Vasodilator drug effects on internal mammary artery and saphenous vein grafts.

The internal mammary artery is a dynamic conduit used for myocardial revascularization in which potential exists for spasm as well as for vasodilation. This study investigated vasodilator drug effects on the mammary artery using nitroprusside and nitroglycerin in vitro to measure the inhibition of contraction of human internal mammary artery and in vivo to examine blood flow through a canine mammary artery. In the in vitro study, ring segments of human internal mammary arteries were suspended on strain gauges in muscle baths containing 37 degrees C Krebs solution for measurement of isometric tension in vitro. Arterial contraction was stimulated with 70 mM potassium and 10 microM norepinephrine, and inhibition of contraction by vasodilators was measured. Nitroprusside was more potent and effective than was nitroglycerin in inhibiting potassium and norepinephrine contraction. The in vivo study utilized a canine (n = 8) right heart bypass preparation that allowed precise control of cardiac output, blood pressure and heart rate, which were maintained constant. The internal mammary artery graft and the saphenous vein graft perfused the same coronary artery bed. Electromagnetic flow probes measured graft flow (with the other graft occluded) before and after 15 min of drug infusion (1 microgram/kg per min). Nitroglycerin significantly increased mammary artery flow 36 +/- 13%, whereas nitroprusside significantly decreased it 12 +/- 2%. Saphenous vein grafts responded differently; graft blood flow decreased with nitroglycerin and increased with nitroprusside. Thus, although nitroprusside was more effective than nitroglycerin in inhibiting mammary artery contraction in vitro, it decreased internal mammary artery graft flow measured in vivo. Nitroglycerin had the opposite effect, increasing mammary graft flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine sensitivity and membrane potentials of caudal arterial muscle in DOCA-salt, Dahl, and SHR hypertension in rat.

Comparison of norepinephrine (NE) sensitivity in caudal arterial muscle of rats with three forms of hypertension showed that there was no increase in either DOCA-salt or Dahl genetic hypertension, in contrast to the increased NE sensitivity found in spontaneously hypertensive rats (SHR). In hypertension induced by deoxycorticosterone acetate (DOCA)-salt treatment, as in Dahl genetic hypertension, there was also no difference in membrane potential (Em) between hypertensive and normotensive rats. By comparison to the SHR membrane alterations reported previously, any increased NE sensitivity might have been associated with altered Em electrogenesis which is triggered by a trophic factor of the sympathetic nervous system. SHR have a lower intracellular K+ free ion concentration and thus a smaller contribution of the ion gradient generated voltage which appears to be compensated for at rest by more active electrogenic ion transport. While SHR show greater depolarization and contraction than Wistar-Kyoto (WKY) rats in response to midrange NE concentrations, DOCA-salt and Dahl hypertensive rat caudal arterial muscle showed neither NE hypersensitivity nor any evidence of altered Em mechanisms. Ion transport in isolated peripheral arteries in DOCA-salt hypertension may only secondarily be altered in response to primary changes in humoral factors and altered neural control mechanism.

Enhanced release of endothelium-derived relaxing factor in mineralocorticoid hypertension.

Ring segments of superior mesenteric arteries studied in vitro were used to determine the role of the vascular endothelium in regulating vascular contractile and relaxant sensitivity in deoxycorticosterone acetate (DOCA)-salt hypertension. Wistar rats were given DOCA (20 mg/kg s.c. twice per week) and 1% NaCl drinking water for 5 weeks. In ring segments containing endothelium, there was a decrease in contractile sensitivity to arginine vasopressin, no change in contractile sensitivity to norepinephrine and KCl, and no change in relaxant sensitivity to acetylcholine or isoproterenol in arteries from hypertensive rats compared with normotensive controls. Removal of the vascular endothelium by rubbing had no effect on the contractile response to arginine vasopressin and KCl or the relaxant response to isoproterenol in control arteries. In arteries without endothelium, DOCA-salt hypertension caused a threefold increase in contractile sensitivity for arginine vasopressin, norepinephrine, and KCl; a 50% reduction in maximal relaxation to isoproterenol; and a threefold decrease in relaxant sensitivity to sodium nitroprusside. Indomethacin (10 microM) had no effect on contraction or relaxation. However, N-monomethyl L-arginine unmasked altered contractile sensitivity to norepinephrine in arteries from DOCA-salt hypertensive rats. These data show that the endothelium compensates for increased contractile and reduced relaxant responses of vascular muscle in DOCA-salt hypertension by increasing the release of endothelium-derived relaxing factor. These data suggest that altered vascular responsiveness is masked by the endothelium, thus preventing these alterations from contributing to increased peripheral resistance during the development of DOCA-salt hypertension.

