Mobilization of Ca2+ by cyclic ADP-ribose from the endoplasmic reticulum of cauliflower florets

The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling.     

Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae.

Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+.     

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.     

Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of oat roots

In plant cells, transient changes in cytoplasmic Ca2+ levels can modulate numerous developmental processes. Ca2+ is accumulated in the vacuole via a H+/Ca2+ antiport system that is energized by the tonoplast H+-pumping ATPase. Inositol 1,4,5-triphosphate (InsP3), but not inositol 1,4-bisphosphate, myo-inositol 1-phosphate, or fructose 2,6-bisphosphate, caused a transient reduction of Ca2+ levels in tonoplast vesicles. The decrease was dependent on InsP3 concentration (Km apparent = 0.6 microM). The InsP3-induced Ca2+ release was blocked by the Ca2+ antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl. These results suggest that the vacuolar membrane is one target site for InsP3 action and that InsP3 may operate as a second messenger in the mobilization of intracellular Ca2+ in plant cells.     

Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Translocation of Ca2+ into mitochondria from a functionally discrete portion of the nonmitochondrial store

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.     

Identification of intracellular calcium pools. Selective modification by thapsigargin

Recent studies have identified inositol 1,4,5-tris-phosphate(InsP3)-sensitive and -insensitive Ca2+ pools and a GTP-dependent mechanism that transfers Ca2+ between them. Here, the Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to distinguish these two Ca2+ pools and identify a third Ca2+ pumping pool unresponsive to InsP3 or GTP. Using saponin-permeabilized DDT1MF-2 smooth muscle cells, approximately 75% of total intracellular ATP-dependent Ca2+ accumulation is blocked by thapsigargin with an IC50 of 30 nM. In contrast, 1 mM vanadate or 5 microM A23187 block 100% of Ca2+ accumulation. The thapsigargin-responsive Ca2+ pool corresponds exactly to that released by 10 microM InsP3 in the presence of 10 microM GTP. Indeed, addition of InsP3 with GTP has no effect on Ca2+ accumulated in the presence of 3 microM thapsigargin whereas A23187 releases all the remaining Ca2+. Added after maximal Ca2+ uptake, thapsigargin induces only slow Ca2+ release consistent with blockade of pumping activity. Unlike InsP3, the action of thapsigargin is entirely heparin insensitive. The large increment in Ca2+ uptake caused by 12 mM oxalate is completely reversed by thapsigargin, indicating that thapsigargin functions on an oxalate-permeable pool. Moreover, the still larger uptake induced by GTP in the presence of oxalate is also completely reversed by either thapsigargin or InsP3. The results indicate that thasigargin blocks Ca2+ uptake into two discrete pools: the InsP3-sensitive, oxalate-permeable Ca2+ pool and the InsP3-insensitive, oxalate-impermeable Ca2+ pool that can be "recruited" into the InsP3-sensitive pool by GTP-dependent Ca2+ translocation (Ghosh, T. K., Mullaney, J.M., Tarazi, F.I., and Gill, D.L. (1989) Nature 340, 236-239). Additionally, a third Ca2+ pool is defined, unreleasable by InsP3 or GTP, and containing a thapsigargin-insensitive Ca2+ pump.     

Subcellular localization of a high affinity binding site for D-myo-inositol 1,4,5-trisphosphate from Chenopodium rubrum

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the binding of D-myo-inositol 1,4,5-trisphosphate (InsP(3)) to a receptor located on the endoplasmic reticulum (ER) triggers an efflux of calcium release from the ER. Sites that bind InsP(3) with high affinity and specificity have also been described in plant cells, but their precise intracellular locations have not been conclusively identified. In contrast to animal cells, it has been suggested that in plants the vacuole is the major intracellular store of calcium involved in signal induced calcium release. The aim of this work was to determine the intracellular localization of InsP(3)-binding sites obtained from 3-week-old Chenopodium rubrum leaves. Microsomal membranes were fractionated by sucrose density gradient centrifugation in the presence and absence of Mg(2+) and alternatively by free-flow electrophoresis. An ER-enriched fraction was also prepared. The following enzymes were employed as specific membrane markers: antimycin A-insensitive NADH-cytochrome c reductase for ER, cytochrome c oxidase for mitochondrial membrane, pyrophosphatase for tonoplast, and 1,3-beta-D-glucansynthase for plasma membrane. In all membrane separations, InsP(3)-binding sites were concentrated in the fractions that were enriched with ER membranes. These data clearly demonstrate that the previously characterized InsP(3)-binding site from C. rubrum is localized on the ER. This finding supports previous suggestions of an alternative non-vacuolar InsP(3)-sensitive calcium store in plant cells.     

Ca2+ currents and acid secretion in the isolated parietal cell involved in response to gastrin, compound 48/80, and ethylenediamine tetraacetic acid

In intact guinea pig parietal cells, gastrin or compound 48/80 caused an initial increase in cytosolic Ca2+ concentration and subsequent acid secretion, owing to release of intracellulary stored Ca2+ besides the Ca2+ entry from the extracellular space. However, the maximum gastrin-induced Ca2+ entry into the cell was delayed by 60 min, a time which coincided with sustained acid secretion (by gastrin) that was dependent on medium Ca2+. On the other hand, there are two ATP-dependent Ca2+-removal systems detected in either plasmalemma or smooth surfaced membrane besides that of mitochondria. The plasmalemmal Ca2+-removal system was dependent on calmodulin. Smooth surfaced membrane vesicles caused an ATP-dependent Ca2+ uptake that was almost similar to that taken by saponin-permiabilized cell. In this system (permeable cell), myo-inositol 1,4,5-triphosphate (InsP3) caused the release of ATP-accumulated Ca2+ into the cytosol, suggesting an ATP-dependent and InsP3-sensitive Ca2+ pool(s) is in or near the smooth surfaced membranes. The ATP-dependent Ca2+ uptake by vesicles was markedly enhanced by the stimulation of cells with gastrin, compound 48/80, or EDTA. The increase of this Ca2+ uptake in stimulated cells by plasmalemmal vesicles exceeded that by smooth surfaced ones. The increase of the Ca2+ uptake by plasmalemmal vesicles was abolished by the cease of intracellular Ca2+ release without Ca2+ entry. In addition, gastrin or compound 48/80 evoked an early Ca2+ efflux across the plasma membrane owing to a pump that was independent of medium Ca2+ in intact cells. These results suggest that in the first acid secretion by gastrin or others, the Ca2+ released, which may be derived from an ATP-dependent and InsP3-sensitive Ca2+ pool, is mainly pumped out by the plasmalemmal Ca2+-removal system rather than the intracellular Ca2+-removal system; whereas the sustained acid secretion by gastrin required medium Ca2+ and in this phase, Ca2+ efflux across the plasma membrane became lower, suggesting that an ATP-dependent Ca2+ pool may be replenished by Ca2+ entering from the extracellular space.     

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from the InsP3-sensitive store and, additionally, that the Ca2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca2+]i to approximately 50 nM above basal that was independent of extracellular Ca2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.     

Inositol polyphosphates regulate Ca2+ efflux in a cardiac membrane subtype distinct from junctional sarcoplasmic reticulum

The membrane location and mechanism of inositol 1,3,4,5-tetrakisphosphate (InsP4)-regulated Ca2+ uptake in cardiac membrane vesicles was investigated. In canine and rat membranes separated by sucrose density gradient centrifugation, InsP4-regulated Ca2+ uptake was slightly more enriched in low density than in higher density membranes. Membranes supporting InsP4-regulated Ca2+ uptake were correspondingly enriched in type 1 InsP3 receptors. Junctional sarcoplasmic reticulum (J-SR), enriched in sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and ryanodine receptors, separated predominantly with higher density membranes. In membranes supporting InsP4-regulated Ca2+ uptake, Ca2+ uptake was facilitated by a high Ca2+ affinity carrier that was insensitive to thapsigargin. Ca2+ uptake in J-SR was mediated by thapsigargin-sensitive SERCA2a. Net Ca accumulation was enhanced by oxalate in both SR subtypes. Although Ca2+-carrier-mediated Ca2+ uptake was ATP independent, ATP indirectly regulated net Ca2+ accumulation by modifying Ca2+ efflux via a Ca2+ channel with properties of type 1 InsP3 receptors. In the presence of < or = 0.1 mM ATP, InsP4 enhanced Ca2+ accumulation whereas InsP4 inhibited Ca2+ uptake at higher ATP concentrations. In the presence of 0.15 mM ATP, InsP4 stimulated Ca2+ efflux from vesicles preloaded with Ca. Several other InsP4 isomers and 1,3,4-InsP3 also stimulated Ca2+ efflux but with slightly less potency than 1,3,4,5-InsP4. Ruthenium red enhanced net Ca accumulation by the Ca2+ carrier and reduced the potency of ATP, InsP4, and InsP3 to stimulate Ca2+ efflux in vesicles. In summary, this investigation shows that a Ca2+ carrier facilitates Ca loading in a sarcoplasmic reticulum subtype distinct from J-SR. InsP4 and InsP3 are proposed to regulate Ca2+ efflux in low density SR by acting on an ATP-modulated Ca2+ channel with properties of type 1 InsP3 receptors.     

