Effects of ras-encoded proteins and platelet-derived growth factor on inositol phospholipid turnover in NRK cells.

The role of ras-encoded proteins and platelet-derived growth factor (PDGF) in inositol phospholipid metabolism has been studied. PDGF stimulates inositol phospholipid turnover in confluent normal rat kidney (NRK) cells and enhances hydrolysis of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in NRK cell membranes in the presence of guanosine 5'-[gamma-thio]triphosphate. The stimulatory effect of PDGF on phosphatidylinositol bisphosphate hydrolysis is not inhibited by pretreatment of NRK cells with pertussis toxin, implying that PDGF-stimulated phospholipase C activity of NRK cells is regulated by a pertussis toxin-insensitive guanine nucleotide-binding protein (G protein) that is different from Gi (inhibitory G protein) or Go (G protein of unknown function). When bacterially made human normal or oncogenic T24 ras protein is added to 32P-labeled NRK cell membranes in the presence of guanosine 5'-[gamma-thio]triphosphate, normal ras protein increases by 3-fold the formation of inositol trisphosphate, whereas T24 ras protein has no significant effect. In addition, normal ras protein and PDGF have additive effects on inositol trisphosphate production. Taken together, these data suggest that normal ras protein stimulates inositol phospholipid turnover in NRK cells by means of a pathway different from the PDGF-regulated one and that oncogenic ras protein is without significant stimulatory effect in this action.     

Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals.

Synaptosomes, purified from rat cerebral cortex, were prelabeled with [3H]inositol to study phosphatidylinositol turnover in nerve terminals. Labeled synaptosomes were either depolarized with 40 mM K+ or exposed to carbamoylcholine (carbachol). K+ depolarization increased the level of inositol phosphates in a time-dependent manner. The inositol trisphosphate concentration increased rapidly and transiently, reaching maximum (250% of control) in less than 3 sec and returning to near basal levels by 30 sec. The inositol bisphosphate level also increased rapidly, but its elevated level (220% of control) was sustained during continued depolarization. The elevated level of inositol bisphosphate was reversed upon repolarization of the synaptosomes. The level of inositol monophosphate increased slowly to 120-130% of control. These effects of K+ depolarization depended on the presence of Ca2+ in the incubation medium. Carbachol stimulated the turnover of phosphatidylinositol in a dose- and time-dependent manner. The level of inositol trisphosphate increased only slightly (120-130% of control) during carbachol stimulation. The level of inositol bisphosphate increased to 210% of control, and this maximal response was seen from 15 to 60 min. Accumulation of inositol monophosphate (250% of control) was larger than that of inositol bisphosphate, but its time course was slower. Atropine and pirenzepine inhibited the carbachol effect with high affinities of 0.8 nM and 16 nM, respectively, indicating that the effect of carbachol was mediated by activation of a M1 muscarinic receptor. Incubation of synaptosomes in Ca2+-free buffer reduced the response to carbachol by 30%, and addition of EGTA abolished it. These data show that both Ca2+ influx and M1 muscarinic receptor activation stimulate phospholipase C activity in synaptosomes, suggesting that phosphatidylinositol turnover may be involved in regulating neurotransmitter release from nerve terminals.     

Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene.

Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.     

Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart

In this study, we examined phosphoinositide metabolism during ischemia and reperfusion using an isolated and perfused rat heart. When myocardial phosphoinositides were prelabeled with [3H]inositol, reperfusion after 30 minutes of normothermic global ischemia resulted in significant accumulations of radiolabeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly in the heart reperfused with [3H]inositol after 30 minutes of ischemia compared with that perfused with [3H]inositol after 30 minutes of nonischemic perfusion. However, isotopic incorporation of [3H]glycerol into diacylglycerol, phosphatidic acid, and all of the three phosphoinositides was diminished in the reperfused hearts. Reperfusion of the ischemic heart prelabeled with [14C]arachidonic acid resulted in significant increases in [14C]diacylglycerol and [14C]phosphatidic acid. The enhanced accumulations of [3H]inositol phosphates during reperfusion were not affected by treatment with prazosin plus atropine or indomethacin, but were inhibited by hypoxic reperfusion, reperfusion with Ca2+-free buffer, or by mepacrine. These results suggest that myocardial reperfusion stimulates phosphodiesteratic breakdown and turnover of phosphoinositides, and increased Ca2+ influx caused by reperfusion may be involved in the mechanism of stimulation of phosphatidylinositol-specific phospholipase C activity in the rat heart.     

Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH

GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2-3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone-stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH-treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release.     

Light-stimulated inositolphospholipid turnover in Samanea saman leaf pulvini

Leaflets of Samanea saman open and close rhythmically, driven by an endogenous circadian clock. Light has a rapid, direct effect on the movements and also rephases the rhythm. We investigated whether light signals might be mediated by increased inositolphospholipid turnover, a mechanism for signal transduction that is widely utilized in animal systems. Samanea motor organs (pulvini) labeled with [³H]inositol were irradiated briefly (5-30 sec) with white light, and membrane-localized phosphatidylinositol phosphates and their aqueous breakdown products, the inositol phosphates, were examined. After a 15-sec or longer light pulse, labeled phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate decreased and their labeled metabolic products inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate increased, changes characteristic of inositolphospholipid turnover. We conclude that inositolphospholipid turnover may act as a phototransduction mechanism in Samanea pulvini in a manner that is similar to that reported in animal systems.     

Localization of phosphatidylinositol signaling components in rat taste cells: role in bitter taste transduction.

To assess the role of phosphatidylinositol turnover in taste transduction we have visualized, in rat tongue, ATP-dependent endoplasmic reticular accumulation of 45Ca2+, inositol 1,4,5-trisphosphate receptor binding sites, and phosphatidylinositol turnover monitored by autoradiography of [3H]cytidine diphosphate diacylglycerol formed from [3H]cytidine. Accumulated 45Ca2+, inositol 1,4,5-trisphosphate receptors, and phosphatidylinositol turnover are selectively localized to apical areas of the taste buds of circumvallate papillae, which are associated with bitter taste. Further evidence for a role of phosphatidylinositol turnover in bitter taste is our observation of a rapid, selective increase in mass levels of inositol 1,4,5-trisphosphate elicited by low concentrations of denatonium, a potently bitter tastant.     

Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe

Previous studies have demonstrated that TRH is a potent stimulator of alpha-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked alpha-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol. TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of alpha-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/l respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of alpha-MSH release from perifused neurointermediate lobes but did not impair TRH-induced alpha-MSH secretion. In the presence of LiCl, TRH (1 mumol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (-70%). These results indicate that, in frog pars intermedia, TRH-evoked alpha-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.     

Increased inositol phosphate accumulation in platelets from patients with NIDDM.

We evaluated thrombin-induced inositol phosphate accumulation in [3H]inositol-labeled platelets prepared from patients with non-insulin-dependent diabetes mellitus. There were no significant differences in [3H]inositol incorporation into and contents of phosphoinositides between the diabetic patients and their age-matched control subjects. Thrombin induced a dose- and time-dependent accumulation of inositol phosphate. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate by thrombin stimulation were significantly enhanced in platelets from the diabetic patients, although the accumulation of [3H]inositol monophosphate did not differ between the diabetic patients and the control subjects. In addition, the platelet aggregation rate induced by thrombin was also significantly enhanced in the diabetic patients in correlation with the enhanced inositol phosphate accumulation. These results suggest that increased inositol phosphate accumulation may cause accelerated platelet functions in diabetes mellitus.     

Effect of growth hormone-releasing factor on phosphoinositide hydrolysis in somatotrophs

We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.     

Relationship of polyphosphoinositide metabolism to the hormonal activation of the inset salivary gland by 5-hydroxytryptamine

Incubation of the insect gland with [3H]inositol results in the incorporation of label into both phosphatidylinositol (PI) and the two polyphosphoinositides (PIP and PIP2). Upon stimulation with 5-HT the initial water-soluble metabolites released are inositol trisphosphate and inositol bisphosphate with no change in the level of inositol monophosphate, suggesting that the primary lipid substrate used by the receptor is one of the polyphosphoinositides (most likely PIP2) rather than PI. This conclusion was substantiated by showing that 5-HT was not able to release inositol or inositol monophosphate when the levels of the two polyphosphoinositides were reduced by lowering the level of ATP. The rate of breakdown of the polyphosphoinositides, as measured by the appearance of inositol phosphates, occurred with no apparent lag whereas the onset of the calcium-dependent change in transepithelial potential had a latency of approximately 1 sec. It is concluded that the primary action of 5-HT is to stimulate the hydrolysis of PIP2 into diacylglycerol and inositol trisphosphate. The latter may function as a second messenger to mobilize the calcium responsible for initiating some of the ionic events responsible for fluid secretion.     

Adrenocorticotropic hormone inhibits angiotensin II-stimulated inositol phosphate accumulation in rat adrenal glomerulosa cells

Rat adrenal glomerulosa cells labelled for 18 h with [3H]inositol responded to angiotensin II with a dose-dependent stimulation of the accumulation of inositol monophosphate, inositol bisphosphate and inositol trisphosphate. Addition of adrenocorticotropic hormone (ACTH) (10(-7)M) reduced the maximum responses without altering the EC50 values for angiotensin II. Thus, ACTH acted as a non-competitive inhibitor with respect to angiotensin II. No inhibition was observed in cells labelled for 2 h with [3H]inositol. Detailed examination of the inhibition showed that ACTH(1-24) was the most potent inhibitor, with ACTH(1-39) being 10-fold less potent. A mixture of alpha-melanocyte-stimulating hormone (alpha-MSH) (ACTH(1-13] and corticotropin-like intermediate lobe peptide (ACTH(18-39] was similarly inactive. ACTH(5-24) did not produce detectable inhibition. In terms of specificity, the receptor mediating ACTH inhibition of phosphatidylinositol turnover was similar to the receptor which mediated stimulation of aldosterone synthesis. Inhibition by ACTH was additive with inhibition produced by dibutyryl cAMP demonstrating that it was not mediated by rises in intracellular cAMP. ACTH inhibition also was additive with inhibition by the calcium channel blocker, nifedipine. These results demonstrate an interaction between ACTH receptors and angiotensin II receptors in adrenal glomerulosa cells at the level of their receptor-second messenger pathways.     

Permissive Role of Calcium in α1-Adrenergic Stimulation of Pineal Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity

Activation of alpha 1-adrenergic receptors increases [Ca+2]i and phosphatidylinositol phosphodiesterase (phospholipase C) activity in the pinealocyte. In this report the receptor involved in the stimulation of phospholipase C activity was further characterized, and the role of Ca2+ in this effect was investigated in some detail. Phospholipase C activity was estimated by measuring the production of [3H]inositol phosphates by [3H]inositol-labelled dispersed pinealocytes in suspension culture. Norepinephrine stimulated [3H]inositol monophosphate production severalfold; this was blocked by alpha 1-adrenergic antagonists, including prazosin, WB 4101, and phenoxybenzamine, but by neither an alpha 2- nor a beta-adrenergic antagonist, confirming that an alpha 1-adrenoceptor is involved in the regulation of phosphatidylinositol hydrolysis. Treatment with the Ca2+ chelator, EGTA, or with inorganic Ca2+ blockers, including Co2+, Mn2+, and La3+, reduced the norepinephrine-stimulated response, suggesting that the alpha 1-adrenergic stimulation of phospholipase C activity is Ca2+ dependent. However, phospholipase C activity was not increased by elevating intracellular Ca2+ with either the Ca2+ ionophore A23187 or with depolarizing concentrations of K+. These results indicate that although Ca2+ is necessary for alpha 1-adrenergic stimulation of phospholipase C activity, an increase in [Ca2+]i alone is not sufficient to stimulate the activity of this enzyme, and that effects which A23187 and depolarizing concentrations of K+ have on pineal function probably do not involve stimulation of phospholipase C activity.     

Regulation of cytosolic pH in bovine parathyroid cells: effect of fluoride

In the present investigation, sodium fluoride (NaF) was employed to explore the role of guanine nucleotide-binding proteins (G-proteins), protein kinase-C, or cytosolic calcium [( Ca]i) in the regulation of cytosolic pH [( pH]i) in dispersed bovine parathyroid cells, using the pH-sensitive fluorescent dye BCECF. When cells acidified by nigericin in Na-free medium were resuspended in Na-containing buffer, [pH]i returned to basal levels. This recovery was blocked by continued removal of Na+ or the addition of amiloride. NaF (10 mM) increased [32P]phosphate incorporation into phosphatidylinositol bisphosphate, suggesting an increase in phosphatidylinositol bisphosphate turnover. NaF caused an initial acidification, followed by an alkaline recovery in a dose-dependent manner (1-10 mM). Amiloride blocked the NaF-induced alkaline recovery. The protein kinase-C activator phorbol 12-myristate 13-acetate (10(-7) M) caused cytosolic alkalinization, while the protein kinase-C inhibitor H7 (6 x 10(-5) M) significantly inhibited the NaF-induced alkaline recovery. Pertussis toxin (1 microgram/ml) did not affect the NaF-induced changes in [pH]i. Removal of extracellular Ca2+ with EGTA blocked the NaF-induced increase in [Ca]i and alkaline recovery. Ionomycin (5 x 10(-7) M) caused cytosolic alkalinization, but pretreatment with EGTA inhibited the ionomycin-induced cytosolic alkalinization. The present studies clearly demonstrated the presence of an amiloride-sensitive Na+/H+ exchanger in parathyroid cells. Our findings suggest that the NaF-induced cytosolic alkaline recovery was via two complementing pathways: 1) activation of protein kinase-C, followed by stimulation of a Na+/H+ exchanger, and 2) existence of extracellular calcium and/or an increase in [Ca]i.     

