InsP3-induced Calcium Release Research Articles

Inositol trisphosphate receptor in higher plants: is it real?

The receptor for D-myo-inositol 1,4,5-trisphosphate (InsP3-R) has been well documented in animal cells. It constitutes an important component of the intracellular calcium signalling system. Today the corresponding genes in many species have been sequenced and the antibodies against some of the InsP3-Rs are available. In contrast, very little is known about its plant counterpart. Only a few published works have dealt directly with this topic. This review summarizes the available relevant data and determines some properties of putative plant receptor(s) including the in silico search for its gene in plant genomes, in vivo evidence, its electrophysiology, the parameters of InsP3-induced calcium release and InsP3 binding, immunological cross-reactivity, and subcellular localization. Future progress in this area seems to be inevitable as, despite the efforts, its gene in plants has not been identified yet.

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate.

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.

2-Aminoethoxydiphenyl borate inhibits phototransduction and blocks voltage-gated potassium channels in Limulus ventral photoreceptors

2-Aminoethoxydiphenyl borate (2-APB) is a membrane-permeable modulator that inhibits the activation of inositol (1,4,5) trisphosphate (InsP 3) receptors, store operated channels (SOCs) and TRP channels in cells that utilize the phosphoinositide cascade for cellular signaling. In Limulus ventral photoreceptors, light-induced calcium release via the phosphoinositide cascade is thought to activate the photocurrent. Injection of either exogenous InsP 3 or calcium ions can therefore mimic excitation by light. One hundred micromolar 2-APB reversibly inhibited the photocurrent of ventral photoreceptors in a concentration-dependent manner, acting on at least two processes thought to mediate the visual cascade. 2-APB reversibly inhibited both light and InsP 3-induced calcium release, consistent with its role as an inhibitor of the InsP 3 receptor. In addition, 2-APB reversibly inhibited the activation of depolarizing current flow through the plasma membrane caused by pulsed pressure injection of calcium ions into the light-sensitive lobe of the photoreceptor. We also found that 100 μM 2-APB reversibly inhibited both transient and sustained voltage-activated potassium current during depolarizing steps. 2-APB has previously been shown to block phototransduction in Drosophila photoreceptors. The lack of specificity of the action of 2-APB in Limulus indicates that this blockade need not necessarily arise from inhibition of InsP 3-induced calcium release.

Amphetamine selectively blocks inhibitory glutamate transmission in dopamine neurons

Amphetamine is a highly addictive psychostimulant that promotes the release of the catecholamines dopamine and norepinephrine. Amphetamine-induced release of dopamine in the midbrain inhibits the activity of dopamine neurons through activation of D2 dopamine autoreceptors. Here we show that amphetamine may also excite dopamine neurons through modulation of glutamate neurotransmission. Amphetamine potently inhibits metabotropic glutamate receptor (mGluR)-mediated IPSPs in dopamine neurons, but has no effect on ionotropic glutamate receptor-mediated EPSCs. Amphetamine desensitizes the mGluR-mediated hyperpolarization through release of dopamine, activation of postsynaptic alpha1 adrenergic receptors, and suppression of InsP3-induced calcium release from internal stores. By selectively suppressing the inhibitory component of glutamate-mediated transmission, amphetamine may promote burst firing of dopamine neurons. Through this mechanism, amphetamine may enhance phasic release of dopamine, which is important in the neural processing of reward.

