The cholecystokinin-induced Ca2+ shuttle from the inositol trisphosphate-sensitive and ATP-dependent pool, and initial pepsinogen release connected with cytoskeleton of the chief cell

In guinea pig chief cells, inositol 1,4,5-trisphosphate (IP3) caused release of Ca2+, which was accumulated by ATP, from an endoplasmic reticulum-enriched fraction in both the permeable system and the cell-free system. This was mimicked with the Ca2+ ionophores A23187 and ionomycin on a large scale since an IP3-sensitive Ca2+ pool might be a subset of the Ca2+ ionophore-sensitive Ca2+ pool. The permeable chief cells, but not the cell-free system, retained the ability to react to synthetic cholecystokinin octapeptide (CCK-OP) with Ca2+ release from an IP3-sensitive pool due to of the non-additive but constant effect in exerting Ca2+ release from the store(s) induced by the combination with IP3 and CCK-OP. The increase in the cytosolic free Ca2+ concentration of intact chief cells responding to CCK-OP or the Ca2+ ionophore, ionomycin, comprised two components, namely, that by the Ca2+ entry from the extracellular space, and that by the Ca2+ release from the intracellular space(s) (as measured by fura-2). When CCK-OP or ionomycin was added, there was a biphasic response of pepsinogen secretion. An initial but transient response reaching a peak in 5 min was followed by a sustained response reaching a peak in 30 min. The initial pepsinogen release was independent of medium Ca2+, whereas the sustained one was dependent on medium Ca2+. The results suggest that the intracellular Ca2+ release from the store(s), presumably endoplasmic reticulum, may trigger the initial pepsinogen release, whereas the sustained pepsinogen secretion may be caused by acting in concert with the initial response and external Ca2+ entry. On the other hand, the disruption of the microtubular-microfilamentous system by colchicine or cytochalasin D failed to cause the Ca2+ release evoked by either IP3, CCK-OP or Ca2+ ionophores and to cause the CCK-OP- or ionomycin-induced initial pepsinogen release. These findings suggest that the IP3-sensitive pool is the same Ca2+ store which is completely or partially sensitive to CCK-OP and Ca2+ ionophores, respectively, and that the assembly of the cytoskeletal system is involved in initial intracellular Ca2+ metabolism and the following initial pepsinogen release. The assembly of the cytoskeletal system may be an early event in mediating the CCK-OP-induced initial pepsinogen release, perhaps by causing the Ca2+ release from an IP3-sensitive pool of the chief cell. The translocation or attachment of the IP3-sensitive pool brought about by cytoskeletal system might be necessary to cause Ca2+ release after the cell stimulation with CCK-OP.     

Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.

The effects of hepatocyte growth factor (HGF) on intracellular Ca2+ mobilization were studied using fura-2-loaded single rat hepatocytes. Hepatocytes microperfused with different amounts of HGF responded with a rapid concentration-dependent rise in the cytosolic free Ca2+ concentration with a maximum increase of 142% at 80 ng/ml of HGF. The lag period of the Ca2+ response was decreased with increasing HGF concentrations, being 64 +/- 12 s, 42 +/- 6 s, and 14 +/- 2 s, respectively, with 8, 20, and 80 ng/ml of HGF. The detailed pattern of Ca2+ transients, however, was variable. Out of 16 cells tested using 20 ng/ml of HGF, 68% showed sustained oscillatory responses, whereas other cells showed a sustained increase in the cytosolic-free Ca2+ upon exposure to HGF, which was dependent on the presence of extracellular Ca2+. HGF also induced Ca2+ entry across the plasma membrane. Mobilization of Ca2+ by HGF was accompanied by a rapid accumulation of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). The effects of HGF and epidermal growth factor (EGF) were comparable and partly additive for Ins 1,4,5-P3 production and for the sustained phase of Ca2+ mobilization. Preincubation of cells with 10 microM of genistein to inhibit protein tyrosine kinases abolished the HGF-induced Ca2+ response and also inhibited HGF-induced Ins 1,4,5-P3 production in rat liver cells. These data indicate that early events in the signal transduction pathways mediated by HGF and EGF have in common the requirements for tyrosine kinase activity, Ins 1,4,5-P3 production, and Ca2+ mobilization.     

