Age-related decrease in beta adrenergic receptor-mediated vascular smooth muscle relaxation

Beta adrenergic receptor-mediated vascular smooth muscle relaxation decreases with increasing age. We have examined the mechanism responsible for this phenomenon using rat mesenteric arteries from young (5-6 weeks) and older (10-12 months) rats. The beta adrenergic agonist isoproterenol produced a dose-dependent relaxation of serotonin-constricted mesenteric artery rings from young rats, whereas the maximal ability of isoproterenol to relax arterial rings from the older rats was found to be reduced markedly (92.7 vs. 27.6%, P less than .0001). The relaxation responses caused by acetylcholine and nitroglycerin, which appear to act independently of cyclic AMP (cAMP), are similar in the two groups. The loss in responsiveness of the mesenteric artery to isoproterenol was not explained by a change in beta receptor number in the vessels (29 +/- 4 in young rats vs. 31 +/- 7 fmol/mg of protein in the older rats). The maximal stimulation of cAMP accumulation by isoproterenol was lower in the older vessels; forskolin activated cAMP accumulation equally in the two groups. However, the vessels from the older rats were less sensitive to forskolin-induced vascular relaxation. Also, the ability of dibutyryl cAMP to promote vascular relaxation was diminished in the older vessels. These data suggest that the diminished cAMP accumulation in older vessels in response to isoproterenol might not necessarily in itself explain completely the reduced physiological response and that an additional defect in the beta adrenergic-mediated relaxation in the vascular smooth muscle of older rats may lie at the level of cAMP-dependent protein kinase activation or more distally.     

Responsiveness of superficial hand veins to phenylephrine in essential hypertension. Alpha adrenergic blockade during prazosin therapy.

Patients with essential hypertension show an increase in vascular resistance. It is unclear whether this is caused by structural changes in the arterial wall or by hyperresponsiveness of vascular smooth muscle to endogenous alpha adrenergic agonists. Using the dorsal hand vein compliance technique we compared the changes in diameter of superficial veins in response to phenylephrine, an alpha 1 adrenergic receptor agonist, and to nitroglycerin, a venorelaxant, in patients with essential hypertension and in normotensive subjects. The dose of phenylephrine that produced 50% of maximal venoconstriction (ED50) in the hypertensive subjects was 257 ng/min (geometric mean; log mean +/- SD was 2.41 +/- 0.54). In the control subjects the ED50 was 269 ng/min (geometric mean; log mean was 2.43 +/- 0.43). Maximal response (Emax) for phenylephrine was 84 +/- 13% in the hypertensive subjects and 90 +/- 6% in the control subjects. Differences in the group means of the ED50 (P = 0.92) or the Emax (P = 0.27) were not significant. There were no significant differences in the ED50 (P = 0.54) or the Emax (P = 0.08) for nitroglycerin between the two groups. These results show no evidence for a generalized change in alpha adrenergic responsiveness in hypertension and support the concept that increased blood pressure responses to alpha adrenergic stimulation in hypertensives are due to structural and geometric changes in the arterial wall rather than to an increased responsiveness of postsynaptic alpha adrenergic receptors. The phenylephrine studies were repeated in seven hypertensive patients during treatment with prazosin, an alpha 1 adrenergic antagonist. The mean dose ratio of the shift in phenylephrine ED50 (ED50 during prazosin therapy/ED50 before prazosin therapy) was 6.1. This indicates that small doses of prazosin (1-2 mg) cause significant in vivo shifts in the dose-response relationship of alpha adrenergic agonists. The dorsal hand vein compliance technique is useful in detecting systemic effects of alpha adrenergic antagonists.     

