Enantiomers of myo-inositol-1,3,4-trisphosphate and myo-inositol-1,4,6 -trisphosphate: stereospecific recognition by cerebellar and platelet myo-inositol-1,4,5-trisphosphate receptors

The naturally occurring tetrakisphosphate myo-inositol-1,3,4, 6-tetrakisphosphate [Ins(1,3,4,6)P4] was able to release Ca2+ from the intracellular stores of permeabilized rabbit platelets but was 40-fold less potent than D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ca2+ releasing activity of Ins(1,3,4,6)P4 was rationalized by envisaging two alternative receptor binding orientations in which the vicinal D-1,6-bisphosphate of Ins(1,3,4,6)P4 mimics the D-4,5-bisphosphate in the Ins(1,4,5)P3 binding conformation. This rationalization predicted that Ins(1,4,5)P3 regioisomers [i.e, D-myo-inositol -1,4,6-trisphosphate [D-Ins(1,4,6)P3] and D-myo-inositol-1,3,6 -trisphosphate [D-Ins(1,3,6)P3]] should also possess Ca(2+)-releasing activity. The unambiguous total synthesis of the enatiomers of Ins(1,4,6)P3 [i.e., D-Ins(1,4,6)P3 and D-Ins(3,4,6)P3] and the enatiomers of Ins(1,3,4)P3 [i.e., D-Ins(1,3,6)P3 and D-Ins(1,3,4)P3] allowed an examination of this prediction. D-Ins(1,4,6)P3 released Ca2+ from the intracellular stores of permeabilized platelets and was only 2-3-fold less potent than Ins(1,4,5)P3. D-Ins(1,3,6)P3 [alternative nomenclature, L-Ins(1,3,4)P3] also released Ca2+ but was 12-fold less potent than Ins(1,4,5)P3. Both D-Ins(1,4,6)P3 and D-Ins(1,3,6)P3 displaced specifically bound [3H]Ins(1,4,5)P3 from the Ins(1,4,5)P3 receptor on rat cerebellar membranes. In contrast, however, D-Ins(3,4,6)P3 [alternative nomenclature, L-Ins(1,4,6)P3] and D-Ins(1,3,4)P3 neither possessed Ca(2+)-releasing activity nor displaced [3H]Ins(1,4,5)P3. The ability of D-Ins(1,3,6)P3 to release Ca2+ in permeabilized platelets is in contrast to its apparent lack of Ca(2+)-mobilizing activity previously reported in rat basophilic leukemic cells. The possibility that this is a reflection of the different Ins(1,4,5)P3 receptor subtypes possessed by these two cell types is discussed.     

Growth factors increase pericellular proteoglycans independently of their mitogenic effects on A10 rat vascular smooth muscle cells

Several reports suggest that serotonin2A (5HT2A) receptors and this receptor-mediated phosphatidyl inositol (PI) hydrolysis signal transduction system are altered in platelets of depressed patients. Inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), an important component of the PI signaling system, plays a crucial role in various physiological processes by releasing Ca2+ from intracellular stores after binding with Ins(1,4,5)P3 receptors. To examine the role of Ins(1,4,5)P3 receptors in depression, we determined [3H]Ins(1,4,5)P3 binding sites and expressed protein levels of Ins(1,4,5)P3 receptors in platelets of depressed patients (n=15) and normal control subjects (n=17). We observed that the mean Bmax of [3H]Ins(1,4,5)P3 binding to Ins(1,4,5)P3 receptors was significantly higher in platelets of depressed subjects compared with normal control subjects, whereas there was no significant difference in K(D) between these two groups. The immuno-detectable expressed level of Ins(1,4,5)P3 receptor protein was also significantly increased in depressed patients in contrast to the levels of normal control subjects. Moreover, a significant correlation was observed in Bmax and the protein level of Ins(1,4,5)P3 receptors. The increase in the number of [3H]Ins(1,4,5)P3 binding sites in platelets of depressed subjects appears to be due to an increase in the amount of Ins(1,4,5)P3 receptor proteins. These results suggest that Ins(1,4,5)P3 receptors may be involved in the pathophysiology of depression.     

Membrane potential regulates inositol 1,4,5-trisphosphate-controlled cytoplasmic Ca2+ oscillations in pituitary gonadotrophs

The influence of membrane potential (Vm) on cytoplasmic calcium ([Ca2+]i) oscillations during the sustained extracellular Ca(2+)-dependent phase of the Ca2+ signaling response to gonadotropin-releasing hormone (GnRH) was analyzed in cultured pituitary gonadotrophs. In agonist- and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-stimulated cells, sustained [Ca2+]i oscillations were extinguished by hyperpolarization after 3-15 min despite the availability of Ca2+ in the extracellular medium. Single depolarizing pulses transiently restored the amplitude of the sustained spiking in a dihydropyridine- and extracellular Ca(2+)-sensitive manner. The responses to depolarization showed a marked dependence on Vm that was correlated with the steady-state inward Ca2+ current. In addition, repetitive application of brief depolarizing pulses modulated the frequency of agonist- and Ins(1,4,5)P3-controlled spiking; depolarization pulses at frequencies lower than the intrinsic rate of episodic Ca2+ release triggered large transients between the autonomous spikes, whereas higher frequencies of depolarizing pulses overcame the original Ca2+ spiking frequency. These extrinsically driven and extracellular Ca(2+)-dependent oscillations were sensitive to the Ca(2+)-ATPase blocker, thapsigargin, but not to ryanodine. On the other hand, spontaneous firing and application of depolarizing pulses to nonstimulated cells failed to induce thapsigargin-sensitive oscillations. These findings demonstrate that the pattern of Ca2+ signaling in gonadotrophs does not depend exclusively on the Ins(1,4,5)P3 concentration, but also on the excitable status of the cell. Such modulation of the Ins(1,4,5)P3-controlled Ca2+ signaling system by changes in Vm could provide a mechanism for the integration of multiple inputs that utilize diverse signal transduction pathways.     

Immunohistochemical analysis of rat S-adenosylmethionine synthetase isozymes in developmental liver

Ins(1,4,5)P3 receptors in adrenal cortical and cerebellar membranes can be distinguished by their affinities for Ins(1,4,5)P3 as well as the potencies with which heparin and Mg2+ inhibit binding. We have found that the differences in Ins(1,4,5)P3 affinity and heparin inhibition are maintained upon receptor solubilization and purification. In contrast to this, heparin-agarose affinity purification of solubilized cerebellar receptors reduces the potency of Mg2+ inhibition to that in adrenal cortex. These results suggest that Ins(1,4,5)P3 receptors in adrenal cortex are structurally distinct from those in cerebellum. Monoclonal antibodies raised against C- and N-terminal regions of mouse cerebellar Ins(1,4,5)P3 receptors recognize 250-300-kDa proteins in both rat cerebellum and bovine adrenal cortex.     

Disaccharide polyphosphates based upon adenophostin A activate hepatic D-myo-inositol 1,4,5-trisphosphate receptors

