Depolarizing response of rat parathyroid cells to divalent cations

Depola izing Response o Ra Pa a hy oid Cells o Di alen Ca ions JOSE LOPEZ-BARNEO and CLAY M . ARMSTRONG F om he Depa men o Physiology, Uni e si y o Pennsyl ania Medical School, Philadelphia, Pennsyl ania 19104 ABSTRACT Memb ane po en ials we e eco ded om a pa a hy oid glands con inuously pe used in i o . A 1 .5 mM ex e nal Ca", he es ing po en ial a e ages -73 ± 5 mV (mean ± SD, n = 66) . On exposu e o 2 .5 mM Ca", he cells depola ize e e sibly o a po en ial o -34 ± 8 mV (mean ± SD) . Depola iza ion o his alue is comple e in ^-2-4 min, and epola iza ion on e u n o 1 .5 mM Ca" akes abou he same ime . The depola izing ac ion o high Ca" is mimicked by all di alen ca ions es ed, wi h he ollowing o de o e ec i eness : Ca" > S " > Mg" > Ba++ o alkali-ea h me als, and Ca" > Cd ++ > Mn++ > Co' > Zn ++ o ansi ion me als . Inpu esis ance in 1 .5 mM Ca" was 24 .35 ± 14 MQ (mean ± SD) and inc eased by an a e age ac o o 2 .43 ± 0 .8 a e swi ching o 2 .5 mM Ca++ . The low alue o inpu esis ance sugges s ha cells a e coupled by low- esis ance junc ions . The es ing po en ial in low Ca' is qui e insensi i e o emo al o ex e nal Na + o Cl - , bu e y sensi i e o changes in ex e nal K+ . Cells depola ize by 61 mV o a 10= old inc ease in ex e nal K+ . In high Ca + +, memb ane po en ial is less sensi i e o an inc ease in ex e nal K+ and is unchanged by inc easing K+ om 5 o 25 mM . Depola iza ion e oked by high Ca' may be slowed, bu is unchanged in ampli ude by emo al o ex e nal Na+ o Cl - . O ganic (13600) and ino ganic (Co++, Cd ++ , and Mn++) blocke s o he Ca' channels do no in e e e wi h he elec ical esponse o Ca" changes . Ou esul s show ema kable pa allels o p e ious obse - a ions on he con ol o pa a ho mone (PTH) elease by Ca" . They sugges an associa ion be ween memb ane ol age and sec e ion ha is e y unusual : pa a hy oid cells sec e e when ully pola ized, and sec e e less when depo- la ized . The ex ao dina y sensi i i y o pa a hy oid cells o di alen ca ions leads us o hypo hesize he exis ence in hei memb anes o a di alen ca ion ecep o ha con ols memb ane pe meabili y (possibly o K + ) and PTH sec e ion . INTRODUCTION I is well known ha he a e o pa a hy oid ho mone sec e ion (PTH) is in e sely ela ed o se um Ca ++ le els . In i o (Maye and Hu s , 1978 ; Add ess ep in s o Clay M . A ms ong, Dep . o Physiology, G4, . Uni e si y o Pennsyl- ania School o Medicine, Philadelphia, PA 19104 . D . Lopez-Ba neo's p esen add ess is Depa amen o de Fisiologia, Facul ad de Medicina, Se illa 9, Spain . J . GEN . PHYSIOL . ©The Rocke elle Uni e si y P ess - 0022-1295/83/08/0269/26 $1 .00  269 Volume 82  Augus 1983  269-294 on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 270  THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 Pa and Luckha d , 1942) and in i o (B own e al ., 1976 ; Mo issey and Cohn, 1978 ; She wood e al ., 1970) expe imen s ha e demons a ed ha a sligh dec ease (on he o de o en hs o millimoles) in ex e nal Ca" concen a ion e okes a s ong sec e o y esponse om pa a hy oid cells . Con e sely, an inc ease in ex acellula Ca" educes he a e o PTH sec e ion . The cu e ela ing sec e ion o Ca" concen a ion is e y s eep, wi h he ull sec e ion a e a 0-0 .5 mM and maximal sup- p ession a 2-2 .5 mM (B own e al ., 1976 ; Habene and Po s, 1976 ; Mo issey and Cohn, 1978) . The s eepness o he cu e sugges s he exis ence in pa a hy oid cells o an unusual and e y sensi i e Ca-sensing mechanism, he na u e o which has no been disco e ed . In many sec e o y cells, ex e nal Ca" is essen ial o sec e ion . Phys- iological s imula ion esul s in Ca" en y in o he cell in e io , which is esponsible o igge ing exocy osis (Douglas, 1968 ; Pe e sen, 1980 ; Rubin, 1970) . This gene al scheme may no apply o pa a hy oid cells . An inc ease in ex e nal Ca", as well as Mg", dec eases PTH sec e ion (B own e al ., 1976 ; Habene and Po s, 1976 ; She wood e al ., 1970) and ull sec e o y ac i i y can be obse ed in he absence o ex e nal Ca" (Fische e al ., 1982 ; Pin ado and Wallace, pe sonal communica ion) . In addi ion, applica ion o he calcium ionopho e A23187, which p esum- ably inc eases cy osolic Ca" (Reed and La dy, 1972), inhibi s PTH sec e ion (B own e al ., 1980 ; Fische e al ., 1982 ; Habene e al ., 1977) . Memb ane elec ical ac i i y plays an impo an ole in s imulus-sec e- ion coupling in many gland cells (Pe e sen, 1980) . Elec ophysiological s udies on he pa a hy oid ha e been pe o med only ecen ly and he wo epo s a ailable show con adic o y indings . In mouse pa a hy oid gland, in i o in acellula eco dings by B uce and Ande son (1979) show ha cells hype pola ize when he ex e nal Ca" concen a ion dec eases . In con as , i has been epo ed ha goa pa a hy oid cells main ained in cul u e depola ize in low Ca" and ha ol age-dependen Ca" en y may igge PTH exocy osis (Sand e al ., 1981) . We ha e pe o med expe imen s in a pa a hy oid glands in i o and ha e ound a e y clea and e e sible depola iza ion in esponse o inc eased ex e nal Ca" in he physiological ange o a s (Ro h and Raisz, 1964) . Ou esul s hus ag ee quali a i ely wi h hose ound in he mouse (B uce and Ande son, 1979) . Depola iza ion is also obse ed a e addi ion o all di alen ca ions es ed, bo h alkali-ea h and ansi ion me als, including he Ca" channel blocke s Cd", Mn", and Co" . The depola iza ion e oked by inc eased Ca" is accompanied by an inc ease in inpu esis ance and may be due o a supp ession o K+ pe meabili y . The possibili y ha a di alen ca ion ecep o loca ed on he ex e nal su ace o he memb ane con ols memb ane pe meabili y and, in u n, PTH sec e ion, is conside ed . METHODS P epa a ion Expe imen s we e pe o med on pa a hy oid glands ob ained om adul , male on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEZ-BARNEO AND ARMSTRONG Depola izing Response o Ra Pa a hy oid Cells  271 Wis a a s 100-200 g in weigh . Animals we e killed wi h e he and he gland was excised unde a dissec ing mic oscope . Pa a hy oid glands in he a a e loca ed bila e ally in he la e al bo de o he hy oid gland and appea as pale o oids - 1-1 .5 mm in diame e pa ially embedded in he hy oid issue (G eene, 1935) . The pa a hy oid gland and pa o he hy oid issue adjacen o i we e ans e ed o he eco ding chambe and secu ed o he bo om by small s ainless-s eel pins . The hin connec i e capsule co e ing he gland was emo ed wi h he aid o a s e eomic oscope . Expe imen al Se up The eco ding chambe and pa o he expe imen al se up a e schema ically ep esen ed in Fig . 