The purpose of this study was to investigate the vasorelaxing effect of vasonatrin peptide (VNP) on human intramammary artery (HIMA).The vasorelaxing effect of VNP on HIMA was measured by means of perfusion in vitro. The effects of HS-142-1, TEA, 8-Br-cGMP and methylene blue (MB) were also observed. It was found that VNP caused a concentration-dependent relaxation in HIMA which was independent of the endothelium. 8-Br-cGMP (0.1-1000 micromol/L) also caused a concentration-dependent relaxation in HIMA. The vasorelaxing effect of VNP disappeared in the presence of HS-142-1 (20 micromol/L), an antagonist of the natriuretic peptide guanylate cyclase (GC) receptor. MB (10 micromol/L), an inhibitor of GC, not only blocked completely the relaxation of HIMA, but also enhanced the vascular contraction induced by norepinephrine. TEA (1 mmol/L), an antagonist of calcium activated potassium channels (K(Ca)), reduced but not completely blocked the vasorelaxing effect of VNP. These findings suggest that VNP can relax HIMA, which is independent of the endothelium. This effect is possibly achieved by the binding of VNP with the natriuretic peptide GC receptors in the smooth muscle cells (SMCs), leading to an increase in intracellular cGMP level. Moreover, the vasorelaxing effect of VNP is associated with K(Ca).
Aged ; Atrial Natriuretic Factor ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; In Vitro Techniques ; Mammary Arteries ; drug effects ; physiology ; Middle Aged ; Potassium Channels, Calcium-Activated ; metabolism ; Receptors, Guanylate Cyclase-Coupled ; metabolism ; Vasodilation ; drug effects ; physiology

PURPOSE: The expression of natriuretic peptides in the neural bundles of the anterior portion of the optic nerves and their functions in regulating vessel tone and blood flow may suggest a possible role in the pathogenesis of glaucoma. The purpose of this study was to investigate the association between normal-tension glaucoma and the genetic variations of atrial natriuretic peptide (Nppa) and natriuretic peptide receptor A (Npr1) gene. METHODS: Sixty-seven Korean normal-tension glaucoma (NTG) patients and 100 healthy subjects (as normal controls) were enrolled. DNA from peripheral blood leukocytes was extracted, and the genotypes of five polymorphisms (c.94G>A, c.454T>C, IVS1+16C>T, IVS2+701G>A, and c.-764C>G) in the Nppa gene and one polymorphism (c.1023G>C) in the Npr1 gene were determined using the restriction fragment length polymorphism and the SNaPshot methods. The genotype and allele frequencies of these polymorphisms in patients with NTG and normal controls were compared using the Fisher's exact test and the chi-square test. RESULTS: In both groups, the genotype distributions were in accordance with the Hardy-Weinberg equilibrium. There was no significant difference in the frequency of the Nppa and Npr1 alleles or genotypes in the normal-tension glaucoma group as compared to the control group. CONCLUSIONS: Nppa and Npr1 gene polymorphisms are not associated with normal-tension glaucoma, suggesting that this gene does not have an important role in the pathogenesis of optic neuropathy in this disease.
Receptors, Atrial Natriuretic Factor/*genetics ; *Polymorphism, Single Nucleotide ; Middle Aged ; Male ; *Intraocular Pressure ; Humans ; Guanylate Cyclase/*genetics ; Glaucoma/genetics/*physiopathology ; Genotype ; Gene Frequency ; Female ; Atrial Natriuretic Factor/*genetics ; Adult

The purpose of this study was to investigate the effects of vasonatrin peptide (VNP) on electrically-induced intracellular calcium ([Ca(2+)](i)) transient and mechanism of the effects in the cardiac myocytes. The [Ca(2+)](i) transient was measured with a fluoremetric method. The effects of HS-142-1, 8-Br-cGMP and methylene blue (MB) on [Ca(2+)](i) transient in cardiac myocytes were also determined. Isoproterenol (Iso) at 10(-10)~10(-6) mol/L augmented electrically-induced [Ca(2+)](i) transient dose-dependently, which was (13+/-8)% (P>0.05), (26+/-13)% (P< 0.05), (66+/-10)% (P<0.01), (150+/-10)% (P<0.01) and (300+/-25)% (P<0.01), respectively. These effects were blocked by an beta-adrenergic bloker propranolol (10(-6) mol/L). The effect of Iso (10(-8) mol/L) on [Ca(2+)](i) transient was attenuated in a dose-dependent manner by VNP at 10(-10)~10(-6) mol/L, which was (99+/-3)% (P>0.05), (96+/-2)% (P<0.05), (84+/-6)% (P<0.01), (66+/-3)% (P<0.01) and (62+/-3)% (P<0.01), respectively. 8-Br-cGMP (10(-7)~10(-3) mol/L) aslo attenuated 10(-8) mol/L Iso-induced [Ca(2+)](i) transient dose-dependent. The effect of VNP on [Ca(2+)](i) transient was almost abolished in the presence of HS-142-1 (2x10(-5) mol/L), an antagonist of the natriuretic peptide guanylate cyclase (GC) receptors. MB (10(-5) mol/L), an inhibitor of GC, not only blocked the effect of VNP in myocytes, but also augmented electrically-induced [Ca(2+)](i) transient. VNP and HS-142-1 themselves did not change the [Ca(2+)](i) transient in the cardiac myocytes significantly. But MB augmented the [Ca(2+)](i) transient in the cardiac myocytes significantly. These results suggest that VNP attenuates [Ca(2+)](i) transient induced by Iso. This effect is possibly achieved by binding VNP with the natriuretic peptide GC receptors in the myocytes, leading to an increase in intracellular cGMP.
Animals ; Atrial Natriuretic Factor ; pharmacology ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cyclic GMP ; metabolism ; Depression, Chemical ; Female ; Guanylate Cyclase ; metabolism ; Isoproterenol ; pharmacology ; Male ; Myocytes, Cardiac ; metabolism ; Rats ; Receptors, Atrial Natriuretic Factor ; metabolism

Atrial natriuretic peptide(ANP), a peptide hormone synthesized mainly in the cardiac atrium, has an important physiological role on the regulation of body fluid and electrolyte balance. Extraatrial ANP system has been reported. The presence of ANP in eye has also been reported. ANP in the eye has claimed to control the intraocular pressure. However, the presence of ANP and its receptors in the intraocular tissues are controversial. Therefore, the purpose of the present study was to determine the characteristics of molecular nature of ANP and its receptors in variable intraocular tissues of cow. Immunoreactive ANP was detected in aqueous humor(10+/-1 pg/ml), cornea (3.6+/-0.5 pg/mg), ciliary body(2.62+/-0.6 pg/mg), choroid(2.1+/-0.5 pg/mg), retina (1.7+/-0.2 pg/mg)and iris(1.4+/-0.5 pg/mg). Chromatographic characterization of molecular profile of ANP showed major peak corresponding to small molecular weight forms of ANP and minor peak corresponding to proANP. ANP mRNA was detected in the cornea, retina and ciliary body using reverse transcriptase-polymerase chain reaction. The production of cGMP by the activation of guanylyl cyclase was stimulated by ANP, BNP and CNP in tissue membranes of cornea, ciliary body and iris. Autoradiographic study showed that the corneal endothelium had A, B, and C subtypes of natriuretic peptide receptor. Longitudinal fibers of ciliary muscle and retina had A subtype of natriuretic receptor. These results suggest that the bovine eye has its own ANP system and ANP may have an important paracrine or autocrine function in the eye.
Aqueous Humor ; Atrial Natriuretic Factor ; Autoradiography ; Body Fluids ; Ciliary Body ; Cornea ; Endothelium, Corneal ; Guanylate Cyclase ; Intraocular Pressure ; Iris ; Membranes ; Molecular Weight ; Receptors, Peptide ; Retina ; RNA, Messenger ; Water-Electrolyte Balance

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*

Nitric oxide (NO) and atrial natriuretic peptide (ANP) may induce vascular relaxation by increasing the production of cyclic guanosine monophosphate (cGMP), an important mediator of vascular tone during sepsis. This study aimed to determine whether regulation of NO and the ANP system is altered in lipopolysaccharide (LPS)-induced kidney injury. LPS (10 mg.kg-1) was injected in the tail veins of male Sprague-Dawley rats; 12 hours later, the kidneys were removed. Protein expression of NO synthase (NOS) and neutral endopeptidase (NEP) was determined by semiquantitative immunoblotting. As an index of synthesis of NO, its stable metabolites (nitrite/nitrate, NOx) were measured using colorimetric assays. mRNA expression of the ANP system was determined by real-time polymerase chain reaction. To determine the activity of guanylyl cyclase (GC), the amount of cGMP generated in response to sodium nitroprusside (SNP) and ANP was calculated. Creatinine clearance decreased and fractional excretion of sodium increased in LPS-treated rats compared with the controls. Inducible NOS protein expression increased in LPS-treated rats, while that of endothelial NOS, neuronal NOS, and NEP remained unchanged. Additionally, urinary and plasma NOx levels increased in LPS-treated rats. SNP-stimulated GC activity remained unchanged in the glomerulus and papilla in the LPS-treated rats. mRNA expression of natriuretic peptide receptor (NPR)-C decreased in LPS-treated rats, while that of ANP and NPR-A did not change. ANP-stimulated GC activity reduced in the glomerulus and papilla. In conclusion, enhancement of the NO/cGMP pathway and decrease in ANP clearance were found play a role in the pathogenesis of LPS-induced kidney injury.
Animals ; Atrial Natriuretic Factor ; Creatinine ; Guanosine Monophosphate ; Guanylate Cyclase ; Humans ; Immunoblotting ; Kidney ; Male ; Neprilysin ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type II ; Nitroprusside ; Plasma ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, Peptide ; Relaxation ; RNA, Messenger ; Sepsis ; Sodium ; Veins

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.

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*