Modifications of phospholipid metabolism induced by chlorpromazine, desmethylimipramine and propranolol in C6 glioma cells

The effects of chlorpromazine (CPZ), desmethylimipramine (DMI) and propranolol (PRO) on phospholipid metabolism in C6 glioma cells were studied by following the incorporation of 32Pi, [U-14C]glycerol, [2-3H]glycerol and [1-14C]oleate into lipids. The drugs produced a dose-dependent increase in the incorporation of 32Pi and [U-14C]glycerol, but not of [1-14C] oleate, into total phospholipids, that reached a plateau at 200 microM CPZ and 500 microM DMI and PRO. The three drugs shifted the incorporation of precursors from neutral [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] to acidic phospholipids [phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2)] in a dose-dependent, qualitatively similar manner. The incorporation of [2-3H]glycerol into diacylglycerol was also depressed markedly by CPZ. Addition of 1 mM 1,2-dioleoylglycerol, 1-oleoyl-2-acetylglycerol or oleate only partially reversed the decrease in PC labeling caused by CPZ. 12-O-Tetradecanoylphorbol-13-acetate counteracted this effect of CPZ completely but greatly increased PC labeling even in the absence of the drug. Polyphosphoinositides rapidly incorporated 32Pi at early times reaching a plateau in about 40 min. The labeling rate of PI was not parallel to that of PIP or PIP2 and continued to increase even after the polyphosphoinositides had reached a plateau. CPZ increased PI labeling much more than that of PIP and PIP2. These data suggest that cationic amphiphilic drugs may act by inhibiting CTP:phosphocholine cytidylyltransferase, thus decreasing incorporation of precursors into PC and PE; inhibiting PA phosphohydrolase with increased formation of phosphatidyl-CMP, the intermediate for the synthesis of acidic phospholipids; and stimulating the inositol exchange reaction, forming a pool of PI that is not available for PIP and PIP2 synthesis.     

Nicotinic stimulation of polyphosphoinositide turnover in rat adrenal medulla slices

When rat adrenal medulla slices, treated with atropine (1 X 10(-6) mol X l-1], were stimulated with acetylcholine (1 X 10(-5) mol X l-1), an enhanced 32P incorporation into phosphatidylinositol-bis-phosphate (PIP2) and phosphatidylphosphate (PIP) was observed. On the ather hand, stimulation of myo-[3H] inositol prelabelled slices led to a decline in radioactivity of PIP2, indicating an increased turnover of this phospholipid. A possible affection of monoesterphosphate groups of PIP2 or of both PIP2 and PIP during cholinergic nicotinic stimulation is assumed.     

Muscarinic cholinergic enhancement of inositide turnover in cerebral nerve endings is not mediated by calcium uptake

Muscarinic cholinergic stimulation of rat cerebral nerve endings incubated with 32Pi causes an enhancement of the labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). The involvement of Ca2+ in the stimulation of PA and PI labeling by carbamylcholine (CCh) was investigated. Enhancement of Ca2+-influx with veratridine and the Ca2+-ionophore A23187 caused a vast decrease of the labeling of the polyphosphoinositides, which was not accompanied by an enhancement of the labeling of PA and PI. The dihydropyridine Ca2+-agonist BAY K8644 did not affect phospholipid labeling. A23187, veratridine and BAY K 8644 did not enhance stimulation of the labeling of PA and PI by CCh. When Ca2+ was omitted from the incubation, A23187 caused an enhancement of basal and CCh-stimulated labeling of PA and PI, possibly indicating a particular feature of A23187 unrelated to its iontophoretic properties. The Ca2+-channel antagonists nimodipine, verapamil and flunarizine were virtually without effect on basal and CCh-stimulated labeling of PI and PA. These data support the notion that the muscarinic cholinergic inositide response is not mediated or controlled by Ca2+-flux.     

