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Ras transfection alters IP3CA2+ signalling in human keratinocytes
105
552 EVIDENCEFORTHEOCCURRENCEOF
Na-K-2ClCOTRANSPORTBRSIN ECCRINE CLEAR CELLS AND THEIR REXXJLATlONBY PROTEINKINASE A AND THEIR INHIBITION BY PROTEIN KINASE C. T. Tovomoto. D. Knutsen. M. Qhtsuvama. F. Saw. S. Cavallin. and K. Sam, Marshall Demntological Research Laboratories, University of Iowa College of Medicine, Iowa City, Iowa. USA. The pharmacologicalbasis for the regulation of ion wnspmt during sweat secretion is still poorly understood,particularly the mechanismfor regulation of Na-K-2CI coaanspmters. Although the presenceof Na-K-2Clcotmnsportershas been predicted in the sweat clear cells, direct evidence has been lacking. It has teen puzzling how the drastic cell shrinkageafter cholinergic (ACb) stimulationcan be mitigated for sustained sweat secretion. Na-K-2CIcotramportersare involved in cell volume regulation and thus their regulation can be studied by dewmining cell volume regulation. Using the image analysis m&cd and collagenasedissoci eccrineclear cells, we obsawd fhaf 0.1 mM bum&wide inhibited pseudo-regulatoryvolume increase(PRVI,ncovq of cell volume from cell shrinkage which occurswhen the medium is stitched from hypootonicm isotonic),pardal ceIIvolumrecoveryduring MCh-inducedce.II sbrinkage(VRPMS),and regulatory cell volume increaseduring hyperosmoticshock (RVI). suggesting that coUans*ortersare involved in ceIIvolume xcovery following various osmotic perturbations. Stimulation of cellular CAMPby IO I.IMfmskolin + isopnnerenol significantly enhancedbumetanide-sensitivecell volume recoveryduring PRVI, VRPMS. and RVI. In contrast the protein kinase C @‘KC)activators, phorbol ester WA), and high concentrationsof merhacholineinhibitedconstitutiveand CAMP-stimulatedcell volume recovery. Okadaic acid (at 1 FM), which stimulatesprotein phosphorylation. stimulated RVI. We postulate rhat the Na-K-2Clcotransponerxwhich are involved in cell volume regulation BT~also involved in sweat secretionand that the cohansportersax stimulated by protein phosphorylationby PKA but inhibited by PKC. Thus the periglandular adrenergicinnwvation is instrumentalin activating the cotransportersand alleviating the potential deleteriouseffect of acetylcholine-induceddrastic cell sluinkage.
C~AANDFK~O~EXERTD~FFERENTEFFECTSONTHEPHDSPHORYLATIDNOF~-YROS~NE RESIDUES IN PSORIATIC LYMPHCCYTES.H.M.~ckenfels.~.Schultewoner.P.M.Buraer Protein tyrosnekineses(PTK) are among the molecules thathave been lmpllcated 10 control cellgrowthand differentiation. One of the first stepsinthe activelion of Tlymphocytes fo,,ow~ng theblndmg of antigen. Ihebinding of monoc,ona,antltmdies to the antigen-receptor complex or to othercellsurfacestructures consists of the phosphorylation of proteins at their lyrosine residues. Psoriasis isdiscussedto be an immunological disease.i.e. thatT-lymphocytes (T-lc) praya prominentrolein pathogenesls of psonasw hnmunosuppressive agentswhich mhlbltIL-2secretion, uke cyclospormeA or FK506, are employedfortherapy. We examwed the timecourseof phoephorylated tyrosines as a marker forcellular fymsinekmase actlwty1" T-1,: from psoriatics and controls alterstimulation withphytohemagglutinin (PHA) and CsA or FK506. T-k from co"lro,s show peaks of phosphotyros,"e (p-tyr) after and after4 h o, PHA sbmulef~on: I" T-k from psorat,cs the 15 ml" peak ISsmaller. whereas the 4 h peek issignificantly hfgberthanafter15 min.Additnnaltreatmentof T-k wth CsA reducesthe 15min and 4-h p-tyramounts more in pso~1~11cs. 1.e to almoet60%. than in controls. By contrast FKSOG did not alter the p-tyremoums in controls or in psoriatlcs significantly after15 mm. After4 hours FK506 dlmlnlshedptyrlevels to 70% in controls only.These data indicate. first, an important unpactof TICactivated via,yrosine-phosphorylated protemson the palhcgenes~s of psoraasls and. second,ditfering antipsoriatlc mechanisms 01 thetwo drugsFK506 and CsA.
