Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Progesterone triggers rapid transmembrane calcium influx and/or calcium mobilization from endoplasmic reticulum,via a pertussis-insensitive G-protein in granulosa cells in relation to luteinization process

We investigated the early effects (5–60 s) of progesterone (1 pM–0.1 μM) on cytosolic free calcium concentration ([Ca²⁺]_i) and inositol 1,4,5-trisphosphate (InsP₃) formation in nonluteinized and in vitro luteinized porcine granulosa cells (pGCs). Progesterone increased [Ca²⁺]_i and InsP₃ formation within 5 s in both cell types. Progesterone induced calcium mobilization from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis-insensitive G-protein. This process was controlled by protein kinases C and A. In contrast, only nonluteinized pGCs showed a Ca²⁺ influx via dihydropyridine-insensitive calcium channel. In both cell types, the nuclear progesterone receptor antagonist RU-38486 did not inhibit the progesterone-induced increase in [Ca²⁺]_i; progesterone immobilized on bovine serum albumin, which did not enter the cell, increased [Ca²⁺]_i within 5 s and was a full agonist, but less potent than the free progesterone; pertussis toxin did not inhibit progesterone effect on InsP₃. In conclusion, progesterone may interact with membrane unconventional receptors that belong to the class of membrane receptors coupled to a phospholipase C via a pertussis toxin-insensitive G-protein. The source of the Ca²⁺ for the progesterone-induced increase in [Ca²⁺]_i also depends on the stage of cell luteinization. © 1996 Wiley-Liss, Inc.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

Intracellular Ca2+ in pancreatic acinar cells: Regulation and role in stimulation of enzyme secretion

Evidence for a primary role for intracellular Ca²⁺ in the stimulation of pancreatic enzyme secretion is reviewed. Measurements of cytoplasmic free Ca²⁺ concentration have allowed direct demonstration of its importance in triggering enzyme secretion and defined the concentration range over which membrane Ca²⁺ pumps must work to regulate intracellular Ca²⁺. Current evidence suggests a key role for the Ca²⁺ Mg-ATPase of rough endoplasmic reticulum in regulating intracellular Ca²⁺ and accumulating a Ca²⁺ store which is released by the action of inositol-l,4,5 trisphosphate following stimulation of secretion.Abbreviations Used EGTA (ethylene dioxy) diethylene-dinitrilotetraacetic acid - BAPTA 1,2-bis (2-aminophenoxy) ethane NNN,N-tetracetic acid - InsP₃ inositol trisphosphate - Ins-1,4,5P₃ and Ins-1,3,4P₃ isomers of inositol trisphosphate with the position of phosphate groups assigned - Ins-1,3,4,5P₄ inositol tetrakisphosphate     

Different Patterns of Agonist-Stimulated Increases of 3H-Inositol Phosphate Isomers and Cytosolic Ca2+ in Bovine Adrenal Chromaffin Cells: Comparison of the Effects of Histamine and Angiotensin II

Abstract: Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP₁, InsP₂, and InsP₃, respectively) isomers, intracellular free Ca²⁺ ([Ca²⁺]_i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca²⁺]_i by 200 nM 3–4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P₃ that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P₃ formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P₃ by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P₂ and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P₃, Ins(1,3)P₂, and Ins(3,4)P₂, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P₂ was the only other InsP₂ besides Ins(1,4)P₂ to accumulate within 1 min of agonist treatment [Ins(3,4)P₂ did not increase]. These results support a correlation between the time course of Ins(1,4,5)P₃ formation and the time course of [Ca²⁺]_i transients and illustrate that Ca²⁺-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca²⁺], changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Endothelin-Mediated Calcium Response and Inositol 1,4,5-Trisphosphate Release in Neuroblastoma-Glioma Hybrid Cells (NG108-15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108-15) induced increases in cytosolic free Ca²⁺ ([Ca²⁺]_i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. The increases in [^Ca2+]_i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca²⁺ in the medium by adding excess EGTA decreased the ET-mediated Ca²⁺ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca²⁺]_i was due to influx from an extracellular source. However, the increase in [Ca²⁺]_i was not affected by verapamil or nifedipine (10^?5M). A rank order potency of ET-1 ET-2 ET-3 is shown for the stimulated increase in [Ca²⁺]_i, as well as labeled inositol phosphates, in these cells. ATP (10^?4M) and bradykinin (10^?7M) also induced the increases in [Ca²⁺]_i and Ins(1,4,5)P₃ in NG108-15 cells, albeit to a different extent. When compared at 10^?7M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P₃, but less than a twofold higher increase in [Ca²⁺]_i than those induced by ET-1. Additive increases in both Ins(1,4,5)P₃ and [Ca²⁺]_i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization to the Ca²⁺ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.     

