Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway

Protein kinase D(PKD) is a serine-threonine protein kinase with distinct structuralfeatures and enzymological properties. Herein we demonstrate thatlysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss3T3 and Rat-1 cells. LPA induced PKD activation in aconcentration-dependent fashion with maximal stimulation (7.6-fold)achieved at 5 µM. Treatment of Swiss 3T3 cells with the proteinkinase C (PKC) inhibitors GF-I, Ro-31-8220, and Gö-7874completely abrogated PKD activation induced by LPA at concentrationsthat did not inhibit PKD activity when added directly to the in vitrokinase assays. PKD activation induced by LPA was attenuated markedlyand selectively by prior exposure of either Swiss 3T3 or Rat-1 cells topertussis toxin (PTx) in a concentration-dependent manner. In contrast,treatment with the protein tyrosine kinase inhibitor genistein, the MEKinhibitor PD-098059, or the phosphoinositide 3-kinase inhibitorwortmannin did not affect PKD activation in response to LPA. Theseresults provide the first example of PTx-sensitive and PKC-dependentPKD activation and identify a novelG_i-dependent event in the action of LPA.

     

Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser(916), an autophosphorylation site. An increase in PKD1 phosphorylation at Ser(916) was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser(916) was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.     

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.

     

Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.     

Vasopressin-induced intracellular redistribution of protein kinase D in intestinal epithelial cells

The spatio-temporal changes of signaling molecules in response to G protein-coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady-state distribution of green fluorescent protein (GFP)-tagged PKD indicated that in non-stimulated IEC-18 cells, GFP-PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP-PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)-mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin-induced GPCR activation.     

Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-delta pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H₂O₂, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H₂O₂ in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC- inhibitor, and by small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H₂O₂-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H₂O₂-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants). Rho kinase; protein kinase C-     

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.

     

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.     

Comparison of the responses of three rat intestinal epithelial cell lines to angiotensin II

Like RIE-1 cells, two of the IEC series of rat intestinal epithelial cell lines were found to express functional angiotensin receptors. As in RIE-1 cells, treatment of IEC-6 or IEC-18 cells with angiotensin II (AII) activated phosphatidylinositol-4,5-bisphosphate (PIP₂) hydrolysis although (in contrast to RIE-1 cells) the magnitude of AII-induced PIP₂ hydrolysis was small and not associated with a mitogenic response in either IEC cell line. In terms of their other functional responses to AII (activation of protein kinase C (PKC) and a small elevation of cyclic AMP), IEC-6 cells are otherwise similar to RIE-1 cells whereas IEC-18 cells exhibit some phenotypic differences to the other two cell types. Thus, whereas IEC-6 and RIE-1 cells each express the AT₁ subtype of angiotensin receptor, the higher affinity receptors on IEC-18 cells are 'atypical', being insensitive to both AT₁- and AT₂-specific angiotensin receptor antagonists. Furthermore, in contrast to its effects in IEC-6 and RIE-1 cells, AII neither activates PKC nor modulates cyclic AMP levels in IEC-18 cells. Whereas IEC-18 cells express the myristoylated alanine-rich C-kinase substrate (MARCKS), immunoreactive MARCKS was not detected in IEC-6 or RIE-1 cells.     

Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells

A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptor (GPCR) agonists or growth factors. In this study, we determined whether these stimuli could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.     

ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca2+, PKC, and Rho kinase

The G protein-coupled receptor agonistsangiotensin II (ANG II) and lysophosphatidic acid (LPA) rapidly inducetyrosine phosphorylation of the cytosolic proline-rich tyrosine kinase2 (Pyk2) in IEC-18 intestinal epithelial cells. The combined Pyk2tyrosine phosphorylation induced by phorbol 12,13-dibutyrate, a directagonist of protein kinase C (PKC), and ionomycin, a Ca²⁺ionophore, was equal to that induced by ANG II. Inhibition of eitherPKC or Ca²⁺ signaling attenuated the effect of ANG II andLPA, although simultaneous inhibition of both pathways failed tocompletely abolish Pyk2 tyrosine phosphorylation. Cytochalasin D, whichdisrupts stress fibers, strongly inhibited the response of Pyk2 to ANGII or LPA. The distinct Rho-associated kinase (ROK) inhibitors HA-1077and Y-27632, as well as the Rho inhibitor Clostridiumbotulinum C3 exoenzyme, also significantly attenuated ANG II- andLPA-stimulated Pyk2 tyrosine phosphorylation. Simultaneous inhibitionof PKC, Ca²⁺, and either actin assembly or ROK completelyabolished the Pyk2 response. Together, these results show that ANG IIand LPA rapidly induce Pyk2 tyrosine phosphorylation in intestinalepithelial cells via separate Ca²⁺-, PKC-, and Rho-mediated pathways.

     

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes

We recently identified a cDNA clone frommouse small intestine, which appears to be involved in folate transportwhen expressed in Xenopus oocytes. Theopen reading frame of this clone is identical to that of the reducedfolate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley,and K. H. Cowan. J. Biol. Chem. 269: 17-20,1994). The characteristics of this cDNA clone [previously referred toas intestinal folate carrier 1 (IFC-1)] expressed inXenopus oocytes, however, were foundto be different from the characteristics of folate transport in nativesmall intestinal epithelial cells. To further study these differences,we determined the characteristics of RFC when expressed in anintestinal epithelial cell line, IEC-6, and compared the findings toits characteristics when expressed inXenopus oocytes. RFC was stablytransfected into IEC-6 cells by electroporation; its cRNA wasmicroinjected into Xenopus oocytes.Northern blot analysis of poly(A)⁺RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFCwas found to be fourfold higher than uptake in control sublines. Thisincrease in folic acid and 5-MTHF uptake was inhibited by treatingIEC-6/RFC cells with cholesterol-modified antisense DNAoligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity(V_max) of theuptake process [the apparent Michaelis-Menten constant(K_m) alsochanged (range was 0.31 to 1.56 µM), but no specific trend wasseen]. In both IEC-6/RFC and control sublines, the uptake of bothfolic acid and 5-MTHF displayed 1)pH dependency, with a higher uptake at acidic pH 5.5 compared with pH7.5, and 2) inhibition to the sameextent by both reduced and oxidized folate derivatives. Thesecharacteristics are very similar to those seen in native intestinalepithelial cells. In contrast, RFC expressed inXenopus oocytes showed1) higher uptake at neutral andalkaline pH 7.5 compared with acidic pH 5.5 and2) higher sensitivity to reducedcompared with oxidized folate derivatives. Results of these studiesdemonstrate that the characteristics of RFC vary depending on the cellsystem in which it is expressed. Furthermore, the results may suggestthe involvement of cell- or tissue-specific posttranslationalmodification(s) and/or the existence of an auxiliary proteinthat may account for the differences in the characteristics of theintestinal RFC when expressed inXenopus oocytes compared with whenexpressed in intestinal epithelial cells.

     

Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle

Ca⁺/calmodulin-dependent protein kinase II(CaM kinase II) has been implicated in the regulation of smooth musclecontractility. The goals of this study were to determine: 1) towhat extent CaM kinase II is activated by contractile stimuli in intactarterial smooth muscle, and 2) the effect of a CaM kinase IIinhibitor (KN-93) on CaM kinase II activation, phosphorylation ofmyosin regulatory light chains (MLC₂₀), and force. Bothhistamine (1 µM) and KCl depolarization activated CaM kinase II witha time course preceding maximal force development, and suprabasal CaM kinase II activation was sustained during tonic contractions. CaMkinase II activation was inhibited by KN-93 pretreatment(IC₅₀ ~1 µM). KN-93 inhibited histamine-induced tonicforce maintenance, whereas early force development andMLC₂₀ phosphorylation responses during the entire timecourse were unaffected. Both force development and maintenance inresponse to KCl were inhibited by KN-93. Rapid increases in KCl-inducedMLC₂₀ phosphorylation were also inhibited by KN-93, whereassteady-state MLC₂₀ phosphorylation responses wereunaffected. In contrast, phorbol 12,13-dibutyrate (PDBu) did notactivate CaM kinase II and PDBu-stimulated force development wasunaffected by KN-93. Thus KN-93 appears to target a step(s) essentialfor force maintenance in response to physiological stimuli, suggestinga role for CaM kinase II in regulating tonic contractile responses inarterial smooth muscle. Pharmacological activation of protein kinase Cbypasses the KN-93 sensitive step.

     

Protein kinase D inhibits plasma membrane Na+/H+ exchanger activity

The regulation of plasma membraneNa⁺/H⁺exchanger (NHE) activity by protein kinase D (PKD), a novel proteinkinase C- and phorbol ester-regulated kinase, was investigated. Todetermine the effect of PKD on NHE activity in vivo, intracellular pH(pH_i) measurements were made inCOS-7 cells by microepifluorescence using the pH indicator cSNARF-1.Cells were transfected with empty vector (control), wild-type PKD, orits kinase-deficient mutant PKD-K618M, together with green fluorescentprotein (GFP). NHE activity, as reflected by the rate of acid efflux(J_H), wasdetermined in single GFP-positive cells following intracellularacidification. Overexpression of wild-type PKD had no significanteffect on J_H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control atpH_i 7.0). In contrast,overexpression of PKD-K618M increasedJ_H (5.31 ± 0.57 mM/min at pH_i 7.0;P < 0.05 vs. control). Transfectionwith these constructs produced similar effects also in A-10 cells,indicating that native PKD may have an inhibitory effect on NHE in bothcell types, which is relieved by a dominant-negative action ofPKD-K618M. Exposure of COS-7 cells to phorbol ester significantlyincreased J_H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M(because basal J_Hwas already near maximal). A fusion protein containing the cytosolicregulatory domain (amino acids 637-815) of NHE1 (the ubiquitousNHE isoform) was phosphorylated in vitro by wild-type PKD, but with lowstoichiometry. These data suggest that PKD inhibits NHE activity,probably through an indirect mechanism, and represents a novel pathwayin the regulation of the exchanger.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Protein kinase D2 mediates lysophosphatidic acid-induced interleukin 8 production in nontransformed human colonic epithelial cells through NF-kappaB

The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD₂ activation and the potential contribution of PKD₂ in regulating LPA-induced interleukin 8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD₂, as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD₂ activation induced by LPA was abrogated by preincubation with selective PKC inhibitors GF-I and Ro-31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD₂ activity. LPA induced a striking increase in IL-8 production and stimulated NF-B activation, as measured by NF-B-DNA binding, NF-B-driven luciferase reporter activity, and IB phosphorylation. PKD₂ gene silencing utilizing small interfering RNAs targeting distinct PKD₂ sequences dramatically reduced LPA-stimulated NF-B promoter activity and IL-8 production. PKD₂ activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-B-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent proinflammatory chemokine, by epithelial cells. NCM460 cells; protein kinase C; CXCL8; phorbol esters     

Differential MAP kinase activation and Na(+)/H(+) exchanger phosphorylation by H(2)O(2) in rat cardiac myocytes

Bursts in reactive oxygen species productionare important mediators of contractile dysfunction duringischemia-reperfusion injury. Cellular mechanisms that mediatereactive oxygen species-induced changes in cardiac myocyte functionhave not been fully characterized. In the present study,H₂O₂ (50 µM) decreased contractility of adultrat ventricular myocytes. H₂O₂ caused aconcentration- and time-dependent activation of extracellularsignal-regulated kinases 1 and 2 (ERK1/2), p38, and c-JunNH₂-terminal kinase (JNK) mitogen-activated protein (MAP)kinases in adult rat ventricular myocytes. H₂O₂ (50 µM) caused transient activation of ERK1/2 and p38 MAP kinase thatwas detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned tobaseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. Theprotein kinase C inhibitor chelerythrine completely blocked JNKactivation and reduced ERK1/2 and p38 activation. The tyrosine kinaseinhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38,activation. H₂O₂-inducedNa⁺/H⁺ exchanger phosphorylation was blocked bythe MAP kinase kinase inhibitor U-0126 (5 µM). These resultsdemonstrate that H₂O₂-induced activation of MAPkinases may contribute to cardiac myocyte dysfunction duringischemia-reperfusion.