Signalling routes and developmental regulation of group I metabotropic glutamate receptors in rat auditory midbrain neurons.

Group I metabotropic glutamate receptors (mGluRs) are linked to intracellular Ca(2+) signalling and play important roles related to synaptic plasticity and development. In neurons from the central nucleus of the inferior colliculus (CIC), the activation of these receptors evokes large [Ca(2+) ](i) responses. By using optical imaging of the fluorescent Ca(2+) -sensitive dye Fura-2, we have explored which [Ca(2+) ](i) routes are triggered by group I mGluR activation in young CIC neurons and whether mGluR-induced [Ca(2+) ](i) responses are regulated during postnatal development. In addition, real-time quantitative RT-PCR was used to study the developmental expression of both group I mGluR subtypes, mGluR1 and mGluR5. Application of DHPG, a specific agonist of group I mGluRs, was used on CIC slices from young rats to elicit [Ca(2+) ](i) responses. A majority of responses consisted of an initial thapsigargin-sensitive Ca(2+) peak, related to store depletion, followed by a plateau phase, sensitive to the store-operated Ca(2+) entry blocker 2-APB. During postnatal development, from P6 to P17, DHPG-induced [Ca(2+) ](i) responses changed. The largest Ca(2+) responses were reached at P6, whereas lower peak and plateau responses were found after hearing onset, at P13-P14 and P17. qRT-PCR analysis also revealed important differences in the expression of both mGluR1 and mGluR5 subtypes during development, with the highest levels of both subtypes at P7 and a developmental decrease of both transcripts. Our results suggest both intra- and extracellular routes for [Ca(2+) ](i) increases linked to group I mGluRs in CIC neurons and a regulation of group I mGluR activity and expression during auditory development.

Analysis of IP3 receptors in and out of cells.

BACKGROUND: Inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP3R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca2+ channels. SCOPE OF THE REVIEW: In this brief review, we first consider a variety of complementary methods that allow the links between IP3 binding and channel gating to be defined. How does IP3 binding to the IP3-binding core in each IP3R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP3, Ca2+ signals and IP3R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP3R signalling. MAJOR CONCLUSIONS: All IP3R are regulated by both IP3 and Ca2+. This allows them to initiate and regeneratively propagate intracellular Ca2+ signals. The elementary Ca2+ release events evoked by IP3 in intact cells are mediated by very small numbers of active IP3R and the Ca2+-mediated interactions between them. The spatial organization of these Ca2+ signals and their stochastic dependence on so few IP3Rs highlight the need for methods that allow the spatial organization of IP3R signalling to be addressed with single-molecule resolution. GENERAL SIGNIFICANCE: A variety of complementary methods provide insight into the structural basis of IP3R activation and the contributions of IP3-evoked Ca2+ signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.

Phosphoinositides and phosphatidic acid regulate pollen tube growth and reorientation through modulation of [Ca2+]c and membrane secretion.

The maintenance of a calcium gradient and vesicle secretion in the apex of pollen tubes is essential for growth. It is shown here that phosphatidylinositol-4,5-bisphosphate (PIP2) and D-myo-inositol-1,4,5-trisphosphate (IP3), together with phosphatidic acid (PA), play a vital role in the regulation of these processes. Changes in the intracellular concentration of both PIP2 and IP3 (induced by photolysis of caged-probes), modified growth and caused reorientation of the growth axis. However, measurements of cytosolic free calcium ([Ca2+]c) and apical secretion revealed significant differences between the photo-release of PIP2 or IP3. When released in the first 50 mum of the pollen tube, PIP2 led to transient growth perturbation, [Ca2+]c increases, and inhibition of apical secretion. By contrast, a concentration of IP3 which caused a [Ca2+]c transient of similar magnitude, stimulated apical secretion and caused severe growth perturbation. Furthermore, the [Ca2+]c transient induced by IP3 was spatially different causing a pronounced elevation in the sub-apical region. These observations suggest different targets for the two phosphoinositides. One of the targets is suggested to be PA, a product of PIP2 hydrolysis via phospholipase C (PLC) or phospholipase D (PLD) activity. It was found that antagonists of PA accumulation (e.g. butan-1-ol) and inhibitors of PLC and PLD reversibly halted polarity. Reduction of PA levels caused the dissipation of the [Ca2+]c gradient and inhibited apical plasma membrane recycling. It was also found to cause abolition of the apical zonation. These data suggest that phosphoinositides and phospholipids regulate tip growth through a multiple pathway system involving regulation of [Ca2+]c levels, endo/exocytosis, and vesicular trafficking.

