32P Labeling Of Phosphatidylinositol Research Articles

Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland

The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4–9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2) which reflects PI cycle activation. AVT (10 −5 and 10 −4 M) induced a significant increase in 32P labelling of PI, PIP and PIP 2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V 1 receptor antagonist. The desmopressin and AVP 4–9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 μg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

Stimulatory effect of bombesin on phosphoinositide metabolism in the rat pineal gland

The pineal gland is under complex peptidergic nervous control originating from hypothalamic nuclei. The daily rhythm of bombesin-like peptide in the hypothalamus suggests that this neuropeptide, similarly as other neuropeptides, might be involved in modulation of the physiological activity of the pineal gland. In our experiments we studied the mechanism of signal transduction of bombesin in the isolated pineal glands of rats. The phosphoinositide signalling system was examined by measuring 32P-labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2), which reflects phosphoinositide cycle activation. Bombesin induced a significant increase in 32P-labelling of PI, PIP and PIP 2. The antagonist of this neuropeptide, ( d-Phe 12-Leu 14)-bombesin, suppressed the increase in 32P-labelling of all phosphoinositides. Bombesin was without effect on cAMP dependent protein phosphorylation. The data indicate that bombesin activates the PI signalling system via specific receptors.

Arginine vasopressin stimulates 32P labeling of phosphoinositides in rat pineal gland

The peptidergic innervation of the pineal gland contains arginine vasopressin (AVP) fibers. Since the site and molecular mechanism of AVP action on the pineal signal transduction has not yet been determined, we examined whether the inositol phosphate transduction system is involved. The phosphoinositide signaling system was studied by measuring metabolic turnover of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol biphosphate (PIP 2), which reflects phosphoinositide cycle activation. AVP induced a significant increase in 32P labeling of PI, PIP and PIP 2. The V 1 receptor site(s) blocker for AVP abolished enhanced 32P labeling of PIP and PIP 2. AVP was without effect on cAMP dependent phosphorylation. The data indicate that AVP activates the phosphoinositide signaling system via V 1 receptors and is without effect on the cAMP system.

Carbamoylcholine and gastrin induce inositol lipid turnover in canine gastric parietal cells.

The potential role of inositol phospholipid turnover in mediating acid secretion was examined in a preparation enriched for isolated canine gastric parietal cells. The stimulatory effects of carbamoylcholine (carbachol) and gastrin on parietal cell uptake of [14C]aminopyrine were linked to dose- and time-dependent selective reduction in cellular phosphatidylinositol content, although the specific fatty acid composition of the phosphoinositides was not altered. Analysis of [3H]inositol phosphates accumulated in cells prelabeled with [3H]inositol revealed an increase in labeled inositol trisphosphate by 5 min of incubation with either carbachol or gastrin. Furthermore, after preincubation of parietal cells in medium containing [32P]orthophosphate, the two secretagogues elicited a time-dependent decrease in 32P labeling of phosphatidylinositol 4,5-bisphosphate and concomitant increase in labeling of phosphatidic acid. These data demonstrate that the acid secretagogue actions of carbachol and gastrin are correlated with turnover of cellular inositol phospholipids in a preparation consisting predominantly of parietal cells.

Phosphorylation of phosphoinositides in human platelets

32P-labelling of phosphatidylinositol (PI), PI-4-monophosphate (PIP), PI-4,5-bisphosphate (PIP2) and phosphatidic acid (PA) in 32P-labelled intact human platelets was investigated in the presence of various agents which alter intracellular level of cAMP or Ca 2+. Addition of dibutyryl cAMP to intact platelets pre- or pulse labelled with 32P resulted in increased 32P-labelling of PIP and in concomitant decreased 32P-labelling of PI without affecting that of PIP2 or PA. Similar changes were observed in intact platelets treated by phostaglandin 12 (PGI2) or a new low Km phosphodiesterase inhibitor (DN-9693). When intracellular Ca 2+ was chelated by loading quin 2-AM to intact platelets, 32P-labelling of PIP was significantly increased in a dose related manner. From these observations it was concluded that PI kinase is activated by elevation of cAMP or chelation of Ca 2+ in intact platelets.

