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.     

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.     

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.     

New developments in the molecular pharmacology of the myo-inositol 1,4,5-trisphosphate receptor

Receptor-mediated activation of phospholipase C to generate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a ubiquitous signalling pathway in mammalian systems. A family of three IP3 receptor subtype monomers form functional tetramers, which act as effectors for Ins(1,4,5)P3, providing a ligand-gated channel that allows Ca2+ ions to move between cellular compartments. As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, Ins(1,4,5)P3 is considered to be a second messenger that mobilizes Ca2+ from intracellular stores. Ca2+ store mobilization by Ins(1,4,5)P3 can be shown to contribute to a variety of physiological and pathophysiological phenomena, and therefore the IP3 receptor represents a novel, potential pharmacological target. In this article, Rob Wilcox and colleagues review recent developments in IP3 receptor pharmacology, with particular emphasis on ligand molecular recognition by this receptor-channel complex. The potential for designing non-inositol phosphate-based agonists and antagonists is also discussed.     

Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel

The skeletal and cardiac isoforms of the ryanodine receptor Ca2+ channel (RyRC) constitute the Ca2+ release pathway in sarcoplasmic reticulum of skeletal and cardiac muscles, respectively. A direct mechanical and a Ca(2+)-triggered mechanism (Ca(2+)-induced Ca2+ release) have been respectively proposed to explain the in situ activation of Ca2+ release in skeletal and cardiac muscle. In non-muscle cells, however, where the RyRC also participates in Ca2+ signalling, the mechanism of RyRC activation is unknown. Cyclic adenosine 5'-diphosphoribose (cADPR), which is present in many mammalian tissues, has been reported to induce Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores in sea urchin eggs. Here we provide evidence that cADPR directly activates the cardiac but not the skeletal isoform of the RyRC. This, together with results on sea urchin eggs, suggests that cADPR is an endogenous activator of the non-skeletal type of RyRC and may thus have a role similar to inositol 1,4,5-trisphosphate in Ca2+ signalling.     

Intracellular calcium release channel expression during embryogenesis

The release of intracellular calcium (Ca2+) via either inositol 1,4, 5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) activates a wide variety of signaling pathways in virtually every type of cell. In the present study we demonstrate that at early stages of development IP3R mRNA and functional IP3-gated Ca2+ release channels are widely expressed in virtually all tissues in murine embryos. As organogenesis proceeds, more specialized RyR channels are expressed in many cell types and the triggering mechanisms for intracellular Ca2+ release become more diverse to include IP3-dependent and voltage-dependent and Ca2+-induced Ca2+ release. As development proceeds virtually all cell types continue to express IP3R channels but in excitable cells including skeletal and cardiac muscles the major Ca2+ release channels are RyRs. This developmental switch from predominantly IP3-mediated to both IP3-mediated and IP3-independent pathways for intracellular Ca2+ release is consistent with data showing that IP3R plays an important regulatory role in cellular proliferation and apoptosis, whereas RyR is required for other cellular functions including muscle contraction.     

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.     

Differential localization of phosphoinositide-linked metabotropic glutamate receptor (mGluR1) and the inositol 1,4,5-trisphosphate receptor in rat brain

The type 1 metabotropic glutamate receptor (mGluR1) is through to act via the phosphoinositide (PI) system with the associated formation of inositol 1,4,5-trisphosphate (IP3) and Ca2+ release. Utilizing immunohistochemistry and in situ hybridization, we have localized protein and mRNA, respectively, for the mGluR1 and the IP3 receptor (IP3R). We have also localized glutamate-linked PI turnover by autoradiography with 3H-cytidine. We observe a striking contrast in localizations of mGluR1 and IP3R both for protein and mRNA. For instance, mGluR1 occurs in the apparent absence of IP3R in neurons of the stratum oriens of the CA1 hippocampus, islands of Calleja, anterodorsal nucleus of thalamus, lateral nucleus of hypothalamus, and the granular cell layer and the deep nuclei of cerebellum. mGluR1 actions in these brain regions may primarily be mediated through the protein kinase C limb of the PI system, as they contain moderate amounts of 3H-phorbol ester binding. The subthalamic nucleus, red nucleus, and Darkshevich's nucleus, which possess high levels of mGluR1, are devoid of both IP3R immunoreactivity and 3H-phorbol ester binding. These reciprocal localizations suggest that mGluR1 actions in many brain areas may not primarily involve IP3, reflecting instead influences on protein kinase C or other second messengers.     

