8-bromocyclic-gmp and Hypertension

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

Topics: Animals; Blood Pressure; Cell Line; Coronary Artery Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Fluoresceins; Hypertension; Male; Mesenteric Arteries; Mice; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Peptide Fragments; Protein Kinase Inhibitors; Spodoptera; Vasoconstriction; Vasodilation

Mechanical load and ischemia induce a series of adaptive physiological responses by activating the expression of O(2)-regulated genes, such as hypoxia inducible factor-1alpha (HIF-1alpha). The aim of this study was to explore the interaction between HIF-1alpha and soluble guanylate cyclase (sGC) and its second messenger cGMP in cultured cardiomyocytes exposed to hypoxia and in pressure-overloaded heart. In cultured cardiomyocytes of neonatal rats, either sGC stimulator BAY 41-2272 or cGMP analog 8-bromo-cGMP decreased the hypoxia (1% O(2)/5% CO(2))-induced HIF-1alpha expression, whereas the inhibition of protein kinase G by KT-5823 reversed the effect of BAY 41-2272 on the expression under hypoxic conditions. In pressure-overloaded heart induced by suprarenal aortic constriction (AC) in 7-wk-old male Wistar rats, the administration of BAY 41-2272 (2 mg.kg(-1).day(-1)) for 14 days significantly suppressed the protein expression of HIF-1alpha (P < 0.05), vascular endothelial growth factor (P < 0.01), and the number of capillary vessels (P < 0.01) induced by pressure overload. This study suggests that the pharmacological sGC-cGMP stimulation modulates the HIF-1alpha expression in response to hypoxia or mechanical load in the heart.

Topics: Animals; Animals, Newborn; Carbazoles; Cardiomegaly; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Down-Regulation; Enzyme Activation; Enzyme Activators; Guanylate Cyclase; Hypertension; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Myocytes, Cardiac; Neovascularization, Physiologic; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems; Soluble Guanylyl Cyclase; Time Factors; Vascular Endothelial Growth Factor A; Ventricular Remodeling

Recent studies have shown that tamoxifen, which belongs to a group called selective estrogen receptor modulators (SERM), may exert protective effects against cardiovascular diseases and stroke in postmenopausal women. On the other hand, abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. The present study was performed to investigate the effects of tamoxifen on cell membrane fluidity (a reciprocal value of membrane microviscosity) in normotensive and hypertensive postmenopausal women.. We used an electron paramagnetic resonance (EPR) and spin-labeling method. Tamoxifen significantly decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(o)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in normotensive postmenopausal women (mean +/- SEM, order parameter value; control 0.719 +/- 0.002, n = 41; tamoxifen 1 x 10(-7) mol/L 0.704 +/- 0.002, n = 41, P < .0001; tamoxifen 1 x 10(-6) mol/L 0.696 +/- 0.002, n = 41, P < .0001; tamoxifen 1 x 10(-5) mol/L 0.692 +/- 0.002, n = 41, P < .0001). The finding indicated that tamoxifen increased the membrane fluidity and improved the membrane microviscosity of erythrocytes. The membrane action of tamoxifen was antagonized by the estrogen receptor antagonist ICI 182,780. The effect of tamoxifen was significantly potentiated by the nitric oxide (NO) donors, l-arginine and S-nitroso-N-acetylpenicillamine, and a cGMP analog 8-bromo-cGMP. In contrast, the change evoked by tamoxifen was counteracted by the NO synthase inhibitors N(G)-nitro-l-arginine-methyl-ester and asymmetric dimethyl-l-arginine. In hypertensive postmenopausal women, the membrane fluidity of erythrocytes was significantly lower than in normotensive postmenopausal women. The effect of tamoxifen on the membrane fluidity was more pronounced in hypertensive postmenopausal women than in normotensive postmenopausal women.. These results showed that tamoxifen increased the membrane fluidity of erythrocytes and improved the rigidity of cell membranes in postmenopausal women, to some extent, through the NO- and cGMP-dependent mechanisms. Furthermore, the greater effect of tamoxifen in hypertensive postmenopausal women suggests that tamoxifen could have a beneficial effect in regulating the blood rheologic behavior and in the improvement of the microcirculation in hypertension.

Topics: Aged; Analysis of Variance; Arginine; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Erythrocyte Membrane; Erythrocytes; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Humans; Hypertension; Membrane Fluidity; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Postmenopause; S-Nitroso-N-Acetylpenicillamine; Selective Estrogen Receptor Modulators; Tamoxifen

The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3', 5' monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65+/-4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64+/-6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-4) M), increased both intracellular and extracellular cGMP (from 1.26+/-0.21 to 88.7+/-12.6 pmol/mg protein and from 0.58+/-0.10 to 9.24+/-1.9 pmol/mL, respectively, P<0.01) and decreased cellular Na+ uptake by 37.4+/-6.8% (P<0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10(-6) M)-induced increase in intracellular cGMP accumulation (from 4.9+/-0.9 to 9.8+/-1.5 pmol/mg protein, P<0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07+/-0.4 to 0.37+/-0.1 pmol/L, P<0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10(-5) M) or its membrane-permeable analogue 8-Br-cGMP (10(-5) M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36+/-0.19 to 289.7+/-29.4 pmol/mg protein, P<0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain con

Topics: Arginine; Biological Transport; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Female; Humans; Hypertension; Ion Transport; Kidney Tubules, Proximal; Male; Middle Aged; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Probenecid; S-Nitroso-N-Acetylpenicillamine; Sodium

Pressure-natriuresis is the physiological protective mechanism whereby elevation of blood pressure induces a rapid increase in renal sodium (Na+) excretion. Pressure-natriuresis abnormalities are common to all forms of hypertension. We tested the hypothesis that pressure-natriuresis is mediated by renal interstitial (RI) cGMP and protein kinase G (PKG). We used anesthetized, uninephrectomized Sprague-Dawley rats and a standard pressure-natriuresis model in which bilateral adrenalectomy and renal denervation was done on rats. Renal perfusion pressure (RPP) was adjusted by manipulating clamps above and below the renal artery, and RI cGMP was quantified by microdialysis. RI cGMP increased from 3.1+/-0.5 to 5.5+/-0.4 fmol/min (P<0.05) when RPP was raised from 100 to 140 mm Hg. This increase in RI cGMP was eliminated by RI infusion of soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-alpha]quinoxalin-1-one (ODQ). Raising RPP from 100 to 140 mm Hg increased urinary sodium excretion from 0.2+/-0.1 to 0.8+/-0.1 micromol/min, fractional sodium excretion from 0.2+/-0.1% to 0.8+/-0.1%, and fractional lithium excretion from 20.1+/-3.0% to 62.7+/-3.7% (all P<0.05). These responses were eliminated by RI infusion of nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester, ODQ, and PKG inhibitors Rp-8-pCPT-cGMP and Rp-8-Br-cGMP. Increasing RPP from 100 to 140 mm Hg decreased fractional proximal sodium reabsorption without influencing fractional distal Na+ reabsorption or glomerular filtration rate. In conclusion, pressure-natriuresis is mediated by RI cGMP and a PKG signaling pathway in target renal proximal tubule cells.

Topics: Adrenalectomy; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Denervation; Enzyme Inhibitors; Female; Glomerular Filtration Rate; Guanylate Cyclase; Hypertension; Ion Transport; Kidney; Kidney Cortex; Kidney Medulla; Kidney Tubules, Proximal; Natriuresis; Nephrectomy; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Sodium; Soluble Guanylyl Cyclase; Thionucleotides

Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca2+ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N(omega)-nitro-L-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amides; Angiotensin II; Animals; Cells, Cultured; Cyclic GMP; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Hypertension; Indoles; Intracellular Signaling Peptides and Proteins; Maleimides; Muscle Proteins; Muscle, Smooth, Vascular; Myosin-Light-Chain Phosphatase; NG-Nitroarginine Methyl Ester; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Threonine