1 . The chambe was made o luci e and di ided in o h ee 3 2 FIGURE 1 . Schema ic d awing o he appa a us used o in acellula eco ding in he con inuously pe used pa a hy oid gland . 1 : eco ding chambe . 2 : in e media e ese oi . 3 : ese oi whe e he suc ion (5) is applied . Fluid lows be ween 1, 2, and 3 by way o co on wicks . 4 : s opcock o selec be ween i e di e en solu ions . 6 : mic o he mis o pe manen ly a ached o he eco ding chambe . 7 : hea ing ilamen ha wa ms up he in low solu ion . 8 : glass mic oelec ode . 9 : 3 M KC1 aga b idge . 10 : ol age ollowe . 11 : s age o elec onic compensa ion o elec ode capaci ance and esis ance . 12 : squa e cu en pulses (ob ained om a cons an cu en pump) ha can be injec ed h ough he mic oelec ode . compa men s . The gland was placed in he i s compa men (1), si ing on a laye o Sylga d (Dow Co ning Co p ., Midland, MI) . This compa men con- ained he inle o he ex e nal luid . A hin wall o plas ic a ached o he bo om o he chambe (no illus a ed in he igu e) was placed as a ba le be ween he gland and he inle ape u e o di ec he solu ion s eam away om he mic oelec ode . The eco ding compa men had a olume o -0 .5 cm' and he ba hing solu ion low a e was 1 ml/min . Solu ion lowed om compa men 1 o a bu e compa men (2) and om his o a hi d compa men (3), whe e suc ion was applied . Flow be ween compa men s was by way o co on wicks . A silk h ead glued a he ou le needle (5) was used o b eak he meniscus, making he suc ion egula and smoo h . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 27 2  THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 The ex e nal pe usion sys em was moun ed sepa a ely om he eco ding able . Any one o i e solu ions could be applied while eco ding in a gi en pa a hy oid cell . Swi ching was accomplished wi hou in e up ing he solu ion low, and he le el o solu ion in he eco ding compa men was hus kep cons an . The dead space o he pe usion sys em was <0 .1 ml and i s washou ime was 1 o 2 s . The empe a u e in he eco ding compa men was moni o ed wi h a mic o he mis o (6) and con olled by a ci cui o ou own design . This ci cui passed he app op ia e cu en h ough a coil (7) placed a ound he solu ion inle , hus main aining he eco ding compa men a 37°C . Solu ions Solu ion composi ions a e shown in Table I . Modi ied K ebs solu ions wi h a iable Ca" concen a ions (be ween 0 .5 and 2 .5 mM) we e made by mixing solu ions A and B . In expe imen s whe e he e ec s o di alen ca ions o he han Ca" o Mg" we e es ed, S C1 2 , BaC1 2 , MnC1 2 , CdC1 2 , COC1 2 , o ZnCl 2 we e subs i u ed equimola ly o NaCl . Cl- ee solu ions (98% Cl- ee) wi h a iable K' concen a ions we e made by mixing solu ions E, F, G, and H . In Reco ding TABLE I Composi ion o Solu ions (in mM) some expe imen s a iable amoun s o D600 om a I-mM s ock solu ion we e added o he modi ied K ebs solu ion un il eaching inal concen a ions be ween 30 and 150 uM . The pH o all solu ions was adjus ed o 7 .4 excep o he Na- ee solu ions (C and D), whe e T izma, pH 7 .5, was used as bu e and as Na subs i u e . Solu ions we e con inuously bubbled wi h 100% 02 and had an osmolali y o 270-290 mosmol/kg . The junc ion po en ials o all solu ions we e measu ed wi h he elec ode placed in he eco ding chambe . In solu ions wi h di e ences only in di alen ca ion concen a ion, he junc ion po en ial di e ed by <1 mV . When swi ching om a modi ied K ebs solu ion o a Na- ee solu ion, a junc ion po en ial o +4 mV was obse ed . Pa a hy oid cells we e impaled wi h glass mic oelec odes illed wi h 3 M KCl and ha ing 100-200 MQ DC esis ance . The mic oelec odes we e pulled wi h a e ical pulle om hick-walled bo osilica e glass (OD = 2 mm, ID = 0 .7 mm) wi h an inne ilamen o ease o illing . The mic oelec odes we e elec ically connec ed o an FET ope a ional ampli ie (AD515 ; Analog De ices, Inc ., A K ebs 0 .5 Ca B K ebs 2 .5 Ca C Na- ee 1 .5 Ca D Na- ee 2 .5 Ca E CI- ee, 0K,2Ca F Cl- ee, 0K,4Ca G CI- ee, 100 K, 2 Ca H CI- ee, 100 K, 4 Ca NaCl 120 117 - - - --- KCI 5 5 5 5 - - - - K-glu ama e - - - - - - 100 100 Na- uma a e 2 .7 2 .7 - - 2 .7 2 .7 2 .7 2 .7 Na-py u a e 4 .9 4 .9 - - 4 .9 4 .9 4 .9 4 .9 Na-glu ama e 4 .9 4 .9 - - 130 127 30 27 MgS0 4 1 .1 1 .1 1 .1 1 .1 1 .1 1 .1 1 .1 1 .1 CaC12 0 .5 2 .5 1 .5 2 .5 2 .0 4 .0 2 .0 4 .0 Glucose 2 .8 2 .8 2 .8 2 .8 2 .8 2 .8 2 .8 2 .8 HEPES 10 10 - - 10 10 10 10 T izma (pH 7 .5) - - 142 .5 141 .5 - - - - on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LUPEz-BARNEO AND ARMSTRONG  Depola izing Response o Ra Pa a hy oid Cells  273 No wood, MA) wi ed as a ol age ollowe and placed close o he p epa a ion . Ex e nal ol age was sensed by a 3 M KCl aga b idge placed in he eco ding compa men o he chambe . The memb ane po en ial was simul aneously moni o ed by an oscilloscope and a digi al me e , and eco ded on a cha eco de (B ush Ins umen s, Gould Inc ., Cle eland, OH) wi h a equency esponse o ^ " 100 Hz . Squa e pulses o 200 ms du a ion and a iable ampli ude (be ween 0 .1 and 0 .25 nA) ob ained om a cons an -cu en pump we e injec ed h ough he mic oelec ode o measu e inpu esis ance . S ay capaci ance and ol age d op ac oss he mic oelec ode we e elec onically compensa ed . The eco ding chambe and mic omanipula o we e placed on a hea y g ani e able si ing on inne ubes o shock abso p ion . The eco ding able was elec ically shielded o p e en noise . Expe imen al P ocedu e Cells we e success ully impaled as deep as 250-300 jm om he su ace o he gland . Mos impalemen s only las ed o a ew seconds, bu occasionally he elec ode s ayed in he cell and a s able alue o he memb ane po en ial was eached in 2 o 3 min . Good impalemen s we e cha ac e ized by a sudden change in he memb ane po en ial . In all cases, he eco ded ol age was balanced o ze o be o e en e ing he gland . A e an impalemen , a check o ol age d i was made by wi hd awing he elec ode om he gland . In e e y cell om which a s able eco ding could be made, he esponse o changes in ex e nal Ca" concen a ion was es ed . This was used as an