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.     

Alteration in sensitivity to isoproterenol and acetylcholine in the rat heart after repeated administration of isoproterenol

Alteration of sensitivity to the inotropic responses to isoproterenol and acetylcholine (ACh) and of saturation constants for the specific binding of [3H]dihydroalprenolol and [3H]quinuclidinyl benzilate to particulate fractions were investigated in rat hearts chronically treated with isoproterenol. The EC50 value for the inotropic response to isoproterenol in the atria of rats injected i.p. with isoproterenol (0.01 or 0.1 mg/kg) twice a day for 10 days, was 9.0-fold higher than in controls. Isoproterenol (0.17 mg/kg i.p.)-induced stimulation of cardiac ornithine decarboxylase activity was significantly (P less than .05) attenuated in atria from animals of the treated groups. Scatchard analysis of specific binding of [3H]dihydroalprenolol revealed that repeated isoproterenol injection produced a significant decrease in the maximum number of binding sites (Bmax), but not in the dissociation constant (KD) for this antagonist. In comparison to the isoproterenol-induced changes in beta adrenergic positive inotropic response, repeated treatment with isoproterenol decreased 2-fold the EC50 value for the negative inotropic response to ACh in isolated atria. Scatchard analysis of specific [3H]quinuclidinyl benzilate binding, however, indicated a significant (P less than .01) decrease in Bmax. Repeated isoproterenol injections also elicited a significant increase in cardiac weight. It is suggested that repeated administration of isoproterenol induces a decrease in density of beta adrenergic receptors which may in part underlie a cardiac hyposensitivity to isoproterenol and an increase in the number of muscarinic ACh receptors and inotropic responsiveness to ACh.     

Changes in beta adrenergic receptors in submaxillary glands of chronically reserpine- or isoproterenol-treated rats

Submaxillary glands of rats, chronically treated with isoproterenol or reserpine undergo morphological and functional alterations. These changes have been described to resemble those seen in human cystic fibrosis. Since it has been proposed that the beta adrenergic-mediated response is altered in exocrine glands of cystic fibrosis patients, we have examined whether the drug-induced alterations in rat salivary glands were accompanied by changes in the numbers and affinities of beta adrenergic receptor sites. Beta receptor characteristics were determined by means of direct binding studies with the beta adrenergic antagonist [3H]dihydroalprenolol. Compared to controls, specific binding capacities of [3H]dihydroalprenolol per unit of protein increased by 110 +/- 14% after reserpine treatment and decreased by 34 +/- 11% after isoproterenol administration (P less than .001). The difference in the number of receptor sites remained statistically significant whether expressed per gram of fresh weight or per unit of the membrane marker 5'-nucleotidase activity. Dissociation constants of the binding were not significantly different between the treatment groups. The observed changes in the number of beta receptors showed an inverse relationship to the drug-induced presumed changes of catecholamine concentrations at the receptor sites. This suggests the existence of a feedback system which maintains the balance within the autonomous nervous system. We speculate that in cystic fibrosis this adaptive system is genetically abnormal.     

Biphasic Adrenergic Modulation of β-Adrenergic Receptors in Man: AGONIST-INDUCED EARLY INCREMENT AND LATE DECREMENT IN β-ADRENERGIC RECEPTOR NUMBER

