Regulation of plasma endothelin by salt in salt-sensitive hypertension.

BACKGROUND: Salt dependency of blood pressure (BP) characterizes most models of experimental hypertension in which endothelins play a significant vasoconstrictor role. Despite this, there are no data on the regulation of plasma endothelin by salt balance in human hypertension. METHODS AND RESULTS: Plasma endothelin was measured in 47 patients with essential hypertension. Endothelin, catecholamine, and plasma renin activity (PRA) responses to 24-hour sodium deprivation (decreasing Na) were assessed in 29 of these patients. Endothelin was higher in hypertensive patients (4.6+/-0.2 fmol/mL) than in 20 control subjects (3.3+/-0.3 fmol/mL, P:<0.002), was correlated with BP, and was negatively associated with PRA (P:<0.04). Salt-sensitive, salt-resistant, and indeterminate groups were defined by the tertiles of the t statistic for the difference in BP before and after decreasing Na. Systolic BP falls were -15+/-1, -2+/-2, and -9+/-1 mm Hg, respectively. PRA, its response to decreasing Na, and its level after decreasing Na were lowest (albeit nonsignificant) in salt-sensitive patients. Baseline catecholamine and endothelin levels did not differ among the groups. In response to decreasing Na, catecholamines increased more significantly in salt-sensitive patients (+2.4+/-0.9 nmol/L) than in the other groups (0.4+/-0.2 and 0.7+/-0.2 nmol/L for indeterminate and salt-resistant groups, respectively; P:<0.03), whereas endothelin increased in the salt-sensitive group (0.8+/-0.3 fmol/mL), decreased in the salt-resistant group (-0.4+/-0.3 fmol/mL), and sustained minimal change in the indeterminate group (0.2+/-0.3 fmol/mL) (P:<0.04). Thus, endothelin levels in the salt-depleted state were highest in salt-sensitive patients (5.2+/-0.4 fmol/mL) versus the other groups (3.4+/-0.4 and 4.4+/-0.4 fmol/mL for salt-resistant and indeterminate groups, respectively) (P:<0.02). Changes in endothelin during decreasing Na and levels after decreasing Na were correlated with changes in catecholamines (P:<0.02). CONCLUSIONS: -Our data suggest that salt-depleted salt-sensitive hypertensives with blunted renin responses exhibit enhanced catecholamine-stimulated endothelin levels and may therefore respond better than unselected patients with essential hypertension to endothelin receptor blockers.

Sympathoinhibitory function of the alpha(2A)-adrenergic receptor subtype.

Presynaptic alpha(2)-adrenergic receptors (alpha(2)-AR) are distributed throughout the central nervous system and are highly concentrated in the brain stem, where they contribute to neural baroreflex control of blood pressure (BP). To explore the role of the alpha(2A)-AR subtype in this function, we compared BP and plasma norepinephrine and epinephrine levels in genetically engineered mice with deleted alpha(2A)-AR gene to their wild-type controls. At baseline, the alpha(2A)-AR gene knockouts (n=11) versus controls (n=10) had higher systolic BP (123+/-2.5 versus 115+/-2.5 mm Hg, P<0. 05), heart rate (730+/-15 versus 600+/-18 b/min, P<0.001), and norepinephrine (1.005+/-0.078 versus 0.587+/-0.095 ng/mL, P<0.01), respectively. When submitted to subtotal nephrectomy and given 1% saline as drinking water, both alpha(2A)-AR gene knockouts (n=14) and controls (n=14) became hypertensive, but the former required 15. 6+/-2.5 days versus 29.3+/-1.4 days for the controls (P<0.001). End-point systolic BP was similar for both at 155+/-2.1 versus 152+/-5.2 mm Hg, but norepinephrine and epinephrine levels were twice as high in the knockouts at 1.386+/-0.283 and 0.577+/-0.143 versus 0.712+/-0.110 and 0.255+/-0.032 ng/mL, respectively, P<0.05 for both. We conclude that the alpha(2A)-AR subtype exerts a sympathoinhibitory effect, and its loss leads to a hypertensive, hyperadrenergic state.

Expression of alpha2-adrenergic receptors in normal and atherosclerotic rabbit aorta.