Beta-adrenergic receptor stimulation induces inositol trisphosphate production and Ca2+ mobilization in rat parotid acinar cells

In dispersed rat parotid gland acinar cells, the beta-adrenergic agonist (-)-isoproterenol, but not its stereoisomer (+)-isoproterenol, induced a transient 1.6-fold (at maximum stimulation, 2 x 10(-4) M) increase in cytosolic free calcium ([Ca2+]i) within 9 s, which returned to resting levels (approximately 190 nM) by 60 s. This [Ca2+]i response was not altered by chelating extracellular Ca2+ with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and could be completely blocked by the beta-adrenergic antagonists propranolol (beta 1 + beta 2) and ICI 118,551 (beta 2) but not by atenolol (beta 1). The muscarinic-cholinergic agonist carbachol (at maximum stimulation, 10(-5) M) induced a 3-4-fold elevation in [Ca2+]i within 6 s, which slowly returned to resting levels by 8-10 min. The peak carbachol [Ca2+]i response was not substantially altered by the addition of EGTA to the extracellular medium. However, if the cells were first stimulated with isoproterenol in the EGTA-containing medium, the peak carbachol response was decreased approximately 54%. When carbachol was added to cells in the presence of high extracellular calcium, at the isoproterenol-stimulated [Ca2+]i peak, the resulting [Ca2+]i level was equal to that achieved when carbachol was either added alone or added after propranolol and isoproterenol. 8-Bromo-cyclic AMP induced a [Ca2+]i response similar to that elicited by isoproterenol, which was not additive to that by carbachol. Carbachol induced a approximately 3.5-fold increase in inositol trisphosphate (IP3) production in parotid cells within 30 s. 8-Bromo-cAMP, N6,O2'-dioctanoyl-cAMP, and isoproterenol consistently induced a significant stimulation in IP3 production. The half-maximal concentration of isoproterenol required for [Ca2+]i mobilization and IP3 production was comparable (approximately 10(-5) M). Isoproterenol-induced IP3 formation was blocked by propranolol. The data show that in rat parotid acinar cells, beta-adrenergic stimulation results in IP3 formation and mobilization of a carbachol-sensitive intracellular Ca2+ pool by a mechanism involving cAMP. This demonstrates an interaction between the cAMP and phosphoinositide second messenger systems in these cells.     

Ionizing radiation stimulates existing signal transduction pathways involving the activation of epidermal growth factor receptor and ERBB-3, and changes of intracellular calcium in A431 human squamous carcinoma cells.

Previous studies demonstrated that ionizing radiation activates the epidermal growth factor receptor (EGFR), as measured by Tyr autophosphorylation, and induces transient increases in cytosolic free [Ca2+], [Ca2+]f. The mechanistic linkage between these events has been investigated in A431 squamous carcinoma cells with the EGFR Tyr kinase inhibitor, AG1478. EGFR autophosphorylation induced by radiation at doses of 0.5-5 Gy or EGF concentrations of 1-10 ng/ml is inhibited by >75% at 100 nM AG1478. Activation of EGFR enhances IP3 production as a result of phospholipase C (PLC) activation. At the doses used, radiation stimulates Tyr phosphorylation of both, PLCgamma and erbB-3, and also mediates the association between erbB-3 and PLCgamma not previously described. The increased erbB-3 Tyr phosphorylation is to a significant extent due to transactivation by EGFR as >70% of radiation- and EGF-induced erbB-3 Tyr phosphorylation is inhibited by AG 1478. The radiation-induced changes in [Ca2+]f are dependent upon EGFR, erbB-3 and PLCgamma activation since radiation stimulated IP3 formation and Ca2+ oscillations are inhibited by AG1478, the PLCgamma inhibitor U73122 or neutralizing antibody against an extracellular epitope of erbB-3. These results demonstrate that radiation induces qualitatively and quantitatively similar responses to EGF in stimulation of the plasma membrane-associated receptor Tyr kinases and immediate downstream effectors, such as PLCgamma and Ca2+.     

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.     

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.     

