Ca2+ transients recorded in single voltage clamped vascular smooth muscle cells using the fluorescent Ca2+ indicator indo-1

J Mol

Cell

Cardiol

24 (Supplement

II) (1992)

SIGNAL TRANSDUCTION IN SMOOTH MUSCLE Andrew P. Somlyo and Avril V. Somlyo. Depts. of Physiology, Pathology and Medicine (Cardiology), University of Viinia, Charlottesville, VA 22908, U.S.A. Signal transduction processes in smc& muscle include elcctmmechanical ccupling and pharmzszom&anii coupling (1). Electron probe analysis (*), Ca2’-indieaurs t3) and laser flash photolysis 14) rewaled that the sarcoplasmic telicuhmt is the source. of pharmacomechanical Ca*+-release mediated by the activation of the phosphatidylinosiil cascade coupled by Gis protein(s) to receptors f5! The second mechanism of pharmacomechankxl coupling. modulation of Ca*+-sensitivit associated with changes in myosin light chain (MLC& pho@orylatio~Wphoqhorylatioa. Ca*+-sensitizatiou ta*& is mediated by inhibition of MLC20 phosphatase, through a G-protein wupled pathway. Studies leading to these conclusions, as well as CuReat work related to mechanisms of Ca*+-sensitization. will be reviewed. [Supported by the Human Fmntier for Science Program and PO1 HL 19242-161. References (1) Somlyo, A.V. and Somlyo, A.P. [1968] J. Pliamucol. Eq. Therop. 159129. (2) Bond, M., Kitazawa, T., Somlyo, A.P. and Somlyo, A.V. [1984] 1. Physiol. (Lmdon) 355:677. (3) Himpens, B and Somlyo, AR 119881 J. Physiol. (Lmdon) 395:507. (4) Somlyo, A.P. and Somlyo, A.V. [1990] Ann. Rev. Physbl. 52857. (5) Kobayashi, S.. Somlyo, A.P. and Somlyo, A.V. [1988] J. Physiol. (London) 403:601. (6) Somlyo, A.P., Kitaulwa, T., Himpens, B., Mattiijs, G., Htiuti, K., Kobayashi. S., Goldman, Y.B. aed Somlyo, A.V. [1989] Adv. Prot. Phosphaa.w 5:181. (7) Kilazawa, 2, MSSUO, M. and Somlyo. A.P. [1991] Proc. Nod. Acad. Sci. 88~9307.

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MOBILIZATIONS OF CYTOSOLIC Ca2 + AS ESTIMATED FROM ACTIVITIES OF THE Ca2 +DEPENDENT K+ CHANNEL IN VASCULAR SMOOTH MUSCLE Kenji Kitamma, Zhiling Xiong. Ma&ii Kamouchi, Himsi Kuriyama. DeparVnent of Ptunmacology, Faculty of Medicine, Kyushu Universi~ Fukuoka. 812 Japan. Influxes of Ca + induced by activations of the voltagedependent Ca2+ channelor/wiithe~~nonaelective catiOnchannel,aSWellaSrel Ca2+ from the sarooplasmu’ reticulum (SR) thnntgh activation of the i~~~itol ~&phosphate (ImP.+ensitive or/with CaP +-sensitive Ca2+ channel trigga activation of medmnkal nspanses thmugh formation of Cacahnodulin complex. ln add&m. increased cytosulic Ca2’ modifies Ca2’depmdem K+, Cl- OTNa+ chatmel and mgulate the physiological function of vascular smooth muscle. Rk focused on the activahm of Ca2+ @dmtK+ chmmel in relation to the Ca2+ mobilidon. Using the voltage-clamp pmcedme, applied depolarimd pllses prodmzed two disti~W tmnsient outwardcurrmts. i.e. subsequently gemsWed initial transient outwud cum% (ITo) followed by generation of Ihe volmgedqr&mt Ca2+ current (l& which was tapidly biocked by removal of Ca2+ in the ?xth w by letion of stcnrxl Ca2+ in the SR. Thus. l.R, 9+ release mecbaniis. may have a causal miation IO activations of Ca2+ hased by Ca2+ -m&cedCa ’ Transient oscihtory outwardnments(I& ev~byprolongeddepolarizationwere~bloclredbylerovalofCa2~butrequircdbngsllpafusion. The I, was blocked by tin OTryanodine, and was mom ck%ly related with released Ca2+ l&n the SR thtm influxes of Ca2+. Such maCIOSCOPiCresponses could also be rewrded from the cell-attached m cell-free. SR-at&&al patch membrane. This I, were composed mainly of synchmnized activations of the big K+ (Maxi K+) and partly small K+ (SK+) channels. ‘fbe Iooinduced hypqolarivUion may pFevent activation of the vv CR chaanelandagalist-induccdsymhesisof sP . Measurements of the Ca2’depemimt K+ channel may be a useful tool to e&mate dynamic mobiions of cytosolic Ca 4.-+.

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Ca” TRANSIENTS RECORDED IN SINGLE VOLTAGE CLAMPED VASCULAR SMOOTH MUSCLE CELLS USING THE FLUORESCENT Ca*’ INDICATOR INDONormand L&&n& Yvon Alkud. Division of Cardiovascular !&nces. Dqarbnent of Physiology, University of Manitoba, St. L3oniface General Hospital Rexarch Centre, Wiipeg, Ivlanito& CANADA R2H 2A6. Little is knovm about the interrel&onship existing between free intrxell~ Ca2+ B ’ (rc2’1i). voltagedependent ion channels and Ca*+ hnnspmt systems involved in Ca2+ m ’ invascularsmoothmuscleTbeprrposcofthisstudy was lo examine the voltagedepeo&oce and kinetics of [C!a2’li changes undcx physiological coediticms. Voltage clamp experimentswereumiedoutinsinglcm~tpataiveinsmoothmusclewlbuJingthestaadardwhdecellmodeofthepatch clamp technique. [Ca2+li was &mated using a mtiometric micxoflmrexx~~ technique (4WtW5Wbm). In cells dialysed with a standard pipette sohmon cuataining 140 mM K+, 10 mM Na+, 5 mh4 ATP, aad 50 ph4 Iodo-l(340 nlvl) Q&7.2), and superfused with normal external solution containint 1.8 mM Ca2+. l-2 set step depdarizations from 40 mV to t60 mV (20 mV incre.ments) from HP - -60 mV elicited Ca + transimts of which amplitude exhibited bell-shape dependence with a maximumnearOmV.Insomecclls.anetinwardc~tpreccdedtbeonsctohantarl~traosientoutwardcumnt(I,~f~ potemials between -20 mV and +2O mV. whiih was followed by a sus&ined outward current ($4 Appl$ation of l-H&M Niiedipine abolished the Ca*+ uansiem and It,, and reduced I,. In most cells. rhe induced Ca + traaslcot was biphasic displaying an -lY cumpomnt within the first 100 msec followed by a sioarer componentwithatimecorriEsntintheonlerof seconds,anobsavationnotreportedinviscexalsmoothmusclece~.oUrdarashowthatactivatiooofL-typeCa*+cbannels repmseut the dominant pathway for Ca*+ entry in these vascular cells. It is hypo&&ed that the complii kin& of [C!a*? chaugcs reflect involvement of intracellular Ca*+ release (SR) of which relative co&b&m remains to be defined. sujlponedbydleMRcofcauada S.61