beta-Adrenergic receptors in mononuclear leukocyte preparations were assessed with (-)[(3)H]-dihydroalprenolol binding studies during the infusion of adrenergic agonists into normal human subjects. During the infusion of isoproterenol into seven subjects, mean (+/-SE) (-)[(3)H]dihydroalprenolol binding increased from 25+/-3 fmol/mg protein to 47+/-8 fmol/mg protein (P < 0.02) at 0.5 h and 40+/-3 fmol/mg protein (P < 0.01) at 1 h and decreased to 12+/-1 fmol/mg protein (P < 0.01) at 4-6 h. During the infusion of epinephrine into three subjects, mean (-)[(3)H]dihydroalprenolol binding increased from 32+/-3 to 63+/-3 fmol/mg protein (P < 0.01) at 0.5-1 h. By Scatchard plot analysis, these changes were attributable to changes in the number of available binding sites rather than changes in binding affinity. The observed changes in the number of (-)[(3)H]dihydroalprenolol binding sites were not paralleled by changes in total mononuclear cell counts or in T lymphocyte, B lymphocyte, and monocyte distributions. Thus, we conclude that adrenergic agonists modulate the number of available beta-adrenergic receptors on circulating mononuclear cells in a biphasic manner, with an early increment and a late decrement, in man. Further, the finding that the increase in pulse rate in response to a "pulse" infusion of isoproterenol was significantly greater after 0.5-1 h of agonist infusion suggests that the observed early agonist-induced increment in beta-adrenergic receptor number on circulating cells is paralleled by increments in extra-vascular beta-adrenergic receptor sensitivity.     

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.     

Characterization of a bromoacetylated derivative of pindolol as a high affinity, irreversible beta adrenergic antagonist in cultured cells

We have utilized cultured cell lines to test the utility of N8-(bromoacetyl)-N1-[3-(4-indolyoxy)-2-hydroxypropyl]-(Z)-1,8-diam ino- p-menthane, BIM, a recently synthesized, irreversible beta adrenergic antagonist. Previously available irreversible antagonists of beta adrenergic receptors have generally exhibited low affinity (typical IC50 values greater than or equal to 1 microM). By contrast, S49 lymphoma cells incubated with 10 nM BIM for 120 min and then washed extensively showed a 70% loss in beta adrenergic receptors, as measured by [125I]iodocyanopindolol binding. This loss, which could be prevented by propranolol, represented a decrease in receptor number without a change in affinity of the remaining receptors for [125I]iodocyanopindolol. The BIM-induced decrease in binding sites was persistent in membranes incubated for several hours after BIM treatment. BIM did not inactivate alpha-1 adrenergic receptors on Madin Darby canine kidney cells, alpha-2 adrenergic receptors on human erythroleukemia cells, nor did BIM treatment alter guanyl-5'-yl-imidodiphosphate-mediated regulation of agonist binding to the beta adrenergic receptors in S49 cell membranes. BIM treatment decreased cyclic AMP (cAMP) accumulation in S49 cells in response to the beta adrenergic agonist isoproterenol, but increased prostaglandin E1-stimulated cAMP accumulation (P = .09) without altering cAMP production in response to forskolin. The inactivation of beta receptors in S49 cells by BIM (IC50 = 0.30 nM) correlated closely with the loss in beta adrenergic receptor-mediated cAMP accumulation in these cells (IC50 = 0.59 nM), implying the absence of substantial receptor reserve for this response. We conclude that BIM is a potent, irreversible, selective beta adrenergic antagonist for the study of beta adrenergic receptors in cultured cells.     

Activation of alpha-2 adrenergic receptors augments neurotransmitter-stimulated cyclic AMP accumulation in rat brain cerebral cortical slices

Norepinephrine-stimulated cyclic AMP (cAMP) accumulation in brain is mediated by both alpha and beta adrenergic receptors. The functional activity of these receptors can be differentiated by examining the response to isoproterenol alone and isoproterenol + 6-fluoronorepinephrine, and alpha adrenergic agonist. The present study was undertaken to define the pharmacological characteristics of the alpha adrenergic component associated with cAMP accumulation in brain. Using a prelabeling technique it was found that norepinephrine- or isoproterenol plus 6-fluoronorepinephrine-stimulated cAMP accumulation was only inhibited partially by an alpha-1 adrenergic receptor antagonist. In contrast, alpha-2 adrenergic receptor antagonists inhibited completely that portion of the response exceeding that produced by isoproterenol alone (alpha adrenergic augmentation). Furthermore, selective alpha-1 adrenergic agonists were incapable of enhancing the cAMP response to isoproterenol, whereas alpha-2 adrenergic agonists were active in this regard. Partial agonists for the alpha-2 adrenergic receptor were ineffective as augmentors. The data suggest that the alpha adrenergic component of the norepinephrine response in rat brain slices has characteristics of both alpha-1 and alpha-2 receptors, with the alpha-2 adrenergic component being a major contributor in this regard.     