A model for Ca2+ oscillations stimulated by the type 5 metabotropic glutamate receptor: an unusual mechanism based on repetitive, reversible phosphorylation of the receptor

In parallel with experimental investigations, the molecular mechanisms responsible for Ca²⁺ oscillations have been much investigated with computational models. In the vast majority of cell-types, these oscillations rely on the biphasic regulation of the inositol 1,4,5-trisphosphate (InsP₃) receptor by cytosolic Ca²⁺. However, when Ca²⁺ oscillations are initiated by agonist stimulation of the type 5 metabotropic glutamate (mGlu5) receptor, oscillatory behaviour is tightly controlled by repetitive cycles of receptor phosphorylation/dephosphorylation leading to the periodic activation/deactivation of the G protein-activated signalling cascade downstream of this G protein-coupled receptor. We present a minimal model for mGlu5 receptor-induced Ca²⁺ oscillations, taking into account receptor phosphorylation by a protein kinase C isoenzyme sensitive to diacylglycerol but not to Ca²⁺. Depending on the density of receptors and the level of stimulation, the model reproduces Ca²⁺ oscillations based on either a ‘dynamic uncoupling’ mechanism or InsP₃ receptor dynamics. When based on the former mechanism, Ca²⁺ oscillation frequency is insensitive to the level of stimulation, while the level of receptor expression is a determinant of oscillation frequency. When investigating the conditions for the occurrence of oscillations, the model predicts that dynamic uncoupling likely relies on a steep relationship between the activity of PKC and the amount of phosphorylated mGlu5 receptor. Finally, we use the model to simulate the adaptation of the signalling pathway during periods of prolonged stimulation associated with receptor desensitization/internalization. The model suggests that the existence of both oscillatory mechanisms could allow for a significant lengthening of the repetitive Ca²⁺ responses under these conditions.     

InsP3R-associated cGMP Kinase Substrate Determines Inositol 1,4,5-Trisphosphate Receptor Susceptibility to Phosphoregulation by Cyclic Nucleotide-dependent Kinases

Ca²⁺ release through inositol 1,4,5-trisphosphate receptors (InsP₃R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca²⁺ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca²⁺ release via InsP₃R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP₃R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP₃R-1 subtype resulted in enhanced Ca²⁺ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP₃R subtype, and phosphorylation of IRAG by PKG attenuated Ca²⁺ release through all InsP₃R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca²⁺ release through InsP₃R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP₃R-2. Phosphorylation of IRAG resulted in reduced Ca²⁺ release through all InsP₃R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.     

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP₃) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully. No effect of Ca²⁺ concentration on [³H]-InsP₃ binding to unreconstituted InsP₃ receptor could be observed either at 4°C or at 25°C, whereas the effect of [Ca²⁺] on reconstituted InsP₃ receptor depended on the temperature. The Ca²⁺ concentration outside the proteolipsome ([Ca²⁺]_o) had no detectable effect on InsP₃ binding to InsP₃ receptor at 4°C. In contrast, with increase of [Ca²⁺]_o from 0 to 100 nmol/L at 25°C, the InsP₃ binding activity increased gradually. Then the InsP₃ binding activity was decreased drastically at higher [Ca²⁺]_o and inhibited entirely at 50 μmol/L [Ca²⁺]_o. Conformational studies on intrinsic fluorescence of the reconstituted InsP₃ receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP₃ receptor could not be affected by [Ca²⁺]_o at 4°C. While at 25°C, the effects of 10 μmol/L [Ca²⁺]_o on global, membrane and cytoplasmic conformation of the reconstituted InsP₃ receptor were different significantly from that of 100 nmol/L [Ca²⁺]_o.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca-Release Channels

To clarify the molecular mechanisms behind quantal Ca²⁺ release, the graded Ca²⁺ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP₃R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP₃R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP₃R gene or from cells solely expressing one recombinant InsP₃R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca²⁺ signaling, including quantal Ca²⁺ release, by tuning ligand sensitivities of InsP₃R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca²⁺ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.     

Spatially Defined InsP3-Mediated Signaling in Embryonic Stem Cell-Derived Cardiomyocytes

The functional role of inositol 1,4,5-trisphosphate (InsP₃) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP₃ receptors can translate Ca²⁺ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP₃-mediated Ca²⁺ release. [InsP₃]_i was monitored with the FRET-based InsP₃-biosensor FIRE-1 (Fluorescent InsP₃ Responsive Element) and heterogeneity in sub-cellular [InsP₃]_i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP₃ 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca²⁺ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca²⁺]_i and spontaneous activity was modulated by [InsP₃]_i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP₃ by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP₃ signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca²⁺ released from InsP₃Rs is more effective than Ca²⁺ released from RyRs to enhance I_NCX. The results support the hypothesis that in ESdCs InsP₃Rs form a functional signaling domain with NCX that translates Ca²⁺ release efficiently into a depolarization of the membrane potential.     

Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

The ubiquitous inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) Ca²⁺ release channel plays a central role in the generation and modulation of intracellular Ca²⁺ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP₃, free Ca²⁺, free ATP⁴⁻) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP₃R channel activity by free Ca²⁺ in the ER lumen ([Ca²⁺]_ER) remains poorly understood because of limitations of Ca²⁺ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca²⁺]_ER on homotetrameric rat type 3 InsP₃R channel activity. In optimal [Ca²⁺]_i and subsaturating [InsP₃], jumps of [Ca²⁺]_ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP₃ but restored when [Ca²⁺]_ER was raised to 1.1 mM. In suboptimal [Ca²⁺]_i, jumps of [Ca²⁺]_ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca²⁺]_ER effects on channel activity exhibited a biphasic dependence on [Ca²⁺]_i. In addition, the effect of high [Ca²⁺]_ER was attenuated when a voltage was applied to oppose Ca²⁺ flux through the channel. These observations can be accounted for by Ca²⁺ flux driven through the open InsP₃R channel by [Ca²⁺]_ER, raising local [Ca²⁺]_i around the channel to regulate its activity through its cytoplasmic regulatory Ca²⁺-binding sites. Importantly, [Ca²⁺]_ER regulation of InsP₃R channel activity depended on cytoplasmic Ca²⁺-buffering conditions: it was more pronounced when [Ca²⁺]_i was weakly buffered but completely abolished in strong Ca²⁺-buffering conditions. With strong cytoplasmic buffering and Ca²⁺ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP₃R channel gating by physiological levels of [Ca²⁺]_ER were completely abolished. Collectively, these results rule out Ca²⁺ regulation of channel activity by direct binding to the luminal aspect of the channel.     

Differential functional properties of Ca stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i) are controlled by inositol 1,4,5-trisphosphate (InsP₃) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca²⁺ oscillations are regulated differentially by InsP₃ and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca²⁺ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP₃ production or activation of InsP₃ receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP₃-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca²⁺ free solutions did not prevent the ATP-induced Ca²⁺ release from InsP₃-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP₃- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca²⁺ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

The effect of ischaemia and reperfusion on sarcolemmal inositol phospholipid and cytosolic inositol phosphate metabolism in the isolated perfused rat heart

In this study the mass of polyphosphoinositides as well as the turnover of [³H]inositol phospholipids and [³H]inositol phosphates during ischaemia and short periods of reperfusion were studied in the isolated perfused rat heart. Since the phosphoinositides located within the sarcolemma are precursors for release of inositoltrisphosphate (InsP₃) and diacylglycerol, sarcolemmal membranes (rather than whole tissue) isolated at the end of the experimental procedure, were used. Hearts were prelabelled with [³H]inositol and subsequently perfused with 10 mM LiCI to block the phosphatidylinositol (PI) pathway. The results showed that 20 min of global ischaemia depressed the amount of [³H]inositol present in both sarcolemmal phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂), as well as in the cytosolic [³H]inositol phosphates, [³H]InsP₂ and [³H]InsP₃. The mass of the sarcolemmal inositol phospholipids remained unchanged during ischaemia. Reperfusion caused an immediate (within 30 sec) increase in the amount of [³H]inositol in sarcolemmal PI, PI-4-P and PI-4,5-P₂. PI-4-P levels showed a transient increase after 30 seconds postischaemic reperfusion, while the mass of the other sarcolemmal inositol phospholipids, PI and PI-4,5-P₂, remained unchanged. [³H]Insp, [³H]InsP₂ and [³H]InsP₃ also increased significantly in comparison to ischaemic hearts after only 30 sec postischaemic reperfusion.In summary, the results obtained indicate inhibition of the PI pathway during ischaemia with an immediate significant stimulation upon reperfusion. In view of the capacity of InsP3 to mobilize Ca²⁺ the possibility exists that stimulation of this pathway during reperfusion may play a role in the intracellular Ca²⁺ overload, characteristic of postischaemic reperfusion.     

The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca²⁺ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca²⁺ increases are transient and oscillatory, defining the so‐called Ca²⁺ code that links cell responses to specific Ca²⁺ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca²⁺ signals. Here, we show that by hydrolyzing phosphatidylinositol‐(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol‐(1,4,5)trisphosphate (InsP₃) levels. By modifying InsP₃ dynamics and diffusion, IpgD favors the elicitation of long‐lasting local Ca²⁺ signals at Shigella invasion sites and converts Shigella‐induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP₃‐dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca²⁺ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca²⁺‐dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.