Donald O. Allen

Role of calcium ion in hormone-stimulated lipolysis

Using the flask-incubated fat cell system, the effects of Ca2+ removal from the incubation medium on the lipolytic system were studied. The removal of Ca2+ resulted in a total abolition of the lipolytic response and the increased cyclic AMP accumulation produced by ACTH. The lipolytic response to isoproterenol and forskolin were reduced approximately 40% by Ca2+ removal, but cyclic AMP accumulation was not altered in the presence of either of these agents using a Ca2+-free medium. The lipolytic response to the dibutyryl analog of cyclic AMP was also reduced by omission of Ca2+ from the incubation medium. It is concluded the Ca2+ is required for the interaction of ACTH with its receptor and the resultant activation of adenylate cyclase. Ca2+ also is required at some step in the lipolytic process distal to cyclic AMP.

Antilipolytic Action of Tolbutamide in Isolated Fat Cells of the Rat

Concentrations of tolbutamide from 0.25 to 4.0 mg. per milliliter were found to inhibit epinephrine-stimulated lipolysis in isolated fat cells of the rat. At a concentration of 1 mg. per milliliter, tolbutamide also inhibited lipolysis stimulated by ACTH or dibutyryl cyclic AMP. At the same concentration it failed to alter basal lipolysis, basal or epinephrine-stimulated cyclic AMP levels. Basal and epinephrine-stimulated adenylate cyclase and glycogen phosphorylase as well as binding of cyclic AMP to protein kinase were unaltered. These data are discussed in relation to the antilipolytic mechanism of tolbutamide.

Modulation of β-receptors as adult and neonatal cardiac myocytes progress into culture

SummaryModulation of β-adrenergic receptors and their ability to respond to β-receptor stimulation was studied in cultures of adult and neonatal rat cardiac myocytes. The radioligand iodocyanopindolol (125I-CYP) was used to identify β-adrenoceptors on the intact cells.125I-CYP was found to bind to the receptors in a stereospecific and saturable manner. Freshly isolated neonatal and adult myocytes both had a receptor density of approximately 50 fmol/mg protein. The number of β-receptors per milligram protein was similar during a 10-d culture period for adult myocytes but increased after a 5-d culture period for neonatal myocytes. Both cell types responded to β-receptor stimulation with isoproterenol by a twofold increase in the concentration of cAMP and this response increased with time in culture. The number of receptors as well as the response to isoproterenol was similar for neonatal myocytes cultured on laminin, collagen type I, or on uncoated culture dishes. From these data we conclude that cultured cardiac myocytes maintain functional β-receptors as they progress into culture, and the expression of β-receptors is not influenced by culture substrates.

Rate-limiting steps in isoproterenol and forskolin stimulated lipolysis

Using the flask-incubated fat cell system, effects of isoproterenol and forskolin on glycerol release, cyclic AMP levels and protein kinase were studied. Isoproterenol increased cyclic AMP levels, protein kinase activity and glycerol release over the same concentration range (10(-9) M to 10(-6) M). Forskolin also increased all three variables in a concentration-dependent manner (10(-7) M to 10(-4) M). The maximum response for each variable was significantly greater with forskolin than with isoproterenol. A combination of isoproterenol and forskolin resulted in an additional increase in cyclic AMP over forskolin alone, but no significant increase in protein kinase activity or glycerol release. These results support the concepts that the maximum lipolytic response to isoproterenol is limited by the accumulation of cyclic AMP and the maximum lipolytic response to forskolin is limited by some step distal to cyclic AMP production, possibly activation of protein kinase. At high concentrations of forskolin or with a combination of forskolin and isoproterenol, cyclic AMP levels were in excess of those needed to maximally activate protein kinase and lipolysis.

Antilipolytic action of insulin in the perifused fat cell system.

Abstract The antilipolytic action of insulin was investigated using the perifused fat cell system. Epinephrine (10−5 M) and glucagon (5 × 10−6 M) both stimulated lipolysis by at least 6-fold, and insulin inhibited both responses. The time course and the magnitude of the antilipolytic action of insulin were determined in the perifused fat cell system. At a concentration of 100 μU/ml, insulin inhibited epinephrine-stimulated lipolysis approximately 50 percent, with a half-time response of 4–5 min. The antilipolytic action of insulin persisted for at least 45 min. following the termination of insulin infusion. This prolonged phase of the action of insulin could be terminated by treatment of the cells with trypsin. These results suggest that only small percentage of insulin receptors is occupied during maximum lipolytic activity and that in adipose tissue many spare receptors exist for insulin.

Acetylcholine modulation of phosphorylase and contractility in rat hearts exposed to anoxia or isoproterenol

Abstract The effects of acetylcholine (ACh) on glycogen phosphorylase activated by either a cyclic AMP-dependent (isoproterenol) or a cyclic AMP-independent (anoxia) mechanism were examined. Cyclic adenosine 3 : 5-monophosphate and cyclic guanosine 3 : 5-monophosphate content, protein kinase and glycogen phosphorylase activities, and the contractile force of isolated perfused rat hearts exposed to either isoproterenol or anoxia were determined. Isoproterenol (1.6 × l0 −7 M) produced an increase in cyclic AMP, activated protein kinase and glycogen phosphorylase, and increased intraventricular pressure developed by the myocardium. Acetyleholine did not alter basal phosphorylase activity or contractility. Acetylcholine, infused concurrently with isoproterenol, produced an increase in cyclic GMP and a decrease in cyclic AMP as well as in protein kinase and phosphorylase activity. The effects of ACh on cyclic GMP and phosphorylase were observed at 10 −5 M. Exposure of isolated perfused hearts to anoxia decreased the intraventricular pressure developed. This negative inotropic effect was accompanied by an activation of glycogen phosphorylase that was independent of alterations in cyclic AMP or cyclic GMP. A high concentration of acetylcholine (10 −4 M) further diminished the contractile activity of the heart and abolished the activation of phosphorylase. These effects also occurred in the absence of alterations in cyclic AMP but were coincident with an elevation of cyclic GMP. A lower concentration of ACh (10 −5 M) infused during anoxia, however, elevated cyclic GMP without concurrent effects on cyclic AMP, protein kinase, phosphorylase or contractility. Thus, phosphorylase activated by a cyclic AMP-independent mechanism was not affected by doses of acetvlcholine that were capable of suppressing phosphorylase activated by a cyclic AMP-dependent mechanism. These data support the concept that a reduction in cyclic AMP may be involved in mediating the effects of ACh on catecholamine-stimulated phosphorylase in myocardial tissue. The data do not support a role for cyclic AMP in the inhibition of anoxia-stimulated phosphorylase activation and, thus, some other modulating factor may be operative under these conditions. The role of cyclic GMP in this response remains in question.