Rapid Coupling of Calcium Release to Depolarization inLimulus polyphemusVentral Photoreceptors as Revealed by Microphotolysis and Confocal Microscopy

Microphotolysis and confocal microscopy were used to investigate the timing of calcium release and of the electrical response in Limulus polyphemus ventral photoreceptors. The fluorescent dyes Fluo-3 and Calcium Green-5N were used to monitor local Ca2+ elevations. Photolysis of caged inositol trisphosphate (InsP3) close to the plasma membrane of the light-sensitive rhabdomeral (R-) lobe resulted in Ca2+ elevation within 10-20 msec, 20-45 msec before the physiological response to light normally would be detected. Inward ionic current flow and depolarization followed InsP3-induced calcium release within 2.5 +/- 3.3 msec. Voltage-clamping the cells and removal of extracellular Ca2+ did not affect the timing of the Ca2+ elevation that followed the photolysis of caged InsP3 or its relationship to the electrical response. In contrast to the physiological response to light, which only released calcium within the R-lobe, photolysis of InsP3 elevated Cai in both lobes, although with much greater effect in the R-lobe, as compared with the bulk of the A-lobe, suggesting the presence of InsP3-sensitive calcium stores in both lobes. Photolysis of caged calcium [o-nitrophenyl EGTA (NPE)] at the edge of the R-lobe activated an inward ionic current within 1.8 +/- 0.7 msec. This NPE-induced current reversed at a membrane potential of 10 +/- 6 mV in the range typical of that of the light-activated current under physiological conditions. Calcium release, therefore, activates an inward current rapidly enough to contribute to the electrical response to light.

Reversible Desensitization of Inositol Trisphosphate-induced Calcium Release Provides a Mechanism for Repetitive Calcium Spikes

Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. Here we show, in intact rat basophilic leukemia cells, that calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) was observed for InsP3 increases that resulted in a near maximal calcium spike and was expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse was increased 2-3-fold, desensitization was overcome and a second calcium response of equal amplitude to the first was produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization was abolished by the addition of the calcium buffers BAPTA and EGTA and could be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization was observed at a free calcium concentration of 290 nM and increased with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a "saw-tooth" parameter that controls the termination of each spike and the frequency of calcium spikes.

Regulation of InsP3-induced contractions by myoplasmic calcium in permeabilized atrial muscle

We studied the effect of calcium on inositol 1,4,5-trisphosphate (InsP3)-induced contractions in saponin-permeabilized chick atrial muscle (80-100 microns in diameter). Calcium has been proposed to modulate InsP3-induced response and InsP3 binding in other tissue. A transient increase in tension was used to detect the release of calcium in this multicellular preparation. Pulsed (2-3 s) superfusion of either calcium or InsP3 produced a transient contraction. Pulsed coapplication of both InsP3 and calcium resulted in a contraction greater than that predicted by an additive effect of both intracellular messengers. The release of calcium by InsP3 is positively and negatively modulated by myoplasmic calcium (EC50 congruent to 0.17 microM and IC50 of approximately 1.5 microM). The peak potentiation occurred at approximately pCa = 6.3 (0.51 microM free calcium). These observations indicate that an increase of InsP3 in the heart, as produced by cardiac neurotransmitters and hormones, may regulate the force of contraction by virtue of its synergistic action with calcium. While calcium remains the primary trigger for calcium release during excitation-contraction (E-C) coupling, we propose that changes in cytoplasmic calcium can modulate InsP3-induced calcium release on a beat-to-beat basis. Thus, InsP3 may regulate force generation during E-C coupling by activating calcium release during a heart beat.

Function and characteristics of repetitive calcium waves associated with meiosis

Internal calcium waves and oscillations are now recognized as universal features of cellular activation, but their exact role remains uncertain. In mammalian and ascidian eggs, a large, sperm-triggered calcium activation wave crosses the egg at fertilization, followed by a series of periodic increases in intracellular calcium concentration ([Ca2+]i). We have previously shown that, in eggs of the ascidian Phallusia mammillata, these periodic, post-activation [Ca2+]i increases are in the form of waves, the origin of which relocalizes to a pacemaker region, and that they stop seconds before the completion of meiosis. We show here that the origin of the first one to four post-activation calcium waves in P. mammillata eggs transfers progressively from the site of sperm entry, usually in the animal hemisphere, towards an endoplasmic reticulum (ER)-rich contraction pole in the vegetal hemisphere, a process that takes about five minutes. Once the origin of these repetitive post-activation calcium waves has reached the contraction pole, all subsequent calcium waves originate from the domain of ER concentrated there, which acts as a pacemaker. The first few post-activation calcium waves are faster than the activation wave and, like the activation wave, they propagate homogeneously throughout the cytoplasm. Approximately five to ten minutes after fertilization, the post-activation calcium waves begin to propagate preferentially in the egg cortex. By manipulating intracellular calcium levels with caged inositol 1,4,5 trisphosphate (InsP3) and a competitive inhibitor of InsP3-induced calcium release, we show that the activation wave induced by the sperm is sufficient to induce extrusion of the first polar body, but that additional [Ca2+]i increases are necessary for completion of the second meiotic division. However, periodic calcium waves per se do not seem to be strictly necessary for the completion of meiosis, as a persistent and homogeneous increase in calcium, induced by the calcium ionophore ionomycin, is sufficient to cause second polar body formation and allow completion of meiosis on time. These results clearly show that, in the ascidian egg, post-activation calcium waves are required to complete meiosis. They also show that following a period of progressive relocalization of the wave origin, which lasts approximately five minutes, an ER-rich domain at the contraction pole finally becomes a pacemaker from which the calcium waves originate. Once their origin becomes stably localized, the calcium waves begin to propagate preferentially around the cortex of the egg rather than throughout the egg cytoplasm.

Exit from mitosis induced by a calcium transient: the relation to the MPF and InsP 3 dynamics

Cells divide only after passing through a control point in late G 1. This passage is followed by the accumulation of the mitotic cyclin which binds to p34 cdc2 , allowing for the subsequent phosphorylation and dephosphorylation of the latter protein. It is the active MPF, i.e. the phosphorylated mitotic cyclin-p34 cdc2 kinase complex that triggers entry into mitosis. MPF becomes increasingly active as the cell forwards to anaphase, when a sudden increase in InsP 3 takes place. This in turn induces a large Ca 2+ release in cytosol from internal calcium stores and a calcium-dependent positive feedback control on InsP 3-induced calcium release enhances the effect on InsP 3-mediated Ca 2+ transient. Meanwhile, empty calcium stores signal to plasma membrane for a constant calcium influx at high InsP 3 levels. The cytosolic calcium excess is assumed to activate the CaMK II holloenzyme which involves the production of the ubiquitination complex necessary for cyclin degradation and MPF inactivation. Accordingly, a mathematical model was proposed by means of an eight-dimensional dynamical system that yields the time dependence of the main cellular quantities in a picture of the mitosis specific events.

Ligation of CD23 triggers cyclic AMP generation in human B lymphocytes.

The low affinity IgE receptor CD23 may play a role in several B lymphocyte functions, such as cell activation and multiplication, Ag presentation, and IgE production. We have previously reported that ligation of the CD23 molecule with anti-CD23 mAb, or IgE-anti-IgE complexes, leads to phosphoinositide hydrolysis and calcium mobilization through the generation of Inositol (1,4,5) trisphosphate via a process involving a Pertussis toxin insensitive GTP-binding protein. In our work, we show that anti-CD23 mAb elicit an increase in cAMP concentration in human peripheral blood-derived B lymphocytes. This effect was detected both in resting and in IL-4-stimulated B cells displaying, respectively, low and high levels of CD23. Maximum cAMP accumulation was reached about 20 min after addition of the mAb. Involvement of Fc gamma RII in this process could be excluded because cAMP increase was also triggered by mAb anti-CD23 F(ab')2 fragments. Accumulation of cAMP was also observed when IgE-sensitized activated B lymphocytes were challenged with the specific hapten. Several lines of evidence indicate that the cAMP increase after CD23 ligation may result, in part, from the stimulation of phosphoinositidase C, inasmuch as it was markedly impaired by treatment with TMB-8, an inhibitor of InsP3-induced calcium release from intracytoplasmic stores and with BAPTA, an intracellular calcium chelator. Addition of GTP-gamma S to permeabilized B cells or to membrane preparations did not potentiate the effect of the mAb, suggesting that a Gs protein is not directly implicated in the generation of cAMP. Besides, cAMP accumulation is not due to the production of PG because it is not modified by indomethacin, an inhibitor of the cyclooxygenase pathway. Pretreatment of B lymphocytes with either anti-CD23 mAb or IL-4 led to autologous as well as heterologous desensitization. This negative cross-talk, at the level of cAMP, between the signaling pathways triggered by ligation of CD23 and of the IL-4 receptor, could contribute to the inhibitory effect of anti-CD23 mAb on IL-4-dependent B cell activation and differentiation.

