Rapid increase in inositol pentakisphosphate accumulation by nicotine in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with nicotine (10 microM), a large and transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s, then declined to the basal level at 2 min. Nicotine also induced [3H]inositol tetrakisphosphate (InsP4) and [3H]inositol hexakisphosphate (InsP6) accumulation with a slower time course and a lesser magnitude than [3H]InsP5. The peaks of [3H]InsP4, [3H]InsP5 and [3H]InsP6 coincided with those of 32P radioactivity, when cells were doubly labeled with [3H]inositol and inorganic 32P. These results suggest that inositol pentakisphosphate is rapidly increased by nicotine, a cholinergic agonist, in cultured adrenal chromaffin cells.     

Inositol trisphosphate accumulation by high K+ stimulation in cultured adrenal chromaffin cells

Stimulation with high K+ (KCl, 56 mM) of myo-[3H]inositol-prelabelled cells increased Ca2+ uptake and [3H]inositol trisphosphate (IP3) accumulation in a concentration-dependent manner. Nifedipine, a Ca2+ channel antagonist, inhibited high K+-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Furthermore, ionomycin (1 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation. These results indicate the existence of the Ca2+ uptake-triggered mechanism of IP3 formation in cultured adrenal chromaffin cells.     

Calcium uptake-dependent and -independent mechanisms of inositol trisphosphate formation in adrenal chromaffin cells: comparative studies with high K+, carbamylcholine and angiotensin II

When [3H]inositol prelabelled cultured bovine adrenal chromaffin cells were stimulated with 56 mM KCl (high K+), 300 microM carbamylcholine (CCh) or 10 microM angiotensin II (Ang II), a rapid accumulation of [3H]IP3 was observed. At the same time, high K+ or CCh induced rapid increases in 45Ca2+ uptake, but Ang II did not induce a significant 45Ca2+ uptake. The concentration-response curve for KCl-induced [3H]IP3 accumulation coincided well with that for KCl-induced 45Ca2+ uptake into the cells. Nifedipine, a Ca2+ channel antagonist, inhibited the high K(+)-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Nifedipine at a similar concentration range also inhibited CCh-induced 45Ca2+ uptake. Although nifedipine inhibited CCh-induced [3H]IP3 accumulation, the potency was approximately 300-fold less than that for the inhibition of 45Ca2+ uptake. Nifedipine failed to affect the Ang II-induced [3H]IP3 accumulation. BAY K 8644 (2 microM), a Ca2+ channel activator, plus partially depolarizing concentration of KCl (14 mM), induced 45Ca2+ uptake and [3H]IP3 accumulation. Ionomycin (1 microM and 10 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation in a concentration-dependent manner. Pretreatment of the cells with protein kinase C activator, 100 nM 12-O-tetradecanoyl phorbol-13-acetate, for 10 min, partially inhibited CCh and Ang II-induced [3H]IP3 accumulation, but failed to inhibit the high K(+)-induced accumulation. Furthermore, the effects of high K+ and Ang II on the IP3 accumulation was additive. Ang II and CCh induced a rapid and transient increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) accumulation (5 s) followed by a slower accumulation of inositol 1,3,4-trisphosphate (1,3,4-IP3). High K+ evoked an increase in 1,3,4-IP3 accumulation but obvious accumulation of 1,4,5-IP3 could not be detected. In Ca2(+)-depleted medium, high K(+)-induced [3H]IP3 accumulation was completely abolished, whereas [3H]IP3 accumulation induced by CCh and Ang II was partially inhibited. These results demonstrate the existence of the Ca2+ uptake-triggered mechanism of IP3 accumulation represented by high K+, and also the Ca2+ uptake-independent mechanism of IP3 accumulation represented by Ang II in cultured bovine adrenal chromaffin cells. Mechanism of CCh-induced IP3 accumulation has an intermediate property between those of high K+ and Ang II.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Stimulus-responsive and rapid formation of inositol pentakisphosphate in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with high K+ (56 mM) and nicotine (10 microM), a large and transient increase in [3H]inositol 1,3,4,5,6-pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s and then declined to the basal level at 2 min. The time course of accumulation of InsP5 was parallel to that of [3H]inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Angiotensin II (Ang II) (10 microM) rapidly accumulated InsP5, but the level was sustained for 2 min. With a slower time course and a lesser amount than InsP5, high K+, nicotine, and Ang II caused an accumulation of [3H]inositol 1,3,4,5-tetrakisphosphate and [3H]inositol hexakisphosphate. Veratridine (100 microM), maitotoxin (10 ng/ml), ATP (30 microM), platelet-derived growth factor (10 ng/ml), and endothelin (10 ng/ml) also induced the InsP5 accumulation. High K+, nicotine, veratridine, and maitotoxin induced an increase in 45Ca2+ uptake, whereas Ang II, ATP, platelet-derived growth factor, and endothelin did not cause 45Ca2+ uptake. Nifedipine, a calcium channel antagonist, inhibited the high K(+)-induced InsP5 accumulation but failed to affect the Ang II-induced InsP5 accumulation. In an EGTA-containing and Ca2(+)-depleted medium, the high K(+)-induced InsP5 accumulation was completely inhibited, whereas the InsP5 accumulation induced by Ang II was not significantly inhibited. 12-O-tetradecanoylphorbol-13-acetate inhibited partially the Ang II-induced InsP5 accumulation but failed to inhibit the high K(+)-induced accumulation. In those experiments, the changes of InsP5 accumulation were closely correlated to those of Ins(1,4,5)P3. In the chromaffin cell homogenate, [3H] Ins(1,4,5)P3 was converted eventually to [3H]InsP5 through [3H]inositol 1,3,4,6-tetrakisphosphate. Taken together, the above results suggest that InsP5 is rapidly formed by a variety of stimulants and that the formation of InsP5 may occur through two mechanisms, i.e. Ca2+ uptake-dependent and Ca2+ uptake-independent ones in cultured adrenal chromaffin cells.     