Antagonism of contractants and relaxants at the level of intracellular calcium and phosphoinositide turnover in the rat uterus

The effects of the uterine relaxants relaxin and isoproterenol on intracellular free calcium and inositol phosphate formation were investigated in rat myometrium. Preincubation of fura-2-loaded myometrial cell suspensions with relaxin and isoproterenol inhibited the oxytocin-induced stimulation of intracellular free calcium, with EC50 values of 0.02 and 1.0 microM, respectively. Pretreatment of cells with pertussis toxin or replacement of extracellular calcium with 2 mM EGTA inhibited the oxytocin-induced increase in intracellular calcium by 47% and 50%, respectively, but did not inhibit the action of relaxin. (Bu)2cAMP and forskolin also inhibited the effect of oxytocin on intracellular calcium. In uterine strips prelabeled with [3H]inositol, oxytocin stimulated a dose-dependent accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, with and EC50 of 0.38 microM, and pertussis toxin inhibited this effect. Relaxin, isoproterenol, chlorophenylthio-cAMP, and forskolin inhibited the oxytocin-stimulated formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. The effect of relaxin on inositol trisphosphate formation was dose dependent, with an EC50 of 0.1 microM. Relaxin and isoproterenol also inhibited inositol phosphate formation in myometrial cells. These data demonstrate the attenuation of contractant-induced elevation in myometrial intracellular calcium and phosphoinositide turnover by uterine relaxants and suggest that these actions may be related. In addition, they provide additional evidence that cAMP-mediated mechanisms may be involved in mediating uterine relaxation.     

The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators

The coupling between GnRH-stimulated phosphoinositide (PI) turnover and LH release has been investigated in rat pituitary cell cultures. Accumulation of [3H]inositol phosphates ([3H]IPs) formed by hydrolysis of PIs was measured in cells that had been preloaded with [3H]myo-inositol. GnRH stimulated both LH release and incorporation of [3H]inositol into total [3H]IPs with similar dose and time dependencies. [3H] IP production in response to GnRH could be blocked by a GnRH antagonist, but was stimulated by a compound that provokes receptor microaggregation. GnRH-stimulated IP production persisted in the presence of either the Ca2+ channel blocker D600 or the calmodulin antagonist pimozide at concentrations that reduced LH release to 60% and 20% of control, respectively. Stimulated [3H]IP production was inhibited at higher concentrations of D600. In 1-h incubations, GnRH-stimulated [3H]IP production, but not LH release, was markedly inhibited by the protein kinase C activators phorbol myristate acetate and 1,2-dioctanoylglycerol. These findings indicate that in the gonadotrope, GnRH-stimulated LH release and [3H]IP production are closely coupled to receptor activation by an agonist; Ca2+ antagonists uncouple stimulated LH release from [3H]IP production; and protein kinase C activators uncouple stimulated PI turnover from LH release. Thus, GnRH-stimulated production of PI metabolites, as measured by [3H]IP accumulation, is apparently not sufficient to support LH release in the absence of Ca2+. In addition, GnRH-stimulated LH release is apparently not dependent on full expression of the PI response.     

Angiotensin II-stimulated phosphatidylinositol turnover in rat adrenal glomerulosa cells has a complex dependence on calcium

