Role of cGMP in relaxation of vascular and other smooth muscle

The hypothesis that the relaxant action of many drugs on vascular and other smooth muscle is mediated by increases in intracellular cGMP, the "cGMP hypothesis," is gaining wide acceptance. While much information supporting this idea can be found in the literature, there is also a significant amount of information indicating that an elevation in the tissue content of cGMP is by itself insufficient to cause smooth muscle relaxation. The literature is reviewed with reference to the criteria that need to be fulfilled to consider cGMP as the second messenger mediating relaxation of smooth muscle by a drug; i.e., activation of guanylate cyclase, elevation of tissue content of cGMP, potentiation by phosphodiesterase inhibitors, antagonism by inhibitors of cGMP synthesis, and production of relaxation by cGMP analogues. For each criterion, key observations supporting the hypothesis are considered, followed by examples of important observations not consistent with the hypothesis. It is concluded that in some smooth muscles, for example, rat myometrium and vas deferens, cGMP is not a mediator of drug-induced relaxation. In other smooth muscles, including vascular smooth muscle, cGMP appears to play an important role in the relaxation process; but current evidence suggests that other factors are also important and that the cGMP hypothesis may need to be modified.     

Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells

In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300-fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle.     

The effects of methylxanthines, ethymizol, ephedrine and papaverine on guinea pig and dog trachea

The study was aimed to compare the effects of pentoxyphylline, aminophylline, choline theophyllinate and ethymizol on guinea pig and dog trachea with those of theophylline, papaverine and ephedrine. The effects of these drugs on the basal tension, on dose-response curves for muscle contraction produced by histamine and on cAMP level were investigated in guinea pig trachea, together with their influence on the resting and histamine-evoked mechanical and membrane activities of dog trachea. Like papaverine, pentoxyphylline did not alter the resting membrane potential, although it relaxed both tracheal preparations, and it antagonised the effects histamine and raised the cAMP level of the smooth muscle. The effects of ethymizol were similar to those of theophylline and its water soluble derivatives (aminophylline and choline theophyllinate). Whereas, ephedrine although it decreased the basal tension and inhibited histamine-evoked responses, also elicited substantial hyperpolarization of the smooth muscle membrane with no effect on the cAMP level. These findings are consistent with the hypothesis that cAMP has an important role in the action of some bronchodilator drugs; however, it is concluded that the possibility of contributing of their action on membrane potential to their action needs to be considered. The similarity of the potencies of ethymizol and pentoxyphylline to that of classical bronchodilators in inhibiting contraction of guinea pig and dog tracheal smooth muscle suggests that they may have a therapeutic value.     

Temporal relationship of cyclic nucleotide levels and calcium exchange to histamine-induced tension development

The purpose of these experiments was to study the temporal relationship between tension development in incubated guinea pig tracheal smooth muscle and changes in tissue levels of cAMP and cGMP, and isotopic Ca. Dose-response studies were performed with increasing concentrations of histamine both in the absence and presence of H1 receptor blockade using 10(-5) M diphenhydramine. The time course of tension development was subsequently determined in the presence of three concentrations of histamine shown to cause 50% (3 X 10(-6) M), 85% (9 X 10(-6) M), and 100% (5 X 10(-5) M) of maximal contraction. Tissue cyclic nucleotide and 45Ca levels were measured 20 sec, 1 min, and 6 min after the onset of contraction. For comparison, the influence of carbachol was also studied. Our findings demonstrate that there were no detectable alterations in tissue cAMP or cGMP levels during the initial phases of contractile change. In contrast, tissue isotopic Ca uptake increased early in histamine-induced contraction and was blocked by the H1 antagonist.     

