CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.     

Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate.

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.     

Antigen-stimulated metabolism of inositol phospholipids in the cloned murine mast-cell line MC9.

Cells of the murine mast-cell clone MC9 grown in suspension culture were sensitized with an anti-DNP (dinitrophenol) IgE and subsequently prelabelled by incubating with [32P]Pi. Stimulation of these cells with DNP-BSA (bovine serum albumin) caused marked decreases in [32P]polyphosphoinositides (but not [32P]phosphatidylinositol) with concomitant appearance of [32P]phosphatidic acid. Whereas phosphatidylinositol monophosphate levels returned to baseline values after prolonged stimulation, phosphatidylinositol bisphosphate levels remained depressed. Stimulation of sensitized MC9 cells with DNP-BSA increased rates of incorporation of [32P]Pi into other phospholipids in the order: phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine. In sensitized cells prelabelled with [3H]inositol, release of inositol monophosphate, inositol bisphosphate and inositol trisphosphate, was observed after stimulation with DNP-BSA. When Li+ was added to inhibit the phosphatase activity that hydrolysed the phosphomonoester bonds in the sugar phosphates, greater increases were observed in all three inositol phosphates, particularly in inositol trisphosphate. The IgE-stimulated release of inositol trisphosphate was independent of the presence of extracellular Ca2+. In addition, the Ca2+ ionophore A23187 caused neither the decrease in [32P]polyphosphoinositides nor the stimulation of the release of inositol phosphates. These results demonstrate that stimulation of the MC9 cell via its receptor for IgE causes increased phospholipid turnover, with effects on polyphosphoinositides predominating. These data support the hypothesis that hapten cross-bridging of IgE receptors stimulates phospholipase C activity, which may be an early event in stimulus-secretion coupling of mast cells. The results with the Ca2+ ionophore A23187 indicate that an increase in intracellular Ca2+ alone is not sufficient for activation of this enzyme.     

Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.

Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Second messenger function of inositol 1,4,5-trisphosphate. Early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells

The second messenger function of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) was investigated in carbamylcholine-stimulated RINm5F cells by analysis of the early changes in inositol phosphates, cytosolic free Ca2+ concentration ([Ca2+]i), and insulin secretion. After a lag of 2 s, [Ca2+]i rose to a peak at 13 +/- 2 s, a response which was due mainly to mobilization from intracellular stores since it persisted even in the absence of extracellular Ca2+. The Ca2+ response had already declined toward prestimulatory levels by the time insulin secretion reached its maximal rate (2-3 min). Although the rises in inositol trisphosphate preceded those of both inositol bisphosphate and monophosphate, all three attained maximal concentrations after 1 min and remained elevated for at least 10 min. The accumulation of inositol trisphosphate was truly Ca2+-independent since it persisted under conditions in which the rise in [Ca2+]i was abolished by prior depletion of intracellular Ca2+ pools. Further analysis by high performance liquid chromatography revealed the presence of the two isomers, Ins-1,4,5-P3 and Ins-1,3,4-P3 in stimulated cells. The latter was virtually absent under nonstimulatory conditions but started to accumulate after a 5-s lag and reached maximal levels after 30 s of stimulation. Ins-1,4,5-P3 doubled within 1 s of carbamylcholine addition, reached a peak after 5 s, and, although declining thereafter, remained slightly elevated for at least 3 min. Hence, both the onset and peak of the rise of Ins-1,4,5-P3 preceded that of [Ca2+]i, which in turn preceded the peak in insulin release. These results strongly suggest that Ins-1,4,5-P3 acts as the second messenger by which carbamylcholine mobilizes intracellular Ca2+ during the initiation of insulin release.     