Effects of Isoproterenol Pretreatment on Phosphatidylinositol Turnover in Rat Parotid Gland

The effects of isoproterenol pretreatment on phosphatidylinositol turnover in rat parotid slices were studied to elucidate the relationship between β- and α1-adrenoceptors. 32P-Labeling of phosphatidylinositol in parotid slices was increased by an α1- and α2-agcmist (epinephrine and norepinephrine) and α1-agonists (methoxamine and phenylephrine), but not by an α2-agonist (clonidine) and a β-agonist (isoproterenol). Prazosin inhibited the increase in phosphatidylinositol turnover elicited by epinephrine, but propranolol did not. These results indicate that the stimulation of phosphatidylinositol turnover elicited by adrenergic agonists is mediated by activation of α1-adrenoceptors in the parotid glands. Isoproterenol pretreatment of the parotid slices caused a significant increase in 32P-labeling of phosphatidylinositol and a decrease in that of phosphatidic at i The epinephrine- or phenylephrine-induced increase in 32P-labeling of phosphatidylinositol were further enhanced by the isoproterenol pretreatment of the slices. In the isoproterenol-treated membranes of the parotid glands, [3H]prazosin binding to a-i-receptors increased, but [3H]dihydroalprenolol binding to β-receptors did not. These findings indicate that the acceleration of phosphatidylinositol turnover induced by the isoproterenol pretreatment may be associated with an increase in α1-adrenoceptor binding sites which might have appeared as a result of the isoproterenol pretreatment of the parotid slices.

Differential responses to adjuvants of macrophages from young virgin, aging virgin and aging breeder mice

Age related differences in macrophage responsiveness to adjuvants seen previously with thioglycollate induced cells were also found with resident macrophges. Thus, activation of phagocytosis by lipopolysaccharide, polyadenylic-polyuridylic acid complexes and muramyl dipeptides occurred when exposed to macrophages removed from young but not aging C58 and C3H/He mice. However, breeding status differences were observed in that aging virgin mice were unresponsive to these adjuvants, while aging breeder mice responded similarly to young virgin mice. Analysis of any changes in membrane phospholipids was carried out to determine their association with the age related and breeding status differences observed. Significant differences occurred in 32P labelling of phosphatidyl inositol between unstimulated macrophages from young and aging C58 mice. No differences were evident, however, after LPS stimulation. Analysis of macrophage plasma membranes from young and aging mice for cholesterol revealed significant age related differences in their ability to undergo increases in cholesterol content after LPS activation.

Interactions of vasopressin, cAMP, and prostaglandins in toad urinary bladder.

Prostaglandin (PG) inhibits the hydroosmotic effect of vasopressin. We therefore reexamined the interaction of vasopressin (VP), cAMP, and prostaglandins in toad bladder epithelial cells. Vasopressin slightly, but reproducibly, stimulated PGE2 and thromboxane B2 (TXB2) synthesis in cells prepared by the use of collagenase. When cells were prepared in the presence of a readily reversible cyclooxygenase inhibitor, ibuprofen, subsequent PGE2 synthesis was enhanced sevenfold but that of TXB2 was not. Increasing cAMP by either phosphodiesterase inhibition or 8-bromo-cAMP significantly inhibited both basal and VP-stimulated PGE2 synthesis. This inhibition was overcome by addition of arachidonic acid. Future studies employing these agents will have to consider these effects. VP enhanced 32P labeling of phosphatidylinositol (PI) and phosphatidic acid. This effect was prevented by the phosphodiesterase inhibitor, which also decreased phosphatidylcholine labeling. The results indicate that the phosphodiesterase inhibitor for cAMP may decrease PG formation by interfering with phospholipase activation. Furthermore, VP, similar to its effect in the liver, also increases PI turnover in toad bladder. This may initiate PG synthesis and provide a link among VP, cAMP, and calcium. A double-reciprocal feedback is proposed, whereby VP stimulates PG synthesis in a cAMP-independent manner and also inhibits PG synthesis in a cAMP-dependent manner.

Phosphoinositide Turnover in Erythrocyte Membranes in Human and Experimental Hypertension

The metabolism of phosphoinositides, a class of membrane lipids involved in Ca2+ -transport and/or mobilization systems was investigated in patients with moderate essential hypertension and in Sabra rats. Experiments were performed in vitro on isolated erythrocyte membranes by measuring the 32P-labelling of phosphatidylinositol 4,5-bisphosphate (PI-P2) and of phosphatidylinositol 4-phosphate (PI-P) following the incubation of membranes with [gamma-32P] ATP. In untreated essential hypertensives (n = 31) or in hypertensive patients whose blood pressure was controlled by beta-blocker therapy (n = 20), 32P-PI-P2 was significantly higher than in normotensive controls (n = 30); no significant difference was observed between the two groups of hypertensive patients. In Sabra rats fed on a low Na diet, 32P-PI-P2 levels were significantly higher in hypertensive-prone animals (SBH) than in hypertensive-resistant animals (SBN). When the animals were fed a high Na diet or were DOCA/salt treated, 32P-PI-P2 did not change in either substrain, although such conditions differentially affected the blood pressure of SBH and SBN. Our data indicate that the modification of phosphoinositide metabolism is not a consequence of the blood pressure elevation, but can be considered as an intrinsic membrane defect which may be associated with functional alterations of Ca2+ fluxes which in hypertensives result in an enhanced intracellular Ca2+ level.

Nutrient and hormone-neurotransmitter stimuli induce hydrolysis of polyphosphoinositides in rat pancreatic islets.

Preincubation of rat pancreatic islets with 3H-inositol, and subsequent exposure, in the presence of LiCl, to either glucose or carbamylcholine resulted in a rapid stimulation of 3H-inositol 1,4,5-triphosphate and 3H-myo-inositol 1,4-bisphosphate formation, the level of which reached a plateau after about 5 min of stimulation. Both stimuli also caused an approximately linear accumulation of 3H-myo-inositol 1-phosphate. The amounts of 3H-inositol phosphates formed were dependent on the concentration of LiCl. Studies of 32P-labeling of islet ATP, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 4-phosphate revealed that these approached isotopic equilibrium after about 240-min incubation, whereas 32P-labeling of phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine proceeded at a lower rate. Carbamylcholine provoked an immediate fall in 32P-PtdIns(4,5)P2 and, to a lesser extent, 32P-phosphatidylinositol 4-phosphate. Glucose caused a similar response although, in this case, the most marked decline was in a more polar 32P-labeled lipid. Cholecystokinin-pancreozymin was also found to induce 32P-PtdIns(4,5)P2 hydrolysis, although the ionophore A23187 was without effect. Both carbamylcholine and glucose induced an increase in 32P-phosphatidic acid. The results provide two independent pieces of evidence suggesting that phospholipase C-mediated hydrolysis of polyphosphoinositides occurs as an early response in rat islets to either nutrient or neurotransmitter secretagogues.

Studies on the effects of acetylcholine and antiepileptic drugs on 32PI incorporation into phospholipids of rat brain synaptosomes.

Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated 32P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated 32P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the 32P labeling of phospholipids nor on the specific radioactivity of [32P]ATP. Omission of Na+ drastically reduced both the 32P labeling of synaptosomal phospholipids and the specific radioactivity of [32P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate NA+ and Ca2+ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca2+ and Na+ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.U

Effect of insulin, catecholamines and calcium ions on phospholipid metabolism in isolated white fat-cells

The incorporation of [(32)P]P(i) into phosphatidylinositol by rat fat-cells was markedly increased in the presence of adrenaline. Phosphatidic acid labelling was also increased, but to a lesser extent. These effects are due to alpha(1)-adrenergic stimulation since they were unaffected by propranolol, blocked by alpha-blockers in the potency order prazosin<<phentolamine<yohimbine and mimicked by methoxamine. The alpha-adrenergic stimulation of phosphatidylinositol labelling did not require extracellular Ca(2+), which supports the hypothesis that an increased turnover of phosphatidylinositol is involved in alpha-adrenergic activation of Ca(2+) entry. Insulin and the ionophore A23187 gave a small increase in (32)P labelling of phosphatidylinositol in Ca(2+)-free medium containing 1mm-EGTA. The increases due to insulin or ionophore A23187 were abolished if 2.5mm-Ca(2+) was added to medium containing EGTA. However, the increases in labelling of phosphatidylinositol due to alpha-adrenergic amines were still evident in medium containing EGTA and Ca(2+). Lipolytic agents such as corticotropin, dibutyryl cyclic AMP, adrenaline in the presence of phentolamine and isoproterenol decreased [(32)P]P(i) incorporation into phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. This inhibitory effect may be secondary to accumulation of intracellular unesterified fatty acids, since it was decreased by incubating fewer cells in medium with 6 rather than 3% albumin and was restored by the addition of oleate to the medium. The incorporation of [(32)P]P(i) into phosphatidylcholine was unaffected by lipolytic agents. The data suggest that there is an inhibition of the synthesis of certain phospholipids in the presence of lipolytic agents, which may be secondary to intracellular accumulation of unesterified fatty acids.

TRANSYNAPTIC STIMULATION OF PHOSPHATIDYLINOSITOL METABOLISM IN SYMPATHETIC NEURONS IN SITU

An increase in 32P labelling of phosphatidylinositol, associated with synaptic activity, has previously been reported in excised sympathetic ganglia excited by preganglionic nerve impulses. In the present experiments a similar effect occurred in naturally-stimulated ganglia. It occurred when the preganglionic impulses were either evoked by electrical stimulation of the preganglionic nerve or were discharged spontaneously from the central nervous system. Thus increased turnover of phosphatidylinositol appears to be a normal accompaniment of synaptic transmission in these ganglia.