Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.     

Vasopressin decreases total free fatty acids but enhances release of radioactivity from isolated hepatocytes labelled with [3H]arachidonic acid

The short-term effects of vasopressin on free fatty acids and lysophospholipids were investigated in hepatocytes isolated from fed rats. Over the time period 0.25 to 10 min vasopressin decreased the steady-state concentrations of palmitic, stearic and oleic acids measured by gas liquid chromatography in extracts of cells incubated at 0.1 mM extracellular Ca2+. The concentrations of arachidonic and linoleic acids did not change. In hepatocytes labelled with [3H]arachidonic acid and incubated at 1.3 mM extracellular Ca2+ vasopressin or the Ca2+-selective ionophore A23187 increased the rate of accumulation of radioactivity in the incubation medium by 40%. The action of A23187 was dependent on extracellular Ca2+. When hepatocytes labelled with 32Pi were treated with vasopressin, no change in the amounts of [32P]lysophosphatidylethanolamine or [32P]lysophosphatidylcholine was observed. It is concluded that the action of vasopressin on hepatocytes is associated with the release of arachidonic acid or metabolites of arachidonic acid but is not accompanied by a general increase in the steady-state concentrations of free fatty acids and lysophospholipids.     

Influence of Bradykinin on Diacylglycerol and Phosphatidic Acid Accumulation in Cultured Bovine Adrenal Chromaffin Cells

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.     

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Relationship between phosphatidylinositol synthesis and recovery of 5-hydroxytryptamine-responsive Ca2+ flux in blowfly salivary glands.

Each salivary gland contains about 135 pmol of phosphatidylinositol. In glands prelabelled by incubation for 1 h with [32P]Pi or [3H]inositol there was a subsequent breakdown of 80% of the labelled phosphatidylinositol over a 2 h incubation period with 10 micrometer-5-hydroxytryptamine. However, there was no detectable decrease either in total phosphatidylinositol based on phosphorus analysis by chemical estimation or in the radioactivity of [32P]phosphatidylinositol in salivary glands of flies raised from the larval stage on diets containing[32P]Pi and whose phospholipids were uniformly labelled. These results suggest that the pool of phosphatidylinositol involved with Ca2+ gating is a small fraction of the total phosphatidylinositol content. Furthermore it is this small compartment that is preferentially radioactively labelled during short-term incubations with radioactively labelled precursors. In salivary glands incubated for 2 h with 10 micrometer-5-hydroxytryptamine there was a marked decrease in the flux of 45Ca2+ across the gland. After removal of the hormone, incubation of salivary glands for 1 h in the presence of 2mM-inositol, but not choline or ethanolamine, resulted in a recovery of hormone-responsive 45Ca2+ flux. Quantitative studies revealed that less than 9 pmol of phosphatidylinositol must be formed to fully restoret he 5-hydroxytryptamine-responsive 45Ca2+ flux.     

The phosphorylation of an acidic protein of the large ribosomal subunit of Krebs II ascites cells.

The ribosomes of Krebs II ascites cells contain an acidic protein, apparently analogous to proteins L7/12 of Escherichia coli. When ascites cells were incubated with [32P]Pi, this protein became labelled, indicating that it is a phosphoprotein.     

Stimulation, by vasopressin and other agonists, of inositol-lipid breakdown and inositol phosphate accumulation in WRK 1 cells.

WRK 1 cells were labelled to equilibrium with 2-myo-[3H]inositol and stimulated with vasopressin. Within 3 s of hormone stimulation there was a marked accumulation of 3H-labelled InsP2 and InsP3 (inositol bis- and tris-phosphate), but not of InsP (inositol monophosphate). There was an associated, and rapid, depletion of 3H-labelled PtdInsP and PtdInsP2 (phosphatidylinositol mono- and bis-phosphates), but not of PtdIns (phosphatidylinositol), in these cells. Some 4% of the radioactivity in the total inositol lipid pool of WRK 1 cells was recovered in InsP2 and InsP3 after 10 s stimulation with the hormone. The selectivity of the vasopressin receptors of WRK 1 cells for a variety of vasopressin agonists and antagonists revealed these to be of the V1a subtype. There was no receptor reserve for vasopressin-stimulated inositol phosphate accumulation in WRK 1 cells. The accumulation of inositol phosphates was enhanced in the presence of Li+ions. Half-maximal accumulation of InsP, InsP2 and InsP3 in vasopressin-stimulated cells was observed with 0.9, 3.0 and 3.6 mM-Li+ respectively. Bradykinin and 5-hydroxytryptamine also provoked inositol phosphate accumulation in WRK 1 cells. The effects of sub-optimal concentrations of bradykinin and vasopressin upon inositol phosphate accumulation were additive, but those of optimal concentrations of the hormones were not.     