The stimulation of phosphatidylinositol (PI) turnover by angiotensin II in rat adrenal glomerulosa cells has been studied in detail and shown to have a complex dependence on Ca2+. After the addition of angiotensin II, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate increased rapidly and transiently. The transient increase was followed by a slower sustained rise, which continued for up to 30 min. Inositol phosphate accumulation during the sustained phase was decreased when experiments were performed in Ca2+-free medium. The initial transient response was not altered. Addition of the Ca2+ ionophore A23187 enhanced the angiotensin II response at 20 min, but not the 15 sec response. The sustained response, but not the transient response, was attenuated by the Ca2+ channel blocker nifedipine, indicating that the effect of Ca2+ required uptake through voltage-dependent Ca2+ channels. Subsequent studies showed that cAMP decreased inositol phosphate accumulation at 20 min while having no effect at 15 sec. Also, incubation with phorbol 12-myristate 13-acetate produced a more effective inhibition of the sustained response than of the initial transient response. However, while the transient and sustained phases of PI turnover were differently affected by Ca2+ and inhibitory compounds, the profiles of inositol phosphates generated were similar. At both 15 sec and 20 min inositol-(1,4,5) trisphosphate was detected, indicating sustained cleavage of PI-(4,5) bisphosphate. Taken together, the results suggest that while the initial PI turnover response is independent of Ca2+ and presumably initiates the rise in cytosolic Ca2+, sustained response requires entry of Ca2+ to maintain elevated cytosolic Ca2+ concentrations. Thus, while the increase in cytosolic Ca2+ may have a direct role in the stimulation of aldosterone synthesis, it is also required to sustain the PI turnover response to angiotensin II.     

Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.     

Multiple signaling pathways control stimulus-secretion coupling in rat peritoneal mast cells.

Fura-2 and membrane capacitance measurements were performed to investigate intracellular Ca2+ concentration [( Ca2+]i) and secretory responses of rat peritoneal mast cells following secretagogue stimulation. Compound 48/80 and internally applied guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) induced transient rises in [Ca2+]i and caused membrane capacitance increases as secretion occurred. The 48/80-induced Ca2+ transients and secretory responses were blocked by guanosine 5'-[beta-thio]diphosphate and neomycin, indicating that inositolphospholipid breakdown mediated by guanine nucleotide-binding regulatory protein (G protein) plays an important role in stimulus-secretion coupling. However, pertussis toxin did not block Ca2+ transients induced by 48/80 or GTP[gamma-S], whereas secretory responses were either abolished (48/80) or developed only after a considerable delay (GTP[gamma-S]). Similar effects were obtained by perfusing cells with cAMP: (i) Ca2+ transients following stimulation with 48/80 remained unaffected by cAMP, but secretory responses were abolished; (ii) GTP[gamma-S] induced normal Ca2+ transients and degranulation in the presence of cAMP. Pretreatment of mast cells with phorbol 12-myristate 13-acetate (PMA) abolished 48/80- and GTP[gamma-S]-induced Ca2+ transients (but not inositol trisphosphate-induced Ca2+ transients), whereas secretion still occurred. At the same time, the Ca2+ requirement for secretion was reduced by PMA. These results indicate that secretion in mast cells is under control of an as yet unidentified signaling pathway that involves a G protein. This pathway is distinct from inositolphospholipid turnover and may provide the triggering mechanism for secretion, whereas the inositolphospholipid pathway serves to increase [Ca2+]i and renders the secretory process more sensitive to [Ca2+]i by activating protein kinase C. Persistent activation of protein kinase C through phorbol ester imposes negative feedback control on the inositolphospholipid pathway, whereas cAMP may inhibit the unidentified signaling pathway.     

Cholinergic stimulation of phosphoinositide hydrolysis in rat anterior pituitary

The production of inositol phosphates in response to carbachol was studied in rat anterior pituitary tissue prelabelled with [3H]inositol. Carbachol (10 microM) stimulated inositol mono-, bis- and trisphosphate production (IP1, IP2 and IP3) by 360 +/- 49, 338 +/- 49 and 503 +/- 49 (mean +/- SEM, P less than 0.001) percent respectively during a 30 min incubation. Mean basal production was 5.4 +/- 0.3, 4.1 +/- 0.5 and 0.9 +/- 0.3 expressed as a percent of total [3H]inositol lipid for IP, IP2 and IP3 respectively. Stimulated inositol phosphate production was dose dependent and detectable after 5 min. Atropine prevented this stimulation indicating mediation via muscarinic receptors. Removal of extracellular Ca2+ reduced both basal and stimulated total inositol phosphate production by 60% and 56% respectively but did not impair carbachol-induced phosphoinositide hydrolysis per se. Pretreatment of pituitary tissue with either somatostatin (5 micrograms/ml) or pertussis toxin (1 microgram/ml) had no effect on either basal or stimulated inositol phosphate production. These results demonstrate a cholinergic stimulation of phosphatidylinositol bisphosphate (PIP2) hydrolysis in the anterior pituitary which may be important in the action of cholinergic agonists on pituitary hormone secretion.