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.     

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.     

Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF.

The effects of low density lipoprotein (LDL) and high density lipoprotein (HDL3) on second messenger systems were investigated in cultured human vascular smooth muscle cells (VSMC) and compared with those of angiotensin II (Ang II) and platelet-derived growth factor (PDGF-BB). Phosphoinositide metabolism was studied in myo-[2-3H]-inositol prelabelled VSMC using high performance liquid anion-exchange chromatography. The spectra of inositol phosphate isomers increased after stimulation with either Ang II, LDL, HDL3 or PDGF-BB were qualitatively identical. Major increases occurred in 4-IP1, 1,4-IP2, 1,3,4-IP3 and 1,3,4,5-IP4. These are metabolic conversion products of 1,4,5-IP3 for which only a minor increase was found. Thus lipoproteins, like Ang II and PDGF-BB, activate polyphosphatidylinositol-specific phospholipase C. Intracellular Ca2+ concentrations ([Ca2+]i) were studied in fura-2 loaded VSMC. In monolayer cultures LDL and HDL3 increased [Ca2+]i with kinetics comparable to those for Ang II. Relative to the effects of these agonists, the PDGF-BB-induced increase in [Ca2+]i was slower in onset and the decay from peak [Ca2+]i levels more gradual. Fluorescence recordings from single cells exposed to LDL and HDL3 revealed a prolonged series of transient oscillations of [Ca2+]i, a phenomenon typical for calcium-mobilizing hormones. Additionally, as found for Ang II, preincubation of VSMC with either phorbol 12-myristate, 13-acetate, forskolin or 8-bromo-cyclic GMP inhibited LDL- and HDL-induced accumulation of [3H]-inositol monophosphate. We propose that LDL and HDL3 stimulate signal transduction in VSMC via mechanisms analogous to those of Ca(2+)-mobilizing hormones.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Muscarinic stimulation of inositol phosphate accumulation and acid secretion in gastric fundic mucosal cells

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time- and dose-dependent: EC50 values for IP1, IP2 and IP3 accumulation were not statistically different. The mean value was 30 +/- 8 microM (n = 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 microM CCh was 160 +/- 15%. CCh-induced IP accumulation was abolished by atropine (Ki = 0.32 +/- 0.18 nM (n = 3)). The CCh concentrations leading to half-maximal inhibition of N-[3H]methylscopolamine binding and half-maximal IP accumulation were similar. The half-maximal value for CCh-induced aminopyrine accumulation was 8-times lower. These results indicate that IP3-mediated mobilization of intracellular Ca2+ might be involved in CCh-induced acid secretion by parietal cells.     

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.     

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.     

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.     

The fucose-sulfate glycoconjugate that induces an acrosome reaction in spermatozoa stimulates inositol 1,4,5-trisphosphate accumulation

The hydrolysis of phosphatidylinositol may generate multiple second messengers, including inositol phosphates, 1,2-diacylglycerol, arachidonic acid, and phosphatidic acid. Here, we describe for the first time in spermatozoa that accumulation of one of these potential second messengers, inositol 1,4,5-trisphosphate (1,4,5-IP3), can be stimulated by the fucose-sulfate glycoconjugate (FSG) that induces an acrosome reaction. Sea urchin spermatozoa were labeled with myo-[3H]inositol and incubated with FSG. The amount of [3H]1,4,5-IP3 obtained from FSG-treated cells was up to 10 times that from untreated cells. Increases in the amount of [3H]1,4,5-IP3 were detected within 30 s after addition of FSG (2.5-fold) and were highest at 2 min after addition. Previously, it was shown that FSG induces Ca2+-dependent increases in cyclic AMP concentrations (Kopf, G. S., and Garbers, D. L. (1980) Biol. Reprod. 22, 1118-1126). Increases in [3H]1,4,5-IP3 accumulation caused by FSG were also dependent on extracellular Ca2+. The Ca2+ channel blockers, verapamil and nifedipine, inhibited increases in both [3H]1,4,5-IP3 and cyclic AMP, and the addition of concentrations of extracellular Ca2+ higher than 9.6 mM could reduce the inhibition. When spermatozoa were incubated in Ca2+-free seawater, FSG-induced increases in [3H]1,4,5-IP3 and cyclic AMP concentrations were blocked; addition of extracellular Ca2+ restored the responses. Other treatments that result in the induction of an acrosome reaction, including the addition of monovalent cation H+ exchangers, nigericin and gramicidin S, and incubation in seawater at alkaline pH (pH 8.8), also stimulated accumulation of [3H]1,4,5-IP3 and cyclic AMP.     

