Differences in both inositol 1,4,5-trisphosphate mass and inositol 1,4,5-trisphosphate receptors between normal and dystrophic skeletal muscle cell lines

Human normal (RCMH) and Duchenne muscular dystrophy (RCDMD) cell lines, as well as newly developed normal and dystrophic murine cell lines, were used for the study of both changes in inositol 1,4,5-trisphosphate (IP₃) mass and IP₃ binding to receptors. Basal levels of IP₃ were increased two- to threefold in dystrophic human and murine cell lines compared to normal cell lines. Potassium depolarization induced a time-dependent IP₃ rise in normal human cells and cells of the myogenic mouse cell line (129CB3), which returned to their basal levels after 60 s. However, in the human dystrophic cell line (RCDMD), IP₃ levels remained high up to 200 s after potassium depolarization. Expression of IP₃ receptors was studied measuring specific binding of ³H-IP₃ in the murine cell lines (normal 129CB3 and dystrophic mdx XLT 4-2). All the cell lines bind ³H-IP₃ with relatively high affinity (K_d: between 40 and 100 nmol/L). IP₃ receptors are concentrated in the nuclear fraction, and their density is significantly higher in dystrophic cells compared to normal. These findings together with high basal levels of IP₃ mass suggest a possible role for this system in the deficiency of intracellular calcium regulation in Duchenne muscular dystrophy. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:902–909, 1998.     

Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]i) was studied in both ³²P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A₂ analogue (STA₂). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca²⁺]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca²⁺]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA₂. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca²⁺]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.     

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex.

Subcellular localization of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex was studied immunohistochemically using a monoclonal antibody. Electron microscopy revealed intense immunoreactivity in the dendritic spines of Purkinje cells forming synapses with the parallel fibers, climbing fibers and recurrent collaterals of Purkinje cell axons. The labelling was associated with the hypolemmal cisternae, surrounding matrix and plasmalemma including the postsynaptic densities. Weaker immunoreactivity was present in the dendritic spines of basket cells and in certain segments of Purkinje cell recurrent collaterals. The postsynaptic regions of the dendritic trunks of Purkinje and basket cells were negative. These results indicate that inositol 1,4,5-trisphosphate 3-kinase is distributed amongst the spines of various synaptic relations with different electrophysiological properties, and that axon terminals of certain cell types are another functional site for the enzyme.     

Localization of inositol 1,4,5-trisphosphate receptors in mouse retinal ganglion cells

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are ligand-gated intracellular Ca(2+)channels that mediate release of Ca(2+) from intracellular stores into the cytosol on activation by second messenger IP(3.). Similarly, IP(3)R mediated changes in cytosolic Ca(2+) concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. IP(3)R-generated cytosolic Ca(2+) transients also control intracellular Ca(2+) release and subsequent retinal ganglion cell (RGC) physiology and pathophysiology. The distribution of IP(3)R isotypes in primary adult mouse RGC cultures was determined to identify molecular substrates of IP(3)R mediated signaling in these neurons. Immunocytochemical labeling of IP(3)Rs in retinal sections and cultured RGCs was carried out using isoform specific antibodies and was detected with fluorescence microscopy. RGCs were identified by the use of morphologic criteria and RGC-specific immunocytochemical markers, neurofilament 68 kDa, Thy 1.1, and Thy 1.2. RGC morphology and immunoreactivity to neurofilament 68 kDa and Thy 1.1 or Thy 1.2 were identified in both RGC primary cultures and tissue cryosections. RGCs showed localization on intracellular membranes with a differential distribution of IP(3)R isoforms 1, 2, and 3. IP(3)R Types 1 and 3 were detected intracellularly throughout the cell whereas Type 2 was expressed predominantly in soma. Expression of all three IP(3)Rs by RGCs indicates that all IP(3)R types potentially play a role in Ca(2+) homeostasis and Ca(2+) signaling in these cells. Differential localization of IP(3) receptor subtypes in combination with biophysical properties of IP(3)R types may be an important molecular mechanism by which RGCs organize their cytosolic Ca(2+) signals.     

Specific expression of inositol 1,4,5-trisphosphate 3-kinase in dendritic spines

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase (IP3K) in the rat cerebral cortex and hippocampus was studied immunohistochemically. In both regions, the major structure expressing a high level of IP3K was the dendritic spines of pyramidal neurons, where immunoreactivity was associated with the spine apparatuses and plasmalemma. The postsynaptic densities showed the most intense labelling. Taking into account the results of our previous observations, which demonstrated the restricted localization of the enzyme in the dendritic spines of Purkinje and basket cells in cerebellum, IP3K may be localized specifically in dendritic spines in various regions of the central nervous system, and involved in synaptic signal transduction at the spines.     

