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cAMP and Ca2+ control of the transepithelial C1- transport
26
Pfmmacolo~ical Research, PH. 26, Supplement I, 1992
TRANSFECI-ION OF SWISS 313 CBLLS WITH CALBtNDlN DZ8k MODtFtES INTRACXLLIJLAR CALCIUM HOMBOSTASiS A.Bassi*, LIJavisoo, R.Bunting, J.Morton, P.Bm.eo~and W.T.Masoo *Unit of Phtumac010~y.2nd Ycnwl UPMedlciua, Univcmiry Pcumico n, Naples, ItaJy. am1 uexpi. UT Neurobiology, APRC Institute of Animal ~ysiotO&y and GeneticsRes., Babrauam.Cambridge. U.K. This study was prompted iu au attempt to find a physiolagical role for the calcium-hiding protcitt. Calbindlu D28k (CaBP) in tttc regulation of intracellular calcium ion homcostasis. A mtrovims containing the full lengUt cDNA codirrg for &BP sssociatedwith a neomycin rcsisrancegene wan used to oanxfcct a cluual celI line (Swiss 3T3) which doss not oormally rxprcss the protein it4 which because it la&t voltage-dependentcaicium ion charm& in its plasma membrane - appears to mgularc intracellular cslcinm predotniniunly from intraceUnlat pwls.Expctimcnts comparcd the cffcas of various substanceson intrac&ular &5um in CaBP ttanslctcd (31’3~CFJ)and wild type (3T3-WT) utttmnsfccted 3T3 cells, using Fma2 as a pmbc for intraccltuiar calcium measuredusing quantitative real time video Uuoresccncemicroscopy (MagiCal. AppUcd Imagiug. Sundcrtand, UK). Both ccl1 types showed typical basal values ofintncellular calcium coocemation and wcrc not stnti,wicaUydiffcrcnt. Eombesi~~.a mitogenic peptidc fur 3T3 4s. slimulirres the rapid hydrnlysis of in&to1 phosphates, catting au ticreaae in ionic cllfcium due to rclcasc Uom intracellular poola. In 3’T3-CD cc&, lmmbcsin w&$ less potent iu elevnting intracellul~ calcium lcvcls tbau iu wild type cc&. In 3T3-WT ceils. 30 pM botnbesii produced il con&tent increa&?of calcium while the 3T3-CD cells needed a higher dose (BUM) Ca++-ATP~sc,~89 also different in both cell types. Iu X3-WT cells. thapsiyargitt at IuM elcvatct~ lnttacellulrr calcium to a higher level than in 3T3-CB cells. Moreover, the tcspousc to 1uM iouomycin was also larger in 3T3-W’T cells. The results can be explanted by the increased in@aceUularcalcium bufferlug capacity of 3T3-CB cells, afforded by calblndin exptussion.
GONADOTROF’lN4tELEASlNG HORMONE SECRETION B Y Byp(yFHALAMIC PULSATILITY AND ROLE OF CALCIUM IN NEUROFEPTIDE RELEASE.
NEURONSz
*FrancescoMerelli, Larar Krsmanovic, Stanko Stnjilkovic and Kevin Catt *Dept. of pharmacology, Catholic University, Rome, Italy and Endocrinology and Reproduction Research Branch, NICHD, NIH, Bethesda,USA CbRH is the centi neurpeptidein the control of gonadotropin release.Studieson the physiological aspectsof GoRH secretionhave been complicatedby the fact that G&I-l-producing cells are not conc=entrated in a discrete region of hypothalamus,but are distributi in the preoptic area and adjacent sites in the mstral position of the hypothalamus.Despite their scatteredlocations, thesecells are interconnectedto form a functional unit termed the “GnRH pulse generator” that is responsible for the episode release of pituitary gonadotropins. Recent advancesin neurone culture and the developmentof a transgenicG&H neuronal cell line have permitted more detded studieson the GnRH puke. generator. Perfusedprimary c&urea of rat hypott@&c neurons, as weU GTl transgenicGnRH cell lines, were found to exhibit spontaneousepisodic G&H secretion. This fmdig in GTl cells indicatesthat GnRH neurons generaterhythmic SecnetoTy activity in the absenceof input from other cdl types. The pulsatiIe secretionof Cl&H increasedin frequency with the duration of the culture and was abolished In extracelhrlarCa2+-de.fIcient medium and in the presenceof the voltage-sensitiveCa2f-channel blocker, n&&pine. In GTl cells cultured on cover slips, depolarixationwith high K+ increased[C!a2+]; and GnRH release in an extracellular Ca2+-dependent and nifediltin+3sensitivemanner. In addition, the dihydmpyridine Ca2+-channel agonist, Bay K %44, increased basal and K-induced Ca2+ elevation and G&H secretionis an intrinsic pmperty of GnRH neurons, and is dependenton voltag~sensitive calcium influx for its maintenance.