Alpha2-adrenergic receptors (alpha2-ARs) in vascular smooth muscle cells are known to mediate vasoconstriction; however, it is unknown which of the 3 subtypes of alpha2-AR (alpha2A, alpha2B, or alpha2C) is expressed in vascular tissue. We have used subtype-specific probes in in situ hybridization and RNase protection assays to analyze the expression of alpha2-AR in the thoracic aorta of New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits, a model for atherosclerosis. We found that the alpha2A-AR mRNA was in endothelial and smooth muscle cells in both NZW and WHHL aorta. In addition, the shoulders and subendothelial regions of the atherosclerotic lesions in WHHL aorta showed abundant expression of alpha2A-AR mRNA. Antibodies to macrophage (RAM-11) and smooth muscle cell (HHF-35) antigens were used to localize macrophage and smooth muscle cells in aortic sections from WHHL rabbits. The expression of alpha2A-AR mRNA within the lesions of WHHL rabbits correlated with the presence of infiltrating macrophages. We discuss the potential role of alpha2A-ARs in macrophage function and in promoting atherosclerosis.

Diverse tissue expression of rat alpha 2-adrenergic receptor genes.

Previously, we have reported two major alpha 2-adrenergic receptor transcripts in rat brain of 3.8 and 3.0 kb and the cloning and characterization of the rat brain complementary DNA (cDNA) (RB alpha 2C) specific for the 3.0-kb messenger RNA. In this report, we used rat brain cDNAs specific for the 3.0 and 3.8 kb transcripts, which encode the alpha 2C- and alpha 2A-adrenergic receptors, respectively, and the RNG alpha 2 cDNA, which encodes for the nonglycosylated alpha 2B-adrenergic receptor in rat, to study tissue-specific expression of the three alpha 2-adrenergic receptor genes in rat. To eliminate cross-hybridization of probes with transcripts from other alpha 2 genes, we subcloned fragments that encode for the highly divergent third cytoplasmic loop of each rat alpha 2-adrenergic receptor cDNA and used RNase protection analysis to detect specific transcripts. We show that the three rat alpha 2-adrenergic receptor genes have diverse patterns of tissue expression, and although transcripts specific for each alpha 2-adrenergic receptor gene are found in brain and kidney, the levels of expression of each subtype differ in these tissues. We speculate on the significance of tissue-specific expression of the alpha 2-adrenergic receptor genes.

Cloning and expression of a rat brain alpha 2B-adrenergic receptor.

We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors.

High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line.

We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.

Effect of arginine vasopressin on coronary and systemic hemodynamics in man.

Arginine vasopressin is a potent constrictor of isolated arterial segments in vitro. However, it is disputed whether, in vivo, arginine vasopressin acts as either a systemic or coronary vasoconstrictor. The purpose of this study was to determine the effect of arginine vasopressin on coronary and systemic vascular tone in man. Six patients undergoing routine diagnostic cardiac catheterization were studied. At angiography, all patients were found to have severe coronary artery disease. Cardiac and systemic pressures and coronary blood flow as well as serum arginine vasopressin and osmolality levels were measured before and after administration of hypertonic contrast dye and then following intravenous injection of an analog inhibitor of arginine vasopressin. At baseline, serum arginine vasopressin levels were within the normal range in all patients, but, following infusion of contrast dye, rose above physiological levels in 5 patients. In these latter patients, there were no significant changes in coronary and systemic hemodynamics between baseline (condition 1), following administration of contrast dye (condition 2) and 10 minutes after injection of arginine vasopressin inhibitor (condition 3). Mean coronary sinus blood flow (ml/min) and mean coronary vascular resistance (dyne.sec.cm-5) were 184 +/- 49 and 41,235 +/- 8275 during condition 1, 204 +/- 62 and 39,442 +/- 8781 during condition 2, and 192 +/- 75 and 44,930 +/- 11455 during condition 3. Mean arterial pressure (mm Hg) and mean systemic vascular resistance (dyne.sec.cm-5) during conditions 1, 2 and 3 were 105 +/- 12 and 1454 +/- 174, 114 +/- 21 and 1432 +/- 210, and 114 +/- 15 and 1436 +/- 147, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Central alpha-adrenoceptors during the development of hypertension in rats on high and low salt intake.

Our purpose was to investigate the binding characteristics of central alpha-adrenoceptors during the early stages of the development of hypertension in rats on high and low salt (NaCl) intake. We measured alpha 1-[( 3H]prazosin) and alpha 2-[( 3H]rauwolscine) binding in membranes of the hypothalamus and medulla oblongata of six groups of young Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and subtotally nephrectomized WKY (SN) rats with mean arterial blood pressure (MAP) ranging from normotensive to hypertensive levels after 1 week of salt restriction or loading. In the hypothalamus the SN-high salt rats and both SHR groups had elevated alpha 1-number but there was no change in alpha 2-number. Moreover, MAP was positively correlated with mean hypothalamic alpha 1-number in the six groups. In the medulla oblongata alpha 1-number was unaffected. However, high salt diet influenced medullary alpha 2-binding in the opposite manner in WKY rats versus SN rats and SHR. In these latter groups the affinity was increased and the number decreased in response to high salt intake. Furthermore, a positive correlation between MAP and mean alpha 1:alpha 2 ratio existed in both the hypothalamus and the medulla of the six groups. The data suggest that hypothalamic alpha 1-binding capacity was increased in SHR due principally to a genetic condition which is mimicked by salt loading in the SN rats. Medullary alpha 2-adrenoceptors of WKY, which remained normotensive despite salt loading, responded differently to high salt intake than those of the SN and SHR, whose blood pressure rose significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a sodium-induced activation of central neurogenic mechanisms in one-kidney, one-clip renal hypertensive rats.

