A B S T R A C T Human platelets aggregate and undergo a release reaction wvhen inetubated wvith catecholamines. Indirect evidence indicates that these events are mediated through a-adrenergic receptors. We used [3H]dihydroergocryptine, an a-adrenergic antagonist, to identify binding sites on platelets that have the characteristics of a-adrenergic receptors. Catecholamines compete for the binding sites in a stereo-specific manner with the potency series of (-) epinephrine > (-) norepinephrine > (+) phenylephrine > (-) isoproterenol. The dissociation constant (Kd) of (-) ,M. Phentolamine and dihydroergocyrptine blocked this response, whereas (±) propranolol had no effect. (-) Epinephrine and (-) norepiniephrine inhibited basal and prostaglandin El-stimulated adenylate cyclase in a dose-related manner. The conicentrationi of( -) epiinephrinie inhibiting adenvlate cyclase 50% was 0.7 ,uM. This inhibitioni was also blocked by phentolamiiine and dihydroergocryptine btut not by (±) propranolol. The specificity of binding and the close correlationi with a-adrenergic receptor-mediated biochemical and physiological responses suggest that the [3H]dihydroergocryptine binding site represents, or is closely related to, the human platelet a-adrenergic receptor. The ability to assay this receptor directly and to correlate these data with independently measured se(uelae of receptor activation should facilitate increased understandinig of the physiology and pathophysiology of the hulmlani platelet a-adrenergic receptor.
A B S T R A C T Prostaglandin (PG)D2
A B S T R A C T The possibility has been explored that decreases of adenylate cyclase may explain diminished hormone sensitivity of adipose tissue with aging. Isolated cells were prepared from epididymal fat pads of rats 1-, 2-, 6-, 12-, and 24-mo-old, fixed in OsO, and sized and counted with a Coulter apparatus. Adenylate cyclase was assayed in cell membranes (ghosts) using [ra-P] 6 and 24 mo per cell. Glucagon stimulated adenylate cyclase 4.5-fold relative to basal in the 1-mo-old rat, but its effect then rapidly decreased and was absent by 12 mo. The fourfold stimulation by ACTH noted in the 1-mo-old animals decreased gradually with age but was still twice basal at 24 mo. Since no significant change of cell size occurred after 6 mo, diminished hormone sensitivity with senescence cannot be related to cell size. Similar age-related patterns of hormonal activation were evoked by 5'-guanylyl-imidodiphosphate [GMP-P(NH)P], a nucleotide analogue which increased both basal-and hormone-activated enzyme at all ages studied. Dose-response curves to hormones, fluoride, and GMP-P(NH)P were not affected by age. High Mg++ (50 mM) in the presence of GMP-P-(NH) P stimulated adenylate cyclase to levels greater than with fluoride, but a similar loss of activity with aging was still observed. Loss of hormone receptors may partially explain the age-related decreases of glucagon and ACTH-sensitive adenylate cyclase, but decreased basal-, epinephrine-, fluoride-, and GMP-P-(NH)P-stimulated responses suggest loss of the catalytic component of the adenylate cyclase enzyme complex in the aging fat cell membranes. INTRODUCTIONIn the past, most studies of aging and hormones have dealt with effects of hormones on the processes of aging or, more often, with effects of aging on hormone secretion and metabolism (1). More recently, however, effects of aging on hormone responsiveness or sensitivity have come under investigation. A number of postmaturational changes of this type have now been identified. In view of the variety of mechanisms involved in the actions of hormones of different types, it is not surprising that a mixed picture has emerged (2). For the many hormones whose mechanism of action involves activation of adenylate cyclase, relatively little information on age relationships is as yet available. To extend our information in this area, and with the hope of being able eventually to relate the findings to physiologic events, we have undertaken studies of adenylate cyclases in a variety of animal and human tissues.
A B S T R A C T Age-related decreases of hormonesensitive adenylate cyclase activities of rat fat cell plasma membranes (ghosts) have been recently described. Glucagon-sensitive activity was completely lost between 1 and 6 mo, an interval in which fat cell size increases rapidly, while decreased activation by ACTH was gradual over the entire life span of the animal (24 mo), and epinephrine-sensitive enzyme diminished modestly and only during senescence. In the present studies an attempt was made by restricting food intake to assess the importance of changing cell size in the age-related alterations of hormonesensitive enzyme activities. Enzyme activities were determined before restriction and at monthly intervals for 3 mo for the unstimulated enzyme (basal) though declining, were still 1.5-3 times greater than the unrestricted controls, regardless of whether activity was expressed as total activity per milligram ghost protein or per cell, or as fold-increase. In the young animals, basal and fluoride-sensitive activities after a 3-mo restriction were unchanged or had decreased only slightly, depending on the base line used. Dietary restriction of adult animals for 3 mo, in contrast to the results in the young, did not increase total hormone-stimulated activity but rather produced either 0% (per milligram protein) or 25% decrease (per cell) for epinephrine-sensitive enzyme, 25 or 50% decrease of ACTH response, and 40 or 60% decreases of basal-and fluoride-stimulated activities. Expression of activities of restricted adults as fold-increase (stimulate/basal) showed an "increase of responsiveness" for all three hormones, but this was a reflection of the marked decrease of basal activity. Nonetheless, the restricted adults showed significant restoration of a small amount of glucagon-sensitive activity (1.8-fold over basal). These results indicate that cell size, per se, is not a dominant factor affecting hormoneresponsive adenylate cyclase under conditions of dietary restriction. The data do not support the postulation that increasing cell size is directly related to the enzyme's decreased activity during aging. Hormonesensitive adenylate cyclase is, however, clearly under dietary control, while age determines the pattern of the observed response during dietary restriction.
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