Pfmmacolo~ical Research, PH. 26, Supplement I, 1992
TRANSFECI-ION OF SWISS 313 CBLLS WITH CALBtNDlN DZ8k MODtFtES INTRACXLLIJLAR CALCIUM HOMBOSTASiS A.Bassi*, LIJavisoo, R.Bunting, J.Morton, P.Bm.eo~and W.T.Masoo *Unit of Phtumac010~y.2nd Ycnwl UPMedlciua, Univcmiry Pcumico n, Naples, ItaJy. am1 uexpi. UT Neurobiology, APRC Institute of Animal ~ysiotO&y and GeneticsRes., Babrauam.Cambridge. U.K. This study was prompted iu au attempt to find a physiolagical role for the calcium-hiding protcitt. Calbindlu D28k (CaBP) in tttc regulation of intracellular calcium ion homcostasis. A mtrovims containing the full lengUt cDNA codirrg for &BP sssociatedwith a neomycin rcsisrancegene wan used to oanxfcct a cluual celI line (Swiss 3T3) which doss not oormally rxprcss the protein it4 which because it la&t voltage-dependentcaicium ion charm& in its plasma membrane - appears to mgularc intracellular cslcinm predotniniunly from intraceUnlat pwls.Expctimcnts comparcd the cffcas of various substanceson intrac&ular &5um in CaBP ttanslctcd (31’3~CFJ)and wild type (3T3-WT) utttmnsfccted 3T3 cells, using Fma2 as a pmbc for intraccltuiar calcium measuredusing quantitative real time video Uuoresccncemicroscopy (MagiCal. AppUcd Imagiug. Sundcrtand, UK). Both ccl1 types showed typical basal values ofintncellular calcium coocemation and wcrc not stnti,wicaUydiffcrcnt. Eombesi~~.a mitogenic peptidc fur 3T3 4s. slimulirres the rapid hydrnlysis of in&to1 phosphates, catting au ticreaae in ionic cllfcium due to rclcasc Uom intracellular poola. In 3’T3-CD cc&, lmmbcsin w&$ less potent iu elevnting intracellul~ calcium lcvcls tbau iu wild type cc&. In 3T3-WT ceils. 30 pM botnbesii produced il con&tent increa&?of calcium while the 3T3-CD cells needed a higher dose (BUM) Ca++-ATP~sc,~89 also different in both cell types. Iu X3-WT cells. thapsiyargitt at IuM elcvatct~ lnttacellulrr calcium to a higher level than in 3T3-CB cells. Moreover, the tcspousc to 1uM iouomycin was also larger in 3T3-W’T cells. The results can be explanted by the increased in@aceUularcalcium bufferlug capacity of 3T3-CB cells, afforded by calblndin exptussion.
GONADOTROF’lN4tELEASlNG HORMONE SECRETION B Y Byp(yFHALAMIC PULSATILITY AND ROLE OF CALCIUM IN NEUROFEPTIDE RELEASE.
NEURONSz
*FrancescoMerelli, Larar Krsmanovic, Stanko Stnjilkovic and Kevin Catt *Dept. of pharmacology, Catholic University, Rome, Italy and Endocrinology and Reproduction Research Branch, NICHD, NIH, Bethesda,USA CbRH is the centi neurpeptidein the control of gonadotropin release.Studieson the physiological aspectsof GoRH secretionhave been complicatedby the fact that G&I-l-producing cells are not conc=entrated in a discrete region of hypothalamus,but are distributi in the preoptic area and adjacent sites in the mstral position of the hypothalamus.Despite their scatteredlocations, thesecells are interconnectedto form a functional unit termed the “GnRH pulse generator” that is responsible for the episode release of pituitary gonadotropins. Recent advancesin neurone culture and the developmentof a transgenicG&H neuronal cell line have permitted more detded studieson the GnRH puke. generator. Perfusedprimary c&urea of rat hypott@&c neurons, as weU GTl transgenicGnRH cell lines, were found to exhibit spontaneousepisodic G&H secretion. This fmdig in GTl cells indicatesthat GnRH neurons generaterhythmic SecnetoTy activity in the absenceof input from other cdl types. The pulsatiIe secretionof Cl&H increasedin frequency with the duration of the culture and was abolished In extracelhrlarCa2+-de.fIcient medium and in the presenceof the voltage-sensitiveCa2f-channel blocker, n&&pine. In GTl cells cultured on cover slips, depolarixationwith high K+ increased[C!a2+]; and GnRH release in an extracellular Ca2+-dependent and nifediltin+3sensitivemanner. In addition, the dihydmpyridine Ca2+-channel agonist, Bay K %44, increased basal and K-induced Ca2+ elevation and G&H secretionis an intrinsic pmperty of GnRH neurons, and is dependenton voltag~sensitive calcium influx for its maintenance.