Increase In Cyclic GMP Levels Research Articles

Influence of the Endothelium on Vascular Responses of Aortae from Endotoxic Rats

Intraperitoneal injection of endotoxin diminished the in-vitro contractile response of rat aorta to phenylephrine or clonidine, whether the intimal layer was disrupted or not. The relaxing responses to acetylcholine and sodium nitroprusside in aorta precontracted with 10(-6) M phenylephrine were similar between control and endotoxic groups. However, when the precontractile force following phenylephrine was adjusted to an equivalent level, the relaxing responses to acetylcholine and sodium nitroprusside were diminished in the endotoxic aorta compared with the controls. There was no significant difference between the two groups in the increase in cyclic GMP levels induced by acetylcholine or by sodium nitroprusside. These results suggest that aortae from endotoxic rats show decreased responsiveness to alpha-adrenoceptor stimulation not because of enhancement of the endothelium-derived relaxing factor but because of abnormality in the vascular smooth muscle which is not specific for subtypes of the alpha-adrenoceptor.

Raloxifene protects endothelial cell function against oxidative stress

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress. Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis. In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or LY294002. Raloxifene also protected endothelial cell function against H2O2. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase. Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.

Nitric oxide regulates the proliferation of chick embryo retina cells by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l-arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [3H]-thymidine incorporation after drug treatment. Incubation for 24h with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [3H]-thymidine incorporation in a dose-dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8-bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), indicating that the effect was not due to cell death or mediated by increases of cyclic GMP levels. The inhibition was completely prevented by dithiotreitol (DTT), strongly indicating the participation of an S-nitrosylation mechanism. SNAP blocked the increase of [3H]-thymidine incorporation induced by ATP. Using purified cultures of glial cells we showed that the NO donor SNAP produced an inhibition of 50% in cell proliferation and did stimulate ERK1/2 phosphorylation, indicating that the inhibition of this pathway was not involved in its cytostatic effect. [3H]-Thymidine autoradiography of mixed cultures showed labeling of oval nuclei of glial flat cells. The injection of eggs with SNAP also did promote an intense inhibition of [3H]-thymidine incorporation in retinas from 9-day-old embryos. These data suggest that nitric oxide affects the proliferation of chick embryo retina glial cells in culture or “in vivo” through cyclic GMP and ERK-independent pathways.

Antiplatelet effects of KW-7, a new inhibitor of cyclic nucleotide phosphodiesterases

The antiplatelet effect of a new synthetic compound, 8,9-dimethoxyl-1-(4-methoxy-phenyl)-5,6-dihydro-pyrrolo[2,1- a]isoquinoline-2,3-dione (KW-7), was determined in rabbit platelets. KW-7 concentration-dependently prevented platelet aggregation caused by arachidonic acid, collagen, platelet-activating factor, and thrombin. KW-7 induced a substantial increase in cyclic AMP levels and a smaller increase in cyclic GMP levels in platelets. In platelet homogenates, KW-7 inhibited both cyclic AMP- and cyclic GMP-phosphodiesterase activities. The antiplatelet effect of KW-7 was reversed by SQ22536 (an inhibitor of adenylate cyclase) and H89 (an inhibitor of protein kinase A) but not by ODQ (an inhibitor of soluble guanylate cyclase). These data suggest that the antiplatelet effect of KW-7 is cyclic AMP-dependent, and is through inhibition of platelet phosphodiesterases. In addition, KW-7 inhibited arachidonic acid-stimulated thromboxane production; this effect was associated with an increase in prostaglandin D 2 levels indicating KW-7 is also an inhibitor of thromboxane synthase. The dual inhibition of KW-7 on phosphodiesterase and thromboxane synthase might provide an attractive target in developing antiplatelet drugs.

