Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland

Thepurpose of this study was to determine the role of p42/p44mitogen-activated protein kinase (MAPK) in₁-adrenergically and cholinergically stimulated proteinsecretion in rat lacrimal gland acinar cells and the pathways used bythese agonists to activate MAPK. Acini were isolated by collagenasedigestion and incubated with the ₁-adrenergic agonistphenylephrine or the cholinergic agonist carbachol, and activation ofMAPK and protein secretion were then measured. Phenylephrine andcarbachol activated MAPK in a time- and concentration-dependent manner.Inhibition of MAPK significantly increased phenylephrine- andcarbachol-induced protein secretion. Inhibition of EGF receptor (EGFR)with AG1478, an inhibitor of the EGFR tyrosine kinase activity,significantly increased phenylephrine- but not carbachol-inducedprotein secretion. Whereas phenylephrine-induced activation of MAPK wascompletely inhibited by AG1478, activation of MAPK by carbachol wasnot. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60^Src, and possibly Pyk2, toactivate MAPK. We conclude that, in the lacrimal gland, activation ofMAPK plays an inhibitory role in ₁-adrenergically andcholinergically stimulated protein secretion and that these agonistsuse different signaling mechanisms to activate MAPK.

     

p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells

We havepreviously shown that Ca²⁺-dependent Clsecretion across intestinal epithelial cells is limited by a signalingpathway involving transactivation of the epidermal growth factorreceptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK inregulation of Ca²⁺-dependent Cl secretion.Western blot analysis of T₈₄ colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 µM)stimulated phosphorylation and activation of p38 MAPK. The p38inhibitor SB-203580 (10 µM) potentiated and prolonged short-circuitcurrent (I_sc) responses to CCh acrossvoltage-clamped T₈₄ cells to 157.4 ± 6.9% of thosein control cells (n = 21; P < 0.001).CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitortyrphostin AG-1478 (0.1 nM-10 µM) and by the Src family kinaseinhibitor PP2 (20 nM-2 µM). The effects of CCh on p38phosphorylation were mimicked by thapsigargin (TG; 2 µM), whichspecifically elevates intracellular Ca²⁺, and wereabolished by the Ca²⁺ chelator BAPTA-AM (20 µM), implyinga role for intracellular Ca²⁺ in mediating p38 activation.SB-203580 (10 µM) potentiated I_sc responses toTG to 172.4 ± 18.1% of those in control cells (n = 18; P < 0.001). When cells were pretreated withSB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs,respectively, I_sc responses to TG and CCh weresignificantly greater than those observed with either inhibitor alone.We conclude that Ca²⁺-dependent agonists stimulate p38 MAPKin T₈₄ cells by a mechanism involving intracellularCa²⁺, Src family kinases, and the EGFR. CCh-stimulated p38activation constitutes a similar, but distinct and complementary,antisecretory signaling pathway to that of ERK MAPK.

     

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     

Vasopressin-mediated mitogenic signaling in intestinal epithelial cells

The role of G protein-coupled receptorsand their ligands in intestinal epithelial cell signaling andproliferation is poorly understood. Here, we demonstrate that argininevasopressin (AVP) induces multiple intracellular signal transductionpathways in rat intestinal epithelial IEC-18 cells via aV_1A receptor. Addition of AVP to these cells induces arapid and transient increase in cytosolic Ca²⁺concentration and promotes protein kinase D (PKD) activation through aprotein kinase C (PKC)-dependent pathway, as revealed by in vitrokinase assays and immunoblotting with an antibody that recognizesautophosphorylated PKD at Ser⁹¹⁶. AVP also stimulates thetyrosine phosphorylation of the nonreceptor tyrosine kinaseproline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinasephosphorylation at Tyr⁴¹⁸, indicative of Src activation.AVP induces extracellular signal-related kinase (ERK)-1(p44^mapk) and ERK-2 (p42^mapk) activation, aresponse prevented by treatment with mitogen-activated protein kinasekinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors(GF-I and Ro-31-8220), depletion of Ca²⁺ (EGTA andthapsigargin), selective epidermal growth factor receptor (EGFR)tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or theselective Src family kinase inhibitor PP-2. Furthermore, AVP acts as apotent growth factor for IEC-18 cells, inducing DNA synthesis and cellproliferation through ERK-, Ca²⁺-, PKC-, EGFR tyrosinekinase-, and Src-dependent pathways.

     

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     

EGF inhibits muscarinic receptor-mediated calcium signaling in a human salivary cell line

The effects of epidermal growth factor(EGF) on intracellular calcium ([Ca²⁺]_i)responses to the muscarinic agonist carbachol were studied in a humansalivary cell line (HSY). Carbachol (10⁴ M)-stimulated[Ca²⁺]_i mobilization was inhibited by 40%after 48-h treatment with 5 × 10¹⁰ M EGF. EGF alsoreduced carbachol-induced [Ca²⁺]_i inCa²⁺-free medium and Ca²⁺ influx followingrepletion of extracellular Ca²⁺. UnderCa²⁺-free conditions, thapsigargin, an inhibitor ofCa²⁺ uptake to internal stores, induced similar[Ca²⁺]_i signals in control and EGF-treatedcells, indicating that internal Ca²⁺ stores were unaffectedby EGF; however, in cells exposed to thapsigargin, Ca²⁺influx following Ca²⁺ repletion was reduced by EGF.Muscarinic receptor density, assessed by binding of the muscarinicreceptor antagonistL-[benzilic-4,4'-³HCN]quinuclidinyl benzilate([³H]QNB), was decreased by 20% after EGF treatment.Inhibition of the carbachol response by EGF was not altered by phorbolester-induced downregulation of protein kinase C (PKC) but was enhancedupon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of thecarbachol response by EGF were both blocked by the MAP kinase pathwayinhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca²⁺ release from internal stores andalso exerts a direct inhibitory action on Ca²⁺ influx. Adecline in muscarinic receptor density may contribute to EGF inhibitionof carbachol responsiveness. The inhibitory effect of EGF is mediatedby the MAP kinase pathway and is potentiated by a distinct modulatorycascade involving activation of PKC. EGF may play a physiological rolein regulating muscarinic receptor-stimulated salivary secretion.

     

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and proliferation in pancreatic -cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca²⁺]_i involves Ca²⁺-induced Ca²⁺ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca²⁺]_i and insulin release in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca²⁺]_i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K⁺ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca²⁺]_i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K⁺. Ovine prolactin increased [Ca²⁺]_i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca²⁺]_i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca²⁺]_i. Our study indicates that hGH stimulates rise in [Ca²⁺]_i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells. c-Src; growth hormone receptor; prolactin receptor; Ca²⁺-induced Ca²⁺ release     

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G_q-coupled P2Y₂ receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of - and -actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of - or -actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca²⁺-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis. lung; mucus; exocytosis     

EGF stimulates proliferation of mouse embryonic stem cells: involvement of Ca2+ influx and p44/42 MAPKs

We examined the effect of EGF on the proliferation of mouse embryonic stem (ES) cells and their related signal pathways. EGF increased [³H]thymidine and 5-bromo-2'-deoxyuridine incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). Inhibition of EGFR tyrosine kinase with AG-1478 or herbimycin A, inhibition of PLC with neomycin or U-73122, inhibition of PKC with bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²⁺ channels with nifedipine or methoxyverapamil prevented EGF-induced [³H]thymidine incorporation. PKC-, -_I, -, -, and - were translocated to the membrane and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased in response to EGF. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²⁺]_i. EGF also increased inositol phosphate levels, which were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and pretreatment with antioxidant (N-acetyl-L-cysteine) inhibited EGF-induced increase of [Ca²⁺]_i. In addition, we observed that p44/42 MAPK phosphorylation by EGF and inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²⁺ channels blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs with PD-98059 (MEK inhibitor) attenuated EGF-induced increase of [³H]thymidine incorporation. Finally, inhibition of EGFR tyrosine kinase, PKC, Ca²⁺ channels, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4, respectively. In conclusion, EGF partially stimulates proliferation of mouse ES cells via PLC/PKC, Ca²⁺ influx, and p44/42 MAPK signal pathways through EGFR tyrosine kinase phosphorylation. calcium; epidermal growth factor; mitogen-activated protein kinases; protein kinase C     

Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.     

Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells

We have examined theinteraction between muscarinic and ₂-adrenergic receptoractivation on inositol phosphate (IP) formation in the nonpigmentedcells of the ciliary body epithelium (NPE cells) of the rabbit. We havecompared these changes with those previously observed in theintracellular free Ca²⁺ concentration. Whereas muscarinicreceptor activation causes an increase in intracellularCa²⁺ and IP formation, activation of₂-receptors does not significantly increase eitherintracellular Ca²⁺ or IPs over basal levels. However,simultaneous activation of muscarinic and ₂-adrenergicreceptors with the specific agonists carbachol and UK-14304 producesmassive Ca²⁺ increases and results in a synergisticincrease in IP formation. This synergistic IP formation is inhibited byboth muscarinic and ₂-adrenergic receptor antagonists aswell as by pertussis toxin and an inhibitor of phospholipase C. IPformation is predominantly independent of intracellularCa²⁺, because it is decreased but not prevented by blockingthe entry of Ca²⁺ with LaCl₃ or chelatingintracellular Ca²⁺ with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca²⁺ resulting fromsimultaneous activation of muscarinic and ₂-adrenergic receptors.

     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells

We have characterized the muscarinic AChreceptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK)strain II epithelial cells. Binding studies with themembrane-impermeable antagonist N-[³H]methylscopolaminedemonstrated that mAChRs are ~2.5 times more abundant on thebasolateral than on the apical surface. Apical, but not basolateral,mAChRs inhibited forskolin-stimulated adenylyl cyclase activity inresponse to the agonist carbachol. Neither apical nor basolateralmAChRs exhibited detectable carbachol-stimulated phospholipase Cactivity. Carbachol application to the apical or the basolateralmembrane resulted in a threefold increase in intracellularCa²⁺ concentration, which wascompletely inhibited by pertussis toxin on the apical side andpartially inhibited on the basolateral side. RT-PCR analysis showedthat MDCK cells express the M₄ and M₅ receptor mRNAs. These datasuggest that M₄ receptors reside on the apical and basolateral membranes of polarized MDCK strain IIcells and that the M₅ receptor mayreside in the basolateral membrane of a subset of cells.

     

Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells

Mucin secretion by airway goblet cells is under the control ofapical P2Y₂, phospholipaseC-coupled purinergic receptors. In SPOC1 cells, the mobilization ofintracellular Ca²⁺ by ionomycin orthe activation of protein kinase C (PKC) by phorbol 12-myristate13-acetate (PMA) stimulates mucin secretion in a fully additive fashion[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):L201-L210, 1997]. This apparent independence between PKC andCa²⁺ in the stimulation of mucinsecretion was tested in streptolysin O-permeabilized SPOC1 cells. Thesecells were fully competent to secrete mucin whenCa²⁺ was elevated from 100 nM to3.1 µM for 2 min following permeabilization; theCa²⁺EC₅₀ was 2.29 ± 0.07 µM.Permeabilized SPOC1 cells were exposed to PMA or 4-phorbol atCa²⁺ activities ranging from 10 nMto 10 µM. PMA, but not 4-phorbol, increased mucin release at allCa²⁺ activities tested: at 10 nMCa²⁺ mucin release was 2.1-foldgreater than control and at 4.7 µM Ca²⁺ mucin release was maximal(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µMthan at 10 nM Ca²⁺. Hence, SPOC1cells possess Ca²⁺-insensitive,PKC-dependent, and Ca²⁺-dependentPKC-potentiated pathways for mucin granule exocytosis.

     

Muscarinic modulation of Cav2.3 (R-type) calcium channels is antagonized by RGS3 and RGS3T

Ca²⁺ influx through voltage-gated R-type (Ca_V2.3) Ca²⁺ channels is important for hormone and neurotransmitter secretion and other cellular events. Previous studies have shown that Ca_V2.3 is both inhibited and stimulated through signaling mechanisms coupled to muscarinic ACh receptors. We previously demonstrated that muscarinic stimulation of Ca_V2.3 is blocked by regulator of G protein signaling (RGS) 2. Here we investigated whether muscarinic inhibition of Ca_V2.3 is antagonized by RGS3. RGS3 is particularly interesting because it contains a lengthy (380 residue) amino-terminal domain of uncertain physiological function. Ca_V2.3, M₂ muscarinic ACh receptors (M₂R), and various deletion mutants of RGS3, including its native isoform RGS3T, were expressed in HEK293 cells, and agonist-dependent inhibition of Ca_V2.3 was quantified using whole cell patch-clamp recordings. Full-length RGS3, RGS3T, and the core domain of RGS3 were equally effective in antagonizing inhibition of Ca_V2.3 through M₂R. These results identify RGS3 and RGS3T as potential physiological regulators of R-type Ca²⁺ channels. Furthermore, they suggest that the signaling activity of RGS3 is unaffected by its extended amino-terminal domain. Confocal microscopy was used to examine the intracellular locations of four RGS3-enhanced green fluorescent protein fusion proteins. The RGS3 core domain was uniformly distributed throughout both cytoplasm and nucleus. By contrast, full-length RGS3, RGS3T, and the amino-terminal domain of RGS3 were restricted to the cytoplasm. These observations suggest that the amino terminus of RGS3 may serve to confine it to the cytoplasmic compartment where it can interact with cell surface receptors, heterotrimeric G proteins, and other signaling proteins. calcium channels; regulator of G protein signaling proteins; muscarinic acetylcholine receptors; enhanced green fluorescent protein-fusion proteins; voltage-gated R-type calcium channels     

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     

Involvement of stretch-activated cation channels in hypotonically induced insulin secretion in rat pancreatic beta-cells

In isolated rat pancreatic -cells, hypotonic stimulation elicited an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) at 2.8 mM glucose. The hypotonically induced [Ca²⁺]_c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca²⁺ channel blocker, and by Gd³⁺, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca²⁺]_c elevation was not inhibited by suramin, a P₂ purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of -cells and produced outwardly rectifying cation currents; Gd³⁺ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd³⁺ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic -cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca²⁺ channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium     

The calcium-dependent activity of large-conductance, calcium-activated K+ channels is enhanced by Pyk2- and Hck-induced tyrosine phosphorylation

Recent results showing that large-conductance, calcium-activated K⁺ (BK_Ca) channels undergo direct tyrosine phosphorylation in the presence of c-Src tyrosine kinase have suggested the involvement of these channels in Src-mediated signaling pathways. Given the important role for c-Src in integrin-mediated signal transduction, we have examined the potential regulation of BK_Ca channels by proline-rich tyrosine kinase 2 (Pyk2), a calcium-sensitive tyrosine kinase activated upon integrin stimulation. Transient coexpression of murine BK_Ca channels with either wild-type Pyk2 or hematopoietic cell kinase (Hck), a Src-family kinase, led to an enhancement of BK_Ca channel activity over the range of 1–10 µM free calcium, whereas coexpression with catalytically inactive forms of either kinase did not significantly alter BK_Ca gating compared with channels expressed alone. In the presence of either wild-type Pyk2 or Hck, BK_Ca -subunits were found to undergo tyrosine phosphorylation, as determined by immunoprecipitation and Western blotting strategies. However, tyrosine phosphorylation of the BK_Ca -subunit was not detected for channels expressed alone or together with inactive forms of either Pyk2 or Hck. Interestingly, wild-type, but not inactive, Pyk2 was also present in BK_Ca channel immunoprecipitates, suggesting that Pyk2 may coassociate with the BK_Ca channel complex after phosphorylation. Collectively, the observed modulation and phosphorylation of BK_Ca channels by Pyk2 and a Src-family kinase may reflect a general cellular mechanism by which G protein-coupled receptor and/or integrin activation leads to the regulation of membrane ion channels. BK channels; tyrosine kinase; calcium; immunoprecipitation     

Regulation of intracellular calcium in human esophageal smooth muscles

We have investigated sources ofCa²⁺ contributing to excitation ofhuman esophageal smooth muscle, using fura 2 to study cytosolic freeCa²⁺ concentration([Ca²⁺]_i)in dispersed cells and contraction of intact muscles. Acetylcholine (ACh) caused an initial peak rise of[Ca²⁺]_ifollowed by a plateau accompanied by reversible contraction. Removal ofextracellular Ca²⁺ or addition ofdihydropyridine Ca²⁺ channelblockers reduced the plateau phase but did not prevent contraction.Caffeine also caused elevation of[Ca²⁺]_iand blocked responses to ACh. Undershoots of[Ca²⁺]_iwere apparent after ACh or caffeine. Blockade of the sarcoplasmic reticular Ca²⁺-ATPase bycyclopiazonic acid (CPA) reduced the ACh-evoked increase of[Ca²⁺]_iand abolished the undershoot, indicating involvement ofCa²⁺ stores. When contraction wasstudied in intact muscles, removal ofCa²⁺ or addition of nifedipinereduced, but did not abolish, carbachol (CCh)-induced contraction.Elevation of extracellular K⁺caused contraction that was inhibited by nifedipine, although CCh stillelicited contraction. CPA caused contraction and suppressed theCCh-induced contraction, whereas ryanodine reduced CCh-induced contraction. Our studies provide evidence that muscarinic excitation ofhuman esophagus involves both release ofCa²⁺ from intracellular stores andinflux of Ca²⁺.