Involvement of phosphodiesterase-cGMP-PKG pathway in intracellular Ca2+ oscillations in pituitary GH3 cells.
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The present study investigates the potential role of the Ca2+-calmodulin-dependent type I phosphodiesterase (PDE)-cGMP-protein kinase G (PKG) pathway in spontaneous [Ca2+]i oscillations in GH3 cells using fura-2 single cell videoimaging. Vinpocetine (2.5-50 microM), a selective inhibitor of type I PDE, induced a concentration-dependent inhibition of spontaneous [Ca2+]i oscillations in these pituitary cells, and at the same time produced an increase of the intracellular cGMP content. The cell permeable cGMP analog N2,2'-O-dibutyryl-cGMP (dB-cGMP) (1 mM) caused a progressive reduction of the frequency and the amplitude of spontaneous [Ca2+]i oscillations when added to the medium. KT5823 (400 nM), a selective inhibitor of cGMP-dependent protein kinase (PKG), produced an increase of baseline [Ca2+]i and the disappearance of spontaneous [Ca2+]i oscillations. When KT5823 was added before vinpocetine, the PKG inhibitor counteracted the [Ca2+]i lowering effect of the cGMP catabolism inhibitor. Finally, the removal of extracellular Ca2+ or the blockade of L-type voltage-sensitive calcium channels (VSCC) by nimodipine produced a decrease of cytosolic cGMP levels. Collectively, the results of the present study suggest that spontaneous [Ca2+]i oscillations in GH3 cells may be regulated by the activity of type I PDE-cGMP-PKG pathway. (+info)
Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues.
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We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism. (+info)
Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation.
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Although the cGMP-dependent relaxation of contractile cells seems to depend on the ability of the cyclic nucleotide to interfere with intracellular calcium, this does not appear to be the only mechanism involved. The present experiments were designed to analyse alternative mechanisms, trying to test the hypothesis that cGMP could relax rat mesangial cells by activating myosin light-chain phosphatase (MLC-PP), with the subsequent dephosphorylation of myosin light chain (MLC). The effect of a cGMP analogue, dibutyryl cGMP (dbcGMP), on angiotensin II-(AII) and PMA-induced MLC phosphorylation (MLCP) was tested, in the presence of calyculin A (CA), an inhibitor of MLC-PP. MLCP was measured, after cell labelling with (32)P, by immunoprecipitation. dbcGMP prevented the increased MLCP induced by AII or PMA, and this inhibition was blocked by CA. dbcGMP also increased the MLC dephosphorylation observed in cells incubated with AII and in which MLC kinase and protein kinase C activities were blocked. The AII-elicited increased intracellular calcium concentration was only partially inhibited by dbcGMP. These results suggest that the cGMP-induced mesangial-cell relaxation could be due, at least partially, to the stimulation of MLC-PP. (+info)
Membrane hyperpolarization removes inactivation of Ca2+ channels, leading to Ca2+ influx and subsequent initiation of sperm motility in the common carp.
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Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca(2+)-free hypoosmotic solutions, and significant increase in the intracellular Ca(2+) level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10(-7) to 10(-5) M Ca(2+). Ca(2+) channel blockers such as verapamil and omega-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca(2+) influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca(2+) channels, leading to Ca(2+) influx at the initiation of carp sperm motility. (+info)
Activation of the particulate and not the soluble guanylate cyclase leads to the inhibition of Ca2+ extrusion through localized elevation of cGMP.
