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Objective: To examine the frequency of distribution of allelic polymorphisms of the alpha 2-adrenoceptor gene in normotensive and hypertensive humans., Design: The frequency of alpha 2-adrenoceptor genotypes was compared in the two groups using the chi 2-test., Setting: The Midwest Hypertension Research Center Outpatient Clinic of Creighton University School of Medicine., Study Participants: History was taken from and physical examination performed on each of the 60 hypertensive and 47 normotensive adults., Methods: DNA was extracted from leukocytes from each participants. Twenty restriction endonucleases were used and one restriction fragment length polymorphism (RFLP) was found using a 950-bp restriction fragment from the coding region of the human platelet alpha 2-adrenoceptor gene (ADRA2R) and Bsu36I restriction endonuclease. This probe and Bsu36I restriction endonuclease, in addition to another restriction endonuclease (Dra I), were then used in the study., Results: Three genotype patterns were found. Homozygotes for the Bsu36I RFLP have either a unique 12-kb or a unique 5.8-kb band. Heterozygotes have both bands. The frequency of this alpha 2-adrenoceptor RFLP was calculated. In hypertensives the frequencies of the 12- and 5.8-kb alleles were 0.52 and 0.48, compared with 0.45 and 0.55, respectively, in normotensive, a difference that was not statistically significant., Conclusions: The frequency of the Bsu36I RFLP involving an alpha 2-adrenoceptor gene in hypertensives did not differ significantly from that in normotensives. A genetic linkage study is now under way to test for an association of the Bsu36I RFLP of the alpha 2-adrenoceptor gene with essential hypertension in families.

We examined the functional significance of the presynaptic alpha-adrenergic receptor, to stimulus-induced norepinephrine release. The effects of prazosin and phentolamine, alpha-adrenergic receptor antagonists with different in vitro selectivities for the presynaptic alpha-receptor, on mean arterial pressure and serum renin activity were determined in the conscious rat. Both drugs resulted in dose-related reductions in mean arterial pressure and dose-related increase in heart rate and serum renin activity. However, consistent with the greater selectivity of prazosin for the postsynaptic alpha-receptor, a given reduction in arterial pressure was associated with a lesser increment in heart rate and serum renin activity after prazosin than after the nonselective agent phentolamine. The differential effects of these agents on heart rate and serum renin activity are consistent with different degrees of blockade of a functionally significant presynaptic alpha-receptor.

Circulating angiotensin II is said to inhibit renin release by a direct, intrarenal action. This effect of angiotensin was studied indirectly using the selective angiotensin II antagonist saralasin (1Sar-8-Ala-angiotensin II) in conscious normal, sodium-depleted, and sodium-loaded rats. Saralasin caused a dose-related increase in plasma renin concentration (PRC) in normal and sodium-depleted rats, but had no effect on PRC in sodium-loaded animals. However, saralasin was 300 times more active in sodium-depleted rats than in normal rats. Saralasin caused hypotension and tachycardia in sodium-depleted rats, but not in normals. Propranolol inhibited saralasin-induced renin release by 99% in normal rats and by 75% in sodium-depleted rats but not alter the hypotensive effect of saralasin in the latter. Saralasin potentiated phentolamine-induced renin release, hypotension, and tachycardia in normal rats, and this potentiated renin release was blocked by propranolol. We conclude that a portion of saralasin-elicited renin release in sodium-depleted rats is mediated by hypotensive activation of the carotid baroreceptor reflex which increases sympathetic nervous activity in the kidney. However, in sodium-depleted rats saralasin induced a 42-fold increase in PRC, whereas an equipotent hypotensive dose of the vasodilator hydralazine caused only a 3.5-fold increase in PRC. Thus, we find that saralasin appears to have a selective effect on renin release over and above its hypotensive effect, which suggests an angiotensin-mediated, feedback mechanism inhibitory to renin release. Thus, we have come to the conclusion that for part of saralasin-induced renin release appears to be caused by disinhibition of angiotensin suppression of renin secretion. This "short-loop" feed-back mechanism is closely associated with intrarenal beta-adrenergic receptors, since propranolol impaired saralasin-induced renin release under all circumstances in our experiments.

[3H]Dihydroergocryptine, a nonselective alpha adrenergic antagonist, the alpha-1 selective antagonist, [3H]prazosin and the alpha-2 selective antagonist, [3H]yohimbine, were used to study binding sites in rat renal membranes. To establish a correlation between binding and a biological function, the ability of alpha adrenergic agents to stimulate or inhibit vasoconstriction was quantified in vitro using an isolated perfused kidney preparation. Binding with each radioligand was rapid, saturable and specific. Moreover, the order of potencies of a variety of adrenergic agents, determined by competitive inhibition studies, suggested that the binding of each radioligand was to sites with alpha adrenergic specificity. The total number of binding sites in these rat renal membranes. determined with [3H]dihydroergocryptine (Bmax, 212 fmol/mg of protein; KD, 12.8 nM) was approximately equal to the sum of binding site concentrations determined with the alpha-1 and alpha-2 selective radioligands (Bmax, 57 and 170 fmol/mg of protein; KD, 0.85 and 20 nM, respectively). However, the alpha receptor mediating renal arteriolar vasoconstriction appeared to be of the alpha-1 subtype as there was a close correlation between the in vitro results and the binding data determined with [3H]prazosin (r = 0.93). In addition, in both the functional and [3H]prazosin binding studies, unlabeled prazosin ws 5 to 40-fold more potent than the nonselective antagonist, phentolamine, and 400- to 1500-fold more potent than the alpha-2 antagonist, yohimbine. These studies suggest that rat renal plasma membranes contain binding sites with both alpha-1 and alpha-2 adrenergic receptor specificity, in a ratio of approximately 1:3. Despite the preponderance of alpha-2 receptors, the alpha receptor mediating renal vasoconstriction appears to be of the alpha-1 type.

The mechanism by which clonidine suppresses renin release was investigated in conscious rats. This suppression was studied by means of selected autonomic interventions in conjunction with changes in sodium balance. Serum renin activity and direct arterial pressure were monitored. Clonidine administration suppressed basal (by 68-85%), diuretic-induced (by 89%), and sympathetic nervous system-mediated (by 75-100%) renin release. Cholinergic, ganglionic, and peripheral sympathetic neuronal blockade did not prevent this inhibitory effect of clonidine. These results indicate a peripheral site of action for suppression of renin release by clonidine. The alpha-adrenergic blocking drug phentolamine prevented clonidine suppression of renin release in sodium-depleted rats and was partially effective in normal rats. Phentolamine blocked the decrease in renin caused by clonidine in ganglion-blocked rats. Clozapine, a new neuroleptic agent with alpha-adrenergic blocking activity, or phenoxybenzamine blocked the effect of clonidine on renin release in both sodium-depleted and normal rats. After ganglionic blockade in sodium-depleted rats, clonidine caused a significantly greater suppression of renin release than did an equipressor dose of methoxamine. These data, combined with hemodynamic correlates, suggest that clonidine inhibits renin release by activation of an intrarenal alpha-adrenergic receptor.

During signal transduction, renal adrenoceptors associate with (as in other tissues) specific members of a family of GTP-binding (G) proteins. Renal beta-adrenoceptors activate adenylate cyclase in glomeruli and distal nephrons via an interaction with the stimulatory G protein Gs. alpha 1-Adrenoceptors bind to an as yet uncharacterized G protein and activate phospholipase C to produce the putative second messengers 1,4,5-inositol trisphosphate and diacylglycerol. alpha 1-Adrenoceptors are found in most segments of the nephron where they regulate sodium reabsorption, and metabolic functions such as gluconeogenesis. Renal alpha 2-adrenoceptors which inhibit adenylate cyclase through the inhibitory G protein Gi are located mainly in the proximal convoluted tubule and the collecting ducts. Another population of alpha 2-adrenoceptors has been identified which enhances the sodium-hydrogen exchange across the luminal membrane of proximal tubules. Ongoing research is clarifying the locations and functions of these renal adrenoceptor subtypes.

Catecholamines possessing alpha adrenergic receptor agonist properties induce lightening or reverse melanocyte stimulating hormone darkening of frog skin in vitro. The capacity to activate this alpha receptor by the methyldopa metabolites methyldopamine and methylnorepinephrine was compared with the capacity of the naturally occurring dopa metabolites, dopamine and norepinephrine. Melanocyte stimulating hormone-induced darkening or dispersion of the granules was reversed by each of these metabolites. Methylnorepinephrine was 10 times as potent as norepinephrine, and methyldopamine was 30- to 100-fold more potent than the naturally occurring dopamine. These inhibitory effects on melanocyte stimulating hormone could be blocked or partially impaired using the alpha adrenergic blocker, phentolamine. They were not affected by pretreatment of frogs with the monoamine oxidase inhibitor pheniprazine (Catron) nor by the application of pheniprazine, angiotensin or serotonin in vitro. This neuroendocrine model has alpha adrenergic receptor relationships analogous to those described in the central nervous system for methyldopa metabolites.

Renal alpha 1 and alpha 2-adrenergic receptors were quantified in Dahl salt-sensitive and salt-resistant rats, in Okamoto-Aoki spontaneously hypertensive rats (SHR), in Wistar Kyoto "normotensive" (WKY), and in Charles River rats made hypertensive by the Grollman ligature technique and by DOC-NaCl administration after unilateral nephrectomy. The effect of high dietary NaCl on renal alpha receptors was studied in Dahl, SHR, and WKY rats. Renal alpha 1 and alpha 2 receptor densities were higher (p less than 0.05) in SHR and in Dahl salt-sensitive rats than in their normotensive controls. High dietary sodium increased renal alpha 2 receptors and blood pressure in SHR, WKY and Dahl salt-sensitive, but not in resistant Dahl rats. A study of time relationships revealed that the increase in renal alpha 2 receptors preceded most of the blood pressure elevation due to high dietary sodium. Renal alpha-adrenergic receptor densities of surgical (Grollman) and endocrine (DOC-NaCl) forms of rat hypertension were not different from normotensive controls. Thus, renal alpha 2 receptor density and increase thereof by dietary sodium may be: 1) a biochemical marker for genetic forms of hypertension in the rat, and 2) closely linked to the basic mechanism of high blood pressure.

Dahl sodium-sensitive and resistant rats were fed low and high sodium diets. Renal plasma membrane alpha 2-adrenergic receptor concentrations were greater (p less than .05) in sensitive than in resistant rats while ingesting a low sodium diet. On a high sodium diet the alpha 2 receptor concentration increased (p less than .01) in sensitive but not in resistant rats. Since alpha 2 receptors are located in proximal tubules at sites where norepinephrine induces sodium reabsorption, we postulate that this genetically determined abnormal receptor regulation could lead to exaggerated sodium retention and possibly high blood pressure.

Activation of renal alpha-adrenergic receptors induces vasoconstriction, proximal tubular reabsorption of sodium, and inhibition of renal release. Excesses of these effects are present in varying degrees in animal models of, and in patients with, "essential" hypertension. Since essential hypertension is genetically determined, we sought abnormalities of renal alpha-adrenergic receptors in the Okamoto-Aoki strain of spontaneously hypertensive rats (sr-SHR) and their stroke-prone variant (sp-SHR). Total alpha-adrenergic receptor concentrations were determined by Scatchard analysis of [3H]dihydroergocryptine binding to a renal membrane fraction and were found to be increased (p less than 0.02) in male sr-SHR at 4, 16, and 32 weeks of age and in female sr-SHR at 16 weeks of age as compared to age- and sex-matched Wistar-Kyoto controls. They were also increased in 9-week-old sp-SHR renal membranes (p less than 0.005). Further studies revealed that this increase in renal alpha-adrenergic receptors was due entirely to an increase in alpha 2-receptors as measured by [3H]yohimbine binding rather than to an increase in alpha 1-receptors as quantitated by [3H]prazosin binding. No difference in binding affinities of the various radioligands could be demonstrated between any of the hypertensive and normotensive groups of rats. Plasma norepinephrine levels were elevated (p less than 0.01) in the 4-, 9- and 16-week-old SHR, but not in the 32-week-old hypertensive rats. Thus, high renal alpha 2-adrenergic receptor number is coupled with a significant increase in plasma norepinephrine concentrations during the development of hypertension in SHR. By mediating an enhanced receptor-coupled response, such as increased proximal tubular sodium reabsorption, this abnormality of renal alpha-adrenergic receptors may contribute to some or all of the pathophysiologic derangements leading to hypertension in SHR.