On the relationship between the inhibition of thrombin stimulated aggregation and thromboxane formation in isolated platelets treated with beta-adrenoceptor blocking drugs.

Thromboxane B2 (TXB2) formation in isolated, thrombin-stimulated rat platelets was time dependent and appeared after 5 s of incubation. Beta-adrenoceptor blocking (BAB) drugs inhibited thrombin-stimulated TXB2 formation in the following rank order of potency: metipranolol approximately alprenolol approximately propranolol > oxprenolol > practolol. Atenolol was ineffective in inhibiting TXB2 production in stimulated platelets. The inhibition of thrombin-stimulated TXB2 formation by BAB drugs correlated with their inhibitory effect on thrombin-stimulated platelet aggregation, arachidonic acid liberation from membrane phospholipids and with their membrane fluidization. The higher was the liposolubility of the beta-adrenoceptor blocking drugs investigated, the higher was their inhibition of stimulated TXB2 formation. Hydrophilic, selective atenolol and practolol revealed slight or no inhibitory effect on stimulated thromboxane production.     

Development of inositol 1,4,5-trisphosphate 3-kinase immunoreactivity in cerebellar Purkinje cells in vivo and in vitro.

Development profiles in vivo and in vitro of inositol 1,4,5-trisphosphate 3-kinase (IP3K) were investigated immunohistochemically in the cerebellar Purkinje cells. In in vivo preparations of rat cerebellum, IP3K immunoreactivity appeared in Purkinje cell bodies and dendrites shortly after birth, increased rapidly by postnatal day 5, and was subsequently confined to their dendritic processes by day 20. The appearance and shift of IP3K immunoreactivity in Purkinje cells showed an identical time course even when Purkinje cells were placed under culture conditions commencing on day 0, suggesting that Purkinje cells have their own biological clock on the expression of IP3K in the absence of external influences.     

Impaired thromboxane synthesis in preactivated human blood platelets: agonist-specific, irreversible desensitization to thrombin

The causes for reduced platelet thromboxane synthesis in patients with acquired platelet storage pool disease are incompletely understood. The present study was designed to define the nature of the defect(s) underlying diminished thromboxane synthesis in human platelets previously exposed to thrombin in vitro. Platelets pretreated with high concentrations of thrombin were unable to form measurable amounts of thromboxane in response to a second stimulation with thrombin. In contrast, thrombin-pretreated platelets formed additional thromboxane in response to arachidonate, collagen, or A23,187. Thrombin-pretreated platelets did not recover with respect to thrombin-inducible thromboxane synthesis when incubated for two hours in plasma either in the presence or absence of added arachidonic acid. These observations suggest that neither inactivation of cyclooxygenase nor depletion of endogenous arachidonic acid is responsible for the impaired thrombin-induced thromboxane synthesis in thrombin-prestimulated platelets. Impaired thrombin-induced thromboxane synthesis in these platelets may be due to agonist-specific, irreversible receptor uncoupling.     

A study of cerebellar inositol 1,4,5-trisphosphate receptor following climbing and parallel fibre deafferentation

We have examined the influence of climbing fibre and parallel fibre afferent inputs on the inositol 1,4,5-trisphosphate (InsP₃) receptor in rat cerebellum. Lesions of the interior olive-climbing fibre projections to Purkinje cells by 3-acetylpyridine (3-AP) significantly reduced the [³H]InsP₃ binding density (−20%) with not apparent changes in the binding affinity 21 days after treatment. No futher reduction in binding density was found in rats given a second dose 7 days after the initial injection. A significant reduction in the binding density was evident as earlyas 7 days post-lesion. However, 3-AP (0.5 mM) failed to inhibit [³H]InsP₃ binding in vitro. Cerebellar granule cells were lesioned by two consecutive injections of methylazoxymethanol acetate at birth. In these 60-day-old granuloprival rats, the density and affinity of [³H]InsP₃ binding sites in the cerebellum remained comparable to the controls. Since lesions of the climbing fibres increase Purkinje cell activity, we suggest that changes in InsP₃ receptor density may reflect an adaptive response to the heightened Purkinje cell activity. In addition, the results also indicate that expression of the InsP₃ receptor in the cerebellum is largely independent of the presence of granule cell-parallel fibre synaptic innervation onto the Purkinje cells.     

Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP(3)R1) is an intracellular calcium (Ca(2+)) release channel that plays an important role in neuronal function. In yeast two-hybrid screen of rat brain cDNA library with the InsP(3)R1 carboxy-terminal bait we isolated multiple clones of neuronal cytoskeletal protein 4.1N. We mapped the 4.1N-interaction site to a short fragment (50 amino acids) within the carboxy-terminal tail of the InsP(3)R1 and the InsP(3)R1-interaction site to the carboxy-terminal domain (CTD) of 4.1N. We established that InsP(3)R1 carboxy-terminal binds selectively to the CTDDelta alternatively spliced form of the 4.1N protein. In biochemical experiments we demonstrated that 4.1N and InsP(3)R1 specifically associate in vitro. We showed that both 4.1N and InsP(3)R1 were enriched in synaptic locations and immunoprecipitated the 4.1N-InsP(3)R1 complex from rat brain synaptosomes. In biochemical experiments we demonstrated a possibility of InsP(3)R1-4.1N-CASK-syndecan-2 quaternary complex formation. From our findings we hypothesize that InsP(3)R1-4.1N association may play a role in InsP(3)R1 localization or Ca(2+) signaling in neurons.     

Distribution of the inositol 1,4,5-trisphosphate receptor,P400, in adult rat brain

The distribution of the inositol 1,4,5-trisphosphate receptor protein, P₄₀₀, was investigated in adult rat brain by immunocytochemistry with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactive neuronal cell bodies were detected in the cerebral cortex, the claustrum, the endopiriform nucleus, the corpus callosum, the anterior olfactory nuclei, the olfactory tubercle, the nucleus accumbens, the lateral septum, the bed nucleus of the stria terminalis, the hippocampal formation, the dentate gyrus, the caudate-putamen, the fundus striatum, the amygdaloid complex, the thalamus, the caudolateral part of the hypothalamus, the supramammillary nuclei, the substantia nigra, the pedunculopontine tegmental nucleus, the ventrotegmental area, the Purkinje cells in the cerebellum, the dorsal cochlear nucleus, the subnucleus oralis and caudalis of trigeminal nerve, and the dorsal horn of the spinal cord. Immunoreactive fibres were found in the midial forebrain bundle, the globus pallidus, the stria terminalis, the pyramidal tract, the spinal tract of trigeminal nerve, and the ventral horn of spinal cord. Nerve fibres forming a dense plexus ending in terminal-like boutons were detected in relation to nonimmunoreactive neurons of the dentate, interpositus, and fastigial nuclei of the cerebellum and around neurons of the vestibular nuclei. This receptor protein binds a specific second messenger, inositol 1,4,5-trisphosphate, which produces a mobilization of intracellular Ca²⁺ and a modulation of transmitter release. © 1993 Wiley-Liss, Inc.     

Saccharin activates cation conductance via inositol 1,4,5-trisphosphate production in a subset of isolated rod taste cells in the frog

The transduction mechanism of the conductance activated by saccharin was analysed in isolated bullfrog taste cells under whole-cell voltage-clamp. Bath application of 30 mM saccharin induced an inward current of -34 +/- 12 pA (mean +/- SEM, n = 10) at a membrane potential of -50 mV in 10 (23%) of 44 rod cells. The concentration-response relationship for the saccharin-gated current was consistent with that of the gustatory neural response. The saccharin-induced current was accompanied with a conductance increase under internal low Cl- condition (E(Cl) = -56 mV), suggesting that saccharin activated a cation conductance. The reversal potential of the saccharin-induced current was -17 +/- 2 mV (n = 10). Intracellular dialysis of 0.5 mM guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) completely blocked the saccharin-induced response, suggesting the involvement of a G protein in the transduction. The dialysis of heparin (1 mg/mL) also inhibited the response almost completely, but the dialysis of 1 mM 8-Br-cAMP did not affect the response significantly. Intracellular 50 microM inositol 1,4,5-trisphosphate (1,4,5 InsP(3)) also induced the inward current in five (38%) of 13 rod cells, but intracellular Pasteurella multocida toxin (5 microg/mL, G alpha q-coupled PLC activator) did not elicit any response in the cells. The results suggest that saccharin mainly activates a cation conductance in frog taste cells through the mediation of IP3 production.     

Guanosine 5'-0-(3-thiotriphosphate) induced calcium release in human platelets is mediated by inositol 1,4,5-triphosphate.

