Elevated cGMP Levels Research Articles

Differential Amplification of Intron-containing Transcripts Reveals Long Term Potentiation-associated Up-regulation of Specific Pde10A Phosphodiesterase Splice Variants

We employed differential display of expressed mRNAs (Liang, P., and Pardee, A. B. (1992) Science 257, 967-971) to identify genes up-regulated after long term potentiation (LTP) induction in the hippocampus of awake adult rats. In situ hybridization confirmed the differential expression of five independently amplified clones representing two distinct transcripts, cl13/19/90 and cl95/96. Neither cl13/19/90 nor cl95/96 showed significant sequence homology to known transcripts (mRNA or expressed sequence tag) or to the mouse or human genome. However, comparison with the rat genome revealed that they are localized to a predicted intron of the phosphodiesterase Pde10A gene. cl13/19/90 and cl95/96 are likely to be part of the Pde10A primary transcript as, using reverse transcriptase-PCR, we could specifically amplify distinct introns of the Pde10A primary transcript, and in situ hybridization demonstrated that a subset of Pde10A splice variants are also up-regulated after LTP induction. These results indicate that amplification of a primary transcript can faithfully report gene activity and that differential display can be used to identify differential expression of RNA species other than mRNA. In transiently transfected Cos7 cells, Pde10A3 reduces the atrial natriuretic peptide-induced elevation in cGMP levels without affecting basal cGMP levels. This cellular function of LTP-associated Pde10A transcripts argues for a role of the cGMP/cGMP-dependent kinase pathway in long term synaptic plasticity.

Burn-induced lung damage in rat is mediated by a nitric oxide/cGMP system.

This study was conducted to demonstrate the burn-induced lung neutrophil deposition and damage in rats is affected by the nitric oxide (NO)-dependent downstream cGMP signaling. In experiment 1, 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ) was given (20 mg/kg i.p.) to specific pathogen-free Sprague-Dawley rats immediately postburn to suppress the guanylate cyclase (GC) activity. At 8 h after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation and lung tissues were harvested for myeloperoxidase (MPO) determination and histological studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. In experiment 2, Sodium nitroprusside (SNP) was given (2 mM, i.p.) to elevate cGMP levels and ODQ (20 mg/kg, i.p.) or methylene blue (100 microM, i.p.) or saline was given. The animals were sacrificed 4 h after injection and lung tissues were harvested for iNOS mRNA study. The MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased up to 2-fold, 4-fold, and 2.5-fold after burn. Inhibition of GC by ODQ administration significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 55%, 66%, and 53%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. The protective effects of ODQ were comparable to the use of selective iNOS inhibitor as demonstrated previously. Furthermore, ODQ decreased the burn or SNP-induced iNOS mRNA levels at 4 h after burn. These findings suggest that burn-induced lung dysfunction is mediated by the NO/cGMP system because it is abolished by application of either iNOS inhibitor or GC inhibitor. Also, the beneficial effect of ODQ is partly due to the attenuation of burn-induced iNOS expression by GC inhibition.

Kallikrein gene delivery attenuates cardiac remodeling and promotes neovascularization in spontaneously hypertensive rats.

Hypertension that results in left ventricular (LV) hypertrophy and/or fibrosis can lead to cardiac dysfunction. Spontaneously hypertensive rats (SHR) develop high blood pressure and LV hypertrophy at an early age and are a popular model of human essential hypertension. To investigate the role of the tissue kallikrein-kinin system in cardiac remodeling, an adenovirus containing the human tissue kallikrein gene was injected intravenously into adult SHR and normotensive Wistar-Kyoto (WKY) rats. The blood pressure of WKY rats remained unchanged throughout the experiment. Alternatively, kallikrein gene transfer reduced blood pressure in SHR for the first 2 wk, but had no effect from 3 to 5 wk. Five weeks after kallikrein gene delivery, SHR showed significant reductions in LV-to-heart weight ratio, LV long axis, and cardiomyocyte size; however, these parameters were unaffected in WKY rats. Interestingly, cardiac collagen density was decreased in both SHR and WKY rats receiving the kallikrein gene. Kallikrein gene transfer also increased cardiac capillary density in SHR, but not in WKY rats. The morphological changes after kallikrein gene transfer were associated with decreases in JNK activation as well as transforming growth factor (TGF)-beta 1 and plasminogen activator inhibitor-1 levels in the heart. In addition, kallikrein gene delivery elevated LV nitric oxide and cGMP levels in both rat strains. These results indicate that kallikrein-kinin attenuates cardiac hypertrophy and fibrosis and enhances capillary growth in SHR through the suppression of JNK, TGF-beta 1, and plasminogen activator inhibitor-1 via the nitric oxide-cGMP pathway.

