alpha-2-Adrenergic activation of proopiomelanocortin-containing neurons in the arcuate nucleus causes opioid-mediated hypotension and bradycardia.

Treatment of rats for 4 days with alpha-methyldopa, 200 mg/kg/day i.p., increases steady state levels of proopiomelanocortin (POMC) mRNA in the mediobasal hypothalamus, as measured by DNA excess solution hybridization. The increase is prevented by parallel treatment with yohimbine, 2 mg/kg/day i.p., but not by naltrexone, 2 mg/kg/day i.p. Treatment with the peripheral vasodilator hydralazine, 2 mg/kg/day, does not affect POMC mRNA levels. In situ hybridization histochemistry with a cRNA probe for POMC indicates that POMC-containing cells are located within the confines of the arcuate nucleus both in control and in alpha-methyldopa-treated rats, and confirms the increase in POMC mRNA in the latter. Microinjection of 2 micrograms of alpha-methylnorepinephrine unilaterally into the arcuate nucleus of urethane-anesthetized rats causes hypotension and bradycardia, which can be inhibited by 200 ng of yohimbine microinjected into the same site, or by 100 ng l-naloxone microinjected into the ipsilateral nucleus tractus solitarii, but not into the arcuate nucleus. These findings are interpreted to indicate that activation of alpha 2-adrenergic receptors located on POMC-containing neurons in the arcuate nucleus causes beta-endorphin release and stimulation of opiate receptors in the NTS, which results in hypotension and bradycardia, and that this mechanism contributes to the hypotensive action of alpha-methyldopa.

Proopiomelanocortin messenger RNA is decreased in the mediobasal hypothalamus of rats made dependent on ethanol.

It is thought that certain actions of ethanol involve an interaction with endogenous opioids, including proopiomelanocortin-derived peptides such as beta-endorphin. To examine this possibility, we used a sensitive and specific assay for proopiomelanocortin mRNA to obtain an estimate of the activity of the endorphinergic system in the mediobasal hypothalamus and the pituitary of rats exposed for 10 days in an inhalation chamber to either ethanol or water. This protocol causes dependence in the ethanol-exposed group, as demonstrated by the presence of withdrawal seizures after cessation of treatment. While ethanol treatment did not affect proopiomelanocortin mRNA levels in the pituitary, the level in hypothalamus was significantly lower in the ethanol-treated animals than in controls. These results suggest that some effect of ethanol may involve the hypothalamic endorphinergic system.

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy.

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.

The relationship between homotropic and heterotropic cooperativity for angiotensin receptors in smooth muscle.

1. Angiotensin-induced contraction of smooth muscle is accompanied by both homotropic (receptor-receptor) and heterotropic (receptor-G protein) cooperativity. 2. Binding constants for angiotensins II and III at uterine smooth muscle receptors have been compared in bioassays and binding assays, using the competitive antagonist Sarmesin to verify the binding assay/bioassay interrelationship. 3. Agonist affinities determined from binding studies in the presence of GTP/S were found to be similar to the affinities observed in responding rat uterine tissues under conditions which eliminate positive homotropic cooperativity, suggesting that heterotropic cooperativity and homotropic cooperativity are interdependent events for smooth muscle contraction. 4. The data are consistent with an allosteric or autosteric mechanism of receptor function involving cooperativity between two agonist binding sites on the receptor. 5. The model has been used to calculate homotropic efficacies for angiotensins II and III from bioassay data and binding data, respectively.

Methods for analyzing and interpreting cooperativity in dose-response curves--I. Antagonist effects on angiotensin receptors in smooth muscle.

1. Dose-response curves for angiotensins II and III in the rat isolated uterus, portal vein and aorta, in the absence and presence of the antagonist [Sar1Ile8]ANG II, have been analyzed by methods which detect cooperativity. 2. Hanes-Woolf transformations of dose-response data were used to define phases of positive homotropic cooperativity, and this information was applied to the interpretation of Hill plots. 3. The level of positive cooperativity was found to be higher for ANG II (nH = 1.35-2.00) than ANG III (nH = 1.02-1.43) in the three tissues studied. 4. In the presence of the antagonist [Sar1Ile8]ANG II a decrease in the level of cooperativity produced by both agonists was observed, and high concentrations of the antagonist could induce negative cooperativity. 5. These findings have been interpreted on the basis of a 2-site agonist binding mechanism involving receptor dimers, in which angiotensin agonists induce an increase in their own receptor binding affinity, whereas the antagonist [Sar1Ile8]ANG II decreases the affinity of angiotensin receptors for ANG II and ANG III.

Classification of angiotensin receptors in rat isolated uterus, portal vein, and aorta using a slowly dissociating antagonist [Sar1,Ile8]ANG II for receptor blockade.

Receptor blockade with the slowly dissociating angiotensin (ANG) antagonist, [Sar1,Ile8]ANG II, was used to determine dissociation constants (Kd) for angiotensins II and III at receptors in rat isolated uterus, portal vein and aorta. The Kd values obtained were 1) ANG II: uterus, 2.2 +/- 1.2 X 10(-8)M; portal vein, 8.5 +/- 2.5 X 10(-9)M; aorta, 7.8 +/- 1.7 X 10(-9)M; and 2) ANG III: uterus, 5.7 +/- 1.7 X 10(-7)M; portal vein, 4.5 +/- 3.1 X 10(-7)M; aorta, 1.4 +/- 0.5 X 10(-7)M. For either ANG II or ANG III, there were no clearly significant differences in the Kd values obtained for the smooth muscle tissues investigated. The derived Kd values provided a relationship between receptor occupancy and response that illustrated that the two peptides had similar efficacies in all three tissues. The method provides a quantitative description of receptors and suggests that the angiotensin receptor is of the same type in all three tissues.

Classification of angiotensin receptors in rat isolated uterus, portal vein, and aorta with the novel competitive antagonist sarmesin.

Blockade of in vitro contractile responses to angiotensins II and III by the reversible competitive angiotensin antagonist [Sar1, Tyr(Me)4]ANG II (sarmesin) was investigated in 3 rat smooth muscle tissues. The pA2 values for sarmesin were: rat uterus, 7.46 +/- 0.04 versus ANG II and 7.46 +/- 0.07 versus ANG III; rat aorta, 7.98 +/- 0.07 versus ANG II and 7.67 +/- 0.08 versus ANG III; rat portal vein, 7.75 +/- 0.05 versus ANG II and 7.41 +/- 0.08 versus ANG III. Statistical analysis revealed that the pA2 values in each tissue were not significantly different, suggesting that ANG II and ANG III interact with the same receptors in each tissue. This conclusion was supported by cross-tachyphylaxis studies. Further statistical analysis revealed that pA2 values were not significantly different between tissues, suggesting that there are no readily discernible differences between angiotensin receptors in the 3 smooth muscle preparations investigated.

Synthesis and biological activities of analogues of angiotensins II and III containing O-methyltyrosine and D-tryptophan.

Analogues of angiotensin II and III (ANG II and ANG III) in which the tyrosine and/or phenylalanine residues were substituted have been synthesized by the solid-phase method and purified by (carboxymethyl)cellulose chromatography and reversed-phase HPLC. The antagonist and agonist potencies of these peptides were determined in the rat isolated uterus assay. [Sar1,Tyr(Me)4]ANG II, [Tyr(Me)3]ANG III, [Sar1,D-Trp4]ANG II, [D-Trp3]ANG III, [Sar1,D-Trp8]ANG II, [D-Trp7]ANG III, [Sar1,Tyr(Me)4,Ile8]ANG II, [Tyr(Me)3,Ile7]ANG III, [Sar1,D-Trp4,Ile8]ANG II, [D-Trp3,Ile7]ANG III, [Sar1,Tyr(Me)4,D-Trp8]ANG II, and [Tyr(Me)3,D-Trp7]ANG III had antagonist activities (pA2) respectively of 8.1, less than 6, less than 6, less than 6, (7.7), (6.7), 7.2, less than 6, less than 6, less than 6, 7.1, and less than 6. The agonist activity of each peptide was less than 0.1% of that of ANG II. Analogues in which only the Phe residue was substituted were not readily reversible in the bioassay, whereas analogues in which only the Tyr residue or both the Tyr and Phe residues were substituted were reversible antagonists. Peptides that were twice substituted had lower antagonist activities than peptides having a single aromatic residue substitution. Substitution of the Tyr residue in ANG II, but not ANG III, provides a new route for the synthesis of potent and competitive angiotensin antagonists. Differences in the biological properties of ANG II and ANG III analogues substituted at the Tyr residue suggest different binding/conformation requirements for the two endogenous ligands at angiotensin receptors in smooth muscle.

A cyclic angiotensin antagonist: [1,8-cysteine]angiotensin II.

[Cys(S-acetamidomethyl)1,8]angiotensin II has been synthesized by the solid-phase method and purified by carboxymethylcellulose chromatography and reversed-phase HPLC. Treatment of this peptide with iodine gave the cyclic octapeptide [Cys1,8]angiotensin II, which was isolated by Sephadex G-25 chromatography and reversed-phase HPLC. Comparison of the circular dichroism spectra of the open-chain and cyclic peptides, respectively, demonstrated the existence of a disulfide bond with right-handed chirality in the cyclic peptide. In the isolated rat uterus assay, the open-chain peptide was found to be a potent antagonist of angiotensin II, having approximately 10% of the activity of [Sar1, Ile8]angiotensin II, and the cyclic peptide had about 10% of the antagonist potency of its open-chain synthetic precursor.

A new approach to angiotensin antagonists: methylation of the tyrosine hydroxyl in angiotensin II.

[Sar1, Tyr(Me)4]angiotensin II, synthesized by the solid phase method and purified by ion-exchange chromatography and reversed-phase HPLC, was found to inhibit the contractile response to angiotensin II in the rat isolated uterus and inhibit the pressor response to angiotensin II in the vagotomized ganglion-blocked rat. In the rat isolated uterus Schild plots gave a pA2 of 8.1, and a slope of 0.9 indicative of competitive inhibition. In the rat pressor assay, infusion of the analogue at a rate of 500ng/kg/min caused a parallel displacement of the dose-response curve to ANG II to the right. In contrast, the classical angiotensin inhibitor [Sar1, Ile8] ANG II appeared to demonstrate non-competitive inhibition in both the rat isolated uterus and the pressor assays. The phenolic hydroxyl of phenoxide anion of Tyr4 in angiotensin II appears to be critical for the activation of angiotensin receptors in smooth muscle. Alkylation of the tyrosine residue in angiotensin analogues provides a new route for the synthesis of potent competitive antagonists of angiotensin.