Multiple signaling pathways involved in the effect of endothelin type B receptor in rat median eminence

We assessed the possible link between endothelin receptor mediated phosphoinositide breakdown and NO/cGMP signaling pathways in rat arcuate nucleus-median eminence fragments (AN-ME), brain structures known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers, together with densely arranged endothelin ETB-receptors-like immunoreactive fibres. Our data show that ET-1, ET-3 and the ETB-receptors agonist, IRL 1620, increased inositol monophosphate (InsP1) accumulation, NOS activity and cGMP formation, in a similar degree. The stimulatory effect of ETs on InsP1 accumulation and cGMP formation was inhibited by the phospholipase C (PLC) inhibitor, neomycin, and the absence of extracellular calcium, suggesting that calcium is involved in endothelin receptor-induced PLC activation. The L-arginine analog, L-NAME, inhibited ET-1 or IRL1620-stimulated cGMP formation. The ETA receptor antagonists BQ 123, did not alter, while the ETB receptor antagonists BQ788 inhibited ETs-induced increase in the PI metabolism, NOS activity and cGMP generation. Our data indicate that in AN-ME, ETB receptor signals through receptor-mediated calcium dependent-stimulation of phosphoinositide breakdown and activation of NOS/cGMP signaling pathway.     

H(1)-Receptor activation triggers the endogenous nitric oxide signalling system in the rat submandibular gland

BACKGROUND: Histamine is released from mast cells by immunologic and non-immunologic stimuli during salivary gland inflammation, regulating salivary secretion. The receptor-secretory mechanism has not been studied in detail. AIMS: The studies reported were directed toward elucidating signal transduction/second messenger pathways within the rat submandibular gland associated with 2-thiazolylethylamine (ThEA)-induced H(1)-receptor responses. MATERIALS AND METHODS: To assess the H(1) receptor subtype expression in the rat submandibular gland, a radioligand binding assay was performed. The study also included inositolphosphates and cyclic GMP accumulation, protein kinase C and nitric oxide synthase activities, and amylase release. RESULTS: The histamine H(1) receptor subtype is expressed on the rat submandibular gland with high-affinity binding sites. The ThEA effect was associated with activation of phosphoinositide-specific phospholipase C, translocation of protein kinase C, stimulation of nitric oxide synthase activity and increased production of cyclic GMP. ThEA stimulation of nitric oxide synthase and cyclic GMP was blunted by agents able to interfere with calcium movilization, while a protein kinase C inhibitor was able to stimulate ThEA action. On the other hand, ThEA stimulation evoked amylase release via the H1 receptor but was not followed by the L-arginine/nitric oxide pathway activation. CONCLUSIONS: These results suggest that, apart from the effect of ThEA on amylase release, it also appears to be a vasoactive chemical mediator that triggers vasodilatation, modulating the course of inflammation.     

Prostaglandin E2 and dexamethasone regulate eosinophil differentiation and survival through a nitric oxide- and CD95-dependent pathway.

Apoptosis, involving both CD95/CD95L interactions and their modulation by nitric oxide (NO), is central to regulation of mature eosinophil numbers. However, its role in regulating eosinophil production from bone-marrow precursors is unknown. We examined the effects of prostaglandin E2 (PGE2) and dexamethasone on eosinophil differentiation and survival in murine bone-marrow cultures, and their relationship to: NO production as well as CD95/CD95L-dependent apoptosis. Bone-marrow cultures were established with IL-5, alone or in association with PGE2, dexamethasone or both. PGE2 (10(-7)M) inhibited eosinophil differentiation by selectively inducing apoptosis in developing eosinophils. Dexamethasone (10(-7)M) protected developing eosinophils from PGE2-induced apoptosis. Since dexamethasone prevents induction of nitric oxide synthase (NOS), we evaluated the role of NO in the effects of both PGE2 and dexamethasone. NO donors (SNAP and SNP) down-modulated eosinophil precursor responses to IL-5. SNAP induced apoptosis through a dexamethasone-resistant mechanism. The NOS inhibitors, Nomega-nitro-L-arginine and aminoguanidine, blocked the effects of PGE2 on developing eosinophils. PGE2 was ineffective in bone-marrow from knockout mice lacking inducible NOS. PGE2 up-regulated CD95 and CD95L expression in developing eosinophils. Neither PGE2 nor SNAP were effective in cultures from CD95L-deficient gld mice. These data suggest that PGE2 induces apoptosis in developing eosinophils through inducible NOS, leading to NO-dependent activation of the CD95L/CD95 pathway, while dexamethasone antagonizes the effects of PGE2 on the same targets.     

Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands

We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE2 levels and a positive (r=0.8870) and significant (p=0.0077) correlation between PGE2 production and mucin released was determined. The participation of PGE2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M1 and M4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M1 and M4 mAChR subtypes induces an increase of intracellular Ca2+ concentration ([Ca2+]I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE2 released induces cAMP accumulation which, together with PKC are involved in the PGE2 increased Ca2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production.     

Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)-1-aminocyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans -ACPD were independent of extracellular calcium blocked by N ^ω-nitro- l -arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans -ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 n M free calcium, whereas at 25–70 n M free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.     

Nitric oxide synthase and cGMP activity in the salivary glands of the American dog tick Dermacentor variabilis

We colocalized nitric oxide synthase (NOS) activity in epithelial cells that surround the salivary gland duct in female Dermacentor variabilis with NADPH diaphorase histochemistry and immunohistochemistry using a polyclonal anti-endothelial NOS. Using size-exclusion chromatography, a fraction with a molecular mass of about 185 kDa that had diaphorase activity was eluted from tick salivary gland homogenate. This fraction converted arginine to citrulline with the production of nitric oxide (NO), which was detected by using electron spin resonance spectroscopy. The complete activity of the diaphorase fraction was dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, (CaM), and Ca(2+), but was not dependent on dithiothreitol. The arginine analog N(G)-monomethyl-L-arginine inhibited the activity of this fraction. NO and arginine activated soluble guanylate cyclase to produce cGMP in dopamine-stimulated isolated salivary glands. Dopamine-stimulated isolated salivary glands treated with tick saline containing either EDTA, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, or the calcium/CaM binding inhibitor W-7 showed no increase in cGMP. The NO donor sodium nitroprusside significantly increased cGMP levels in unstimulated isolated salivary glands. A possible function for NO in salivation by this ixodid tick is discussed.     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

Dehydroepiandrosterone prevents the aggregation of platelets obtained from postmenopausal women with type 2 diabetes mellitus through the activation of the PKC/eNOS/NO pathway

The steroid hormone dehydroepiandrosterone (DHEA), suggested to be a cardioprotector, prevents platelet aggregation in healthy humans. This hormone is reduced in postmenopausal women by 60% of its normal value. Platelets in patients with type 2 diabetes (T2D) are more sensitive to aggregation, which has been attributed to a reduced ability to produce nitric oxide (NO). In light of these precedents and considering that DHEA is able to increase the production of NO in cultured endothelial cells, we suggest that DHEA prevents the aggregation of platelet from postmenopausal women with T2D through the activation of PKC/eNOS/NO/cGMP pathway. To determine the effect of DHEA in platelet aggregation, platelet-rich plasma (PRP) obtained from postmenopausal women with T2D was preincubated with DHEA, and aggregation induced by ADP was determined in the presence or absence of L-NNA (LNG-nitroarginine), Rottlerin, NOS, or PKC delta inhibitors, respectively. Platelet NO production was measured with the fluorescent probe DAF2DA and eNOS activation was determined by Western blot, using an anti-p-eNOS (ser 1177) antibody. DHEA 1) prevented platelet aggregation by 40% compared to control, 2) increased NO production by 63%, 3) increased p-eNOS (phosphorylated endothelial nitric oxide synthase) levels, and 4) increased cGMP production. These effects were reduced in the presence of L-NNA or Rottlerin. DHEA prevents platelet aggregation induced by ADP. This effect is mediated by the activation of the PKCδ/eNOS/NO/cGMP pathway. Our results suggest that DHEA could be considered to be a potential therapeutic tool in the prevention of atherothrombotic processes in postmenopausal women with T2D.     

Involvement of multiple protein kinases in cPLA2 phosphorylation, arachidonic acid release, and cell death in in vivo and in vitro models of 1-methyl-4-phenylpyridinium-induced parkinsonism – the possible key role of PKG

The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP⁺-induced cytosolic phospholipase A₂ (cPLA₂) activation of dopaminergic neurons. MPP⁺ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca²⁺]_i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA₂, and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF₃), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA₂ on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA₂ phosphorylation at Ser505. Inhibitors of cPLA₂ and PKG increased viability and reduced MPP⁺-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA₂ phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA₂.     

Peripheral neuronal nitric oxide synthase activity mediates the antinociceptive effect of Crotalus durissus terrificus snake venom, a delta- and kappa-opioid receptor agonist

Previous work has shown that nitric oxide (NO) mediates the antinociceptive effect of Crotalus durissus terrificus venom on carrageenin-induced hyperalgesia. In the present study the role of constitutive neuronal or of inducible form of nitric oxide synthase on venom effect was determined. The rat paw prostaglandin E(2) (PGE(2))-induced mechanical hyperalgesia model was used for nociceptive evaluation. The venom (200 microg/kg) administered per oz immediately before prostaglandin induced antinociception that persisted for 120 h. The characterisation of the antinociceptive effect of the venom in this model of hyperalgesia showed that kappa and delta-opioid receptors are involved in this effect. 7-nitroindazole (7-NI), a neuronal nitric oxide synthase (NOS) inhibitor, but not L-N(6)-(1-iminoethyl)lysine (L-NIL), an inhibitor of the inducible form of NOS, injected by intraplantar (i.pl.) route, antagonized the antinociceptive effect of the venom. The i.pl. administration of 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a selective guanylate cyclase inhibitor, blocked antinociception, whereas Rp-cGMP triethylamine, a cGMP-dependent protein kinase inhibitor, partially reversed this effect. These data indicate that peripheral kappa- and delta-opioid receptors are involved in the antinociceptive effect of Crotalus durissus terrificus on prostaglandin E(2)-induced hyperalgesia. Peripheral nitric oxide, generated by neuronal NO synthase, and cGMP/PKc are responsible, at least partially, for the molecular mechanisms of venom effect.     

Involvement of phosphoinositide-3-kinase and phospholipase C transduction systems in the non-genomic action of progesterone in vascular tissue

We investigate the participation of tyrosine kinase, phosphatidylinositol-3-kinase, phospholipase C systems in the intracellular transduction pathways involved in the non-genomic stimulation of vasodilators compounds synthesis induced by progesterone (Pg). Using aortic strips isolated from female fertile Wistar rats, we showed that physiological concentrations of progesterone markedly increase prostacyclin synthesis in a very short time interval (45 s to 10 min) as well as nitric oxide release (5-30 min). The stimulatory action of progesterone on nitric oxide synthase (NOS) activity was maintained even in the presence of an antagonist of progesterone receptor, compound RU486. In contrast, in the presence of tyrosine kinase inhibitor (1 microM genistein) or phosphatidylinositol-3-kinase inhibitor (1 microM LY294002), the enhancement of nitric oxide elicited by 10-100 nM progesterone was completely suppressed. The steroid stimulates phopholipase C activity, inducing significant increase in diacylglycerol generation (5-15-min treatment). The presence of an inhibitor of protein kinase C (PKC) impaired the anti-aggregatory action of the hormone. Due to the fact that phospholipase C activation implies calcium mobilization, we investigate the role of changes in calcium fluxes on progesterone nitric oxide generation. We found that calcium influx from extracellular medium and calcium mobilization from internal pools was required. The present results suggest that, tyrosine kinase and phosphatidylinositol-3-kinase cascades are involved in progesterone nitric oxide synthase stimulation and that diacilglicerol/protein kinase C system may be relevant for physiological regulation of platelet aggregation process.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.     

Novel action of estrone on vascular tissue: regulation of NOS and COX activity

The hypothesis tested in the present work is that estrone non-genomically regulates aortic nitric oxide synthase (NOS) and cyclooxygenase (COX) activities in female rats, and that such regulation depends on ovarian function. We found that physiological concentrations of estrone (E(1)) (0.1-10nM) significantly increased nitric oxide (NO) production (133 and 163% above control). The stimulatory action of E(1) on NOS activity was independent of calcium influx since the increase in NO elicited by the hormone was not affected by EGTA or verapamil. When COX activity was measured, we observed that estrone enhanced thromboxane (TXB(2)) production and prostacyclin (PGI(2)) release, but not prostaglandin (PGF(2), PGD(2), and PGE(2)) synthesis. Finally we demonstrated that the hormonal effect on NOS activity was not detected in rat aortic strips (RAS) isolated from animals deprived of ovarian activity (FR(-)) or ovariectomized rats (OVX). These results suggest that estrone exerts a direct, non-genomic action on rat aortic metabolism, which involves NOS and COX activation and depends on ovarian activity.     

Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.     

Role of endothelin type B receptor in NO/cGMP signaling pathway in rat median eminence

We studied the effect of endothelins (ETs) on receptor-mediated NO/cGMP signaling in rat arcuate nucleus–median eminence (AN-ME) fragments, an hypothalamic structure known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers together with densely arranged ET_B-receptor-like immunoreactive fibers. NOS activity was determined measuring the conversion of [³H] arginine to [³H] citrulline, as an index of NO produced. cGMP production was determined by radio immunoassay. ET-1, ET-3, and the selective ET_B receptor agonist, IRL1620, significantly increased cGMP formation and NOS activity. Preincubation of AN-ME fragment with L-arginine analog, N-nitro-L-arginine (L-NAME), inhibited ET-1 or IRL1620-stimulated cGMP formation. The addition of the selective ET_B receptor antagonist, BQ788, blocked ET-1-, ET-3-, or IRL1620-induced increase in NOS activity and cGMP generation, while BQ123, a selective ET_A receptor antagonist, was ineffective. Our results demonstrate that in whole rat AN-ME fragments, ETs stimulate NO/cGMP signaling pathway through the interaction with the ET_B receptor subtype, supporting the concept that ETs may represent an important regulator of reproductive and neuroendocrine function.     

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.     

Angiotensin type 1 receptor is linked to inhibition of nitric oxide production in pulmonary endothelial cells

We previously demonstrated that angiotensin II (Ang II) stimulates an increase in nitric oxide synthase (NOS) mRNA levels, eNOS protein expression and NO production via the type 2 (AT2) receptor, whereas signaling via the type 1 (AT1) receptor negatively regulates NO production in bovine pulmonary artery endothelial cells (BPAECs). In the present study, we investigated the components of the AT1 receptor-linked signaling pathway(s) that are involved in the downregulation of eNOS protein expression in BPAECs. Treatment of BPAECs with either AT1 receptor antagonists or an anti-AT1 receptor antibody induced eNOS protein expression. Furthermore, intracellular delivery of GP-Antagonist-2A, an inhibitor of Galphaq proteins, and treatment of BPAECs with U73122, a phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, enhanced eNOS protein expression. Treatment of BPAECs with the cell-permeable calcium chelator, BAPTA/AM, increased eNOS protein expression at 8 h, while increasing intracellular calcium with either thapsigargin or A23187 prevented Ang II-induced eNOS protein expression. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, completely prevented Ang II-stimulated eNOS protein expression at 8 h, whereas depletion of PKC by long-term treatment with PMA, induced eNOS protein expression. Treatment of BPAECs with a PKCalpha-specific inhibitor or transfection of BPAECs with an anti-PKCalpha neutralizing antibody stimulated eNOS protein expression. Conversely, rottlerin, a PKCdelta specific isoform inhibitor had no effect on basal or Ang II-stimulated eNOS protein expression. Moreover, treatment of BPAECs with U73122, BAPTA/AM and PKCalpha-specific inhibitors increased NO production at 8 h. In conclusion, Ang II downregulates eNOS protein expression via an AT1 receptor-linked pathway involving Galphaq/PLC/calcium/PKCalpha signaling pathway in BPAECs.     

Involvement of the nitric oxide-cyclic GMP pathway in the desensitization of bradykinin responses of cultured rat sensory neurons.

Bradykinin (BK) excites a subset of dorsal root ganglion neurons by inducing an inward cation current (IBK) that strongly desensitizes and is accompanied by elevations in cGMP. We have examined the links between cGMP metabolism and IBK. The BK dose dependencies of IBK activation, desensitization, and cGMP production are comparable. Stimulation (with sodium nitroprusside [NP] or 8-bromo-cGMP [8Br-cGMP]) or inhibition (with methylene blue, hemoglobin, and nitric oxide synthase [NOS] inhibitors) of cGMP levels did not mimic or diminish IBK. However, desensitization was affected by the following agents: first, desensitization was enhanced by NP and reduced by NOS inhibitors. Second, the effects of NOS inhibitors could be overcome by 8Br-cGMP or L-arginine. Third, 8Br-cGMP modification of desensitization required receptor occupancy. We conclude that the NO-cGMP pathway affects a component of IBK desensitization at the receptor or G protein level.