The effects of amino acid-reactive reagents on the functioning of the inositol 1,4,5-trisphosphate-sensitive calcium channel from rat cerebellum

Covalent modification of arginine residues with phenylglyoxal and lysine residues with 4,4′-diisothiocyano-2,2′-disulphonic acid stilbene (DIDS) was carried out in preparations of rat cerebellar microsomes, as was the interaction of silver ions (silver nitrate) with cysteine residues. The effects of these amino acid-reactive agents on [ 3H]inositol 1,4,5-triphosphate (InsP 3) binding and InsP 3-induced calcium release were assessed. Both phenylglyoxal and DIDS inhibited [ 3H]InsP 3 binding and InsP 3-induced calcium release (IC 50 = 2.0 mM and 18 μM, respectively). Silver ions inhibited InsP 3-induced Ca 2+ release alone (IC ≈ 1 μM). These results suggest that arginine and lysine residues may be located at or close to the InsP 3 binding site of the InsP 3 receptor, while cysteine residues are important in channel opening.

Intracellular Signaling Events Associated with the Induction of DNA Synthesis in Human B Lymphocytes: I. Stimulation of PKC-Dependent and -Independent Pathways by LMW-BCGF

The low molecular weight B cell growth factor (LMW-BCGF) induces the G1 → S transition in human B lymphocytes activated by a first signal, Stāphylococcus aureus Cowan (SAC) or anti-μ antibody. It also stimulates proliferation of normal long-term B cell lines and some B cell tumors. We have previously reported that LMW-BCGF induces the hydrolysis of polyphospho-inositides (PI) and a rise in intracellular free calcium concentration, through the generation of inositol trisphosphate (InsP 3) (Renard et al., Eur, J. Immunol. 18, 1705, 1988). In the present work we have analyzed the possible association between early signaling events elicited by LMW-BCGF in SAC-activated B cells and its ability to provoke DNA synthesis, notably at the level of phospholipase C (PLC) and protein kinase C (PK-C) activation. Inhibitors of PLC and of InsP 3-induced calcium release were found to block LMW-BCGF-dependent DNA synthesis. An increase in membrane-associated protein kinase C (PK-C) activity was detected after the addition of the growth factor and the mitogenic effect of LMW-BCGF was partially suppressed when B cell blasts were incubated with staurosporine or H-7, two inhibitors of PK-C activity. In addition, the mitogenic effect due to the addition of LMW-BCGF was not modified by the incubation of B cell blasts with high concentrations of TPA, even if this treatment inhibited cellular response to a low concentration of TPA. LMW-BCGF also increased intracellular pH in B cell blasts and lymphokine-induced mitogenic activity was reduced when the Na +/H + amiloride or ethylisopropyl amiloride (EIPA) antiport blockers were added. These results suggest that (i) LMW-BCGF-induced PI breakdown and Ca 2+ mobilization and cell alkalinization are associated with the induction of cell proliferation, and (ii) the activation of PK-C does not appear to be the sole pathway activated by LMW-BCGF.

Intracellular Signaling Events Associated with the Induction of DNA Synthesis in Human B Lymphocytes: II. Different Pathways Triggered by IL-2 and IL-4

In attempts to detect associations between early signaling events triggered by interleukins and the induction of DNA synthesis, inhibitors of various second messenger pathways were tested for their effects on IL-2- and IL-4-elicited mitogenesis in preactivated human B lymphocytes. Inhibitors of phosphoinositidase C and of InsP 3-induced calcium release suppressed IL-4- but not IL-2-mediated proliferation. The response to both lymphokines was also impaired by an inhibitor of the calcium/calmodulin complex and was modulated by variations of the [Ca 2+] i. PKC inhibitors and PK-C depletion did not significantly alter the response to IL-2 and IL-4. The response to IL-2, but not to IL-4, was inhibited by cAMP analogues or by agents that raise cAMP. In contrast, IL-4, but not IL-2, stimulated cAMP accumulation in activated B cells. Taken together, these observations indicate that IL-2 and IL-4 use different signaling pathways to induce the G1 → S transition in these cells and suggest that the IL-4 inhibition of the B cell response to IL-2 may result from its effect on cAMP generation.

Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum.

The calcium indicator dye fluo-3/AM was loaded into the ER of isolated cortices of unfertilized eggs of the sea urchin Arbacia punctulata. Development of the fluorescent signal took from 8 to 40 min and usually required 1 mM ATP. The signal decreased to a minimum level within 30 s after perfusion with 1 microM InsP3 and increased within 5 min when InsP3 was replaced with 1 mM ATP. Also, the fluorescence signal was lowered rapidly by perfusion with 10 microM A23187 or 10 microM ionomycin. These findings demonstrate that the cortical ER is a site of ATP-dependent calcium sequestration and InsP3-induced calcium release. A light-induced wave of calcium release, traveling between 0.7 and 2.8 microns/s (average speed 1.4 microns/s, N = 8), was sometimes observed during time lapse recordings; it may therefore be possible to use the isolated cortex preparation to investigate the postfertilization calcium wave.

Feedback inhibition by calcium limits the release of calcium by inositol trisphosphate in Limulus ventral photoreceptors

Injection of inositol 1,4,5 trisphosphate (InsP 3) into Limulus ventral photoreceptors elevates the concentration of intracellular calcium ions and as a consequence depolarizes the photoreceptor. This InsP 3-induced elevation can be inhibited by a prior injection of calcium or InsP 3 delivered 1 s earlier. Recovery from this inhibition has a half-time of between 1.5 and 5 s at 20°C. Calcium released by (InsP 3) therefore inhibits further release of calcium from InsP 3-sensitive calcium stores. This feedback inhibition may protect the calcium stores from depletion during prolonged bright illumination. Feedback inhibition, rather than periodic depletion of calcium stores, may also underlie the oscillatory bursts of InsP 3-induced calcium release that have been observed in many cell types.

The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.

An examination of the ability ofinositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea

We have examined the ability of inositol 1,4,5-trisphosphate (InsP 3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP 3 (10–500 μM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impenneant anion by Cl −, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP 3. InsP 3 did not modify responses to caffeine or calcium-induced calcium release. Free Mg 2+ was lowered to 40 μM and 15 mM D-2,3-diphosphoglycerate was added in order to inhibit the possible breakdown of InsP 3 by inositol trisphosphatase. Neither measure revealed a response to InsP 3. Arsenazo III absorbance measurements failed to detect any binding of Mg 2+ (0–0.5 mM) by 0.35 mM InsP 3 in our solutions. Inhibitors of SR calcium uptake (cadmium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4° to 22°C also failed to reveal a response of frog skinned fibres to InsP 3. The nucleotide GTP, which has been reported to enhance InsP 3-induced calcium release from rat liver microsomes, had no effect at 50 μM on the response of frog fibres to InsP 3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP 3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP 3 as an essential link in excitation-contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.