guanylyl-imidodiphosphate and Hypertension

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) >> Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.

Topics: Animals; Binding Sites; Dose-Response Relationship, Drug; Dynorphins; Estrenes; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; Heart; Hypertension; Male; Myocardium; Neomycin; Nociceptin; Opioid Peptides; Pyrrolidinones; Rats; Rats, Inbred SHR; Reverse Transcriptase Polymerase Chain Reaction; Synaptosomes; Time Factors

Renal dopamine-1 (DA-1) receptors are involved in the regulation of sodium transport in several nephron segments, including the proximal convoluted tubule (PCT). DA-1 receptors in the PCT and cortical collecting duct of normotensive rats are linked to the stimulation of adenylyl cyclase (AC). We have reported a defect in the DA-1 receptor/AC coupling in the PCT of the spontaneously hypertensive rat (SHR) of the Okamoto-Aoki strain. Hyperactivity and hypertension are both expressed in the SHR. To determine if the DA-1 receptor coupling defect is associated with hyperactivity or hypertension, we studied the DA-1 receptor in the PCT of two new inbred rat strains derived from the SHR: the hyperactive WKHA and the hypertensive WKHT rat. Tail-cuff blood pressures taken at 4 weeks indicated that WKHT rats were not hypertensive (86 +/- 3 mm Hg, n = 6), whereas at 12 weeks systolic pressures in both SHR and WKHT rats exceeded 150 mm Hg. Hyperactivity, however, was noted in WKHA rats even at this early age. Basal AC activity was similar in WKHA and WKHT PCT in either age group. In the older rats, the DA-1 agonist fenoldopam (10(-7) mol/L) stimulated AC activity in WKHA (70.6 +/- 16.1 fmol per 3 mm PCT per 20 minutes, n = 3) but not in WKHT PCT (43.3 +/- 5.3 fmol per 3 mm PCT per 20 minutes, n = 4). Gpp(NH)p (10(-5) mol/L), a nonhydrolyzable GTP analogue, stimulated AC activity to a similar extent in WKHA and WKHT PCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Adenylyl Cyclases; Aging; Animals; Benzazepines; Blood Pressure; Colforsin; Dopamine Agents; Fenoldopam; Guanylyl Imidodiphosphate; Hypertension; Iodine Radioisotopes; Kidney; Kidney Cortex; Kidney Tubules, Collecting; Kidney Tubules, Proximal; Kinetics; Male; Motor Activity; Nephrons; Radioligand Assay; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY; Receptors, Dopamine D1; Sodium

The present study investigated whether high salt intake (8%) in Dahl salt-sensitive and salt-resistant rats with and without hypertension produces a heterologous desensitization of cardiac adenylyl cyclase as observed in various types of hypertension and human heart failure. In membranes from Dahl salt-sensitive rats on a high-salt diet (8%) basal, isoproterenol-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylyl cyclase was reduced compared with the low-salt (0.4%) group and Dahl salt-resistant rats on either 0.4% or 8% sodium chloride. The activity of the catalyst was depressed, and the expression of the immunodetectable inhibitory G proteins Gi alpha was increased in Dahl salt-sensitive rats on 8% sodium chloride, whereas the density of beta-adrenergic receptors and the activity of the stimulatory G protein Gs alpha reconstituted into Gs alpha-deficient S49 cyc- mouse lymphoma cell membranes were unchanged in any condition studied. We conclude that high salt intake in salt-sensitive hypertensive Dahl rats produces hypertension, cardiac hypertrophy, and heterologous desensitization of cardiac adenylyl cyclase. The latter alteration is due to an increase of Gi alpha proteins and a depressed catalyst activity of adenylyl cyclase. The results demonstrate that heterologous adenylyl cyclase desensitization can precede the development of contractile dysfunction in later stages and can occur independently of changes in beta-adrenergic receptors.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Amino Acid Sequence; Animals; Blood Pressure; Cardiomegaly; Cell Membrane; Colforsin; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; Hypertension; Immune Sera; Kinetics; Lymphoma; Male; Mice; Molecular Sequence Data; Myocardium; NAD; Oligopeptides; Radioimmunoassay; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta; Recombinant Proteins; Sodium Chloride; Transducin; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