15mn
551 RAS TRANSFECTION ALTERS
IPJ CA- SIGNALLING IN HUNAN KERATINOCYTES. Biao Shi. and R. Rivkah Isseroff, Dept. of Dermatology, University of California, Davis, Davis, CA Cultured keratinocytes respond to increases in the Ca'* concentration in the medium by acquiring the differentiated phenotype; this is prevented by ras-transfection. To determine hy ras-Lransfection perturFs the transductiqn of the extracellularCa (Ca, ) srgnal in keratlnocytes, rp+examlq,edrespons$+sqf lnosltol phosphates and intracellular Ca (ca, ) to Ca transfected Jjne I-7 and its non-transfected pare&l ;I?neatlac:;: Switch of Ca from 0.05 UH to 1.5 n+linduced a 1.5-Z fold increase in IP relea& in HaCaT, with little effect on IP level in l-7. The downstream pathway for IP, generation rppearrsintact in the I-7 line, since either the receptor agonist bradykinin or the G rotein activator GTP S] induce equivalent increases in IP, in bot HacaT and l-7 ccl s: Fluorimetric measurement, of fur-P/AH-labelej monolayer keratlnocyt$> demonstrated that stlmulatlon of 1.0 nt4Ct. induced a similar Ca. increase in HaCaT and I-7 cells: a initial transient rise 5 8 'fold in HaCaT,,9 -15 fold in l-7 cells above brsl level) fo lowed by a sustalned lower lateau. However, the aCaT cells while in Ca. plateau was q?lntalned for 24 hours I" PI I-f cells the !concentration fell to near baseline ieve over this time'periad.'Furtpermore, stimulation of I-7 myolayer cells with 0.25 0.5 m# Ca, * induced an intracellular Ca * oscillation (frequency of spike/4 - 5 min, magnitude of 40 I.00nM) lasting >I ix$$, $,seen I" HaCaT cells. These,dlfferences I" the $$sponse,to lndlcate that ras transfectlon alters the Ca sens,ng mechanism and subsequent downstream transduction system in keratinocytes.
f
R
552 EVIDENCEFORTHEOCCURRENCEOF
Na-K-2ClCOTRANSPORTBRSIN ECCRINE CLEAR CELLS AND THEIR REXXJLATlONBY PROTEINKINASE A AND THEIR INHIBITION BY PROTEIN KINASE C. T. Tovomoto. D. Knutsen. M. Qhtsuvama. F. Saw. S. Cavallin. and K. Sam, Marshall Demntological Research Laboratories, University of Iowa College of Medicine, Iowa City, Iowa. USA. The pharmacologicalbasis for the regulation of ion wnspmt during sweat secretion is still poorly understood,particularly the mechanismfor regulation of Na-K-2CI coaanspmters. Although the presenceof Na-K-2Clcotmnsportershas been predicted in the sweat clear cells, direct evidence has been lacking. It has teen puzzling how the drastic cell shrinkageafter cholinergic (ACb) stimulationcan be mitigated for sustained sweat secretion. Na-K-2CIcotramportersare involved in cell volume regulation and thus their regulation can be studied by dewmining cell volume regulation. Using the image analysis m&cd and collagenasedissoci eccrineclear cells, we obsawd fhaf 0.1 mM bum&wide inhibited pseudo-regulatoryvolume increase(PRVI,ncovq of cell volume from cell shrinkage which occurswhen the medium is stitched from hypootonicm isotonic),pardal ceIIvolumrecoveryduring MCh-inducedce.II sbrinkage(VRPMS),and regulatory cell volume increaseduring hyperosmoticshock (RVI). suggesting that coUans*ortersare involved in ceIIvolume xcovery following various osmotic perturbations. Stimulation of cellular CAMPby IO I.IMfmskolin + isopnnerenol significantly enhancedbumetanide-sensitivecell volume recoveryduring PRVI, VRPMS. and RVI. In contrast the protein kinase C @‘KC)activators, phorbol ester WA), and high concentrationsof merhacholineinhibitedconstitutiveand CAMP-stimulatedcell volume recovery. Okadaic acid (at 1 FM), which stimulatesprotein phosphorylation. stimulated RVI. We postulate rhat the Na-K-2Clcotransponerxwhich are involved in cell volume regulation BT~also involved in sweat secretionand that the cohansportersax stimulated by protein phosphorylationby PKA but inhibited by PKC. Thus the periglandular adrenergicinnwvation is instrumentalin activating the cotransportersand alleviating the potential deleteriouseffect of acetylcholine-induceddrastic cell sluinkage.
