Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

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.     

Ca2(+)-sensitivity of inositol 1,4,5-trisphosphate-mediated Ca2+ release in permeabilized pancreatic acinar cells.

Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.     

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

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.     

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release from permeabilized mastocytoma cells

The effect of inositol 1,4,5-trisphosphate (IP₃) on Ca²⁺ release in the transformed murine mast cells, mastocytoma P-815 cells permeabilized with digitonin was studied. Ca²⁺ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca²⁺ concentration was lowered to a new steady-state level. The subsequent addition of IP₃ caused a rapid Ca²⁺ release, which was followed by a slow re-uptake of Ca²⁺. Fifty percent of the sequestered Ca²⁺ was released by 10 μM IP₃. Maximal Ca²⁺ release occurred at 10 μM and half maximal activity was at 1.3 μM. These results indicate that IP₃ may function as a messenger of intracellular Ca²⁺ mobilization in mastocytoma cells.     

Ca2+ release by inositol trisphosphate from Ca2+-transporting microsomes derived from uterine sarcoplasmic reticulum

Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 microM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that by mobilizing intracellular Ca2+, inositol trisphosphate may be the link between hormonal stimuli and smooth muscle contraction.     

Ca2+ uptake and IP3-induced Ca2+ release in permeabilized human lymphocytes

The 45Ca2+ uptake and 45Ca2+ release in saponin-permeabilized human lymphocytes were studied. An ATP-dependent Ca2+ uptake into a nonmitochondrial, intracellular Ca2+ store is observed which is approx. 2 orders of magnitude greater than the ATP-independent Ca2+ uptake. The Ca2+ uptake is inhibited by vanadate, but it is insensitive to oligomycin and ruthenium red. IP3 induces dose-dependent 45Ca2+ release. For half-maximum Ca2+ release 0.25-0.5 microM IP3 is required. The results of our studies suggest that 45Ca2+ is predominantly stored within the endoplasmic reticulum of the lymphocytes.     

Ca2+ dependence of Ca2+ release from intracellular stores of intact and permeabilized Ehrlich ascites tumour cells

Effects of Ca2+ ions on the mobilization of Ca2+ from intracellular stores of intact and permeabilized (15 microM digitonin) Ehrlich ascites tumour cells (EATC) have been compared. For permeabilized cells, the dependences of the initial rate and amplitude of Ca2+ mobilization evoked by the addition of 100 nM inositol 1,4,5-trisphosphate (IP3) on preexisting [Ca2+] were bell-shaped within a [Ca2+] range 10(-7)-10(-6) M with the maxima at [Ca2+] = 166 nM. In intact cells, different concentrations of free cytosolic Ca2+ ([Ca2+]i) were produced using low (up to 0.005%) concentrations of digitonin which selectively increased the permeability of the plasma membrane. Stimulation of cells by exogenous ATP at [Ca2+]i = 10(-8)-10(-6) M resulted in Ca2+ mobilization the rate and amplitude of which were maximal at 102-115 nM Ca2+. The experimental Ca2+ dependences were fit by a model which includes channel opening upon Ca2+ binding and transition to the inactive states upon Ca2+ binding to the closed and open channel forms. Three inactivation types (including two particular cases) demonstrate a slight priority of inhibitory binding of Ca2+ only to the open channel, but predict markedly different parameter values. We conclude that an increase in [Ca2+] can stimulate IP3-induced mobilization, but in intact EATC, deviations of [Ca2+]i from the resting level (about 100 nM) attenuate responses to the agonist stimulation.     

