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Separate genes for alpha-1A and alpha-1B adrenoceptors have now been identified. Whereas alpha-1 adrenoceptors are known to mediate rat renal vasoconstriction, the relative importance of these alpha-1 adrenoceptor subtypes was unknown. We cannulated the right suprarenal artery of anesthetized male Sprague-Dawley rats to permit administration of the alpha-1A and alpha-1B alkylating antagonists, SZL-49 (SZL) and chloroethylclonidine (CEC), respectively, directly into the right kidney. Treated kidneys were homogenized to identify the doses of SZL and CEC that caused the maximum reductions in Bmax for [3H]prazosin, the relatively nonselective alpha-1 adrenoceptor antagonist. In other rats, a Doppler flow probe was placed around the right renal artery, and dose-peak response curves for boluses of the alpha-1 adrenoceptor agonist phenylephrine (PHE) were generated before and after supramaximal dosages of SZL or CEC. Renal vasoconstriction to PHE was nearly obliterated by SZL. In contrast, CEC caused only a modest rightward shift in the PHE DRC. SZL also abolished the renal vascular response to two other alpha-1 adrenoceptor agonists, cirazoline and methoxamine. Our data support the conclusion that the alpha-1 adrenoceptors at the level of the rat renal resistance vessels are predominantly alpha-1A adrenoceptors.

alpha 1-Adrenoceptors are expressed in high density in the rat kidney. Recent studies have shown that alpha 1-adrenoceptors are heterogeneous and can be subtyped based on their affinity for the antagonists WB 4101 (alpha 1A greater than alpha 1B) and chloroethylclonidine (alpha 1B-selective). We therefore investigated the distribution of alpha 1-adrenoceptor subtypes using [3H]prazosin binding in membranes prepared from cortex, the outer stripe of the outer medulla (OSOM), the inner stripe of the outer medulla (ISOM) and inner medulla dissected from rat kidney. [3H]Prazosin binding was detectable in each region with the following maximum binding site values for [3H]prazosin (femtomoles per milligram of protein): cortex, 186 +/- 20; OSOM, 76 +/- 14; ISOM, 34 +/- 2; and inner medulla, 8 +/- 2. Pretreatment of membranes with chloroethylclonidine (10 microM for 10 min) reduced maximum binding sites to 41 +/- 4% (75 +/- 9 fmol/mg) of control in cortex, 41 +/- 9% of control in OSOM (29 +/- 8 fmol/mg) and 15 +/- 3% of control in ISOM (5 +/- 1 fmol/mg). In competition studies, WB 4101 labeled both high and low affinity sites in cortex (respective pKi values = 10.01 +/- 0.3 and 8.23 +/- 0.1) and OSOM (pKi values = 9.6 + 0.3 and 8.3 +/- 0.5), but only low affinity sites in ISOM (pKi = 8.41 +/- 0.1). The relative prevalence of high:low affinity sites revealed by WB 4101 was 53:47 for cortex, 52:48 for OSOM and virtually all low affinity for ISOM. Prior treatment with chloroethylclonidine greatly reduced the low affinity component of the WB 4101 competition curve in cortex and OSOM. We conclude that: 1) the density of alpha 1-adrenoceptors is highest in the cortex and decreases from cortex to papilla and 2) the alpha 1A and alpha 1B subtypes are approximately equally distributed in the cortex and OSOM, but the alpha 1B subtype predominates in ISOM.

The dose-dependent selectivity of an irreversible binding antagonist, phenoxybenzamine (POB), for renal alpha-1 adrenoceptors, was biochemically and physiologically characterized. Receptors were quantified with the radioligands [3H]prazosin and [3H]rauwolscine for alpha-1 and alpha-2 adrenoceptors, respectively. Alpha-1 adrenoceptor function was quantified by the shift of the norepinephrine and phenylephrine pressor response in vivo and the vasoconstrictor response in the isolated perfused kidney preparation. A renal alpha-2 adrenoceptor response was demonstrated by showing that epinephrine could reverse the effect of vasopressin on water and sodium in the presence of beta blockade and alpha-1 destruction by POB. Doses of POB from 0.1 to 10.0 mg/kg progressively reduced [3H]prazosin binding to renal alpha-1 adrenoceptors until there was no specific binding at the 10.0-mg/kg dose. At this dose more than 60% of [3H]rauwolscine binding to renal alpha-2 adrenoceptors was still present. POB 1.0 mg/kg/hr decreased specific binding to renal alpha-1 adrenoceptors by approximately 40% (P less than .05) but reduced the alpha-1 adrenoceptor-induced vasoconstriction in the nonrecirculating isolated perfused kidney to 10 to 20% of the control. This was the maximal dose of POB studied which did not affect the alpha-2 adrenoceptor-induced antagonism of vasopressin. Higher doses of POB (3.0 mg/kg/hr) demonstrated some alpha-2 adrenoceptor binding as indicated by an attenuation of the antagonism by alpha-2 adrenoceptors. Thus, POB displays selectivity for renal alpha-1 over alpha-2 adrenoceptors. Our data indicate that a dose of 1.0 mg/kg/hr of POB will leave alpha-2 adrenoceptors intact while functionally incapacitating alpha-1 adrenoceptors to 10 to 20% of the control value.

Des-Asp angiotensin II (des-Asp AII) is a naturally occurring heptapeptide metabolite of angiotensin II (AII) which is formed by the enzymatic action of aminopeptidase A. Angiotensin II and des-Asp AII were infused into unanesthetized rats while direct mean arterial pressure, serum aldosterone and serum corticosterone were measured. Both AII and des-Asp AII caused a dose-related increase in serum aldosterone with a significant increase occurring with a dose as low as 1 ng/min. This effect was blocked by pretreatment with 1-Sar-8-Ala-angiotensin II, a competitive inhibitor of AII; however, the inhibitor was more effective in blocking the effects of AII (101%) than of des-Asp AII (82%). Both angiotensins induced a dose-related increase in serum corticosterone and mean arterial pressure. Des-Asp AII was however only 1/10 as potent as AII in elevating mean arterial pressure. 1-Sar-8-Ala-AII was also effective in inhibiting the pressor effects of AII and des-Asp AII. These data illustrate a high degree of organ specificity or selectivity for des-Asp AII and a low specificity for AII. Aminopeptidase A and leucine aminopeptidase were identified in the adrenal cortex and medulla in large amounts. Des-Asp AII may thus be formed from AII locally in the adrenal gland prior to exerting its action at that site.

[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.

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.

The vasodilatory drugs, minoxidil and hydralazine, induce renin release in the rat, man and the dog. Previous reports suggest that the rat adrenal cortex was insensitive to angiotensin stimulation. As a result these studies were designed to obtain evidence for or against the hypothesis that the control of aldosterone release in the rat is unique among mammalian species. Minoxidil and hydralazine induced a time-related increase in both serum renin activity and serum aldosterone. Minoxidil caused a dose-related, proportional increase in serum renin and aldosterone. This response was blocked by prior bilateral nephrectomy but was not affected by hypophysectomy. A competitive angiotensin antagonist, saralasin (1-Sar-8-Ala angiotensin II), impaired minoxidil-induced aldosterone release in a dose-related manner while potentiating minoxidil-induced renin release. Pretreatment with propranolol, a beta adrenergic blocking drug, impaired minoxidil-induced renin and aldosterone release. Only small changes in serum corticosterone occurred after minoxidil or hydralazine administration. These results indicate that minoxidil-induced aldosterone release was mediated by the endogenous angiotensin II formed from renin release. They also support the unanesthetized rat as an appropriate animal model for study of the renin-angiotensin-aldosterone axis and its modification by drugs.