The stimulatory effects of cationic amphiphilic drugs on human platelets treated with thrombin

The actions of eight cationic amphiphilic drugs on human platelets displayed three different effects according to drug concentration ranges. At lower concentrations (below approximately 25 microM), the drugs stimulated secretory responses induced by 0.2 U/mL of thrombin, while at concentrations in the 25-50 microM range they inhibited these responses. Above 50-100 microM, the drugs caused permeabilization of the platelet plasma membrane as measured by leakage of cytoplasmic adenine nucleotides. The effects of these agents on phosphoinositide metabolism were monitored in platelets prelabeled with (32)P-inorganic phosphate, such that phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP(2)), but not phosphatidylinositol (PI), were labeled to equilibrium. In unstimulated platelets, the level of labeled PA decreased slightly (about 25%), with corresponding increases in PIP(2) labeling up to drug concentrations of about 50 microM. In contrast to the relatively small changes in PI and PIP(2), the levels of labeled PIP, precursor to PIP(2), increased 2- to 4-fold in both resting and thrombin-treated platelets from 5 microM up to about 50-100 microM of drugs and remained elevated throughout the permeabilization concentrations. [(32)P]PA increased 20-fold over control upon thrombin activation and 5-30 microM of drugs caused [(32)P]PA to increase 30-37 times over that seen in control, resting platelets; the concentration of drugs that potentiated thrombin-induced [(32)P]PA elevation corresponded to that causing the potentiation of platelet secretion. Higher drug concentrations decreased [(32)P]PA elevation. [(32)P]PIP(2) levels increased about 25% in response to thrombin treatment alone; low concentrations of drugs led to another 25% elevation. A significant decrease in [(32)P]PIP(2) was seen above 30 microM, corresponding to inhibition of platelet secretion. Concentrations of 5-30 microM of several psychoactive agents, both neuroleptics and antidepressants, potentiated the thrombin-induced activation of platelets as measured by dense granule secretion and increased turnover of phosphoinositides. Remarkably, all of the drugs increased the levels of PIP even in resting platelets, indicating that they have common effects apart from the specific receptor interactions currently attributed to them. These common effects, e.g. an increase in membrane fluidity such as is known to be caused by amphipathic agents, may be in part responsible for the observed overlapping psychotropic effects of tricyclic antidepressants and phenothiazines.     

Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells.

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [32P]phosphoinositide breakdown, the first peak of carbachol (1 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2). The second peak was found to be related to [32P]PIP2 breakdown. Carbachol stimulated the release of [3H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [3H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [3H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-microM)-induced release of [3H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.     

M1 acetylcholine receptor-mediated phosphatidylinositol turnover in adult and senescent rat brain slices.

Phosphatidylinositol (PI) turnover via muscarinic acetylcholine (mACh) receptor was investigated using the cerebral cortex from adult rats. Activities in the cerebral cortex, hippocampus and striatum from senescent rats were compared with those from adult rat. Carbachol (1 mM)-stimulated [3H]IP accumulation in the presence of 10 mM LiCl was inhibited by pirenzepine more potently than by AF-DX 116. Although the displacing activity of carbachol for [3H]pirenzepine binding was decreased by 50 microM GTP gamma S, pretreatment of slices with pertussis toxin (PTX, 0.01-1.0 micrograms/ml) did not affect the carbachol-induced [3H]IP accumulation. In the slices from all 3 tissues, cerebral cortex, hippocampus and striatum, both incorporation of [3H]inositol and carbachol-stimulated [3H]IP accumulation were reduced at 28 months compared to those at 2 months. Furthermore, the Bmax values of [3H]pirenzepine binding in membranes from these three regions were diminished at the senescent stage. Taken together, the results suggest that an M1-subtype of muscarinic acetylcholine receptor could be involved in PI turnover via GTP-binding proteins insensitive to PTX. Age-related changes in M1-receptor mediated PI turnover seem to be in part due to the decreased number of M1-receptors with increasing age in the cerebral cortex, hippocampus and striatum; and some qualitative changes also seem to have occurred in the hippocampus of senescent rats in the mACh receptor-PI turnover system.     

Enhancement of phosphatidylinositol turnover and cyclic nucleotide accumulation by chronic anethole trithione treatment in rat submaxillary glands

The effect of chronic treatment with anethole trithione (ANTT) on the phosphatidylinositol (PI) turnover and cyclic (c)AMP and cGMP accumulation in rat submaxillary glands (SMG) has been compared with the effect of chronic treatment with atropine and a cholinesterase inhibitor, diisopropylfluorophosphate (dyflos, DFP). Experiments were performed 24, 48 and 24 h after the last dose of ANTT, atropine and dyflos, respectively. ANTT and atropine enhanced carbachol-stimulated [32P] incorporation into phosphatidic acid in the SMG slices, while dyflos showed no effect. Pilocarpine-stimulated in-vivo incorporation of [3H]myoinositol into inositol phosphates was significantly enhanced by ANTT, but not by atropine or by dyflos. Phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by ANTT and atropine, but not by dyflos. Pilocarpine-stimulated in-vivo accumulation of cAMP and cGMP was enhanced by ANTT and atropine, but dyflos reduced cAMP accumulation without affecting cGMP accumulation. The enhancement of PI turnover and cyclic nucleotide accumulation seems to contribute to the development of supersensitivity of the salivary gland caused by chronic treatment with ANTT and atropine, while reduction of cAMP accumulation may be responsible for the subsensitivity caused by dyflos.     

The effect of isoproterenol on the metabolism of phosphatidylinositol by rat heart in vitro

The effect of epinephrine and isoproterenol (10^?4 M) on the incorporation of ³²P orthophosphate and [1-¹⁴C] palmitic acid into rat heart slices was investigated in pulse and chase experiments. Epinephrine stimulated the uptake of ³²P into phosphatidic acid and phosphatidyl-inositol. Isoproterenol stimulated the labelling of phosphatidic acid but had no effect on phosphatidyl-inositol metabolism in the first 60 min of the incubation. Propranolol stimulated the incorporation of ³²P and [1-¹⁴C] palmitic acid into phospholipids in the first 20–30 min of the incubation but counteracted isoproterenol thereafter. These results indicate that the main effect of isoproterenol on phospholipid metabolism in rat heart is to increase the turnover of phosphatidic acid and to slow down the conversion of phosphatidic acid into phosphatidylinositol.     

Alpha1-adrenergic stimulation of phosphatidylinositol-phosphatidic acid turnover in rat parotid cells

The regulation of phosphatidylinositol turnover by alpha-adrenergic agonists in rat parotid acinar cell aggregates was examined with respect to kinetics and agonist-antagonist interactions. Phosphatidylinositol turnover was followed by the changes in the specific activities of [32P]phosphatidic acid and [32P]phosphatidylinositol. The specific activity of phosphatidic acid increased rapidly (within 1 min) after addition of epinephrine (10(-5) M), reached a maximal level within 12-16 min, and then decreased. Incorporation of 32P into phosphatidylinositol exhibited a lag phase of about 5 min and then increased continuously for an additional 40 min. The absolute amounts of phosphatidic acid and phosphatidylinositol did not change. The concentrations of epinephrine needed to stimulate 32P incorporation into phosphatidic acid and phosphatidylinositol, measured at 15 and 30 min, respectively, were similar; Ka values of 2.05 +/- 0.46 X 10(-6) M for phosphatidic acid and 2.98 +/- 0.30 X 10(-6) M for phosphatidylinositol were found. The effects of agonists on 32P labeling of phosphatidylinositol, in order of potency, were epinephrine greater than or equal to norepinephrine greater than phenylephrine much greater than normetanephrine. When various adrenergic antagonists were evaluated for their ability to inhibit 10(-5) M epinephrine-stimulated 32P incorporation into both phosphatidic acid and phosphatidylinositol, the order of antagonist potency was prazosin greater than or equal to phenoxybenzamine greater than phentolamine greater than or equal to yohimbine greater than much greater than propranolol. These findings indicate that phosphatidylinositol-phosphatidic acid turnover in the rat parotid gland is mediated by the alpha 1-adrenergic receptor system.     

Involvement of alpha 1-adrenergic receptors in stimulation of phosphatidylinositol metabolism by catecholamines in mouse thyroids

The effects of alpha-adrenergic agonists and thyroid stimulating hormone on the incorporation of radioactive phosphate into phosphatidylinositol were investigated in mouse thyroids in vitro. The incorporation of 32P orthophosphate into phosphatidylinositol was stimulated by thyroid stimulating hormone, norepinephrine (a mixed alpha 1- and alpha 2-adrenergic agonist), methoxamine and phenylephrine (alpha 1-agonists) and slightly by clonidine and oxymetazoline (alpha 2-agonists) but not by isoproterenol (beta-agonist). Prazosin (alpha 1-antagonist) inhibited the stimulation by norepinephrine of 32P incorporation into phosphatidylinositol, but yohimbine (alpha 2-antagonist) was less effective. Although norepinephrine inhibits the thyroid stimulating hormone-induced release by activating alpha-, especially alpha 1-adrenoceptors in mouse thyroids [M. L. Maayan et al., Metabolism 26, 473 (1977); M. L. Maayan et al., Endocrinology 101, 284 (1977); T. Muraki et al., Endocrinology 110, 51 (1982)] alpha 1-agonists did not decrease the stimulation of turnover elicited by thyroid stimulating hormone and did not have additive action with it. These results suggest that (1) the stimulation of phosphatidylinositol turnover of mouse thyroids elicited by adrenergic agonists is mediated by activation of alpha 1-adrenoceptors and (2) the inhibitory effect of norepinephrine on the thyroid stimulating hormone-induced release of thyroxine is not mediated by norepinephrine-inhibition of phosphatidylinositol-turnover stimulated by thyroid stimulating hormone.     

Phosphoinositide hydrolysis is correlated with agonist-induced calcium flux and contraction in the rabbit aorta

In the present study changes in the extent of 32P labelling of membrane phospholipids were correlated with the alpha 1-adrenoceptor-induced events of increased 45Ca influx, 45Ca release and contraction in the rabbit aorta. Under basal conditions 32P incorporation into all phospholipids proceeded without saturation through 80 min of labelling. During a 5 min exposure to 10(-5) M norepinephrine (NE) after 25 min of prelabelling the incorporation of 32P into certain phospholipids was substantially increased. Phosphatidic acid (PA) labelling was increased above basal levels by 4.1 fold, phosphatidylinositol (PI) 2.5 fold and phosphatidylcholine (PC) 1.8 fold. Half maximal stimulation of 32P labelling of PA occurred at 2.0 microM, which was similar to the EC50 value for stimulation of 45Ca influx (2.5 microM) and 45Ca release (2.1 microM) but slightly higher than the value for contractile response (0.9 microM). Antagonist sensitivity studies reinforced the alpha 1 receptor subtype character of the rabbit aorta. Prazosin (10(-7) M) reduced agonist-induced events by 63-82% while yohimbine (10(-7) M) was without influence. Phenoxybenzamine (10(-8) M) reduced agonist-induced events by 56-76%. A temporal comparison showed that agonist stimulation of PA labelling was slower than 45Ca release, but similar to the time course of 45Ca influx. Hydrolysis of 32P-labelled phosphatidylinositol diphosphate (PIP2) was more rapid and paralleled 45Ca release. These findings suggest that PIP2 hydrolysis may account for the rapid phase of norepinephrine-induced contraction in rabbit aorta while PA or its immediate precursor diacylglycerol may account for receptor-induced Ca2+ influx.     

Bradykinin-stimulated differential incorporation of arachidonic acid into lipids of kidney cortex and medulla

We investigated bradykinin-induced changes in the turnover of arachidonate in renal lipids of the perfused rabbit kidney. Upon hormone stimulation, this cellular system undergoes only transient dynamic changes in arachidonic acid metabolism; no loss of bradykinin effect on arachidonate release and prostaglandin generation is shown upon repeated hormone administrations during 8-9 hr of perfusion. Ureter-obstructed rabbit kidneys were perfused for 5-6 hr and then saline or bradykinin in saline was administered, followed after 10 sec by pulse labelling with [14C]arachidonate. The pattern of distribution of [14C]arachidonate in lipid fractions of the cortex showed that bradykinin caused a 2 to 2.5-fold increase in the relative incorporation of arachidonic acid into phosphatidylinositol (PI), phosphatidic acid (PA), diglyceride (DG) and triglyceride (TG) fractions and a concomitant decrease in its incorporation into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In contrast, in the medulla hormone administration caused a marked increase of arachidonate incorporation into PI and PC, and a decrease in incorporation into PE, PA, DG and TG. This differential arachidonate labelling of cortical vs medullary lipids following bradykinin stimulation suggests that the hormone activates different lipolytic processes in cortex and medulla, and promotes hydrolysis of arachidonic acid from different phospholipid pools.     

Influence of chronic ethanol consumption on muscarinic cholinergic receptors and their linkage to phospholipid metabolism in mouse synaptosomes

C57/BL mice rendered physically dependent to ethanol exhibited an increase in specific binding of [3H]QNB to a cholinergically enriched synaptosomal fraction of brain without a parallel increase in muscarinic receptor-stimulated incorporation of [32P] into phosphatidylinositol and phosphatidic acid. Receptor-enhanced phospholipid labeling was reduced by the addition of ethanol (100mM) in vitro. In chronically treated mice, tolerance developed to the inhibitory effects of ethanol on receptor-enhanced phospholipid labeling. It is suggested that the inhibition of this response by ethanol added in vitro may be associated with alterations in neuronal function during alcohol intoxication.     

The human astrocytoma cell line 1321 N1 contains M2-glandular type muscarinic receptors linked to phosphoinositide turnover.

1. Muscarinic receptors present in the human astrocytoma cell line 1321 N1 were characterized in radioligand binding studies and in functional studies of carbachol-stimulated phosphatidylinositol (PI) turnover. 2. In radioligand binding studies the muscarinic receptor in intact cells could be labelled using [3H]-N-methylscopolamine ([3H]-NMS) but not by [3H]-pirenzepine. In the intact cells these receptors displayed low pirenzepine affinity (pKi = 6.83) indicating that they were not of the M1 subtype. Furthermore, the 1321 N1 muscarinic receptors displayed low affinity for the two M2-cardiac selective ligands methoctramine (pKi = 5.82) and AF-DX 116 (pKi = 6.29). This pharmacology was consistent with the 1321 N1 cells containing a single population of muscarinic receptors that displayed a similar pharmacology to the M2-receptor present in exocrine gland tissue. 3. The M2-gland nature of the receptors was further indicated in the functional studies where antagonist affinities were determined from their ability to antagonize carbachol-stimulated PI turnover in 1321 N1 cells. pA2 values for pirenzepine (7.31), methoctramine (6.10) and AF-DX 116 (6.52) were similar to those determined in the binding studies. 4. From these studies we conclude that 1321 N1 astrocytoma cells contain an M2-gland muscarinic receptor which mediates muscarinic receptor-mediated stimulation of PI turnover in these cells.     

Ethanol stimulates basal and serotonin-induced formation of [32P]phosphatidic acid in human platelets

The addition of serotonin to preparations of 32P-labelled human platelets resulted in a time- and dose-dependent hydrolysis of [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) and formation of [32P]phosphatidic acid (PA). This response was inhibited by the serotonin2 receptor antagonist ritanserin, indicating that the stimulation was mediated via the serotonin2 receptor. The addition of 50-150 mM of ethanol prior to stimulation with 10(-5) M serotonin resulted in an increased accumulation of [32P]PA, but had no effect on [32P]PIP2. Ethanol stimulated [32P]PA formation at all serotonin concentrations studied (10(-7)-10(-5) M). Furthermore, in the absence of serotonin, ethanol increased basal [32P]PA formation.     

Increased formation of phosphatidic acid induced with vasopressin or Ca2+ ionophore A23187 in rat hepatocytes

The effects of vasopressin and Ca2+ ionophore A23187 on phospholipid metabolism were investigated in rat hepatocytes. Vasopressin stimulated the incorporation of [32P]Pi into phosphatidic acid within 2 min but then it returned to control level after 10 min. On the other hand, the stimulation of the incorporation of [32P]Pi into phosphatidylinositol continued with incubation times up to 20 min. The Ca2+ ionophore A23187 also increased the 32P-labeling in phosphatidic acid, although it had no effect on [32P]Pi incorporation into phosphatidylinositol. Concerning the incorporation of [3H]glycerol, vasopressin did not enhance its incorporation into phosphatidic acid and phosphatidylinositol. The Ca2+ ionophore A23187 increased the incorporation into phosphatidic acid without significant effects on that into phosphatidylinositol. In the hepatocytes prelabeled with [3H]arachidonic acid, stimulated degradation of phosphatidylinositol with the addition of vasopressin and resultant formation of phosphatidic acid were observed within 5 min. The transient accumulation of diacylglycerol, the product of phosphatidylinositol hydrolysis, also occurred within 5 min with vasopressin. On the other hand, with the Ca2+ inophore A23187, stimulated degradation of triacylglycerol to diacylglycerol and the consequent formation of phosphatidic acid were observed. The Ca2+ ionophore A23187 caused a significant release of free [3H]arachidonic acid, although vasopressin had no effect.     

Studies on the mechanism of alteration by propranolol and mepacrine of the metabolism of phosphoinositides and other glycerolipids in the rabbit iris muscle

We have investigated the effects and mechanism of action of propranolol and mepacrine, two drugs with local anesthetic-like properties, on phospholipid metabolism in rabbit iris and iris microsomal and soluble fractions. In the iris, propranolol, like mepacrine [A. A. Abdel-Latif and J. P. Smith, Biochim, biophys. Acta 711, 478 (1982)], stimulated the incorporation of [14C]arachidonic acid ( [14C]AA) into phosphatidic acid (PA), CDP-diacylglycerol (CDP-DG), phosphatidylinositol (PI), the polyphosphoinositides (poly PI) and DG, and it inhibited that of phosphatidylcholine (PC), phosphatidylethanolamine (PE), triacylglycerol (TG) and the prostaglandins. Similarly, mepacrine, like propranolol [A. A. Abdel-Latif and J. P. Smith, Biochem. Pharmac. 25, 1697 (1976)], altered the incorporation of [14C]oleic acid, [3H]glycerol, 32Pi and [14C]choline into glycerolipids of the iris. Time-course studies in iris muscle prelabeled with [14C]AA showed an initial decrease in the production of DG and a corresponding increase in that of PA by the drugs, followed by an increase in accumulation of DG at longer time intervals (60-90 min). The above findings are in accord with the hypothesis that these drugs redirect glycerolipid synthesis by inhibiting PA phosphohydrolase. Propranolol and mepacrine stimulated the activities of DG kinase and phosphoinositide kinases and inhibited that of DG cholinephosphotransferase. The drugs had little effect on the activity of DG acyltransferase. It is concluded that propranolol and mepacrine redirect glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in phospholipid biosynthesis. We suggest that these drugs could exert their local anesthetic-like effects by effecting an increase in the synthesis of the acidic phospholipids (PA, PI and the poly PI) and subsequently the binding of Ca2+- to the cell plasma membrane.     

Effects of pentobarbital and veratridine on phosphatidylinositol and phosphatidate metabolism in rat parotid acinar cells

The metabolism of phosphatidylinositol and phosphatidate was studied in isolated rat parotid cells, incubated in a physiological buffer containing [32P]phosphate or [3H]glycerol. Carbamylcholine and epinephrine stimulated 32P incorporation into both of these phospholipids, causing their half-maximal effects at 2 and 0.8 microM respectively. The former concentration is much lower than that anticipated from binding studies. The Hill coefficients for carbamylcholine activation of 32P incorporation were 0.61 +/- 0.05 for phosphatidate and 0.64 +/- 0.05 for phosphatidylinositol. Pentobarbital (0.58 mM) inhibited the increased 32P incorporation caused by 5 microM carbamylcholine but not 100 microM carbamylcholine. Pentobarbital inhibited the incorporation of 3H equally in the presence and absence of epinephrine, indicating that the effect of pentobarbital on 32P incorporation is on turnover and not on de novo synthesis. Veratridine (200 microM) had no effect on phospholipid metabolism in the presence and absence of either carbamylcholine or epinephrine, which contrasts with our previous findings in synaptosomes [J.C. Miller and I. Leung, Biochem. J. 178, 9 (1979)].     

Chlorpromazine increases the turnover of metabolically active phosphoinositides and elevates the steady-state level of phosphatidylinositol-4-phosphate in human platelets.

Non-permeabilizing concentrations (< 40 microM) of chlorpromazine (CPZ) increase the radioactivity of phosphatidylinositol-4-phosphate (PIP) in platelets pre-labelled with [32P]Pi, but the biochemical mechanisms underlying this increase are poorly understood. Incubation of [32P]Pi-labelled, gel-filtered platelets with 25 microM CPZ for 10 min increased: (1) the mass of PIP from 315 to 476 nmol/10(11) platelets but not the total inositol phospholipid mass, (2) the specific phosphodiester radioactivities in phosphatidylinositol (PI), PIP and phosphatidylinositol-4,5-bisphosphate (PIP2) by 34, 63 and 37%, respectively, and (3) the specific phosphomonoester radioactivities in PIP and PIP2 by 53 and 10%, respectively. In control platelets (no CPZ) the specific radioactivity of the phosphodiester was the same in PI, PIP and PIP2, and the specific radioactivity in the phosphomonoester in PIP and PIP2 was 55% of that of the gamma-phosphoryl in ATP, measured as metabolically active, actin-bound ADP. These results suggest that 55% of each of PI, PIP and PIP2 constitutes a metabolic pool which is labelled by 32P in the platelets, while the remainder is in a metabolically inactive pool and not labelled. CPZ has two major effects: (1) CPZ interferes with the kinase and phosphohydrolase reactions that maintain the steady-state level of PIP in the metabolic phosphoinositide pool, resulting in a 92% increase in the PIP level of this pool, and (2) CPZ causes synthesis (45% in 10 min) of new phosphodiester in the metabolically active phosphoinositides by tentative stimulation of the turnover of the phosphoinositide cycle, de novo phosphoinositide synthesis and/or diacylglycerol formation through phospholipases C and D. The marked alteration by CPZ of phosphoinositide metabolism may be part of the mechanism by which this drug effects its psychotropic action.     

Acetylcholine receptor agonists stimulate [H]taurine release from rat sympathetic ganglia

The endogenous taurine content, and the uptake and release of [³H]taurine were examined using the rat superior cervical ganglion. Taurine was found to be one of the most abundant amino acids in the superior cervical ganglion, and the superior cervical ganglion took up [³H]taurine from the incubation medium. Carbachol stimulated the release of [³H]taurine in a concentration-dependent manner with an EC₅₀ of 26 μM and maximal stimulation at 100 μM. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium stimulated release with the same potency but with greater efficacy than carbachol. The nicotinic receptor antagonist hexamethonium (1 mM) inhibited carbachol-stimulated release by 74% (±)-Muscarine stimulated release with an EC₅₀ of 8 μM but with a maximal effect of only 32% of that produced by 100 μM carbachol. Oxotremorine another muscarinic receptor agonist, was ineffective, even at 1 mM. The muscarinic receptor antagonist atropine inhibited carbachol-stimulated release by 30% at 10μM. These results show that ³H taurine release from rat superior cervical ganglion can be stimulated by cholinergic receptor agonists. Release is mediated predominantly by a nicotinic receptor and partially by a muscarinic receptor.