Guanosine 5'-triphosphate (GTP) and its nonhydrolyzable analogs, such as guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), induce several responses in platelets including secretion, production of inositol 1,4,5-triphosphate (IP3) and mobilization of Ca2+ from intracellular sites. Because IP3 is well established as a second messenger in mobilizing Ca2+ from intracellular stores it has been generally assumed that Ca2+ release by GTP/GTP gamma S in platelets is mediated by IP3. However, studies in neuronal, hepatic and smooth muscle cells have suggested that IP3 and GTP/GTP gamma S activate Ca2+ release by distinct mechanisms and that IP3-independent mechanisms mediate GTP/GTP gamma S-induced Ca2+ release. In several tissues heparin inhibits binding of IP3 and blocks IP3-stimulated Ca2+ release in a competitive and specific manner. In the present studies, IP3 and GTP gamma S induced Ca2+ release and their relationship was examined in human platelets using heparin as a probe. In saponin permeabilized platelets, IP3 (0.05-5 microM) induced a prompt, dose-dependent release of Ca2+ (EC50 0.5 microM). GTP gamma S (1-50 microM) released Ca2+ in a dose-dependent manner with EC50 of 2 microM but with a time lag of 30-90 seconds. Exposure of platelets to 1 microM IP3 following a submaximal response with GTP gamma S (1 microM) resulted in a further increase in Ca2+ release but no further increase was noted on adding 1 microM IP3 following a maximal response with GTP gamma S (10 microM); similar findings were noted on reversing the order of addition of GTP gamma S and IP3 suggesting that these effectors release Ca2+ from the same source. IP3 (0.5 microM) induced Ca2+ release was blocked by low molecular weight (4000-6000) heparin (IC50 30 micrograms/ml). More importantly, heparin abolished GTP gamma S (2.5 microM) induced Ca2+ release (IC50 10 micrograms/ml). These results indicate that, in contrast to the findings in some other cells, in human platelets GTP gamma S-induced Ca2+ release is mediated largely by a mechanism involving IP3.     

Developmental profile of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex: light and electron microscopic immunohistochemical studies

Developmental expression and intracellular distribution of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex were studied immunohistochemically. Immunoreactivity appeared first at postnatal day 1 in the rostral region of the cerebellum and by day 15 had extended throughout the whole cerebellum, being localized in the Purkinje cell layer. Shortly after the expression of the enzyme in each Purkinje cell, the labelling showed a tendency to accumulate in the dendrites in a fine granular pattern. Electron microscopy revealed that immunoreactivity was present in the Purkinje dendritic trunks with accentuation in the distal segments during the early postnatal period, thereafter becoming concentrated in the dendritic spines at later developmental stages. Labelling was associated mainly with the plasmalemma, including the postsynaptic densities and open coated vesicles, and the subplasmalemmal vesicles of the smooth endoplasmic reticulum. Immunoreactivity was also evident in the perisomatic processes of immature Purkinje cells, which are transient projections synapsing with climbing fibers. In developing Purkinje axons, immunoreactivity was accentuated in the distal segments, associated with the plasmalemma and synaptic vesicles. These results suggest that inositol 1,4,5-trisphosphate 3-kinase is involved in the dendritic arborization and subsequent spine synaptogenesis of Purkinje cells, and that the developing presynaptic nerve endings of these cells are another functional site for the enzyme.     

Spatial learning enhances the expression of inositol 1,4,5-trisphosphate 3-kinase A in the hippocampal formation of rat

Calcium-mediated signaling is crucial for the synaptic plasticity and long-term memory storage, which requires de novo protein synthesis. Inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A) is an enzyme, which is involved in the maintenance of intracellular calcium homeostasis by converting inositol 1,4,5-trisphosphate (IP(3)) to inositol 1,3,4,5-tetrakisphosphate (IP(4)). Because IP(3)K-A is enriched in the dendritic spines of hippocampal neurons, it has been speculated that this enzyme is involved in the memory formation. In the present study, we demonstrated that the expression of IP(3)K-A is increased in the hippocampal formation of the rats during the Morris water maze training. Immunohistochemical analysis indicated the specific induction of IP(3)K-A protein in the hippocampal formation following 5-day water maze training. Furthermore, in situ hybridization histochemistry showed that the induction of IP(3)K-A mRNA in the hippocampal formation was observed on the first day of training, and the induced level of IP(3)K-A mRNA was maintained until the fifth day of training. These results suggest that IP(3)K-A plays a role in the processing of spatial memory, most likely by regulating the calcium signaling in the dendritic spines of hippocampal formation.     

Effect of cyclic amp on cytoplasmic free calcium in human platelets stimulated by thrombin: Direct measurement with quiǹ2

With the fluorescent Ca²⁺ indicator quin2, we measured directly cytoplasmic free Ca²⁺ ([ Ca²⁺ ]_i) in washed human platelets stimulated by thrombin and examined the effect of cyclic AMP on [ Ca²⁺ ]_i levels and ¹⁴C-serotonin release. Thrombin (0.2 U/ml) evoked a rise in [ Ca²⁺ ]_i from the basal level of about 100 nM to ～3 μM which was fast enough to trigger serotonin release. This rise was inhibited in a dose-dependent manner by preincubation with prostaglandin E_l (PGE_l) (0.1–10 μM) or dibutyryl cyclic AMP (0.01–1 mM). Parallel to this, serotonin release was also inhibited by these drugs. When added to platelets after stimulation by thrombin, PGE_l caused the rapid decrease of elevated [ Ca²⁺ ]_i. These results provide direct evidence that [ Ca²⁺ ]_i levels in platelets are regulated by cyclic AMP.     

Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation

In a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 microM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA [( Ca2+o] less than 100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists.