Chronic norepinephrine elicits desensitization by uncoupling the beta-receptor.

The goal of this study was to determine the mechanism of beta-adrenergic receptor desensitization after chronic elevation of circulating NE levels. Osmotic minipumps containing either NE or saline were implanted subcutaneously in dogs for 3-4 wk. Physiologic desensitization to isoproterenol was confirmed in conscious dogs, i.e., left ventricular dP/dt increased in response to isoproterenol (0.4 micrograms/kg per min) by 5,625 +/- 731 mmHg/s in control dogs with saline pumps, and significantly less, P less than 0.01, by 2,093 +/- 263 mmHg/s in dogs with NE pumps. Myocardial beta-adrenergic receptor density as determined with 125I-cyanopindolol binding was 49% higher (p less than 0.05) in the NE pump group. However, beta-adrenergic receptor agonist binding with isoproterenol demonstrated a significant shift into the low affinity state for the animals with NE pumps. Basal, GTP plus isoproterenol, 5'-guanylylimidodiphosphate, sodium fluoride, and forskolin-stimulated adenylate cyclase activity in the NE pump group were significantly depressed (P less than 0.05) by amounts ranging from 20 to 40%. The functional activity of the guanine nucleotide binding protein Gs was also reduced (P less than 0.05) in animals with NE pumps. Thus, the process of desensitization in response to chronic elevation of NE levels in intact, normal dogs does not involve a decrease in beta-adrenergic receptor density. Rather, it is characterized by reduced adenylate cyclase activation and uncoupling of the beta-adrenergic receptor in association with decreased activity of the GTP-coupling protein Gs.

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.

Translocation and uncoupling of the beta-adrenergic receptor in rat lung after catecholamine promoted desensitization in vivo.

Beta-adrenergic agonists and antagonists are widely used in many clinical situations to regulate beta-adrenergic stimulation. However, responsiveness to beta-stimulation may be reduced by the process of desensitization. We have established an in vivo mammalian model system, the rat lung, to study the cellular and biochemical basis for beta-agonist induced desensitization. After in vivo administration of a beta-agonist [(-)isoproterenol] the adenylate cyclase becomes rapidly insensitive to further stimulation by beta-agonists with no change in basal or NaF-stimulated activity. The in vivo desensitization can be blocked by the simultaneous administration of a beta-antagonist [(+/-)propranolol] and the process displays the pharmacological characteristics typifying the beta 2 receptor of rat lung. This indicates that the in vivo desensitization is itself a receptor-mediated event. The processes of de- and resensitization are very rapid with onset within 5 min, maximal effect at 10 min, and complete reversal by 2-3 h. The change of adenylate cyclase sensitivity is paralleled by a translocation of approximately 40% of the beta-receptors from the plasma membrane fraction to a light membrane fraction, which has very low activities of plasma membrane marker enzymes. The receptors translocated to the light membrane fraction as well as those remaining in the plasma membranes are uncoupled with loss of their ability to form the high affinity, nucleotide sensitive, physiologically active state of the receptor. During resensitization the receptors in the plasma membrane fraction are recoupled before all the translocated receptors have returned. This suggests that translocation and uncoupling of the receptors are two distinct, probably independent processes. During the entire process of de- and resensitization no structural change of the receptor protein residing in the plasma membranes or light membrane fraction can be demonstrated as visualized by photoaffinity labeling.

Effects of N-ethylmaleimide on gonadotropin and beta-adrenergic receptor function coupled to rabbit luteal adenylyl cyclase.