Modulation of Xenopus laevis Ca-activated Cl currents by protein kinase C and protein phosphatases: implications for studies of anesthetic mechanisms.

Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1alpha and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP(3)), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl(2) were directly injected into the oocyte. Responses to agonists (LPA 10(-6) M, IP(3) 10(-4) M, CaCl(2) 0.5 M) were measured at a holding potential of -70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 alpha and PP2A inhibited I(Cl(Ca)) from 7.6 +/- 0.9 microC to 2.5 +/- 0.9 microC and 3.2 +/- 1.4 microC, respectively. PP inhibition enhanced I(Cl(Ca)) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 alpha or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl(2)-induced I(Cl(Ca)). Our data indicate that the Xenopus I(Cl(Ca)) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.

Acetyl-L-carnitine requires phospholipase C-IP3 pathway activation to induce antinociception.

The cellular events involved in acetyl-L-carnitine (ALCAR) analgesia were investigated in the mouse hot plate test. I.c.v. pretreatment with aODNs against the alpha subunit of G(q) and G(11) proteins prevented the analgesia induced by ALCAR (100 mg kg(-1) s.c. twice daily for 7 days). Administration of the phospholipase C (PLC) inhibitors U-73122 and neomycin, as well as the injection of an aODN complementary to the sequence of PLCbeta(1), antagonized the increase of the pain threshold induced by ALCAR. Pretreatment with U-73343, an analogue of U-73112 inactive on PLC, did not modify ALCAR analgesic effect. In mice undergoing treatment with LiCl, which impairs phosphatidylinositol synthesis, or pretreatment with TMB-8, a blocker of Ca(++) release from intracellular stores, the antinociception induced by ALCAR was dose-dependently antagonized. I.c.v. treatment with heparin, an IP(3) receptor antagonist, prevented the increase of pain threshold induced by the investigated compound, analgesia that was restored by co-administration of D-myo-inositol. On the other hand, i.c.v. pretreatment with the selective protein kinase C (PKC) inhibitors calphostin C and cheleritryne, resulted in a dose-dependent potentiation of ALCAR antinociception. The administration of PKC activators, such as PMA and PDBu, dose-dependently prevented the ALCAR-induced increase of pain threshold. Neither aODNs nor pharmacological treatments produced any behavioral impairment of mice as revealed by the rota-rod and hole board tests. These results indicate that central ALCAR analgesia in mice requires the activation of the PLC-IP(3) pathway. By contrast, the simultaneous activation of PKC may represent a pathway of negative modulation of ALCAR antinociception.

The phospholipase C-IP3 pathway is involved in muscarinic antinociception.

The cellular events involved in muscarinic analgesia were investigated in the mouse hot-plate test. Intracerebroventricular (i.c.v.) pretreatment with antisense oligonucleotides (aODNs) against the alpha subunit of G(q) and G(11) proteins prevented the analgesia induced by physostigmine and oxotremorine. Furthermore, administration of the phospholipase C (PLC) inhibitor U-73122, as well as the injection of an aODN complementary to the sequence of PLCbeta(1), antagonized the increase of the pain threshold induced by both cholinomimetic drugs. In mice undergoing treatment with LiCl, which impairs phosphatidylinositol synthesis, or treatment with heparin, an IP(3) receptor antagonist, the antinociception induced by physostigmine and oxotremorine was dose-dependently antagonized. I.c.v. pretreatment with TMB-8, a blocker of Ca(2+) release from intracellular stores, prevented the increase of pain threshold induced by the investigated cholinomimetic drugs. Coadministration of Ca(2+) restored the muscarinic analgesia in LiCl, heparin, and TMB-8-preatreated mice. On the other hand, i.c.v. pretreatment with the selective protein kinase C (PKC) inhibitor calphostin C, resulted in a dose-dependent enhancement of physostigmine- and oxotremorine-induced antinociception. The administration of PKC activators, such as PMA and PDBu, dose dependently prevented the cholinomimetic drug-induced increase of pain threshold. Neither aODNs nor pharmacological treatments employed produced any behavioral impairment of mice as revealed by the rota-rod and hole-board tests. These results indicate a role for the PLC-IP(3) pathway in central muscarinic analgesia in mice. Furthermore, activation of PKC by cholinomimetic drugs may represent a pathway of negative modulation of muscarinic antinociception.

