ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.     

Convulxin induces platelet shape change through myosin light chain kinase and Rho kinase.

Once platelets are activated, the first event to occur is a rapid change in shape, associated with Ca2+/calmodulin-dependent myosin light chain (MLC) phosphorylation and with Rho kinase activation. The purpose of this study was to investigate which is the biochemical pathway that leads to platelet shape change in response to convulxin, a selective GpVI activator, and to verify whether MLC phosphorylation is essential for this process. The inhibition of the Ca2+-dependent pathway by means of the Ca2+ chelator BAPTA, the Ca2+/calmodulin inhibitor W-7 or the cAMP enhancing drug iloprost reduced about 50% of platelet shape change in response to convulxin. The treatment with either the Rho kinase inhibitors Y27632 or HA 1077 had no effect on platelet shape change induced by convulxin. When both Ca2+/calmodulin-dependent and Rho kinase-dependent pathways were concomitantly inhibited by the combined use of Y27632 plus BAPTA, W-7 or iloprost, platelet shape change was completely abolished. Our findings suggest that convulxin-induced platelet shape change occurs via both pathways, the Ca2+/calmodulin-dependent, which appears to be more important, and the Rho kinase-dependent one. The pattern of MLC phosphorylation was not modified by Rho kinase inhibitors. Conversely, the inhibition of the Ca2+-dependent pathway caused a strong reduction of MLC phosphorylation in BAPTA-treated platelets, and a total inhibition in W-7 or iloprost-treated platelets. Our results demonstrate that following Rho kinase-dependent pathway platelet shape change can occur without the involvement of MLC phosphorylation.     

Effects of extracellular nucleotides in the thyroid: P2Y2 receptor-mediated ERK1/2 activation and c-Fos induction in PC Cl3 cells

Aim of the present paper was to investigate the signaling pathways of P2Y2 in rat thyroid PC Cl3 cell line and its effects on proliferation. This study demonstrates that P2Y2 activation provoked: (a) a cytosol-to-membrane translocation of PKC-alpha, -betaI and -epsilon; (b) the phosphorylation of the extra cellular signal-regulated kinases 1 and 2 (ERK1/2); (c) the expression of c-Fos protein; (d) no effects on the G1/S progression and overall cell proliferation. The P2Y2-stimulated ERK1/2 phosphorylation was: (a) completely blocked by PD098059, a mitogen-activated protein kinase (MEK) inhibitor or by W-7, a Ca2+-calmodulin (CaM) antagonist; (b) reduced by GF109203X, inhibitor of PKCs, or AG1478, inhibitor of EGFR tyrosine kinase, or LY294002/wortmannin, inhibitors of phosphoinositide 3-kinases, or cytochalasin D, inhibitor of actin microfilament bundles polymerization. The c-Fos induction was greatly diminished by Go6976 or PD098059, and completely abolished when combined. In conclusion, data indicate that the P2Y2-induced phosphorylation of ERK1/2 and the induction of c-Fos are due to the operation of CaM, with PKC, PI3K, EGFR and receptor endocytosis mechanisms endorsing the signalling. On the other hand, no mitogenic effects of P2Y2 are whatsoever noticed in PC Cl3 cells.     

Calmodulin is involved in the Ca2+-dependent activation of ceramide kinase as a calcium sensor

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca(2+) (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca(2+) as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca(2+)-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca(2+). The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM.CERK complexes. An in vitro CaM binding assay also demonstrated Ca(2+)-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca(2+) sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca(2+)-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca(2+)-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells.     

Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts.

When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI-38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y-27632), myosin light chain kinase inhibitor (ML-7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf-enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf-enhanced MLC phosphorylation was inhibited by Y-27632 and ML-7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation.     

The involvement of Ca2+ and myosin light chain kinase in collagen-induced platelet activation.

In this study we have used several complementary biochemical and immunological techniques to examine the involvement of Ca2+ and myosin light chain kinase in collagen-induced platelet activation. Our results indicate that collagen stimulates a rapid influx of external Ca2+ (within the first 1-5 min of treatment) which is followed by phosphorylation of myosin light chains (within 10 min of treatment) and granule secretion (within 15 min of treatment). In addition, we have found that certain Ca2+ channel entry blockers (e.g. nifedipine and bepridil) or calmodulin antagonists (e.g. W-7) specifically inhibit collagen-induced Ca2+ influx, myosin light chain phosphorylation and subsequent granule secretion. These data suggest that Ca2+/calmodulin-dependent myosin light chain kinase-mediated myosin light chain phosphorylation is necessary for regulating the actomyosin-related contractility required for normal platelet function.     