Comparison of beta adrenergic receptor binding characteristics and coupling to adenylate cyclase in rat pulmonary artery versus aorta

In an effort to define the mechanisms regulating pulmonary vasodilatation and explain the greater in vitro response to iso-proterenol in the pulmonary artery (PA) vs. aorta (AO), we compared beta adrenergic receptor binding characteristics and coupling to adenylate cyclase in PA and AO obtained from adult male rats. Beta adrenergic receptor binding characteristics and affinity for agonists were determined with [125I]-iodocyanopindolol. Agonist displacement studies were characteristic of a beta-2 adrenergic receptor subtype. Receptor density (44.7 +/- 7.3 vs. 39.6 +/- 0.8 fmol/mg of protein means +/- S.E.M., PA vs. AO) and the dissociation constant for the radioligand (10.3 +/- 2.6 vs. 13.4 +/- 3.5 pM) were similar in the two arteries. However, affinity for l-isoproterenol was greater (the inhibition constant was lower) in PA compared to AO (0.08 +/- 0.03 vs. 1.20 +/- 0.18 microM, P less than .05), as was affinity for l-epinephrine (0.89 +/- 0.20 vs. 3.87 +/- 0.62 microM, P less than .05). Affinity was similar for l-norepinephrine (18.93 +/- 3.63 vs. 13.49 +/- 3.12 microM). Base-line cyclic AMP (cAMP) content, basal adenylate cyclase activity and adenylate cyclase activity stimulated by GTP, isoproterenol plus GTP and forskolin were measured by radioimmunoassay for cAMP. Base-line cAMP content was greater in PA than in AO (513.5 +/- 46.9 vs. 125.5 +/- 19.1 pmol of cAMP per mg of protein, P less than .001), as was basal adenylate cyclase activity (10.8 +/- 1.2 vs. 5.7 +/- 1.3 pmol of cAMP per mg of protein per min, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta Adrenergic Receptors of Polymorphonuclear Particulates in Bronchial Asthma

We have tested the beta adrenergic receptor theory of bronchial asthma by determining the number and affinity of binding sites of the beta adrenergic radioligand [(3)H]dihydroalprenolol (DHA) and the activity of adenylate cyclase in broken cell preparations of polymorphonuclear leukocytes (PMN). We studied 31 control subjects (group 1), 30 asthmatics receiving no systemic adrenergic medication (group 2), and 17 asthmatics receiving adrenergic agonists systemically (group 3). Control subjects and asthmatics taking no adrenergic drugs bound similar amounts of DHA at 0.5 nM and 30 nM DHA and had about 900 binding sites per PMN. In contrast, asthmatics receiving adrenergic agonists had a >70% decrease in their number of DHA binding sites per PMN (254+/-57). In a subset of our three groups of subjects (eight from group 1, six from group 2, and five from group 3) we measured DHA binding at several DHA concentrations and found similar values (0.4-0.7 nM) for the dissociation constant of DHA among these subjects.In further studies we examined the interaction of the agonist (-)-isoproterenol with beta adrenergic receptors in 8 normal subjects and 10 asthmatics not receiving adrenergic medication. We tested the ability of isoproterenol to compete for DHA binding sites and to stimulate adenylate cyclase in sonicates prepared from PMN and examined under identical conditions. The dissociation constants for the competition of isoproterenol for DHA binding sites in normal and asthmatic subjects were virtually identical ( approximately 1.0 muM). In addition, the (activation constant) values for stimulation of adenylate cyclase were similar (0.16-0.19 muM) in the two groups of subjects.Thus, these data suggest that asthma per se is not associated with alteration in either the number or affinity of beta adrenergic receptors in PMN. Our findings indicate that previous reports of abnormal beta adrenergic receptor function in asthmatic patients may in part be explained by prior treatment of such patients with adrenergic agonists. Because the asthmatics who received adrenergic agonists in our study tended to be more ill and to receive additional medication compared to subjects in group 2, we cannot rule out unequivocally that severe asthma may be associated with decreased binding to beta adrenergic receptors. Nevertheless, we conclude that beta adrenergic receptors on PMN from asthmatics are relatively normal unless such patients are treated with adrenergic agonists.     