C~AANDFK~O~EXERTD~FFERENTEFFECTSONTHEPHDSPHORYLATIDNOF~-YROS~NE RESIDUES IN PSORIATIC LYMPHCCYTES.H.M.~ckenfels.~.Schultewoner.P.M.Buraer Protein tyrosnekineses(PTK) are among the molecules thathave been lmpllcated 10 control cellgrowthand differentiation. One of the first stepsinthe activelion of Tlymphocytes fo,,ow~ng theblndmg of antigen. Ihebinding of monoc,ona,antltmdies to the antigen-receptor complex or to othercellsurfacestructures consists of the phosphorylation of proteins at their lyrosine residues. Psoriasis isdiscussedto be an immunological disease.i.e. thatT-lymphocytes (T-lc) praya prominentrolein pathogenesls of psonasw hnmunosuppressive agentswhich mhlbltIL-2secretion, uke cyclospormeA or FK506, are employedfortherapy. We examwed the timecourseof phoephorylated tyrosines as a marker forcellular fymsinekmase actlwty1" T-1,: from psoriatics and controls alterstimulation withphytohemagglutinin (PHA) and CsA or FK506. T-k from co"lro,s show peaks of phosphotyros,"e (p-tyr) after and after4 h o, PHA sbmulef~on: I" T-k from psorat,cs the 15 ml" peak ISsmaller. whereas the 4 h peek issignificantly hfgberthanafter15 min.Additnnaltreatmentof T-k wth CsA reducesthe 15min and 4-h p-tyramounts more in pso~1~11cs. 1.e to almoet60%. than in controls. By contrast FKSOG did not alter the p-tyremoums in controls or in psoriatlcs significantly after15 mm. After4 hours FK506 dlmlnlshedptyrlevels to 70% in controls only.These data indicate. first, an important unpactof TICactivated via,yrosine-phosphorylated protemson the palhcgenes~s of psoraasls and. second,ditfering antipsoriatlc mechanisms 01 thetwo drugsFK506 and CsA.
15mn
551 RAS TRANSFECTION ALTERS
IPJ CA- SIGNALLING IN HUNAN KERATINOCYTES. Biao Shi. and R. Rivkah Isseroff, Dept. of Dermatology, University of California, Davis, Davis, CA Cultured keratinocytes respond to increases in the Ca'* concentration in the medium by acquiring the differentiated phenotype; this is prevented by ras-transfection. To determine hy ras-Lransfection perturFs the transductiqn of the extracellularCa (Ca, ) srgnal in keratlnocytes, rp+examlq,edrespons$+sqf lnosltol phosphates and intracellular Ca (ca, ) to Ca transfected Jjne I-7 and its non-transfected pare&l ;I?neatlac:;: Switch of Ca from 0.05 UH to 1.5 n+linduced a 1.5-Z fold increase in IP relea& in HaCaT, with little effect on IP level in l-7. The downstream pathway for IP, generation rppearrsintact in the I-7 line, since either the receptor agonist bradykinin or the G rotein activator GTP S] induce equivalent increases in IP, in bot HacaT and l-7 ccl s: Fluorimetric measurement, of fur-P/AH-labelej monolayer keratlnocyt$> demonstrated that stlmulatlon of 1.0 nt4Ct. induced a similar Ca. increase in HaCaT and I-7 cells: a initial transient rise 5 8 'fold in HaCaT,,9 -15 fold in l-7 cells above brsl level) fo lowed by a sustalned lower lateau. However, the aCaT cells while in Ca. plateau was q?lntalned for 24 hours I" PI I-f cells the !concentration fell to near baseline ieve over this time'periad.'Furtpermore, stimulation of I-7 myolayer cells with 0.25 0.5 m# Ca, * induced an intracellular Ca * oscillation (frequency of spike/4 - 5 min, magnitude of 40 I.00nM) lasting >I ix$$, $,seen I" HaCaT cells. These,dlfferences I" the $$sponse,to lndlcate that ras transfectlon alters the Ca sens,ng mechanism and subsequent downstream transduction system in keratinocytes.
f
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