Nitrate tolerance (NT) in hypertension is attributed to reduced activity of soluble guanylyl cyclase (sGC). We examined NT in basilar artery vascular smooth muscle cells (VSMCs) from control rats, rats infused with angiotensin II (Ang; 240 microg/kg per hour for 4 days), which were normotensive, and Ang-hypertensive rats (AHR; 240 microg/kg per hour for 28 days). Ca2+-activated K+ (Maxi-K) channels in VSMCs from AHR showed reduced activation by NO donor, consistent with NT. The concentration-response relationship for 8-Br-cGMP was shifted 2.5-fold to the right, indicating that abnormal sGC alone could not account for NT. Inside-out patches from AHR showed normal activation with exogenous cGMP-dependent protein kinase I (cGKI), suggesting no abnormality downstream of cGKI. We hypothesized that the reduction in apparent affinity of 8-Br-cGMP for cGKI in AHR might be due to a change in relative amounts of cGKIalpha versus cGKIbeta, since cGKIbeta is less sensitive to cGMP activators than cGKIalpha. This was substantiated by showing the following in AHR: (1) reduced effect of the cGKIalpha-selective activator 8-APT-cGMP; (2) reduced total cGKI protein (both isoforms), but an increase in cGKIbeta protein in quantitative immunofluorescence and Western blots; (3) similar changes in cGKI isoforms immunoisolated with Maxi-K channels; and (4) a large increase in cGKIbeta mRNA and a decrease in cGKIalpha mRNA in real-time PCR and Northern blots. Upregulation of cytosolic cGKIbeta was evident 4 days after Ang infusion, before development of hypertension. Our data identify a functional role for cGKIbeta in VSMCs previously ascribed exclusively to cGKIalpha. Ang-induced alternative splicing of cGKI represents a novel mechanism for reducing sensitivity to NO/cGMP.

Topics: Alternative Splicing; Angiotensins; Animals; Blood Pressure; Cell Separation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Drug Tolerance; Female; Guanylate Cyclase; Hypertension; Isoenzymes; Large-Conductance Calcium-Activated Potassium Channels; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitric Oxide Donors; Patch-Clamp Techniques; Phosphoric Diester Hydrolases; Potassium Channels, Calcium-Activated; Protein Kinase C; Rats; Rats, Inbred WKY; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Soluble Guanylyl Cyclase

Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension.. Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 micro m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and K(Ca) channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining.. Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156+/-2 and 155+/-1 mm Hg, respectively) than that of control offspring (137+/-1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A(2) mimetic and dilation to carba-prostaglandin I(2) did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of K(Ca) (another important mediator of NO action) and relaxation to the K(Ca) opener NS1619 were unchanged by antenatal diet.. Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.

Topics: Animals; Chronic Disease; Cyclic GMP; Dietary Proteins; Disease Models, Animal; Endothelium, Vascular; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypertension; In Vitro Techniques; Microcirculation; Neurotransmitter Agents; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Pia Mater; Pregnancy; Prenatal Exposure Delayed Effects; Protein Deficiency; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Vasodilation; Vasodilator Agents; Vasomotor System

Many studies have shown that estrogen may exert cardioprotective effects and reduce the risk of hypertension and coronary events. On the other hand, it has been proposed that cell membrane abnormalities play a role in the pathophysiology of hypertension, although it is not clear whether estrogen would influence membrane function in essential hypertension. The present study was performed to investigate the effects of 17beta-estradiol (E(2)) on membrane fluidity of erythrocytes in normotensive and hypertensive postmenopausal women. We determined the membrane fluidity of erythrocytes by means of an electron paramagnetic resonance and spin-labeling method. In an in vitro study, E(2) significantly decreased the order parameter for 5-nitroxide stearate and the peak height ratio for 16-nitroxide stearate obtained from electron paramagnetic resonance spectra of erythrocyte membranes in normotensive postmenopausal women. The finding indicates that E(2) might increase the membrane fluidity of erythrocytes. The effect of E(2) was significantly potentiated by the NO donor, S-nitroso-N-acetylpenicillamine, and a cGMP analogue, 8-bromo-cGMP. In contrast, the change in the membrane fluidity evoked by E(2) was attenuated in the presence of the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and asymmetric dimethyl-L-arginine. In hypertensive postmenopausal women, the membrane fluidity of erythrocytes was significantly lower than that in normotensive postmenopausal women. The effect of E(2) on membrane fluidity was significantly more pronounced in the erythrocytes of hypertensive postmenopausal women than in the erythrocytes of normotensive postmenopausal women. The results of the present study showed that E(2) significantly increased the membrane fluidity and improved the microviscosity of erythrocyte membranes, partially mediated by an NO- and cGMP-dependent pathway. Furthermore, the greater action of E(2) in hypertension might be consistent with the hypothesis that E(2) could have a beneficial effect in regulating rheological behavior of erythrocytes and could have a crucial role in the improvement of the microcirculation in hypertension.

Topics: Aged; Arginine; Cyclic GMP; Electron Spin Resonance Spectroscopy; Erythrocytes; Estradiol; Female; Humans; Hypertension; In Vitro Techniques; Membrane Fluidity; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Postmenopause; S-Nitroso-N-Acetylpenicillamine

1. Cyclic guanosine monophosphate (cyclic GMP)-mediated mechanism plays an important role in vasodilatation and blood pressure regulation. We investigated the effects of high salt intake on the nitric oxide (NO) - cyclic GMP signal transduction pathway regulating relaxation in aortas of spontaneously hypertensive rats (SHR). 2. Four-week-old SHR and normotensive Wistar-Kyoto rats (WKY) received a normal salt diet (0.3% NaCl) or a high salt diet (8% NaCl) for 4 weeks. 3. In aortic rings from SHR, endothelium-dependent relaxations in response to acetylcholine (ACh), adenosine diphosphate (ADP) and calcium ionophore A23187 were significantly impaired by the high salt intake. The endothelium-independent relaxations in response to sodium nitroprusside (SNP) and nitroglycerin were also impaired, but that to 8-bromo-cyclic GMP remained unchanged. On the other hand, high salt diet had no significant effects on the relaxations of aortic rings from WKY. 4. In aortas from SHR, the release of NO stimulated by ACh was significantly enhanced, whereas the production of cyclic GMP induced by either ACh or SNP was decreased by the high salt intake. 5. Western blot analysis showed that the protein level of endothelial NO synthase (eNOS) was slightly increased, whereas that of soluble guanylate cyclase (sGC) was dramatically reduced by the high salt intake. 6. These results indicate that in SHR, excessive dietary salt can result in downregulation of sGC followed by decreased cyclic GMP production, which leads to impairment of vascular relaxation in responses to NO. It is notable that chronic high salt intake impairs the sGC/cyclic GMP pathway but not the eNOS/NO pathway.

Topics: Acetylcholine; Adenosine Diphosphate; Animals; Aorta, Thoracic; Blood Pressure; Calcimycin; Cyclic GMP; Dose-Response Relationship, Drug; Down-Regulation; Endothelium, Vascular; Guanylate Cyclase; Heart Rate; Hypertension; In Vitro Techniques; Ionophores; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroglycerin; Nitroprusside; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sodium Chloride, Dietary; Solubility; Species Specificity; Vasodilation; Vasodilator Agents

It has been shown that rheological abnormality might be an etiological factor in hypertension. Recent studies have revealed that human erythrocytes possess a nitric oxide (NO) synthase and that this activation might be involved in the regulation of rheological properties of erythrocytes. The present study was undertaken to investigate the role of NO in the regulation of membrane functions of erythrocytes in patients with essential hypertension by means of an electron paramagnetic resonance (EPR) and spin-labeling method. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(0)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner. The finding indicated that the NO donor increased the membrane fluidity of erythrocytes. In addition, the effect of SNAP was significantly potentiated by 8-bromo-cyclic guanosine monophosphate. By contrast, the change of the fluidity induced by SNAP was reversed in the presence of L-N(G)-nitroarginine methyl ester and asymmetric dimethyl L-arginine. In patients with essential hypertension, the membrane fluidity of erythrocytes was significantly lower than in the normotensive subjects. The effect of SNAP was more pronounced in essential hypertension than in normotensive subjects. These results showed that NO increased the membrane fluidity and decreased the rigidity of cell membranes. Furthermore, the greater effect of NO on the fluidity in essential hypertension suggests that NO might actively participate in the regulation of rheological behavior of erythrocytes and have a crucial role in the improvement of microcirculation in hypertension.

Topics: Arginine; Blood Pressure; Calcimycin; Cell Membrane; Cyclic GMP; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocytes; Hemorheology; Humans; Hypertension; Membrane Fluidity; Middle Aged; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Penicillamine; Spin Labels

In the glycerol model of renal injury we describe an acute rise in systemic arterial pressure which is attended by a reduced vasodilatory response to acetylcholine in vivo; vasodilatory responses to verapamil, however, were not impaired. Neither arginine nor sodium nitroprusside diminished this rise in blood pressure; N(omega)-nitro-L-arginine methyl ester (L-NAME) elevated basal mean arterial pressure and markedly blunted the rise in mean arterial pressure following the administration of glycerol. Aortic rings from the glycerol-treated rat demonstrate an impaired vasodilatory response to acetylcholine, an effect not repaired by arginine; the vasodilatory responses to nitric oxide donors, sodium nitroprusside and SIN-1, were also impaired; 8-bromo-cGMP, at higher doses, evinced a vasodilatory response comparable to that observed in the control rings. This pattern of responses was not a nonspecific effect of aortic injury, since aortic rings treated with mercuric chloride, a potent oxidant, displayed an impaired vasodilatory response to acetylcholine but not to sodium nitroprusside. We conclude that in the glycerol model of heme protein-induced tissue injury, there is an acute elevation in mean arterial pressure attended by impaired endothelium-dependent vasodilatation in vitro and in vivo. We suggest that the acute scavenging of nitric oxide by heme proteins depletes the blood vessel wall of its endogenous vasodilator and permeation of heme proteins into the blood vessel wall may contribute to such sustained effects as observed in vitro.

Topics: Acetylcholine; Acute Disease; Animals; Blood Pressure; Cyclic GMP; Disease Models, Animal; Glycerol; Hemeproteins; Hypertension; Kidney; Male; Nitroprusside; Rats; Rats, Sprague-Dawley; Vasodilator Agents

Nitric oxide (NO) plays an important role not only in the regulation of blood vessel tone, but also in the growth of vascular smooth muscle cells (VSMC). The precise mechanism involved in the inhibition of VSMC growth by NO is not known. To further explore the effect of NO on VSMC growth, we examined the effect of NO on the expression of angiotensin II type 1 receptor (AT1-R) that is important for hypertrophy and hyperplasia of VSMC. S-nitroso acetyl DL-penicillamine (SNAP; 200 micromol/L), a potent NO donor, suppressed expression level of AT1-R mRNA by 90% and AT1-R number by 60% after 24 hours of stimulation. The suppressive effect was dose-dependent. Actinomycin D, which is an inhibitor of gene transcription, did not affect the decrease of AT1-R mRNA by NO. Cyclic guanosine monophosphate (cGMP) analogue, 8 bromo-cGMP, did not affect AT1-R mRNA level. Deletion mutants of the promoter region of rat AT1a-R gene were fused to luciferase reporter gene and introduced to VSMC. Transfected cells were stimulated with SNAP, and luciferase activity was measured. Inhibitory effect of NO was still observed in the shortest deletion mutant that contained 61 bp upstream from transcription start site. In this DNA segment, two DNA binding protein were observed by gel mobility shift assay, and one of these binding proteins was decreased on stimulation by NO. NO downregulates AT1-R gene expression independently of cGMP. A DNA binding protein that binds to the proximal promoter region of AT1-R gene may be responsible for this inhibitory effect. The inhibition of AT1-R gene expression may be implicated in the anti-atherogenic property of NO.

Topics: Angiotensin II; Animals; Aorta, Thoracic; Cells, Cultured; Cyclic GMP; Down-Regulation; Hypertension; Kinetics; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; Recombinant Fusion Proteins; RNA, Messenger; S-Nitroso-N-Acetylpenicillamine; Transcription, Genetic; Transfection

In vascular smooth muscle cell (VSMC) cultures from Sprague-Dawley (SD) and hypertensive transgenic rats for the mouse renin gene Ren-2 (TGR), the DNA synthesis, which was analyzed by the uptake of [3H]thymidine, was higher in TGR than SD VSMCs (2.5- to 8-fold, mean of 5.6-fold) under basal conditions. DNA synthesis was increased by fetal calf serum (10%) in SD cells more than in TGR VSMCs, and was decreased by heparin (400 micrograms/ml) and by phorbol-12,13-dibutyrate (10(-7) M) in TGR VSMCs to a higher degree than in SD cells. Neither endothelin (10(-7) M), angiotensinogen (10(-8) M), the renin inhibitor CGP 29,287 (10(-4) M), angiotensin I (10(-7) M), captopril (10(-5) M), angiotensin II (10(-7) M), nor saralasin (10(-6) M) modified DNA synthesis in either type of VSMCs. Sodium nitroprusside (10(-4) and 10(-3) M) increased DNA synthesis in both kinds of VSMCs but in TGR cultures it became toxic at 10(-3) M. 8-Bromocyclic GMP (10(-7) to 10(-5) M) reduced DNA synthesis in SD cells more than in TGR VSMCs. These results suggest that (a) cellular mechanisms of proliferation appear to be more activated in TGR VSMCs, likely involving a protein kinase C-dependent pathway but not the renin-angiotensin system, and (b) in both type of cells, sodium nitroprusside possesses proliferative properties whereas 8-bromocyclic GMP has antiproliferative properties.

Topics: Animals; Animals, Genetically Modified; Captopril; Cell Division; Cells, Cultured; Cyclic GMP; DNA; Endothelins; Femoral Artery; Heparin; Hypertension; Male; Muscle, Smooth, Vascular; Nitroprusside; Phorbol 12,13-Dibutyrate; Rats; Rats, Sprague-Dawley

ANF(99-126) (1 microgram/kg/min x 30 min iv) lowered BP from 198 +/- 3 to 140 +/- 4 mmHg (P less than .05; N = 7), in association with marked diuresis (372.2 +/- 33.9 vs 48.2 +/- 11.5 microliters/kg/min in the control) and natriuresis (62.7 +/- 6.4 vs 6.6 +/- 1.7 muEq/kg/min) in anesthetized SHR. Concomitantly, great increases in plasma cGMP levels and urinary cGMP excretion occurred. Elevation in plasma cGMP due to ANF persisted in SHR with bilateral nephrectomy. SNP (4 micrograms/kg/min x 30 min iv) decreased BP from 192 +/- 3 to 158 +/- 5 mmHg (P less than .05; N = 7). In contrast to ANF, this occurred without significant changes in urine and sodium excretion; alterations in plasma and urinary cGMP were also absent. Furthermore, 8-Br-cGMP (0.3 mg/kg/min x 30 min iv) also lowered BP from 164 +/- 9 to 129 +/- 7 mmHg (P less than .05; N = 6) in the absence of diuresis (8.5 +/- 1.3 vs 19.8 +/- 4.1 microliters/kg/min). Intravenous infusion of 8-Br-cAMP at the same rate did not affect BP and produced a modest but significant increase in sodium and water excretion. Our results indicate that the renal excretory responses to exogenous cGMP or SNP differ from those to ANF. The findings are consistent with a mediating role of cGMP in the vascular but not the renal effects of ANF, since at equally effective hypotensive doses both SNP and 8-Br-cGMP failed to register any significant renal excretory effects.

Topics: Animals; Atrial Natriuretic Factor; Blood Vessels; Cyclic GMP; Hypertension; Kidney; Male; Nitroprusside; Rats; Rats, Inbred SHR

Responsiveness to endothelium-dependent (acetylcholine and A23187) and endothelium-independent (nitroprusside and 8-bromo cyclic guanosine 3',5'-monophosphate [cGMP]) vasodilators was examined in two vascular preparations from hypertensive and normotensive mice. CBA Agouti mice were made hypertensive by exposure to social stress in a complex population cage. After 2 months, the hindquarter vascular bed was pump-perfused at a constant flow with plasma substitute to evaluate changes in perfusion pressure, and helical strips of aorta were suspended in muscle baths for measurement of isometric force generation. Tissues were treated with methoxamine to induce contractile tone. Threshold dilator responses to acetylcholine were elicited at a significantly lower dose in the hindquarters of hypertensive mice than in those from normotensive mice, indicating increased vasodilator sensitivity. In contrast, vasodilator responsiveness to nitroprusside in hindquarters of hypertensive mice did not differ from that in hindquarters of normotensive mice. Aortas from hypertensive mice were more sensitive (lower ED50) to the relaxant effects of acetylcholine and A23187 than those from normotensive mice. The relaxant effects of nitroprusside and 8-bromo cGMP on aortas from hypertensive mice were not significantly different from those in normotensive aortas. Aortic strips that had been rubbed on the lumen surface with a wooden stick did not relax to acetylcholine or A23187. In aortas that were not initially contracted with methoxamine, acetylcholine and A23187 caused small contractions from baseline. The magnitude of these contractile responses were potentiated after removal of the endothelium, and the potentiation was greater in aortas from hypertensive mice. These results demonstrate an increased responsiveness to endothelium-dependent vasodilators in this psychosocial model of hypertension.

Topics: Acetylcholine; Animals; Aorta; Calcimycin; Crowding; Cyclic GMP; Hypertension; Male; Methoxamine; Mice; Nitric Oxide; Nitroprusside; Vasodilation; Vasodilator Agents

Blood vessel responses to relaxant drugs have been reported to change with aging and with the development of hypertension. In view of the requirement of endothelial cells for the activity of many relaxant drugs, we examined the role of the endothelium in the relaxation response of vascular tissue. Aortic and mesenteric ring segments from normotensive and hypertensive rats, ages 5 to 6, 15 to 18 and 30 to 31 weeks, were examined for relaxation to sodium nitroprusside, sodium nitrite, atrial natriuretic factor and 8-bromo-cyclic GMP. Relaxation responses to the nitrovasodilators were reduced progressively with aging in ring segments of Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) with intact endothelium; however, intact SHR ring preparations displayed less relaxation to nitrovasodilators at 15 to 18 and 30 to 31 weeks than those of WKYs. Rubbed (endothelium denuded) ring preparations displayed greatest relaxation to nitrovasodilators with no difference being observed between SHR and WKY preparations at any age tested. Relaxation to atrial natriuretic factor and 8-bromo-cyclic GMP was not different between rubbed and unrubbed ring segments or between SHRs and WKYs, indicating no detectable impairment of the overall relaxation response in the vascular smooth muscle of SHRs. These results suggest that the total functional capacity of vascular smooth muscle to relax to nitrovasodilators is not changed with aging or hypertension. However, the endothelial cells exert modulatory influences upon the vascular smooth muscle to reduce overall responsiveness to nitrovasodilators, an effect that is enhanced with aging and the development of genetic hypertension.

Topics: Aging; Animals; Atrial Natriuretic Factor; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium; Hypertension; Male; Methylene Blue; Muscle Contraction; Muscle Relaxation; Nitroprusside; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sodium Nitrite; Vasodilator Agents