Inhibition of lipolysis by adenosine is potentiated with age.

The ability of a variety of hormones to activate cells declines with age. We have investigated the mechanism for the reduced ability of beta adrenergic stimulation to activate lipolysis in fat cells from older rats. Previously, we have found that these cells have an intact lipolytic response to a cAMP analogue but diminished cAMP accumulation after isoproterenol stimulation, suggesting that the blunted cAMP response is rate limiting. In the present study we have tested the hypothesis that enhanced inhibition of lipolysis by endogenously released adenosine accounts for the diminished lipolysis. Adenosine deaminase was added to media containing the adipocytes from older rats to remove endogenous adenosine. Under these conditions beta adrenergic stimulation of lipolysis is intact in fat cells from older rats. The adenosine analogue N6-phenylisopropyladenosine more effectively inhibited lipolysis in the older group (77 +/- 6%) than in the younger group (46 +/- 5%), suggesting that enhanced efficacy of endogenous adenosine may account for the reduced lipolytic response to catecholamines. When pertussis vaccine was used to functionally inactivate adenosine receptors in adipocytes from the younger and older rats, the ability of isoproterenol to activate lipolysis was restored in the older group. All the data are consistent with the hypothesis that enhanced inhibitory effects of adenosine explain the diminished ability of beta adrenergic agonists to activate lipolysis. It is possible that enhanced inhibitory pathways may be involved in blunting responses to stimulatory hormones in other tissues from older animals.     

Defective beta adrenergic response of cystic fibrosis sweat glands in vivo and in vitro.

Abnormal ductal NaCl absorption has been known as the only defect in cystic fibrosis (CF) sweat glands. We have fortuitously found that the secretory portion of CF sweat glands is also abnormal in that it failed to show a sweating response to beta adrenergic stimulation (isoproterenol, [ISO]) both in vivo and in vitro. For the in vitro sweat test, eccrine sweat glands were isolated from skin biopsy specimens of the forearm, cannulated, and stimulated to secrete sweat. All 14 isolated CF sweat glands failed to respond to ISO + theophylline (TH, as aminophylline), but 17 of 18 control glands responded with a mean rate (SR) of 1.1 nl/min per gland. Cholinergic responsiveness of isolated CF sweat glands was comparable with that of control glands. The in vivo sweat test was performed by intradermal injection in the forearm of 0.2 ml of 2.4 or 8 X 10(-5) M ISO with or without 10(-2) M TH (and 1.4 X 10(-4) M atropine as a necessary anticholinergic agent). The beads of sweat secreted into the oil-filled sweat collection ring glued to the skin were then collected with a glass capillary under a stereomicroscope. Of 28 CF patients, 26 failed to show a secretory response to intradermal injection of ISO + TH, and 2 CF patients gave SR of less than 0.007 nl/min per gland in the first test but no response in the repeat test performed later. In contrast, all 35 age- and sex-matched control subjects responded with the mean SR of 0.72 nl/min per gland. Response of CF patients to epinephrine and phenylephrine was comparable with control, indicating that the alpha adrenergic responsiveness of CF sweat glands is not defective. A preliminary attempt was made to determine tissue cyclic AMP accumulation by radioimmunoassay in isolated sweat glands. No significant difference was observed between CF and control glands in their maximal accumulation of tissue cAMP in response to ISO or ISO + TH, except that the rise time of ISO + TH-induced cAMP accumulation in CF glands was significantly slower during the first 5 min of incubation. The data suggest that beta adrenergic regulation is abnormal in CF sweat glands and justifies further investigations into the mechanism of beta adrenergic regulation of the eccrine sweat gland in both normal and CF subjects.     