Regenerative release of calcium from functionally discrete subcellular stores by inositol trisphosphate.

Fluorescence imaging was used to determine the spatial and temporal patterns of subcellular calcium (Ca2+) liberation induced in Xenopus oocytes by photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. Increasing levels of InsP3 evoked Ca2+ release that began in a graded manner but, at varying threshold levels of InsP3, localized sites then showed transient and asynchronous 'puffs' of Ca2+ release. With higher levels of InsP3, Ca2+ from adjacent sites formed a focus for initiation of a propagating Ca2+ wave. The results show that InsP3-sensitive Ca2+ stores are arranged as distinct and functionally independent units, and that Ca2+ is released in both graded and regenerative fashions.     

Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage protein chromogranin A

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.     

Distinct occurrence of phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release and inositol 1,4,5-triphosphate-induced release in ATP-dependent Ca2+-transporting platelet microsomes

The effects of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and inositol 1,4,5-triphosphate(InsP3) on the Ca2+ release from ATP-dependent Ca2+-transporting microsomes prepared from ox platelets were investigated. Under optimal conditions, both PtdInsP2 and InsP3 released Ca2+ from the microsomes in a similar dose-dependent manner. However, the maximal amount of Ca2+ released by InsP3 was almost one-fourth of that released by PtdInsP2. Neither PtdInsP2 nor InsP3 appeared to act as a Ca2+ ionophore since they showed no effect on the Ca2+ content of liposomes prepared from platelet microsomal lipids. InsP3-induced but not PtdInsP2-induced Ca2+ release was decreased with increasing extravesicular Ca2+ from 0.1 microM to 10 microM and it was completely inhibited by 10 microM Ca2+. PtdInsP2-induced but not InsP3-induced Ca2+ release was markedly inhibited by Mg2+, ruthenium red and neomycin. In addition, InsP3 could induce no additional Ca2+ release after the accumulated Ca2+ had been maximally released by PtdInsP2. These results indicate that PtdInsP2 releases Ca2+ from platelet microsomes more effectively than InsP3 by a mechanism distinct from that of InsP3-induced release, and further that InsP3-sensitive microsomes are included within the population of PtdInsP2-sensitive microsomes.     

NAADP and InsP3 play distinct roles at fertilization in starfish oocytes

NAADP participates in the response of starfish oocytes to sperm by triggering the fertilization potential (FP) through the activation of a Ca2+ current which depolarizes the membrane to the threshold of activation of the voltage-gated Ca2+ channels. The aim of this study was to investigate whether this Ca2+ influx is linked to the onset of the concomitant InsP3-mediated Ca2+ wave by simultaneously employing Ca2+ imaging and single-electrode intracellular recording techniques. In control oocytes, the sperm-induced membrane depolarization always preceded by a few seconds the onset of the Ca2+ wave. Strikingly, the self-desensitization of NAADP receptors either abolished the Ca2+ response or resulted in abnormal oocyte activation, i.e., the membrane depolarization followed the Ca2+ wave and the oocyte was polyspermic. The inhibition of InsP3 signaling only impaired the propagation of the Ca2+ wave and shortened the FP. The duration of FP was also reduced in low-Na+ sea water. Finally, uncaged InsP3 produced a Ca2+ increase, which depolarized the membrane upon the activation of a Ca2+-sensitive cation current. These results support the hypothesis that Ca2+ entry during the NAADP-triggered FP is required for the onset of the Ca2+ wave at fertilization. The InsP3-mediated Ca2+ wave, in turn, may interact with the NAADP-evoked depolarization by activating a Ca2+-dependent Na+ entry.     

Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes

We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.     

Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes.

Stimuli which act through the second messenger inositol 1,4,5-trisphosphate (InsP3) often increase free intracellular Ca2+ concentration ([Ca2+]i) in a localized subcellular area. Actively propagated Ca2+ waves then extend this focal Ca2+ signal to other parts of the cell. To understand how cells may control the spatial distribution of Ca2+, we investigated the mechanism by which Ca2+ waves propagate through the cytoplasm of Xenopus oocytes. Heparin, which inhibits the binding of InsP3 to its receptor, prevented the migration of Ca2+ waves induced by a poorly metabolized InsP3 (InsP3S3). This result suggested that Ca2+ waves move through the cell via the serial release of Ca2+ from InsP3-sensitive stores. Interventions which caused a localized increase in [Ca2+]i without elevations of InsP3 did not trigger Ca2+ waves. In the presence of a Ins-P3S3, however, endogenously released or locally injected Ca2+ elicited Ca2+ waves. A cooperative interaction between Ca2+ and InsP3 may therefore be responsible for the propagation of Ca2+ waves.     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.