Matthew E. Conolly

Reversal of human lymphocyte β‐adrenoceptor desensitization by glucocorticoids

To investigate the effect of glucocorticoids on β‐agonist‐induced desensitization, we studied the effect of a single intravenous dose of methylprednisolone (2 mg/kg) on β‐receptor density and affinity in lymphocytes from four normal and four mildly asthmatic subjects at the end of 3 to 5 wk of terbutaline therapy and from four normal subjects taking no other drug. Terbutaline decreased (‐) [3H]‐dihydroalprenolol binding sites by 53% in normal and by 42% in asthmatic subjects. Methylprednisolone restored the number of binding sites to levels statistically indistinguishable from the preterbutaline values in both groups of subjects. In subjects not exposed to terbutaline beforehand there was no significant alteration in receptor density after methylprednisolone, nor in normal lymphocytes incubated in vitro for 90 min with hydrocortisone (10−5M). No significant change in the dissociation constant was observed in any situation. A single intravenous dose of methylprednisolone reverses terbutaline‐induced down‐regulation of β‐adrenoceptors. This may provide a mechanism for the beneficial effect of steroids in restoring catecholamine responsiveness in asthmatic subjects.

Selective subsensitization of beta-adrenergic receptors in central airways of asthmatics and normal subjects during long-term therapy with inhaled salbutamol

In five subjects with mild asthma and in five normal subjects, we determined the effect of a 4 wk course of inhaled salbutamol (albuterol), 200 micrograms q.i.d., on (1) acute bronchodilator responsiveness, (2) bronchial sensitivity to inhaled histamine, (3) beta-adrenergic protection against histamine-induced bronchospasm, and (4) beta-receptor density of peripheral blood lymphocytes. We observed a diminution in central airway bronchodilator responsiveness (as measured by airway conductance responses) to acutely inhaled salbutamol and to subcutaneous terbutaline in both groups of subjects, although only the response to subcutaneous terbutaline was statistically significant (p less than 0.02). On the other hand, no impairment of small airway bronchodilator responsiveness was noted in either group of subjects when responses were measured as partial expiratory flow rates at 60% below total lung capacity. These findings suggest the development of selective subsensitization of beta-receptors in the larger central airways, where a proportionately greater amount of the inhaled beta-agonist aerosol would necessarily be deposited. A greater loss of protection against histamine-induced bronchospasm was seen in asthmatics than in normals (approximately twofold), although the difference was not significant. A modest but not significant reduction in peripheral blood lymphocyte beta-receptor density was observed by the end of the 4 wk treatment period. The possibility that the observed changes in bronchodilator responsiveness might influence the morbidity and mortality associated with bronchial asthma is discussed.

Beta-adrenergic receptors on human lymphocytes: comparison of down-regulation in vivo and in vitro.

beta-Adrenergic receptors on isolated human lymphocytes were enumerated using 125I-cyanopindolol (125I-CYP), after a 90-min exposure to 50 mumols/l l-isoproterenol in vitro. No change in receptor density could be shown in assays performed at 37 degrees C, although a 40% reduction was apparent in binding studies carried out at 4 degrees C. In contrast, beta-adrenergic receptors on lymphocytes from mild asthmatics after a 3-week course of oral terbutaline showed a 40% reduction in receptor density regardless of the assay temperature, in addition to a 2.5-fold reduction in the receptor affinity for isoproterenol. The data are consistent with reports that a fraction of receptors are sequestered during short-term exposure to agonists. Sequestered receptors may or may not be detected by radioligand binding assays depending on the ligand of choice, temperature of the binding assay and duration of prior exposure to the agonist. After extended exposure to an agonist in vivo, the number of surface receptors was reduced, and sequestered receptors were not present, presumably as a result of degradation.

Calcium dependence of beta-adrenoceptor mediated cyclic AMP accumulation in human lymphocytes.

In intact human lymphocytes, cyclic AMP accumulation in response to isoproterenol was inhibited by 5 mM EDTA, by deletion of calcium ions from the medium and by 1 mM lanthanum chloride, but not by 1 microM verapamil or by 10 microM nifedipine. A23187 caused a modest increase in cyclic AMP content. Exposure of lymphocytes to 5 microM 1-isoproterenol desensitized the cells to subsequent beta-adrenergic stimulation, reducing cyclic AMP accumulation. With higher concentrations of 1-isoproterenol (50 microM), receptor density was reduced as well. None of the above agents attenuated losses in agonist-stimulated cyclic AMP accumulation induced by treatment with 5 microM isoproterenol for 90 min. These data suggest that calcium ions, both those present in the extracellular medium and those bound to the plasma membrane, are required for isoproterenol-stimulation of adenylate cyclase. In addition, it appears that neither the presence of extracellular calcium ions nor full activation of adenylate cyclase are required for desensitization.

Extracellular magnesium is not required for beta adrenergic drug-receptor interactions in intact human lymphocytes

Abstract We have investigated the effect of extracellular magnesium ions on the function of beta adrenergic receptors in intact human lymphocytes. We examined adenylate cyclase stimulation by isoproterenol displacement of (-) (3H) dihydroalprenolol from beta receptor sites, and down regulation (desensitization) of beta receptors by prolonged exposure of the cells to isoproterenol. Contrary to results obtained using broken cell preparation, in none of these situations did the presence or absence of extracellular magnesium ions make any difference. The importance of selecting the most nearly physiological preparations for conducting in vitro studies that may be extrapolated to the whole organism is stressed.

Lymphocyte β-adrenergic receptors are not altered in hyperthyroidism

Binding of (‐)[3H]‐dihydroalprenolol (3H‐DHA) to lymphocytes from five thyrotoxic patients and five age‐ and sex‐matched controls were examined to ascertain whether β‐adrenergic receptor number or binding affinity were altered in thyrotoxicosis. Whereas an increase in β‐adrenoceptor density has been reported in triiodothyronine‐induced hyperthyroidism, we did not find changes in β‐adrenergic receptor density or binding affinity. In one patient studied sequentially, we did not find alteration in 3H‐DHA binding before or after the subject was rendered euthyroid. We conclude that lymphocyte β‐adrenergic receptor density and affinity are not altered in spontaneous hyperthyroidism.

Workshop 2: Special pharmacologic considerations

Abstract Because NBAAD are designed to either prevent release or antagonize the effects of cellular mediators, special pharmacologic problems during their development and clinical evaluation phases are bound to arise. Many of these drugs exert optimal effects in local tissue sites, where the adverse effects of delayed onset asthmatic response are most apparent. Thus controlled bioavailability studies often have to be extended for longer periods of time. For similar reasons, evaluation of tachyphylaxis and interactions with other active drugs may be more difficult to interpret in this category of drugs. Whenever possible, detailed pharmacokinetic investigations of these drugs should include determination of both single- and multiple-dose absorption assays, protein binding, distribution, rate of elimination, and extent of absorption. The steadystate equilibrium status of these drugs is particularly relevant to evaluate their durations of actions. Thus dose response effects of longer acting drugs may only become apparent after multiple, chronic dosing studies. For these reasons, new agents in the NBAAD category must often be evaluated in the context of unique drug effects combined with generally accepted pharmacodynamic techniques. In addition, the development of an NBAAD will almost always require consultation with clinical pharmacologists.