Age-related changes in relaxant response of vascular smooth muscles to atrial natriuretic peptide

The effect of aging on responses of vascular smooth muscles to atrial natriuretic peptide (ANP) and other vasodilator substances was investigated in isolated rat aortae, rat renal arteries and monkey renal arteries which were precontracted with norepinephrine. There was no significant difference in the ANP-induced maximum relaxation between young and old rat aortae. However, the concentration of agonists causing a 50% relaxation (ED50) value for the old rats was 7.3 times greater than that for the young ones. In rat and monkey renal arteries, the ED50 ratios were 6.2 and 3.8, respectively. The relaxant responses of the rat aortae to isoproterenol and acetylcholine also decreased with increasing age. The ED50 ratios for isoproterenol and acetylcholine were more than 40 and 17, respectively. The maximum relaxation induced by 10(-5) M isoproterenol also decreased significantly in the aortae from the older rats. On the other hand, the ED50 for nitroprusside, nifedipine- and potassium-induced relaxation was not affected by increasing age. These results suggest that ANP-induced relaxation of vascular smooth muscles is reduced with increasing age in rat aortae, rat renal arteries and monkey renal arteries. The mechanisms by which the ANP-induced relaxation decreased in association with the aging process may be quite different from those in acetylcholine-induced and beta adrenoceptor-induced relaxation.     

Increased vascular alpha1-adrenergic sensitivity in patients with renal failure: receiving recombinant erythropoeitin

End stage renal disease (ESRD) is associated with altered hemodynamic regulation as a result of the pathophysiology or treatment of renal failure. Hypertension, common among dialysis patients, is a recognized complication of recombinant human erythropoietin (rHuEPO) therapy. We determined vascular adrenergic and nitric-oxide-mediated responsiveness in 7 patients with established ESRD on rHuEPO treatment and in 13 healthy volunteers using the dorsal hand vein technique. Sensitivity to the alpha1-adrenergic selective agonist phenylephrine was significantly increased in patients with ESRD on rHuEPO. The mean dose of phenylephrine producing 50% venoconstriction (ED50) was 38 +/- 1.6 ng/min in patients with ESRD and 135 +/- 1.3 ng/min in healthy volunteers-almost a 4-fold increase in dose, P = 0.01. In contrast, maximal venodilation mediated by bradykinin, an endothelium-dependent vasodilator, was not different in the 2 groups. To determine whether rHuEPO has a direct vasoconstrictor effect, we studied venous responsiveness to local infusions of rHuEPO in healthy volunteers. Increasing concentrations of rHuEPO produced no vasoconstriction in hand veins of healthy volunteers. These results suggest that vascular responsiveness to alpha-adrenergic stimulation in patients with ESRD on rHuEPO is increased whereas bradykinin-mediated venodilation remains intact. This increase in vascular alpha-adrenergic responsiveness may contribute to the increased peripheral vascular resistance and hypertension seen in patients with ESRD on rHuEPO.     

Functional antagonism in canine tracheal smooth muscle: inhibition by methacholine of the mechanical and biochemical responses to isoproterenol

The biochemical basis for the functional interaction between bronchoconstricting and bronchodilating pathways was investigated. Contracting canine trachealis strips with increasing concentrations of methacholine resulted in a progressive shift to the right of isoproterenol concentration-response curves. Thus, the EC50 for the relaxant response to isoproterenol was nearly 500-fold higher in preparations exposed to 3.0 microM methacholine than in tissues exposed to 0.03 microM methacholine. The maximum relaxation produced by isoproterenol was also dependent upon the initial muscarinic cholinergic tone. For example, isoproterenol reversed completely the contraction induced by 0.03 microM methacholine but did not relax trachealis strips contracted with 30 microM methacholine. To identify the molecular mechanism responsible for this functional antagonism, experiments were conducted to determine the effect of methacholine on isoproterenol-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation. Methacholine did not alter basal cyclic AMP content but did reduce cyclic AMP accumulation in response to isoproterenol. Furthermore, the ability of isoproterenol to activate cyclic AMP-dependent protein kinase was inhibited by methacholine in a concentration-dependent manner. This inhibition paralleled the decrease in mechanical responsiveness to isoproterenol. These results suggest that muscarinic cholinergic stimulation of canine tracheal smooth muscle functionally antagonizes the relaxant responses to beta adrenergic agonists and that a portion of this antagonism may be due to a suppression of catecholamine-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation.     

On the use of functional antagonism to estimate dissociation constants for beta adrenergic receptor agonists in isolated guinea-pig trachea.

In guinea-pig trachea, the maximum degree of relaxation that can be elicited by beta adrenergic receptor agonists depends upon the degree of contraction of the smooth muscle induced by cholinergic agonists. In these studies, it is shown that increasing concentrations of carbamylcholine (carbachol) result in a shift to the right of the dose-response curves to (-)-isoproterenol and (-)-soterenol and a reduction of the maximum degree of relaxation produced by these agonists relative to that produced by papaverine. Soterenol is demonstrated to be a partial agonist relative to isoproterenol since its maximum response is reduced to a greater extent by carbachol and it displaces the carbachol dose-response curves to the right less than does isoproterenol. The data were used to calculate, by three different theoretical models of drug-receptor interactions, a dissociation constant (KA) for soterenol. All values obtained were within 2-fold differences. The range of KA values for soterenol was from 5.4 to 9.6 times 10(-8) M. These values are about 100 times larger than the ED50 value for soterenol obtained in the absence of carbachol. The KA value estimated for (-)-isoproterenol (about 3 times 10(-8) M) by one of the models is also around 100 times larger than its ED50 value. This demonstrates further that ED50 values are unreliable indicators of the affinities of agonists for receptors.     

Impaired beta adrenergic receptor binding and function in cystic fibrosis neutrophils.

Cystic fibrosis (CF), a genetic disease characterized by abnormalities of exocrine gland and mucociliary function, has recently been shown to be associated with abnormal adrenergic and cholinergic physiologic responses in addition to decreased beta adrenergic-induced cyclic AMP generation in human leukocytes. In this study we have attempted to elucidate the nature of this hyporesponsiveness by assessing beta adrenergic receptor number and affinity (KD) in the intact neutrophil using the antagonist ligand [3H] dihydroalprenolol and cyclic AMP responses to isoproterenol in addition to histamine, and prostaglandin E1 in CF subjects, CF obligate heterozygotes (CFH), and normal control subjects. CF patients had significantly less (p less than 0.025) cyclic AMP stimulation above basals levels with isoproterenol (0.1 microM to 0.1 mM), compared with control values, but no consistent differences between groups were noted with histamine or PGE1. CF neutrophils had significantly fewer (p less than 0.005) beta adrenergic receptors per neutrophil (398.0 +/- 54.2 vs. 819.4 +/- 67.2) compared with control neutrophils, but the KD (0.740 +/- 0.11 vs. 0.630 +/- 0.05 nM) did not differ significantly (p greater than 0.05). There was no correlation between clinical severity and either cyclic AMP generation or dihydroalprenolol binding (r = 0.27 and 0.24, respectively, p greater than 0.05). The CFH group had approximately 50% of the cyclic AMP stimulation compared with controls, but the number (909.8 +/- 89.3) and KD (0.710 +/- 0.09 nM) of their beta adrenergic receptors were indistinguishable from control subjects. These findings suggest "down regulation" of the beta receptor in the CF patient. The cause of this remains unknown. Although the etiology of the decreased cyclic AMP responses in CFH was not due to decreased beta adrenergic receptors as assessed by antagonist ligand binding, further studies inthe CFH group to include agonist binding, receptor-adenylate cyclase coupling, intrinsic adenylate cyclase activity, and catecholamine metabolism may help determine the basic cause of beta adrenergic hyperesposiveness in both CFH and CF.     