Subtype-selective regulation of beta adrenergic receptor-adenylyl cyclase coupling by phorbol esters in 3T3-L1 fibroblasts

To study the epigenetic regulation of beta adrenergic receptor subtypes, we examined the effects of phorbol esters on beta adrenergic receptor coupling to adenylyl cyclase in 3T3-L1 fibroblasts, which express both beta-1 and beta-2 adrenergic receptor subtypes. Pretreatment of intact 3T3-L1 cells with the protein kinase C activator phorbol dibutyrate caused a dose- and time-dependent decrease in subsequent cyclic AMP (cAMP) accumulation mediated by the beta adrenergic agonist isoproterenol. This effect was selective for beta-adrenergic receptor-mediated responses because there was a potentiation of cAMP accumulation caused by other activators such as prostaglandin E1, forskolin or cholera toxin. The inactive phorbol, alpha-phorbol dibutyrate was ineffective at 1 microM in attenuating isoproterenol stimulation, and 25 nM of the protein kinase C inhibitor staurosporine blocked the effects of phorbol ester on beta adrenergic agonist responses. Stimulation of cAMP accumulation by isoproterenol occurred through a greater proportion of beta-2 adrenergic receptors in phorbol dibutyrate-treated cells than in control cells. This was demonstrated using the beta-1 adrenergic selective antagonist ICI 89.406 and the beta-2 adrenergic selective antagonist ICI 118.551 to inhibit competitively isoproterenol-stimulated cAMP accumulation. Beta-2 adrenergic receptor number and subtype in these cells are regulated by glucocorticoids and butyrate. Decreasing the proportion of beta-1 adrenergic receptors and concomitantly increasing beta-2 adrenergic receptors with either glucocorticoids or butyrate decreased the ability of phorbol ester pretreatment to attenuate cAMP accumulation by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Norepinephrine- and isoproterenol- induced changes in cardiac contractility and cyclic adenosine 3':5' -monophosphate levels during early development of the embryonic chick.

Changes in contractility and cyclic adenosine 3':5'-monophosphate (cAMP) levels in response to norepinephrine and isoproterenol were monitored in isolated 4-day-old (noninnervated) and 7-day-old (innervated) embryonic hearts to determine whether a relationship between beta adrenergic receptor, adenylate cyclase and altered cardiac function is established at a very early stage in embryonic development before innervation takes place. Norepinephrine and isoproterenol promoted rapid time- and dose-dependent rises in cAMP levels which were greater in the 4-day-old hearts. These increases paralleled observed functional alterations within a specific range of drug concentrations and time. The elevation of cAMP levels and effect on cardiac function produced by isoproterenol (10(-7)M) were blocked by propranolol (10(-6)M). Dissociations between changes in tissue cAMP levels and cardiac function were also uncovered. Maximal increases in contractility were achieved with lower drug concentrations than were required to promote maximal accumulation of cAMP. Relatively high concentrations of norepinephrine or isoproterenol were less effective than lower concentrations in stimulating contractility but were more effective in promoting cAMP accumulation, The results indicate that both cardiac beta receptors adenylate cyclase are present and functionally related early in embryogenesis before sympathetic innervation occurs and that cAMP accumulation is associated with modulation of contractile activity whithin a certain concentration range and time course.     