Inhibition of nitric oxide/cyclic GMP-mediated relaxation by purified flavonoids, baicalin and baicalein, in rat aortic rings

The dried roots of Scutellaria baicalensis Georgi (Huangqin) are widely used in traditional Chinese medicine. We purified two flavonoids, baicalin and baicalein from S. baicalensis Georgi and examined their effects on isolated rat aortic rings. Baicalin (3–50 μM) inhibited endothelium/nitric oxide (NO)-dependent relaxation induced by acetylcholine (Ach) or cyclopiazonic acid (CPA). Baicalein at 50 μM abolished Ach-induced relaxation and markedly reduced CPA-induced relaxation. Treatment with 1 mM l-arginine partially but significantly reversed the effects of baicalin (50 μM) or baicalein (50 μM) on Ach-induced relaxation. In endothelium-denuded rings, treatment with baicalin, baicalein or methylene blue partially inhibited relaxations induced by the NO donors, sodium nitroprusside (SNP) and hydroxylamine. Both flavonoids markedly reduced the increase in cyclic GMP levels stimulated by Ach in endothelium-intact rings and by SNP in endothelium-denuded rings. In contrast, exposure of endothelium-denuded rings to baicalin or baicalein did not affect relaxations induced by pinacidil or NS 1619, putative K + channel activators. Neither flavonoids affected agonist-induced increase in the endothelial [Ca 2+] i. Our results indicate that baicalin and baicalein attenuated NO-mediated aortic relaxation and cyclic GMP increases, likely through inhibition of NO-dependent guanylate cyclase activity.

Amlodipine activates the endothelial nitric oxide synthase by altering phosphorylation on Ser1177 and Thr495.

The Ca2+ antagonist amlodipine increases the generation of nitric oxide (NO) from native and cultured endothelial cells. The aim of this investigation was to determine whether or not the activation of the endothelial NO synthase (eNOS) by this Ca2+ antagonist is related to alterations in eNOS phosphorylation. In isolated, pre-contracted, endothelium-intact porcine coronary arteries, amlodipine elicited an NO-mediated relaxation and a leftward shift in the concentration-relaxation curve to bradykinin. Moreover, the Ca2+ antagonist increased the generation of NO from native endothelial cells, as detected by electron spin resonance spectroscopy and stimulated an 8-fold increase in cyclic GMP levels in cultured endothelial cells. In unstimulated endothelial cells, eNOS was not phosphorylated on Ser1177 but was phosphorylated on Thr495. Amlodipine elicited the phosphorylation of Ser1177 and attenuated Thr495 phosphorylation, with a time course similar to that of eNOS activation. The amlodipine-induced relaxation of porcine coronary arteries was attenuated by the B2 kinin receptor antagonist, icatibant, but this antagonist did not affect amlodipine-induced changes in eNOS phosphorylation in cultured endothelial cells. Moreover, amlodipine elicited the NO-mediated relaxation of rat aortic rings which do not express the B2 receptor. Amlodipine time-dependently attenuated the phosphorylation of protein kinase C (PKC) in endothelial cells, with a time course similar to the changes in eNOS phosphorylation, and prevented the phorbol-12-myristate-13-acetate-induced activation of PKC. The PKC inhibitor, Ro 31-8220, also elicited the phosphorylation of Ser1177 and the dephosphorylation of Thr495 in cultured cells and induced a leftward shift in the concentration-relaxation curve to bradykinin in rings of porcine coronary artery. The Ca2+ antagonist, amlodipine, enhances endothelial NO generation by inducing changes in the phosphorylation of eNOS. Although the activation of eNOS was related to the activation of the B2 kinin receptor in the porcine coronary artery, a B2 receptor-independent mechanism involving the inhibition of PKC appears to account for the effects observed in the rat aorta as well as in cultured endothelial cells.

YC-1 inhibits proliferation of human vascular endothelial cells through a cyclic GMP-independent pathway

This study was designed to investigate the effect of YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole, in human umbilical vein endothelial cells (HUVECs) proliferation and its underlying mechanism. YC-1 at a range of concentrations (5–50 μM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a dose- and time-dependent manner. YC-1 was not cytotoxic at these concentrations. [ 3 H ]thymidine incorporation and flow cytometry analyses revealed that YC-1 treatment decreased DNA synthesis and arrested the cells at the G0/G1 phase of the cell cycle. Western blot analysis demonstrated that YC-1 (5–50 μM) increased the levels of cyclin-dependent kinase (CDK)-inhibitory proteins (CKIs), p21 and p27, but did not induce any significant changes of cyclins and CDKs. In the YC-1-treated HUVEC, the formation of CDK2–p21 complex, but not CDK2–p27 complex, was increased and the assayable CDK2 kinase activity was decreased. These changes were in a dose-dependent manner. In contrast, the formations of CDK4–p21 and CDK4–p27 complex were slightly increased and the assayable CDK4 kinase activity was slightly decreased (if there were any changes). Pretreatment with guanylyl cyclase inhibitors, 1 H-(1,2,4)oxadiazolo[4,3- a]quinozalin-1-one (ODQ) and methylene blue, inhibited the YC-1-induced increase of cyclic GMP level, but did not change significantly the magnitude of the YC-1-induced inhibition of thymidine incorporation and cell number in HUVEC. These results indicate that YC-1-induced cell cycle arrest in HUVEC occurred when the cyclin–CDK system was inhibited just as p21 and p27 protein levels were augmented. This YC-1-induced antiproliferation effect in HUVEC is via a cyclic GMP-independent pathway.

YC-1 potentiates the nitric oxide/cyclic GMP pathway in corpus cavernosum and facilitates penile erection in rats

The aim of present study was to characterize the in vitro and in vivo pharmacological effects of YC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole), a soluble guanylate cyclase activator, on corpus cavernosal smooth muscle and penile erectile activity. YC-1 relaxed phenylephrine precontracted cavernosal smooth muscle (EC 50=4.4 μM) and this effect was partially antagonized by 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ). ODQ is a selective soluble guanylate cyclase inhibitor that completely blocked the relaxation induced by sodium nitroprusside, suggesting that YC-1 binds to soluble guanylate cyclase at a different site from nitric oxide (NO). Both YC-1 and sodium nitroprusside, but not sildenafil (1–100 μM) caused concentration-dependent increases in cyclic GMP levels in cultured rabbit cavernosal smooth muscle cells and produced synergistic effects. Intraperitoneal administration of YC-1 (10 μmol/kg) evoked penile erection in rats with 70% incidence. More importantly, YC-1 was able to significantly augment the pro-erectile effects of a suboptimal dose of apomorphine. These results suggest that the soluble guanylate cyclase activator YC-1 increases cyclic GMP levels, leading to relaxation of cavernosal smooth muscle. These biochemical events may be related to the pro-erectile properties of YC-1 in vivo.

Vasodilating procyanidins derived from grape seeds.

We have shown in previous work that extracts of grape seeds (GSE) and skins, grape juice, and many red wines exhibit endothelium-dependent relaxing (EDR) activity in vitro. This EDR activity involves endothelial nitric oxide (NO) release and subsequent increase in cyclic GMP levels in the vascular smooth muscle cells. The NO/cyclic GMP pathway is known to be involved in many cardiovascular-protective roles. The current study focuses on the isolation and identification of EDR-active compounds (procyanidins) from GSE. Crushed Concord grape seeds were extracted with methanol and the extract was separated into seven fractions (A-G) on a Toyopearl TSK-HW-40 column. EDR-active fractions (D-G) were further separated into 25 individual compound peaks by HPLC, 16 of which were EDR active (threshold for relaxation ranged between less than 0.5 microg/mL and greater than 4 microg/mL). Procyanidin identification was accomplished by electrospray-ion trap mass spectrometry (ES-ITMS), MS/MS, and by tannase treatment and acid thiolysis, followed by HPLC and ES-ITMS of the products. Activity of isolated procyanidins tended to increase with degree of polymerization, epicatechin content, and with galloylation. These EDR-active compounds (many of which also possess antioxidant activity), individually or in the form of wines, juices, or nutritional supplements, may be useful in preventing or treating cardiovascular diseases.

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K ATP-channels

Recent studies have shown that nitric oxide (NO) modulates K +-channel activity which play an important role in controlling v̀ascular tone. The formation of cyclic guanosine 3′,5′-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K +-current by activating Ca 2+-activated K +-channels (K Ca-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K +-channels [K ATP channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K ATP-channels, in addition to K Ca-channels, in the rat aorta.

Glutamate release is involved in PAF-increased cyclic GMP levels in hippocampus.

The involvement of glutamate in PAF-increased cyclic GMP levels was studied. Glutamate treatment caused a dose-response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10(-7)M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK-801, a NMDA receptor antagonist. Results indicate that PAF-increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF-induced increase of cyclic GMP levels in hippocampus.

Nitric oxide synthesis in myocardium following burn injury in rats

We investigated nitric oxide and cyclic GMP production in myocardium early after burn injury in rats. Nitric oxide synthase activity was measured in cytosol from the left ventricular wall of burned rats. Cytosol from control group animals was shown to contain mainly Ca 2+-dependent nitric oxide synthase (cNOS) with a small amount of Ca 2+-independent nitric oxide synthase (iNOS). Following burn injury, there was a marked increase in iNOS activity with a peak at 8 h post-burn, however, myocardial cNOS activity was found to decline. Parallel to iNOS induction there was a significant increase in myocardial nitric oxide and cyclic GMP production. All these changes were alleviated by treatment of the rats with dexamethasone. Since increases in cyclic GMP levels in the hart were associated with reduced myocardial contractility, it is possible that enhanced production of nitric oxide by a Ca 2+-independent NO synthase accounts, at least in part, for the depression of myocardial contractility seen in burn animals and patients.

Hypothermia During or After Severe Perinatal Asphyxia Prevents Increase in Cyclic Gmp Levels - in the Newborn Rat Striatum 1882

In the rat striatum, the enzyme nitric oxide synthase (NOS) is present in a dense fiber network and in few medium sized non-spinal interneurons. This enzyme is activated by glutamate throughout the influx of Ca2+ to the cell. A sustained activation of NOS caused by excessive release of glutamate, results in strongly increased NO that is a potent activator of the soluble guanylyl cyclase resulting in the synthesis of intracellular cGMP. Previous work showed that NO-containing striatal neurons suffer short-and long-term changes after perinatal asphyctic insult and that changes were prevented by hypothermic exposure. In order to know if this morphological changes were due to an excess of the NOS activity we designed the meassurements of striatal cGMP levels, with the aim to evaluate NOS activity in asphyctic and hypothermic treated pups using a model of severe perinatal asphyxia (SPA). The levels of striatal cGMP of normal pups (PN10) using a RIA procedure were compared to those subjected to SPA and SPA treated with hypothermia during or after insult. cGMP levels in striatal tissue from control pups (PN10) were 25.8 pmol/mg protein and in the SPA group 38.1 pmol/mg which is an increase of approx 70%(p<0.01). Hypothermia during as well as after insult was capable to prevent this increase of cGMP. L-NAME at 0.1 mM decreased cGMP levels in control, SPA and hypothermic treated pups whithout differences between them. Nitroprusside stimulated cGMP showed no differences between groups. These findings indicate that high cGMP levels in the striatum of rats subjected to SPA are under control of an hyperactive NOS leading to excessive NO production which could be harmful to the cerebral tissue. Hypothermia may prove a useful therapeutic agent against cerebral oxidative stress as it reduces NOS activity.

Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion

The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief (15 min) exposure of the neurones to glutamate caused a dose-dependent (0.01–1 mM) increase in cyclic GMP levels together with delayed (24 h) neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide ( ·NO) synthase (NOS) inhibitor N ω-nitro- l-arginine methyl ester (NAME; 1 mM) or by the N-methyl- d-aspartate (NMDA) glutamate-subtype receptor antagonist d-(−)-2-amino-5-phosphonopentanoate (APV; 0.1 mM). Glutamate exposure (0.1 mM and 1 mM) followed by 24 h of incubation caused the inhibition of succinate–cytochrome c reductase (20–25%) and cytochrome c oxidase (31%) activities in the surviving neurones, without affecting NADH–coenzyme-Q 1 reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose (by 26%) or succinate (by 39%) as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion (20%) by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving ·NO biosynthesis.

Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP.

1. There is conflicting evidence in the literature concerning the role of cyclic GMP in the regulation of myometrial contractility and the importance of hormonal status on the uterine response to cyclic GMP-elevating agents. The objective of the present study was to investigate further the importance of cyclic GMP in the control of uterine contractility, by monitoring the effects of cyclic GMP-elevating agents on spontaneous contractions and cyclic GMP levels in myometrial strips from pregnant rats and from ovariectomized rats under the influence of oestrogen and/or progesterone. 2. Sodium nitroprusside (SNP) 5 mM, atrial natriuretic peptide (ANP) 100 nM, L-arginine 1 mM and 8-bromo-cyclic GMP 100 mM had no relaxant effect on the spontaneous contractions of myometria from pregnant rats or from ovariectomized rats under the influence of oestrogen or progesterone. 3. Tissue levels of cyclic GMP were significantly elevated by SNP in all treatment groups, including pregnant animals. For example, in ovariectomized, progesterone-treated rats, SNP raised cyclic GMP levels approximately 8 fold from a basal level of 2.9 +/- 0.4 pmol mg(-1) protein to 24.8 +/- 4.0 pmol mg(-1) protein. ANP increased cyclic GMP levels approximately 2 fold in all treatment groups, except in the pregnant animals. L-Arginine elevated cyclic GMP significantly only in ovariectomized, vehicle-treated myometria. 4. The activity of cyclic GMP-dependent protein kinase (PKG) was significantly increased (3 fold) in myometria exposed to SNP (5 mM). Thus, the inability of SNP to relax uterine preparations was not due to a failure of SNP-elevated cyclic GMP to activate PKG. 5. The more potent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), at a concentration of 100 microM was able to inhibit spontaneous contractions significantly in myometrial preparations from both non-ovariectomized and ovariectomized rats treated with oestrogen or progesterone. 6. Tissue levels of cyclic GMP were markedly increased by SNAP at concentrations of 10, 30 and 100 microM. At 100 microM, cyclic GMP levels increased from 1.9 +/- 0.2 pmol mg(-1) protein to 74.0 +/- 18.0 pmol mg(-1) protein. However, complete or partial blockade of SNAP-induced increases in cyclic GMP levels by the soluble guanylyl cyclase inhibitor, ODQ (25 microM), had no effect on the relaxant response to SNAP. Thus, the relaxant effect of SNAP in this tissue appears to be mediated via a mechanism independent of cyclic GMP. 7. Taken as a whole, the results of the present study indicate that cyclic GMP does not play an important role in the control of contractility in the rat uterus.

Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells.

1. The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC). 2. As determined by cyclic GMP accumulation, substrate (L-arginine)- and inhibitor (N(G)-monomethyl-L-arginine, NMMA)-sensitive iNOS activity was significantly elevated in CH- or ANI-treated RASMC after 24 h. 3. Lipopolysaccharide (LPS) produced a time-dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS-induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration-dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 microM and inhibition at higher concentrations. 4. Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS- and CH-treated cells. 5. CH-induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor. 6. RT-PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS- or CH stimulation alone. 7. These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine-treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins.

Cyclic GMP-dependent protein kinase activation in the absence of negative inotropic effects in the rat ventricle.

1. It has been suggested that activation of cyclic GMP-dependent protein kinase (PKG) is a necessary step in the chain of events leading to the production of negative inotropy by muscarinic receptor agonists in mammalian ventricles, and that some cyclic GMP-elevating agents, such as sodium nitroprusside (SNP), fail to exert a negative inotropic effect because they elevate cyclic GMP levels in a pool that does not activate the kinase. This hypothesis was tested in the present study by monitoring the effects of carbachol, SNP and atrial natriuretic peptide (ANP) on contractility, cyclic GMP content and PKG activity in rat intact ventricular preparations and freshly isolated ventricular cardiomyocytes. 2. The presence of PKG in both the intact vehicle and in isolated ventricular cardiomyocytes was confirmed by MonoQ anion exchange chromatography and Western blotting. The elution profile indicated that the conditions of the PKG assay were selective for measuring PKG activity. 3. Carbachol induced a marked negative inotropic effect in intact, perfused hearts and ventricular strips in the presence of isoproterenol. The negative inotropic effect of carbachol was not associated with significant changes in cyclic GMP content or PKG activity in intact ventricular tissue, or in PKG activity in isolated cardiomyocytes. 4. SNP and ANP significantly increased cyclic GMP levels and activated PKG in intact ventricular preparations. Both drugs also activated PKG in isolated cardiomyocytes. However, neither drug had any negative inotropic effect in isoprenaline-stimulated perfused hearts and ANP did not change the contractility of isoprenaline-stimulated isolated cardiomyocytes. 5. The results of this study demonstrate that the negative inotropic effects of muscarinic receptor agonists can occur in the absence of significant activation of PKG. Conversely, marked increases in ventricular cyclic GMP content and PKG activity caused by SNP or ANP were not accompanied by a negative inotropic effect. 6. These results suggest that increases in cyclic GMP levels and activation of PKG do not play important roles in the regulation of rat ventricular contractility by muscarinic receptor agonists.

Activation of soluble guanylyl cyclase by YC-1 in aortic smooth muscle but not in ventricular myocardium from rat.

1. The effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), an activator of soluble guanylyl cyclase, on tension, levels of cyclic GMP and cyclic AMP, and cardiac L-type Ca2+-current (I[Ca(L)]) were investigated in aortic smooth muscle and ventricular heart muscle from rat. 2. YC-1 (0.1-30 microM) induced a concentration-dependent relaxation in aortic rings precontracted with phenylephrine (3 microM). The relaxant effects of YC-1 were reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (30 microM; ODQ), potentiated by zaprinast (10 microM) and antagonized by Rp-8-Br-cGMPS (100 microM). 3. In ventricular heart muscle strips, YC-1 (30 microM) exhibited no effects on force of contraction (Fc) in the absence or presence of either zaprinast (10 microM) or 3-isobutyl-1-methylxanthine (30 microM). Fc was slightly increased by YC-1 (30 microM) in the presence of isoprenaline (100 nM), but this effect was not influenced by ODQ (30 microM). 4. Cardiac I[Ca(L)] was not significantly affected by YC-1 (30 microM), either in the absence or presence of isoprenaline (30 nM). 5. In aortic rings, cyclic GMP levels were increased almost 3 fold by YC-1 (30 microM); this effect was abolished by ODQ (30 microM). In isolated ventricular cardiomyocytes, cyclic GMP levels were not affected by YC-1 (30 microM) but almost doubled by activation of particular guanylyl cyclase with atriopeptin II (100 nM). 6. YC-1 (30 microM) did not increase cyclic AMP levels either in aortic rings or in ventricular cardiomyocytes. In contrast, isoprenaline (3 microM) increased cyclic AMP levels about two fold in both tissues. In cardiomyocytes, the effect of isoprenaline (3 microM) was slightly enhanced by YC-1 (30 microM). 7. It is concluded that relaxation of smooth muscle preparations by YC-1 is mediated mainly by activation of soluble guanylyl cyclase and subsequent increase in cyclic GMP levels. The failure of YC-1 to affect cardiac Fc, levels of cyclic GMP, and I[Ca(L)] suggests that soluble guanylyl cyclase is not influenced by YC-1 in rat heart muscle or only barely present in this tissue.

Cyclic GMP decreases cardiac myocyte oxygen consumption to a greater extent under conditions of increased metabolism.

We tested the hypothesis that the negative effects of intracellular guanosine 3',5'-cyclic monophosphate (cyclic GMP) were more profound on cardiac myocyte oxygen consumption (VO2) during increased metabolism of the myocytes. The steady state VO2 of a suspension of single myocytes isolated from hearts of New Zealand White rabbits was measured in a glass chamber by using a Clark-type oxygen electrode, and cyclic GMP was determined by using a radioimmunoassay. The cellular cyclic GMP levels were increased either by adding 3-morpholino-sydnonimine (SIN-1), a guanylate cyclase stimulator, or zaprinast (ZAP), a cyclic GMP-phosphodiesterase inhibitor, at various doses. In 0.5 mM Ca2+ medium, average VO2 was 123 +/- 8 nl/min/100,000 cells, and cyclic GMP was 35.4 +/- 9.3 fmol/100,000 cells, and these increased significantly to 182 +/- 9 nl/min/100,000 cells and 78.2 +/- 7.3 fmol/100,000 cells in 2.0 mM Ca2+. There were dose-dependent responses of the VO2 and cellular cyclic GMP levels in responding to both SIN-1 and ZAP. An inverse relation between cellular cyclic GMP level and VO2 existed in the myocytes. The regression equations for the four treatments were log(VO2) = -0.002[cyclic GMP] + 2.19, r = 0.96 for SIN-1 in low (0.5 mM) Ca2+; log(VO2) = 0.005[cyclic GMP] + 1.80, r = 0.38 for ZAP in low Ca2+; log(VO2) = -0.001 [cyclic GMP] + 2.24, r = 0.82 for SIN-1 in high (2.0 mM) Ca2+; and log(VO2) = -0.004[cyclic GMP] + 2.56, r = 0.93 for ZAP in high Ca2+. The slope of ZAP regression line was significantly more negative than that of SIN-1 with high calcium. At any given level of cyclic GMP, ZAP decreased the VO2 to a greater extent than did SIN-1 although the latter caused the maximal increase in cyclic GMP level. The reduction in VO2 caused by a corresponding increase in cellular cyclic GMP was greater in myocytes incubated with high-Ca2+ medium.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M 1 and M 2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M 2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M 2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.