The glyconucleotides adenophostin A and B are the most potent known agonists at type 1 inositol trisphosphate [Ins(1,4,5)P3] receptors, although their stuctures differ markedly from that of Ins(1,4,5)P3. Equilibrium competition binding with [3H]Ins(1,4,5)P3 and unidirectional 45Ca2+ flux measurements were used to examine the effects of adenophostin A in hepatocytes, which express predominantly type 2 Ins(1,4,5)P3 receptors. Both Ins(1,4,5)P3 (Kd = 8.65 +/- 0.98 nM) and adenophostin A (Kd = 0.87 +/- 0.20 nM) bound to a single class of [3H]Ins(1,4,5)P3-binding site and each fully mobilized the same intracellular Ca2+ pool; although, adenophostin A (EC50 = 10.9 +/- 0.7 nM) was more potent than Ins(1,4,5)P3 (EC50 = 153 +/- 11 nM). Working on the assumption that it is the phosphorylated glucose component of the adenophostins that mimics the critical features of Ins(1,4,5)P3, we synthesized various phosphorylated disaccharide analogs containing this structure. The novel disaccharide-based analogs, sucrose 3,4,3'-trisphosphate [Sucr(3,4,3')P3], alpha,alpha'-trehalose 3,4,3',4'-tetrakisphosphate [Trehal(3,4,3',4')P4], alpha,alpha'-trehalose 2,4,3', 4'-tetrakisphosphate [Trehal(2,4,3',4')P4], and methyl 3-O-(alpha-d-glucopyranosyl)-beta-d-ribofuranoside 2,3', 4'-trisphosphate [Rib(2,3',4')P3], were all able to mobilize the same intracellular Ca2+ pool as Ins(1,4,5)P3 and adenophostin A; although, none was as potent as adenophostin A. The rank order of potency of the analogs, adenophostin A > Ins(1,4,5)P3 approximately Rib(2,3',4')P3 > Trehal(2,4,3',4')P4 > Glc(2',3,4)P3 approximately Trehal(3,4,3',4')P4 > Sucr(3,4,3')P3, was the same in radioligand binding and functional assays of hepatic Ins(1,4,5)P3 receptors. Both Rib(2,3',4')P3, which was as potent as Ins(1,4,5)P3, and Trehal(2,4,3',4')P4 bound with significantly higher affinity ( approximately 27 and approximately 3-fold, respectively) than the only active carbohydrate agonist of Ins(1,4,5)P3 receptors previously examined [Glc(2',3,4)P3]. We conclude that phosphorylated disaccharides provide novel means of developing high-affinity ligands of Ins(1,4,5)P3 receptors.     

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.     

Inositol 1,4,5-trisphosphate receptor function in neonatal cardiomyocytes

We have previously reported that the metabolism of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) is altered when rat neonatal ventricular cardiomyocytes are isolated and cultured. In the current study we show that the mass content of Ins(1,4,5)P3 is lower in the isolated cells than in the intact tissue. However, the properties of the Ins(1,4,5)P3 receptors were not different in the two preparations and the isolated cells remained insensitive to Ins(1,4,5)P3 in terms of 45Ca2+ release. Thus, despite the altered pattern of metabolism of Ins(1,4,5)P3 in isolated neonatal cells, the properties of the receptors were similar to those reported in other myocardial preparations.     

Pindolol does not act only on 5-HT1A receptors in augmenting antidepressant activity in the mouse forced swimming test

Hypocapnia produces cerebral vasoconstriction. The mechanisms involved in hypocapnia-induced elevation of vascular smooth muscle tone remain unclear. We addressed the hypothesis that, in cerebrovascular smooth muscle, increases in extracellular pH (pHo) cause increases in Ins(1,4,5)P3 and cytosolic calcium ([Ca2+]c). Superfused primary cultures of piglet cerebral microvascular smooth muscle cells were exposed to artificial CSF (aCSF) of control (pHo 7. 4, PCO2 36 mm Hg), metabolic alkalosis (pHo 7.7, PCO2 36 mm Hg), or respiratory alkalosis (pHo 7.7, PCO2 19 mm Hg). Intracellular pH (pHi) and [Ca2+]c were measured, using BCECF and fura-2, respectively, with dual wavelength spectroscopy. Ins(1,4,5)P3 was determined by a protein binding assay. Both metabolic and respiratory acidosis treatments increased pHi from the control value of about 7.2 to 7.35. Metabolic and respiratory alkalosis increased Ins(1,4,5)P3, as we showed previously. Metabolic and respiratory alkalosis increased [Ca2+]c about 80% and 110%, respectively. Neither Ins(1,4,5)P3 nor [Ca2+]c increased in cells treated with aCSF that produced control pHo with increased pHi (7.3). In contrast, when pHo increased (7.7), but pHi was maintained at control (7.2), Ins(1,4,5)P3 increased from 123 pmol/well to 307 pmol/well and [Ca2+]c increased 46%. However, the increase of [Ca2+]c was less than with either respiratory or metabolic alkalosis. Thus, hypocapnia-induced cerebral vasoconstriction could involve production of Ins(1,4,5)P3 with resultant elevation in [Ca2+]c. While the Ins(1,4,5)P3 signal appears to be dependent on an increase in extracellular pH, a role for intracellular pH cannot be completely excluded.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca2+ and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst5 receptors (CHOsst5 cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC50 7.10) and UTP (pEC50) 5.14) caused concentration-dependent increases in [Ca2+]i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca2+]i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P3 but the SRIF maximum was about 30% of that to UTP. Removal of [Ca2+]e attenuated the SRIF-induced peak rise in [Ca2+]i but had no effect on the peak increases in Ins(1,4,5)P3. UTP-induced increases in [Ca2+]i and Ins(1,4,5)P3 were attenuated in the absence of [Ca2+]e. Following pre-exposure to SRIF (1 microM) or UTP (100 microM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca2+]e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca2+]e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca2+]i in CHOsst5 cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca2+ and release from an Ins(1,4,5)P3 sensitive Ca2+ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca2+]i could be demonstrated in the presence or absence of extracellular Ca2+ respectively, and the latter appeared to involve depletion of a common intracellular Ca2+ store.     

