Formation and metabolism of [3H]inositol phosphates in AR42J pancreatoma cells. Substance P-induced Ca2+ mobilization in the apparent absence of inositol 1,4,5-trisphosphate 3-kinase activity

After 2 days of incubation of AR42J pancreatoma cells with 400 microM [3H]inositol, the specific radioactivity of [3H]phosphatidylinositol 4,5-bisphosphate and the specific radioactivity of [3H]inositol were similar, indicating that isotopic equilibrium had been achieved. The inositol 1,4,5-trisphosphate (1,4,5-IP3) level in cells was estimated to be approximately 2 microM and was increased by substance P receptor activation to about 25 microM. HPLC analysis of [3H]inositol phosphates indicated that only 1,4,5-IP3, inositol 1,4-bisphosphate, and inositol 4-monophosphate were increased upon receptor activation. There was no increase in inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4), or in any of its metabolites. Incubation of [3H]1,4,5-IP3 with a cell homogenate did not result in the formation of [3H]1,3,4,5-IP4. Therefore, it appears that 1,4,5-IP3 3-kinase is either not present or not functional under these assay conditions. Substance P increased cytosolic calcium levels in fura-2-loaded cells from about 600 nM to 2.5 microM. This increase in Ca2+ was partially attenuated in the absence of extracellular calcium, indicating that in AR42J cells, substance P stimulation appears to activate calcium signaling through both Ca2+ entry and intracellular Ca2+ release. These modes of Ca2+ mobilization occur without an increase in 1,3,4,5-IP4 or any of its metabolites.     

Activation of phospholipase D by the fucose-sulfate glycoconjugate that induces an acrosome reaction in spermatozoa

We report for the first time that phospholipase D activity in sea urchin spermatozoa can be regulated by a component of egg jelly known to induce an acrosome reaction. The fucose-sulfate glycoconjugate (FSG) of egg jelly that induces an acrosome reaction in spermatozoa caused Ca2+-dependent increases in 1,2-diacylglycerol and phosphatidic acid. Diacylglycerol concentrations were increased 2-fold, and phosphatidic acid concentrations were elevated up to 10-fold 2 min after the addition of FSG to spermatozoa. FSG also caused increases in choline, but not in choline phosphate concentrations. Neither phosphorylation of diacylglycerol nor de novo synthesis from glycerol were significant routes of synthesis of phosphatidic acid during the acrosome reaction. When spermatozoa were incubated with FSG in the presence of ethanol, phosphatidylethanol was produced. As ethanol concentrations in the extracellular medium were increased from 0.1 to 2.5%, the amount of phosphatidylethanol increased, whereas phosphatidic acid concentrations decreased, suggesting a competitive transphosphatidylation reaction catalyzed by phospholipase D. Furthermore, when a phosphatidylcholine pool in spermatozoa was radiolabeled using [3H]1-O-alkyl-2-lyso-glycerol-3-phosphorylcholine, the subsequent addition of FSG caused a 4-fold accumulation of [3H]phosphatidic acid. FSG-induced elevations in [3H]phosphatidic acid were positively correlated with the percent of cells that had undergone an acrosome reaction.     

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.     

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.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y-induced intracellular Ca2+ increases in vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+]i. All peptides induced dose-dependent and transient rises in [Ca2+]i which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]i rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+]i rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation. Although NPY attenuated forskolin-induced accumulation of cyclic AMP, forskolin- and 3-isobutyl-1-methyl-xanthine-induced alterations in intracellular cyclic AMP did not affect the NPY-induced [Ca2+]i rise. These results suggest that NPY increases [Ca2+]i by a pertussis toxin-sensitive GTP binding protein-involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.     

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.     

Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Receptor coupled events in bradykinin action: rapid production of inositol phosphates and regulation of cytosolic free Ca2+ in a neural cell line.

The addition of bradykinin to NG115-401L cells grown on coverslips results in the generation of rapid transient increases in intracellular [Ca2+] and inositol phosphates. Changes in intracellular Ca2+, measured using the fluorescent indicator dye Fura-2, show two components; an initial rapid peak in [Ca2+]i which is essentially independent of extracellular Ca2+, and a sustained plateau dependent on the presence of extracellular Ca2+. Analysis of bradykinin stimulated production of [3H]inositol phosphates, by h.p.l.c., shows a rapid biphasic production of inositol 1,4,5-trisphosphate, inositol tetrakisphosphate and inositol bisphosphates, followed by a sustained rise in inositol 1,3,4-trisphosphate production. Quantitative measurements have indicated the presence of other, more polar, [3H]inositol-labelled metabolites which do not show major changes on bradykinin stimulation. The initial phase of inositol phosphate production parallels the rapid transient increase in intracellular [Ca2+], however, the second phase of inositol phosphate production occurs when intracellular [Ca2+] is declining and implies a complex series of regulatory events following receptor stimulation. Similar time courses of inositol 1,4,5-trisphosphate and Ca2+ signals provides supporting evidence that inositol 1,4,5-trisphosphate is the second messenger coupling bradykinin receptor stimulation to release of Ca2+ from intracellular stores.     

Plasma from uninephrectomized rats stimulates production of inositol trisphosphates and inositol tetrakisphosphate in renal cortical slices.

Metabolism of inositol phosphates in renal cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats stimulated production of InsP3 (inositol trisphosphate) when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response of InsP3 production was obtained. This effect could be prevented by 0.1 microM-TPA (12-O-tetradecanoylphorbol 13-acetate). When analysis of inositol phosphates was performed by h.p.l.c., plasma from uninephrectomized rats stimulated a rapid increase in Ins(1,4,5)P3 radioactivity, whereas the increase in inositol 1,3,4,5-tetrakisphosphate and Ins(1,3,4)P3 radioactivity was slower. Plasma from uninephrectomized rats decreased cyclic AMP concentration in renal cortical slices. Similar effect was obtained when slices were incubated with TPA (0.05 microM). Plasma from uninephrectomized rats increased cyclic GMP concentration in renal cortical slices, but this effect was abolished when extracellular Ca2+ had been chelated with 4 mM-EGTA. Results indicate that plasma from uninephrectomized rats stimulates phospholipase C, increases cyclic GMP concentration and decreases cyclic AMP concentration in renal cortical slices. Increases in cyclic GMP depend on extracellular Ca2+, whereas the decrease in cyclic AMP concentration is mediated by protein kinase C.     

K+-Evoked Depolarization Stimulates Cyclic AMP Accumulation in Photoreceptor-Enriched Retinal Cell Cultures: Role of Calcium Influx Through Dihydropyridine-Sensitive Calcium Channels

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.     

Isomers of inositol trisphosphate in exocrine pancreas.

In rat pancreatic acinar cells, the Ca2+-mobilizing receptor-agonist, caerulein, at both maximal and submaximal concentrations, stimulated a rapid, transient, increase in [3H]inositol 1,4,5-trisphosphate [(1,4,5)IP3], followed by a slower, sustained, increase in [3H]inositol 1,3,4-trisphosphate [(1,3,4)IP3]. Neither activation of protein kinase C by phorbol dibutyrate nor prevention of the caerulein-stimulated elevation of cytosolic [Ca2+] significantly affected the pattern of formation of the two isomers of IP3. Although carbachol evoked an increase in cytosolic [Ca2+], it did not significantly stimulate [3H](1,4,5)IP3 accumulation, but did promote [3H](1,3,4)IP3 accumulation. Moreover, both carbachol and caerulein maintained hormone-sensitive intracellular Ca2+ pools in a Ca2+-depleted state after [3H](1,4,5)IP3 had returned to basal concentrations. One interpretation of these findings is that total cellular concentrations of [3H](1,4,5)IP3 may not accurately reflect the concentration of this putative mediator in biologically relevant compartments.     

Cyclic GMP Formation and Inositol Phosphate Accumulation Do Not Share Common Origins in Rat Brain Slices

Cyclic GMP formation and inositol phospholipid hydrolysis were studied in rat brain slices to determine if the two processes have common origins. Muscarinic cholinergic stimulation enhanced [3H]inositol phosphate ([ 3H]IP) accumulation from slices prelabelled with [3H]inositol but did not affect cyclic GMP formation in the cortex, striatum, or cerebellum. An elevated level of extracellular K+ stimulated accumulation of both cyclic GMP and [3H]IP in cortex slices. The former, but not the latter, was reduced by lipoxygenase and phospholipase A2 inhibition. Calcium channel activation enhanced and blockade reduced K+-stimulated [3H]IP formation without affecting the cyclic GMP level, and there were differences in the Ca2+ requirements for the two responses. Thus, there is no support for the concept that guanylate cyclase activation inevitably accompanies inositol phospholipid breakdown, and the evidence presented demonstrates that K+ stimulation promotes cyclic GMP and [3H]IP accumulation by different transducing pathways.     

The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages.

The haemopoietic growth factors interleukin 3 (IL-3) and colony stimulating factor-1 (CSF-1) stimulate the survival and proliferation of murine normal bone-marrow-derived macrophages. To establish whether these growth factors elicit their effects via the hydrolysis of phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form the second messengers inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and diacylglycerol, macrophages were labelled with tracer quantities of [3H]inositol. Treatment of these cells with either IL-3 or CSF-1 did not alter the levels of PtdIns(4,5)P2 or Ins(1,4,5)P3. However, addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) which does not stimulate proliferation in macrophages caused a marked and rapid increase in the levels of Ins(1,4,5)P3, inositol bisphosphate and inositol monophosphate, and a decrease in the amount of PtdIns(4,5)P2. FMLP also evoked a rapid increase in intracellular cytosolic Ca2+ levels, as measured with quin 2 the Ca2+-sensitive fluorescent probe, whereas IL-3 and CSF-1 had no such effect. These results suggest that FMLP stimulates the hydrolysis of PtdIns(4,5)P2 to form the second messenger Ins(1,4,5)P3 which acts to increase the levels of cytosolic Ca2+, and that IL-3- and CSF-1-stimulated proliferation in macrophages is not associated with the formation of PtdIns(4,5)P2-derived second messengers.     

Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.     

The role of cytosolic free calcium in the generation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in HL-60 cells. Differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.     

Regulation of inositol phosphate metabolism in chemoattractant-stimulated human polymorphonuclear leukocytes. Definition of distinct dephosphorylation pathways for IP3 isomers

The metabolism of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer was studied in myo-[3H]inositol labeled, chemoattractant-stimulated human polymorphonuclear neutrophils (PMNs), and in PMN lysates. It was determined that 1,4,5-IP3 is metabolized in vitro by two distinct pathways: 1) by sequential dephosphorylation to 1,4-IP2, 4-IP1, and inositol or 2) by ATP dependent conversion to 1,3,4,5-IP4, followed by dephosphorylation to form 1,3,4-IP3, 3,4-IP2, 3-IP1, and inositol. In PMNs stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), 1,4-IP2, 1,4,5-IP3, and IP4, were elevated by 5 s; whereas production of 1,3,4-IP3, 3,4-IP2, and IP1 occurred only after an initial lag (approximately 15 s). The predominant IP1 isomer formed in fMet-Leu-Phe-stimulated cells was 4-IP1. Production of 1,3,4-IP3 and 3,4-IP2 was markedly reduced (17 and 35% of control, respectively) in fMet-Leu-Phe-stimulated cells pretreated to prevent a rise in intracellular calcium ([Ca2+]i). PMNs were also stimulated with leukotriene B4 (LTB4) since this agent is a poor activator of the respiratory burst compared to fMet-Leu-Phe. Peak levels (5 s) of 1,4,5-IP3 were equivalent after stimulation with 0.1 microM fMet-Leu-Phe versus 0.1 microM LTB4 (320 +/- 38% versus 378 +/- 38% of control values, respectively; n = 5); however, at 30 s, 1,4,5-IP3 remained elevated only in fMet-Leu-Phe-stimulated cells. Similarly, elevation of [Ca2+]i was more prolonged in response to 0.1 microM fMet-Leu-Phe (greater than 3 min) versus LTB4 (1 min). Thus, signal transduction in PMNs may be modulated by both the duration of the initial 1,4,5-IP3 signal and by the metabolic pathway(s) utilized to convert this IP3 isomer to other, potentially active inositol phosphate products.     

Recovery of hepatocytes from attack by the pore former amphotericin B.

Factors underlying the transience of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation following muscarinic stimulation of RINm5F cells were examined. Transience was not due to a protein kinase C-mediated stimulation of Ins(1,4,5)P3 dephosphorylation, since pretreatment of cells with tetradecanoyl-phorbol acetate (TPA) did not alter the rate of this conversion. However, preincubation with TPA did inhibit carbamoylcholine-stimulated Ins(1,4,5)P3 formation. In permeabilized cells, the conversion of Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was slightly enhanced in the presence of TPA or cyclic AMP, but much more markedly by raising the Ca2+ concentration from 10(-7) M to 10(-6) or 10(-5) M. In intact cells the most rapid rate of accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 occurred in the first 2 s following stimulation, whereas the levels of inositol 1,4-bisphosphate were not increased until after 5 s. This suggests that Ins(1,4,5)P3 kinase is chiefly responsible for the early disposal of Ins(1,4,5)P3 following cellular stimulation. The results are consistent with the proposal that the transient accumulation of Ins(1,4,5)P3 is due both to its enhanced metabolism via the Ca2+-calmodulin-sensitive Ins(1,4,5)P3 kinase, as well as a down-regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis.     

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.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.