Inducible expression of the alpha2-macroglobulin signaling receptor in response to antigenic stimulation: a study of second messenger generation.

Thioglycollate (TG)-elicited murine, peritoneal macrophages express two receptors for activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*)--namely, the low density lipoprotein receptor-related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). We now report that resident peritoneal macrophages express only 400+/-50 alpha2MSR receptors/cell compared to 5000+/-500 receptor/TG-elicited macrophage. By contrast, LRP expression is only 2-2.5-fold greater on elicited cells. The low level of alpha2MSR expression by resident cells is insufficient to trigger signal transduction in contrast to TG-elicited cells which when exposed to alpha2M* demonstrate a rapid rise in inositol 1,4,5-trisphosphate and a concomitant increase in cytosolic free Ca2+. We then studied a variety of preparations injected subcutaneously for their ability to upregulate alpha2MSR. Macroaggregated bovine serum albumin (macroBSA) injection upregulated alpha2MSR and triggered signaling responses by splenic macrophages. Nonaggregated BSA injection alone or in the presence of alum, by contrast, did not alter alpha2MSR expression. Recombivax (hepatitis B antigen adsorbed to alum) injection also upregulated alpha2MSR on splenic macrophages while the alum carrier had no effect. We conclude that macrophage alpha2M* receptors are inducible and their expression may be regulated, in part, by potential antigens.

Inositol 1,4,5-trisphosphate and reperfusion arrhythmias.

1. The present review focuses on the role of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate (IP3) in initiating arrhythmias during early reperfusion following a period of myocardial ischaemia. 2. Evidence for an arrhythmogenic action of IP3 was provided by studies showing a correlation between the extent of the increase in IP3 and the incidence of arrhythmias in early reperfusion. In addition, phospholipase C inhibitors selective for thrombin receptor stimulation were anti-arrhythmic only when arrhythmias were thrombin initiated. 3. Mechanisms by which IP3 could initiate arrhythmias are discussed, with particular emphasis on the role of slow and unscheduled Ca2+ release. 4. The reperfusion-induced IP3 and arrhythmogenic responses can be initiated through either alpha1-adrenoceptors or thrombin receptors, but endothelin receptor stimulation was ineffective. Further studies have provided evidence that the noradrenaline-mediated response was mediated by alpha1A-receptors, while the alpha1B-adrenoceptor subtype appeared to be protective. 5. Reperfusion-induced IP3 responses could be inhibited by procedures known to reduce the incidence of arrhythmias under these conditions, including preconditioning, inhibiting Na+/H+ exchange or by dietary supplementation with n-3 polyunsaturated fatty acids. 6. Inositol 1,4,5-trisphosphate generation in cardiomyocytes can be facilitated by raising intracellular Ca2+ and it seems likely that the rise in Ca2+ in ischaemia and reperfusion is responsible for the generation of IP3, which will, in turn, further exacerbate Ca2+ overload.

ATP-Induced Ca(2+) release in cochlear outer hair cells: localization of an inositol triphosphate-gated Ca(2+) store to the base of the sensory hair bundle.

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Effects of fish oil supplementation on the changes in myocardial cyclic adenosine monophosphate, inositol 1,4,5-triphosphate and mitochondrial calcium levels during acute coronary occlusion-reperfusion in cholesterol-fed rabbits.