8Br-cGMP mediates relaxation of tracheal smooth muscle through PKA

In this study, guinea pig tracheal smooth muscle pre-contracted with histamine was relaxed by the addition of 100microM 8Br-cGMP, a non-hydrolyzable and cell-permeable analog for cGMP. This effect was not sensitive to cGMP-dependent protein kinase (PKG) inhibitors, whereas it was partially blocked by cAMP-dependent protein kinase (PKA) inhibitors. The relaxation observed was also reverted up to 50+/-8.5% by iberiotoxin, a selective inhibitor of large conductance, calcium-activated potassium channels (BK(Ca)). Our results indicate that there exists a crosstalk mechanism between cAMP and cGMP signaling pathways which lead to relaxation of guinea pig tracheal smooth muscle and also that BK(Ca) channels are involved to a certain extent in this phenomenon.     

Zmu-1：DHP和DHP两个品系豚鼠组胺激发试验气道反应性的比较

目的研究Zmu-1：DHP和DHP豚鼠组胺激发试验气道反应性的差异,为哮喘研究提供具有较好反应性的动物模型。方法通过活体气管插管及雾化组胺气体吸入,测定豚鼠气道阻力和肺动态顺应性;采用离体气管片组胺滴定法,通过气管片收缩测定气管平滑肌对组胺的敏感性;采用同位素标记药物配位法,测定豚鼠气道组织组胺H1受体的密度及平衡解离常数。结果当豚鼠吸人大于0．5mg／mL雾化组胺时,Zmu-1：DHP豚鼠气道阻力上升率和肺动态顺应性下降率与DHP豚鼠相比,其速度有增大趋势,但未达到显著水平（P〉0．05）;两个品系豚鼠雄性个体比雌性的敏感性显著增加（P〈0．01）。当组胺浓度为10-5mol／L和10μmol／L时,Zmu-1：DHP豚鼠气管片平滑肌的收缩率显著大于DHP豚鼠（P〈0．01～0．05）。两个品系豚鼠气道组胺Hl受体的密度及其平衡解离常数无显著差异（P〉0．05）。结论在一定的组胺浓度范围内,Zmu-1：DHP豚鼠气道平滑肌对组胺的敏感性大于DHP豚鼠,雄性豚鼠的敏感性大于雌性豚鼠。两个品系豚鼠气道组胺H1受体数量无差异,提示气道反应可能还受到平滑肌膜上其它受体和化学介质的作用。     

Actions of histamine on muscle and ganglia of the guinea pig gallbladder

Histamine is an inflammatory mediator present in mast cells, which are abundant in the wall of the gallbladder. We examined the electrical properties of gallbladder smooth muscle and nerve associated with histamine-induced changes in gallbladder tone. Recordings were made from gallbladder smooth muscle and neurons, and responses to histamine and receptor subtype-specific compounds were tested. Histamine application to intact smooth muscle produced a concentration-dependent membrane depolarization and increased excitability. In the presence of the H(2) antagonist ranitidine, the response to histamine was potentiated. Activation of H(2) receptors caused membrane hyperpolarization and elimination of spontaneous action potentials. The H(2) response was attenuated by the ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide in intact and isolated smooth muscle. Histamine had no effect on the resting membrane potential or excitability of gallbladder neurons. Furthermore, neither histamine nor the H(3) agonist R-alpha-methylhistamine altered the amplitude of the fast excitatory postsynaptic potential in gallbladder ganglia. The mast cell degranulator compound 48/80 caused a smooth muscle depolarization that was inhibited by the H(1) antagonist mepyramine, indicating that histamine released from mast cells can activate gallbladder smooth muscle. In conclusion, histamine released from mast cells can act on gallbladder smooth muscle, but not in ganglia. The depolarization and associated contraction of gallbladder smooth muscle represent the net effect of activation of both H(1) (excitatory) and H(2) (inhibitory) receptors, with the H(2) receptor-mediated response involving the activation of K(ATP) channels.     

