Characteristics of bile acid-mediated Ca2+ release from permeabilized liver cells and liver microsomes

Saponin-treated liver cells and a microsomal fraction were used to characterize the mechanism of the Ca2+ release induced by different bile acids. The saponin-treated cells accumulated 0.8-1 nmol/mg of protein of the medium Ca2+ in a nonmitochondrial, high affinity, and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool. Three of five bile acids tested, lithocholate and the conjugates taurolithocholate and taurolithocholate sulfate, released 85% of the Ca2+ pool within 45-60 s and with ED50 from 16 to 28 microM. Ins(1,4,5)P3 released 80% from the same Ca2+ pool with an ED50 of 0.3 microM. The Ca2+-Mg2+-ATPase inhibitor vanadate (1 mM) had no effect on the Ca2+ released by the bile acids and Ins(1,4,5)P3. The Ins(1,4,5)P3-binding antibiotic neomycin (1 mM) and the receptor competitor heparin (16 micrograms/ml) abolished the releasing effect of Ins(1,4,5)P3 but had no effect on the bile acid-mediated Ca2+ release. The 45Ca2+ accumulated by the microsomal fraction (8 nmol of 45Ca2+/mg of protein) was released by the bile acids within 45-90 s and with an ED50 of 17 microM. In contrast, the bile acids had no effect on the Ca2+ permeability of other natural and artificial membranes. The resting 45Ca2+ influx of intact cells (0.45 nmol/mg of protein/min), the 45Ca2+ accumulated by mitochondria (2-13 nmol of 45Ca2+/mg of protein), and the 45Ca2+ trapped in sonicated phosphatidylcholine vesicles (5 mM 45Ca2+) were not altered by the different bile acids. These results suggest that the Ca2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool. It is not mediated by Ins(1,4,5)P3 or via activation of the Ins(1,4,5)P3 receptor, and it is specific for the membrane of the internal pool.     

Bile acids mobilise internal Ca2+ independently of external Ca2+ in rat hepatocytes

In the present study, we investigated the possible role of external Ca2+ in the rise of the cytosolic Ca+ concentration induced by the monohydroxy bile acid taurolithocholate in isolated rat liver cells. The results showed that: (a) the bile acid promotes the same dose-dependent increase in the cytosolic Ca+ concentration (half-maximal effect at 23 microM) in hepatocytes incubated in the presence of 1.2 mM Ca2+ or 6 microM Ca2+; (b) taurolithocholate is able to activate the Ca2(+)-dependent glycogen phosphorylase a by 6.3-fold and 6.0-fold in high and low Ca2+ media, respectively; (c) [14C]taurolithocholate influx is not affected by external Ca2+, and 45Ca2+ influx is not altered by taurolithocholate. These results establish that the effects of taurolithocholate on cell Ca2+ do not require extracellular Ca2+ and are consistent with the view that monohydroxy bile acids primarily release Ca2+ from the endoplasmic reticulum in the liver.     

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.     

Inositol trisphosphate, calcium and muscle contraction

The identity of organelles storing intracellular calcium and the role of Ins(1,4,5)P3 in muscle have been explored with, respectively, electron probe X-ray microanalysis (EPMA) and laser photolysis of 'caged' compounds. The participation of G-protein(s) in the release of intracellular Ca2+ was determined in saponin-permeabilized smooth muscle. The sarcoplasmic reticulum (SR) is identified as the major source of activator Ca2+ in both smooth and striated muscle; similar (EPMA) studies suggest that the endoplasmic reticulum is the major Ca2+ storage site in non-muscle cells. In none of the cell types did mitochondria play a significant, physiological role in the regulation of cytoplasmic Ca2+. The latency of guinea pig portal vein smooth muscle contraction following photolytic release of phenylephrine, an alpha 1-agonist, is 1.5 +/- 0.26 s at 20 degrees C and 0.6 +/- 0.18 s at 30 degrees C; the latency of contraction after photolytic release of Ins(1,4,5)P3 from caged Ins(1,4,5)P3 is 0.5 +/- 0.12 s at 20 degrees C. The long latency of alpha 1-adrenergic Ca2+ release and its temperature dependence are consistent with a process mediated by G-protein-coupled activation of phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) hydrolysis. GTP gamma S, a non-hydrolysable analogue of GTP, causes Ca2+ release and contraction in permeabilized smooth muscle. Ins(1,4,5)P3 has an additive effect during the late, but not the early, phase of GTP gamma S action, and GTP gamma S can cause Ca2+ release and contraction of permeabilized smooth muscles refractory to Ins(1,4,5)P3. These results suggest that activation of G protein(s) can release Ca2+ by, at least, two G-protein-regulated mechanisms: one mediated by Ins(1,4,5)P3 and the other Ins(1,4,5)P3-independent. The low Ins(1,4,5)P3 5-phosphatase activity and the slow time-course (seconds) of the contractile response to Ins(1,4,5)P3 released with laser flash photolysis from caged Ins(1,4,5)P3 in frog skeletal muscle suggest that Ins(1,4,5)P3 is unlikely to be the physiological messenger of excitation-contraction coupling of striated muscle. In contrast, in smooth muscle the high Ins(1,4,5)P3-5-phosphatase activity and the rate of force development after photolytic release of Ins(1,4,5)P3 are compatible with a physiological role of Ins(1,4,5)P3 as a messenger of pharmacomechanical coupling.     

The inositol 1,4,5-trisphosphate-binding site in adrenal cortical cells is distinct from the endoplasmic reticulum

The distribution of binding sites for the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was investigated in subcellular fractions of bovine adrenal cortex. The [3H]Ins(1,4,5)P3-binding capacity was enriched in the microsomal fraction, which contained a single class of high affinity binding sites with a Kd of 21.6 +/- 3.0 nM. The specific [3H]Ins(1,4,5)P3 binding appeared to be sharply pH dependent and was inhibited by millimolar concentrations of ATP. Upon fractionation of microsomes on sucrose density gradient there was a clearcut separation of the Ins(1,4,5)P3 receptor-containing fractions from those enriched in specific endoplasmic reticulum markers such as sulfatase C activity or RNA content. The microsomes enriched in Ins(1,4,5)P3-binding sites were of lower density than the endoplasmic reticulum and co-purified partly with the plasma membrane. In addition, Ins(1,4,5)P3-sensitive 45Ca2+ uptake into the microsomes was maximal in the lighter fractions. This distinction between Ins(1,4,5)P3-binding sites and endoplasmic reticulum-derived microsomes was confirmed upon fractionation according to their electrophoretic mobilities by free flow electrophoresis. These results indicate that in adrenal cortical cells, the source of Ca2+ mobilized by Ins(1,4,5)P3 upon stimulation with an agonist is not located in the endoplasmic reticulum. Our data support the hypothesis that a specialized vesicular organelle, distinct from endoplasmic reticulum and in close apposition with the plasma membrane, is involved in intracellular Ca2+ homeostasis.     

Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells

The effects of four bile acids on cell Ca2+ were examined in suspensions of isolated rat hepatocytes. Taurolithocholate and lithocholate which inhibit bile secretion increased the cytosolic Ca2+ concentration (ED50, 25 microM), as measured by the fluorescent indicator quin2, and promoted a net loss of Ca2+ from the cells. This effect resulted from rapid mobilization of Ca2+ from an intracellular Ca2+ store. This store corresponds to the one that is permeabilized by the inositol (1,4,5)trisphosphate-dependent hormone vasopressin. However, taurolithocholate and lithocholate, unlike the hormone, did not induce a significant accumulation of inositol trisphosphate fraction in isolated hepatocytes. In addition, these agents did not alter the cell and the mitochondria membrane permeability to ions. When applied to saponin-permeabilized cells, taurolithocholate and lithocholate released Ca2+ (ED50, 20 microM) from an ATP-dependent, nonmitochondrial pool which is sensitive to inositol (1,4,5)trisphosphate. In contrast, the bile acids taurocholate and cholate, which increase bile secretion, had no effect on cell Ca2+ in intact hepatocytes or in saponin-permeabilized hepatocytes. It is suggested that taurolithocholate and lithocholate permeabilize the endoplasmic reticulum to Ca2+ and that the resulting permeabilization of this compartment may be involved in the inhibition of bile secretion in mammalian liver.     

Enhancement of the inositol 1,4,5-trisphosphate-releasable Ca2+ pool by GTP in permeabilized hepatocytes

Permeabilized rat hepatocytes were used to study the effects of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP on Ca2+ uptake and release by ATP-dependent intracellular Ca2+ storage pools. Under conditions where these Ca2+ pools were completely filled, maximal doses of Ins(1,4,5)P3 released only 25-30% of the sequestered Ca2+. The residual Ca2+ was freely releasable with the Ca2+ ionophore ionomycin. Addition of GTP in the absence of Ins(1,4,5)P3 did not cause Ca2+ release and had no effect on the steady-state level of Ca2+ accumulation by intracellular storage pools. However, after a 3-4-min treatment with GTP the size of the Ins(1,4,5)P3-releasable Ca2+ pool was increased by about 2-fold, with a proportional decrease in the residual Ca2+ available for release by ionomycin. In contrast to the situation with freshly permeabilized cells, permeabilized hepatocytes from which cytosolic components had been washed out exhibited direct Ca2+ release in response to GTP addition. The potentiation of Ins(1,4,5)P3-induced Ca2+ release by GTP in permeabilized hepatocytes was concentration-dependent with half-maximal effects at about 5 microM GTP. The dose response to Ins(1,4,5)P3 was not shifted by GTP; instead GTP increased the amount of Ca2+ released at all Ins(1,4,5)P3 concentrations. The effects of GTP were not mimicked by other nucleotides or nonhydrolyzable GTP analogues. In fact, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) inhibited the actions of GTP. However, this inhibition only occurred when GTP gamma S was added prior to GTP, suggesting that the GTP effect is not readily reversible once the cells have been permeabilized. Experiments using vanadate to inhibit the ATP-dependent Ca2+ uptake pump showed that Ins(1,4,5)P3 releases all of the Ca2+ within the Ins(1,4,5)P3-sensitive Ca2+ pool even in the absence of GTP. The increase of Ins(1,4,5)P3-induced Ca2+ release brought about by GTP was also unaffected by vanadate. It is concluded that GTP increases the proportion of the sequestered Ca2+ which is available for release by Ins(1,4,5)P3, either by unmasking latent Ins(1,4,5)P3-sensitive Ca2+ release sites or by allowing direct Ca2+ movement between Ins(1,4,5)P3-sensitive and Ins(1,4,5)P3-insensitive Ca2+ storage pools.     

Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group.

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.     

Caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release from the endoplasmic reticulum in honeybee photoreceptors

Light stimulation of invertebrate microvillar photoreceptors causes a large rapid elevation in Cai, shown previously to modulate the adaptational state of the cells. Cai rises, at least in part, as a result of Ins(1,4,5)P3-induced Ca2+ release from the submicrovillar endoplasmic reticulum (ER). Here, we provide evidence for Ca(2+)- induced Ca2+ release (CICR) in an insect photoreceptor. In situ microphotometric measurements of Ca2+ fluxes across the ER membrane in permeabilized slices of drone bee retina show that (a) caffeine induces Ca2+ release from the ER; (b) caffeine and Ins(1,4,5)P3 open distinct Ca2+ release pathways because only caffeine-induced Ca2+ release is ryanodine sensitive and heparin insensitive, and because caffeine and Ins(1,4,5)P3 have additive effects on the rate of Ca2+ release; (c) Ca2+ itself stimulates release of Ca2+ via a ryanodine-sensitive pathway; and (d) cADPR is ineffective in releasing Ca2+. Microfluorometric intracellular Ca2+ measurements with fluo-3 indicate that caffeine induces a persistent elevation in Cai. Electrophysiological recordings demonstrate that caffeine mimics all aspects of Ca(2+)-mediated facilitation and adaptation in drone photoreceptors. We conclude that the ER in drone photoreceptors contains, in addition to the Ins(1,4,5)P3-sensitive release pathway, a CICR pathway that meets key pharmacological criteria for a ryanodine receptor. Coexpression of both release mechanisms could be required for the production of rapid light-induced Ca2+ elevations, because Ca2+ amplifies its own release through both pathways by a positive feedback. CICR may also mediate the spatial spread of Ca2+ release from the submicrovillar ER toward more remote ER subregions, thereby activating Ca(2+)-sensitive cell processes that are not directly involved in phototransduction.     

Rapid formation of inositol 1,4,5-trisphosphate in rat pancreatic acini stimulated by carbamylcholine

Cholinergic stimulation of inositol phosphate formation was studied in isolated rat pancreatic acini, prelabelled with myo-[2-3H]inositol. Carbamylcholine increased incorporation of radioactivity into Ins(1,4,5)P3 and InsP4 within 5 s. Increases in [3H]Ins(1,3,4)P3 were delayed with marked stimulation occurring between 10 s and 1 min. Inositol polyphosphate formation was less sensitive to carbamylcholine concentration than was stimulation of amylase release. At a low (0.3 microM) carbamylcholine concentration, no increase in inositol polyphosphate formation was detected, whereas stimulation of amylase release, which was not dependent on extracellular calcium, was observed. Ins(1,4,5)P3 was shown to release actively accumulated 45Ca2+ from isolated rough endoplasmic reticulum membranes to a similar extent as that released from rough endoplasmic reticulum following cholinergic stimulation of pancreatic acini (Richardson, A.E. et al. (1984) Biochem. Soc. Trans. 12, 1066-1067). The data is consistent with Ins(1,4,5)P3 being produced rapidly enough to release sufficient calcium from the rough endoplasmic reticulum to cause an observed increases in cytoplasmic free Ca2+.     

Inositol tetrakisphosphate-induced sequestration of Ca2+ replenishes an intracellular pool sensitive to inositol trisphosphate

In a permeable neoplastic rat liver epithelial (261B) cell system, inositol 1,3,4,5-tetrakisphosphate--Ins(1,3,4,5)P4--induces sequestration of Ca2+ released by inositol 2,4,5-trisphosphate--Ins(2,4,5)P3; a non-metabolized inositol trisphosphate (InsP3) isomer--and Ca2+ added exogenously in the form of CaCl2. Studies were performed to identify the Ca2+ pool filled after Ins(1,3,4,5)P4 treatment. Both Ins(2,4,5)P3 and inositol 1,4,5-trisphosphate--Ins(1,4,5)P3--dose-dependently release Ca2+ from permeable 261B cells--Ins(1,4,5)P3 having a threefold greater potency--but differ in that Ca2+ released by Ins(1,4,5)P3 is readily sequestered, while the Ca2+ released by Ins(2,4,5)P3 is not. Maximal release of Ca2+ by 6 microM Ins(2,4,5)P3 blocked the action of Ins(1,4,5)P3, demonstrating that these two isomers influence the same intracellular Ca2+ pool through a shared membrane receptor. Addition of 2 microM Ins(2,4,5)P3 to discharge partially the Ca2+ pool reduced the amount of Ca2+ released by a maximal dose of Ins(1,4,5)P3 (2 microM). Ins(1,3,4,5)P4 combined with Ins(2,4,5)P3 produced a Ca2+ release and sequestration response, which replenished the InsP3-sensitive pool as indicated by a recovery of full Ca2+ release by 2 microM Ins(1,4,5)P3. Induction of Ca2+ sequestration by Ins(1,3,4,5)P4 occurred dose-dependently, with a half-maximal response elicited at a dose of 0.9 microM. Further studies of the effect of Ins(1,3,4,5)P4 apart from the influence of Ins(2,4,5)P3 using a model in which the Ca2+ levels are raised by an exogenous addition of CaCl2 showed that Ins(1,4,5)P3 released twice the amount of Ca2+ from the storage pool following Ins(1,3,4,5)P4-induced Ca2+ sequestration. These results demonstrate that the Ca2+ uptake induced by Ins(1,3,4,5)P4 preferentially replenishes the intracellular Ca2+ storage sites regulated by Ins(1,4,5)P3 and Ins(2,4,5)P3.     

InsP(3)-induced Ca(2+) release in permeabilized invertebrate photoreceptors: a link between phototransduction and Ca(2+) stores

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.     

Characterization of inositol 1,4,5-trisphosphate-stimulated calcium release from rat cerebellar microsomal fractions. Comparison with [3H]inositol 1,4,5-trisphosphate binding.

The abilities of D-myo-inositol phosphates (InsPs) to promote Ca2+ release and to compete for D-myo-[3H]-inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) binding were examined with microsomal preparations from rat cerebellum. Of the seven InsPs examined, only Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 stimulated the release of Ca2+. Ca2+ release was maximal in 4-6 s and was followed by a rapid re-accumulation of Ca2+ into the Ins(1,4,5)P3-sensitive compartment after Ins(1,4,5)P3, but not after Ins(2,4,5)P3 or Ins(4,5)P2. Ca2+ re-accumulation after Ins(1,4,5)P3 was also faster than after pulse additions of Ca2+, and coincided with the metabolism of [3H]Ins(1,4,5)P3. These data suggest that Ins(1,4,5)P3-induced Ca2+ release and the accompanying decrease in intraluminal Ca2+ stimulate the Ca2+ pump associated with the Ins(1,4,5)P3-sensitive compartment. That this effect was observed only after Ins(1,4,5)P3 may reflect differences in either the metabolic rates of the various InsPs or an effect of the Ins(1,4,5)P3 metabolite Ins(1,3,4,5)P4 to stimulate refilling of the Ins(1,4,5)P3-sensitive store. InsP-induced Ca2+ release was concentration-dependent, with EC50 values (concn. giving half-maximal release) of 60, 800 and 6500 nM for Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 respectively. Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 also competed for [3H]Ins(1,4,5)P3 binding, with respective IC50 values (concn. giving 50% inhibition) of 100, 850 and 13,000 nM. Comparison of the EC50 and IC50 values yielded a significant correlation (r = 0.991). These data provide evidence of an association between the [3H]Ins(1,4,5)P3-binding site and the receptor mediating Ins(1,4,5)P3-induced Ca2+ release.     

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.     

GTP- and inositol 1,4,5-trisphosphate-induced release of 45Ca2+ from a membrane store co-localized with pancreatic-islet-cell plasma membrane.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], arising from hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is proposed as the link between membrane-receptor activation and mobilization of Ca2+ from intracellular sites in hormone-secreting cells. The location of Ins(1,4,5)P3-sensitive membranes was investigated in cultured neonatal beta-cells. Membranes were obtained after lysis of cells attached to positively charged Sephadex. After lysis the presence of the enzyme markers 5'-nucleotidase, glucose-6-phosphatase, NADH-cytochrome c reductase, UDP-galactosyltransferase and succinate dehydrogenase indicated the mixed nature of the preparation. After sonication, however, UDP-galactosyltransferase and succinate dehydrogenase activities were undetectable, but 4.8% of total cellular glucose-6-phosphatase and 3.4% of total cellular NADH-cytochrome c reductase remained with 5'-nucleotidase in the preparation, indicating endoplasmic-reticulum association. ATP-dependent 45Ca2+ accumulation was shown in this preparation (410 +/- 24 pmol/mg of protein at 150 nM free Ca2+) and was inhibited by vanadate (100 microM). Ca2+ release was effected by Ins(1,4,5)P3, with half-maximal release at 0.5 +/- 0.14 microM-Ins(1,4,5)P3, t1/2 11.2 +/- 1.1 s. GTP- and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG)-promoted release of 45Ca2+ was demonstrated in this preparation, but the kinetics of release (half-maximal Ca2+ release at 5.4 +/- 0.7 microM, with t1/2 77.3 +/- 6.9 s, and at 51.1 +/- 4.2 microM, with t1/2 19.0 +/- 2.2 s, for GTP and p[NH]ppG respectively), and the ability of neomycin sulphate to block p[NH]ppG-induced release only, are indicative of separate release mechanisms after treatment with these agents. A close association between plasma membrane and elements of the endoplasmic reticulum is indicated in this model, providing a possible mechanism for local alterations in free Ca2+ in the sub-plasma-membrane region.     