Adrenomedullin induces direct (endothelium-independent) vasorelaxations and cyclic adenosine monophosphate elevations that are synergistically enhanced by brain natriuretic peptide in isolated rings of rat thoracic aorta.

Our laboratory previously demonstrated that nitric oxide and natriuretic peptides can synergistically enhance cAMP elevations and vasorelaxations in rat aortic rings induced by calcitonin gene-related peptide, likely involving cyclic guanosine monophosphate (cGMP)-mediated inhibition of type-3 phosphodiesterase (PDE3). It was predicted that this cellular mechanism may also serve as a point of synergism between adrenomedullin (ADM) and brain natriuretic peptide (BNP) in aortic smooth muscle cells. The current study shows that ADM (100 nM)-induced vasorelaxations in isolated aortic rings of Sprague-Dawley rats are dependent on endothelium (34.1 +/- 4.2% relaxation with endothelium versus 3.0 +/- 0.6% relaxation without endothelium; P < 0.001). To determine interactions between ADM and BNP in smooth muscle cells without interference from endothelium-derived factors, further studies used aortic rings denuded of endothelium. Pretreatment with BNP (1 nM), which elevated cGMP levels 1.6 fold, uncovered direct vasorelaxant effects of ADM in endothelium-denuded rings, showing 5.6 +/- 1.8%, 20.9 +/- 6.1%, and 55 +/- 9.4% relaxations with ADM at 1, 10, and 100 nM, respectively (n = 6). ADM (100 nM) significantly (P < 0.05) increased cyclic adenosine monophosphate (cAMP) levels in denuded aortic rings pretreated with BNP (1 nM), but not in denuded rings without BNP. Quazinone (20 microM), a PDE3 inhibitor, caused similar enhancement of direct cAMP elevations to ADM (100 nM). The data indicate vasodilatory synergism between ADM and BNP in aorta, likely mediated by enhanced accumulation of cAMP in smooth muscle cells resulting from BNP/cGMP-induced inhibition of PDE3. This synergistic mechanism may be especially important in subjects with dysfunctional endothelium, in which BNP may uncover direct vasorelaxant effects of ADM in arteries that normally require healthy (nitric oxide-releasing) endothelium for ADM-induced vasorelaxations to occur.

Actin Binding of Human LIM and SH3 Protein Is Regulated by cGMP- and cAMP-dependent Protein Kinase Phosphorylation on Serine 146

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.

Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes

Objectives: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. Background: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. Methods: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3′,5′-monophosphate (cGMP) levels was also measured. Results: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 μM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of d-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. Conclusions: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets. Condensed Abstract The thrombotic process involves platelets and polymorphonuclear leukocytes (PMNs). PMNs release both reactive oxygen species (ROS) and nitric oxide (NO). The present study was conducted to investigate the physiological significance of the ascorbate in circulation, by using rat, human and monkey platelets and PMNs. Ascorbate significantly potentiated the PMNs-mediated inhibition of aggregation. Ascorbate-mediated effects were mediated by dehydroascorbate and subsequently releases NO. NO, then activates the guanylyl cyclase in the platelets and elevates the platelet cGMP levels. It seems likely that ascorbic acid in PMNs play a significant role in vascular homeostasis by elevating levels of NO and subsequently maintaining the platelet in an inactivated state, preventing the initiation of thrombotic process.

Atrial Natriuretic Peptides Elevate Cyclic GMP Levels in Primary Cultures of Rat Ependymal Cells

The aim of this study was to examine the effect of atrial natriuretic peptides on primary cultures of ependymal cells, as measured by changes in intracellular levels of cyclic GMP. Incubation of ependymal cells with rat atrial natriuretic peptide-(1-28) (rANP) elicited a 30-fold increase in ependymal cGMP content within 1 min and more than a 100-fold increase within 10 min to a plateau value of approximately 30 pmol/mg protein. The C-type natriuretic peptide (CNP) elicited a similar increase in cGMP levels; however the maximal effect was observed within 1 min and the levels subsequently dropped by 90% to a low plateau within 10 min. A comparison of the concentration-response curves for rANP, human ANP-(1-28) (hANP) and CNP showed that rANP, hANP and CNP had similar effects, with regards to elevation of cGMP levels at high concentrations, but with differing EC50 values. These results demonstrate the presence of a heterogenous population of functional ANP receptors i n cultured ependymalcells suggesting that ANP may regulate specific ependymal cell activity.