Regulation of phosphatidate synthesis by secretagogues in parotid acinar cells.

The metabolism of phosphatidate in rat parotid acinar cells was investigated, particularly with regard to the actions of agonists known to act by mobilizing Ca2+. When cells were incubated in medium containing 10 microM-[32P]Pi, phosphatidate was rapidly labelled, approaching an apparent steady-state with a half-time of approx. 20 min. Methacholine provoked a more than doubling of phosphatidate radioactivity, which was reversed by the muscarinic antagonist atropine. These results suggest that phosphatidate labels to near steady-state rapidly and that in cells prelabelled for 60 min the increase in radioactivity induced by agonists probably reflects net synthesis rather than an increase in specific radioactivity. Phosphatidate synthesis in response to methacholine was rapid and occurred, within the resolution of a few seconds, with no measurable latency. Adrenaline and substance P also stimulated phosphatidate synthesis but both agonists were less efficacious than methacholine. A Ca2+ ionophore, ionomycin, did not provoke phosphatidate synthesis. By using a protocol that eliminates the receptor-regulated Ca2+ pool, it was demonstrated that methacholine-induced phosphatidate formation does not come about as a consequence of Ca2+ influx nor of Ca2+ release. These results indicate that the phosphatidate synthesis response has characteristics compatible with its previously suggested role as a primary mediator of membrane Ca2+-gating.     

Phospholipids of Trypanosoma cruzi: increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation.

We have studied the effect of carbamoylcholine in Trypanosoma cruzi epimastigote forms prelabelled with [32P]-Pi. Suspensions of cells were incubated at 28 degrees C to measure changes in the levels of [32P]-labelled phospholipids after stimulation. The presence of this cholinergic agonist induced changes in the phosphoinositide metabolism; a shift in the levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) was observed, whereas the levels of the other glycerophospholipids were not changed. This study shows that carbamoylcholine either directly or indirectly influences changes in phosphoinositide metabolism.     

Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures

The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84-88% of the labeled adenine nucleotides, ADP 11-13%, and AMP 1-3%. The turnover of 32P-adenine nucleotides and 3H-nucleotides was biphasic and virtually identical; there was an initial fast phase with a t1/2 of 3.5-4.5 h and a slow phase with a half-life varying from 7 to 17 days, depending upon the particular cell preparation. The t1/2 of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half-life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half-lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P-labeled compounds and relatively three times as much catecholamine as 3H-labeled compounds.     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

Stimulated cholesterol-enriched platelets display increased cytosolic Ca2+ and phospholipase A activity independent of changes in inositol trisphosphates and agonist/receptor binding.

To investigate the mechanism of enhanced responsiveness of cholesterol-enriched human platelets, we compared stimulation by surface-membrane-receptor (thrombin) and post-receptor (AlF4-) G-protein-directed pathways. Platelets were labelled with [32P]Pi and [methyl-3H] choline chloride, incubated with sonicated lipid dispersions of various ratios of cholesterol and phospholipid, and loaded with the fluorescent Ca2+ indicator fura-2. We report the following. (1) Cholesterol enrichment enhances cytosolic Ca2+ accumulation and phospholipase A activation in response to both receptor-directed and post-receptor-directed agonists. No enhancement by cholesterol of phospholipase A activity at fixed Ca2+ concentrations is observed in lysed platelets, implying that no perturbation by cholesterol of phospholipase A/substrate interaction occurs in our preparations. (2) In both normal and cholesterol-enriched platelets, Ca2+ mobilization is promoted by a factor(s) apart from InsP3 that appear(s) to be modulated by cholesterol. A disproportionate increase in cytosolic Ca2+ relative to [32P]InsP3 is observed with increasing doses of thrombin in normal, and to a larger extent in cholesterol-enriched, platelets. When AlF4- is the agonist, there is no cholesterol-associated enhancement in [32P]InsP3 to account for the heightened Ca2+ rise seen with cholesterol enrichment. (3) Enhanced phospholipase A activation is not necessarily proportional to cytosolic Ca2+ increase. The magnitude of the increase in phospholipase A activity for a given rise in cytosolic Ca2+ is greater in cholesterol-enriched platelets that are stimulated by AlF4- than in those stimulated by thrombin. We conclude that increased membrane microviscosity associated with cholesterol enrichment may promote G-protein/phospholipase A interaction as well as the Ca2(+)-release mechanism, without significantly altering G-protein/phospholipase C interaction.     

Functional heterogeneity of polyphosphoinositides in human erythrocytes.

After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosphomonoester groups of PtdIns4P and of PtdIns(4,5)P2 approached equilibrium values which were close to that of the gamma-phosphate of ATP (78-85%), showing that almost all of these phosphate groups were metabolically active. Phosphoinositidase C (PIC) activation, using Ca2+ and the ionophore A23187, of 32P-prelabelled erythrocytes was used to investigate a possible functional heterogeneity of the phosphoinositides. Hydrolysis of PtdIns(4,5)P2, measured from its radioactivity, decreased as function of the time of prelabelling up to a constant value equal to that measured from its content. In contrast, hydrolysis of PtdIns4P, determined both from radioactivity and from content, was always the same. These data suggest that newly labelled molecules of PtdIns(4,5)P2, initially accessible to PIC, then moved towards a PIC-resistant pool. This was further confirmed by measuring the fraction of labelled PtdIns(4,5)P2 molecules accessible to PIC after a prelabelling period of 5 min and different times of reincubation. Hydrolysis by PIC was also measured in erythrocytes in which the phosphoinositide content had been modified by activation (Mg2+-enriched cells) or inhibition (ATP-depleted cells) of the phosphoinositide kinases. The sizes of the PIC-resistant pools of polyphosphoinositides were not affected by these treatments, indicating that the kinases (and the phosphatases) act on the PIC-sensitive pools. This was also shown by the decrease in the production of Ins(1,4,5)P3 upon PIC activation in ATP-depleted erythrocytes. A model is presented in which the PIC-sensitive pools of polyphosphoinositides are those which are accessible to the kinases and the phosphatases and are rapidly turned over.     

Characterization of a proteinase inhibitor isolated from the fungal pathogen Coccidioides immitis.

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.     

Turnover of the phosphomonoester groups of polyphosphoinositol lipids in unstimulated human platelets

The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by short-term labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] had the same specific 32P radioactivity as the gamma-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4-13% compared to that of the ATP-gamma-phosphate. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)P2, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)P2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)P2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the gamma-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.     

Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase

Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.     

Thyrotropin-releasing hormone receptor occupancy determines the fraction of the responsive pool of inositol lipids hydrolysed in rat pituitary tumour cells.

We report that there are distinct thyrotropin-releasing hormone (TRH)-responsive and -unresponsive pools of inositol (Ins) lipids in rat pituitary tumour (GH3) cells, and present evidence that the size of the responsive pool is determined by the number of activated TRH-receptor complexes. By use of an experimental protocol in which cycling of [3H]Ins is inhibited and resynthesis occurs with unlabelled Ins only, we were able to measure specifically the effects of TRH on the hydrolysis of the Ins lipids present before stimulation. A maximally effective dose of TRH (1 microM) caused a time-dependent decrease in 3H-labelled Ins lipids that attained a steady-state value of 42 +/- 1% of the initial level between 1.5 and 2 h. After 2 h, even though there was no further decrease in 3H-labelled Ins lipids, and no increase in [3H]Ins or [3H]Ins phosphates, turnover of Ins lipids, as assessed as incorporation of [32P]Pi into PtdIns, continued at a rate similar to that in cells incubated without LiCl or unlabelled Ins. These data indicate that Ins lipid turnover was not desensitized during prolonged TRH stimulation. Depletion of lipid 3H radioactivity by TRH occurred at higher TRH doses on addition of the competitive antagonist chlordiazepoxide. Addition of 1 microM-TRH after 3 h of stimulation by a sub-maximal (0.3 nM) TRH dose caused a further decrease in 3H radioactivity to the minimum level (40% of initial value). We propose that the TRH-responsive pool of Ins lipids in GH3 cells is composed of the complement of Ins lipids that are within functional proximity of activated TRH-receptor complexes.