We studied the changes in myocardial second messengers and mitochondrial calcium levels during acute coronary occlusion-reperfusion in New Zealand white male rabbits fed a high cholesterol diet with or without fish oil supplementation. Group I, control rabbits, were fed a standard laboratory rabbit chow. In addition to the standard chow, Group II rabbits received a 1% cholesterol-enriched diet for 2 weeks, while Group III rabbits were fed a 1% cholesterol and 10% fish oil supplemented diet for 2 weeks. Acute coronary occlusion for 10 min or 1 h was induced by ligating the marginal branch of the left circumflex coronary artery. The vessel was then reperfused for 1 or 4 h in short- and long-term ischemia studies respectively. In the short-term ischemia study, myocardial samples taken from the cholesterol-fed rabbits had the highest cyclic adenosine monophosphate, inositol 1,4,5-triphosphate and mitochondrial calcium levels among the normal (nonischemic) and the ischemic areas of the three groups. The cholesterol and fish oil treated rabbits significantly suppressed the elevation of cyclic adenosine monophosphate (P < 0.05 compared with the cholesterol-fed rabbits in normal and ischemic areas respectively), but did not significantly attenuate the elevation of inositol 1,4,5-triphosphate and calcium levels. In the long-term ischemia study, the cholesterol-fed rabbits had the highest levels of these three messengers among the normal areas. However, only inositol 1,4,5-triphosphate level reached statistical significance (P < 0.05 compared with control). This group of rabbits had the lowest level of cyclic adenosine monophosphate, but the highest inositol 1,4,5-triphosphate and calcium levels among the ischemic areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the second-messenger systems in the rat spinal cord during prolonged peripheral inflammation.

Unilateral intraplantar injection of Freund's complete adjuvant (FCA) into 1 hind paw of rats was used as a model of peripheral inflammation and persistent pain in order to examine time course effects of a continuous barrage of nociceptive input on the second-messenger transducing systems in the spinal cord. cAMP, cGMP and inositol 1,4,5-trisphosphate (insP3) were extracted from the lumbosacral cord at days 1, 7, 14, 21 and 42 following FCA injection and quantified by either radioreceptor-assay (RRA) or radioimmunoassay (RIA). The lumbosacral contents of cAMP and cGMP when quantified in whole lumbosacral cord segment were not significantly changed by FCA treatment at all time points. InsP3 accumulation was significantly increased on days 14, 21 and 42 following FCA injection relative to sham-treated time-matched controls. However, cGMP and insP3 contents were significantly increased in the left longitudinal half of the lumbar enlargement ipsilateral to the injected paw on day 21 following FCA treatment, but not in the sham-treated time-matched controls. With [3H]insP3 as a ligand, Scatchard (Rosenthal) analyses of the concentration-dependent saturation curves showed that the densities (Bmax) of insP3 receptors (insP3R) were significantly increased throughout the time course of adjuvant-induced peripheral inflammation. The binding affinities (KD) for insP3R were significantly decreased on days 7, 14 and 21 following FCA injection corresponding to the times of most stable and peak inflammation. InsP3R from the cerebelli of the same rats as used in the lumbosacral insP3R characterization was used as a positive control in this study and did not show any change in both Bmax and KD as a result of FCA treatment, thus demonstrating that the changes in lumbosacral insP3R characteristics might be specific to the nociceptive sensory pathway such as the spinal cord. Thus it appears that sustained afferent nociceptive input induced by FCA injection increased the accumulation of cGMP, insP3 and insP3R density in the spinal cord through increased neuronal activities of functional receptors coupled to major classes of chemical mediators of nociception including neuropeptides and excitatory aminoacids. Changes in insP3 accumulation in the lumbosacral cord following FCA injection were significantly correlated with changes in insP3R density. Changes in the ratios of lumbosacral insP3 contents and insP3R density were also significantly correlated with changes in body weight and hind paw size induced by FCA injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms and function of intercellular calcium signaling.

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.

Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs.

Cell stimulation causes diacylglycerol kinase (DGK) to convert the second messenger diacylglycerol into phosphatidate, thus initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity. Of the DGK isoforms so far reported, only porcine DGK from lymphocytes has been characterized in detail. Here we report the isolation and sequencing of complementary DNA clones that together cover the entire region encoding porcine DGK (relative molecular mass 80,000 (80K)). The deduced primary structure of this DGK contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences similar to those found in protein kinase C, and two E-F hand motifs, typical of Ca2(+)-binding proteins like calmodulin. Indeed, we find that the activity of this DGK isoform is enhanced by micromolar concentrations of Ca2+ in the presence of deoxycholate or sphingosine. These properties of 80K DGK indicate that its action is probably linked with both of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate.