Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca²⁺ ([Ca²⁺]_i) by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca²⁺]_i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M₃) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M₁) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M₃, but not M₁, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms—pilocarpine acting via M₃ receptors and Src-dependent transactivation of EGF receptors, and carbachol via M₁/M₃ receptors and PKC—converging on the ERK pathway. muscarinic receptor; epidermal growth factor receptor; protein kinase C     

The Ca2+-sensing receptor: a target for polyamines

The Ca²⁺-sensing receptor(CaR) is activated at physiological levels of externalCa²⁺(Ca_o) but is expressed in anumber of tissues that do not have well-established roles in thecontrol of Ca_o, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown byour current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic freeCa²⁺(Ca_i), inositol phosphateproduction, and the activity of a nonselective cation channel. Sperminestimulated Ca_i transients inCaR-transfected HEK cells, with a concentration producing ahalf-maximal response (EC₅₀) of ~500µM in the presence of 0.5 mMCa²⁺, whereas sustained increasesin Ca_i had anEC₅₀ of ~200 µM. The order ofpotency was spermine > spermidine >> putrescine. Elevation ofCa_o shifted theEC₅₀ for spermine sharply to theleft, with substantial stimulation below 100 µM. Addition ofsubthreshold concentrations of spermine increased the sensitivity ofCaR-expressing HEK cells to Ca_o.Parathyroid hormone secretion from bovine parathyroid cells wasinhibited by 50% in the presence of 200 µM spermine, a responsesimilar to that elicited by 2.0 mMCa_o. These data suggest thatpolyamines could be effective agonists for the CaR, and severaltissues, including the brain, may use the CaR as a target for theactions of spermine and other endogenous polycationic agonists.

     

Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor

We determined the effect of aromatic aminoacid stimulation of the human extracellular Ca²⁺-sensingreceptor (CaR) on intracellular Ca²⁺ concentration([Ca²⁺]_i) in single HEK-293 cells. Additionof L-phenylalanine or L-tryptophan (at 5 mM)induced [Ca²⁺]_i oscillations from a restingstate that was quiescent at 1.8 mM extracellular Ca²⁺concentration ([Ca²⁺]_e). Each[Ca²⁺]_i peak returned to baseline values, andthe average oscillation frequency was ~1 min¹ at37°C. Oscillations were not induced or sustained if the[Ca²⁺]_e was reduced to 0.5 mM, even in thecontinued presence of amino acid. Average oscillation frequency inresponse to an increase in [Ca²⁺]_e (from 1.8 to 2.5-5 mM) was much higher (~4 min¹) than thatinduced by aromatic amino acids. Oscillations in response to[Ca²⁺]_e were sinusoidal whereas those inducedby amino acids were transient. Thus both amino acids andCa²⁺, acting through the same CaR, produce oscillatoryincreases in [Ca²⁺]_i, but the resultantoscillation pattern and frequency allow the cell to discriminate whichagonist is bound to the receptor.

     

Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria

In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca²⁺ ([Ca²⁺]_m), which fluctuates in coordination with intracellular Ca²⁺ ([Ca²⁺]_i). Polyunsaturated FA (PUFA) have been shown to cause [Ca²⁺]_i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP₃) receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca²⁺]_m with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, blocks PUFA-induced Ca²⁺ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca²⁺ release from mitochondria, possibly via the Ca²⁺ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells. arachidonic acid; mitochondrial Ca²⁺ uniporter; G protein-coupled receptor; IP₃ receptor     

Plasma and intracellular membrane inositol 1,4,5-trisphosphate receptors mediate the Ca(2+) increase associated with the ATP-induced increase in ciliary beat frequency

An increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP₃) receptor-dependent Ca²⁺ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP₃ peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca²⁺ from internal stores and an influx of extracellular Ca²⁺, respectively. In addition, ATP-dependent Ca²⁺ mobilization required protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP₃ receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca²⁺]_i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca²⁺ influx is also dependent on IP₃ receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP₃ receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP₃ receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP₃ receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP₃ receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP₃ receptor can directly trigger Ca²⁺ influx in response to ATP. transduction mechanisms; P2Y receptor; calcium influx     

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs. calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling     

Calcium-sensing receptor activation induces intracellular calcium oscillations

Parathyroid hormone secretion isexquisitely sensitive to small changes in serum Ca²⁺concentration, and these responses are transduced via theCa²⁺-sensing receptor (CaR). We utilized heterologousexpression in HEK-293 cells to determine the effects of small,physiologically relevant perturbations in extracellularCa²⁺ on CaR signaling viaphosphatidylinositol-phospholipase C, using changes in fura 2 fluorescence to quantify intracellular Ca²⁺. Chronicexposure of CaR-transfected cells to Ca²⁺ in the range from0.5 to 3 mM modulated the resting intracellular Ca²⁺concentration and the subsequent cellular responses to acute extracellular Ca²⁺ perturbations but had no effect onthapsigargin-sensitive Ca²⁺ stores. Modest,physiologically relevant increases in extracellular Ca²⁺concentration (0.5 mM increments) caused sustained (30-40 min) low-frequency oscillations of intracellular Ca²⁺ (~45 speak to peak interval). Oscillations were eliminated by 1 µMthapsigargin but were insensitive to protein kinase inhibitors (staurosporine, KN-93, or bisindolylmaleimide I). Staurosporine didincrease the fraction of cells oscillating at a given extracellular Ca²⁺ concentration. Serum Ca²⁺ concentrationsthus chronically regulate cells expressing CaR, and small perturbationsin extracellular Ca²⁺ alter both resting intracellularCa²⁺ as well as Ca²⁺ dynamics.

     

Soybean β51–63 peptide stimulates cholecystokinin secretion via a calcium-sensing receptor in enteroendocrine STC-1 cells

We previously demonstrated that intraduodenal administration of an arginine-rich β51–63 peptide in soybean β-conglycinin suppresses food intake via cholecystokinin (CCK) secretion in rats. However, the cellular mechanisms by which the β51–63 peptide induces CCK secretion remain to be clarified. In the present study, we examined whether the extracellular calcium-sensing receptor (CaR) mediates β51–63-induced CCK secretion in murine CCK-producing enteroendocrine cell line STC-1. CCK secretion and changes in intracellular Ca²⁺ concentration in response to β51–63 peptide were measured in STC-1 cells under various extracellular Ca²⁺ concentrations and after treatment with a CaR antagonist. Intracellular Ca²⁺ concentrations in response to β51–63 peptide and extracellular Ca²⁺ were also measured in CaR-expressing human embryonic kidney (HEK-293) cells. The β51-63 peptide induced CCK secretion and intracellular Ca²⁺ mobilization in STC-1 cells under normal (1.2 mM) extracellular Ca²⁺ conditions in a dose-dependent manner. These responses to β51–63 peptide were reduced by the removal of intra- or extracellular Ca²⁺ but enhanced by increasing extracellular Ca²⁺ concentrations. Intracellular Ca²⁺ mobilization induced by extracellular Ca²⁺ was also increased by the pretreatment with β51–63 peptide. Treatment with a specific CaR antagonist (NPS2143) inhibited β51–63-induced CCK secretion and intracellular Ca²⁺ mobilization. In addition, HEK-293 cells transfected with CaR acquired sensitivity to the β51–63 peptide. From these results, we conclude that CaR is the β51–63 peptide sensor responsible for the stimulation of CCK secretion in enteroendocrine STC-1 cells.     

Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy

Agonist-inducedhypertrophy of cultured neonatal rat ventricular myocytes (NRVM) hasbeen attributed to biochemical signals generated during receptoractivation. However, NRVM hypertrophy can also be induced byspontaneous or electrically stimulated contractile activity in theabsence of exogenous neurohormonal stimuli. Using single-cell imagingof fura 2-loaded myocytes, we found that low-density, noncontractingNRVM begin to generate intracellularCa²⁺ concentration([Ca²⁺]_i)transients and contractile activity within minutes of exposure to the₁-adrenergic agonistphenylephrine (PE; 50 µM). However, NRVM pretreated with verapamiland then stimulated with PE failed to elicit[Ca²⁺]_itransients and beating. We therefore examined whether PE-induced [Ca²⁺]_itransients and contractile activity were required to elicit specificaspects of the hypertrophic phenotype. PE treatment (48-72 h)increased cell size, total protein content, total protein-to-DNA ratio,and myosin heavy chain (MHC) isoenzyme content. PE also stimulatedsarcomeric protein assembly and prolonged MHC half-life. However,blockade of voltage-gated L-typeCa²⁺ channels with verapamil,diltiazem, or nifedipine (10 µM) blocked PE-induced total protein andMHC accumulation and prevented the time-dependent assembly ofmyofibrillar proteins into sarcomeres. Inhibition of actin-myosincross-bridge cycling with 2,3-butanedione monoxime (7.5 mM) alsoprevented PE-induced total protein and MHC accumulation, indicatingthat mechanical activity, rather than[Ca²⁺]_itransients per se, was required. In contrast, blockade of[Ca²⁺]_itransients and contractile activity did not prevent the PE-induced increase in cell surface area, activation of the mitogen-activated protein kinases ERK1 and ERK2, or upregulation of atrial natriuretic factor gene expression. Thus contractile activity is required to elicitsome but not all aspects of the the hypertrophic phenotype induced by₁-adrenergic receptoractivation.

     

The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase and activate ERK, G_q to stimulate phospholipase C and phospholipase A₂, and G to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G_12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G_i and G_q, and a p115RhoGEF construct containing the RGS domain for G_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT inhibited extracellular Ca²⁺-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G_i and G_q. This suggests that the CaR may regulate cytoskeleton via G_12/13, Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins     

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na(+)-K(+)-ATPase activity

The Na⁺-K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺-K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na⁺-K⁺-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na⁺-K⁺-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^–6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺-K⁺-ATPase inhibition by lowering extracellular K⁺, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ 100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity (measured as stimulation of QO₂ by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na⁺-K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type. protein kinase C; intracellular Ca²⁺ concentration; muscarinic receptor; ₁-subunit; potassium removal     

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca²⁺, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca²⁺ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca²⁺ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X₃R > P2X_2b + X₄R > P2X_2bR > P2X_2a + X₄R > P2X₄R > P2X_2aR > P2X₇R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca²⁺ influx because of the coactivation of endogenously expressed Ca²⁺-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca²⁺ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca²⁺ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca²⁺ signals were not affected by voltage-gated Ca²⁺ influx and removal of extracellular sodium. Ca²⁺ signals reflected well the receptor-specific EC₅₀ values for ATP and the extracellular Zn²⁺ and pH sensitivities of P2XRs. These results indicate that intracellular Ca²⁺ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals     

Protein kinase C-mediated desensitization of the neurokinin 1 receptor

An understanding of the mechanisms that regulate signaling bythe substance P (SP) or neurokinin 1 receptor (NK1-R) is of interestbecause of their role in inflammation and pain. By using activators andinhibitors of protein kinase C (PKC) and NK1-R mutations of potentialPKC phosphorylation sites, we determined the role of PKC indesensitization of responses to SP. Activation of PKC abolishedSP-induced Ca²⁺ mobilization in cells that expresswild-type NK1-R. This did not occur in cells expressing aCOOH-terminally truncated NK1-R (NK1-R324), which may correspond toa naturally occurring variant, or a point mutant lacking eightpotential PKC phosphorylation sites within the COOH tail (NK1-RSer-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392,Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, thet_1/2 of SP-induced Ca²⁺mobilization was seven- and twofold greater in cells expressing NK1-R324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in thet_1/2 of SP-induced Ca²⁺mobilization. Neither inhibition of PKC nor receptor mutation affecteddesensitization of Ca²⁺ mobilization to repeated challengewith SP or SP-induced endocytosis of the NK1-R. Thus PKC regulatesSP-induced Ca²⁺ mobilization by full-length NK1-R and doesnot regulate a naturally occurring truncated variant. PKC doesnot mediate desensitization to repeated stimulation or endocytosis ofthe NK1-R.

     

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca²⁺ level ([Ca²⁺]_i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca²⁺]_i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca²⁺]_i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca²⁺]_i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca²⁺]_i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca²⁺ signaling and degranulation. mast cell; immunoglobulin E; cytochalasin D; Y-27632; wortmannin     

Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells

Astrocytes are involved in normal andpathological brain functions, where they become activated and undergoreactive gliosis. Astrocytes have been shown to respond toextracellular nucleotides via the activation of P2 receptors, either Gprotein-coupled P2Y receptors or P2X receptors that are ligand-gatedion channels. In this study, we have examined the manner in whichactivation of the P2X₇ nucleotide receptor, anextracellular ATP-gated ion channel expressed in astrocytes, can leadto the phosphorylation of ERK1/2. Results showed that theP2X₇ receptor agonist2',3'-O-(4-benzoyl)benzoyl-ATP induced ERK1/2phosphorylation in human astrocytoma cells overexpressing therecombinant rat P2X₇ receptor (rP2X₇-R), aresponse that was inhibited by the P2X₇ receptorantagonist, oxidized ATP. Other results suggest thatrP2X₇-R-mediated ERK1/2 phosphorylation was linked to thephosphorylation of the proline-rich/Ca²⁺-activated tyrosinekinase Pyk2, c-Src, phosphatidylinositol 3'-kinase, and proteinkinase C activities and was dependent on the presence ofextracellular Ca²⁺. These results support the hypothesisthat the P2X₇ receptor and its signaling pathways play arole in astrocyte-mediated inflammation and neurodegenerative disease.

     

Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells

Although it iswell known that progesterone alters uterine contractility and plays animportant role in maintenance of pregnancy, the biochemical mechanismsby which progesterone alters uterine contractility in human gestationare less clear. In this investigation we sought to identifyprogesterone-induced adaptations in human myometrial smooth musclecells that may alter Ca²⁺signaling in response to contractile agents. Cells were treated withvehicle or the progesterone analog medroxyprogesterone acetate (MPA)for 5 days, and intracellular freeCa²⁺ concentration([Ca²⁺]_i)was quantified after treatment with oxytocin (OX) or endothelin (ET)-1.OX- and ET-1-induced increases in[Ca²⁺]_iwere significantly attenuated in cells pretreated with MPA in adose-dependent manner. Progesterone receptor antagonists prevented theattenuated Ca²⁺ transients inducedby MPA. ET_A andET_B receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted insignificant downregulation of ET_Aand ET_B receptor binding. MPA didnot alter ionomycin-stimulated increases in[Ca²⁺]_iand had no effect on inositol trisphosphate-dependent or -independent release of Ca²⁺ from internalCa²⁺ stores. We conclude thatadaptations of Ca²⁺ homeostasis inmyometrial cells during pregnancy may include progesterone-inducedmodification of receptor-mediated increases in[Ca²⁺]_i.     

Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+ signaling

Astrocytes represent a major target for endothelins (ETs), afamily of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in thebrain. In the present study we have investigated the involvement ofdifferent ET receptor subtypes on intercellular dye diffusion, intracellular Ca²⁺homeostasis, and intercellularCa²⁺ signaling in cultured ratastrocytes from hippocampus and striatum. Depending on the ETconcentration and the receptor involved, ET-1- and ET-3-inducedintracellular Ca²⁺ increases withdifferent response patterns. Both ET-1 and ET-3 are powerful inhibitorsof gap junctional permeability and intercellular Ca²⁺ signaling. The nonselectiveET receptor agonist sarafotoxin S6b and theET_B receptor-selective agonist IRL1620 mimicked these inhibitions. The ET-3 effects were only marginallyaffected by an ET_A receptorantagonist but completely blocked by anET_B receptor antagonist. However,the ET-1-induced inhibition of gap junctional dye transfer andintercellular Ca²⁺ signaling wasonly marginally blocked by ET_A orET_B receptor-selective antagonistsbut fully prevented when these antagonists were applied together. TheET-induced inhibition of gap junction permeability and intercellularCa²⁺ signaling indicates thatimportant changes in the function of astroglial communication mightoccur when the level of ETs in the brain is increased.

     

PAF receptor antagonist modulates neutrophil responses with thermal injury in vivo

The role of platelet-activating factor (PAF) inCa²⁺ signaling and Ca²⁺-related enhancement ofreactive oxygen intermediate (ROI) generation in neutrophils ofburn-injured rats was ascertained by evaluating the effect of treatmentof the rats with a PAF receptor antagonist. The treatment of rats withthe antagonist also allowed us to evaluate the role of PAF in thepriming of neutrophil ROI response with burn in vivo. A full skinthickness burn injury was produced in anesthetized rats by exposing30% of total body surface area to 98°C water for 10 s. Sham andburn rats were killed 1 day later, and their blood was collected toobtain neutrophils. Fluorescence-activated cell sorter analysis wasused to quantify ROI production by the neutrophils. Cytosolic-freeCa²⁺ concentration ([Ca²⁺]_i)imaging technique was employed to measure neutrophil[Ca²⁺]_i in individual cells andmicrofluorometry for the assessment of[Ca²⁺]_i responses in suspensions ofneutrophils. There was an overt enhancement of ROI generation by burnrat neutrophils. ROI release was accompanied by a marked elevation of[Ca²⁺]_i signaling. The treatment of rats withPAF receptor antagonist before burn prevented the upregulation of both[Ca²⁺]_i and ROI generation in neutrophils.These studies indicate that enhanced ROI production in neutrophils inthe early stages after burn injury results from a PAF-mediated primingof the [Ca²⁺]_i signaling pathways in vivo.

     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca²⁺ concentration ([Ca²⁺]_o) and modulation of cellular processes associated with acute or sustained changes in [Ca²⁺]_o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca²⁺]_o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca²⁺]_o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca²⁺]_o required influx of Ca²⁺through Ni²⁺-sensitive Ca²⁺channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca²⁺]_o. Activation of ERK1/2 by high [Ca²⁺]_o was not necessary for the [Ca²⁺]_o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca²⁺]_o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.