The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback

Microvillar photoreceptors of invertebrates exhibit a light-induced rise in the intracellular concentration of free calcium (Cai) that results in part from release of calcium from an intracellular compartment. This light-induced release of calcium appears to result from a cascade of reactions that involve rhodopsin, a GTP-binding protein and a phospholipase-C which releases inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) from the plasma membrane; the Ins(1,4,5)P3 acts to release calcium from smooth endoplasmic reticulum. In the ventral photoreceptor of the horseshoe crab Limulus polyphemus not all of the endoplasmic reticulum is subject to calcium release by Ins(1,4,5)P3. Only endoplasmic reticulum in the light-sensitive region of the cell is competent to release calcium in response to Ins(1,4,5)P3. The release of calcium by Ins(1,4,5)P3 in ventral photoreceptors appears to be subject to feedback inhibition through elevated Cai. We suggest that this feedback inhibition contributes to sensory adaptation in the photoreceptor and may account for oscillatory membrane responses sometimes observed with large injections of Ins(1,4,5)P3.     

The effect of inositol 1,4,5-trisphosphate and GTP on calcium release from rat liver microsomes

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP mobilized 8% and 90% of the ionophore-releaseable Ca2+ pool from rat liver microsomes, respectively. In contrast to GTP, which acted after a lag-time, the Ins(1,4,5)P3-induced Ca2+ release was immediate. Poly(ethylene glycol) inhibited the effect of Ins(1,4,5)P3 and enhanced that of GTP. Ins(1,4,5)P3 accelerated and enhanced the GTP-induced Ca2+ release. Guanylyl imidodiphosphate inhibited competitively the GTP stimulated Ca2+ release, but not the GTP-dependent phosphorylation of the Mr 17,000 and 38,000 protein bands.     

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.     

Inositol 1,3,4,5-tetrakisphosphate and inositol 1,4,5-trisphosphate act by different mechanisms when controlling Ca2+ in mouse lacrimal acinar cells

In internally perfused single lacrimal acinar cells the competitive inositol 1,4,5-trisphosphate (Ins 1,4,5-P3)-antagonist heparin inhibits the ACh-evoked K+ current response mediated by internal Ca2+ and also blocks both the Ins 1,4,5-P3-evoked transient as well as the sustained K+ current increase evoked by combined stimulation with internal Ins 1,4,5-P3 and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). When, during sustained stimulation with both Ins 1,4,5-P3 and Ins 1,3,4,5-P4, one of the inositol polyphosphates is removed, the K+ current declines; whereas removal of Ins 1,4,5-P3 results in an immediate termination of the response, removal of Ins 1,3,4,5-P4 only causes a very gradual and slow reduction in the current. Ins 1,3,4,5-P4 is therefore not an acute controller of Ca2+ release from stores into the cytosol, but modulates the release of Ca2+ induced by Ins 1,4,5,P3 by an unknown mechanism, perhaps by linking Ins 1,4,5 P3-sensitive and insensitive Ca2+ stores.     

Characteristics of inositol trisphosphate-mediated Ca2+ release from permeabilized hepatocytes

Ca2+ release triggered by inositol trisphosphate (Ins(1,4,5)P3) has been measured in saponin-permeabilized hepatocytes with 45Ca2+ or Quin 2. The initial rate of Ca2+ release was not greatly affected by the incubation temperature (175 +/- 40 pmol X s-1 X mg dry weight-1, at 30 degrees C versus 133 +/- 24 pmol X s-1 X mg dry weight-1 at 4 degrees C). The amount of Ca2+ released by Ins(1,4,5)P3 was not affected by pH (6.5-8.0). La3+ (100 microM) markedly inhibited the effect of 1 microM Ins(1,4,5)P3. The possibility that La3+ chelates Ins(1,4,5)P3 cannot be excluded since the effect of La3+ could be overcome by increasing the Ins(1,4,5)P3 concentration. Ins(1,4,5)P3-mediated Ca2+ release showed a requirement for permeant cations in the incubation medium. Optimal release was observed with potassium gluconate. Other monovalent cations, with the exception of Li+, can substitute for K+. Permeant anions, at concentrations above 40 mM, inhibited Ca2+ release produced by Ins(1,4,5)P3. Cl-, Br-, I-, and SO2-4 were equally effective as inhibitors. Ins(1,4,5)P3 also caused the release of 54Mn2+ and 85Sr2+ accumulated by the permeabilized hepatocytes. Our results are consistent with Ins(1,4,5)P3 promoting the membrane translocation of divalent cations through an ion channel rather than an ion carrier. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membranes. The transport systems responsible for these compensatory ion movements may represent a potential site for the regulation of the hormone-mediated Ca2+ signal.