The effects of the sulfhydryl-reactive alkylating agent N-ethylmaleimide (NEM) on the rabbit luteal adenylyl cyclase system were studied. Treatment of luteal membranes with NEM revealed three activities with differing sensitivities to NEM treatment. When luteal membranes were treated with NEM on ice for 30 min, it was found that NaF-stimulated adenylyl cyclase activity in cholate extracts of these membranes was most sensitive to this treatment. Half-maximal inhibition was obtained at 0.09 mM NEM. The activity of the stimulatory guanine nucleotide- and Mg-binding regulatory component (Ns), as assessed by functional reconstitution of NaF-stimulated adenylyl cyclase activity into membranes from the cyc-variant of the S49 mouse lymphoma, was less sensitive to this treatment, with half-maximal inhibition occurring at 0.69 mM NEM. In contrast, high affinity gonadotropin and beta-adrenergic binding, as assessed by competitive displacement of [125I]iodo-hCG by bovine LH and (-)3-[125I]iodocyanopindolol by isoproterenol, was unaffected by NEM concentrations up to 50 mM when membranes were treated on ice. However, when membranes were treated with NEM at 25 C for 30 min, high affinity gonadotropin and beta-adrenergic binding demonstrated similar sensitivities to NEM treatment, such that 50 mM NEM completely inhibited high affinity binding to both receptors. Under either of the conditions described above, neither the number of receptors nor the affinities of the labeled probes for their receptors were altered by NEM treatment. Thus, there appears to be at least three NEM-sensitive sites necessary for the functioning of the rabbit luteal adenylyl cyclase system, one associated with the catalytic component, one on Ns which interacts with the catalytic component, and one involved in high affinity agonist binding. Furthermore, it appears that formation of the high affinity binding state is regulated similarly for gonadotropin and beta-adrenergic receptors.

Beta-adrenergic receptors, glucagon receptors, and their relationship to adenylate cyclase in rat liver during aging.

The beta-adrenergic and glucagon receptor-binding capacities in rat livers from 6-27 months of age were measured to investigate the mechanism of a previously observed rise in beta-adrenergic stimulated adenylate cyclase with increasing age. There was no concomitant increase in glucagon-stimulated adenylate cyclase. In the present study neither glucagon-binding capacity nor glucagon-stimulated adenylate cyclase changed with age. In contrast, the beta-adrenergic receptor capacity, measured in the same membranes by [125I]iodopindolol binding, increased nearly 3-fold from 6.6 +/- 0.6 fmol/mg at 6 months to 19.1 +/- 3.3 fmol/mg at 18-19 months. The increase was directly proportional to the maximum isoproterenol-stimulated adenylate cyclase activity in livers of rats up to 19 months of age. By 24-27 months the binding capacity had increased to 24.9 +/- 3.3 fmol/mg, but there was no further increase in adenylate cyclase activity. Thus, there appeared to be a beta-receptor-adenylate cyclase uncoupling in livers from the senescent animals (25-27 months). The defect could not be demonstrated by studies examining isoproterenol competition of [125I]iodopindolol from agonist-induced high affinity sites on the membranes, a procedure that examines receptor-Ns protein coupling. Activation of adenylate cyclase by the nonhormonal stimulators F- and forskolin did not change with age, indicating that the catalytic unit was not a limiting factor. Since the relationship between the glucagon receptor and adenylate cyclase also remained unaltered, the uncoupling apparently lies in an alteration of the interaction between the beta-adrenergic receptor and the guanine nucleotide-sensitive Ns protein.

Use of electrofocusing for the analysis and purification of turkey erythrocytes beta 1-adrenoceptors.

We show that following one cycle of alprenolol affinity chromatography of turkey erythrocyte beta 1-adrenoceptors, electrofocusing on polyacrylamide gels in digitonin, followed by electroelution, results in complete receptor purification. The overall yield from the electrofocusing-electroelution step of turkey erythrocyte beta-adrenoceptor is 75 +/- 3%. In addition, we are able to demonstrate that receptor-binding assays can be performed directly on the polyacrylamide gel, using 125I-cyanopindolol. This method can be employed for minute quantities of receptor which is an advantage when one wishes to characterize rapidly the beta-adrenoceptor in its native state from tissues that may be available only in limited amounts. We also report, for comparison, on the behavior of the turkey erythrocyte beta 1-adrenoceptor on immobiline polyacrylamide gels and the ability to purify only partially the receptor on these gels.

Characteristics of the beta-adrenergic receptor in the rat ventral prostate using [125I]cyanopindolol.

The binding characteristics of the beta-adrenergic receptor in the rat ventral prostate homogenate have been studied using the highly potent beta-adrenergic antagonist [125I]cyanopindolol (CYP) as ligand. The bound ligand was separated from the free moiety by precipitation with polyethylene glycol (PEG-6000). This technique is simple, accurate, fast and more advantageous than filtration of the hormone-receptor complex on glass fiber filters or direct centrifugation. [125I]CYP binds to a single class of high affinity sites at an apparent KD value of 23 pM. Using 0.1 microM (-)propranolol to determine non-specific binding, a number of sites of 600 fmol/mg protein were measured. The observed order of potency of adrenergic agonists (KD values) in competing for [125I]CYP binding was: (-)isoproterenol (25 nM) greater than (-)epinephrine (74 nM) much greater than (-)norepinephrine (1900 nM). Detailed study of the binding potency of a large series of beta 1- and beta 2-adrenergic agonists and antagonists showed the presence of a typical beta 2-subtype adrenergic receptor in the rat ventral prostate. The best estimate indicates that the proportion of beta 2-adrenergic receptors in rat ventral prostate is more than 95% of the total population of beta-adrenergic receptors in this tissue. The high selectivity and density of beta 2-adrenergic receptors in rat ventral prostate suggest a physiological role of circulating and/or locally secreted catecholamines in the control of prostatic growth and function.

Effect of physical training on ventricular beta-adrenergic receptor adenylate cyclase system of diabetic rats.

This study was designed to assess the effect of physical training on the ventricular beta-adrenergic receptor adenylate cyclase system of diabetic rats. Mild diabetes mellitus was induced by an intravenous (IV) injection of streptozotocin (45 mg/kg). Rats were randomized into a group submitted to a progressive 10-week running program on a treadmill, while another group was kept sedentary. A group of sedentary nondiabetic rats was used as normal controls. Results showed a similar reduction in the density of beta-adrenergic receptors in sedentary diabetic (P less than .05) and trained diabetic rats (P less than .01) compared with controls, without any significant alteration in the dissociation constant. The basal and the sodium fluoride-stimulated maximal adenylate cyclase activities were similar in the three groups. However, the maximal response of adenylate cyclase to isoproterenol was significantly reduced in the two diabetic groups compared with controls (P less than .01). The decrease in adenylate cyclase response to isoproterenol observed in the diabetic groups appeared to be associated with a reduction in the total number of beta-adrenergic receptors and more specifically in those existing in the high-affinity state. On the other hand, the hyperglycemia and hyperglucagonemia present in sedentary diabetic rats was improved by training. These data suggest that the beneficial effects observed in response to training in experimental diabetes are not associated with changes in beta-adrenergic receptor adenylate cyclase system on membranes from ventricular tissue.

Beta-adrenergic receptor activity of cerebral microvessels in experimental diabetes mellitus.

The effect of diabetes mellitus on beta-adrenergic receptor number (B(max)), receptor-cyclase coupling and adenylate cyclase (AC) activity was determined in cerebral microvessels isolated from control and streptozotocin induced diabetic rats after 5 weeks of induction of diabetes. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated that the B(max) (fmol/mg) in diabetic rat cerebral microvessels (63.8 +/- 4.8) (mean +/- S.E.M.) was not significantly different from the B(max) in control rats (56.5 +/- 6.9). Isoproterenol competition of [125I]ICYP binding sites indicated that the percentage of beta-receptors expressing high affinity binding was 53.9 +/- 0.45% in control rats and 47.5 +/- 2.3% in diabetic rats. The total isoproterenol-stimulated AC activity (pmol cAMP/mg) in diabetic rats (76.7 +/- 6.1) was significantly lower than that in control rats (118.4 +/- 11.2) (P less than 0.01). However, the net isoproterenol-stimulated AC activity (i.e. total minus GTP-stimulated AC activity) was not altered in diabetes. The net sodium fluoride (NaF) stimulated AC activity in diabetic rats (109.5 +/- 11.4) was significantly lower than the control rats (154.3 +/- 16.3) (P less than 0.05). It is concluded that diabetes mellitus in rats is associated with reduced post receptor activation of adenylate cyclase in cerebral microvessels while the beta-adrenergic receptor density, affinity and receptor-cyclase coupling are not significantly altered.

3,5,3'-L-triiodothyronine regulation of beta-adrenergic receptor density and adenylyl cyclase activity in synaptosomal membranes of aged rats.

To determine the effect of aging on T3 up-regulation of beta-adrenergic receptor activity of purified synaptosomal membranes (SPM), beta-adrenergic receptor density (Bmax) and adenylyl cyclase (AC) activity were measured in young (6 months old) and aged (26 months old) male F-344 rats at baseline and after treatment with triiodothyronine (T3), 15 micrograms/100 g, intraperitoneally for 10 days. The Bmax (fmol/mg protein) as measured by 125I-iodocyanopindolol binding was reduced in aged rats (70.8 +/- 4.1) compared to young rats (93.5 +/- 11.2). T3 treatment resulted in a significant increase in Bmax of young rats but not in aged rats (P < 0.05). The AC activity (pmol cAMP/mg) in response to prostaglandin E1 (PGE1) was reduced in aged rats compared to young rats (67.8 +/- 2.9 vs 108.3 +/- 18.6; P < 0.01). The sodium fluoride (NaF) stimulated AC activity was not altered with age. A net isoproterenol stimulated AC activity could not be demonstrated in any age group. T3 treatment did not alter AC activity of SPM. It is concluded that aging is associated with reduced responsiveness to T3-stimulated up-regulation of beta-adrenergic receptor number in synaptosomal membranes.

Activated charcoal and syrup of ipecac in prevention of cimetidine and pindolol absorption in man after administration of metoclopramide as an antiemetic agent.

The effects of activated charcoal and ipecac syrup by mouth on cimetidine and pindolol absorption were studied in seven subjects, who had ingested 20 mg metoclopramide 1 h earlier, and compared with the adsorption capacity of charcoal in vitro. Activated charcoal, 50 g, given 5 min after 400 mg cimetidine + 10 mg pindolol, reduced their absorption by 99% or more, based on AUC0-48h and the 48-h urinary excretion of the drugs. Syrup of ipecac caused emesis on each occasion. On the average, ipecac reduced the absorption of cimetidine and pindolol by 75% and 60%, respectively. Based on studies in vitro it seems probable that the adsorbing capacity of charcoal for cimetidine but not for pindolol will be saturated if 50 g charcoal is given after an overdose of about 100 fold the therapeutic dose. Because the use of ipecac allowed an absorption of the drugs at least 30 fold that allowed by charcoal, the immediate administration of activated charcoal, without preceding lavage or emesis, should be considered in such poisonings where the adsorption capacity of high charcoal doses will not be saturated.

Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors.

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.

Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity.

The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258-270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.

Endurance training, not acute exercise, differentially alters beta-receptors and cyclase in skeletal fiber types.

beta-Adrenergic receptor binding characteristics and adenylate cyclase activity were examined in rat skeletal muscle membranes to determine if acute exercise or endurance training altered beta-receptors or adenylate cyclase activity in different muscle fiber types. Binding characteristics and adenylate cyclase activity were examined in type IIA [red fast-twitch, red vastus (RV)], type IIB [white fast-twitch, white vastus (WV)], and type I [red slow-twitch, soleus (S)] muscles. Acute exercise involved a 20-min run on a treadmill at 20 m/min and did not alter beta-receptor density or adenylate cyclase activity in any of the fiber types examined. Endurance training consisted of a progressive treadmill protocol that involved increasing intensity and duration of exercise for 18 wk. beta-Adrenergic receptor density increased in skeletal muscle fiber types primarily recruited during submaximal training (types I and IIA), whereas nonreceptor-mediated adenylate cyclase activity was altered in the three fiber types. Endurance training significantly increased beta-receptor density in RV by 25% and in S by 19% (P less than 0.05), whereas in WV beta-receptor density was not altered. Basal adenylate cyclase activity in RV was increased approximately 2.5 fold by endurance training. Nonreceptor-mediated adenylate cyclase activity, stimulated by NaF and forskolin, increased by approximately twofold in both RV and WV as a result of endurance training. The data support and extend previous observations to show greater effects of endurance training in types I and IIA fibers with respect to alterations in beta-receptor density and alterations in adenylate cyclase activity in each fiber type. Acute exercise did not alter these parameters either in trained or untrained rats.

The mammalian beta 2-adrenergic receptor: reconstitution of functional interactions between pure receptor and pure stimulatory nucleotide binding protein of the adenylate cyclase system.