Effects of protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, on beta-2 adrenergic receptor activation and desensitization in intact human lymphocytes

To investigate the role of protein kinases in agonist-mediated beta-2 adrenergic receptor regulation, the effects of the protein kinase A and C inhibitor, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], on isoproterenol-induced beta adrenoceptor activation and desensitization have been studied in intact human lymphocytes. In the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, H-7 potentiated 10(-8) to 10(-4) M isoproterenol or prostaglandin E1-induced cyclic AMP (cAMP) accumulation in a dose-dependent manner. We failed to observe any effect of H-7 on forskolin-induced cAMP accumulation. These effects of H-7 are probably not due to its inhibition of phosphodiesterase. In addition, whereas up to 10(-3) M H-7 had no beta adrenergic receptor blocking effect, preincubation of intact cells with 10(-3.5) M H-7 partially prevented 50 nM isoproterenol-induced beta-2 adrenergic receptor desensitization in terms of decreases in beta adrenoceptor density (maximum binding), isoproterenol-mediated cAMP responsiveness and high affinity receptor binding for agonist. Interestingly, 10(-3.5) M H-7 alone treated cells also showed an up-regulation of cell surface beta receptor density (maximum binding) and increased cAMP responsiveness to isoproterenol stimulation. The mechanisms are unclear. If these effects occur as a result of inhibition by H-7 of protein kinase A and/or C, it may suggest an important role of protein kinase A and/or C in agonist-induced beta-2 adrenergic receptor regulation.     

Relaxation of intrapulmonary artery and vein by nitrogen oxide-containing vasodilators and cyclic GMP

The present study examines the relationship between tissue cyclic nucleotide levels and relaxation of bovine intrapulmonary arterial and venous smooth muscle in response to nitroglycerin, nitroprusside, S-nitroso-N-acetylpenicillamine and isoproterenol. Recent studies have suggested that cyclic GMP may be involved in the relaxation of vascular smooth muscle produced by nitrogen oxide-containing vasodilators and that S-nitrosothiols may act as intermediates of the latter agents. In the present study, nitroglycerin, nitroprusside and S-nitroso-N-acetylpenicillamine were more potent as relaxants of venous than arterial segments. Each of these agents elevated tissue cyclic GMP levels, but not cyclic AMP levels, before relaxation. These nitrogen oxide-containing agents were more potent as elevators of cyclic GMP levels in venous than arterial tissue and this correlated generally with their effects on vascular smooth muscle tone. Methylene blue antagonized both relaxation and increased cyclic GMP levels elicited by nitroglycerin, nitroprusside and S-nitroso-N-acetylpenicillamine. In contrast to the nitrogen oxide vasodilators, 8-bromo-cyclic GMP was equally effective in reducing induced tone in arterial or venous segments. Similarly, isoproterenol relaxed arterial and venous segments with equivalent sensitivities. Relaxation by isoproterenol was preceded by or occurred concomitantly with increased levels of cyclic AMP but not cyclic GMP and both effects were antagonized by propranolol. These findings are consistent with the hypothesis that vascular smooth muscle relaxation in response to nitrogen oxide-containing vasodilators or isoproterenol may be mediated or modulated by the intracellular accumulation of cyclic GMP or cyclic AMP, respectively.     

Functional interactions in smooth muscle: kinetic characterization of the relaxation and desensitization responses to a beta adrenergic agonist in the rabbit aorta

Vascular smooth muscle tone is continuously modulated in vivo by the functional interaction of a variety of vasoconstrictor and vasodilator stimuli. Endogenous substances such as epinephrine simultaneously activate alpha adrenergic receptors that elicit muscle contraction and beta adrenergic receptors that relax the muscle. This study characterizes the beta adrenergic response in the isolated rabbit aorta precontracted with 1 microM phenylephrine. The beta adrenergic agonist isoproterenol (0.03-10 microM) produces a biphasic response that is composed of a rapid relaxation followed by a slower regaining of tension, which is identified as desensitization. An exploratory kinetic model that describes both the relaxation and the desensitization as first-order processes provides a good fit to the experimental data. The parameters used to describe the isoproterenol response are: 1) the observed rate constant for relaxation and its magnitude (krel and R, respectively), 2) the observed rate constant for desensitization and its magnitude (kdes and D, respectively) and 3) the observed delay in the onset of the desensitization response (td). Both the krel and the fractional relaxation were dependent on concentration of isoproterenol in a saturable manner (EC50 = 0.017 and 0.067 microM, respectively). No concentration dependence was observed for kdes, fractional desensitization and td (the average values +/- S.E.M. of these parameters are (4.7 +/- 0.2). 10(-3) sec-1, 0.83 +/- 0.02 and 191 +/- 6 sec, respectively). This work demonstrates that a kinetic approach is necessary to characterize the desensitization response and is also very useful in characterizing the kinetic and steady-state parameters of the relaxation response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of age on beta adrenergic relaxation of the rat jugular vein