Fast biphasic regulation of type 3 inositol trisphosphate receptors by cytosolic calcium

In cytosol-like medium (CLM) with a free [Ca(2+)] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP(3)) (30 microm) evoked (45)Ca(2+) release from type 3 IP(3) receptors only after a latency of 48 +/- 6 ms; this latency could not be reduced by increasing the IP(3) concentration. In CLM containing a low free [Ca(2+)] ( approximately 4 nm), 300 microm IP(3) evoked (45)Ca(2+) release after a latency of 66 +/- 11 ms; this was reduced to 14 +/- 3 ms when the [Ca(2+)] was 1 mm. Preincubation with CLM containing 100 microm Ca(2+) caused a rapid (half-time = 33 +/- 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP(3) (300 microm). Hepatic (type 2) IP(3) receptors were not inhibited by Ca(2+) once they had bound IP(3), but 100 microm Ca(2+) rapidly inhibited type 3 IP(3) receptors whether it was delivered before addition of IP(3) or at any stage during a response to IP(3). Ca(2+) increases the affinity of IP(3) for hepatic receptors by slowing IP(3) dissociation, but Ca(2+) had no effect on IP(3) binding to type 3 receptors. The rate of inhibition of type 3 IP(3) receptors by Ca(2+) was faster than the rate of IP(3) dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP(3)) agonist. Dissociation of agonist is not therefore required for Ca(2+) to inhibit type 3 IP(3) receptors. We conclude that type 2 and 3 IP(3) receptors are each biphasically regulated by Ca(2+), but by different mechanisms. For both, IP(3) binding causes a stimulatory Ca(2+)-binding site to be exposed allowing Ca(2+) to bind and open the channel. IP(3) binding protects type 2 receptors from Ca(2+) inhibition, but type 3 receptors are inhibited by Ca(2+) whether or not they have IP(3) bound. Increases in cytosolic [Ca(2+)] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP(3) has dissociated.     

Phosphoinositide signalling system in platelets of schizophrenic patients and the effect of neuroleptic therapy.

Alterations in the phosphoinositide signalling system have been proposed as a possible biological marker of schizophrenia. We studied the levels of inositol 1,4,5-trisphosphate (IP3), cytosolic Ca2+ concentrations ([Ca2+]i), and the incorporation of [32P]-orthophosphate into inositol phospholipids and phosphatidic acid (PA) in blood platelets of neuroleptic-treated schizophrenics in comparison with controls. The [Ca2+]i was significantly higher in platelets of one month neuroleptic-treated patients (155+/-5.8 nM) in comparison with controls (95+/-5.4 nM). Neuroleptic therapy decreased the [Ca2+]i, but even after long-term therapy it remained significantly higher (114+/-5.7 nM) than in controls. Differences were also found in the level of IP3 between controls (30+/-4.0 pmol/10(9) platelets), drug-free schizophrenics (52+/-9.0 pmol/10(9) platelets) and treated patients (50+/-6.0 pmol/10(9) platelets). The increased turnover of PA was observed in platelets of neuroleptic-treated schizophrenic patients. The study suggests that the regulation of calcium homeostasis and pathways involved in the phosphoinositide signalling system are altered in the platelets of schizophrenics. Neuroleptic therapy did not remove the observed changes in [Ca2+]i and IP3 levels.     

Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

At maximally effective concentrations, vasopressin (10(-7) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by 100% at 3 s and 150% at 6 s, while adrenaline (epinephrine) (10(-5) M) produced a 17% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least 10 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with angiotensin II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with 10(-9) M-vasopressin and 10(-7) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by 10mM-Li+, after a lag of approx. 1 min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, angiotensin II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.     