indica ion ha he eco dings we e made in a quali a i ely homogeneous popula ion o pa a hy oid cells . A e his es , he es o he expe imen al p o ocol was pe o med . We ha e eco ded mo e han 60 pa a hy oid cells o pe iods om 10 min o >3 h . In long pene a ions i was possible o es se e al solu ions wi h di e en ionic compo- si ions . In many o he cells, s able eco dings las ing se e al minu es we e ob ained, bu he elec ode came ou o he cell be o e a solu ion change could be accomplished . RESULTS Res ing Po en ial in Pa a hy oid Cells Mos cells we e impaled while he gland was ba hed wi h a solu ion con aining 1 .5 mM Ca" . In many o hese cells, he es ing po en ial s abilized in he i s minu e a e he pene a ion, eaching an a e age alue o -73 ± 5 mV (mean ± SD, n = 66) . Memb ane Po en ial Depends on Ex e nal Ca In all pa a hy oid cells exposed o mo e han one solu ion, he magni ude o he memb ane po en ial was s ongly dependen on he concen a ion o ex e nal Ca" . This is illus a ed in Fig . 2 by a con inuous eco ding o memb ane ol age om a single cell . The cell was ini ially impaled in K ebs 2 .5 mM Ca", yielding a es ing po en ial o -27 mV . Solu ion changes a e indica ed by he a ows . The eco ds show e y clea ly ha memb ane po en ial depends s ongly and e e sibly on ex e nal Ca" . The cell depola izes in esponse o an inc ease in ex e nal Ca" a e a on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 27 4 memb o~e po en ial (mV)  elec ode in, 2 .5mMCa " I - I -257 -50 7 -75-~ THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 delay equi ed o washou and equilib a ion . Once he memb ane po en- ial s a ed o change, he ime equi ed o a ain he new s eady s a e le el was be ween 1 .5 and 3 min, depending on each pa icula cell . Memb ane ol age is sensi i e o ex e nal Ca" in a na ow concen a- ion ange, be ween 1 and 2 .5 mM Ca" . Ca" concen a ions o <1 mM we e only es ed in a ew cells because, o unknown easons, pe haps chance, i was di icul o ob ain s able in acellula eco dings . In he lowe pa o Fig . 2, swi ching om 1 .5 o 0 .5 mM Ca" solu ion does 0- -25- _50- -75- -100i  2 5Co  1 SCa  0 5Ca  2 .5Co 5min FIGURE 2 .  Modi ica ions o memb ane po en ial by changes o ex e nal Ca" concen a ion . The aces a e a con inuous in acellula eco ding om a pa a hy oid cell exposed o di e en ex e nal Ca" concen a ions a he ime indica ed . The cell was impaled in a solu ion con aining 2 .5 mM Ca" . The s eepes ol age esponse occu s be ween 1 .5 and 2 .5 mM Ca" . In 0 .5 mM Ca +, he memb ane po en ial is abou he same as in 1 .5 mM Ca", o sligh ly mo e posi i e . Di e en Ca" concen a ions we e ob ained by mixing solu ions A and B o Table 1 . Osmolali y o all solu ions was be ween 270 and 290 mosmol/kg, pH 7 .4, empe a u e 37°C . no ha e an app eciable e ec on he memb ane po en ial . Some cells e en showed a sligh depola iza ion when he ba hing solu ion was changed om 1 .5 o 0 .5 mM Ca" . The mean es ing po en ial o se e al cells as a unc ion o ex e nal Ca" concen a ion is shown in Fig . 3 . The alues in A a e om cells con inuously eco ded o 10-120 min and exposed o a leas wo solu ions wi h di e en Ca" concen a ions . Ex eme alues o po en ial we e -34 ± 8 mV (mean ± SD) in 2 .5 mM Ca" and -80 .5 ± 2 mV (mean ± SD) in 1 .0 mM Ca" . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEZ-BARNEO AND ARMSTRONG Depola izing Response o Ra Pa a hy oid Cells  275 Fig . 3B shows he same cu e compiled om pene a ions main ained o only 1-10 min, oo sho a ime o a solu ion change o be accom- plished . The Ca" concen a ion in he ba hing solu ion o each pene a- ion is gi en on he axis . The measu emen s by he wo me hods a e in excellen ag eemen and clea l~ demons a e ha in pa a hy oid cells a 1 .5 mM change in ex e nal Ca + in a de ini e ange (be ween 1 and 2 .5 mM) e okes a mean po en ial change o 46 mV . The ela ionship be ween memb ane po en ial and ex e nal Ca" in ha ange is e y s eep . One o he mos s iking cha ac e is ics o his elec ical esponse is i s nea ly pe ec e e sibili y . Some cells we e exposed se e al imes o A _20- . 30- .40- . 50- .W -70 -80 mamb po en ial W  g  al jm l 0  0- . 10 i  -1 _20- . 30- .40-  1101 -50J 131 -6 -70 . 80 131 1341 QS  1  5 2 2S (Ca-1  OS  1  1 .5  2 2 .51Ca**I FIGURE 3 .  Memb ane po en ial in pa a hy oid cells as a unc ion o ex- e nal Ca" . (A) Da a om single pene a ions ha we e held long enough (10 min o 3 h) o eco d es ing po en ial in a leas h ee solu ions wi h di e en Ca" concen a ions . Each poin ep esen s he mean ± SD o 3- 27 measu emen s as gi en by he numbe s in pa en heses . (B) Da a om pene a ions oo b ie o allow a chan e o he ba hing solu ion . The pene a ions we e pe o med in he Ca ' concen a ion speci ied on he axis, and he es ing po en ial emained s able o 1-10 min . Each poin ep esen s he mean ± SD o 1-66 measu emen s, and he numbe s o measu emen s a e in pa en heses . Composi ion o solu ions is he same as in Fig . 2 . solu ions wi h 1 .5 and 2 .5 mM Ca" and he memb ane po en ial was measu ed a a gi en ime a e he solu ion change . In a ypical cell he mean memb ane po en ial alues o eigh measu emen s done 5-6 min a e swi ch4 o he new solu ion we e -79 .7 ± 2 .4 mV (mean ± SD) in 1 .5 mM Ca + and -32 .2 ± 2 .5 mV (mean ± SD) in 2 .5 mM Ca" . The ex emely small dispe sion a ound he mean indica es a nea ly pe ec e e sibili y o he esponse . On h ee occasions s able in acellula eco dings om he issue su - ounding he pa a hy oid gland (p esumably om hy oid cells) we e ob ained . The es ing po en ial o hese cells was abou -65 mV and did on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 276  THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 no show any app eciable modi ica ion in esponse o changes in ex e nal Ca" concen a ion in he ange o 0 .5-2 .5 mM Ca" . Absence o Spike Ac i i y The elec ical esponse e oked by inc easing o dec easing ex e nal Ca" always showed he quali a i e ea u es e e ed o abo e and no spiking ac i i y was e e seen . In some cells ha showed a e y low es ing po en ial (-40 mV in 1 .5 mM Ca"), i egula jumps and spikes we e obse ed in he i s seconds a e he impalemen ha we e p obably due o an impe ec pene a ion o he cell, since ei he he mic oelec ode came ou o he cell sho ly a e he impalemen o he memb ane sealed a ound he elec ode and he "spikes" disappea ed as memb ane po en ial inc eased . Calcium E ec s A e Mimicked by O he Di alen Ca ions O he alkali-ea h ca ions ha e he same quali a i e e ec on pa a hy oid cell memb ane po en ial as Ca" . This is illus a ed in Fig . 4A, which shows a con inuous in acellula eco ding om a single cell . The ini ial impalemen was done in a solu ion con aining 1 .5 mM Ca", yielding a es ing po en ial o -76 mV . A he ime indica ed by he a ows, he ba hing solu ion was changed o one con aining, in addi ion o 1 .5 mM Ca", 1 mM o ano he di alen ca ion . All he ca ions caused a e e sible depola iza ion, bu wi h di e en ime cou ses and magni udes . The e ec i eness o he esponse was Ca' > S ++ > Mg++ > Ba++ . None o hese di alen ca ions was as e ec i e as Ca++ in depola izing pa a hy oid cells . S ++ and Ba ++ esponses we e, espec i ely, ^-60 and ^-30% o he depola iza ion obse ed wi h Ca ++ . Mg + yielded an in e media e alue . The di e ence in he ime cou se is also e y appa en . Whe eas he depola iza ion induced by 1 mM addi ional Ca ++ eached he s eady s a e in 2 .5 min, he Ba ++ -induced depola iza ion was h ee imes smalle and equi ed ^ 6 min o a ain he s eady s a e . The same expe imen al p o ocol shown in Fig . 4A has been epea ed in h ee di e en cells wi h simila esul s . We ha e also s udied he e ec o o he di alen ca ions, and an example is shown in Fig . 4B . The eco d belongs o ano he pa a hy oid cell impaled in 1 .5 mM Ca ++ , wi h an ini ial memb ane po en ial o -75 mV . The expe imen al p o ocol was he same as be o e : in addi ion o 1 .5 mM Ca + , 1 mM o a es di alen ca ion was added o he ba hing solu ion . In all cases he cell depola ized when he es ca ion was added . In his case he e ec i eness o he esponse is Ca ++ > Cd ++ > Mn++ > Co ++ . Cd ++ and Mn ++ seem o be sligh ly mo e e ec i e han S ++ , and he e ec i eness o Co ++ is abou he same as wi h Ba ++ . The e ec s o Cd ++ , Mn ++ , and Co ++ ha e been es ed in wo di e en cells wi h iden ical quali a i e esul s . Zn ++ was es ed in a cell (no illus a ed) and e oked a depola iza ion o abou he same magni ude as Co ++ . Fig . 4A and B shows unequi ocally ha memb ane po en ial in pa a hy oid cells is a ec ed no only by ex e nal Ca ++ bu by all he di alen ca ions es ed . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEz-BARNEO AND ARMSTRONG  Depola izing Response o Ra Pa a hy oid Cells  277 Inpu Resis ance and Cell- o-Cell Coupling Inpu esis ance has been measu ed in se e al cells by passing hype pola - izing squa e cu en pulses h ough he eco ding mic oelec ode . Fo hese measu emen s he ol age d op ac oss he mic oelec ode esis ance memb ape po en i o l elec ode in  (mV) o, _ . 1 .5mMCa+* B 1mm g o ++ 5 min    2 .5MM 1 .5nW 1 .5mm  1 .5mm 1 .5MM 1 .SMM 1 .5 nM 1 .5mm Ca ++ Ca ++ co" , Ca+' Ca ++ Ca ++ Ca+ + C a '+ 1mMCd ++ 1mmco ++ 1 nMMn++ 5min FIGURE 4 .  E ec o a ious di alen ca ions on memb ane po en ial o pa a hy oid cells . A shows a con inuous in acellula eco ding om a cell ba hed wi h solu ions con aining he indica ed concen a ion o di alen ca ions . The cell was impaled in 1 .5 mM Ca ++ , and swi ches o o he solu ions a e indica ed by he a ows . Solu ion composi ion is he same as in Fig . 1, excep ha MgC1 2 , S Cl 2 , BaC1 2 , o addi ional CaC1 2 we e subs i u ed equimola ly o NaCl . The dashed ho izon al line a -50 mV memb ane po en ial is d awn o con enience o compa ison . B shows a con inuous eco ding o ano he cell exposed o he indica ed ansi ion elemen s . The ime be ween he pa allel ba s in he eco d was 2 .5 min, and he dashed line a -57 mV memb ane po en ial is d awn o con en- ience . was sub ac ed ou elec onically . The sub ac ion ci cui was adjus ed be o e pene a ing he cell ; he adequacy o he sub ac ion was es ed a e he elec ode was wi hd awn . The measu emen was disca ded i elec ode esis ance changed signi ican ly du ing he pene a ion . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 28 4  THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 5 mM K + solu ion, he cell epola izes o app oxima ely he o iginal le el . In all cells depola ized by ele a ed ex e nal K+, he epola iza ion showed a slowe ime cou se han he depola iza ion . Fig . 9A also shows ha he same cell was depola ized e e sibly by 4 mM Ca" a a cons an (5 mM) K+ concen a ion by ^-36 mV . Fig . 9B illus a es a con inuous in acellula eco ding om ano he cell, which was ini ially impaled in 5 mM K, 4 mM Ca and had a es ing po en ial o -33 mV . V m wen nega i e by 34 mV on changing o 5 mM K, 2 mM Ca . The addi ion o 25 mM K, 2 mM Ca e e sibly depola ized he cell by 28 mV . A e eco e y in 5 mM K, 2 mM Ca, he cell was subsequen ly depola ized by 43 mV in 5 mM K, 4 mM Ca, eaching a memb ane po en ial o abou -30 mV . A his poin he addi ion o 25 and 50 mM K+, keeping cons an he ex e nal Ca", had li le, i any, e ec on he memb ane po en ial . The ein oduc ion o 5 mM K, 2 mM Ca solu ion epola ized he cell o he p e ious es ing po en ial le el (-74 mV) . Fig . 9B also shows ha he ime cou se o memb ane depola - iza ion and hype pola iza ion induced by Ca" is e y simila o he ol age changes obse ed by he in oduc ion o he wi hd awal o high K + solu ion . The eco ds in Fig . 9 a e ypical examples o he esponse o pa a hy- oid cells o changes in ex e nal Ca" and K' in Cl- ee solu ions, and hey sugges ha ex e nal Cl - does no make an impo an con ibu ion o ei he he es ing po en ial o he Ca"-induced elec ical esponse . In low Ca", he memb ane po en ial is highl~ sensi i e o ex e nal K+, bu once he cells a e depola ized by high Ca ', ex e nal K + seems o ha e li le e ec on he memb ane po en ial . The e ec s o ex e nal Ca + and K + in Cl- ee solu ions ha e been s udied in many cells and a summa y is gi en in Fig . 