Sensitive Calcium Stores Research Articles

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells.

The properties of the calcium stores coupled to a depletion-operated cation current (IDOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 microM) activated an initial, large ( approximately 200 pA), transient, calcium-dependent chloride current (IClCa) followed by a smaller ( approximately 10 pA) sustained, non-selective cation current (IDOC). Intracellular application of heparin (5 mg ml-1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 microM) activated IDOC but not IClCa; ryanodine (30 microM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of IClCa by carbachol. In the presence of 30 microM, but not 3 microM, ryanodine, carbachol was able to activate IDOC. Cyclopiazonic acid (CPA; 10 microM) abolished the response to carbachol; however, caffeine was still able to activate IClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 microM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4, 5-trisphosphate-(IP3-) sensitive or ryanodine-sensitive calcium stores is able to activate IDOC, and, by extension, capacitative calcium entry in this tissue.

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells.

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

Immunohistochemical localization of inositol 1,4,5-trisphosphate receptors in non-neural tissues, with special reference to epithelia, the reproductive system, and muscular tissues.

Although the pharmacological properties and distribution of inositol 1,4,5-trisphosphate (IP3)-sensitive calcium stores vary considerably among tissues, studies of its localization have been devoted mostly to the central nervous system. In this report we have analyzed the localization of IP3 receptors in diverse non-neural tissues of the mouse, using polyclonal antibodies raised against purified IP3 receptors through immunoblotting and immunohistochemistry. These antibodies mainly recognized type 1 IP3 receptors, although they also reacted with other types of IP3 receptors. The receptors were localized in the apical surface areas of highly polarized epithelia, such as the choroid plexus, retinal pigment epithelium, and columnar epithelium lining the interlobular ducts in the sublingual and submaxillary salivary glands. Immunoelectron microscopy of choroidal cells revealed that IP3 receptors are localized on the surfaces of several structures, including clear vesicles, tubules and vesicular profiles of smooth endoplasmic reticulum, rough endoplasmic reticulum and a part of the nuclear envelope, as well as clusters of ribosomes in the cytoplasmic matrix. The surface areas of ciliated epithelia, such as those of the trachea and oviduct, also stained positive. The cytoplasmic distribution was homogeneous in mesangial cells, myoepithelial cells, and endothelial cells accompanying muscle, as well as in a population of endocrine cells. Intense immunostaining was found in the surface areas as well as in the cytoplasm of diverse smooth muscle cells. Staining intensity of smooth muscle varied among cells in the same tissue. Staining was intense in the cytoplasm of premature oocytes in the primary follicle, but then attenuated as these cells matured. The Sertoli cells with clusters of elongating maturing sperm were stained, but mature sperm were unstained. These results indicate a heterogeneous cellular distribution, and possibly a heterogeneous subcellular distribution, of a population of IP3 receptors in a variety of non-neural tissues.

The enhancement and the inhibition of noradrenaune-induced cyclic amp accumulation in rat brain by stimulation of metabotropic glutamate receptors