Using the Fisher 344 rat model and blood vessel ring segments in vitro, age-related changes in vascular beta adrenergic relaxation were investigated. In the pulmonary artery and aorta, maximum isoproterenol-induced relaxation and sensitivity to isoproterenol declined from 1 to 3 months of age confirming previous reports. In animals 6 months of age, these vessels no longer relaxed to isoproterenol. In the jugular vein, in which beta adrenergic mechanisms predominate, there was no change in maximum relaxation to isoproterenol or in EC50 values in animals 3 to 27 months of age. Furthermore, determination of propranolol dissociation constants (KB) showed no change in affinity up to 27 months of age. Thus, in venous smooth muscle, in contrast to arteries, beta adrenergic relaxation is well maintained through senescence.     

Comparison of signal transduction mechanisms of alpha-2C and alpha-1A adrenergic receptor-stimulated prostaglandin synthesis.

Prostaglandin (PG) synthesis elicited by adrenergic transmitter in the vascular smooth muscle cells (VSMC) of rabbit aorta is primarily mediated through activation of alpha-2C and alpha-1A adrenergic receptors (ARs). We have now investigated and compared the signal transduction mechanisms involved in alpha-2C and alpha-1A AR-stimulated prostacyclin (PGI2) production, measured as 6-keto-PGF1 alpha, in vascular smooth muscle cells. Norepinephrine, methoxamine (an alpha-1 AR agonist) and UK-14304 (an alpha-2 AR agonist) enhanced 6-keto-PGF1 alpha production. UK-14304 and norepinephrine (in the presence of propranolol), but not methoxamine, reduced basal adenosine 2':3'-cyclic monophosphate (cyclic AMP) as well as forskolin- and isoproterenol-stimulated cyclic AMP accumulation. Forskolin and isoproterenol did not alter basal 6-keto-PGF1 alpha production and alpha AR agonist-induced 6-keto-PGF1 alpha production. Alpha-2C and alpha-1A AR-stimulated 6-keto-PGF1 alpha production was independent of cyclic AMP levels in vascular smooth muscle cells. Both alpha-2C and alpha-1A AR-stimulated 6-keto-PGF1 alpha production required extracellular Ca++. Pertussis toxin prevented inhibition of cyclic AMP accumulation and reduced 6-keto-PGF1 alpha production in response to AR agonists. Guanosine 5'-O-(3-thiotriphosphate) potentiated 6-keto-PGF1 alpha production induced by norepinephrine and UK-14304 but not by methoxamine, whereas at a higher Mg++ concentration (4 mM), guanosine 5'-O-(3-thiotriphosphate) potentiated 6-keto-PGF1 alpha production by all three agonists. In contrast, the effect of UK-14304 on cyclic AMP was prevented in the presence of 4 mM Mg++. These data suggest that the pertussis toxin-sensitive G protein(s) mediated the stimulation of PG synthesis by alpha-1A and alpha-2C AR activation and the decrease in cyclic AMP accumulation by alpha-2C AR activation.     

Responsiveness of superficial hand veins to adrenergic stimuli in patients with cystic fibrosis