Familial amyloid polyneuropathy type IV (Finnish type)--a clinicopathological study

Experimental observations on rat glomerulosa cells inspired a model which postulates that plasmalemmal dihydropyridine receptors are in juxtaposition and interaction with inositol 1,4,5-trisphosphate receptors in subplasmalemmal calciosomes. Activation of dihydropyridine receptors promotes the Ca2+ releasing effect of inositol 1,4,5-trisphosphate. The most important observations compatible with the model are the following: (1) angiotensin II does not influence Ca2+ influx during the peak phase of Ca2+ signal; (2) dihydropyridine drugs modify the initial peak of the Ca2+ signal induced by angiotensin II; (3) inhibitors of the dihydropyridine receptor reduce the initial Ca2+ signal also in the presence of 5 mM Ni2+, an inhibitor of voltage dependent Ca2+ influx; and (4) changes in extracellular K+ concentration within the physiological range also modify the cytoplasmic Ca2+ response to angiotensin II.     

A cytosolic sperm protein factor mobilizes Ca2+ from intracellular stores by activating multiple Ca2+ release mechanisms independently of low molecular weight messengers

Ca2+ oscillations can be induced in mammalian eggs and somatic cells by microinjection of a cytosolic sperm protein factor. The nature of the sperm factor-induced Ca2+ signaling was investigated by adding sperm protein extracts to homogenates of sea urchin eggs, which contain multiple classes of Ca2+ release mechanisms. We show that the sperm factor mobilizes Ca2+ from non-mitochondrial Ca2+ stores in egg homogenates after a distinct latency. This latency is abolished by preincubation of sperm extracts with egg cytosol. The preincubation step is highly temperature-dependent and generates a high molecular weight, protein-based Ca2+-releasing agent that can also mobilize Ca2+ from purified egg microsomes. This Ca2+ release appears to be mediated via both inositol 1,4,5-trisphosphate and ryanodine receptors, since homologous desensitization of these two release mechanisms by their respective agonists inhibits further release by the sperm factor. However, sperm factor-induced Ca2+ release by these channels is independent of inositol 1,4, 5-trisphosphate or cADPR since antagonists of either of these two messengers did not block the Ca2+ release effected by the sperm factor. The sperm protein factor may cause Ca2+ release via an enzymatic step that generates a protein-based Ca2+-releasing agent.     

Inositol 1,4,5-trisphosphate receptors and calcium signaling

Many cellular responses to extracellular stimuli are mediated by the second messenger inositol 1,4,5-trisphosphate (InsP3). InsP3 releases Ca2+ from intracellular stores by binding to an InsP3 receptor (InSP3R), which is an InsP3-gated Ca2+ release channel. The resultant increase in the cytoplasmic Ca2+ concentration modulates various cellular functions, such as gene expression, metabolism, proliferation, secretion, and neural excitation. In these signaling cascades, InsP3R works as a signal converter from InsP3 to Ca2+. We describe here structural and functional properties and localization of InsP3R, a key molecule in the Ca2+ signaling pathway.     

Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.     

IP3-activated calcium-permeable channels in the inside-out patches of cultured cerebellar Purkinje cells

Inositol-1,4,5-trisphosphate (IP3)-activated calcium-permeable channels were recorded from inside-out patches of cultured cerebellar Purkinje cells. When 2-5 microM of IP3 was applied to the internal surface of the inside-out patches, inward Ba2+ currents were activated within 10 sec following the application in 11 out of 24 patches. In the presence of heparin (100 micrograms/ml), activation of Ba2+ currents by IP3 was inhibited. Unitary currents with different amplitudes and kinetics were observed; small and large unitary currents, and rapid fluctuations with various amplitudes. The small unitary currents (single channel conductance; 5.6 pS) were most frequent. Addition of inositol 1,3,4-trisphosphate (2-5 microM) slightly activated Ba2+ currents in 2 out of 10 patches, but the amount of the increment was much smaller than that produced by IP3. These results suggest a possibility that IP3 directly activates Ca(2+)-permeable channels in the plasma membrane of cerebellar Purkinje cells.     

Adenophostin A and inositol 1,4,5-trisphosphate differentially activate Cl- currents in Xenopus oocytes because of disparate Ca2+ release kinetics

Depletion of endoplasmic reticulum Ca2+ stores induces Ca2+ entry from the extracellular space by a process termed "store-operated Ca2+ entry" (SOCE). It has been suggested that the novel fungal metabolite adenophostin-A may be able to stimulate Ca2+ entry without stimulating Ca2+ release from stores. To test this idea further, we compared Ca2+ release, SOCE, and the stimulation of Ca2+-activated Cl- currents in Xenopus oocytes in response to inositol 1,4,5-trisphosphate (IP3) and adenophostin-A injection. IP3 stimulated an outward Cl- current, ICl1-S, in response to Ca2+ release from stores followed by an inward current, ICl2, in response to SOCE. In contrast, low concentrations of adenophostins (AdAs) activated ICl2 without activating ICl1-S, consistent with the suggestion that AdA can activate Ca2+ entry without stimulating Ca2+ release. However, when Ca2+ entry has been stimulated by AdA, Ca2+ stores are largely depleted of Ca2+, as assessed by the inability of ionomycin to release additional Ca2+. The Ca2+ release stimulated by AdA, however, was 7 times slower than the release stimulated by IP3, which could explain the minimal activation of ICl1-S; when Ca2+ is released slowly, the threshold level required for ICl1-S activation is not attained.     

Biochemical and functional heterogeneity of rat cerebrum microsomal membranes in relation to SERCA Ca(2+)-ATPases and Ca2+ release channels

Rat cerebrum microsomes were subfractionated on isopycnic linear sucrose (20-42%) density gradients. The Ca2+ loading/release properties and the distribution of intracellular Ca2+ store channels, inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine (Ry) receptor, and SERCA pumps, were monitored in each subfraction by ligand binding and 45Ca2+ loading/release assays. Three different classes of vesicles were identified: (i) heavy density vesicles with high content of Ry receptors and Ca2+ pumps and high thapsigargin (TG)-sensitivity of Ca2+ loading; (ii) intermediate sucrose density vesicles with high content of IP3 receptor, high IP(S)3-sensitivity of Ca2+ loading and low content of Ry receptors; and (iii) light sucrose density vesicles with high content of Ry receptors, low content of IP3 receptors and low content of SERCA pumps highly sensitive to TG. Isolation of molecularly heterogeneous rat cerebrum microsomes and identification of specific Ca2+ loading/release properties support the presence of multiple, potentially active, heterogeneous rapidly exchanging Ca2+ stores in rat cerebrum.     

Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes

Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca2+ indicator, indo-1 to measure the concentration of cytosolic free Ca2+ ([Ca2+]i), we show that AVP triggers a transient and plateau pattern of Ca2+ signal. The [Ca2+]i elevation activates the apamin-sensitive Ca2+-activated K+ current, which, in turn, causes membrane hyperpolarization. The Ca2+ signal can be elicited in the absence of extracellular Ca2+ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP3). Both GDP-beta-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca2+ release from the (IP3)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca2+]i elevation. Exocytosis can also be triggered when Ca2+ is released directly from the IP3-sensitive store via flash photolysis of caged IP3. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release from the IP3-sensitive store.     

In situ characterization of nonmitochondrial Ca2+ stores in individual pancreatic beta-cells

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.     

The effect of mersalyl on inositol trisphosphate receptor binding and ion channel function

A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 degrees C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups.