Membrane-permeable Cyclic GMP Analogue Research Articles

Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3',5'-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K(+) channels by 4-aminopyridine, a K(+) channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca(2+) channels by Cd(2+), a general voltage-dependent Ca(2+) channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca(2+) channels in the presynaptic nerve terminals of A1 neurons.

Up-regulation of Na +-dependent Mg 2+ transport by nitric oxide and cyclic GMP pathway in renal epithelial cells

A putative, Na +-dependent Mg 2+ transport pathway controls the intracellular free Mg 2+ concentration ([Mg 2+] i) in various mammalian cells. The characteristics of this Mg 2+ transport pathway have not been clarified. Herein, we examined the regulatory mechanism of Na +-dependent Mg 2+ efflux in renal epithelial NRK-52E cells. Mg 2+ removal from the extracellular bathing solution induced an Na +-dependent [Mg 2+] i decrease in Mg 2+ (5 mM)-loaded cells but not in control cells. Amiloride inhibited the [Mg 2+] i decrease in a dose-dependent manner (IC 50=3 μM). Similarly, atomic absorption spectrophotometry showed that Mg 2+ removal decreased intracellular Mg 2+ content, while it increased Na + content. Calphostin C (1 μM), a protein kinase C inhibitor, and genistein, a tyrosine kinase inhibitor (10 μM), blocked the [Mg 2+] i decrease. The [Mg 2+] i decrease was accompanied by an increase in intracellular nitric oxide (NO) and cyclic GMP contents. ( E)-4-methyl-2-[( E)-hydoxyimino]-5-nitro-6-methoxy-3-hexenamide (0.1 mM), an NO donor, and 8-bromo-cyclic GMP (0.1 mM), a membrane-permeable cyclic GMP analogue, accelerated the [Mg 2+] i decrease. In contrast, N G-monomethyl- l-arginine ( l-NMMA, 0.1 mM), an NO competitive inhibitor, and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxaline-1-one (ODQ, 10 μM), an NO-sensitive guanylate cyclase inhibitor, significantly blocked the [Mg 2+] i decrease. These results indicate that a decrease in extracellular Mg 2+ concentration induces the production of NO and cyclic GMP, which leads to the up-regulation of Na +-dependent Mg 2+ efflux.

Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP >> CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.

Nitric Oxide Inhibits Inducible Nitric Oxide Synthase mRNA Expression in RAW 264.7 Macrophages

Using cultured murine RAW 264.7 macrophages, the present study investigates the influence of nitric oxide (NO) on the expression of the inducible NO synthase (iNOS) enzyme at the transcriptional level. Incubation of cells with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) led to a marked increase in iNOS mRNA levels. Inhibition of LPS/IFN-γ-induced NO synthesis with the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA) was accompanied by a significant up-regulation of iNOS mRNA that was reversed in the presence of the NO donor sodium nitroprusside (SNP). Treatment of cells with SNP alone decreased LPS/IFN-γ-induced iNOS mRNA levels in a concentration-dependent manner. The inhibitory effect of SNP on iNOS mRNA expression was not prevented by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase. In agreement with this finding, incubation of cells with the membrane-permeable cyclic GMP analogue 8-bromo cyclic GMP left LPS/IFN-γ-induced iNOS mRNA expression virtually unaltered. Together, our results demonstrate that both iNOS-derived and exogenous NO exert an inhibitory effect on the expression of iNOS by a mechanism independent of the soluble guanylyl cyclase/cyclic GMP pathway. In conclusion, NO may control the extent of iNOS mRNA expression by a negative autoregulatory feedback.

Amyloid β-Peptide-induced Inhibition of MTT Reduction in PC12h and C1300 Neuroblastoma Cells: Effect of Nitroprusside

Takenouchi T. and E. Munekata. Amyloid β-peptide induced inhibition of MTT reduction in PC12h and C1300 neuroblastoma cells: effect of nitroprusside. Peptides 19(2) 365–372, 1998.—We have investigated the effect of amyloid β-peptide (Aβ) in rat pheochromocytoma PC12h and murine C1300 neuroblastoma cells by using MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} reduction assay. Exposure of the cells to Aβ peptides, Aβ1–40 and its fragment Aβ25–35, induced a concentration-dependent inhibition of MTT reduction in both cell lines, and MTT-dependent LDH release due to cell lysis in PC12h cells. We also found that sodium nitroprusside (SNP), a spontaneous nitric oxide (NO) generator, significantly prevented the inhibition of MTT reduction and MTT-dependent LDH release caused by Aβ peptides at 10–100 μM, although a high concentration of SNP (≥333 μM) was remarkably toxic by itself. Since the inhibition of MTT reduction caused by Aβ is known as one of the first indicators of its toxicity, these findings suggest that Aβ peptides have a toxic effect in these cell lines, and SNP may attenuate the Aβ peptide-induced toxicity. In regard the mechanisms of the actions of SNP, hydroxylamine which also generates NO and 8-Br-cGMP, a membrane-permeable analogue of cyclic GMP (cGMP), failed to prevent the inhibition of MTT reduction caused by Aβ25–35 in PC12h cells, implying that the effect of SNP may be mediated by the NO-independent pathway. Since potassium ferrocyanide showed a significant prevention at 333 μM although it had toxic effect at this concentration, it is considered that the ferrocyanide portion of the SNP metabolite may be partially involved. The cell death induced by other oxidative insults, such as glutamate and hydrogen peroxide (H 2O 2), could not be attenuated by SNP in both cell lines. Thus, the observed effect of SNP might not be due to its direct antioxidative action.

Role of nitric oxide, cyclic GMP and superoxide in inhibition by adenosine of calcium current in rabbit atrioventricular nodal cells.

To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) in atrioventricular (AV) nodal myocytes. The whole-cell patch-clamp technique was used to record ICa from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Isoprenaline, 0.1 microM, increased peak ICa from 0.58 +/- to 1.23 +/- 0.1 nA, and this increase was reversibly inhibited by adenosine, 10 microM (83 +/- 6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 microM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on ICa-Methylene blue (10 microM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (.02-), did not prevent, but increased, the inhibiting action of adenosine (49.5 +/- 6.6%, P < 0.01). Methylene blue (50 microM) caused a reduction of ICa, with further inhibition when combined with adenosine. A .O(2-)-generating system, xanthine oxidase (0.02 U/ml) and purine (2.3 mM), also increased the inhibitory action of adenosine on ICa. Inhibition of ICa by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 microM) and was not influenced by pre-incubation of cells with a NO synthase inhibitor, L-NAME (0.5 mM). The inhibitory effect of adenosine on ICa in rabbit AV nodal myocytes can be mediated by two mechanisms--stimulation of cGMP-stimulated phosphodiesterase by NO-induced cGMP, and a mechanism which involves interaction with .O2- production.

Nitric oxide depolarizes Type II paraventricular nucleus neurons in vitro

Nitric oxide is a labile gas which has been implicated in neuronal signalling. The enzyme responsible for the production of this molecule is present in the paraventricular nucleus of the hypothalamus, yet a specific role for nitric oxide in neurotransmission within this nucleus remains unclear. Using whole-cell patch-clamp recordings from paraventricular nucleus neurons in a coronal hypothalamic slice, we have assessed the acute effects of nitric oxide on membrane potential and ionic conductance. Recordings were obtained from 78 neurons with a mean resting membrane potential of −57.8±0.6 mV and a mean input resistance of 972±46 M Ω. Cells were electrophysiologically classified into Type I or Type II according to previously established criteria. Bath application of nitric oxide (delivered either as a gas dissolved in solution, or liberated from the donor compound, N-acetyl- S-nitroso- d-penicillamine) elicited reversible membrane depolarizations (>3 mV) in 14 of the 19 Type II cells tested. These cells also exhibited a decrease in input resistance following nitric oxide application. Similar effects were observed in response to bath application of l-arginine, with 11 of 14 cells displaying depolarizations and accompanying decreases in input resistance. Inhibition of nitric oxide synthase abolished the responses to l-arginine ( n=2). The nitric oxide effects persisted when voltage-activated Na + channels were blocked by tetrodotoxin ( n=6). The depolarizations observed in Type II cells were mimicked by bath application of a membrane permeable cyclic GMP analogue (8-bromo-cyclic GMP) ( n=8). Furthermore, nitric oxide depolarizations were abolished by pre-treatment of the slice with the guanylate cyclase inhibitor, LY83583 ( n=4). Type I cells did not depolarize in response to nitric oxide ( n=11). It is concluded that nitric oxide specifically depolarizes parvocellular neurons within the paraventricular nucleus via a mechanism that requires activation of guanylate cyclase and subsequent production of cyclic GMP. These findings provide the first insight into the cellular mechanisms underlying the acute effects of nitric oxide on neurons in the paraventricular nucleus.

Nitric oxide and excitatory postsynaptic currents in immature rat sympathetic preganglionic neurons in vitro

Neuronal nitric oxide synthase immunoreactivity was localized to sympathetic preganglionic neurons of the intermediolateral cell column and cyclic GMP immunoreactivity to nerve fibers projecting into the intermediolateral cell column of 20–25-day-old rats. Whole-cell patch-clamp recordings were made from sympathetic preganglionic neurons in spinal cord slices of immature rats and the role of nitric oxide and cyclic GMP on excitatory postsynaptic currents was studied. Superfusing the slices with the nitric oxide precursor l-arginine (300 μM) increased the amplitude of evoked excitatory postsynaptic currents as well as the frequency of spontaneous miniature excitatory postsynaptic currents in some neurons from minutes to over 1 h. The nitric oxide synthase inhibitor N W-nitro- l-arginine (100 μM) and the nitric oxide scavenger hemoglobin (100 μM) antagonized the potentiating effect of l-arginine. The nitric oxide donor sodium nitroprusside (100 μM) potentiated the synaptic currents in a manner similar to that of l-arginine and this effect was blocked by hemoglobin. The membrane-permeable cyclic GMP analogue dibutyryl guanosine 3′,5′-cyclic monophosphate (350 μM), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (750 μM), potentiated the evoked excitatory postsynaptic currents and increased the frequency of miniature excitatory postsynaptic currents; these effects were not prevented by hemoglobin. The results indicate that nitric oxide may facilitate the release of excitatory transmitters, possibly through a presynaptic cyclic GMP-dependent mechanism.

Nucleotide receptor-site on the labellar sugar receptor cell of the blowfly Phormia regina

Previously we reported that, among nucleotides applied extracellularly to the chemosensillum of the blowfly Phormia regina, a membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP, was particularly effective, inducing much impulse-discharge. From the results obtained, we discussed the possibility that a nucleotide site exists on the sugar receptor cell. Here we report results of our electrophysiological experiments to investigate this putative nucleotide receptor-site. As previously reported, when dibutyryl cyclic GMP was applied with sucrose, the impulse-discharge was depressed. Cyclic GMP or another membrane-permeable cyclic GMP analogue, 8-bromo cyclic GMP, enhanced the impulse frequency when applied to a chemosensillum with sucrose. However, 8-bromo cyclic GMP partly depressed the impulse-discharge evoked by dibutyryl cyclic GMP. If we hypothesize that the sugar receptor-site has a nucleotide receptor-site separated from the receptor site for sucrose (P-site) on the membrane and that the cell uses cyclic GMP as an intrinsic intracellular messenger mediating the taste transduction, then, these results can be explained as follows. Dibutyryl cyclic GMP inhibits the P-site with its butyryl groups. On the other hand, it binds the nucleotide receptor-site and stimulates the cell like other nucleotides. However, 8-bromo cyclic GMP, which does not affect the P-site, binds the nucleotide receptor-site like a competitive inhibitor. Thus, the impulses induced by 8-bromo cyclic GMP are due to the 8-bromo cyclic GMP acting in place of cyclic GMP as the intracellular messenger. As for the responses induced by dibutyryl cyclic GMP, a part which can be reduced by 8-bromo cyclic GMP is due to the dibutyryl cyclic GMP binding the nucleotide receptor-site, and other part is due to the dibutyryl cyclic GMP acting as the intracellular messenger.

Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.

Prevention of natural motoneurone cell death by dibutyryl cyclic GMP.

Natural neuronal cell death is a well-described developmental phenomenon common to many nerve centres in a variety of animal species. Neuronal survival has been shown to depend on the presence and size of the available target tissue and it has been suggested that neuronal survival is dependent on successful competition for either a limited number of synaptic sites or a limited amount of trophic factor(s). In the lateral motor column of the lumbar spinal cord in the chick embryo, the period of axon elongation and innervation of the periphery has been shown to precede that of natural motoneurone cell death. While muscle contractile activity appears to regulate the extent of motoneurone death, to date the intracellular molecular events that initiate and regulate the developmental process of natural neuronal cell death or, more importantly, neuronal survival are unknown. Our earlier studies suggested that either contact or association between spinal cord processes and muscle cells during neuromuscular junction formation in vivo leads to an increase in cyclic GMP in whole spinal cord. We now show that treatment of chick embryos with the membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP during the period of natural motoneurone cell death prevents greater than 58% of natural motoneurone cell death in the lumbar lateral motor column.