BAY 41-2272: a stimulator of soluble guanylyl cyclase induces nitric oxide-dependent penile erection in vivo

Objectives To determine the effectiveness of BAY 41-2272 on penile erections in an in vivo rabbit model. The nitric oxide (NO)-dependent increase of intracellular cyclic guanosine monophosphate (cGMP) by cGMP-phosphodiesterase (PDE5) inhibition has been shown to be an effective mechanism in the treatment of erectile dysfunction. Direct, NO-independent stimulation of soluble guanylyl cyclase should also lead to elevated cGMP levels in tissues and could be an attractive alternative therapeutic option for the treatment of erectile dysfunction. BAY 41-2272 is a novel non-NO-based direct stimulator of soluble guanylyl cyclase that activates purified enzyme in a synergistic fashion with NO. Methods BAY 41-2272 was administered to conscious rabbits intravenously (IV) and orally (PO). Erection was assessed in a time-dependent manner by measuring the length of the uncovered penile mucosa. Erections were evaluated in the absence and presence of NO (with intravenous sodium nitroprusside [SNP] as the NO donor). Results BAY 41-2272 only induced weak penile erections in conscious rabbits after IV (1 mg/kg) and PO (10 mg/kg) administration in the absence of an NO donor. However, the efficacy of BAY 41-2272 was potentiated by the simultaneous administration of SNP. Through simultaneous SNP administration, the effective doses of BAY 41-2272 were reduced significantly (minimal effective dose 0.1 mg/kg IV and 1 mg/kg PO). Conclusions The results of this study clearly demonstrated the effect of BAY 41-2272 on penile erection in the conscious rabbit model after PO and IV administration. The time-course and onset of erection was concurrent with the stimulation by exogenous NO (SNP), suggesting that this new pharmacologic mechanism of soluble guanylyl cyclase stimulation could be used in the treatment of erectile dysfunction. Because the effect is increased by SNP, it can be expected that BAY 41-2272 would have enhanced activity during sexual arousal, when NO is produced endogenously.

Glucose-6-phosphate dehydrogenase overexpression decreases endothelial cell oxidant stress and increases bioavailable nitric oxide.

Glucose-6-phosphate dehydrogenase (G6PD), the principal source of NADPH, serves as an antioxidant enzyme to modulate the redox milieu and nitric oxide synthase activity. Deficient G6PD activity is associated with increased endothelial cell oxidant stress and diminished bioavailable nitric oxide (NO.). Therefore, we examined whether overexpression of G6PD would decrease reactive oxygen species accumulation and increase bioavailable NO. in endothelial cells. Adenoviral-mediated gene transfer of G6PD increased G6PD expression, activity, and NADPH levels in bovine aortic endothelial cells (BAECs). BAECs overexpressing G6PD demonstrated a significant reduction in reactive oxygen species accumulation when exposed to hydrogen peroxide, xanthine-xanthine oxidase, or tumor necrosis factor-alpha compared with BAECs with basal levels of G6PD. BAECs overexpressing G6PD maintained intracellular glutathione stores when exposed to oxidants because of increased activity of glutathione reductase, an effect that was not observed in endothelial cells with normal G6PD activity. Overexpression of G6PD was also associated with enhanced nitric oxide synthase activity, resulting in elevated levels of cGMP, nitrate, and nitrite, and this response was increased after stimulation with bradykinin. Overexpression of G6PD in vascular endothelial cells decreases reactive oxygen species accumulation in response to exogenous and endogenous oxidant stress and improves levels of bioavailable NO.

The NO - K+ channel axis in pulmonary arterial hypertension. Activation by experimental oral therapies.