Effects of adrenergic stimulation on ventilation in man

The mechanism by which catecholamines affect ventilation in man is not known. Ventilatory responses to catecholamines were observed in normal subjects before and after adrenergic receptor blockade. Intravenous infusions of norepinephrine and isoproterenol caused significant increases in minute volume and decreases in end-tidal P(Co2) which were blocked by the administration of propranolol, a beta adrenergic receptor blocker. The hyperventilatory response to hypoxia was not altered by propranolol.Intravenous infusion of phenylephrine caused a small but significant decrease in minute volume which was antagonized by phentolamine, an alpha adrenergic receptor blocker. Angiotensin, a nonadrenergic pressor agent, also decreased minute volume significantly.100% oxygen was administered to suppress arterial chemoreceptors. Increases in minute volume and decreases in arterial P(Co2) in response to norepinephrine and isoproterenol were blocked by breathing 100% oxygen. The decrease in minute volume during phenylephrine was not altered by 100% oxygen.THE RESULTS INDICATE THAT: (a) beta adrenergic receptors mediate the hyperventilatory response to norepinephrine and isoproterenol but not to hypoxia. (b) the pressor agents phenylephrine and angiotensin decrease ventilation, and (c) suppression of chemoreceptors blocks the ventilatory response to norepinephrine and isoproterenol but not to phenylephrine. Implications concerning the interaction of adrenergic receptors and chemoreceptors with respect to the hyperventilatory response to catecholamines are discussed.     

Adrenergic Modulation of Pancreatic Glucagon Secretion in Man

In order to characterize the influence of the adrenergic system on pancreatic glucagon secretion in man, changes in basal glucagon secretion during infusions of pure alpha and beta adrenergic agonists and their specific antagonists were studied. During infusion of isoproterenol (3 mug/min), a beta adrenergic agonist, plasma glucagon rose from a mean (+/-SE) basal level of 104+/-10 to 171+/-15 pg/ml, P < 0.0002. Concomitant infusion of propranolol (80 mug/min), a beta adrenergic antagonist, prevented the effects of isoproterenol, although propranolol itself had no effect on basal glucagon secretion. During infusion of methoxamine (0.5 mg/min), an alpha adrenergic agonist, plasma glucagon declined from a mean basal level of 122+/-15 to 75+/-17 pg/ml, P < 0.001. Infusion of phentolamine (0.5 mg/min), an alpha adrenergic antagonist, caused a rise in plasma glucagon from a mean basal level of 118+/-16 to 175+/-21 pg/ml, P < 0.0001. Concomitant infusion of methoxamine with phentolamine caused a reversal of the effects of phentolamine.The present studies thus confirm that catecholamines affect glucagon secretion in man and demonstrate that the pancreatic alpha cell possesses both alpha and beta adrenergic receptors. Beta adrenergic stimulation augments basal glucagon secretion, while alpha adrenergic stimulation diminishes basal glucagon secretion. Furthermore, since infusion of phentolamine, an alpha adrenergic antagonist, resulted in an elevation of basal plasma glucagon levels, there appears to be an inhibitory alpha adrenergic tone governing basal glucagon secretion. The above findings suggest that catecholamines may influence glucose homeostasis in man through their effects on both pancreatic alpha and beta cell function.     

The Glucose Receptor A DEFECTIVE MECHANISM IN DIABETES MELLITUS DISTINCT FROM THE BETA ADRENERGIC RECEPTOR

Acute serum insulin responses in 10 normal subjects after rapid intravenous injection of glucose (5 g) or isoproterenol (2 μg) were of similar magnitude and timing (glucose: 431±349%; mean Δ3-5′ insulin (IRI)±SD, per cent basal and isoproterenol: 359±216%; mean Δ2-4′ IRI±SD, per cent basal). To elucidate the relationship of glucose-induced insulin secretion to pancreatic beta adrenergic receptors and the implications of this relationship with regards to abnormal insulin secretion in diabetes mellitus, two questions were studied. (a) To determine whether glucose-induced insulin secretion is dependent upon beta adrenergic activity, the effect of beta adrenergic blockade with intravenous propranolol (0.08 mg/min) upon acute insulin responses to isoproterenol and glucose were compared in normal subjects. (b) To determine whether acute insulin responses to beta adrenergic stimulation were intact in diabetes mellitus, the effect of isoproterenol upon serum insulin levels was studied in diabetic subjects. Beta adrenergic blockade in the normal subjects obliterated acute insulin responses to isoproterenol (before: 361±270%, during: - 31±15%; n = 6, P < 0.001) but did not significantly affect responses to glucose (before; 311±270%; during: 284±206%; n = 5). The mean acute insulin response after isoproterenol in the diabetic group was significantly elevated over basal levels (152±74%; n = 10, P < 0.001) but the response after glucose was not (- 11±11%). These data suggest that insulin responses to glucose in normal subjects are mediated by specific pancreatic glucose receptors which are independent from beta adrenergic receptors and that abnormal glucose-induced insulin secretion in diabetics is due to defects within glucose receptors and not beta adrenergic receptors as has been previously hypothesized.     

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.     

Characterization of beta adrenoceptor subtypes in canine airway smooth muscle by radioligand binding and physiological responses

Beta adrenoceptor subtypes in canine tracheal smooth muscle have been investigated by radioligand binding and by physiological responses to beta agonists and sympathetic nerve stimulation in vitro. Specific binding of [3H]dihydroalprenolol to tracheal smooth muscle membranes was of high affinity (Kd = 1.0 +/- 0.08 nM), as in peripheral lung membranes from the same animals, but the concentration of binding sites (95.6 +/- 4.7 fmol/mg of protein) was much lower than in lung (532 +/- 48 fmol/mg of protein). Binding was stereoselective and agonists competed with the rank order of potency isoproterenol greater than epinephrine greater than norepinephrine, signifying a preponderance of beta-2 receptors. Using selective beta antagonists, we determined the ratio of beta-1/beta-2 receptors in tracheal smooth muscle membranes to be 1:4. The relaxation response of tracheal smooth muscle strips to exogenous beta agonists was mediated by beta-2 receptors, with a very small contribution from beta-1 receptors. However, the relaxation response to electrical field stimulation of sympathetic nerves was mediated predominantly by beta-1 receptors. Our results suggest that most beta receptors in dog tracheal smooth muscle are of the beta-2 subtype and mediate responses to circulating catecholamines, but there is a small concentration of beta-1 receptors which mediate the response to neurally released norepinephrine.     

Decline in beta adrenergic receptor-mediated vascular relaxation with aging in man

Beta adrenergic relaxation of vascular smooth muscle, mediated by cyclic AMP, is blunted with age in a variety of experimental animals. The applicability of these observations to man is uncertain. The dorsal hand vein technique provides an excellent method to examine the direct effects of aging on vascular responsiveness. Thirty-nine healthy male volunteers over the age range of 19 to 79 were studied. No differences in vascular responsiveness to phenylephrine, an alpha adrenergic agonist, were found for either the ED50 (dose producing 50% vasoconstriction) or Emax (maximum vasoconstriction attained). In marked contrast, vascular relaxation induced by isoproterenol, a beta adrenergic agonist, was significantly different in both the ED50 (dose producing 50% of maximum relaxation from a preconstricted state) and Emax (maximum relaxation attained). ED50 +/- S.E.M. for the youngest and oldest deciles were 8.9 +/- 2.3 and 60 +/- 17.0 ng/min, respectively (P less than .05); Emax +/- S.E.M. were 96.7 +/- 3.3 and 37.7 +/- 8.7%, respectively (P less than .001). Nitroglycerin, a smooth muscle relaxant whose effects are not mediated through the cyclic AMP system, was also used to examine the specificity of this blunted response to isoproterenol. Almost complete relaxation was achieved with the infusion of nitroglycerin in the older group. These results suggest that aging is associated with a specific decrease in beta adrenoreceptor-mediated vascular relaxation.