Glycosyl phosphatidylinositol in thyroid: cell signalling or protein anchor?

During the last 10 years, attention has been focused on the stimulation by various agonists of the hydrolysis of phosphatidylinositol bis-phosphate into the second messengers inositol tris-phosphate and diacylglycerol. Two other aspects of the metabolism of phosphoinositides were therefore not paid sufficient attention. The first one was the release by insulin of a glycosyl inositol-phosphate from a glycosyl phosphatidylinositol, the hydrosoluble product being able to reproduce some of the hormone effects; the second was the discovery that several membrane proteins were anchored via a glycosyl phosphatidylinositol. For over 20 years, we have been interested in the effect of thyreostimulin (TSH) on the turnover of phosphatidylinositol in pig thyrocyte. As this effect did not seem to result from the hydrolysis of phosphatidylinositol bis-phosphate we explored another possibility, the synthesis of glycosyl inositol-phosphate. We have shown that, in cultured pig thyrocytes, TSH stimulates the release of the polar head of a glycosyl phosphatidylinositol. This soluble glycosyl inositol-phosphate which acts as insulin on adipocyte, modulates the cAMP accumulation and iodine metabolism in thyrocytes and could be held responsible for the cAMP independent effects of TSH. However, we do not yet know if there is a link between the glycosyl phosphatidylinositol sensitive to TSH and the anchor membrane protein. To date, the amount of 2 of these proteins: NAD glyco-hydrolase in thyroid cell membranes and heparan sulfate proteoglycan have been shown to be increased by TSH treatment of thyroid cells.     

Evidence for glucose-responsive and -unresponsive pools of phospholipid in pancreatic islets

The effect of glucose on the metabolism of phospholipids in pancreatic islets was studied with three radioactive phospholipid precursors, [32P]orthophosphate, [3H]myoinositol, and [3H]arachidonic acid, to determine the conditions necessary for studying the breakdown of prelabeled phospholipids. Islets were incubated in the presence of a radioactive precursor for 60 or 90 min and in the presence of either 3.3 or 16.7 mM glucose to prelabel phospholipids. To study the breakdown of prelabeled phospholipid, the unincorporated precursor was removed and the islets were reincubated for 15 or 20 min under conditions that either did or did not stimulate insulin release. Prelabeling in the presence of a noninsulinotropic concentration of glucose (3.3 mM) supported the incorporation of precursors into almost all islet phospholipids studied. Prelabeling in an insulinotropic concentration of glucose (16.7 mM) increased the incorporation of precursors into a number of phospholipids even more; and reincubation in 16.7 mM glucose caused a rapid loss of radioactivity from specific phospholipids (phosphatidylinositol and/or phosphatidylcholine, depending on the precursor). This breakdown was observed only when islets had been prelabeled in 16.7 mM glucose. The amount of radioactivity lost from phospholipid corresponded roughly to the additional amount incorporated during the prelabeling in the high concentration of glucose. Radioactivity in phospholipids in islets prelabeled in 3.3 mM glucose or in nonsecretagogue metabolic fuels, such as malate plus pyruvate, did not decrease when the islets were subsequently exposed to 16.7 mM glucose, nor did it decrease in 3.3 mM glucose when these islets had been prelabeled in 16.7 mM glucose. Glyceraldehyde, an insulin secretagogue, but not galactose or L-glucose which are not insulin secretagogues, stimulated phospholipid breakdown in islets that had been prelabeled in 16.7 mM glucose. Depriving islets of extracellular calcium, a condition that inhibits insulin release, inhibited phospholipid breakdown. The results suggest that pancreatic islets contain a glucose-responsive and a glucose-unresponsive phospholipid pool. The glucose-responsive pool becomes labeled and undergoes rapid turnover only under stimulatory conditions and may play a role in the stimulus-secretion coupling of insulin release.     

Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils

The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP-induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP-induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30-s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phospholipid activation of the insulin receptor kinase: regulation by phosphatidylinositol

A soybean phospholipid mixture produced a concentration-dependent enhancement of beta subunit autophosphorylation of the detergent-soluble, purified human placental insulin receptor. Although phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine also increased insulin receptor autophosphorylation, only phosphatidylinositol (PtdIns) stimulated to a similar extent as the phospholipid mixture. The effect of PtdIns was biphasic, stimulating at low concentrations (75 microM), but having no stimulatory effect at high concentrations (1.0 mM). Phospholipids also stimulated the exogenous protein kinase activity of the insulin receptor toward histone H2B. Phosphorylation of PtdIns occurred with these purified insulin receptor preparations, but this activity was insulin-independent, and the turnover number for PtdIns phosphorylation in the presence of soybean phospholipid was 1/220th as small as the turnover number for the autophosphorylating activity. These results suggest that although PtdIns can modulate the activity of the insulin receptor kinase, PtdIns phosphorylation itself is not directly involved in this regulation.     

Effect of leptin on insulin, glugacon and somatostatin secretion in the perfused rat pancreas.

We have investigated the effect of rat leptin as well as the 22-56 fragment of this molecule on pancreatic hormone secretion in the perfused rat pancreas. In pancreases from fed rats, leptin failed to alter the insulin secretion elicited by glucose, arginine or tolbutamide, but inhibited the insulin response to both CCK-8 and carbachol, secretagogues known to act on the B-cell by increasing phospholipid turnover. This insulinostatic effect was also observed with the 22-56 leptin fragment. In pancreases obtained from 24-hour fasted rats, no effect of leptin on carbachol-induced insulin output was found, perhaps as a consequence of depressed B-cell phospholipid metabolism. Leptin did not influence glucagon or somatostatin release. Our results do not support the concept of leptin as a major regulator of B-cell function. Leptin inhibition of carbachol-induced insulin output might reflect a restraining effect of this peptide on the cholinergic stimulation of insulin release.     

Possible role of phosphatidylinositol turnover in insulin release from isolated rat pancreatic islets

We have previously reported occurrence of Ca2+-activated, phospholipid-dependent protein kinase (referred as protein kinase C) in the rat pancreatic islets. It has been suggested that unsaturated diacylglycerol which results from hydrolysis of phosphatidylinositol by phospholipase C-like enzyme activates protein kinase C. Therefore, we studied the effect of exogenous phospholipase C on insulin release from isolated islets of rat pancreas. Bacterial phospholipase C enhanced insulin release induced by glucose in a dose dependent manner. The effect, however, was decreased in the islets pretreated with colchicine. Both phospholipase C and glucose caused an increase in 32p incorporation into phosphatidylinositol. These results indicate that phospholipid metabolism is linked to the insulin release mechanism.     

Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism

We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.     

Pancreatic islets synthesize phospholipids de novo from glucose via acyl-dihydroxyacetone phosphate

There is considerable evidence that an increased turnover of phosphoinositides and phosphatidic acid accompanies stimulus-induced insulin release. As glucose metabolism via glycolysis produces precursors for phospholipid synthesis, the time course of incorporation of [U14C] labelled glucose was measured to determine the pathways of triose carbon incorporation into phospholipids in the islet. Cultured islets were stimulated with glucose 2.7 or 33 mM. The labelled phospholipids present after stimulation were acyldihydroxyacetone phosphate, lysophosphatidic acid, phosphatidic acid and phosphatidylinositol. Acyl-dihydroxyacetone phosphate rose promptly within 1 minute of raising the glucose concentration and was the primary acylated triose labelled during the first 15 minutes. It was possible to show in vitro conversion of [U14C] glucose-derived acyl-dihydroxyacetone phosphate to lysophosphatidic acid and phosphatidic acid in the presence of NADPH (100 microM), indicating the presence in the islet of acyl-dihydroxyacetone phosphate: NADP oxidoreductase and acyl CoA:1 acylglycerol-3-phosphate acyl transferase, respectively. This study suggests that de novo synthesis of phosphatidic acid provides a link between glucose metabolism and the release of insulin.     

Stimulation of phospholipid methylation by glucose in pancreatic islets

A two fold stimulation in the incorporation of [3H-methyl] groups from [3H-methyl] methionine into phospholipids was seen in intact pancreatic islets within six minutes of exposure to a glucose concentration that stimulates insulin release. Nonstimulatory sugars, L-glucose and D-galactose, as well as dibutyryl cAMP, did not affect phospholipid methylation in islet cells. A calcium channel blocker, verapamil, inhibited methylation. These studies suggest that the signal for glucose-induced insulin release could involve phospholipid methylation.     

In vitro study of adrenergic stimulation of 32P incorporation into phospholipids of brown adipose tissue of control and cold-acclimated rats

1. 32P-labelled inorganic phosphate incorporation into total and mitochondrial phospholipids was studied, in vitro, on brown adipose tissue (BAT) of control and cold-acclimated rats. 2. It was found that norepinephrine acts as in vivo, on BAT phospholipid metabolism via alpha 1 adrenergic receptors specifically increasing phosphatidic acid and phosphatidylinositol turnover with the same magnitude in both groups. 3. Cold-induced alpha 1 adrenergic desensitization is not as important as cold-induced beta adrenergic desensitization. 4. No specific effect of norepinephrine was seen in mitochondrial phospholipid turnover.     

Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus

We have previously reported that atrial natriuretic factor (ANF) increased neuronal norepinephrine (NE) uptake and reduced basal and evoked neuronal NE release. Changes in NE uptake and release are generally associated to modifications in the synthesis and/or turnover of the amine. On this basis, the aim of the present work was to study ANF effects in the rat hypothalamus on the following processes: endogenous content, utilization and turn-over of NE; tyrosine hydroxylase (TH) activity; cAMP and cGMP accumulation and phosphatidylinositol hydrolysis. Results showed that centrally applied ANF (100 ng/microl/min) increased the endogenous content of NE (45%) and diminished NE utilization. Ten nM ANF reduced the turnover of NE (53%). In addition, ANF (10 nM) inhibited basal and evoked (with 25 mM KCl) TH activity (30 and 64%, respectively). Cyclic GMP levels were increased by 10 nM ANF (100%). However, neither cAMP accumulation nor phosphatidylinositol breakdown were affected in the presence of 10 nM ANF. The results further support the role of ANF in the regulation of NE metabolism in the rat hypothalamus. ANF is likely to act as a negative putative neuromodulator inhibiting noradrenergic neurotransmission by signaling through the activation of guanylate cyclase. Thus, ANF may be involved in the regulation of several central as well as peripheral physiological processes such as cardiovascular function, electrolyte and fluid homeostasis, endocrine and neuroendocrine synthesis and secretion, behavior, thirst, appetite and anxiety that are mediated by central noradrenergic activity.     

Regional differences in the effects of insulin-induced hypoglycemia on phospholipid metabolism of rat brain

Insulin-induced hypoglycemia in rats may lead to stimulated brain activity and if severe enough, they may develop a stupor-coma condition. In this study, the effects of insulin-induced hypoglycemia on brain phospholipid metabolism were examined in rats which were prior injected with ³²Pi. Three hours after insulin injection (1 or 5 units/100 g body wt, i.p.), there was an increase (25%) in radioactivity of the lipid phase of cerebral cortex, but radioactivity in the cerebellum tended to decrease instead. Radioactivity in the aqueous phase of cortex was not altered after insulin injection, but that in the cerebellum was decreased by 30%. Differences were observed in labeling of individual phospholipids in response to the hypoglycemic treatment. A marked decrease in labelled phosphatidate was observed in the cerebellum from the hypoglycemic samples, but not in the cerebral cortex. In the cortex, hypoglycemic condition resulted in an increase in ³²Pi uptake into the phospholipids. However, the differences in the amount of label among individual phospholipids suggest that phosphatidylinositol and phosphatidylcholine are turning over more rapidly than other phospholipids. The hypoglycemic rats also showed a 3-fold increase in the brain free fatty acid level, but the level of diacylglycerol was not changed. Results thus suggested a correlation between the free fatty acid release and the increased turnover of phosphatidylinositol and phosphatidylcholine during brain stimulation due to insulin-induced hypoglycemia.     

Lithium increases turnover of phospholipids in flight muscles of Periplaneta americana L

The effect of prolonged lithium administration on the phospholipid metabolism of flight muscles of the cockroach Periplaneta americana has been studied. Following daily injections of LiCl in a dose of 19.25 mumol LiCl per gram of wet weight [32P]- orthophosphate were injected and its incorporation into the phospholipids was measured 2, 12 and 24 h later. Lithium administration did not change the content of phospholipids but increased the 32P incorporation into phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and sphingomyeline 1.87, 2.13, 2.02 and 1.87 times, respectively, as compared with the control values. These increases were neither due to an increased permeability of the tissue for inorganic phosphate nor to an increased turnover of gamma-P-ATP. It is concluded that prolonged lithium treatment increases the turnover of all phospholipids in insect flight muscle tissue.     

Thyrotropin and norepinephrine stimulate the metabolism of phosphoinositides in FRTL-5 thyroid cells

Hormone-induced changes in phospholipid metabolism were examined in a functioning rat thyroid cell line (FRTL-5). Stimulation of FRTL-5 cells, prelabeled with 32P, with TSH or NE resulted in a rapid decrease in the radioactivity of both phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-monophosphate (PIP). The effects of TSH on phospholipid metabolism and calcium mobilization are independent of those on adenylate cyclase. This suggests that the TSH receptor may be unique in that it activates enzyme cascades involved in cAMP production and Ca2+ mobilization.     

Mechanisms of gonadotropin-releasing hormone and prostaglandin action on luteal cells

Both gonadotropin-releasing hormone (GnRH) and prostaglandin F2 alpha (PGF2 alpha) can inhibit cAMP and progesterone production in the corpus luteum; however, their mechanism of action is not known. GnRH or PGF2 alpha causes a rapid and marked increase of labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min into PA and PI, respectively. The labelling of the other phospholipids is not affected. GnRH and PGF2 alpha exert their stimulatory effects on PA-PI turnover at a mean effective dose value of ca. 15 and 100 nM, respectively. Their effects appeared to be additive when both agents were present in the same incubations. Interestingly, addition of the calcium ionophore A23187 also causes a dramatic increase of PA-PI turnover in luteal cells. By contrast, human chorionic gonadotropin and isoproterenol, agents that stimulate cAMP and progesterone production in luteal cells, as well as PGE2 (1 microM), all fail to alter phospholipid labelling; dibutyryl or 8-bromo-cAMP (2-5 mM) actually attentuates the GnRH or PGF2 alpha effect on PI and PA. A very similar PA-PI response to GnRH and PGF2 alpha has also been observed using rat granulosa cells in culture. It seems that following their binding to membrane receptors, GnRH and PGF2 alpha may share a common mechanism in the ovarian cell, possibly involving the stimulation of PA-PI metabolism.     

Effects of luteinizing hormone on phosphoinositide metabolism in rat granulosa cells

Rat granulosa cells isolated from mature Graafian follicles were incubated with luteinizing hormone under various conditions in order to follow the synthesis and degradation of phospholipids. During acute incubations, luteinizing hormone provoked rapid and concentration-dependent increases in the incorporation of 32PO4 into phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides. Similarly, luteinizing hormone provoked increases in labeling of phosphatidylinositol and the polyphosphoinositides when granulosa cells were incubated with myo-[2-3H]inositol. When granulosa cells were prelabeled with 32PO4 in order to label phosphatidylinositol to constant specific radioactivity (4 h), luteinizing hormone treatment significantly increased 32P-phosphatidylinositol levels (23%). Comparable increases (27%) in the cellular concentrations of phosphatidylinositol were observed in response to luteinizing hormone. In pulse-chase experiments employing 32PO4 - or [3H]inositol-prelabeled cells, luteinizing hormone did not alter phospholipid degradation. In addition, luteinizing hormone did not stimulate degradation of polyphosphoinositides. These results demonstrate that: (a) luteinizing hormone has selective effects on phospholipid metabolism in rat granulosa cells which involve phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides, (b) luteinizing hormone increases net levels of phosphatidylinositol and presumably phosphatidic acid and the polyphosphoinositides, and (c) luteinizing hormone does not increase phospholipid degradation. Our findings suggest that luteinizing hormone provokes increases in de novo synthesis of phosphatidylinositol in rat granulosa cells. These changes in phospholipid metabolism may be important for steroidogenesis and other enzymatic processes during treatment with luteinizing hormone.     

Mechanisms of the stimulation of insulin release by arginine-vasopressin in normal mouse islets

The mechanisms by which arginine-vasopressin (AVP) affects pancreatic B-cell function were studied in normal mouse islets. AVP produced a dose-dependent (0.1-1000 nM; EC50 approximately 1-2 nM) amplification of glucose-induced insulin release. This amplification was of slow onset and reversibility. AVP was ineffective when the concentration of glucose was less than 7 mM, but was still very effective in 30 mM glucose. The increase in insulin release produced by AVP was accompanied by small accelerations of 86Rb and 45Ca efflux from islet cells. Omission of extracellular Ca2+ accentuated the effect of AVP on 86Rb efflux, attenuated that on 45Ca efflux, and abolished that on release. Under no condition did AVP inhibit 86Rb efflux. AVP did not significantly affect cAMP levels, but increased inositol phosphate levels in islet cells, even in the absence of extracellular Ca2+. AVP did not affect the membrane potential in unstimulated B-cells and augmented glucose-induced electrical activity only slightly. This was not due to a direct action on ATP-sensitive K+ channels as revealed by patch-clamp recordings (whole cell and outside-out patches). In conclusion, AVP is not an initiator of insulin release, but it potently amplifies glucose-induced insulin release in normal mouse B-cells. This effect involves a stimulation of phosphoinositide metabolism, and presumably an activation of protein kinase C, rather than a change in cAMP levels or a direct control of the membrane potential.     

Evidence Against Dopaminergic and Further Support For α-Adrenergic Receptor Involvement in the Pineal Pho sphatidy lino sitol Effect

Abstract: Several α-adrenergic receptor agonists and antagonists were used to strengthen the earlier findings that the stimulation by (-)-norepinephrine of ³²P₁ incorporation into acidic phospholipids, especially phosphatidylinositol, in the rat pineal gland is mediated through α-adrenergic receptors. Dopamine was able to induce similar stimulation, although always to a smaller extent than equimolar concentrations of norepinephrine. The dopaminergic agonists apomorphine and piribedil did not increase phosphatidylinositol labeling. A number of antagonists considered to act primarily at dopaminergic or α-adrenergic receptors respectively completely prevented dopamine from exerting its effect. Both types of antagonists also were able to inhibit in varying degree the elevation of phospholipid labeling induced by norepinephrine. Dopamine increased phosphatidylinositol turnover without first being converted to norepinephrine, inasmuch as dopamine β-hydroxylase inhibitors had no influence on dopamine activity. Dopamine and α-agonists competitively activated the receptors involved in the phospholipid effect. The conclusion drawn from the several lines of evidence is that only α-adrenergic receptors are concerned with the changes in pineal phospholipid metabolism brought about by the various agonists used and that the action of dopamine occurs through these receptors rather than through discrete dopaminergic receptors.     

Inositol phospholipid turnover in platelets of schizophrenic patients.

Abnormalities in blood cell membrane phospholipid composition and metabolism from schizophrenic patients have been reported by many groups of investigators. Among membrane phospholipids, inositol phospholipids are of special importance as they are involved in transduction system that generates second messengers such as inositol trisphosphate and diacylglycerol. Our studies on platelet inositol phospholipid turnover suggest a significant increase in platelet phosphatidylinositol 4,5-bisphosphate levels, an increased production of inositol trisphosphate in neuroleptic-treated and neuroleptic-free schizophrenic patients platelets and a reduced calcium release by thrombin in neuroleptic-treated schizophrenic patients platelets. The enhanced production of inositol trisphosphate may be due to an increase in its precursor phosphatidylinositol 4,5-bisphosphate with an associated desensitisation of the intracellular inositol trisphosphate receptor by neuroleptics, which may explain the diminished calcium response to thrombin in schizophrenic patients platelets.     

Phospholipid metabolism in rat kidney cortical tubules. II. Effects of hormones on 32P incorporation

To evaluate the effect of hormones on renal phospholipid metabolism and turnover, we studied the changes in 32P-labeling of phospholipids in rat cortical tubule suspension. Angiotensin II, phenylephrine and parathyroid hormone (PTH) stimulate 32P incorporation into PC by 25, 29 and 26% and into PI by 189, 328 and 33% above control rates, respectively, whereas phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labeling was not affected. However, when phospholipids were prelabeled with [32P]Pi, addition of angiotensin II led to a significant decrease in phosphatidylinositol 4,5-bisphosphate labeling in the first 2 min with no effect on the other phospholipid fractions. The phenylephrine effect on phospholipid labeling was blocked by prazosin but not by yohimbine, indicating an alpha 1-mediated action. In contrast, the effect of angiotensin II was not inhibited by either antagonist. The stimulating effect of substrates on 32P incorporation reported in the preceding paper was additive to that of hormones. Our results confirm previous studies on renal gluconeogenesis that catecholamines act by an alpha 1-type receptor on proximal tubules, and indicate that phenylephrine and angiotensin II act by different receptor sites exerting the same metabolic effect. The additivity of hormone effects with that of renal substrates indicates that the former are not secondary to release of precursors for phospholipid biosynthesis. The rapid decrease in phosphatidylinositol 4,5-bisphosphate labeling after angiotensin II suggests that the polyphosphoinositide is degraded after hormone binding to the receptor and that PI labeling is a secondary event.