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.     

Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells

We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signal-regulated kinase-1/2 (ERK1/2) that is normally mediated by AMP-activated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca(2+)/calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X(7) receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP.     

Role of calmodulin and myosin light chain kinase in the activation of carbachol-activated cationic current in murine ileal myocytes

We investigated the effect of calmodulin (CaM) and myosin light chain kinase (MLCK) on murine ileal myocytes using the whole-cell patch-clamp technique. Under the voltage clamp, at the holding potential of -60 mV, 50 micromol/L carbachol (CCh) induced inward currents (I CCh), and spontaneous decay of I CCh occurred. The peak inward currents induced by the repetitive application of CCh (50 micromol/L) tended to decrease in amplitude. Intracellular application of 0.2 mmol/L guanosine 5'-O-(gamma-thio)triphosphate (GTP gammaS) from the patch electrode induced an inward current at a holding potential of -60m V, and the peak inward currents induced by the repetitive application of Cs tended to decrease slightly in amplitude. The amplitude of I CCh was reduced by pretreatment either with W-7, trifluoroperazine, W-5, and melittin (CaM inhibitors) or with ML-7 and ML-9 (selective MLCK inhibitors), and the inhibitory effects were reversible. However, when we pretreated with 50 micromol/L W-7 or 5 micromol/L ML-7 on GTP gammaS-induced inward currents, almost no inhibition was observed in the inward currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTP gammaS-induced currents. We conclude that CaM and MLCK modulate the activation process of I CCh in murine ileal myocytes and suggest that the classical type transient receptor potential (TRPC) channel 5 might be a candidate for nonselective cationic currents (NSCC) activated by muscarinic stimulation in gastrointestinal smooth muscle cells.     

平滑肌细胞迁移的肌球蛋白轻链非磷酸化途径

为了阐明平滑肌细胞迁移存在肌球蛋白轻链非磷酸化调节途径,研究花生四烯酸(arachidonicacid,AA)对肌球蛋白轻链非磷酸化状态下平滑肌细胞迁移的影响及其相关的信号传导途径.经Boyden小室跨膜迁移实验发现,AA对培养的兔血管平滑肌SM3细胞具有明显的诱导迁移作用.然而,当预先用10μmolL肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)特异性抑制剂ML7作用SM3细胞后,发现AA对SM3细胞仍然具有明显的诱导迁移作用,并呈剂量依赖性,这种诱导作用可被细胞外信号调节激酶12(ERK12)的特异性抑制剂PD98059或磷脂酶C(PLC)的特异性抑制剂U73122所拮抗.此外,Ⅱ型肌球蛋白抑制剂blebbistatin(BLB)可部分抑制“非磷酸化”状态下AA的诱导迁移作用.经Western印迹检测显示,10μmolLML7可完全抑制SM3细胞中20kD肌球蛋白轻链(MLC20)磷酸化,并且加入AA后MLC20仍为非磷酸化状态.应用免疫荧光染色法观察肌动蛋白在SM3细胞中分布的变化,发现在AA作用下肌动蛋白呈细胞边缘聚集现象,有伪足形成,细胞形态表现为迁移状态.预先用ML7作用后再加入AA,肌动蛋白的分布与上述结果相同.研究结果初步表明,在平滑肌细胞迁移的作用途径中,在MLC磷酸化调节途径受到抑制时,AA可诱导MLC非磷酸化的平滑肌细胞发生迁移,其分子机理可能与ERK12和PLC信号传导途径有关,非磷酸化的肌球蛋白直接参与了该迁移过程.     

Calmodulin/calmodulin-dependent protein kinase II mediates SAAF-induced motility activation of ascidian sperm

Ca2+-influx and membrane hyperpolarization by sperm-activating and -attracting factor (SAAF) released from the unfertilized egg of the ascidians Ciona cause a transient increase in cAMP, which triggers activation of sperm motility. We demonstrated here the presence of Ca2+-binding protein, calmodulin (CaM), and CaM-dependent kinase II (CaMKII) in the sperm. CaM antagonist, W-7, and CaMKII inhibitor, KN-93, suppressed SAAF-induced membrane hyperpolarization, increase in cAMP, and activation of sperm motility, but inactive analogues of W-7 and KN-93, namely W-5 and KN-92, respectively, did not. Subsequent addition of K+ ionophore, valinomycin, hyperpolarized the plasma membrane, increased cAMP, and conferred motility to the immotile sperm even in the presence of W-7 and KN-93. Addition of IBMX activated motility of sperm, which has been immobilized by W-7 and KN-93. These suggest that increased [Ca2+]i through influx of Ca2+ by SAAF binds to CaM to activate CaMKII. The activated CaMKII may cause membrane hyperpolarization to increase cAMP, which triggers the activation of sperm motility in Ciona.     

Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change.

Platelets undergo shape change upon activation with agonists. During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia. When agonist-induced cytosolic Ca(2+) increases were prevented using the cytosolic Ca(2+) chelator, 5, 5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5, 5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic Ca(2+), agonist-stimulated myosin light chain phosphorylation was significantly inhibited. The myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, thrombin, or U46619-induced Ca(2+)-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and myosin light chain phosphorylation. Our results indicate that Ca(2+)/calmodulin-stimulated myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to myosin light chain phosphorylation and platelet shape change, through Ca(2+)-sensitive and Ca(2+)-insensitive pathways, respectively.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Activation of calcium-dependent kinases and epidermal growth factor receptor regulate muscarinic acetylcholine receptor-mediated MAPK/ERK activation in thyroid epithelial cells

We previously showed that stimulation of muscarinic acetylcholine receptors (mAChR) by carbachol (Cch) caused a time- and dose-dependent increase of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) phosphorylation in thyroid epithelial cells. In this study, we demonstrated that mAChR stimulation also induced a time-dependent increase in the tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), which was prevented by pretreatment of thyroid epithelial cells with the specific Src-family tyrosine kinase inhibitor PP2. Besides, phosphorylation of Pyk2 was attenuated by chelation of extracellular Ca(2+) or inhibition of phospholipase C (PLC), and was evoked by thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor. Incorporation of Pyk2 antisense oligonucleotides in thyroid epithelial cells to down-regulated Pyk2 expression or pretreatment of cells with the Ca(2+)/calmodulin protein kinase II (CaM kinase II) inhibitor KN-62 significantly reduced Cch-induced MAPK/ERK phosphorylation. In addition, Cch-induced MAPK/ERK phosphorylation was partially inhibited by LY294002 and wortmannin, two selective inhibitors of phosphatidylinositol 3-kinase (PI3K), tyrphostin AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, and (-)-perillic acid, a post-translational inhibitor of small G-proteins isoprenylation. Taken together, our data suggest that Pyk2, CaM kinase II and Src-family tyrosine kinases are key molecules for the activation of MAPK/ERK cascade through the EGFR/Ras/Raf pathway in thyroid epithelial cells in response to mAChR stimulation.     

Collagen-induced platelet shape change is not affected by positive feedback pathway inhibitors and cAMP-elevating agents

Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK.     

A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase

Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13- dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4- diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.     

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.     

Involvement of CaM kinase II in gonadotropin-releasing hormone-induced activation of MAP kinase in cultured hypothalamic neurons

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons. There is accumulating evidence that GnRH neurons have GnRH receptors and that the autocrine action of GnRH activates MAP kinase. In this study, we found that KN93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases), inhibited the GnRH-induced activation of MAP kinase in immortalized GnRH neurons (GT1-7 cells). Immunoblot analysis indicated that the CaM kinase IIdelta2 isoform (CaM kinase IIdelta2) and synapsin I were expressed in GT1-7 cells. GnRH treatment rapidly increased phosphorylation of synapsin I at serine 603, a specific phosphorylation site for CaM kinase II, suggesting that GnRH treatment rapidly activated CaM kinase IIdelta2. In addition, when we stably overexpressed CaM kinase IIdelta2 in GT1-7 cells, the activation of MAP kinase was strongly enhanced. These results suggest that CaM kinase IIdelta2 was involved in the GnRH-induced activation of MAP kinase in GT1-7 cells.