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We examined whether localized increases in cytosolic cGMP have distinct regulatory effects on the concentration of cytosolic free Ca(2+) in ECV304 cells. Stimulation of the particulate guanylate cyclase by brain-type natriuretic peptide in fura-2-loaded cells caused a profound potentiation of the ATP-stimulated and thapsigargin-stimulated rise in cytosolic free Ca(2+). This effect is mediated by the inhibition of Ca(2+) extrusion via the plasma membrane Ca(2+)-ATPase pump. Furthermore, the addition of brain-type natriuretic peptide caused the partial inhibition of cation influx in ATP-stimulated cells. In contrast, elevation of cytosolic cGMP by activation of the soluble guanylate cyclase induced by the addition of sodium nitroprusside causes an increased reuptake of Ca(2+) into the intracellular stores without affecting cation influx or Ca(2+) efflux. Thus, localized pools of cGMP play distinct regulatory roles in the regulation of Ca(2+) homeostasis within individual cells. We define a new role for natriuretic peptides in the inhibition of Ca(2+) efflux that leads to the potentiation of agonist-evoked increases in cytosolic free Ca(2+). (+info)
We have previously shown that cyclooxygenase-2 (COX-2) is localized to the cortical thick ascending limb of the loop of Henle (cTALH)/macula densa of the rat kidney, and expression increases in response to low-salt diet and/or angiotensin-converting enzyme (ACE) inhibition. Because of the localization of neuronal nitric oxide synthase (nNOS) to macula densa and surrounding cTALH, the present study investigated the role of nitric oxide (NO) in the regulation of COX-2 expression. For in vivo studies, rats were fed a normal diet, low-salt diet or low-salt diet combined with the ACE inhibitor captopril. In each group, one-half of them were treated with the nNOS inhibitors 7-nitroinidazole (7-NI) or S-methyl-thiocitrulline. Both of these NOS inhibitors inhibited increases in COX-2 mRNA and immunoreactive protein in response to low salt and low salt+captopril. For in vitro studies, COX-2 expression was studied in primary cultures of rabbit cTALH cells immunodisssected with Tamm-Horsfall antibody. Basal COX-2 immunoreactivity expression was stimulated by S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, and intracellular cGMP concentration. The cultured cells expressed immunoreactive nNOS, and 7-NI inhibited basal COX-2 immunoreactivity expression, which could be partially overcome by cGMP. In summary, these studies indicate that NO is a mediator of increased renal cortical COX-2 expression seen in volume depletion and suggest important interactions between the NO and COX-2 systems in the regulation of arteriolar tone and the renin-angiotensin system by the macula densa. (+info)
Guanosine 3',5'-cyclic monophosphate reduces the response of the Moth's olfactory receptor neuron to pheromone.
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The effects of the membrane-permeable dibutyryl guanosine 3', 5'-cyclic monophosphate (db-cGMP) on the bombykol-elicited receptor current and nerve impulse activity were studied using the open sensillum recording technique. db-cGMP was applied to the outer dendritic membrane of the olfactory receptor neuron of the moth Bombyx mori. db-cGMP reduced the amplitude of the overall receptor current activated by a pulse of strong pheromone stimuli as well as diminished the nerve impulse frequency elicited by continuously applied weak pheromone stimuli. The observed inhibition of the response to pheromone was due to size reduction of an elementary receptor current that elicits the nerve impulses and underlies the overall receptor current. It is suggested that cGMP is a factor which may adjust cell sensitivity to odour and play a role in olfactory adaptation. (+info)
Nitric oxide modulation of interleukin-1[beta]-evoked intracellular Ca2+ release in human astrocytoma U-373 MG cells and brain striatal slices.
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Intracellular Ca(2+) mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1beta (IL-1beta) and other pyrogens. The source and mechanism of IL-1beta-induced intracellular Ca(2+) mobilization was investigated using two experimental models. IL-1beta (10 ng/ml) treatment of rat striatal slices preloaded with (45)Ca(2+) elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous (45)Ca(2+) release that was potentiated by l-arginine (300 microm) and counteracted by N-omega-nitro-l-arginine methyl ester (l-NAME) (1 and 3 mm). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mm) and spermine/NO (0.1 and 0.3 mm) mimicked the effect of IL-1beta on Ca(2+) release. IL-1beta stimulated tissue cGMP concentration, and dibutyryl cGMP enhanced Ca(2+) release. The guanyl cyclase inhibitors 1H-[1,2, 4]oxadiazole[4,3-a] quinoxalin-1-one (100 microm) and 6-[phenylamino]-5,8 quinolinedione (50 microm) counteracted Ca(2+) release induced by 2.5 but not 10 ng/ml IL-1beta. Ruthenium red (50 microm) and, to a lesser extent, heparin (3 mg/ml) antagonized IL-1beta-induced Ca(2+) release, and both compounds administered together completely abolished this response. Similar results were obtained in human astrocytoma cells in which IL-1beta elicited a delayed (30 min) increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (402 +/- 71.2% of baseline), which was abolished by 1 mm l-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1beta-evoked Ca(2+) mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca(2+) stores are involved. (+info)