Adrenergic and cholinergic stimulation of 32P-labeling of phospholipids in rabbit iris muscle

Abstract

The phospholipid and cholesterol composition of the iris muscle of the rabbit was determined, and the incorporation of 32Pi into the individual phospholipids of irises incubated in Krebs Ringer bicarbonate buffer that contained glucose in the presence or absence of adrenergic or cholinergic neurotransmitters and their agonists and antagonists was investigated. The results of studies on the characteristics and the effects of norepinephrine and other neuropharmacological agents on the 32P-la-beling of phosphatidic acid (PhA). phosphatidylinositol (PhI), phosphatidylcholine (PhC). phosphatidyl-ethanolamine (PhE) and phosphatidylserine (PhS) in the iris muscle are reported. Addition of l-nore-pinephrine. l-epinephrine, dopamine or acetylcholine (0.003 to 0.3 mM) to irises which were preincubated for 20 min in 32P-Krebs-Ringer without cold phosphate stimulated significantly the isotope incorporation into PhA and Phi. but not PhC. In contrast histamine, noremetanephrine. metanephnnc. adrenochrome. 6-hydroxydopamine and eserine (0.003 to 0.3 mM) had a negligible effect on the isotope incorporation. At higher concentrations of norepinephrine (10mM), labeling of PhA, PhI and PhC was elevated to 989. 630 and 185 per cent of that of the control respectively. It was concluded that in the iris muscle α-receptors and not β-receptors are involved in the stimulatory action of norepinephrine on 32Pi incorporation into phospholipids. This conclusion is based on the following findings. (a) Only α-stimulators. such as norepinephrine and phenylephrine. increased significantly the labeling of PhA and PhI. This effect was blocked by the α-blockers. phenoxybenzamine and phentolamine. (b) Isoproterenol, a β-stimulator, had no effect on the labeling of PhA and PhI. Sotalol and propranolol, both β-blockers, did not block the norepinephrine stimulation of 32Pi incorporation, (c) Cyclic AMP (or dibutyryl cyclic AMP), which has been suggested as a β-receptor mediator, exerted no influence on the phospholipid effect. Atropine blocked completely the acetylcholine-stimulated 32P-labeling of PhA and PhI. At 0.3 mM, phentolamine and propranolol increased by several-fold the isotope labeling of PhA, PhI and, unexpectedly, of CDP-diglyceride and inhibited that of PhC. The properties of the norepinephrine stimulation of 32P-labeling in the iris muscle can be summarized as follows, (a) It is concentration dependent; but this dependence varies with the condition of incubation, (b) It is temperature dependent, but the effect is lost upon freezing and thawing, (c) It can be demonstrated during a period of 3 days of aging at 4. (d) Glucose is required for maximal stimulation, (e) The phospholipid effect is not specific to a particular subcellular fraction, (f) Addition of norepinephrine brought about a 25 per cent decrease in the level of PhI and a 63 per cent increase in the level of PhA. (g) Time-course studies on the 32P-labeling of phospholipids in the presence and absence of norepinephrine revealed that, in contrast to PhI and PhC, the specific radioactivity of PhA increased with time of incubation (0.90 min) only when the neurotransmitter was added, (h) Direct activation of the enzyme diglyceride kinase by norepinephrine does not appear to be the molecular mechanism underlying the phospholipid effect. Our data suggest that in the iris muscle norepinephrine increases the turnover of PhA and PhI phosphorus by stimulating the hydrolysis of endogenous PhA or PhI. or both, to form more membraneous diglyceride. The latter is then rephosphorylated by diglyceride kinase to form more labeled PhA.