1. The basic biochemical defect of cystic fibrosis (CF) remains undetermined, but impaired function of the beta-adrenoceptor-mediated adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent regulatory pathway in secretory cells is likely to be involved in the pathophysiology of the disease. 2. We have compared responsiveness to beta-adrenergic stimulation in vivo by infusing isoprenaline locally into peripheral veins of CF patients and control subjects; the dorsal hand vein technique was used to measure the vascular response to isoprenaline. 3. CF patients required significantly higher doses of isoprenaline for half-maximal dilatation of the preconstricted veins (ED50) than controls (geometric mean: 44.5 ng/min in CF patients compared with 14.8 ng/min in controls; P less than 0.05). Maximal venodilatation was 74 +/- 30% of baseline in CF patients compared with 94 +/- 50% in controls (NS between groups). 4. The clinical score of CF patients was uncorrelated with the ED50 of isoprenaline. Thus the decreased beta-adrenergic responsiveness does not seem to be related to the severity of the disease. 5. Our results indicate a defect in the cyclic-AMP-dependent pathway in vascular smooth muscle cells of CF patients. Whether this is associated with the CF gene defect itself requires further study.     

Binding and functional characteristics of beta adrenergic receptors in the intact neutrophil

Beta adrenergic receptors have been previously characterized in human neutrophil sonicates. In the present study the intact neutrophil has been assessed for the number and affinity of beta adrenergic binding sites by using the antagonist DNA. Agonist and antagonist potencies, characterized by their effect on DHA binding and cyclic AMP accumulation, are compared with agonist inhibition of lysosomal enzyme (beta glucuronidase) release. Criteria for beta adrenergic receptor identification were successfully demonstrated. At 30 degrees C, beta adrenergic binding was rapid (t 1/2 2 min) and reversible (t 1/2 9 min). Receptor binding was saturable, revealing approximately 900 high-affinity receptors per neutrophil with DHA concentrations of 0.1 to 10 nM. By utilizing both equilibrium and kinetic techniques, the KD was determined to be approximately 0.6 nM. Agonists and antagonists competed for DHA binding in a manner consistent with their effect on cyclic AMP generation. Rank order potency was suggestive of a beta-2 receptor: isoproterenol greater than epinephrine greater than norepinephrine. Stereoselectivity was shown by the greater potency of L-propranolol compared to the D isomer. A high degree of receptor-adenylate cyclase coupling efficiency was suggested by the observation that with only 1% receptor occupancy isoproterenol stimulated 50% maximal cyclic AMP generation. Finally, there was an excellent correlation between the isoproterenol concentration which resulted in 50% of maximal inhibition of beta glucuronidase release (Ki) and that causing 50% maximal cyclic AMP stimulation (Kact), suggestive of a close relationship between beta adrenergic-induced adenylate cyclase activation and beta adrenergic regulation of neutrophil lysosomal enzyme release. The data presented suggest that the use of the intact neutrophil for study of the beta adrenergic receptor is feasible and may provide information which is considerably more closely related to modulation of physiological function by neurohormones than is possible with disrupted cell preparations.     

Bronchodilator mechanisms in bullfrog lung: differences in response to isoproterenol, theophylline and papaverine

The effects of bronchodilators and smooth muscle relaxants on mechanical responses and lung cyclic nucleotide levels in the isolated hemilung of Rana catesbeiana demonstrate striking differences in intensity and time course of drug action in an unstimulated preparation of airway smooth muscle. Isoproterenol, nitroprusside and nitroglycerin elicit a fast onset relaxation (minutes) with ceiling effects at 20, 22 and 43%, respectively, of maximal relaxation. Theophylline, dibutyryl cyclic AMP and papaverine produce maximal or near maximal relaxation, but require 8 to 32 hr for peak effect. Papaverine-induced relaxation is accompanied by a slow increase in lung cyclic AMP and cyclic GMP and is markedly accelerated by isoproterenol. Theophylline (10(-3) M) produces no change in cyclic nucleotide levels and its relaxant effect is not accelerated by isoproterenol. The hierarchy of relaxant responses suggests drug action at discrete loci in a highly compartmentalized effector chain, with cyclic AMP-dependent mechanisms separable into at least two components. The first is activated by isoproterenol and elicits a rapid, limited response, presumably reflecting an increase in cyclic AMP in a relatively restricted pool. The second is activated by papaverine and elicits a very slow, but complete relaxation, presumably reflecting a more pervasive or diffuse accumulation of cyclic AMP secondary to phosphodiesterase inhibition. The major portion of theophylline-induced relaxation in this preparation appears to be independent of changes in cyclic nucleotide metabolism.     

