The aim of present study is to investigate the effects of cGMP on hyperpolarization activated inward current (If), pacemaker current of the heart, in rabbit sino-atrial node cells using the whole-cell patch clamp technique. When sodium nitroprusside (SNP, 80 muM), which is known to activate guanylyl cyclase, was added, If amplitude was increased and its activation was accelerated. However, when If was prestimulated by isopreterenol (ISO, 1 muM), SNP reversed the effect of ISO. In the absence of ISO, SNP shifted activation curve rightward. On the contrary in the presence of ISO, SNP shifted activation curve in opposite direction. 8Br-cGMP (100 muM), more potent PKG activator and worse PDE activator than cGMP, also increased basal If but did not reverse stimulatory effect of ISO. It was probable that PKG activation seemed to be involved in SNP-induced basal If increase. The fact that SNP inhibited ISO-stimulated If suggested cGMP antagonize cAMP action via the activation of PDE. This possibility was supported by experiment using 3-isobutyl-1-methylxanthine (IBMX), non-specific PDE inhibitor. SNP did not affect If when If was stimulated by 20 muM IBMX. Therefore, cGMP reversed the stimulatory effect of cAMP via cAMP breakdown by activating cGMP-stimulated PDE. These results suggest that PKG and PDE are involved in the modulation of If by cGMP: PKG may facilitate If and cGMP-stimulated PDE can counteract the stimulatory action of cAMP.
1-Methyl-3-isobutylxanthine ; Guanylate Cyclase ; Heart ; Nitroprusside ; Sinoatrial Node*

The Obligatory Role of the Chorionic Membranes in the Synthesis of Myometrial cGMP during Pregnancyand Its Inhibition by Maternal and Fetal Plasma The mechanism of uterine quiescence during pregnancy and initiation of labor is unknown. In previousreport, we demonstrated myometrial cGMP rises dramatically during pregnancy and then declines just priorto the onset of labor. Further, pregnancy decrease myometrial soluble guanylate cyclase activity whileenhancing particulate activity. Theses findings suggest a natriureitc peptide(NP) is responsible for theincrease in cGMP. This study was undertaken to evaluate the source of that NP. We incubatedmyometrium from term guinea pigs in oxygenated buffer in the absence/presence of either amnionicmembranes(AM, 400mg), chorionic membrane(CM, 400mg), fetal guinea pig plasma(FP, 1ml), maternalguinea pig plasama(MP, 1ml), or a combination. After incubation, cGMP(mol/mg portein) was measuredby radioimmuno assay. Both AM and CM(CM>AM, significantly) increased myometrial cGMP. Thestimulation of myometrial cGMP by the CM has a significant linear relationship between fetal weight andthe greatest cGMP increase occurred between 51~60 days after which it showed a trend to decrease. BothFP and MP decrease myometrial cGMP and inhibition of cGMP by MP increase significantly withadvancing gestational age.Our date indicate that myometrial cGMP is stimulated by a compound produced by the CM and the actionof this compound is inhibited by a substance produced by the mother and fetus at the end of a normalpregnancy.
Animals ; Chorion* ; Fetal Weight ; Fetus ; Guanylate Cyclase ; Guinea Pigs ; Humans ; Membranes* ; Mothers ; Oxygen ; Plasma* ; Pregnancy*

Furosemide acts primarily on the thick ascending limb of the loop of Henle and inhibits the chloride transport in this site, which is the main mechanism of diuretic action of furosemide However, the precise molecular mechanism of diuretic action of furosemide is still unknown. Recent studies have shown that cGMP might be involved in diuretic effect of furosemide. In this study, the effects of furosemide on the renal tissue level of cGMP in vivo and on the renal guanylate cyclase in vitro were investigated. Also, the influence of aspirin on these effects was examined. The results were as follows: 1. The renal tissue level of cGMP was increased after administration of furosemide, but decreased after administration of aspirin. A combined administration of furosemide and aspirin increased the renal tissue level of cGMP, but the degree of elevation was less than those of the furosemide group. 2. The renal guanylate cyclase activity was slightly increased by furosemide, but this increase was not significant. The renal guanylate cyclase activity was significantly increased by arachidonic acid. Furosemide potentiated the effect of arachidonic acid on renal guanylate cyclase activity, which was inhibited by aspirin. These results indicate that effect of furosemide on renal tissue level of cGMP may be indirect effect that furosemide activates guanylate cyclase by means of increasing prostaglandin synthesis.
Animals ; Arachidonic Acid ; Aspirin ; Diuretics ; Extremities ; Furosemide* ; Guanylate Cyclase* ; Loop of Henle ; Rats*

BACKGROUNDThe role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear. This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body, trabecular meshwork and the Schlemm's canal.

METHODSTwelve eyes after corneal transplantation were used. Expression of three NOS isoforms (i.e. neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC. Ciliary bodies were dissected free and the total proteins were extracted. Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC.

RESULTSExpression of 3 NOS isoforms and GC were observed in the ciliary epithelium, ciliary muscle, trabecular meshwork and the endothelium of the Schlemm's canal. Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells. Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting.

CONCLUSIONSThe expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP, cGMP) signaling pathway in the ciliary body, and may play a role in both processes of aqueous humor formation and drainage.

The aim of this study was to clarify the mechanism of the inhibitory action of carbon monoxide (CO) on contraction, by measuring cytosolic Ca2+ level ((Ca2+)i) and ionic currents in guinea-pig ileum. CO (10%) inhibited 40 mM KCl-induced contraction and this effect was blocked by ODQ (1 micrometer), a soluble guanylyl cyclase (sGC) inhibitor. CO inhibited the 40 mM KCl-induced contraction without changing (Ca2+)i. Cumulative addition of KCl induced a graded increase in (Ca2+)i and muscle tension. In the presence of CO, cumulative addition of KCl induced smaller contraction than in the absence of CO. On the other hand, the increase in (Ca2+)i induced by cumulative addition of KCl was only slightly decreased in the presence of CO, and the (Ca2+)i-tension relationship shifted downwards. Using the patch clamp technique with a holding potential of -60 mV, we found that CO had little effect on the peak Ba currents (IBa) when voltage was stepped from -60 mV to 0 mV. In addition, CO showed no effect on the depolarization-activated outward K+ currents in the all potential ranges. We conclude that CO inhibits smooth muscle contraction mainly by decreasing the Ca2+ sensitivity of contractile elements via a cGMP-dependent pathway, not by involving L-type Ca2+ and outward-potassium currents in guinea-pig ileum.
Animals ; Carbon Monoxide* ; Carbon* ; Cytosol* ; Guanylate Cyclase ; Guinea Pigs* ; Guinea* ; Hand ; Ileum ; Muscle Tonus ; Muscle, Smooth*

Pathophysiological implications of the vascular nitric oxide (NO)/cGMP pathway in hypertension were investigated. Sprague-Dawley rats were made deoxycorticosterone acetate (DOCA)-salt hypertensive for six weeks. The protein expression of endothelial constitutive NO synthase (ecNOS) and the tissue content of NO were determined in the thoracic aorta. The protein expression and catalytic activity of soluble guanylyl cyclase (GC) were also determined. Systolic blood pressure measured on the day of experiment was significantly higher in the experimental group than in the control. The hypertension was associated with decreases in the vascular tissue content of NO metabolites, concomitantly with the expression of ecNOS proteins. The protein expression of GC was not affected, while its catalytic activity was significantly decreased in hypertension. These results indicate that the high blood pressure is associated with a decreased activity of vascular NO/cGMP pathway in DOCA-salt hypertension.
Aorta, Thoracic ; Blood Pressure ; Desoxycorticosterone* ; Guanylate Cyclase* ; Hypertension* ; Nitric Oxide ; Nitric Oxide Synthase ; Rats, Sprague-Dawley

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on contraction, cytosolic Ca2+ level ((Ca2+)i), and ionic currents in guinea-pig ileum. SIN-1 (0.01~100 micrometer) inhibited 25 mM KCl- or histamine (10 micrometer)-induced contraction in a concentration-dependent manner. SIN-1 reduced both the 25 mM KCl- and the histamine-stimulated increases in muscle tension in parallel with decreased (Ca2+)i. Using the patch clamp technique with a holding potential of -60 mV, SIN-1 (10 micrometer) decreased peak Ba currents (IBa) by 30.9+/-5.4% (n=6) when voltage was stepped from -60 mV to +10 mV and this effect was blocked by ODQ (1 micrometer), a soluble guanylyl cyclase inhibitor. Cu/Zn SOD (100 U/ml), the free radical scavenger, had little effect on basal IBa, and SIN-1 (10 micrometer) inhibited peak IBa by 32.4+/-5.8% (n=5) in the presence of Cu/Zn SOD. In a cell clamped at a holding-potential of -40 mV, application of 10 micrometer histamine induced an inward current. The histamine-induced inward current was markedly and reversibly inhibited by 10 micrometer SIN-1, and this effect was abolished by ODQ (1 micrometer). In addition, SIN-1 markedly increased the depolarization-activated outward K+ currents in the all potential ranges. We concluded that SIN-1 inhibits smooth muscle contraction mainly by decreasing (Ca2+)i resulted from the inhibition of L-type Ca2+ channels and the inhibition of nonselective cation currents and/or by the activation of K+ currents via a cGMP-dependent pathway.
Cytosol* ; Guanylate Cyclase ; Histamine ; Humans ; Ileum ; Muscle Tonus ; Muscle, Smooth* ; Nitric Oxide ; Tissue Donors*

Cyclosporin A (CsA), a widely used immunosuppressant, is well known to cause nephrotoxicity and hypertension as major side effects. The present study was aimed at investigating the effects of CsA-pretreatment on the activities of cytosolic guanylate cyclase (cGC) in relation to the alteration of relaxant responses in the rat thoracic aorta. CsA (10 micrometer)-preincubation for 90 min significantly attenuated the vasodilatation induced by sodium nitroprusside (SNP), a cytosolic guanylate cyclase activator, shifting the dose-response curve to the right. The increase in cGMP contents induced by SNP was markedly attenuated by CsA. SNP (1 micrometer apprx 1 mM) increased the cGC activity dose-dependently, and the increase was completely abolished by CsA. CsA attenuated the SNP-induced cGC activation dose-dependently. The abolishing effect of CsA-pretreatment on the SNP-induced cGC activation was not affected by washing the preparation, suggesting that the inhibition is irreversible. When CsA was added simultaneously with SNP, cGC activation was not attenuated. 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H-7), a protein kinase C (PKC) inhibitor, decreased SNP-induced cGC activation and blocked the CsA-attenuation of cGC activation. These results suggest that CsA directly inhibits cGC participating in the CsA-induced impairment of vasodilatation, and that PKC is involved in the inhibitory action of CsA on cGC.
Animals ; Aorta, Thoracic* ; Cyclosporine* ; Cytosol* ; Guanylate Cyclase ; Hypertension ; Nitroprusside ; Protein Kinase C ; Rats* ; Vasodilation*

Animals ; Guanylate Cyclase ; metabolism ; Hypercapnia ; metabolism ; Hypoxia ; metabolism ; Lung ; metabolism ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Soluble Guanylyl Cyclase

Vasoplegic syndrome occurs in 8~10% of patients following cardiac surgery, and this happens in part because of inducing the inflammatory response. Nitric oxide and guanylate cyclase play an important role in this response, and this is associated with increased morbidity and mortality. For our case, we administered methylene blue (MB), an inhibitor of guanylate cyclase, early after performing cardiopulmonary bypass in a patient with vasoplegic syndrome. The patient recovered immediately after MB administration and maintained an optimal blood pressure without the aid help of any vasopressors.
Blood Pressure* ; Cardiopulmonary Bypass ; Endocarditis* ; Guanylate Cyclase ; Heart* ; Humans ; Methylene Blue* ; Mortality ; Nitric Oxide ; Thoracic Surgery* ; Vasoplegia*