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.     

Inhibition by nicardipine of endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell

We have investigated the effects of endothelin on phosphoinositide metabolism and Ca2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphate formation in a time- and dose-dependent manner. IP3 was significantly elevated by 30 sec and reached a 2.0-fold above control at 1 min. The EC50 for endothelin was 0.5 nM. The initiation of inositol phosphate formation was independent of extracellular Ca2+, and the Ca2+ ionophore, A23187, did not stimulate IP3 formation. However, the sustained elevation of inositol phosphates was partially inhibited by incubating cells in buffer lacking Ca2+ or in buffer containing nicardipine. Endothelin mobilized both intracellular and extracellular Ca2+ reaching a peak intracellular concentration of 350 +/- 11 nM by 1 min when cells were bathed with Ca2+-complete buffer. Intracellular Ca2+ remained 2-fold above baseline for at least 15 min. In contrast, when cells were exposed to endothelin in Ca2+-free buffer, the peak value of [Ca2+]i was 195 +/- 20 nM and returned to baseline by 2 min. Nicardipine completely blocked the influx of extracellular Ca2+ but did not interfere with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP3 is consistent with the time course for mobilization of intracellular Ca2+. Elevated cytosolic Ca2+ levels are maintained by the influx of extracellular Ca2+ through a nicardipine-sensitive Ca2+ channel and are involved in the sustained formation of inositol phosphates. These data provide an explanation for the sustained, nicardipine-inhibitable contraction of coronary artery strips induced by endothelin.     

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.     

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.     

Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils

The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP-induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP-induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30-s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.     

Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production

The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production. In conclusion: 1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.     

Regulation of calcium influx across the plasma membrane of the human T-leukemic cell line, JURKAT: dependence on a rise in cytosolic free calcium can be dissociated from formation of inositol phosphates

A rise in the cytosolic free Ca2+ concentration due to both mobilization of Ca2+ from internal stores and influx of extracellular Ca2+ across the plasma membrane through 'second messenger-operated Ca2+ channels' is one of the first transmembrane signals detected following activation of CD2 or CD3 receptors on T-cells. In this study, we have further elucidated the regulation of these channels in the human T-leukemic cell line, JURKAT. Stimulation with either OKT3 or PHA induced a prompt influx of Ca2+ as assessed by MN2+ quenching of intracellular fura-2 fluorescence. When cytosolic free Ca2+ transient was partially buffered by loading the cells with BAPTA, neither agonist could induce Ca2+ entry into the cells as depicted by the lack of quenching of the fluorescence signal by Mn2+. This is in good agreement with our previous data on agonist-induced 45Ca2+ influx demonstrating that a rise in cytosolic free Ca2+ due to agonist-induced mobilization of Ca2+ from intracellular stores, could, directly or indirectly via the inositol cycle, initiate Ca2+ influx in these cells. Further support of this idea comes from the data demonstrating that agonist-induced mobilization of Ca2+ precedes the influx of Ca2+ across the plasma membrane. The present findings show that agonist-stimulation significantly increased the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after only 5 s, indicating that one or both of these substances could play a role in the regulation of Ca2+ influx. However, when agonist-induced Mn2+ influx was totally abolished, by partially buffering the cytosolic free Ca2+ rise, the formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was not affected. Consequently, the dependence of an initial rise in cytosolic free Ca2+ for the subsequent regulation of Ca2+ influx across the plasma membrane, can be dissociated from the formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Ion channels and regulation of intracellular calcium in vascular endothelial cells

Endothelial cells in vivo form an interface between flowing blood and vascular tissue, responding to humoral and physical stimuli to secrete relaxing and contracting factors that contribute to vascular homeostasis and tone. The activation of endothelial cell-surface receptors by vasoactive agents is coupled to an elevation in cytosolic Ca2+, which is caused by Ca2+ entry via ion channels in the plasma membrane and by Ca2+ release from intracellular stores. Ca2+ entry may occur via four different mechanisms: 1) a receptor-mediated channel coupled to second messengers; 2) a Ca2+ leak channel dependent on the electrochemical gradient for Ca2+; 3) a stretch-activated nonselective cation channel; and 4) internal Na+-dependent Ca2+ entry (Na+-Ca2+ exchange). The rate of Ca2+ entry through these ion pathways can be modulated by the resting membrane potential. Membrane potential may be regulated by at least two types of K channels: inwardly rectifying K channels activated upon hyperpolarization or shear stress; and a Ca2+-activated K channel activated upon depolarization, which may function to repolarize the agonist-stimulated endothelial cell. After agonist stimulation, cytosolic Ca2+ increases in a biphasic manner, with an initial peak due to inositol 1,4,5-trisphosphate-mediated Ca2+ release from intracellular stores, followed by a sustained plateau that is dependent on the presence of [Ca2+]o and on membrane potential. The delay in agonist-activated Ca2+ influx is consistent with the coupling of receptor activation to Ca2+ entry via a second messenger. Oscillations in [Ca2+]i, which may involve both Ca2+ entry and release, have been observed in isolated and confluent endothelial cell monolayers stimulated by histamine and bradykinin. Receptor-mediated Ca2+ entry, release, and refilling of intracellular stores follows a cycle that involves the plasma membrane.     

Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

In the presence of ouabain, prostaglandin (PG) E2 stimulated a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells. PGE2 or ouabain alone evoked a marginal secretory response. The synergism of ouabain was also observed with muscarine. PGE2, like muscarine, induced a concentration-dependent formation of inositol phosphates: rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of inositol monophosphate. This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. The potency of PGs (PGE2 greater than PGF2 alpha greater than PGD2) to stimulate catecholamine release was well correlated with that to affect phosphoinositide metabolism and that to increase the level of intracellular Ca2+. PGE2 did not stimulate cAMP generation significantly in bovine chromaffin cells. The effect of PGE2 on catecholamine release was mimicked by 12-O-tetradecanoylphorbol 13-acetate and A23187, but not by the cAMP analogue dibutyryl cAMP nor by forskolin. These results indicate that PGE2 may enhance catecholamine release from chromaffin cells by activating protein kinase C in concert with the increment of intracellular Ca2+.     

Differential activation of phosphatidylinositol deacylation and a pathway via diphosphoinositide in macrophages responding to zymosan and ionophore A23187

The inositol phospholipids of peritoneal macrophages were prelabeled with [3H]inositol to enable studies on the enzymatic mechanisms of stimulus-induced phosphatidylinositol breakdown. Ionophore A23187 induced a rapid breakdown of phosphatidylinositol in the presence of Ca2+ with 25% loss occurring within 5 min. The main water-soluble product of this breakdown was identified as inositol diphosphate. Since the accumulation of inositol diphosphate far exceeded the concomitant decrease in polyphosphoinositides, an increased phosphorylation of phosphatidylinositol must have preceded, or accompanied, the degradation of diphosphoinositide. The degradation of phosphatidylinositol induced by A23187 was shown to be strictly dependent on Ca2+. The monovalent cation ionophore monensin and platelet-activating factor increased the level of diphosphoinositide but caused no net degradation of inositol phospholipids. The same effect was seen with ionophore A23187 in the absence of Ca2+. Zymosan particles also induced extensive degradation of phosphatidylinositol. Products of phosphodiesterase-catalyzed cleavage of inositol lipids were observed, but the pathway of deacylation dominated as evidenced by the accumulation of lysophosphatidylinositol and glycerophosphoinositol. Deacylation was also enhanced in response to concanavalin A. Thus, in mouse peritoneal macrophages phosphatidylinositol breakdown occurred primarily by deacylation or via diphosphoinositide, depending on the stimulus, rather than through a phosphatidylinositol phosphodiesterase reaction.     

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.     

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.     

Inhibition of inositol phospholipids metabolism and calcium mobilization by cyclic AMP-increasing agents and phorbol ester in neutrophils

Cyclic AMP-increasing agents such as PGE2 and dibutyryl cAMP inhibited the fMLP-induced inositol phospholipids metabolism mainly through the suppression of the conversion of phosphatidylinositol(PI) to phosphatidylinositol 4,5-bisphosphate(PIP2). A part of this inhibition was found to be caused by the inhibitory effect of cAMP on PI kinase using isolated plasma membranes. On the other hand, 12-O-tetradecanoyl phorbol acetate(TPA) mainly inhibited the conversion of phosphatidylinositol 4-phosphate(PIP) to PIP2 without a significant effect on the fMLP-induced breakdown of PIP2, though direct effect of TPA on PI and PIP kinases was not demonstrated in isolated plasma membranes. Concerning Ca2+ mobilization, both cAMP-increasing agents and TPA inhibited the fMLP-induced second phase of Ca2+ elevation, while they did not affect the first phase of Ca2+ rapid increase. However, Ca2+ ionophore ionomycin-induced Ca2+ elevation was only inhibitable by TPA but not PGE2. These results suggest that cAMP inhibits the fMLP-induced Ca2+ influx, while TPA stimulates Ca2+ removal from cytosol.     

Increased plasma membrane permeability to Ca2+ in anti-Ig-stimulated B lymphocytes is dependent on activation of phosphoinositide hydrolysis

The biochemical basis of Ca2+ mobilization after anti-Ig binding to B cell Ag-R has been further characterized by flow cytometric analysis of indo-1-loaded B cells. The ability to distinguish intracellular Ca2+ release from extracellular Ca2+ influx by using an extracellular calcium depletion-repletion approach has allowed us to study the relationship between the mobilization of Ca2+ from these sources. Studies involving manipulation of the Ca2+ gradient across the plasma membrane indicate that a significant portion of the Ca2+ mobilization response is preserved even when the normal inwardly directed Ca2+ gradient is reversed. In the presence of an extracellular calcium concentration ([Ca2+]o) of 10 microM, the response to anti-Ig is not blocked by the organic Ca2+ channel blockers. This response is not reduced by further depletion of [Ca2+]o by EGTA Ca2+-binding buffers. Thus, the Ca2+ response that occurs when [Ca2+]o less than or equal to 10 microM represents intracellular calcium release. Analysis of B cells stimulated with anti-Ig in low Ca2+ medium ([Ca2+]o = less than 10 microM) followed by repletion of [Ca2+]o to 1 to 5 mM reveals that a significant increase in permeability of the plasma membrane to Ca2+ develops in the stimulated cells. The resultant Ca2+ influx is nimodipine (20 microM) sensitive. Both intracellular Ca2+ release and Ca2+ influx are reduced in parallel as the concentration of anti-Ig stimulus is decreased, suggesting that Ca2+ influx may be coupled to the release of intracellular stores. Neomycin blocks anti-Ig-stimulated formation of inositol trisphosphate, which mediates release of Ca2+ from the endoplasmic reticulum. It also blocks the anti-Ig-induced release of intracellular Ca2+ stores as well as Ca2+ influx, indicating that both responses may be dependent upon phosphatidylinositol 4,5-bisphosphate hydrolysis.     

The route of Ca2+ entry during reloading of the intracellular Ca2+ pool in pancreatic acini

To trace the route of Ca2+ entry and the role of the cytosolic Ca2+ pool in reloading of the internal stores of pancreatic acinar cells, Mn2+ influx into Fura 2-loaded cells and the effect of 1,2-bis(2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA) on Ca2+ storage in intracellular stores and reloading were examined. Treatment of acini suspended in Ca2(+)-free medium with carbachol (cell stimulation) or carbachol and atropine (reloading period) resulted in 2-fold increase in the rate of Mn2+ influx. Increasing Ca2+ permeability of the plasma membrane by elevation of extracellular pH from 7.4 to 8.2 further increased the rate of Mn2+ influx observed during cell stimulation and the reloading period. Loading the acini with BAPTA by incubation with 50 microM of the acetomethoxy form of BAPTA (BAPTA/AM) was followed by a transient reduction in free cytosolic Ca2+ concentration ((Ca2+]i). To compensate for the increased Ca2+ buffering capacity in the cytosol the acini incorporated Ca2+ from the external medium. Although BAPTA prevented changes in free cytosolic Ca2+ concentration during carbachol and atropine treatment, it had no apparent effect on Ca2+ content of the internal stores or the ability of agonists to release Ca2+ from these stores. Loading the cytosol with BAPTA considerably reduced the rate of Ca2+ reloading. These observations are not compatible with direct communication between the medium and the inositol 1,4,5-trisphosphate releasable pool and provide direct evidence for Ca2+ entry into the cytosol prior to its uptake into the intracellular pool, both during cell stimulation and the Ca2+ reloading.     

Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H2O2. The present study shows that upon activation of death response by H2O2, a dose- and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. Partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.     

Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)