Uncoupling of the beta-adrenergic receptor as a mechanism of in vitro neutrophil desensitization

Human leukocytes have been useful in studying desensitization phenomena to beta-adrenergic agonists in a number of clinical conditions. For example, we have previously shown that oral terbutaline causes a time-dependent decrease in neutrophil (PMN) beta receptor number, using the beta antagonist ligand [3H]dihydroalprenolol (DHA), in conjunction with a significant loss of isoproterenol-induced adenylate cyclase activity. In the present in vitro study we have explored the mechanism for beta-adrenergic desensitization and have compared conditions for homologous and heterologous desensitization, using the intact PMN model. PMN preincubated with isoproterenol (10(-4)M), washed thoroughly, then restimulated, desensitize rapidly so that within 10 min 80% of control isoproterenol-induced cyclic AMP stimulation is lost. Cells washed free of isoproterenol recover full responsiveness in 1 to 2 hr. The estimated isoproterenol desensitization EC50 in cells washed and then restimulated is 1 X 10(-5)M, and the EC50 in unwashed cells that are restimulated is 9 X 10(-8)M. Rank-order potency studies of catecholamine desensitization show isoproterenol greater than epinephrine greater than norepinephrine, a beta-2 pattern. Isoproterenol-induced desensitization results in a small reduction in [3H]DHA binding sites, which becomes statistically significant (p less than 0.05) from control values at 1 hr (67% of control) and 3 hr (64%). Since the change in number of beta receptors did not explain the profound, rapid loss of beta agonist-induced cyclic AMP responsiveness, we explored the possibility of an uncoupling phenomenon. In the absence of GTP, isoproterenol binding is characterized by an EC50 of 6.6 +/- 2.6 X 10(-7)M, which is significantly different (p less than 0.05) from the EC50 of 38.1 +/- 9.1 X 10(-7)M found when cells are previously desensitized with isoproterenol for 10 min. GTP does not affect the EC50 of desensitized cells. These findings are consistent with the uncoupled receptor state fitting the model described by Su et al. Finally, prolonged (3 hr) isoproterenol preincubation results in a small but significant (p less than 0.05) loss of cyclic AMP responsiveness to histamine (67.7% +/- 11.7 of control) and PGE1 (59.3% +/- 7.4), suggesting heterologous desensitization. These studies suggest that the human PMN is a suitable model to study both homologous and heterologous desensitization in vitro.     

Beta-2 adrenergic control of ornithine decarboxylase activity in brain regions of the developing rat

Development of brain tissue is thought to be regulated, in part, by biogenic amines. We examined the role of noradrenergic stimulation in regulation of ornithine decarboxylase (ODC), an enzyme whose activity is obligatory for neuronal development and which has been used as a biochemical marker for cellular maturation. Intracisternal administration of adrenergic agonists produced a prompt increase in ODC in neonatal rat cerebellum, an effect mediated through beta-2 receptors: the rank order of activity was isoproterenol greater than epinephrine greater than norepinephrine greater than methoxamine; the effect could be blocked by propranolol but not phenoxybenzamine; and zinterol (a beta-2 selective agonist) was equipotent to isoproterenol whereas prenalterol (a beta-1 agonist) was ineffective. The elevation of ODC caused by adrenergic stimulation was cyclic AMP-dependent, as evidenced by: direct measurement of cyclic AMP levels after isoproterenol administration; comparisons of the dose-response curve for stimulation of cyclic AMP with that of ODC; examination of the time course of effect on the two variables; stimulation of ODC by administration of cyclic AMP analogs; demonstration of identical kinetic mechanisms for ODC stimulation by dibutyryl-cyclic AMP and isoproterenol; and potentiation of the actions of isoproterenol on both cyclic AMP and ODC by RO-201724, a specific inhibitor of phosphodiesterase. Examination of the ontogenetic pattern of phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic regulation of cyclic adenosine 3'',5'' monophosphate accumulation in human gastric epithelial glands. Inhibitory effect of somatostatin

The action of catecholamines and somatostatin on cyclic adenosine 3',5' monophosphate (cyclic AMP) formation in human isolated gastric glands is reported. We show that: (1) there is a beta 2 receptor-mediated stimulation of cyclic AMP production in fundus. Catecholamines act with the order of potencies isoproterenol (ED50 = 50 nmol 1(-1) greater than epinephrine (ED50 = 0.1 mumol 1(-1] greater than norepinephrine (ED50 = 5 mumol 1(-1]. Their action is completely reversed by propranolol at doses 10(3) times lower than practolol, while unaffected by phentolamine; (2) isoproterenol and Vasoactive Intestinal Peptide (VIP) have additive effects on cyclic AMP in fundic glands whereas no additivity is observed between histamine and isoproterenol; this, together with the absence of catecholamine effect in antral glands, suggests that the beta 2 receptor is located on parietal cells; (3) somatostatin (1 mumol 1(-1] non-competitively inhibits the stimulation by catecholamines but does not affect VIP and histamine stimulations. These results suggest a physiological stimulatory effect of catecholamines on gastric acid secretion in man, through a beta 2 receptor coupled to the cyclic AMP system, regulated by somatostatin.     

Enhancement of alpha-1 and alpha-2 adrenergic agonist-induced vasoconstriction by removal of endothelium in rat aorta

It is well known that the vascular endothelial cell layer plays an essential role in the vasodilatory response of several agents. In this study we have investigated the possibility that the endothelium may also modulate alpha adrenergic agonist-induced vasoconstriction. The responses of rat aortae to selective alpha-1 and alpha-2 adrenergic agonists were studied. Removal of the endothelium did not significantly alter the maximum contractile response to norepinephrine. However, the maximum responses to selective alpha-1 agonists (phenylephrine and methoxamine) were increased 2-fold. The vasoconstrictor effects of both clonidine and B-HT920 (selective alpha-2 agonists) were enhanced 5- to 7-fold after removal of the endothelial cell layer. The sensitivity of the tissue, as reflected by the EC50 value, to each alpha adrenergic agonist was enhanced in the absence of endothelium. An explanation for the present results is that alpha-1 and alpha-2 adrenergic agonists activate adrenoceptors in the endothelial cells and thereby may promote the release of a relaxing factor to inhibit vascular smooth muscle contraction. Removal of the endothelium would abolish release of this putative inhibitory substance and adrenergic agonist would activate only adrenoceptors in the muscle to cause vasoconstriction. On the other hand, endothelial cells may function as an uptake site for the various adrenergic agonists. Ablation of this uptake process could conceivably result in a greater effective concentration of the agonist in the receptor area and thus promote a stronger vasoconstrictor effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of imipramine and adrenocorticotropin administration on the rat brain norepinephrine-coupled cyclic nucleotide generating system: alterations in alpha and beta adrenergic components

Continuous treatment (1-3 weeks) with imipramine or adrenocorticotropin (ACTH) decreases the responsiveness of the norepinephrine-coupled cyclic nucleotide generating system in rat brain cerebral cortex. Experiments were undertaken to determine which component of the second messenger system is influenced by the hormone and antidepressant. Neither treatment modified the amount or function of extractable stimulatory guanine nucleotide binding protein or the activities of adenylate cyclase or phosphodiesterase. While both imipramine and ACTH treatment decreased the cyclic AMP response to norepinephrine, only imipramine administration influenced the response to isoproterenol. ACTH treatment was found to reduce the alpha adrenergic potentiation of isoproterenol- and 2-chloroadenosine-stimulated cyclic AMP production, as well as reduce the sensitivity of the norepinephrine response to prazosin. These findings indicate that imipramine and ACTH treatments decrease the responsiveness of the rat brain norepinephrine-stimulated cyclic AMP generating system through actions on the alpha and beta adrenergic receptor components. The results suggest that noradrenergic receptor activity may be under the control of adrenal and/or pituitary hormones.