Calcium-Dependent Nerve Growth Factor-Stimulated Hydrolysis of Phosphoinositides in PC12 Cells

The effect of nerve growth factor (NGF) on the hydrolysis of phosphoinositides in PC12 cells was examined. Addition of NGF to PC12 cells prelabeled with [3H]-inositol resulted in an increase in the formation of labeled inositol trisphosphate ([3H]IP3), inositol bisphosphate ([3H]IP2), and inositol monophosphate ([3H]IP), an observation indicating that NGF stimulated hydrolysis of the polyphosphoinositides. The increase in these inositol phosphates was detected as early as 15 s after addition of NGF. In the presence of LiCl, the accumulation of [3H]IP was linear for at least 20 min. The NGF-stimulated accumulation of [3H]IP was dose-dependent with a Kact of 0.17 nM and was dependent on the presence of extracellular calcium. In a calcium-free buffer containing EGTA, the NGF-dependent increase in accumulation of [3H]IP was not seen, and the basal level of [3H]IP accumulation was lower than that observed in the presence of extracellular calcium. Lanthanum inhibited both the basal and NGF-stimulated accumulation of [3H]IP, whereas the calcium ionophore A23187, in the absence of NGF, stimulated an accumulation of [3H]IP. The maximal accumulation of [3H]IP in the presence of A23187 was the same as that observed in the presence of NGF. Incubation of the cells with both A23187 and NGF resulted in an accumulation of [3H]IP that was not significantly different from the effect of either agent alone. These results suggest that NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells and that this NGF-stimulated hydrolysis of phosphoinositides occurs by a calcium-dependent mechanism.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.     

Luteinizing hormone-releasing hormone enhances polyphosphoinositide breakdown in rat granulosa cells

A 2-min addition of LHRH to [3H]inositol-prelabeled rat granulosa cells in primary culture evoked significant increases in the accumulation of [3H]inositol phosphates, i.e. radiolabeled inositol monophosphate (IP), inositol diphosphate (IP2), and inositol triphosphate (IP3) levels increased to 210, 590 and 520%, respectively, when compared to control cultures. By contrast, addition of FSH failed to elicit such a response. The effect of LHRH was completely blocked by the concomitant presence of a specific LHRH antagonist. LHRH evoked increase in [3H]IP3 and [3H]IP2 accumulation as early as 30 sec, while the increase in [3H]IP became significant at 2 min. These data support the hypothesis that polyphosphoinositide breakdown may be an early step in the intracellular signal mechanism which mediates the action of LHRH.     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinizing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells

The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.     

Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+-dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

Angiotensin-induced formation and metabolism of inositol polyphosphates in bovine adrenal glomerulosa cells

The actions of angiotensin II (AII) on inositol polyphosphate production and metabolism were analyzed in cultured bovine adrenal glomerulosa cells. In cells labeled for 24 hr with [3H]inositol, AII caused a rapid and prominent rise in formation of Ins-P3 (mainly the Ins-1,3,4,-P3 isomer) and of Ins-P4, with marked increases in two isomers of Ins-P2 and Ins-P. These findings are consistent with rapid formation and turnover of Ins-1,4,5-P3, partly via conversion to Ins-1,3,4,5-P4 with subsequent metabolism to Ins-1,3,4-P3 and lower inositol phosphates. The demonstration of a cytosolic Ins-P3-kinase gave further evidence for the presence of the tris/tetrakisphosphate pathway and Ins-P4 synthesis during AII action in the bovine adrenal cortex.     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.     

Binding sites for inositol trisphosphate in the bovine adrenal cortex

Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.     

Structures and metabolism of inositol tetrakisphosphates and inositol pentakisphosphate in bovine adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II stimulates rapid increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), followed by slower increases in two additional inositol tetrakisphosphate (InsP4) isomers. One of these InsP4 isomers was previously identified as Ins-1,3,4,6-P4 and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4 isomer, purified from cultured bovine adrenal cells labeled with [3H]inositol and stimulated by angiotensin II, revealed that the polyol produced by periodate oxidation, borohydrate reduction, and dephosphorylation was [3H]iditol. This finding is consistent with precursor structures of either Ins-1,4,5,6-P4 or Ins-3,4,5,6-P4 (= L-Ins-1,4,5,6-P4) for the third InsP4 isomer. The [3H]iditol was readily converted to [3H]sorbose by the stereospecific enzyme, L-iditol dehydrogenase, indicating that it originated from Ins-3,4,5,6-P4. Chicken erythrocytes labeled with [3H]inositol also contained high levels of Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4, as well as InsP5, but only small amounts of Ins-1,3,4,5-P4. Both [3H]Ins-1,3,4,6-P4 and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5 in permeabilized bovine glomerulosa cells. In addition, InsP5 itself was slowly dephosphorylated to Ins-1,4,5,6-P4, indicating that its structure is Ins-1,3,4,5,6-P5. These results demonstrate that the higher inositol phosphates are metabolically interrelated and are linked to the receptor-regulated InsP3 response by the conversion of Ins-1,3,4-P3 through Ins-1,3,4,6-P4 to Ins-1,3,4,5,6-P5. The source of Ins-3,4,5,6-P4, the other precursor of InsP5, is not yet known but its elevation in angiotensin II-stimulated glomerulosa cells suggests that its formation is also influenced by agonist-regulated processes.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

Gonadotropin releasing hormone enhances polyphosphoinositide hydrolysis in rat pituitary cells

Addition of gonadotropin releasing hormone to myo-[2-3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate within 30 sec of stimulation, followed by a rise in inositol diphosphate and inositol monophosphate. Inositol phosphate accumulation was enhanced up to 5-to-8-fold and was time-dependent between up to 15 min incubation without further increase beyond this time period. Without preincubation with LiCl2, there was no measurable increase of GnRH-induced inositol phosphate accumulation compared to controls. The presence of calcium in the incubation medium did not affect the increase of inositol phosphates. These data give evidence, that polyphosphoinositide breakdown may be an early step in the action of gonadotropin releasing hormone on gonadotropin secretion.