The prognosis of patients with pulmonary arterial hypertension (PAH) is poor. Available therapies (Ca(++)-channel blockers, epoprostenol, bosentan) have limited efficacy or are expensive and associated with significant complications. PAH is characterized by vasoconstriction, thrombosis in-situ and vascular remodeling. Endothelial-derived nitric oxide (NO) activity is decreased, promoting vasoconstriction and thrombosis. Voltage-gated K+ channels (Kv) are downregulated, causing depolarization, Ca(++)-overload and PA smooth muscle cell (PASMC) contraction and proliferation. Augmenting the NO and Kv pathways should cause pulmonary vasodilatation and regression of PA remodeling. Several inexpensive oral treatments may be able to enhance the NO axis and/or K+ channel expression/function and selectively decrease pulmonary vascular resistance (PVR). Oral L-Arginine, NOS' substrate, improves NO synthesis and functional capacity in humans with PAH. Most of NO's effects are mediated by cyclic guanosine-monophosphate (c-GMP). cGMP causes vasodilatation by activating K+ channels and lowering cytosolic Ca++. Sildenafil elevates c-GMP levels by inhibiting type-5 phosphodiesterase, thereby opening BK(Ca). channels and relaxing PAs. In PAH, sildenafil (50 mg-po) is as effective and selective a pulmonary vasodilator as inhaled NO. These benefits persist after months of therapy leading to improved functional capacity. 3) Oral Dichloroacetate (DCA), a metabolic modulator, increases expression/function of Kv2.1 channels and decreases remodeling and PVR in rats with chronic-hypoxic pulmonary hypertension, partially via a tyrosine-kinase-dependent mechanism. These drugs appear safe in humans and may be useful PAH therapies, alone or in combination.

Brain Natriuretic Peptide (BNP)‐Induced Cyclic GMP Elevations Synergistically Enhance the Vasodilatory and Cyclic AMP Responses to Adrenomedullin in Rat Aortic Rings

Introduction: BNP, a hypotensive and natriuretic factor derived from cardiac muscle, acts similar to the cardiac hormone atrial natriuretic peptide (ANP), causing endothelium-independent vasorelaxations in rat aortic rings by activating particulate guanylyl cyclase (pGC), elevating cyclic GMP (cGMP) levels and activating protein kinase G (PKG) in vascular smooth muscle cells (VSMCs) [1,2]. Recently, we have shown that BNP [3], like nitric oxide (NO) [4,5], synergistically potentiates the vasorelaxant and cAMP-elevating actions of calcitonin gene-related peptide (CGRP), an important neuropeptide mediating inflammatory vasodilations. Both BNP and NO, via cGMP elevations, inhibit the type-3 phosphodiesterase (PDE3) in VSMC, thus enhancing cAMP accumulation and potentiating the vasorelaxations induced by CGRP [3-5]. We hypothesized that BNP may also synergistically enhance cAMP elevations and vasorelaxations induced by another important peptide, adrenomedullin (ADM), which has vascular properties similar to that of CGRP. Methods R p < 0.001). To uncover interactions between ADM and BNP in VSMCs without the interference from endothelium-derived agents, aortic rings were denuded of endothelium. After precontraction with phenylephrine (100 nM), BNP (1 nM) was added to the endothelium-denuded aortic rings, resulting in vasorelaxations of 20.1 ± 3.7%. This was followed by addition of log incremental concentrations of ADM (1, 10 & 100 nM), resulting in further vasorelaxations of 5.6 ± 1.8%, 20.9 ± 6.1% and 55 ± 9.4%, respectively (n = 6). ADM (100 nM) significantly (p < 0.05) elevated cAMP levels in endothelium-denuded rings pretreated with BNP (1 nM), but had no effect in rings without BNP. By itself, BNP significantly (p < 0.05) elevated cGMP levels by 1.6 folds and had no affect on cAMP levels. Conclusion: BNP synergistically enhances ADM-induced elevations of intracellular cAMP levels in VSMCs, significantly potentiating the endothelium-independent vasorelaxant effects of ADM. This synergistic interaction between BNP and ADM may be especially relevant to certain cardiovascular pathologies, such as septic shock and heart failure, in which both ADM and BNP levels are known to be elevated. 1 Fiscus, Rapoport, Waldman and Murad. Biochim. Biophys. Acta 846:179-184, 1985. 2 Zhou and Fiscus. Neuropeptides 14:161-169, 1989. 3 Fiscus, Lu, Tu, Hao, Yang and Wang. Neuropeptides 32:499-509, 1998. 4 Fiscus, Hao, Wang, Arden and Diana. Neuropeptides 26:133-144, 1994. 5 Lu and Fiscus, Eur. J. Pharmacol. 376:307-314, 1999. (Supported by a Direct Grant for Research)

Blocking Cyclic GMP Synthesis Enhances the Pro‐Apoptotic Actions of Nitric Oxide (NO) in the NG108‐15 Cholinergic Neuronal Cell Line

Introduction: Little is known about the regulation of apoptosis in sympathetic and parasympathetic nerves that innervate the cardiovascular system. Our previous studies have shown that PC12 cells, a cell culture model of sympathetic nerves, respond to NO or atrial natriuretic peptide (ANP) with increases in production of cyclic GMP (cGMP), resulting in increased levels of cGMP in both the intracellular and extracellular spaces (Fiscus, Robles, Waldman and Murad, J. Neurochem. 48:522-528, 1987). These cGMP elevations resulted from NO- and ANP-induced activation of soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC), respectively. However, at the time, the biological function of this signaling pathway in neural cells was unknown. In collaboration with Mark Mattson's laboratory at the University of Kentucky, we found that cGMP elevations in hippocampal neurons increased their survival, specifically blocking the cell death induced by stress, such as glutamate toxicity (Barger, Fiscus, Ruth, Hofmann and Mattson, J. Neurochem. 64:2087-2096, 1995). Recently, we showed that cGMP inhibits the onset of apoptosis and prolongs the survival of stressed PC12 cells (Fiscus, Tu and Cheng Chew, NeuroReport (Neurochemistry) 12:185-189, 2001). Specifically, elevating cGMP levels with either ANP or a related peptide, brain natriuretic peptide (BNP), inhibited the apoptotic DNA fragmentation induced by serum deprivation in the PC12 cells. Objectives: We determined if the elevation of cGMP levels induced by an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), has protective (anti-apoptotic) effects in the NG108-15 cholinergic neuronal cell line. Methods: NG108-15 cells were plated at 5 x 106 cells/dish in 100mm culture dishes. After 24 hr, the cells were exposed for 24 hr to SNAP (1-1000 μM), with or without co-incubation with ODQ (40 μM), a selective sGC inhibitor. DNA was extracted from the cells and apoptotic DNA fragmentation (DNA laddering) was analyzed on 2% agarose gels. Results: SNAP, at 500 and 1000 μM, caused apoptotic DNA fragmentation. Inhibition of sGC with ODQ (40 μM) completely blocked the cGMP elevations and exaggerated the pro-apoptotic action of SNAP. Interestingly, ODQ (40 μM), by itself, caused noticeable apoptotic DNA fragmentation, suggesting that even basal levels of cGMP (i.e. in the absence of added NO) may be important for protecting neural cells against onset of apoptosis. Similar results were obtained in 4 other experiments with NG108-15 cells. Conclusions: These data suggest that low basal levels of cGMP (0.1–1 pmol/106 cells) may protect cells against apoptosis and that the elevated levels of cGMP induced by NO may serve as an important counter-balance to the toxic/pro-apoptotic actions of NO. (Supported by a Direct Grant for Research)

Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.

Inhibition of platelet P2Y12 and α2A receptor signaling by cGMP-dependent protein kinase

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G i-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G i-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G i-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G i-signaling for platelet activation, we propose that inhibition of G i-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

35] Determination of angiogenesis-regulating properties of NO

Publisher Summary Most used in vivo bioassay in laboratory is the rabbit cornea assay. Because the cornea is an avascular tissue, this assay avoids the problems of interpretation inherent in the other bioassays, such as the chick chorion allantoic membrane (CAM). The outgrowth of new vessels into the cornea stroma can be readily identified and their morphology documented. The corneal assay is performed in New Zealand white rabbits. Nitric oxide (NO) directs endothelial cells in each step of angiogenesis and its role is twofold. In vitro administration of NO donor drugs to sparse coronary endothelium increases their mitotic index and favors cell migration and matrix degradation. As a result, in vivo NO donors speed up and potentiate neovascular growth. Thus, NO acts as the true effector of angiogenesis. However, mediators of angiogenesis signal the angiogenic switch in endothelium by releasing free NO and elevating cGMP levels. The development of NO-targeting drugs with higher selectivity for the different isoforms, combined with targeted tissue delivery to minimize side effects, as well as qualitative assessment of NO function from activity, to genomic expression, is a promising strategy for the exploitation of the findings on the role of NO on angiogenesis.

Nocturnal accumulation of cyclic 3',5'-guanosine monophosphate (cGMP) in the chick pineal organ is dependent on activation of guanylyl cyclase-B.

The role of cGMP in the avian pineal is not well understood. Although the light-sensitive secretion of melatonin is a well-known output of the circadian oscillator, pharmacologically elevated cGMP levels do not result in altered melatonin secretory amplitude or phase. This suggests that pineal cGMP signalling does not couple the endogenous circadian oscillator to the expression of melatonin rhythms. Nonetheless, the free-running rhythm of cGMP signalling implies a link to the circadian oscillator in chick pinealocytes. As the circadian rhythm of cGMP levels in vitro is not altered by pharmacological inhibition of phosphodiesterase activity, we infer that the synthesis, rather than the degradation of cGMP, is under circadian control. In vitro experiments with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine as well as with an inhibitor of the NO-sensitive soluble guanylyl cyclase (sGC), showed that the NOS-sGC pathway does not play a major role in the circadian control of cGMP generation. In organ culture experiments, we demonstrated that C-type natriuretic peptide (CNP), but not atrial natriuretic peptide (ANP), elevated daytime levels of cGMP. As CNP acts on the membrane guanylyl cyclase isoform B (GC-B), which is expressed at very high levels in mammalian pineals, we investigated the effect of the membrane GC-specific inhibitor HS-142 on chick pineal cGMP levels. CNP-induced daytime cGMP levels were reduced by HS-142. More importantly, 'spontaneously' high nocturnal levels of cGMP in vitro were reduced to daytime levels by acute addition of HS-142. These data implicate endogenous nocturnal CNP release and subsequent activation of GC-B as the major input driving cGMP synthesis in the chick pineal organ.

Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle.

The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.

Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle

The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.

Inhibitory effects of melatonin on vascular reactivity: possible role of vasoactive mediators

Melatonin (MEL), the principal hormone of the vertebral pineal gland, elicits several neurobiological effects. However, the effects of MEL on vascular tissues are still vague. The first goal of this study was to investigate the effect of MEL on isolated rabbit aortic rings and its role in the vascular reactivity to contractile agents, noradrenaline (NA) and phenylephrine (PHE) and relaxant agents (acetylcholine and sodium nitroprusside). In addition, the levels of nitric oxide (NO), cGMP, total calcium, lipid peroxides, superoxide dismutase (SOD) and glutathione (GSH) were also investigated in tissue homogenates of rabbit aortic rings preincubated (20 min) in MEL with and without contractile agents. Our results revealed that MEL has an endothelium-dependent vaso-relaxant effect and potentiated significantly the vaso-relaxant effect of acetylcholine. Moreover, MEL (10 −4 M) had a significant inhibitory effect on the contractile responses of aortic rings to both NA and PHE. In comparison with control tissue rings, the levels of lipid peroxides were significantly increased while the levels of GSH, and SOD activities were significantly decreased in tissue homogenates of aortic rings pre-incubated (20 min) in NA or PHE. In addition, the levels of NO and cGMP were significantly lower in tissue rings pre-treated with NA and PHE, respectively. Also, the levels of total calcium were significantly increased only in tissue rings pre-treated with NA. The levels of lipid peroxides were significantly decreased, while the levels of GSH, NO and cGMP and SOD activities were significantly increased in tissue homogenates of aortic rings incubated (20 min) in MEL (10 −4 M) in comparison to ring tissues incubated in NA or PHE alone. In aortic rings incubated in MEL+PHE, the levels of lipid peroxides were significantly lower while the levels of GSH and cGMP and SOD activities were significantly higher than their levels in ring tissues incubated in PHE. In aortic rings incubated in MEL+NA, the levels of lipid peroxides and total calcium were significantly lower while the levels of NO were significantly higher than their levels in ring tissues incubated in NA alone. We conclude that MEL has an endothelium dependent vasorelaxant effect and potentiates the endothelium dependent vasorelaxation induced by acetylcholine. MEL inhibits the contractile responses of aortic rings to NA and PHE. These effects may be, in part, due to re-balancing the pro-oxidant/antioxidants system, lowered calcium content and elevated NO and cGMP levels in vascular tissue.