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCK(A) receptor

1. Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCK(A)) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCK(A)WT) and mutant (CCK(A)MT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. 2. Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCK(A)WT cells and CHO-CCK(A)MT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively). 3. The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCK(A)MT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCK(A)WT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-beta involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCK(A) receptor in CCK-8-stimulated CHO-CCK(A)WT cells. 4. The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCK(A)WT cells but not CHO-CCK(A)MT cells. This is in agreement with the idea that PKC acts directly at the CCK(A) receptor to attenuate adenylyl cyclase activation. 5. In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site. 6. TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCK(A)MT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas-7. These findings are in agreement with the idea that partial reversal of agonist-induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. 7. The data presented demonstrate that the predicted sites for PKC action on the CCK(A) receptor are the only sites involved in TPA-induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post-receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C-beta activation by receptors that are not phosphorylated by PKC.     

Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine

In mammalian fertilization, inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+ release is a crucial signaling event that originates from the vicinity of sperm-egg interaction and spreads as a wave throughout the egg cytoplasm. While it is known that Ca2+ is released by the type 1 IP3R in the egg cortex, the potential involvement of other isoform types responsible for the Ca2+ rise in the mouse egg (interior) and their spatial distribution are not known. In addition, the biochemical basis has not been definitively established for the development of increased sensitivity to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation. Using specific antibodies to the type 1, 2, and 3 IP3R, we tested the hypotheses that different IP3R isoforms are responsible for the internal Ca2+ elevation and that they contribute to the maturation-associated acquisition of IP3 sensitivity. In both preovulatory oocytes and ovulated eggs of CF-1 mice, immunofluorescence revealed that types 1 and 2 isoforms were present in the cell cortex and interior. Type 1 was observed throughout the cytoplasm, and Western analysis indicated a 1.9-fold maturation-associated increase. In contrast, the signals detected for the type 2 (high-affinity) isoform and type 3 were present to a lesser extent, with type 2 restricted to isolated islands (similar to aggregates of vesicles detected by electron microscopy), which, in the cortex, may amplify early sperm-egg signaling events. The cortical-to-perinuclear localization of the receptor and cortical vesicle aggregates imply an efficient mechanism for propagating Ca2+ release from the cortex into the interior of the egg to activate development, and the isoform localization analysis indicates a clear spatial and biochemical heterogeneity. Types 1 and 2 isoforms were also present in granulosa cells.     

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.     

Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C

The effects of adrenomedullin (AM), a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2+ concentration ([Ca2+]i) at concentrations greater than 10(-7) M. Human calcitonin gene-related peptide (CGRP), a peptide structurally related to AM, also increased [Ca2+]i with a potency similar to that of AM. AM increased [Ca2+]i in the absence of extracellular Ca2+. Further, AM increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) level in a concentration-dependent manner similar to that of AM-induced [Ca2+]i, suggesting that AM-induced elevation of [Ca2+]i is due to Ca2+ release from Ins(1,4,5)P3-sensitive stores. AM (10(-9) to 10(-6) M) increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the [Ca2+]i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases [Ca2+]i through a mechanism independent of cAMP.     

Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca2+ safeguards against spontaneous activity

BACKGROUND: Ca2+ waves allow effective delivery of intracellular Ca2+ signals to cytosolic targets. Propagation of these regenerative Ca2+ signals probably results from the activation of intracellular Ca2+ channels by the increase in cytosolic [Ca2+] that follows the opening of these channels. Such positive feedback is potentially explosive. Mechanisms that limit the spontaneous opening of intracellular Ca2+ channels are therefore likely to have evolved in parallel with the mechanism of Ca2+-induced Ca2+ release. RESULTS: Maximal rates of 45Ca2+ efflux from permeabilised hepatocytes superfused with medium in which the [Ca2+] was clamped were cooperatively stimulated by inositol 1,4,5-trisphosphate (IP3). A minimal interval of approximately 400 msec between IP3 addition and the peak rate of Ca2+ mobilisation indicate that channel opening does not immediately follow binding of IP3. Although the absolute latency of Ca2+ release was unaffected by further increasing the IP3 concentration, it was reduced by increased [Ca2+]. CONCLUSIONS: We propose that the closed conformation of the IP3 receptor is very stable and therefore minimally susceptible to spontaneous activation; at least three (probably four) IP3 molecules may be required to provide enough binding energy to drive the receptor into a stable open conformation. We suggest that a further defence from noise is provided by an extreme form of coincidence detection. Binding of IP3 to each of its four receptor subunits unmasks a site to which Ca2+ must bind before the channel can open. As IP3 binding may also initiate receptor inactivation, there may be only a narrow temporal window during which each receptor subunit must bind both of its agonists if the channel is to open rather than inactivate.     

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.     

Regulation of cyclooxygenase-2 pathway by HER2 receptor

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.     

Regulation of nerve growth mediated by inositol 1,4,5-trisphosphate receptors in growth cones

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) acts as a Ca2+ release channel on internal Ca2+ stores. Type 1 IP3R (IP3R1) is enriched in growth cones of neurons in chick dorsal root ganglia. Depletion of internal Ca2+ stores and inhibition of IP3 signaling with drugs inhibited neurite extension. Microinjection of heparin, a competitive IP3R blocker, induced neurite retraction. Acute localized loss of function of IP3R1 in the growth cone induced by chromophore-assisted laser inactivation resulted in growth arrest and neurite retraction. IP3-induced Ca2+ release in growth cones appears to have a crucial role in control of nerve growth.     

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-induced calcium waves in oscillatory cells: a biological example of Burgers' equation

Oscillations in cytosolic Ca2+ concentrations in living cells are often a manifestation of propagating waves of Ca2+. Numerical simulations with a realistic model of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ wave trains lead to wave speeds that increase linearly at long times when (a) IP3 levels are in the range for Ca2+ oscillations, (b) a gradient of phase is established by either an initial ramp or pulse of IP3, and (c) IP3 concentrations asymptotically become uniform. We explore this phenomenon with analytical and numerical methods using a simple two-variable reduction of the De Young-Keizer model of the IP3 receptor that includes the influence of Ca2+ buffers. For concentrations of IP3 in the oscillatory regime, numerical solution of the resulting reaction diffusion equations produces nonlinear wave trains that shows the same asymptotic growth of wave speed. Due to buffering, diffusion of Ca2+ is quite slow and, as previously noted, these waves occur without appreciable bulk movement of Ca2+. Thus, following Neu and Murray, we explore the behavior of these waves using an asymptotic expansion based on the small size of the buffered diffusion constant for Ca2+. We find that the gradient in phase of the wave obeys Burgers' equation asymptotically in time. This result is used to explain the linear increase of the wave speed observed in the simulations.     

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.