10 . In he igu e, memb ane po en ial in 2 mM ( illed symbols) and 4 mM (open symbols) Ca" has been plo ed s . he loga i hm o ex e nal K+ . The e ical line ep esen s he s anda d de ia ion o he mean and he numbe s o obse a ions a e in pa en heses . In low Ca", he memb ane po en ial is e y sensi i e o ex e nal K+ . Fi ing by eye, he s aigh pa o he cu e (abo e 25 mM K+) yields an ^-61 mV change in memb ane po en ial o a 10- old change in ex e nal K + , which is simila o he alue p edic ed by he Ne ns equa ion o a po assium elec ode a 37'C . Ex apola ing he s aigh line o 0 mV memb ane po en ial gi es an es ima e o he in acellula po assium concen a ion o 160 mM . A high Ca", he numbe o measu emen s is smalle , bu i is e y clea ha memb ane po en ial is less sensi i e o ex e nal K+ . Thus, i seems ha a low Ca", pa a hy oid cells beha e as a po assium elec ode and P K is he dominan pe meabili y . A high Ca", he a io o P K o o he pe meabili ies p ob- ably dec eases and hus memb ane po en ial is less sensi i e o ex e nal po assium . The possible pa icipa ion o di e en ionic pe meabili ies in he ol age change seen in pa a hy oid cells will be analyzed in he Discussion . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEz-BARNEO AND ARMSTRONG Depola izing Response o Ra Pa a hy oid Cells  285 E ec s o Isop o e enol I is known ha he be a agonis isop o e enol, a concen a ions be ween 1 and 10 gym, inc eases PTH sec e ion a e in dispe sed bo ine pa a hy- oid cells (B own e al ., 1977) . We ha e s udied he e ec s o a iable concen a ions o isop o e enol (be ween 10 and 40 uM) added o he modi ied K ebs solu ion in ou cells whose elec ical esponse o ex e nal Ca" had p e iously been obse ed . These concen a ions o isop o e - enol did no ha e an impo an e ec on he memb ane po en ial ei he a low o a high Ca" . In one cell he e was no ol age change a all . In ano he cell memb ane po en ial hype pola ized by 6-7 mV, and in he memb ane po en ial InIVI 0- -------------------------------------------------- -10- -20-  / . a(l) /ool .~h) b(3) _40 (,3)  / - so - _60 - -70 . ' ~ . 1(34) -80 .  IF 5  10  25  50  100  160 mM log CKa7 FIGURE 10 .  Dependence o memb ane po en ial (o dina e) on he ex e - nal concen a ion o K' ep esen ed in a semiloga i hmic scale (abscissa) . Filled ci cles a e measu emen s in low Ca" (2 mM) and open squa es a e measu emen s in high Ca" (4 mM) . E e y poin ep esen s he mean and he e ical ba s ep esen he s anda d de ia ion o se e al measu emen s ob ained om eco ds like hose shown in Fig . 10 . The numbe o meas- u emen s is in pa en heses . The s aigh line has been d awn by eye and has a slope o ^-61 mV pe 10- old inc ease in ex e nal K+ concen a ion . The in e cep o his line wi h he 0-mV axis gi es an es ima e o he in e nal concen a ion o K+ . o he wo cases a 4-5-mV depola iza ion was obse ed . Al hough he numbe o obse a ions is small and no de ini i e conclusion can be d awn, ou da a sugges ha he mechanism o isop o e enol s imula ion o pa a hy oid cells is di e en om ha e oked by lowe ing di alen ca ions (see Discussion) . DISCUSSION The e ec s o ex e nal calcium concen a ion on pa a hy oid cell mem- b ane po en ial ha e been' epo ed only wice, wi h con adic o y conclu- sions . Mouse pa a hy oid cells hype pola ize on lowe ing ex e nal Ca" on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 28 6)  THE JOURNAL OF GENERAL PHYSIOLOGY - VOLUME 82 - 1983 (B uce and Ande son, 1979), bu cul u ed goa pa a hy oid cells a e said o do he opposi e, ha ing a es ing po en ial o -29 .2 mV in 0 .8 mM Ca" and -44 .7 mV in 2 .4 mM Ca + (Sand e al ., 1981) . Ou measu e- men s on a pa a hy oid ag ee quali a i ely wi h obse a ions in he mouse, bu he magni ude o ou es ing po en ials is -1 .5 imes la ge han epo ed o mouse, and wice as la ge as he epo ed alues o cul u ed goa cells . The e ha e been no epo s o spon aneous spike ac i i y, ega dless o Ca" concen a ion, and we ha e ound none, e en hough ou cells had es ing po en ials o e y heal hy magni ude . In cul u ed cells, e- gene a i e esponses a e said o be igge ed by injec ion o depola izing cu en h ough he mic oelec ode (Sand e al ., 1981) . The high elec- ode esis ance and low inpu esis ance o he gland p e en ed us om s imula ing e ec i ely h ough he mic oelec ode . Pa a hy oid cells in he a a e ^, 8 ,um in diame e (Ro h and Raisz, 1964) . Using 1,000 SZ cm' as a guess o memb ane esis ance (Cole, 1968), he inpu esis ance would be 498 MR we e he cells no coupled o each o he by junc ions o lowe esis ance . Fo goa pa a hy oid cells in cul u e, he epo ed igu e is 503 ± 218 MR in 2 .4 mM Ca" and 290 ± 198 MR a 0 .8 mM Ca" . We epo a much lowe alue o inpu esis ance o cells in he gland, 24 .3 ± 14 MO . This s ongly sugges s ha in he pa a hy oid, as in o he glands (Pe e sen, 1980), cells a e in e connec ed by low- esis ance junc ions . F eeze- ac u e eplicas o a pa a hy oid cells ha e con i med he exis ence o nume ous gap junc ions (Ra azzola and O ci, 1977 ; Lopez-Ba neo and F anzini-A ms ong, 1982) . Possible O igins o he Depola iza ion Induced by Di alen Ca ions I is i s necessa y o conside possible bu unin e es ing explana ions o he sensi i i y o memb ane po en ial o Ca" concen a ion . The la ge magni ude o he es ing po en ial in low Ca" makes i unlikely ha we ha e p oblems wi h he seal be ween memb ane and mic oelec ode . Fu he , he inpu esis ance is highe in 2 .5 mM Ca", when he cell is depola ized . The depola iza ion is hus unlikely o esul om a ailu e o he memb ane-elec ode seal . A second possibili y is ha aising Ca" causes su icien Ca" en y o educe coupling be ween cells (De Mello, 1975 ; Iwa suki and Pe e sen, 1977 ; Rose and Loewens ein, 1975) . This could lead o depola iza ion o he impaled cell by he shun esis ance a ound he elec ode, when he impaled cell is no longe suppo ed by i s neighbo s . This possibili y can be disca ded on he g ounds ha Mg", which is no mally p esen in he cell a high concen a ion and does no cause uncoupling (De Mello, 1975 ; Iwa suki and Pe e sen, 1977), has an e ec on memb ane po en ial simila o Ca" . T ansi ion elemen s also depola ize pa a hy oid cells, al hough hey a e e y impe mean and do no di ec ly a ec cell coupling . On occasion, hey e en compe e wi h Ca" and es o e cell coupling (Pe e sen, 1980) . Thy oid ollicula cells on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEz-BARNEO AND ARMSTRONG  Depola izing Response o Ra Pa a hy oid Cells  287 om he a a e coupled and ha e an inpu esis ance alue o 11 M12 (G een and Pe e sen, 1981) . Bo h in he mouse (B uce and Ande son, 1979) and in he a (p esen expe imen s), he es ing po en ial o hy oid cells was no a ec ed by changes in ex e nal Ca" in he ange o in e es . Thus, bo h o hese unin e es ing explana ions a e unlikely . The po en ial changes obse ed a e