1. 1. The actions of several metabotropic glutamate receptor agonists and antagonists on noradrenaline (NA)-stimulated [ 3H]-cyclic AMP accumulation were investigated in rat cerebral cortical slices. 2. 2. Quisqualate (QUIS), L-2-amino-3-phosphonopropionic acid (L-AP3) and glutamate (GLU) elicited concentration-dependent inhibition of (NA)-stimulated [ 3H]-cyclic AMP accumulation, with IC 50 values of 105 ± 29, 275 ± 36 and 944 ± 150 μM respectively. In contrast (Rs)-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) (0.5 mM) and N-methyl-D-aspartic acid (NMDA) (0.5 mM) had no effect. 3. 3. (2S,3S,4S)-α-(Carboxycyclopropyl)glycine (L-CCGI), 1-Aminocyclo-pentane-lS,3R-dicarbo-xylate (1S,3R-ACPD), ibotenate (IBO) and (RS)-4-carboxy-3-hydroxy-phenylglycine (CHPG)elicited a concentration-dependent enhancement ofNA-stimulated [ 3H]-cyclic AMP accumulation, with EC 50 values of 2.5 ± 0.11, 42 ± 1.3, 97.8 ± 2.1 and 157 ± 13.4 μM, respectively. 4. 4. (S)-3-carboxy-4-hydroxyphenylglycine (3C4HPG) and (S)-4-carboxy-3-hydroxyphenyl-glycine (4C3HPG) produced a biphasic effect, at concentrations up to 100 and 500 μM, respectively, they significantly enhanced the action of NA (100 μM), at ImM concentration both compounds as well as α-methyl-4-carboxyphenylglycine (MCPG) produced a significant inhibition of NA-stimulated cyclic AMP accumulation. 5. 5. A putative mGluR antagonist — L-AP3, inhibited the 1S,3R-ACPD (100 μM) induced enhancement of the action of NA (100 μM) on [ 3H]-cyclic AMP accumulation in a biphasic manner with an IC 50 of 4.5 μM for the high affinity site, which represented 65% of the total and an IC 50 of 283 μM for the low affinity site. 6. 6. β-adrenoceptor antagonist propranolol inhibited the interaction between 1S,3R-ACPD (100 μM) and NA (100 μM) on [ 3H]-cyclic AMP accumulation by about 80%, with an IC 50 of 0.52 ± 0.011 μM, to the level observed after 1S,3R-ACPD alone. Prazosin, an α 1-adrenoceptor antagonist was more potent (IC 50 of 0.091 ± 0.012 μM) but less efficacious (60% inhibition) as an inhibitor of the interaction either between NA and 1S,3R-ACPD while yohimbine, na α 2-adrenoceptor antagonist (up to 1 μM) had no effect. 7. 7. Neither the protein kinase C inhibitor — staurosporine (10 μM) nor thapsigargin (1 μM), which depletes IP3 sensitive calcium stores, inhibited significantly the 1S,3R-ACPD (100 μM)-induced enhancement of the action of NA (100 μM) on [ 3H]-cyclic AMP accumulation. 8. 8. Adenosine deaminase (0.5 U/ml) abolished both the 1S,3R-ACPD (100 μM)-induced [ 3H]-cyclic AMP accumulation and the synergistic interaction of this compound with NA (100 μM). 9. 9. These results indicate the existence of different subtypes of metabotropic glutamate receptors in rat brain which either inhibit or enhance the NA-stimulated [ 3H]-cyclic AMP accumulation. The enhancement m cerebral cortical slices is mediated via receptors which are blocked with high affinity by L-AP3 and occurs via interactions with endogenous adenosine; the inhibition is mediated by receptors sensitive to quisqualate, L-AP3 and glutamate and may represent a predominant interaction between NA and excitatory amino acids (EAA), which in cerebral cortical slices is masked by excitatory effects.

α1B, But Not α1A, Adrenoceptor Activates Calcium Influx through the Stimulation of a Tyrosine Kinase/Phosphotyrosine Phosphatase Pathway, Following Noradrenaline-Induced Emptying of IP3 Sensitive Calcium Stores, in PC C13 Rat Thyroid Cell Line

In PC Cl3 rat thyroid cell line noradrenaline-induced Ca2+ response, mainly due to the activation of α1B receptors, is characterized by a rapid peak phase, due to the Ca2+ mobilization from inositol trisphosphate-sensitive internal stores, followed by a sustained plateau, representing the capacitative calcium entry. The plateau phase elicited by noradrenaline returns to the basal value within 100 sec from the removal of agonist. The tyrosine kinases inhibitor genistein completely abolishes the plateau upon noradrenaline withdrawl. On the contrary, the tyrosine phosphatases blocker, vanadate, potentiates the plateau phase of calcium response to noradrenaline and prevents the gradual decrease of [Ca2+]i after removal of noradrenaline. The noradrenaline-induced Ca2+ influx, due to the activation of α1A receptor-operated Ca2+ entry is not affected by vanadate. The treatment with noradrenaline induced the tyrosine phosphorylation of specific substrates in lysates derived from PC Cl3 cells, an effect inhibited by genistein pretreatment. These results show that a balance between tyrosine phosphorylation and dephosphorylation is required for the regulation of capacitative calcium entry following noradrenaline stimulation of α1B receptor, whilst the influx of Ca2+ directly operated by α1A receptor activation seems to be independent of the tyrosine phosphorylating pathway.

