No abstract

Solubilized and partially purified adenylate cyclase from bull sperm was found to be specifically activated (up to 6-fold) by sodium bicarbonate (NaHCO3) and to a lesser extent by NaNO a. Other sodium salts were either ineffective (e.g. NaCOOH) or inhibitory (e.g. NaHSO3, NaHSO 4 and Na2B4OT). Stimulation by NaHCO 3 was dose-dependent in the range of 0--40 mM and was greater when enzyme activity was assayed in the presence of magnesium as compared with manganese ions. Bicarbonate seems to affect maximal enzyme velocity (Vmax) and has no effect on the Km of adenylate cyclase for Mn-ATP. Stimulation of adenylate cyclase by NaHCO a coincided with the elution pattern of the enzyme as recorded following chromatography on DEAE-cellulose or gel filtration on BioGel P-100. These results suggest that in the course of stimulation of sperm adenylate cyclase, bicarbonate is likely to interact directly with the enzyme. Furthermore, this intrinsic and unique property of sperm adenylate cyclase may explain results reported by others on the stimulation of cAMP production by bicarbonate in intact and broken sperm preparations and suggest a biochemical basis for enhanced sperm motility associated with high bicarbonate concentrations.

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulinstimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.Insulin stimulates glucose transport activity in rat adipose cells primarily by inducing the translocation of the transporter isoforms GLUT1 and GLUT4 from an intracellular location to the plasma membrane (1-4). GLUT1 is widely distributed whereas GLUT4, the predominant form in adipose cells (4-6), is found only in tissues where insulin regulates glucose transport activity (i.e., white and brown adipose tissue, heart, and skeletal muscle; for review see refs. 7 and 8). Hereafter, insulin-stimulated glucose transport activity will be abbreviated as transport activity. A second level of transport regulation is exerted by agents that modulate adenylyl cyclase and lipolysis (9-11). Lipolytic agents (isoproterenol, glucagon, and corticotropin) inhibit transport activity, but only in the absence of antilipolytic agents (adenosine, nicotinic acid, and prostaglandin E1). Further, Smith et al. (12) and Kuroda et al. (13) demonstrated that both the transport inhibition by lipolytic agents and the augmentation by antilipolytic agents did not result from changes in transporter location.An unresolved issue is whether the changes in transport activity mediated by these various agents are related to the changes in cAMP. Currently, two distinct mechanisms have been proposed. Kuroda et al. (13) demonstrated an apparent dissociation between isoproterenol-mediated stimulation of cAMP-dependent protein kinase (A-kinase) and inhibition of transport activity, suggesting that changes in A-kinasemediated phosphorylation are not responsible for transport inhibi...