Activation of calcium mobilization and calcium influx by alpha 1-adrenergic receptors in a smooth muscle cell line

Alpha 1-adrenergic receptor (alpha 1R) mediated increases in the cytosolic levels of free Ca+2 and the inositol phosphates were measured in a smooth muscle cell line, DDT1. Norepinephrine (NE) stimulated a rapid increase in cytosolic Ca+2 by two distinct components: 1) release of Ca+2 from intracellular sites (mobilization), and 2) influx of extracellular Ca+2. The mobilization component was not affected by removal of extracellular Ca+2 or addition of La+3 or Co+2 to the buffer. The influx component was abolished by EGTA, La+3, or Co+2, but was not affected by the voltage-operated Ca+2 channel blockers diltiazem or nifedipine. Depolarization of DDT1 cells with 100 mM KCl or with gramicidin did not induce Ca+2 influx. NE also increased inositol trisphosphate to 78% over basal levels within 1 minute. These results suggest that alpha 1R on DDT1 cells are coupled to both the mobilization of intracellular Ca+2 and to receptor-operated Ca+2 channels in the plasma membrane, and that polyphosphoinositide hydrolysis may play a role in these phenomena.     

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.     

Mitogen-stimulated release of inositol phosphates in human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with a growth factor mixture (consisting of epidermal growth factor (EGF), insulin, bradykinin, and vasopressin) rapidly induces an increase in Na influx via a Ca-mediated activation of an amiloride-sensitive Na/H exchanger. Inositol phosphates (specifically inositol-1',4',5'-phosphate) have been implicated in mediating the mobilization of intracellular Ca stores in other cell types and we have now completed a detailed analysis of the mitogen-induced release of inositol phosphates in HSWP cells. Stimulation of inositol trisphosphate release is rapid (within 5 s) and reaches a maximum level (416-485% basal) within 10-15 s after the addition of growth factor mixture. Inositol bisphosphate and inositol monophosphate reach maximum levels by 30 s (1257% basal) and 60 s (291% basal), respectively. Levels of all three compounds then decay toward basal levels but remain elevated (150-350% of basal levels) after 10 min of incubation with mitogens. The effects of different combinations of these growth factors and of the bee venom peptide, melittin, have also been determined. We have also found that 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, which prevents the mitogen-induced rise in intracellular calcium activity and activation of Na influx, does not alter the mitogen-stimulated accumulation of inositol trisphosphate. In addition, the calcium ionophore A23187, which increases cytosolic Ca activity and induces a Na influx, does not stimulate the release of inositol trisphosphate. Assays performed in the presence of lithium, which inhibits inositol phosphate monophosphatase, promotes the prolonged and enhanced accumulation of inositol monophosphate. Treatment with the phospholipase inhibitor mepacrine or pretreatment with dexamethasone reduces the amount of inositol phosphates released upon mitogenic stimulation. Hence mitogenic stimulation of HSWP cells leads to the rapid stimulation of inositol phosphate release via a calcium-independent mechanism and suggests inositol trisphosphate as a candidate to mediate the release of intracellular calcium stores which is involved in the processes responsible for the activation of the Na/H exchanger.     

Ca2+ release by inositol trisphosphate from Ca2+-transporting microsomes derived from uterine sarcoplasmic reticulum

Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 microM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that by mobilizing intracellular Ca2+, inositol trisphosphate may be the link between hormonal stimuli and smooth muscle contraction.     

Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis.

The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.     

Metabolism and functions of inositol phosphates

Activation of Ca2+-mobilizing receptors rapidly increases the cytoplasmic Ca2+ concentration both by releasing Ca2+ stored in endoplasmic reticulum and by stimulating Ca2+ entry into the cells. The mechanism by which Ca2+ release occurs has recently been elucidated. Receptor activation of phospholipase C results in the hydrolysis of the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield two intracellular messengers, diacylglycerol (DAG) and (1,4,5)inositol trisphosphate [(1,4,5)IP3]. DAG remains in the plasma membrane where it stimulates protein phosphorylation via the phospholipid-dependent protein kinase C. (1,4,5)IP3 diffuses to and interacts with specific sites on the endoplasmic reticulum to release stored Ca2+. Receptor stimulation of phospholipase C appears to be mediated by one or more guanine nucleotide-dependent regulatory proteins by a mechanism analogous to hormonal activation of adenylyl cyclase. The actions of (1,4,5)IP3 on Ca2+ mobilization are terminated by two metabolic pathways, sequential dephosphorylation to inositol bisphosphate (IP2), inositol monophosphate (IP) and inositol or by phosphorylation to inositol tetrakisphosphate (IP4) and sequential dephosphorylation to different inositol phosphates. A sustained cellular response also requires Ca2+ entry into the cell from the extracellular space. The mechanism by which hormones increase Ca2+ entry is not known; a recent proposal involving movement of Ca2+ through the endoplasmic reticulum, possibly regulated by IP4, will be considered here.     

Gonadotropin releasing hormone stimulates the formation of inositol phosphates in rat anterior pituitary tissue.

The production of inositol phosphates in response to gonadotropin releasing hormone (GnRH) was studied in rat anterior pituitary tissue preincubated with [3H]inositol. Prelabelled paired hemipituitaries from prepubertal female rats were incubated in the presence or absence of GnRH in medium containing 10 mM-Li+ X Li+, which inhibits myo-inositol-1-phosphatase, greatly amplified the stimulation of inositol phosphate production by GnRH (10(-7) M) to 159, 198 and 313% of paired control values for inositol 1-phosphate, inositol bisphosphate and inositol trisphosphate respectively after 20 min. The percentage distribution of [3H]inositol within the phosphoinositides was 91.3, 6.3 and 2.4 for phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively and was unaffected by GnRH. The stimulation of inositol trisphosphate production by GnRH was evident after 5 min incubation, was dose-dependent with a half-maximal effect around 11 nM, and was not inhibited by removal of extracellular Ca2+. Elevation of cytosolic Ca2+ by membrane depolarization with 50 mM-K+ had no significant effect on inositol phosphate production. These findings are consistent with the hypothesis that GnRH action in the anterior pituitary involves the hydrolysis of phosphatidylinositol 4,5-bisphosphate. The resulting elevation of inositol trisphosphate may in turn lead to intracellular Ca2+ mobilization and subsequent stimulation of gonadotropin secretion.     

Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 microM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP3, Ins(1,3,4)P3, and Ins(1,4,5)P3 as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P3 occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca2+ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca2+ after chelation of extracellular Ca2+ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca2+ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P3 and the consequential release of Ca2+ from intracellular stores.     

Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca(2+) was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50mum) increased the breakdown of phosphatidylinositol bisphosphate in [(3)H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of (3)H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the (3)H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20mum), but not tubocurarine (100mum); and it was abolished by depletion of extracellular Ca(2+) with EGTA, but restored on addition of low concentrations of Ca(2+) (20mum). 5. Calcium-antagonistic agents, such as verapamil (20mum), dibenamine (20mum) or La(3+) (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (;microsomes') was stimulated by 43% in the presence of 50mum-Ca(2+). 7. The results indicate that increased Ca(2+) influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca(2+) activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the (3)H radioactivity of this phospholipid.     

Inhibition of CD3-induced Ca2+ signals in Jurkat T-cells by myristic acid.

Stimulation of T lymphocytes with antibodies against the T cell receptor/CD3 complex induces within seconds a rise in the concentration of intracellular free Ca2+. Here we show that treatment with 20 microM free myristic acid completely inhibits this Ca2+ signal and the cellular proliferation in Jurkat T cells. Also lauric acid inhibited cell growth while its blocking effect on the Ca2+ signal was weaker than that of myristic acid. Other saturated free fatty acids were inactive. The inhibitory effect of myristic acid could be reversed by the addition of fatty acid free albumin, which will bind the fatty acid. Myristic acid, but not its methyl ester, inhibited both the anti-CD3-induced Ca2+ influx across the cell membrane and Ca2+ release from intracellular stores, but not the formation of inositol phosphates. In contrast, thapsigargin-induced release of Ca2+ from the same intracellular stores was unaffected by myristic acid. Thus, myristic acid specifically blocks T cell antigen receptor-CD3 induced Ca2+ mobilization in T cells.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.     

Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.     

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.     

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min). The heat-induced increase in 45Ca++ influx was linearly related to membrane accumulation of phosphatidic acid, phosphoinositide metabolite that may be involved in Ca++ gating. These results suggest that the membrane may be the proximal target of heat shock; stimulation of rapid breakdown of polyphosphoinositides and subsequent increases in intracellular free Ca++ may provide a mechanistic insight into the pleiotropic effects of heat. In addition, the large increases in Ca++ influx could initiate a Ca++ dependent mechanism of thermal cell killing.