Calcium efflux from an intracellular pool activated by GTP hydrolysis in cultured gastric smooth muscle

In these studies, we have characterized calcium movement due to guanosine triphosphate (GTP) hydrolysis from an ATP-sequestered intracellular calcium pool in cultured gastric smooth muscle. GTP (1-100 microM), when added to an ATP-regenerating medium, resulted in a concentration-dependent and irreversible efflux of calcium from an organellar calcium pool. GTP-induced calcium efflux was not affected by variation of the ATP/ADP ratio (8.5-155.0), indicating that GTP did not act by inhibiting calcium influx via calcium adenosinetriphosphatase. To assess whether the calcium increase was necessarily associated with GTP hydrolysis, experiments were performed with the nonhydrolyzable guanine nucleotide analogues guanosine 5'-[beta-thio]diphosphate (GDP beta S), 5'-guanylyl imidodiphosphate guanosine (GppNHp), and 5'-O-(3-thiotriphosphate) (GTP gamma S). Administration of GDP beta S and GppNHp resulted in no significant calcium efflux. GTP gamma S caused a small steady-state calcium increase (20% of that induced by the hydrolyzable nucleotide) but irreversibly inhibited all subsequent calcium increase due to GTP. The possibility that GTP may either modify the concentration of mobilizable calcium in inositol trisphosphate (IP3)-sensitive calcium stores or the responsivity of IP3-associated calcium channels was assessed by two experiments: 1) prior administration of GTP at concentrations < or = 100 microM had no effect on IP3-induced calcium release, and 2) heparin, which competitively inhibits IP3 binding to its receptor on the endoplasmic reticulum, did not affect GTP-associated calcium increase. These results demonstrate that, in gastric smooth muscle, GTP causes calcium efflux from an intracellular pool that is functionally independent from that pool sensitive to IP3.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic stimulation and cAMP mobilize Ca2+ from an IP3-insensitive pool in rat submandibular granular ducts.

The beta-adrenergic agonist isoproterenol induced an increase in intracellular calcium concentration ([Ca2+]i) in rat submandibular granular ducts that was blocked by beta-adrenergic but not by alpha-adrenergic or muscarinic antagonists. This effect was only partially inhibited by the selective beta 1- and beta 2-adrenergic antagonists atenolol and ICI-118,551, but was completely blocked by the combination of the two, suggesting the involvement of multiple (or atypical) beta-adrenergic receptor subtypes. The response to isoproterenol was mimicked by forskolin, 3-isobutyl-1-methylxanthine, and dibutyryl adenosine 3',5'-cyclic monophosphate, but it was blocked by protein kinase inhibitors. The response of [Ca2+]i to isoproterenol was sustained in Ca(2+)-replete replete medium but transient in Ca(2+)-free medium, indicating the involvement of both Ca2+ entry and release from intracellular stores. However, isoproterenol stimulation produced no increase in ductal inositol phosphate levels. In addition, isoproterenol was still able to increase [Ca2+]i after the carbachol-induced depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive calcium stores. We conclude that isoproterenol, acting through cAMP, releases Ca2+ from an IP3-insensitive intracellular store in salivary granular ducts.

The effects of tetrahexyl ammonium cations (THA +) on inositol 1,4,5-trisphosphate-induced calcium release from porcine cerebellar microsomes: THA + can induce calcium release selectively from the [formula omitted