The mechanisms sustaining high blood pressure in conscious one-kidney, one-clip Goldblatt rats were evaluated with the use of SK&F 64139, a phenylethanolamine N-methyltransferase inhibitor capable of crossing the blood-brain barrier and of captopril, an angiotensin converting enzyme inhibitor. The rats were studied 3 weeks after left renal artery clipping and contralateral nephrectomy. During the developmental phase of hypertension, two groups of rats were maintained on a regular salt (RNa) intake, whereas two other groups were given a low salt (LNa) diet. On the day of the experiment, the base-line mean blood pressure measured in the LNa rats (177.4 +/- 5.2 mm Hg, mean +/- S.E., n = 15) was similar to that measured in the RNa rats (178.7 +/- 5.4 mm Hg, n = 16). SK&F 64139 (12.5 mg p.o.) induced a significantly more pronounced (P less than .001) blood pressure decrease in the RNa rats (-25.6 +/- 3.6 mm Hg, n = 8) than in the LNa rats (-4.3 +/- 3.3 mm Hg, n = 7) during a 90-min observation period. On the other hand, captopril (10 mg p.o.) normalized blood pressure in LNa rats (n = 8), but produced only a 13.4 mm Hg blood pressure drop in RNa rats (n = 8). RNa rats treated with SK&F 64139 were found to have decreased phenylethanolamine N-methyltransferase activity by an average 80% in selected brain stem nuclei when compared with nontreated rats. No significant difference in plasma catecholamine levels was found between the RNa and LNa rats. These results suggest that, in this experimental model of hypertension, the sodium ion might increase the model of hypertension, the sodium ion might increase the vasoconstrictor contribution of the sympathetic system via a centrally mediated neurogenic mechanism while at the same time it decreases the renin-dependency of the high blood pressure.

Blood pressure response to central and/or peripheral inhibition of phenylethanolamine N-methyltransferase in normotensive and hypertensive rats.

We studied the effects on blood pressure and heart rate of two different phenylethanolamine N-methyltransferase (PNMT) inhibitors in normotensive, in two-kidney renal hypertensive, and in deoxycorticosterone-salt (DOC-salt) hypertensive rats. One compound (SK&F 64139) blocks the conversion of norepinephrine to epinephrine in both the central and the peripheral nervous system, whereas the other (SK&F 29661) does not cross the blood-brain barrier and therefore is active mostly in the adrenal glands. In the rats given SK&F 29661, practically no acute blood pressure changes were in the adrenal glands. In the rats given SK&F 64139 induced only a minor blood pressure and heart rate response in normotensive and two-kidney renal hypertensive rats. However, in DOC-salt hypertensive rats, it reduced arterial pressure to approximately normal levels and concomitantly slowed pulse rate. There was a close correlation between the magnitude of the blood pressure response observed in all SK&F 64139-treated animals and the control plasma norepinephrine (4 = -0.795, P less than 0.001) and epinephrine (r = -0.789, P less than 0.001) levels. These results suggest an important role for central epinephrine in regulating the peripheral sympathoadrenomedullary and the baroreceptor reflex activity, particularly when the maintenance of the high blood pressure is not renin-dependent.

Effects of tyrosine infusion in normotensive and hypertensive rats.

To clarify further the action of acute administration of L-tyrosine in lowering blood pressure, L-tyrosine ethylester was infused intravenously into awake [deoxycorticosterone acetate (DOCA)-salt] hypertensive rats, two-kidney Goldblatt hypertensive rats, and normotensive rats. The effects of tyrosine were measured on arterial pressure, heart rate, plasma catecholamine levels, and plasma renin activity. Blood pressure and heart rate were lowered in all groups despite significant elevation of plasma dopamine in all groups and epinephrine in the hypertensive groups, norepinephrine did not rise significantly, and plasma renin activity was always found to be within the ranges expected for each model. It was concluded that tyrosine produced the progressive decline in blood pressure and heart rate by bringing about a sustained state of parasympathetic dominance, as effective sympathetic compensation did not occcur. This could be attributed to increased alpha-adrenergic activity in certain sites in brain secondary to increased catecholaminergic activity in these areas.