Albert O. Davies

In Vitro Desensitization of Beta Adrenergic Receptors in Human Neutrophils. ATTENUATION BY CORTICOSTEROIDS

The receptor alterations involved in catecholamine-induced desensitization of adenylate cyclase in human neutrophils have been investigated as has the ability of hydrocortisone to modify such alterations. Incubation of human neutrophils with isoproterenol for 3 h in vitro resulted in an 86% reduction in the ability of isoproterenol to stimulate cyclic AMP accumulation in the cells. Two types of receptor alterations were documented. There was a 40% reduction in the number of beta adrenergic receptors (42 vs. 25 fmol/mg protein, P < 0.005) present after desensitization as assessed by [(3)H]dihydroalprenolol ([(3)H]DHA) binding. In addition the receptors appeared to be relatively uncoupled from adenylate cyclase. This uncoupling was assessed by examining the ability of the agonist isoproterenol to stabilize a high-affinity form of the receptor, detected by computer modelling of competition curves for [(3)H]DHA binding. Desensitized receptors were characterized by rightward-shifted agonist competition curves. When hydrocortisone was added to the desensitizing incubations (combined treatment) there was a statistically significant attenuation in the desensitization process as assessed by the ability of isoproterenol to increase cyclic AMP levels in the cells. Although combined treatment did not prevent the decline in receptor number, it did attenuate the uncoupling of the receptors. Combined treatment resulted in competition curves intermediate between the control and the rightward-shifted desensitization curves. Prednisolone was similar to hydrocortisone in attenuating isoproterenol-induced uncoupling. Thus, steroids appeared to attenuate agonist-induced desensitization of the beta adrenergic receptor-adenylate cyclase system by dampening the ability of agonists to uncouple receptors without modifying their ability to promote down-regulation of beta adrenergic receptors.

Regulation of Adrenergic Receptors

Transmission of intercellular messages by the adrenergic mediators, epinephrine and norepinephrine, is a ubiquitous phenomenon in vertebrates which continues to invoke investigative effort after many years of research. It has become apparent that both the release and biological actions of these mediators are modulated very specifically by pharmacological and pathological states. An understanding of the mechanisms of modulation of adrenergic actions is fundamental to addressing the relationships between adrenergic responses and other hormonal or cellular responses.

Impaired formation of beta-adrenergic receptor-nucleotide regulatory protein complexes in pseudohypoparathyroidism.

Decreased activity of the guanine nucleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes of some patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity of N (PHP-Ib). Low N activity in PHP-Ia results in a decrease in hormone (H)-stimulatable adenylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared beta-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Competition curves that were generated by increasing concentrations of the beta-agonist isoproterenol competing with [125I]pindolol were shallow (slope factors less than 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist competition curves from the PHP-Ia patients were shifted significantly (P less than 0.02) to the right as a result of a significant (P less than 0.01) decrease in the percent of beta-adrenergic receptors in the high affinity state from 64 +/- 22% in PHP-Ib and 56 +/- 5% in controls to 10 +/- 8% in PHP-Ia. The agonist competition curves were computer fit to a ternary complex model for the two-step reaction: H + R + N in equilibrium HR + N in equilibrium HRN. The modeling was consistent with a 60% decrease in the functional concentration of N, and was in good agreement with the biochemically determined decrease in erythrocyte N protein activity. These in vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP (Carlson, H. E., and A. S. Brickman, 1983, J. Clin. Endocrinol. Metab. 56:1323-1326) are consistent with the notion that N is a bifunctional protein interacting with both R and the adenylate cyclase. It may be that in patients with PHP-Ia a single molecular and genetic defect accounts for both decreased HRN formation and decreased adenylate cyclase activity, whereas in PHP-Ib the biochemical lesion(s) appear not to affect HRN complex formation.

Assessing redox status in human plasma. Experience in critically ill patients

Clinical evaluation of metabolic acidosis has involved measurement of lactate (L), pyruvate (P), beta-hydroxybutyrate (BOHB), and acetoacetate (AcAc). We previously demonstrated that these metabolites are not at equilibrium in plasma. Their degree of disequilibrium is reflected in the ratio of apparent equilibrium constants (KLP/KBA) for the two redox couplets, L-P and BOHB-AcAc. The purpose of the study was to examine how well this ratio reflects disequilibrium in patients with metabolic acidosis. Measurements of the four metabolites were obtained in 23 critically ill patients. Disequilibrium was again observed, as manifested in an inconstant ratio (p < .01). The ratio increased with clinical improvement. Patients were more likely to die during their ICU stay if the estimated ratio was low, particularly if metabolic acidosis was present. Patients with respiratory acidosis had both intermediate probabilities of death and intermediate ratios when compared to inpatient controls (ICU patients without acidosis). Our data indicate that changes in the L-P-BOHB-AcAc cycle reflect the degree of metabolic derangement in critically ill patients.

Nonequilibrium of two redox couplets in human plasma: lactate-pyruvate and beta-hydroxybutyrate-acetoacetate.

We examined the extracellular equilibrium status of two redox couplets normally found in plasma (lactatepyruvate [L-P], beta-hydroxybutyrate, and acetoacetate) during acute metabolic acidosis produced by muscle exertion. Both pre- and postexertion plasma spontaneously underwent loss of acetoacetate and gain of L when compared to the baseline values. Exercise further induced a 332% rise in L (p < .001) and a 102% rise in P (p < .001). The empirically derived ratio of equilibrium constants, KI.P/KBA, fell 50% (p < .001), and the calculated change in free energy (ΔF) fell from 3.6 to 3.1 kcal/mol (p < .001) after exercise. The changes induced by exertion were simulated closely by an in vitro model of a reduced state. Thus, the triad of a) inconstant metabolite concentrations, b) inconstant KI.P/KBA, and c) ΔF both inconstant and non-zero, indicates that there is no state of equilibrium for these metabolite couplets in human plasma. The KI.P/KBA ratio appeared to reflect the degree of deviation from equilibrium and may therefore be useful when investigating altered redox states such as metabolic acidosis.