much oo la ge o be accoun ed o as a di ec esul o a change in he equilib ium po en ial o Ca" . E en i he memb ane we e exclusi ely Ca + pe meable, changing Ca" om 1 .5 o 2 .5 mM would change he memb ane po en ial by only 6 .7 mV, much less han he obse ed change . No does i seem likely ha Ca" channels o he ype epo ed in o he issues play a pa in he esponse . The selec i i y o he Ca" channel o alkali-ea h di alen ca ions (Hagiwa a e al ., 1974 ; Hagiwa a, 1975) con as s wi h ou ind- ings in pa a hy oid cells, whe e no quali a i e dis inc ion be ween he ou ca ions (Ca", S ", Mg ++ , and Ba ++ ) was seen . Ba ++ is no e y e ec i e in depola izing pa a hy oid cells al hough i pe mea es well h ough he Ca channel (Hagiwa a e al ., 1974 ; Reu e , 1973) . In addi- ion, Co', Cd ++ , and Mn ++ , which a e Ca ++ channel blocke s (Bake e al ., 1973 ; Kohlha d e al ., 1973 ; Hagiwa a, 1975), mimic he e ec s o Ca++ a he han blocking he esponse . Finally, he Ca++ channel blocke D600 (Kohlha d e al ., 1972) is wi hou e ec on he esponse o aising Thus, i seems ha pa a hy oid cells possess an unusual calcium-sensing mechanism o ex ao dina y sensi i i y, no obse ed in o he biological p epa a ions . A simila bu much smalle esponse is seen in he es ing sec e o y memb ane o eshwa e lamellib anch man les . A 10- old de- c ease in ex e nal calcium ( om 6 o 0 .6 mM) hype pola izes by 6 mV (So enson e al ., 1980) . The o de o e ec i eness o di alen ca ions in depola izing pa a hy- oid cells is e y simila o he a ini y o Ca-binding p o eins and cal- modulin-like ecep o s o se e al di alen ca ions . I is known ha Mn++, S ++, Mg ++ , and Ba++ can bind o a b ain Ca ++ egula o y p o ein (Wol e al ., 1977) . In molluscan neu ons, ac i a ion o po assium pe meabili y by in e nal Ca++ (an e ec p obably media ed by a Ca++ ecep o loca ed in he inne side o he memb ane) is simula ed by o he di alen ca ions (Meech, 1974 ; Meech and S anden, 1975) . Ca ++ is he mos e ec i e ion, ollowed by Cd++, S ++, and Mn++ . The ca ions Ba++, Co++, M g ++, and Zn ++ ha e been ound o be e y ine ec i e (Go man and He mann, 1979) . Abou he same o de has been ound in he exchange o a ious di alen ca ions o oponin-bound "Ca ++ (Fuchs, 1971) . These indings sugges ha he in e ac ion o di alen ca ions wi h pa a hy oid cells is no based on hei di e en pe meabili y h ough he memb ane and poin owa d he possible exis ence o a di alen ca ion ecep o in he ex e nal su ace o hese cells . This ecep o could be a Ca-binding p o ein wi h a di e en a ini y o each o he di alen ca ions ha a ec memb ane ol age . Modi ica ions in memb ane pe meabili y on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 28 8  THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 by con o ma ional changes o he ecep o a e i binds o one o se e al di alen ca ions would esul in he elec ical e en s associa ed wi h al e a ions in he ex e nal concen a ion o di alen ca ions . Recen mo phological s udies on a pa a hy oid glands using he eeze- ac u e echnique (Lopez-Ba neo and F anzini-A ms ong, 1982 ; C . F anzini- A ms ong, unpublished obse a ions) ha e e ealed he exis ence in he cell memb ane o pa icles ha a e densely packed, egula ly dis ibu ed h oughou he plasmalemma, and unusually uni o m in size . One migh specula e ha hese unusual pa icles a e ecep o s, whose dense packing con e s on hese cells hei ex ao dina y Ca' sensi i i y . Inpu Resis ance and Memb ane Resis ance The inpu esis ance (R ;) in a issue ha is elec ically in e connec ed by gap junc ions depends on bo h junc ional (R j ) and memb ane esis ance (R m) . The change ha we obse e on aising ex e nal Ca" could a ise om al e ing ei he ac o . We know no mechanism by which ex acel- lula Mg" o ansi ion elemen s could aise junc ional esis ance, and he e o e we suspec ha di alen ca ions al e memb ane esis ance . The magni ude o he R mchange signaled by a 2 .4- old inc ease in R ; depends on he a io R ;/Rm (c . Holman and Neild, 1979, o a somewha di e en conclusion) . To app oach his p oblem we used a simple model o he issue, which was adequa e o ou pu poses . The model (Fig . 11 A) assumes ha he pene a ed cell (1) is a sphe e o adius 4 Am, su ounded by sphe ical shells o 8 Am hickness . The shells con ain successi e gen- e a ions o neighbo s . Fig . 11B illus a es he elec ical ci cui ha co - esponds o his h ee-dimensional a ay assuming ha (a) R m ( esis ance o he impaled cell) and R j ( esis ance o i s junc ions o all o i s nea es neighbo s) a e he same o all cells, and (b) he esis i i y o ex acellula and in acellula media is negligible (Benne , 1966 ; Socola and Loew- ens ein, 1979) . To acili a e compu a ions, only h ee shells o cells (in addi ion o he impaled one) ha e been conside ed . The alues o Rm1,2,and9 and RJiandI (see Fig . 11 B) used a e gi en in he igu e legend . Sol ing his equi alen ci cui yields he plo shown in Fig . 11 C . The o dina e ep esen s he a io R ;/R ., a di e en alues o Rj/Rm, as gi en on he abscissa in a semilog scale . The cu e shows ha when R ;/Rm is la ge, R ;/R .. app oaches uni y, since he impaled cell is well isola ed om i s neighbo s and inpu esis ance e lec s only he memb ane esis ance o he impaled cell . When Rj/Rm is e y small, R ;/Rm again app oaches uni y because he eco ding is being made om wha is in essence a la ge single cell . Fo in e media e alues o Rj/Rm, i can be seen ha R ;/RI, is much smalle han uni y and he e o e a gi en inc ease in Rn, is signaled by a smalle change in R ; . The same quali a i e ela ionship be ween R m , R j , and R ; is ob ained wi h a wo-dimensional model, bu he minimal R ;/ R m occu s a a di e en a io o junc ional o memb ane esis ance . Pe meabili y Changes In ol ed in he Response o High Calcium In he absence o o he in o ma ion, he ol age change induced by high on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEz-BARNEO AND ARMSTRONG Depola izing Response o Ra Pa a hy oid Cells  289 Ca" could be due o ei he an inc ease o a depola izing conduc ance o a dec ease in a pola izing conduc ance, e .g ., P K . Neglec ing he con i- bu ion o anions, i can be calcula ed by he Goldman (1943) equa ion ARiARm A B Rm Rj/Rm 5  1 0 .5 0 .1  0 .01  OA01  0 .0001 FIGURE 11 . Cell- o-cell coupling in he pa a hy oid gland . A shows a model o cell opology in he pa a hy oid gland . The impaled cell (I) is su ounded by sphe ical shells o closely packed cells . The i s , second, and hi d shells con ain 25, 97, and 216 cells . B shows he equi alen elec ical ci cui o his model . R m is he memb ane esis ance o a single cell, and R j is he pa allel esis ance o he junc ions o all o i s neighbo s . Rm  RM 2 1 and Rms a e he pa allel memb ane esis ance o he i s , second, and hi d shell su ounding he pene a ed cell and a e, espec i ely, Rm/ 25, R m /97, and R m /216 . R j , and RA Y a e he esul an junc ional esis ance be ween he i s and he second shells and be ween he second and he hi d shells . These alues a e 25R j /97 and 25R ;/216, espec i ely . C illus a es ha he a io o he change in inpu esis ance o he change in memb ane esis ance (R ;/R .) depends on he a io Rj/Rm (abscissa) . The in e up ed line is d awn a a a io R ;/R,,, o 0 .3 (a gi en change in R m esul s in a change in R ; ha is smalle by a ac o o 0 .33) . ha i a depola iza ion o 39 mV (-73 mV a 1 .5 Ca" and -34 mV a 2 .5 mM Ca") we e due o a dec ease in P K , a 7 .9- old inc ease in memb ane pe meabili y would be obse ed ha would be e lec ed in an on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 29 0 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 R  , change o abou he same size . This calcula ion is done assuming 10 mM as he in acellula concen a ion o Na and 160 mM (es ima ed om he plo in Fig . 10) as he in acellula concen a ion o K + . In a coupled issue, an 8- old inc ease in R m can esul in a2 .4- old R ; change, only 30% as la ge as he R m change, as discussed abo e . The in e up ed line in Fig . I 1 C shows ha wi h he h ee-dimensional model, wo di e en Rj/Rm a ios could gi e an R ;/R m a io o his alue . R ;/R m nea 0 .01 seems a easonable a io o a well-coupled issue . The e o e, i is no impossible ha he depola iza ion seen in high Ca" is due o a supp ession o K+ pe meabili y . Cla i ica ion o his poin mus awai mo e explici da a on Rm, and his will necessa ily in ol e elec ically isola ed cells . Rela ionship Be ween Memb ane Elec ical E en s and PTH Sec e ion In he mouse (B uce and Ande son, 1979) and in he a (p esen expe imen s), he ela ionship be ween memb ane po en ial and ex e nal Ca" is e y simila o he ela ionship be ween PTH elease and ex e nal Ca" obse ed in he cow, bo h in i o (Maye and Hu s , 1978) and in i o (B own e al ., 1976 ; Habene and Po s, 1976) . PTH elease and memb ane po en ial as a unc ion o ex e nal Ca" a e illus a ed in Fig . 12 . Pa A has been ed awn om Habene and Po s (1976) and pa B is based on ou expe imen al da a . Bo h cu es a e e y s eep and compa able, al hough he e is a shi o 0 .2-0 .3 mM along he Ca" concen a ion axis when hey a e supe imposed . This shi may be ela ed o di e ences be ween animal species o o di e ences in calcium ac i i y be ween solu ions used in biochemical and elec ophysiological expe i- men s . We a e no awa e o he exis ence o in o ma ion ela ing PTH elease and ex e nal Ca" in he a . Howe e , he Ca" ange in which a s eep change in memb ane ol age is seen (1-2 .5 mM) is e y simila o he ange o e which e e sible mo phological changes a e seen in cul u ed a pa a hy oid glands (1 .3-2 .7 mM ; Ro h and Raisz, 1964) . O he di alen ca ions also ha e pa allel e ec s on memb ane ol age and PTH sec e ion . Mg" is 2 .5 imes less e icien han Ca" in dec easing PTH sec e ion (Habene and Po s, 1976 ; She wood e al ., 1970), and we ind ha i is ^-2 .4 imes less po en han Ca" in depola izing pa a hy oid cells . I has ecen ly been obse ed ha Mn" and S " a e e y e ec i e in inhibi ing PTH sec e ion and ha Ba ++ is a poo Ca" subs i u e in dec easing PTH elease (Wallace and Sca pa, 1982) . This co esponds qui e well wi h hei depola izing po ency in ou expe i- men s . O he poin s o pa allelism be ween memb ane ol age and sec e ion a e ha ouabain inhibi s PTH sec e ion, possibly by depola izing he cells (B own e al ., 1981 ; Fische e al ., 1982), and ha comple e emo al o ex e nal Na' (which we ha e shown does no subs an ially a ec he memb ane po en ial) does no al e he modula ion by ex e nal Ca" o on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEz-BARNEO AND ARMSTRONG  Depola izing Response o Ra Pa a hy oid Cells  291 PTH sec e ion in dispe sed cells om he cow (Fische e al ., 1982 ; Wallace and Sca pa, 1982) . Taken oge he , hese da a s ongly sugges ha in pa a hy oid cells, hype pola iza ion is associa ed wi h inc eased sec e ion, and depola iza ion wi h dec eased sec e ion . A sepa a e mechanism, media ed by CAMP, may also exis . Isop o e - enol is a po en sec e agogue o PTH elease (B own e al ., 1977 ; Fische e al ., 1982), al hough i does no seem o modi y memb ane po en ial . Isop o e enol ac i a ion o PTH elease, in con as o low-Ca"-ac i a ed sec e ion (Fische e al ., 1982), is accompanied by a la ge inc ease in A PTH elease  memb ane N  po en ial (mV) B -70~ -60- -40~ -30- 0 .5  1  1 .5  2  2 .S[Ca++I  0 .5  1  5  2  i5 1C0++1 MM  MM FIGURE 12 .  Ca' al e a ions a ec PTH sec e ion and memb ane po en- ial in pa allel . A has been ed awn om he da a o Habene and Po s (1976) on slices o cal pa a hy oid glands main ained in i o . The pe cen o immuno eac i e PTH is gi en in he o dina e and Ca ++ concen a ion in he abscissa . The Mg++ concen a ion was held cons an a 0 .8 mM . Poin s ep esen he means o ou o six measu emen s . B is om ou esul s on he a . The Mg ++ concen a ion was held cons an a 1 .1 mM . Poin s ep esen he means o 3-66 measu emen s . in acellula cAMP (B own e al ., 1977 ; Fische e al ., 1982) . This co espondence be ween memb ane ol age and sec e ion con- as s wi h he usual sequence o e en s ound in o he glands, whe e he physiological s imulan s o sec e ion e oke a memb ane depola iza ion ha opens ol age-dependen Ca" channels . Ca" en y h ough hese channels seems o be esponsible o igge ing exocy osis, and emo al o di alen ca ions om he ex e nal solu ion u ns o sec e ion (Pe e sen, 1980 ; Rubin, 1970) . The e seem o be some excep ions o his usual pa e n . Fo example, jux aglome ula cells hype pola ize by 8-10 mV on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 29 2  THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 82 - 1983 a e addi ion o ad enaline, which is a s imulan o enin sec e ion (Fishman, 1976) . In hese cells, comple e emo al o ex e nal Ca" and inc ease o Mg" s imula e enin elease (F ay, 1977) . In addi ion o he elec ophysiological indings and he e ec o di alen ca ions on PTH sec e ion, o he e idence suppo s he iew ha he mechanism o s imulus sec e ion-coupling in he pa a hy oid gland is somehow unusual . Fully e e sible sec e o y ac i i y can be obse ed in dispe sed cells in he absence o Ca" . Fu he , la ge doses (up o 10 -4 M) o he Ca" channel blocke D600 ha e no e ec on PTH sec e ion ac i a ed by low Ca" (Wallace e al ., 1983) . Finally, i is known ha he di alen ca ion ionopho e A23187, which p esumably inc eases cy osolic Ca" (Reed and La dy, 1972), inhibi s PTH sec e ion (B own e al ., 1980 ; Fische e al ., 1982 ; Habene e al ., 1977) . In summa y, memb ane po en ial o pa a hy oid cells is s eeply de- penden on ex e nal Ca" in he physiological ange . I seems highly likely ha he memb ane po en ial change is ela ed o a change in sec e o y ac i i y (Fig . 12) . The ela ion be ween memb ane ol age, calcium, and sec e ion is e y unusual and sugges s he exis ence o a s imulus-sec e ion coupling mechanism ha has no p eceden in he li e a u e . The mecha- nism o his coupling and he pe meabili ies in ol ed in he Ca"-induced depola iza ion emain o be sol ed . We a e g a e ul o Lin-E Lin o he con ibu ions o some o he expe imen s . Suppo ed by Na ional Ins i u es o Heal h g an NS 12547, and by an NIH In e na ional Pos doc o al Fellowship o J . Lopez-Ba neo . Recei ed o publica ion 9 Augus 1982 and in e ised o m 29 Ap il 1983 . REFERENCES Bake , P . F ., H . Me es, and E . B . Ridgway . 1973 . E ec s o manganese and o he agen s on he calcium up ake ha ollows depola iza ion o squid axons . J . Physiol . (Lond .) . 231 :511-526 . Benne , M . V . L . 1966 . Physiolog y o elec o onic junc ions . Ann . AT Acad . Sci . 137 :509- 539 . B own, E . M ., D . G . Ga dne , and G . D . Au bach . 1980 . E ec s o he calcium ionopho e A23187 on dispe sed bo ine pa a hy oid cells . Endoc inology . 106 :133-137 . B own, E . M ., S . Hu wi z, and G . D . Au bach . 1976 . P epa a io n o iable isola ed bo ine pa a hy oid cells . Endoc inology . 99 :1582-1588 . B own, E . M ., E . M . Hu wi z, and G . D . Au bach . 1977 . Be a-ad ene gi c s imula ion o cyclic AMP con en and pa a hy oid ho mone elease om isola ed bo ine pa a hy oid cells . Endoc inology . 100 :1696-1702 . B uce, B . R ., and N . C . Ande son . 1979 . Hype pola iza io n in mouse pa a hy oid cells by low calcium . Am . J . Physiol . 236(1) :C15-C21 . Cole, K . S . 1968 . Memb anes, Ions and Impulses . Uni e si y o Cali o nia P ess, Be keley, CA . 569 pp . Douglas, W . W . 1968 . S imulus-sec e ion coupling : he concep and clues om ch oma in and o he cells . B . J . Pha macol . 34 :451-474 . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983 LOPEZ-BARNEO AND ARMSTRONG  Depola izing Response o Ra Pa a hy oid Cells  293 De Mello, W . C . 1975 . E ec o in acellula injec ion o calcium and s on ium on cell communica ion in hea . J . Physiol . (Lond .) . 250 :231-245 . Fische , J . A ., P . Olles, and D . W . Demps e . 1982 . Mechanis m o pa a hy oid ho mone (PTH) elease by Ca, K and isop o e enol (Iso) . Annual Mee ing o he Endoc ine Socie y, San F ancisco, CA . Fishman, M . C . 1976 . Memb ane po en ial in jux aglome ula cells . Na u e (Lond .) . 260 :542-544 . F ay, J . C . S . 1977 . S imula io n o enin elease in pe used kidney by low calcium and high magnesium . Am . J . Physiol . 232(4) :F377-F382 . Fuchs, F . 1971 . Ion exchange p ope ies o he calcium ecep o si e o oponin . Biochim . Biophys . Ac a . 245 :221-229 . Goldman, D . E . 1943 . Po en ial, impedance, and ec i ica ion in memb anes . J . Gen . Physiol . 27 :37-60 . Go man, A . L . F ., and A . He mann . 1979 . In e nal e ec s o di alen ca ions on po assium pe meabili y in molluscan neu ons . J . Physiol . (Lond .) . 296 :393-410 . G een, S . T ., and O . H . Pe e sen . 1981 . Thy oi d ollicula cells : he es ing memb ane po en ial and he communica ion ne wo k . P uge s A ch . Eu . J . Physiol . 391 :119-124 . G eene, E . C . 1935 . Ana omy o he Ra . Ha ne Publishing Company, New Yo k . 370 pp . Habene , J . F ., and J . T . Po s . 1976 . Rela i e e ec i eness o magnesium and calcium on he sec e ion and biosyn hesis o pa a hy oid ho mone in i o . Endoc inology . 98 :197- 202 . Habene , J . F ., T . D . S e ens, T . Ra azzola, L . O ci, and J . T . Po s . 1977 . E ec s o calcium ionopho es on he syn hesis and elease o pa a hy oid ho mone . Endoc inology . 101 :1524-1537 . Hagiwa a, S . 1975 . Ca-dependen ac ion po en ial . In Memb anes : A Se ies o Ad ances . G . Eisenman, edi o . Ma cel Dekke , New Yo k . 3 :359-381 . Hagiwa a, S ., J . Fukuda, and D . C . Ea on . 1974 . Memb an e cu en s ca ied by Ca, S , and Ba in ba nacle muscle ibe du ing ol age clamp . J . Gen . Physiol . 63 :564-578 . Hodgkin, A . L ., and P . Ho owicz . 1959 . The in luence o po assium and chlo ide ions on he memb ane po en ial o single muscle ib es . J . Physiol . (Lond) . 148 :127-160 . Holman, M . E ., and T . O . Neild . 1979 . Memb an e p ope ies . B . Med . Bull . 35 :235- 241 . Iwa suki, N ., and O . H . Pe e sen . 1977 . Ace ylcholine-lik e e ec s o in acellula calcium applica ion in panc ea ic acina cells . Na u e (Lond .) . 268 :147-149 . Kohlha d , M ., B . Baue , H . K ause, and A . Fleckens ein . 1972 . Di e en ia ion o he ansmemb ane Na and Ca channels in mammalian ca diac ib es by he use o speci ic inhibi o s . P luge s A ch . Eu . J . Physiol . 335 :309-322 . Kohlha d , M ., B . Baue , H . K ause, and A . Fleckens ein . 1973 . Selec i e inhibi ion o he ansmemb ane Ca conduc i i y o mammalian myoca dial ib es by Ni, Co and Mn ions . P luge s A ch . Eu . J . Physiol . 338 :115-123 . L6pez-Ba neo, J ., and C . F anzini-A ms ong . 1982 . Pa a hy oid : unusual sec e o y cells . J . Cell Biol . 95 :88a . (Abs .) Maye , G . P ., andJ . G . Hu s . 1978 . Sigmoida l ela ionship be ween pa a hy oid ho mone sec e ion a e and plasma calcium concen a ion in cal es . Endoc inology . 102:1036- 1042 . Meech, R . W . 1974 . The sensi i i y o Helix aspe so neu ones o injec ed calcium ions . J . on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Augus 1, 1983