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

Activation and development of porcine oocytes matured in vitro following injection of inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (IP3) is considered to be important for activation of mammalian oocytes at the time of fertilization, and activation induces a rise in intracellular Ca2+ concentration ([Ca2+]i) by release from the Ca2+ stores in the oocytes. Therefore, IP3 could act as an artificial activator of porcine oocytes. Activation and development, and rise in [Ca2+]i in matured oocytes injected with various concentrations of IP3 were investigated in this study. Porcine oocytes were recovered from the ovaries of prepubertal gilts, matured for 46-48 h and cultured in vitro for 7 days in following treatments as non-injected oocytes (NI), injected with carrier buffer, 2.5, 5 and 500 microM of IP3. The result showed that IP3 activated porcine oocytes matured in vitro (NI 3.8%, buffer 7.1%, 2.5 microM IP3 73.5%, 5 microM IP3 76.2%, 500 microM IP3 85.2%). There was a slight but not significant increase in the proportion of oocytes activated as the level of IP3 increased. The rate of development to the cleavage stage increased remarkably when the concentration of IP3 increased (NI 4.9%, buffer 5.7%, 2.5 microM IP3 30.3%, 5 microM IP3 47.1%, 500 microM IP3 78.1%). Blastocyst development was only observed in oocytes that had been injected with a higher concentration of IP3 (5 microM IP3 6.1% and 500 microM IP3 5.3%). Both the peak value and duration of [Ca2+]i rise also increased as the concentration of IP3 increased. Baseline values (ratio value, R) for [Ca2+]i ranged from 1.51 to 1.57 and was not affected by the buffer treatment. The peak value of [Ca2+]i rose significantly with increasing level of IP3 treatment (2.5 microM IP3, 3.54 +/- 0.32; 5 microM IP3, 7.50 +/- 0.37; 500 microM IP3, 8.54 +/- 0.33). Similarly, the duration of the [Ca2+]i rise increased as the level of IP3 increased (2.5 microM IP3, 43.7+/- 7.00 s; 5 microM IP3, 93.5 +/- 9.17 s; 500 microM IP3, 160.6 +/- 18.9 s). It was concluded that injected IP3 promotes the development of porcine matured oocytes and that their developmental ability is positively correlated with the rise in [Ca2+]i induced by IP3.     

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.     

Cluster formation of inositol 1,4,5-trisphosphate receptor requires its transition to open state

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) Ca(2+) channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP(3)R forms clusters on the endoplasmic reticulum when cytosolic Ca(2+) concentration ([Ca(2+)](C)) is elevated. However, the molecular mechanism of IP(3)R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP(3)R type 1 (GFP-IP(3)R1), evoked by IP(3)-generating agonists, did not correlate with [Ca(2+)](C) but seemed compatible with cytoplasmic IP(3) concentration. IP(3) production alone induced GFP-IP(3)R1 clustering in the absence of a significant increase in [Ca(2+)](C) but elevated [Ca(2+)](C) without IP(3) production did not. Moreover IP(3)R1 mutants that do not undergo an IP(3)-induced conformational change failed to form clusters. Thus, IP(3)R clustering is induced by its IP(3)-induced conformational change to the open state. We also found that GFP-IP(3)R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.     

Inositol 1,4,5-trisphosphate induced calcium release from corn coleoptile microsomes

The effect of inositol 1,4,5-trisphosphate (IP3) on Ca2+ release from microsomes of corn coleoptiles was investigated. Addition of micromolar concentrations of IP3 to Ca2+ loaded microsomes resulted in rapid release of 20-30% of sequestered Ca2+. Maximal and half maximal Ca2+ release occurred at 20 and 8 microM of IP3 respectively. Part of the Ca2+ released by IP3 was reaccumulated into microsomes within 4 min. The amount of Ca2+ released by IP3 was found to be dependent on free Ca2+ concentration in the incubation medium at the time of release. Maximum Ca2+ release was observed around 0.1 microM free Ca2+ concentration in the assay medium. These data suggest that IP3 might act as a second messenger in plants in a manner similar to animal systems by altering cytosolic levels of calcium.     

Modulation of intracellular Ca2+ in the parathyroid cell. Release of Ca2+ from non-mitochondrial pools by inositol trisphosphate

Stimuli which enhance secretion from parathyroid cells such as low extracellular Ca2+ or Mg2+ are associated with a decrease in the cytosolic Ca2+ concentration as measured by quin2. Current evidence suggests that increased production of inositol 1,4,5-triphosphate (IP3) releases Ca2+ from cellular stores thus increasing cytosolic Ca2+. We used saponin-permeabilized dispersed bovine parathyroid cells to study the effect of IP3 on intracellular Ca2+. IP3 released Ca2+ from these cells in a dose-dependent manner; half-maximal response occurred with 0.3 microM IP3 and maximal response with 1.2 microM IP3. Permeabilized cells incubated in the presence of the mitochondrial inhibitor antimycin A released a similar amount of Ca2+ suggesting that IP3 releases Ca2+ from a non-mitochondrial pool. These results suggest that IP3 regulates cytosolic Ca2+ in this system and may function as a second messenger controlling hormone secretion.     

Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.

We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.     

Calcium-dependent oxidation of thioredoxin during cellular growth initiation.

The fraction of cell thiol proteins in the oxidized disulfide form were quantified during mitogen-induced HaCaT keratinocyte growth initiation. Oxidized thioredoxin increased from 11 +/- 1.2% in resting cells to 80 and 61% 2 min after addition of bradykinin or EGF. Thioredoxin oxidation was transient returning toward normal values by 20 min. The disulfide forms of other cellular proteins rose in parallel with thioredoxin oxidation. The oxidation of thioredoxin depended on a rise in cytosolic calcium. It was prevented by preloading cells with BAPTA, a Ca(2+) chelator and induced by addition of Ca(2+)-ionophore A23187 or of thapsigargin. In cell extracts, thioredoxin reductase was inhibited by micromolar calcium. The rise in cytosolic Ca(2+) led to a concomitant burst of H(2)O(2) formation. The oxidizing intracellular milieu suggests that redox regulation actively participates in the growth initiation cascade. The role of peroxiredoxins and ASK 1 cascade activation are discussed in this context.     

Bradykinin-induced changes in phosphoinositides, inositol phosphate production and intracellular free calcium in cultured bovine aortic endothelial cells

Acute hydrolysis of phosphoinositides has been demonstrated in bovine aortic endothelial cells (BAEC) treated with bradykinin (BK) (10(-7)M). The first phosphoinositide to decrease was phosphatidylinositol-4,5-bisphosphate (PIP2) indicating this to be the initial substrate of phospholipase action. Other lipid changes associated with the stimulation of BAEC were an increase in diacylglycerol (DAG) and arachidonic acid (AA) with a sustained production of phosphatidic acid (PA). The changes in cell phospholipids were accompanied by the release of inositol phosphates. Inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) was produced within 10 s of stimulation with BK. There was no evidence for the production of inositol-1,3,4-trisphosphate. The release of ionic calcium (Ca2+) intracellularly was demonstrated. The timecourse of the rise in intracellular Ca2+ was consistent with the timecourse of production of IP3. Intracellular Ca2+ rose from 127 +/- 21 nM to 462 +/- 27 nM. The Ca2+ peak was at 7.0 +/- 0.4 s and took 3 min to reach a steady state which remained above the basal level. When extracellular Ca2+ was depleted in the extracellular medium a spike of intracellular Ca2+ release was measured with an immediate return to basal. Entry of extracellular Ca2+ into the cell after ionophore A23187 treatment does not induce inositol phosphate release, indicating that phosphoinositide hydrolysis is likely to be the cause rather than consequence of the elevation in cytosolic Ca2+. These data indicate action of phospholipase C (PLC) on PIP2 after BK stimulation of BAEC with the subsequent production of InsP3 causing the resulting intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inositol trisphosphate kinase activity in hypertrophied rat heart

In the present experiment, we demonstrated that IP3 kinase activity was increased in SHRSP heart compared to WKY heart. IP3 kinase activity in the heart was highest in the cytosolic fraction in both SHRSP and WKY. Its activity progressively increased with age in 5- to 20-week SHRSP. The activity reached about three times the level of 5-week-old SHRSP in 40-week-old SHRSP. On the other hand, in WKY it was 1.3-fold at 40 weeks compared with that at 5 weeks. We determined the effect of divalent cations on IP3 kinase activity. Ca2+ stimulated its activity in a dose-dependent manner at 10(-9) to 10(-6) M. In SHRSP it was enhanced about 2.1-fold at 10(-6) M of Ca2+, but in WKY it was 1.5-fold at 1.0(-6) M of Ca2+. Mn2+ also stimulated IP3 kinase activity in both groups of animals, while, Fe2+, Zn2+, and Cu2+ inhibited IP3 kinase activity. In our experiment IP3 kinase activity was increased in SHRSP and its activity was markedly affected by divalent cations. These data suggest that the accumulations of IP3 and IP4 after hormonal stimulation play a physiologic role, possibly by alteration of Ca2+ levels in cardiac tissue.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.