Pure beta-adrenergic receptors (beta-AR) isolated from guinea pig lung and pure guanine nucleotide binding regulatory protein (NS) of adenylate cyclase isolated from human erythrocytes have been inserted into phospholipid vesicles, resulting in the functional coupling of these two components. The reconstitution of receptor and NS interactions results in the establishment of a guanine nucleotide sensitive state of the receptor that binds agonists with high affinity. Competition curves of isoproterenol for labeled antagonist binding to vesicles containing both beta-AR and NS are biphasic and reveal two affinity states, one of high (approximately 2 nM) and the other of low affinity (approximately 300 nM). In the presence of guanine nucleotides, the competition curves become monophasic and are shifted to a single low-affinity state for the agonist similar to the situation observed in membrane preparations. In addition, the interactions of the receptor and NS lead to the induction of a GTPase activity in NS. The GTPase activity can be stimulated by beta-adrenergic agonists such as isoproterenol (2-5-fold) and is completely blocked by antagonists such as alprenolol in a stereoselective manner. The established hormone responsive activity retains the beta 2-adrenergic specificity conferred by the pure receptor, and similar extents of stimulation (up to 4-fold) are observed with pure receptor from frog erythrocytes, indicating a similar efficiency of coupling between receptors from different species and NS.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal sensitivity of adenylate cyclase incorporated in proteoliposomes.

Proteoliposomes containing adenylate cyclase (AC) from rabbit heart ventricles were obtained using a novel reconstitution procedure from solubilized state. The enzyme preparation can be stimulated by 5'guanylylimidodiphosphate (GppNHp) and NaF, but not by isoproterenol. Hormonal responsiveness of AC is restored by either isoproterenol trapped by the proteoliposomes during the reconstitution or pretreatment of proteoliposomes with alamethicin. GTP in the presence of alamethicin decreases the affinity of beta-adrenoceptors to the agonist, thus confirming that the properties of reconstituted AC system do not differ from the native one. It is demonstrated that the degree of AC activation by isoproterenol depends strongly on the beta-adrenoceptors content in the proteoliposomes, which in turn can be changed artificially in the process of reconstitution. The described reconstitution technique might be a useful tool for investigating the role of component stoichiometry in the functioning of hormone-regulated AC-system.

Beta-adrenergic receptor activity of cerebral microvessels is reduced in aged rats.

The effect of age on beta- (beta) adrenergic receptor number (Bmax) and adenylate cyclase (AC) activity was determined in microvessels isolated from male F-344 rats at 3, 18, and 24 months of age. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated reduced Bmax (fmol/mg) of microvessels isolated from 24 month old rats (27.2 +/- 4.9) compared with 3 month old (50.4 +/- 5.2) and 18 month old rats (p less than 0.01) (61.4 +/- 7.6). The basal AC activity (pmol cAMP/mg) in 24 month old rats (32.0 +/- 6.7) and in 18 month old rats (30.4 +/- 2.1) were significantly reduced compared to the basal activity in the young (50.1 +/- 4.2). The net isoproterenol or NaF stimulated AC activity in 24 month old rats (zero and 15.6 +/- 8.5 respectively) was also reduced compared to young rats (10.1 +/- 3.9 and 166.0 +/- 21.2 respectively). It is concluded that aging is associated with reduced isoproterenol stimulated AC activity of cerebral microvessels. This reduction is the product of reduced beta-adrenergic receptor number and reduced activity of AC in aged rat cerebral microvessels.

Viral pneumonia attenuates adenylate cyclase but not beta-adrenergic receptors in murine lung.

Respiratory infections provoke increased airway reactivity in both asthmatic and otherwise healthy subjects in part through impaired beta-adrenergic relaxation of bronchial and tracheal muscle. The precise mechanism remains obscure, but some studies report a decrease in the number of beta-adrenergic receptors. The present study was designed to assess the effect of viral respiratory infection with influenza A on lung beta-adrenergic receptors and adenylate cyclase activation in mice under two separate protocols. First, to determine whether changes are due to a local or systemic effect, we compared mice with influenza infections limited to the upper respiratory tract to mice with infection of the total respiratory tract. Four days after upper respiratory tract infection there were no changes in either isoproterenol- or NaF-stimulated adenylate cyclase activity. In contrast, there was an 82% decrease in isoproterenol- and a 25% decrease in NaF-stimulated adenylate cyclase activity on the fourth day after total respiratory tract infection. There were no changes in beta-adrenergic receptors or receptor coupling to adenylate cyclase with either type of infection. Our second protocol compared acutely infected mice to postrecovery mice. Twelve days after infection the virus was no longer present in the lungs, and adenylate cyclase activity was restored to normal. These data suggest that viral respiratory infection may impair airway function through attenuation of receptor and postreceptor activation of adenylate cyclase activity.

Modification of beta-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid.

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.