Metabolic pathway of magnetized fluid-induced relaxation effects on heart muscle

The effect of magnetized physiological solution (MPS) on isolated, perfused snail heart muscle contractility, (45)Ca uptake and intracellular level of cAMP, and cGMP was studied. The existence of the relaxing effect of MPS on heart muscle at room temperature (22 degrees C) and its absence in cold medium (4 degrees C) was shown. The MPS had a depressing effect on (45)Ca uptake by muscles and intracellular cAMP content and an elevating effect on intracellular cGMP level. It is suggested that the relaxing effect of MPS on heart muscle is due to the decrease of intracellular Ca ions as the result of activation of cGMP-dependent Ca efflux. The MPS induced decrease of intracellular cAMP content can be considered as a consequence of intracellular Ca loss, leading to the Na + K-ATPase reactivation, and causing the decrease of the intracellular level of ATP, serving as a substrate and positive modulator of cyclase activity.     

Increase in intracellular calcium induced by stimulating histamine H1 receptors in macrophage-like P388D1 cells.

The addition of histamine to macrophage-like P388D1 cells resulted in a dose-dependent increase in intracellular calcium [Ca2+]i measured by fura-2 in single cells. The maximum level of [Ca2+]i was obtained by addition of 1 x 10(-4) M histamine. The increase was primarily due to release from the intracellular store. The addition of an H1 specific antagonist pyrilamine before histamine treatment inhibited the increase reversibly, while an H2 specific antagonist cimetidine had no inhibitory effect. Histamine also resulted in a dose-dependent increase in cGMP but not in cAMP. These data suggest the existence of histamine H1 receptors in these cells and histamine may have some biological effect on the function of macrophages via [Ca2+]i and cGMP as the second messengers.     

Molecular mechanism of cGMP-mediated smooth muscle relaxation

Contraction and relaxation of smooth muscle is a tightly regulated process involving numerous endogenous substances and their intracellular second messengers. We examine the key role of cyclic guanosine monophosphate (cGMP) in mediating smooth muscle relaxation. We briefly review the current art regarding cGMP generation and degradation, while focusing on the recent identification of the molecular mechanisms underlying cGMP-mediated smooth muscle relaxation. cGMP-induced SM relaxation is mediated mainly by cGMP-dependent protein kinase activation. It involves several molecular events culminating in a reduction in intracellular Ca(2+) concentration and a decrease in the sensitivity of the contractile system to Ca(2+). We propose that the cGMP-induced decrease in Ca(2+) sensitivity is a strategic way to achieve "active relaxation" of the smooth muscle. In summary, we present compelling evidence supporting a key role for cGMP as a mediator of smooth muscle relaxation in physiological and pharmacological settings.     

Several pathogenetic factors of experimental "indomethacin" hypertension]

The content of cyclic nucleotides (cAMP and cGMP) in the blood plasma, urine and tissues, and also morphological changes of the vascular renal bed were studied in rats with arterial hypertension induced by chronic inhibition of prostaglandin synthesis. A considerable thickening of the wall of the interlobular and arcuate arteries with marked lumen narrowing occurred mainly on account of hypertrophy and the swelling of smooth muscle cells. At the same time there was a marked increase in the cGMP concentration, a decrease of cAMP level, and a reduction of the cAMP/cGMP coefficient in the biological fluids. It is suggested that the changed cyclic nucleotides metabolism is associated with organic and functional changes of the peripheral vascular bed underlying an increase of the total vascular resistance in arterial hypertension.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

Participation of cyclic nucleotides and phospholipidis in signal transduction of histamin receptor H3 of the gastric mucosa parietal cells in rats with experimental ulcer

The goal of the work was to evaluate plasma membrane phospholipid composition in rat gastric parietal cells under the histamine H3 receptor activation. The content of cyclic nucleotides was also studied. It was shown that H3-receptor agonist (R)-alpha-methylhistamine increases the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) level and decreases the phoshatidylinositole level in plasma membrane of rat gastric parietal cells and leads to attenuation of the cGMP production and enhancement of the cAMP production under the experimental stress--the induced stomach ulcer formation. The histamine H3-receptor antagonist, thioperamide, insignificantly increases the PC and PE level and increases the phoshatidylinositole level in the plasma membrane of rat gastric parietal cells and leads to cAMP production attenuation, and cGMP contents decreases in the above-stated cells. Thus it was shown that histamine H3 receptor activation causes different effects on polyphosphoinositide and adenylatcyclase cascades in parietal cells under stomach ulcer conditions.     

Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.     

Neuropeptide Y stimulation of myosin light chain phosphorylation in cultured aortic smooth muscle cells

Neuropeptide Y (NPY) is released from an extensive network of postganglionic sympathetic perivascular neurons. NPY has been shown to affect vascular tone postsynaptically by 1) directly stimulating contraction; 2) inhibiting vasorelaxation; and 3) potentiating contraction elicited by exogenous vasoconstrictors. The molecular mechanisms mediating these effects of NPY are undefined. Therefore, we examined the possibility that NPY could stimulate smooth muscle contraction through myosin light chain phosphorylation in cultured porcine aortic smooth muscle cells. NPY (100 nM) caused a rapid, transient increase in myosin light chain (MLC) phosphorylation, an important regulatory event in the initiation of smooth muscle contraction. NPY-stimulated MLC phosphorylation was prevented by preincubation of cells with pertussis toxin and was independent of extracellular Ca2+. In parallel studies, NPY alone had no detectable effect on cellular cAMP or cGMP content; however, NPY potently inhibited forskolin-stimulated cAMP accumulation (IC50 = 0.03 nM) through a pertussis toxin-sensitive pathway. NPY had no detectable effect on basal phosphoinositide hydrolysis or protein kinase C activation but enhanced angiotensin II-stimulated production of inositol phosphates and activation of protein kinase C. These results indicate that NPY-stimulated MLC phosphorylation can occur in the absence of detectable changes in cAMP content, cGMP content, inositol phosphate production, or protein kinase C activation; however, the interactions between NPY and other vasoactive agents may be mediated by the indirect effects of NPY on adenylate cyclase activity and phosphoinositide hydrolysis.     

A semi quantitative method for cyclic nucleotide localization by immunocytochemistry and its application in determining the distribution of cyclic AMP in lungs of normal and pertussis-vaccinated mice following histamine or epinephrine challenge.

A method is described which allows quantitative comparison of cAMP content determined by immunocytochemical procedures. This technique was then employed to localize cAMP in lungs of normal and pertussis-vaccinated mice following saline, histamine, or epinephrine challenge. All primary pulmonary compartments were shown to contain some immunoreactive (cAMP) material. However, epinephrine and histamine challenge selectively increased the cAMP content of the vasculature. No effect of epinephrine or histamine was detected in bronchial smooth muscle or interstitial tissue. This increased cAMP accumulation was observed in both normal and pertussis-vaccinated mice following epinephrine challenge but only in pertussis-vaccinated mice after histamine administration. These results demonstrate that histamine and epinephrine stimulate cAMP accumulation in the same pulmonary compartments supporting earlier speculation that histamine acts indirectly through epinephrine release. Further, primary involvement of the vasculature supports a more prominent role for this tissue in pertussis mediated histamine hypersensitivity.     

Cross talk between NO and cyclic nucleotide phosphodiesterases in the modulation of signal transduction in blood vessel.

An increase in cAMP and/or cGMP induces vasodilation which could be potentiated by endothelium or NO-donors. Cyclic nucleotide phosphodiesterases (PDE) are differently distributed in vascular tissues. cAMP hydrolyzing PDE isozymes in endothelial cells are represented by PDE2 (cGMP stimulated-PDE) and PDE4 (cGMP insensitive-PDE), whereas in smooth muscle cells PDE3 (cGMP inhibited-PDE) and PDE4 are present. To investigate the role of NO in vasodilation induced by PDE inhibitors, we studied the effects of PDE3- or PDE4-inhibitor alone and their combination on cyclic nucleotide levels, on relaxation of precontracted aorta and on protein kinase implication. Furthermore, the direct effect of dinitrosyl iron complex (DNIC) was studied on purified recombinant PDE4B. The results show that: 1) in endothelial cells PDE4 inhibition may up-regulate basal production of NO, this effect being potentiated by PDE2 inhibition; 2) in smooth muscle cGMP produced by NO inhibits PDE3 and increases cAMP level allowing PDE4 to participate in vascular contraction; 3) protein kinase G mediates the relaxing effects of PDE3 or PDE4 inhibition. 4) DNIC inhibits non competitively PDE4B indicating a direct effect of NO on PDE4 which could explain an additive vasodilatory effect of NO. A direct and a cGMP related cross-talk between NO and cAMP-PDEs, may participate into the vasomodulation mediated by cAMP activation of protein kinase G.     