In this study we show that the potassium-channel blocker tetrahexyl ammonium chloride (THA −) is able to inhibit inositol 1,4,5-trisphosphate ( InsP 3)- induced calcium release in an apparently biphasic fashion with a IC 50 of 3 μM. This inhibition was not alleviated by valinomycin and, therefore, is not consistent with the blocking of K + counter-ion movement, an observation initially made by Palade et al. (Palade, P., Dettbarn, C., Volpe, P., Alderson, B. and Otero, A.S (1989) Mol. Pharmacol. 36, 664–672). THA + affected quantal calcium release by reducing the amount of calcium released by InsP 3 , but did not greatly affect the concentration of InsP 3 required to cause half-maximal calcium release. THA + did not affect the metabolism of InsP 3 , or its binding to porcine cerebellar microsomes. THA + could also itself induce calcium release. At concentrations below 100 μM, THA + appears to release Ca 2+ selectively from the InsP 3- sensitive calcium stores , since prior depletion of these stores with supramaximal doses of InsP 3 abolishes this response. At higher THA + concentrations (above 100 μM) Ca 2+ is released non-selectively from all stores. THA + has no effect on the Ca 2+-ATPase activity at concentrations below 100 μM, indicating that selective THA +-induced Ca 2+ release is not due to non-specific inhibition of the microsomal Ca 2+ pumps and does not affect Ca 2+ leakage. A number of pharmacological modulators of intracellular calcium channels were also tested on THA +-induced calcium release with little effect, except for spermidine which reduced this release by up to 50%. Our observations are consistent with the view that THA +, at concentrations below 100 μM, selectively releases calcium from the InsP 3- sensitive calcium stores .

Depolarization promotes caffeine induced [ 3H]-noradrenaline release in calcium-free solution from peripheral sympathetic nerves

The transmitter releasing action of caffeine was studied in the absence of extracellular Ca 2+ from the peripheral sympathetic nerves of the rabbit main pulmonary artery. Caffeine (10 mM) increased the release of [ 3H]-noradrenaline moderately, but not significantly in Ca 2+-free (+1 mM EGTA) Krebs solution. When peripheral nerve endings/varicoslties were depolarized by elevating extracellular K + to 47.2 mM and 70.8 mM in Ca 2+-free solution, the transmitter releasing effect of 10 mM caffeine became significant. Ca 2+ removal itself transiently increased the [ 3H]-noradrenaline outflow. In the individual experiments the amount of the caffeine evoked transmitter release at 47.2 mM and 70.8 mM K +-depolarization was inversely correlated to the release evoked by Ca 2+-removal. Our results suggest that caffeine-sensitive calcium stores are present in peripheral nerve terminals of rabbit pulmonary artery, and part of the caffeine sensitive calcium stores may discharge during Ca 2+-removal from the extracellular solution.

Caffeine-sensitive calcium stores in presynaptic nerve endings: A physiological role?

Ca 2+-sensitive minielectrodes and the fluorescent cytosolic calcium probes, quin2 and fura2, were used to study some aspects of calcium homeostasis in intact and permeabilized synaptosomes from whole rat brain. Experiments in permeabilized synaptosomes revealed the existence of a vesicular, non-mitochondrial, ATP-dependent calcium uptake system with a vanadate sensitivity similar to that of brain microsomes, or endoplasmic reticulum-type calcium sequestering organelles. By using the fluorescent probes it was possible to show that caffeine mobilizes calcium from these internal stores in intact synaptosomes. Our results indicate a role of the caffeine sensitive calcium stores in the buffering of calcium loads elicited by plasma membrane depolarization.

Inositol 1,4,5 trisphosphate releases calcium from specialized sites within Limulus photoreceptors.

We have investigated the subcellular distribution and identity of inositol trisphosphate (InsP3)-sensitive calcium stores in living Limulus ventral photoreceptor cells, where light and InsP3 are known to raise intracellular calcium. We injected ventral photoreceptor cells with the photoprotein aequorin and viewed its luminescence with an image intensifier. InsP3 only elicited detectable aequorin luminescence when injected into the light-sensitive rhabdomeral (R)-lobe where aequorin luminescence induced by light was also confined. Calcium stores released by light and InsP3 are therefore localized to the R-lobe. Within the R-lobe, InsP3-induced aequorin luminescence was further confined around the injection site, due to rapid dilution and/or degradation of injected InsP3. Prominent cisternae of smooth endoplasmic reticulum are uniquely localized within the cell beneath the microvillar surface of the R-lobe (Calman, B., and S. Chamberlain, 1982, J. Gen. Physiol., 80:839-862). These cisternae are the probable site of InsP3 action.