The muscarinic receptors in the aorta of the normo- and hypertensive rats were characterised with tritiated acetylcholine (3H-ACh) and various muscarinic receptor antagonists. The binding of 3H-ACh to the endothelial membranes of the normotensive Wistar Kyoto rats (WKY) and the spontaneously hypertensive rats (SHR) was displaceable by nanomolar range of scopolamine but only by micromolar range of atropine and homatropine. The reverse was observed with the muscle binding sites, i.e. the 3H-ACh was displaceable by nanomolar range of atropine and homatropine but only by micromolar range of scopolamine. Pirenzepine and 4-diphenyl-acetoxy-N-methyl-piperidine metobromide (4-DAMP) displaced the binding of 3H-ACh from both tissues in the nano to micromolar range, with the displacement from the endothelial binding sites occurring at lower concentration range of the ligands. The apparent IC50 values of both compounds for the smooth muscle were 9 and 16 times greater than those for the endothelial binding sites respectively. When saturated with guanylyl-imididiphosphate (GppNHp), conversion of high to low-affinity binding site occurred in both tissues of the WKY but only in the smooth muscle of the SHR. GppNHp had no apparent effect on the binding of 3H-ACh to the endothelial binding sites confirming that the high-affinity site for 3H-ACh was missing in the endothelium of the SHR.

Topics: Acetylcholine; Animals; Aorta; Atropine; Binding, Competitive; Endothelium, Vascular; Guanylyl Imidodiphosphate; Hypertension; In Vitro Techniques; Kinetics; Muscle, Smooth, Vascular; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Muscarinic; Scopolamine; Tropanes

The natriuretic effect of dopamine-1 (DA-1) agonists is reduced in spontaneously hypertensive rat (SHR), partly because of defective DA-1 receptor-adenylate cyclase (AC) coupling in renal proximal convoluted tubules. To investigate this defective coupling, DA-1 dopamine receptors from renal proximal tubules were solubilized and reconstituted into phospholipid vesicles. The binding of DA-1-selective ligand [125I]SCH 23982 was specific and saturable, with no differences in receptor density or Kd between SHR and normotensive rats (Wistar-Kyoto rats; WKY). Competition experiments of the reconstituted DA-1 dopamine receptors in WKY with a DA-1-selective agonist, SKF R-38393, revealed the presence of high- (Kh = 350 +/- 209 nM) and low-affinity (Kl = 70,500 +/- 39,500 nM) binding sites. 100 microM Gpp(NH)p abolished the agonist high-affinity sites, converting them to a low-affinity state (Ki = 33,650 +/- 10,850 nM). In SHR, one affinity site was noted (Ki = 13,800 +/- 500) and was not modulated by Gpp(NH)p (Ki = 11,505 +/- 2,295). The absence of guanine nucleotide-sensitive agonist high-affinity sites may explain the defective DA-1/AC coupling mechanism in the SHR.

Topics: Animals; Benzazepines; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hypertension; In Vitro Techniques; Kidney Tubules, Proximal; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Dopamine; Receptors, Dopamine D1; Solubility

Sodium ions markedly decreased in vitro renal alpha 2-adrenoceptor affinity for epinephrine in Sabra hypertensive (SBH) but not in normotensive (SBN) rats. Under these conditions, affinity of alpha 1-adrenoceptor for epinephrine was unchanged in SBH and SBN rats. If these data could be confirmed in vivo, the sodium ion, by acting as an inhibitor, could modify the effect of agonists on renal alpha 2-adrenoceptors in SBH rats. Conversely, the absence of sodium regulation in SBN rats might represent a genetically mediated change responsible for the resistance to the development of salt-induced hypertension.

Topics: Animals; Drug Resistance; Epinephrine; Guanylyl Imidodiphosphate; Hypertension; In Vitro Techniques; Kidney; Male; Rats; Receptors, Adrenergic, alpha; Sodium; Sodium Chloride; Yohimbine

1. In the normotensive rat (WKY) the guanine nucleotide, Gpp(NH)p, and sodium reduced the ability of the dopaminergic agonist fenoldopam to compete for the [3H]-SKF-38393 (dopamine-1 agonist) binding to the renal proximal tubular DA-1 receptors. 2. In the spontaneously hypertensive rat (SHR) Gpp(NH)p failed to reduce the affinity of fenoldopam for [3H]-SKF-38393 binding to renal tubular DA-1 receptors. 3. Sodium reduced the affinity of fenoldopam for [3H]-SKF-38393 binding in the SHR renal proximal tubular cells, but to a lesser extent than the WKY. 4. We conclude that the SHR has a defective DA-1 receptor or Gs/receptor coupling which interferes with the ability of Gpp(NH)p to act on the DA receptor/G protein complex.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Dopamine Agents; Fenoldopam; Guanylyl Imidodiphosphate; Hypertension; Kidney Tubules, Proximal; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Dopamine; Receptors, Dopamine D1; Sodium

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp[NH]p, isoproterenol plus GTP or Gpp[NH]p was significantly greater in the SHR. Beta-receptor density measured by [3H]DHA binding was unaltered. The dissociation constant, Kd, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 +/- 0.45 nM) compared with the WKY (8.53 +/- 0.82 nM). Activity of Gs was assessed by complementing S49 cyc- acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp[NH]p, and isoproterenol plus Gpp[NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary Gs in the hypertensive rats.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Colforsin; Guanylyl Imidodiphosphate; Hypertension; Isoproterenol; Lung; Male; Rats; Rats, Inbred Lew; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY; Receptors, Adrenergic, beta

The hormone-sensitive adenylate cyclase system of plasma membrane is composed of at least three types of proteins: hormone receptors, activatory (Gs) and inhibitory (Gi) guanine nucleotide-regulatory proteins and the catalytic unit (C). Abnormal hormonal regulations of platelet adenylate cyclase in both humans and experimental animals have been reported to occur in hypertension. However, little is known about the mechanisms for these alterations. The aim of the present study was to compare the activity of C and the inhibitory capacity of Gi in platelet membranes from spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). Adenylate cyclase activity of 40,000 g membranes was assessed at pH 7.5 with 0.1 mM (alpha-32P) ATP and an appropriate bivalent cation (Mn2+ or Mg2+). Under incubation conditions that uncoupled C from Gs and Gi (25 mM MnCL2, 100 microM forskolin), a significantly lower adenylate cyclase activity was measured in membranes from SHR rats (2.07 +/- 0.12 vs 2.36 +/- 0.1 nmol cAMP/mn/mg of protein, p less than 0.05). This difference between the two strains was also observed in platelet homogenates. In a second kind of experiments, membranes were incubated with 2.1 mM MgCl2 instead of MnCl2. In both strains of rats, low concentrations of Gpp (NH)p (10 to 300 nM) inhibited adenylate cyclase activity when stimulated by 50 microM forskolin. However, the maximal extent of inhibition was significantly reduced in hypertensive rats (49.7 +/- 2.4 vs 60.5 +/- 2.3 p. 100, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Animals; Blood Platelets; Cell Membrane; Colforsin; Cyclic AMP; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hypertension; Magnesium; Male; Manganese; Rats; Rats, Inbred SHR; Rats, Inbred WKY

The cardiac adenylate cyclase activity from normotensive and spontaneously hypertensive (SHR) rats was studied as a function of temperature between 17 degrees and 37 degrees C. Arrhenius plots of adenylate cyclase activity displayed a break around 31 degrees C when tested under basal conditions or in the presence of GTP but were linearized after activation with p[NH]ppG, NaF, secretin, glucagon or isoproterenol. The energy of activation of adenylate cyclase activity in the presence of GTP (9.5 +/- 0.5 kcal/mol) was significantly lower than in the presence of p[NH]ppG (17.7 +/- 0.8 kcal/mol). A hormone was without effect on the energy of activation observed with either GTP or p[NH]ppG but the simultaneous presence of hormone and nucleotide increased markedly the activity of the enzyme. The energies of activation were analyzed in terms of variation of enthalpy and entropy and discussed in relation with the process of activation and coupling of the guanine nucleotide regulatory protein. These thermodynamic characteristics were similar in cardiac membranes from normotensive and spontaneously hypertensive rats, suggesting that the impairment of hormone-stimulated adenylate cyclase activity observed in the heart membranes of hypertensive rats was not a consequence of a defect in the activation process of the enzyme.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Guanylyl Imidodiphosphate; Hypertension; Kinetics; Magnesium; Male; Myocardium; Rats; Rats, Inbred Strains; Rats, Mutant Strains; Sodium Fluoride; Species Specificity; Temperature; Thermodynamics

Particulate fractions of myocardium taken from spontaneously hypertensive rats (SHR) contained an adenylate cyclase system that was less responsive than normotensive Wistar Kyoto rats to norepinephrine, isoproterenol (mixed beta-agonist), salbutamol (beta 2-agonist), dobutamine (beta 1-agonist), dopamine, histamine, and glucagon. Addition of 5'-guanylylimidodiphosphate to the SHR myocardial preparation again yielded a lessened sensitivity to all agents except norepinephrine, dopamine, and histamine. Chronic treatment of SHR with clonidine and a high dose of propranolol produced a cardiac enzyme that was insensitive to activation by norepinephrine. Similar treatment with a low dose of propranolol did not alter myocardial responses to norepinephrine.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Albuterol; Animals; Catecholamines; Clonidine; Glucagon; Guanylyl Imidodiphosphate; Histamine; Hypertension; Male; Myocardium; Propranolol; Rats

Topics: Adenylyl Cyclases; Animals; Dose-Response Relationship, Drug; Fluorides; Glucagon; Guanylyl Imidodiphosphate; Hypertension; Isoproterenol; Kinetics; Myocardium; Rats; Secretin

Topics: Adenylyl Cyclases; Animals; Calcium; Guanylyl Imidodiphosphate; Hypertension; In Vitro Techniques; Isoproterenol; Magnesium; Manganese; Membranes; Myocardium; Rats