Vasopressin response in collecting ducts of rats resistant to mineralocorticoid hypertension.

In previous studies we found that vasopressin stimulation of both cyclic AMP (cAMP) formation in cortical collecting tubules (CCT) and sodium reabsorption in isolated perfused kidneys was markedly exaggerated in rats with mineralocorticoid hypertension. In the present study, we tested the response (cAMP accumulation) of cortical and outer medullary collecting tubules (OMCT) to vasopressin in two rat models that are resistant to deoxycorticosterone acetate (DOCA)-induced hypertension, the Wistar-Furth strain and NaCl-deficient rats. The blood pressure of normal outbred Wistar rats rose to hypertensive levels (systolic pressure more than 165 mm Hg) during a 5-week treatment with DOCA (10 mg/week) and 1% saline to drink. Significant hypertrophy of the heart and kidneys was also observed. Vasopressin (10(-8) M)-induced cAMP formation was enhanced 3.4-fold in the CCT (OMCT unchanged) of hypertensive rats compared with normotensive controls. Significant hypertrophy (as indexed by tubule diameter) of the CCT but not the OMCT was also observed in DOCA-salt hypertensive rats. Restriction of dietary NaCl (0.13% in chow, tap water to drink) completely prevented DOCA-induced hypertension, organ and CCT hypertrophy, and enhancement of vasopressin-stimulated cAMP formation in the CCT. In Wistar-Furth rats, DOCA-salt treatment did not alter blood pressure or cause significant organ hypertrophy. However, DOCA-salt treatment enhanced vasopressin-stimulated cAMP formation by 4.1-fold in CCT of Wistar-Furth rats, with significant tubular hypertrophy in the CCT but not the OMCT. We conclude that DOCA-induced hypertension and changes in CCT function are dependent on excess dietary NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological activity of C-terminally truncated fragments of human alpha-calcitonin gene-related peptide.

C-terminally truncated fragments of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) were tested for their ability to stimulate amylase secretion from pancreatic acinar cells and relax precontracted mesenteric arteries. h-alpha-CGRP, h-alpha-CGRP (1-36), h-alpha-CGRP (1-35), and h-alpha-CGRP (1-34) were made by Merrifield's solid-phase peptide synthesis methodology. Peptides were purified by gel filtration, cation-exchange chromatography, and semipreparative reversed-phase high-performance liquid chromatography. The products were characterized by amino acid analysis, mass spectrometry, and tryptic digestion. h-alpha-CGRP stimulated amylase secretion from dispersed guinea pig pancreatic acini in a biphasic concentration-dependent manner. The initial increase in amylase secretion reached 8% of total cellular amylase content with an ED50 value of 7.7 nM, and the second increase reached 11% of total cellular amylase content at a concentration of h-alpha-CGRP of 10(-4)M. h-alpha-CGRP (1-36) caused a small, significant increase in amylase release. C-terminally truncated fragments h-alpha-CGRP (1-35) and h-alpha-CGRP (1-34) did not increase amylase release at concentrations < 10(-5) M. At concentrations > 10(-5) M the fragments h-alpha-CGRP (1-35) and h-alpha-CGRP (1-34) caused a smaller increase in amylase release than that caused by h-alpha-CGRP whereas h-alpha-CGRP (1-36) caused the same increase. h-alpha-CGRP caused a concentration-dependent relaxation of rat mesenteric artery, precontracted with prostaglandin F2 alpha, with an EC50 of 2.9 nM and a maximal relaxation that was 60% of the prostaglandin F2 alpha-induced tone. h-alpha-CGRP (1-35) also relaxed the mesenteric artery in a concentration-dependent manner with a maximum response that was 40% of the prostaglandin F2 alpha-induced tone. The remaining fragments did not relax rat mesenteric arteries. Additionally, h-alpha-CGRP (1-36) and h-alpha-CGRP (1-34) did not block h-alpha-CGRP-induced relaxation of the mesenteric artery. An intact C-terminus is required for h-alpha-CGRP to cause potent biological effects in pancreatic acini and mesenteric artery. The different effects of h-alpha-CGRP (1-35) in mesenteric artery compared with those in pancreatic acini suggest that the CGRP receptors in these two tissues may be different.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide.

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala14]-h-alpha-CGRP, [Aib14]-h-alpha-CGRP, [Asp14]-h-alpha-CGRP, [Asn14]-h-alpha-CGRP, and [Pro14]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala14]-h-alpha-CGRP, [Aib14]-h-alpha-CGRP, [Asp14]-h-alpha-CGRP, and [Asn14]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly14 of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro14]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro14]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Inhibition of human internal mammary artery contractions. An in vitro study of vasodilators.

The internal mammary artery is currently the preferred conduit for myocardial revascularization; however, perioperative vasospasm of the internal mammary artery may limit its use as a bypass graft. The ability of various vasodilators to inhibit internal mammary artery contraction was investigated with the use of discarded segments of human internal mammary artery not used in coronary artery bypass grafting. Ring segments of human internal mammary arteries were suspended on strain gauges in muscle baths containing 37 degrees C Krebs solution for measurement of isometric tension in vitro. Arterial contraction was stimulated by elevating the extracellular potassium concentration to 70 mmol/L or by exposure to a 10 mumol/L concentration of norepinephrine, and inhibition of contraction by vasodilators was measured. The order of potency to inhibit potassium-induced contraction was as follows: nifedipine > verapamil > nitroprusside > papaverine. At maximal effective doses, nifedipine, verapamil, and papaverine almost completely inhibited potassium-induced contraction, whereas nitroprusside inhibited contraction by only 55%. When norepinephrine was used to contract the arteries, a biphasic relaxation curve was seen with nifedipine, but not with other vasodilator drugs. The order of potency to inhibit norepinephrine-induced contraction was as follows: nifedipine > nitroprusside > verapamil > papaverine. Maximal inhibition of norepinephrine contraction by these vasodilators ranged from 68% to 95%. Nitroglycerin, isoproterenol, and adenosine produced little or no inhibition of internal mammary artery contraction caused by potassium or norepinephrine. Although nifedipine was the most potent vasodilator, papaverine produced the greatest maximal inhibition of both potassium- and norepinephrine-induced contraction of human internal mammary artery.

Primary structure and pharmacological activity of a nonapeptide related to neuromedin U isolated from chicken intestine.

An extract of chicken intestine contained neuromedin U-like immunoreactivity (36 pmol/g wet tissue weight). The primary structure of the predominant molecular form (NMU-9), comprising 94% of the total immunoreactivity, was established as: Gly-Tyr-Phe-Phe-Phe-Arg-Pro-Arg- Asn-NH2. This sequence differs from that of pig neuromedin U-8 (NMU-8) by the substitution of Leu3 by Phe and, like the corresponding peptide from the guinea pig, is extended from the NH2-terminus by a Gly residue. A minor component of neuromedin U comprised 25 amino acid residues. An extract of chicken whole brain contained much less NMU-like immunoreactivity (1.5 pmol/g) and the nonpeptide was the only molecular form detected. Synthetic chicken NMU-9 produced a concentration-dependent contraction of smooth muscle from the rat uterus and its effect was unchanged in the presence of tetrodotoxin, atropine and indomethacin. The potency of chicken NMU-9 (EC50 360 +/- 60 nM; mean +/- S.E., n = 6) was approximately 8-fold less than that of pig NMU-8 (EC50 46 +/- 8 nM) but the maximum contraction produced by both agonists was not significantly different.

Isolation, structural characterization and pharmacological activity of dog neuromedin U.

The neuromedin U-like immunoreactivity in an extract of dog small intestine was resolved by reversed-phase HPLC into two molecular forms. The primary structure of the larger form (NMU-25) was established as: Phe-Arg-Leu-Asp-Glu-Glu-Phe-Gln-Gly-Pro10-Ile-Ala-Ser-Gln-Val-Arg- Arg-Gln-Phe- Leu20-Phe-Arg-Pro-Arg-Asn-NH2. This sequence shows five substitutions relative to pig neuromedin U-25. The primary structure of the second peptide (NMU-8) was established as: pGlu-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The sequence contains the substitution pGlu for Tyr1 compared with pig neuromedin U-8. The potency of synthetic dog NMU-8 in contracting smooth muscle from the rat uterus (EC50 10 +/- 2 nM; mean +/- S.E., n = 6) was not significantly different from the corresponding potency of pig NMU-8 (EC50 16 +/- 5 nM) but the maximum response produced by the dog peptide was greater (58%; p less than 0.05) than that produced by pig NMU-8.

Differential actions of lamprey peptide methionine-tyrosine at Y1 and Y2 neuropeptide Y receptors.

Peptide methionine-tyrosine (PMY), a peptide of the neuropeptide Y (NPY) superfamily isolated from the brain and intestine of the sea lamprey, had the same maximum effect but was 11-fold less potent than pig NPY in inhibiting field-stimulated contraction of the rat vas deferens, an effect mediated through the Y2 receptor. In contrast, PMY produced a 9-fold greater maximum effect but was 3-fold less potent than pig NPY in contracting the guinea pig mesenteric artery, an effect mediated through the Y1 receptor. Molecular modelling has suggested that the conformation of PMY is appreciably different from NPY only in the beta-turn region of the molecule (residues 9-14). Our data suggest, therefore, that modifications in this region of NPY may useful in the design of receptor selective analogs.

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio.

Eight peptides with differential growth-inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH(2)) and temporin-1Gd (FILPLIASFLSKFL.NH(2)) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC(50) = 2.4+/-0.1 microM for temporin-1Gb and 2.3+/-0.2 microM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.

Neuropeptide Y: vasoconstrictor effects and possible role in cerebral vasospasm after experimental subarachnoid hemorrhage.

The possible role of neuropeptide Y (NPY) as a vasoconstrictor substance contributing to the development of cerebral vasospasm after experimental subarachnoid hemorrhage was studied. Autologous blood was injected into the cisterna magna of control rabbits and rabbits that had received bilateral superior cervical ganglionectomy 2 days before blood injection. Three days after blood injection the concentration of NPY in cerebrospinal fluid (CSF) was 5971 +/- 551 pg/ml while CSF from control animals contained 992 +/- 162 pg/ml of NPY. The effects of porcine NPY on norepinephrine-induced contraction of rabbit cerebral arteries were also studied in vitro. NPY (1.2 nM) caused a 3-fold potentiation of norepinephrine-induced contraction of cerebral arteries. CSF from control rabbits diluted by 50% with Krebs solution had no significant effect on norepinephrine-induced contraction of cerebral arteries when compared to responses in Krebs solution only; however, diluted CSF from denervated blood-injected rabbits potentiated norepinephrine-induced contraction by 2.6-fold. Antiserum containing NPY specific antibodies was used to immunoprecipitate the peptide from CSF taken from denervated blood-injected rabbits. CSF treated with this antiserum contained less than 40 pg/ml of NPY and had no effect on norepinephrine-induced contraction of cerebral arteries. These results show that the concentration of NPY in CSF of rabbits is elevated after experimental subarachnoid hemorrhage. In addition, NPY in CSF can potentiate the vasoconstrictor effect of norepinephrine which may contribute to the development of cerebral vasospasm.

High levels of NPY in rabbit cerebrospinal fluid and immunohistochemical analysis of possible sources.

We have analyzed rabbit cerebrospinal fluid for neuropeptide Y (NPY)-like immunoreactivity, using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) and examined the anatomical relationship of NPY-containing fibers to the cerebral vasculature and the third cerebral ventricle. Cerebrospinal fluid (CSF) obtained from the cisterna magna of rabbits was injected into a C18 column and subjected to HPLC. The fractions were collected, dried and reconstituted in buffer for NPY radioimmunoassay. A single peak of NPY immunoreactivity was obtained which corresponded in retention time to synthetic porcine NPY. Analysis of CSF samples produced displacement curves parallel to the standard curve. Immunohistochemistry revealed numerous NPY-labeled fibers which penetrated the ependymal lining of the third cerebral ventricle and directly bordered the ventricular lumen. Other fibers were observed in the pia which lines the ventral aspect of the hypothalamus. The basilar artery, its branches and other cerebral vessels were surrounded by NPY-labeled fibers. The results show that: (1) approximately 1 ng/ml of NPY immunoreactivity which corresponds chromatographically to synthetic porcine NPY is present in rabbit CSF; (2) NPY-containing fibers surround the basilar artery and other cerebral vessels; (3) NPY may be released into the CSF from axons in the pia and from axons which penetrate the ependymal lining of the third ventricle. These observations form the basis for our analysis of the vasoconstrictor effects of NPY and its role in cerebrovasospasm after experimental subarachnoid hemorrhage.

Activation of alpha 1-adrenoceptors increases [3H]inositol metabolism in rat vas deferens and caudal artery.

Rings of rat vas deferens and caudal artery were incubated with [3H]inositol in Krebs-Ringer bicarbonate buffer containing 10 mM lithium chloride, and the production of water-soluble [3H]inositol phosphates was monitored. Norepinephrine increased [3H]inositol phosphate accumulation 7-fold in rings from vas deferens and 3-fold in rings from caudal artery. Epinephrine, phenylephrine and methoxamine were as effective as norepinephrine, suggesting that these drugs are full agonists in causing this response. Prazosin, phentolamine and yohimbine completely blocked the stimulation by norepinephrine in both tissues with potencies typical of blockade of alpha 1-adrenoceptors. Despite a substantial receptor reserve for alpha 1-adrenoceptor mediated contractile responses, clonidine, p-amino-clonidine, phenylpropanolamine and ephedrine can only cause a partial contractile response in rat vas deferens. However, all of these partial agonists were either as effective or more effective in increasing [3H]inositol phosphate accumulation in rat vas deferens as they were in activating a contractile response. These data suggest that alpha 1-adrenoceptors increase phosphatidylinositol turnover in rat vas deferens and caudal artery, and that there may be a receptor reserve for alpha 1-adrenoceptor mediated increases in [3H]inositol phosphate accumulation in these smooth muscles.

Vasopressin receptor mediated contraction and [3H]inositol metabolism in rat tail artery.

Inositol phosphates (IP) production and contraction in isolated but otherwise intact rat tail artery were measured in response to stimulation by vasopressin agonists. We have previously studied similar alpha-adrenoceptor responses. Identical rank orders of vasopressin agonists' potency were found for IP accumulation and contraction. The vasopressin analogue [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)-2-(O-methyl)tyrosine,D-arginine8] vasopressin, was shown to be a specific, reversible antagonist for both IP accumulation and contraction by all vasopressin agonists tested. No antagonism of vasopressin induced increases in IP accumulation or contraction were found using phenoxybenzamine. Therefore, in this tissue vasopressin receptors mediate both contraction and IP accumulation; and vasopressin mediated responses appear to be direct effects not mediated via the activation of alpha-adrenoceptors. Demonstration of two entirely different receptors, mediating the same functional response, and which both promote IP production, is consistent with a general obligatory role for phosphoinositide catabolism in receptor mediated vascular smooth muscle contraction.

Regulation of beta-adrenoceptor density and function in rat vas deferens.

beta-Adrenoceptor density and responsiveness were examined in rat vas deferens following surgical and pharmacological treatments. Receptor density was measured by Scatchard analysis of saturation isotherms of specific [125I]pindolol ([125I]PIN) binding in membrane homogenates. Functional responsiveness was measured by isoprenaline-induced inhibition of field stimulated (60 V, 1 ms, 0.1 Hz) or 40 mM K+-induced contractions. Four days following surgical denervation of vas deferens there was no change in the density of [125I]PIN binding sites, suggesting that these sites are not located on prejunctional neurons. Neither 7 day bilateral adrenalectomy, 21 day denervation, nor 7 days treatment with 10 mg/kg per day desmethylimipramine caused changes in either the potency of isoprenaline in inhibiting contraction or the density of [125I]PIN binding sites compared to controls. Infusion of 3 mg/kg per day isoprenaline for 8 days significantly reduced the potency of isoprenaline in inhibiting field stimulated contractions, reduced the maximum degree of inhibition, and reduced the density of [125I]PIN binding sites. These results suggest that beta-adrenoceptor density and responsiveness in rat vas deferens are not affected by removal of adrenal hormones or neuronal stimulation, but that receptor density and responsiveness can be decreased by increasing the concentration of beta-adrenoceptor agonists at the receptor. Therefore, beta-adrenoceptors in rat vas deferens probably receive little tonic stimulation under normal circumstances.

Evidence that neuromedin U may regulate gut motility in reptiles but not in mammals.

Neuromedin U-8 induced a monophasic and concentration-dependent contraction of intact small intestine from the turtle, Pseudemys scripta, whereas the peptide had no effect upon the motility of rat and guinea pig gut. The maximum response produced by neuromedin U-8 was 56% of that produced by acetylcholine and 62% of that produced by potassium chloride. The potency and maximum response to neuromedin U-8 were unaffected by tetrodotoxin and atropine. The data suggest that neuromedin U may play a role in regulation of gut motility in lower vertebrates but not in mammals.

Relationship between alpha 1-adrenoceptor density and functional response of rat vas deferens. Studies with phenoxybenzamine.

The relationship between the density of alpha 1-adrenoceptors and the longitudinal contractile response of rat vas deferens was examined by using phenoxybenzamine to irreversibly decrease alpha 1-adrenoceptor density. Receptor density was measured by Scatchard plots of saturation analysis of specific 125I-BE 2254 (125IBE) binding and compared to the potency of alpha-adrenoceptor agonists in causing contraction and the maximum contraction elicited by these agonists. Treatment of isolated vasa deferentia in organ baths with phenoxybenzamine caused a dose-dependent decrease in the density of 125IBE binding sites, the potency of the full agonist phenylephrine in activating contraction, and the maximum contractile response. The percentage of functional receptors remaining after phenoxybenzamine treatment (q value) was calculated from the contraction data and compared to the percentage of 125IBE binding sites remaining. The reduction in the number of functional receptors was much greater than the reduction in the number of 125IBE binding sites at all doses of phenoxybenzamine. Since the magnitude of the contractile response caused by a weak partial agonist should be approximately proportional to the density of functional receptors in the tissue, partial agonists were used as an independent measure of the degree of functional receptor inactivation. Bath application of two different doses of phenoxybenzamine caused a decrease in the maximal contraction caused by the partial agonists clonidine and ephedrine which was similar to the calculated decrease in the q value, but not to the observed decrease in the density of 125IBE binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of agonists and antagonists to beta-adrenoceptors in rat vas deferens: relationship to functional response.

The properties of beta-adrenoceptors in rat vas deferens were examined using radioligand binding assays of 125I-pindolol(125IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. 125IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of beta 2-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC50 of 18.0 +/- 2.1 nM. KB values for antagonists in competitively antagonizing this response correlated well (r2 = 0.99) with the KD values for inhibition of 125IPIN binding. Inhibition of 125IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl2, agonists inhibited specific 125IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 microM guanosine-5'-triphosphate, agonists inhibited specific 125IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 microM BAAM produced a 45% decrease in the Bmax for 125IPIN with no change in the apparent KD. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol followed by a decreased maximum response. KA values for isoprenaline and salbutamol in activating the functional beta-adrenoceptors were compared with KI values for agonist inhibition of specific 125IPIN binding. The KA values for both agonists were not significantly different from the low affinity KI values, but were significantly different from the high affinity KI values. These data suggest that 1) a homogeneous population of beta 2-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with 125IPIN, 2) there is a substantial beta-adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by beta-adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.

Beta 1- and beta 2-adrenoceptor binding and functional response in right and left atria of rat heart.

The properties of beta 1- and beta 2-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses to beta-adrenoceptor agonists were examined. [125I](-)pindolol (125IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. The k1's for association in right and left atria were 6.5 X 10(9) l/mol-min and 2.3 X 10(9) l/mol-min respectively, while the k-1's for dissociation were 0.20 min-1 and 0.17 min-1. The kinetically determined KD's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibrium KD's determined from Scatchard analysis of saturation isotherms of specific 125IPIN binding. Inhibition of 125IPIN binding by beta-adrenoceptor antagonists was stereoselective and the order of potency was timolol greater than l-propranolol greater than d-propranolol greater than sotalol. Inhibition by beta 1- and beta 2-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement by beta 1-selective (practolol, atenolol and metoprolol) and beta 2-selective (ICI 118,551) antagonists gave estimates of the proportion of beta 1- and beta 2-adrenoceptors present in rat atria. Right atria contained 67 +/- 4.2% beta 1- and 33 +/- 4.2% beta 2-adrenoceptors, while left atria contained 67 +/- 2.8% beta 1- and 33 +/- 2.8% beta 2-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused by beta-adrenoceptor agonists were also measured. pA2 values for non-subtype selective beta-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated with KD values determined for specific 125IPIN binding. pA2 values for beta 1- and beta 2-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pKD values of these drugs in binding to beta 1-adrenoceptors, but not with the pKD values in binding to beta 2-adrenoceptors. Dose-response curves for stimulation of both rate and force by the beta 2-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade of beta 1-adrenoceptors with metoprolol than by selective blockade of beta 2-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activating beta 1-adrenoceptors.(ABSTRACT TRUNCATED AT 400 WORDS)