Inhibition of inositol 1,4,5-trisphosphate-mediated Ca2+ release by Ca2+ in cells from peripheral tissues

Permeabilized cells attached to culture plates were used to evaluate the inhibition of inositol 1,4,5-trisphosphate-mediated release (IPMCR) by Ca2+. In AR42J cells, a pancreatic acinar cell line, when permeabilization and Ca2+ uptake were carried out at low ionized Ca2+ (0.06 microM), Ca2+ had little effect on IPMCR. On the other hand, when permeabilization and Ca2+ uptake were performed at 5 microM Ca2+, IPMCR was inhibited by Ca2+ with an apparent affinity of 0.24 microM. This inhibition could be modified by exposing the cytosol of permeabilized cells to low Ca2+. Hence, permeabilizing the cells in the presence of 5 microM Ca2+ and then exposing them to Ca2+ concentrations between 0.01 and 5 microM before washing and Ca2+ uptake in the presence of 5 microM Ca2+ resulted in a Ca2(+)-dependent loss of inhibitory activity. The loss of inhibitory activity occurred with an apparent affinity for Ca2+ of 0.21 microM. A similar phenomenon with a comparable apparent dissociation constant for Ca2+ was found with three other cell types from peripheral tissues: the osteosarcoma cell line UMR-106-01, the kidney inner medullary cell line IMCD, and primary culture of urinary bladder smooth muscle cells. The properties of inhibition of IPMCR by Ca2+ in cells from peripheral tissues differ from those previously described in neuronal tissues and suggest that a different factor(s) mediates the inhibition of IPMCR by Ca2+ in cells from peripheral and neuronal tissues.     

GTP enhances inositol trisphosphate-stimulated Ca2+ release from rat liver microsomes

Low concentrations of GTP (10-50 microM) greatly enhance the inositol 1,4,5-trisphosphate stimulated Ca2+ release from rat liver microsomal vesicles. The effect of GTP depends on the presence of low concentrations of polyethylene glycol in the incubation medium. Guanylyl imidodiphosphate is ineffective at mimicking the GTP effect and inhibits the action of GTP added subsequently.     

Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release in permeabilized pancreatic acinar cells by hormonal and phorbol ester pretreatment

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 0.9-1.5 nmol of Ca2+/mg acinar protein in an energy-dependent pool. Uptake into this pool was not altered by pretreatment of acinar cells with the Ca2+ mobilizing secretagogues carbamylcholine and cholecystokinin-octapeptide or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, indicating that the Ca2+ pump of the internal Ca2+ store was not affected by prolonged activation of the Ca2+ messenger system. Inositol 1,4,5-trisphosphate (1,4,5-IP3) caused a rapid decrease in Ca2+ content of the internal Ca2+ store. The response was maximal within 30 s following addition of 1,4,5-IP3 and no reuptake of Ca2+ was observed over the next 60 s. Up to 55% of the amount of Ca2+ stored in the energy-dependent pool was 1,4,5-IP3 releasable with an EC50 of 1.0 microM. Pretreatment of acinar cells with carbamylcholine or cholecystokinin-octapeptide significantly reduced the effectivity of 1,4,5-IP3 to release Ca2+ from the internal store. The dose-response curve for 1,4,5-IP3-induced release of actively stored Ca2+ from carbamylcholine-treated acinar cells showed both a rightward shift (EC50 value of 1.7 microM) and a decreased maximal response (maximal release value of 44%), which suggests that the affinity of 1,4,5-IP3 for its receptor as well as the number of 1,4,5-IP3 receptors or 1,4,5-IP3-operated Ca2+ channels was reduced upon prolonged activation of the Ca2+ messenger system. Moreover, analysis of the release values in a Hill plot suggested positive cooperativity for 1,4,5-IP3-induced Ca2+ release from the internal store (n values of 1.3 and 1.7 for saline- and carbamylcholine-treated permeabilized acinar cells, respectively). Pretreatment of acinar cells with 12-O-tetradecanoylphorbol 13-acetate partly mimicked the inhibitory effect of carbamylcholine on 1,4,5-IP3-induced release of actively stored Ca2+ in that the dose-response curve was shifted to the right but the maximal response was not affected, suggesting that the effects of carbamylcholine were at least in part mediated by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression level of inositol trisphosphate and inositol tetrakisphosphate receptors and their influence on Ca2+ release in permeabilized HL-60 and T15 cells

To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL-60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP.     

Ca2+ release from inositol trisphosphate-sensitive stores is not modulated by intraluminal [Ca2+].

In a recent model developed to explain the apparent "quantal" nature of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release from specific intracellular stores, it was proposed that Ca2+ release from the stores may itself be modulated by intraluminal levels of Ca2+, possibly via an action at a binding site on the Ins(1,4,5)P3 receptor/Ca2+ channel complex. Essential predictions of this model include a specific effect of intraluminal Ca2+ levels on the sensitivity of Ins(1,4,5)P3-induced Ca2+ release and a non-exponential decay of passive Ca2+ loss from the store following inhibition of the Ca2+ pump on the store. However, in measurements of Ins(1,4,5)P3-induced Ca2+ release and passive Ca2+ loss in permeabilized preparations of a model exocrine cell under conditions of thapsigargin-induced store depletion, we found that neither of these predicted behaviors could be demonstrated.     

Inositol trisphosphate does not release Ca2+ from permeabilized cardiac myocytes and sarcoplasmic reticulum

The possibility that inositol 1,4,5-trisphosphate (IP3) may act as a Ca2+-mobilizing second messenger in cardiac muscle in a manner analogous to its actions in other cell types has been examined using saponin-permeabilized myocytes and isolated cardiac sarcoplasmic reticulum. Myocytes permeabilized in the presence of MgATP2- sequestered Ca2+ to a level of about 200 nM, similar to the cytosolic free Ca2+ concentration of intact cells, but addition of IP3 was ineffective in causing Ca2+ release from intracellular stores. Similarly, IP3 (up to 50 microM) was unable to inhibit Ca2+ uptake or cause Ca2+ release from isolated canine cardiac sarcoplasmic reticulum vesicles in the presence of either EGTA or sodium vanadate. These results indicate that IP3 is unlikely to mediate mobilization of intracellular Ca2+ stores in myocardial cells.     

Agonist-mediated Ca2+ release in permeabilized UMR-106-01 cells. Transport properties and generation of inositol 1,4,5-trisphosphate

Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.     

Inositol 1,3,4,5-tetrakisphosphate causes release of Ca2+ from permeabilized mouse lymphoma L1210 cells by its conversion into inositol 1,4,5-trisphosphate.

Addition of Ins(1,3,4,5)P4 at micromolar concentrations causes release of Ca2+ from electroporated L1210 cells, but not from digitonin-permeabilized cells. This was shown to be due to its conversion into Ins(1,4,5)P3, because only the electroporated cells convert Ins(1,3,4,5)P4 into Ins(1,4,5)P3. Thus electroporation appears to activate or expose an Ins(1,3,4,5)P4 3-phosphatase.     

Ca2+ depletion and inositol 1,4,5-trisphosphate-evoked activation of Ca2+ entry in single guinea pig hepatocytes

Store-operated Ca(2+) entry was investigated by monitoring the Ca(2+)-dependent K(+) permeability in voltage-clamped guinea pig hepatocytes. In physiological conditions, intracellular Ca(2+) stores are discharged following agonist stimulation, but depletion of this stores can be achieved using Ca(2+)-Mg(2+)-ATPase inhibitors such as 2,5-di(tert-butyl)-1,4-benzohydroquinone and thapsigargin. The effect of internal Ca(2+) store depletion on Ca(2+) influx was tested in single cells using inositol 1,4,5-trisphosphate (InsP(3)) release from caged InsP(3) after treatment of the cells with 2, 5-di(tert-butyl)-1,4-benzohydroquinone or thapsigargin in Ca(2+)-free solutions. We show that the photolytic release of 1-d-myo-inositol 1,4-bisphosphate 5-phosphorothioate, a stable analog of InsP(3), and Ca(2+) store depletion have additive effects to activate a high level of Ca(2+) entry in single guinea pig hepatocytes. These results suggest that there is a direct functional interaction between InsP(3) receptors and Ca(2+) channels in the plasma membrane, although the nature of these Ca(2+) channels in hepatocytes is unclear.