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.     

2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells

2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release in bi-directional Ca2+ -flux conditions. We have now studied the effects of 2APB on the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ efflux from the non-mitochondrial Ca2+ stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3 -induced Ca2+ release, with a half maximal inhibition at 36 microM 2APB, without affecting [3H]IP3 binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+ concentration. The Ca2+ pumps of the non-mitochondrial Ca2+ stores were half-maximally inhibited at 91microM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+ from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3 -induced Ca2+ release. Our results can explain the various effects of 2APB observed in intact cells.     

Differential effects of protein kinase C activators on carbamylcholine- and high K+-induced rises in intracellular free calcium concentration in cultured adrenal chromaffin cells

Pretreatment of adrenal chromaffin cells with protein kinase C activators, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 1-oleoyl 2-acetyl glycerol (OAG), partially inhibited carbamylcholine (CCh)-induced rise in intracellular free Ca2+ concentration ([Ca2+]i). The apparent IC50 values of TPA and OAG were 3 nM and 25 microM, respectively. The effect of TPA on the CCh-induced rise in [Ca2+]i was overcome by pretreatment of the cells with a protein kinase C inhibitor, 1-(5-isoquinidinesulfonyl)-2-methylpiperazine hydrochloride (H-7). In contrast, KCl-induced rise in [Ca2+]i was not affected by pretreating the cells with TPA or OAG. An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate failed to affect the CCh-induced rise in [Ca2+]i. CCh-induced 45Ca2+ uptake was also partially inhibited by pretreatment of the cells with TPA or OAG, but KCl-induced 45Ca2+ uptake was not affected by these pretreatments. These results indicate that protein kinase C activation causes an uncoupling of signal transduction between the nicotinic receptors and Ca2+ channels.     

Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.     

A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland

Electrically permeabilized cells of rat parotid gland, prelabelled with [3H]-inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H]-IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland.     

Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.     

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.     

Metabolism of inositol-1,4,5-trisphosphate in the taste organ of the channel catfish, Ictalurus punctatus.

1. The metabolism of inositol-1,4,5-trisphosphate was studied in the taste organ (barbel) of the channel catfish, Ictalurus punctatus. 2. Homogenates of epithelial barbel scrapings were incubated with [3H]-1,4,5-IP3, whose dephosphorylation or phosphorylation was assayed under first-order conditions by measuring the production of either [3H]-1,4-IP2 (representing the activity of IP3-5-phosphatase) or [3H]-1,3,4,5-IP4 (representing the activity of IP3-3-kinase). 3. Both enzymes were predominantly cytosolic, magnesium-dependent and maximally active at pH 6.4. For IP3-phosphatase, Km = 6 microM and Vmax = 10.5 nmol/min/mg. For IP3-kinase, Km = 0.23 microM and Vmax = 0.05 nmol/min/mg. 4. Neither enzyme was significantly affected by the presence of taste stimuli (amino acids), GTP gamma S, cAMP or phorbol esters. 5. In the presence of physiological levels of free calcium (0.05-12 microM) IP3-phosphatase was moderately activated whereas IP3-kinase was moderately inhibited. 6. IP3-phosphatase was moderately activated by Mn2+, unaffected by LiCl, and strongly inhibited by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, CuCl2, FeCl3 and ZnSO4 7. IP3-kinase was strongly activated by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, FeCl3 and LiCl and inhibited by ZnSO4 and Mn2+. 8. IP3-kinase was significantly activated in a calcium-dependent manner by exogenously-added phosphatidylcholine and sphingomyelin, and to a lesser extent by diacylglycerol. IP3-phosphatase was unaffected by exogenously-added lipids. 9. IP3-phosphatase may participate in taste transduction since calculations based on the first-order rate constant (6.9 sec-1) indicate that it is capable of dephosphorylating basal levels of IP3 with a half-life of 0.1 sec.