Role of histamine and cyclic nucleotides in implantation in the rabbit

Summary The effect of histamine on cAMP and cGMP levels in day 6 (144 h post coitum) rabbit blastocysts was determined. Histamine at 200 M and 1000 M concentrations stimulated the increased formation of cAMP in vitro, whereas stimulation of cGMP occurred only in the presence of 1000 M histamine. Furthermore, intrauterine injection of RMI-12330A (50 g or 500 g/uterine horn), an inhibitor of adenylyl cyclase, on day 5 of pregnancy interrupted embryro development and implantation of the embryo. The drug was also effective in reducing the cAMP level in the endometrial cells in vitro. A relationship between histamine and cyclic nucleotide changes in embryo development and implantation is suggested.     

Effect of sildenafil on renin secretion in human subjects

Sildenafil is a potent and selective inhibitor of the cyclic GMP-specific phosphodiesterase (PDE5) that is very effective in the treatment of male impotence. It inhibits breakdown of cyclic guanosine monophosphate (cGMP) formed in penile smooth muscle cells in response to stimulation by nitric oxide resulting in muscle relaxation. PDE5 is widely distributed in the body, being present in the vasculature, platelets, and kidneys. In the kidney, PDE5 is involved in the regulation of sodium excretion and renin secretion. The aim of the present investigation was to investigate the effect of sildenafil, in doses used clinically, on renin secretion in human subjects. The studies were performed in two groups of healthy normotensive subjects: one in which sodium intake was unrestricted, and one in which sodium intake was restricted to 600 mg/day. Blood pressure and heart rate were monitored throughout the study, and blood samples for the measurement of plasma cGMP and cAMP concentrations and plasma renin activity (PRA) were collected. After control measurements, the subjects ingested a capsule containing sildenafil or placebo. Cardiovascular measurements and blood sampling continued for the next 120 min. Sildenafil had only minor cardiovascular effects. Diastolic pressure tended to be lower and heart rate was generally higher after sildenafil than after placebo, but the differences were small. Sildenafil caused a prompt and sustained increase in plasma cGMP concentration and a more gradual increase in plasma cAMP concentration. After the subjects received placebo, there was a progressive decrease in PRA during the 2-hr observation period, presumably reflecting the circadian rhythm in renin secretion. In contrast, PRA failed to decrease after the subjects received sildenafil, thus indicating that sildenafil exerts a stimulatory action on renin secretion. This action on renin secretion may help explain why sildenafil only has minor effect on blood pressure despite the widespread distribution of PDE5 in vascular tissues.     

Dynamics of the change in cyclic nucleotide content in wound tissues]

Changes in the content of cyclic nucleotides (cAMP and cGMP) in the wound tissues (muscles and granulations) were studied in experiments on rats. A wound with skin defect in the dorsal area with a crushed underlying muscle served as an experimental model. It was shown that the cAMP content in the muscular tissue rises twice: during the 1st day and to a greater measure on the 7th day. The cGMP content slightly increases on the 1-4th day, drops on the 7th day and increases cAMP concentration changes similarly to that inthe muscular tissue: it increases on the 7th day and drops on the 14th day. On the contrary, the cGMP content curve in granulations is more monotonous, only a slight increase being observed on the 7th day.