relaxant agents (e.g. nitric oxide), and via injury to cells important for the regulation of vascular tone (e.g. endothelium). We have found in a variety of preparations that reexposure to O2 after a brief period of severe hypoxia produces vascular responses that appear to be mediated by intracellular H2O2 generation. Thus, active O2 species may contribute to vascular responses in pathophysiological situations associated with their formation (e.g. inflammation, ischemia/reperfusion, etc.) and to the physiological regulation of vascular tone produced by changes in O2 tension (e.g. reactive hyperemia, hypoxic vasoconstriction, etc).