Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation

P oc. Na l. Acad. Sci. USA Vol. 92, pp. 4715-4719, May 1995 Physiology Oxygen-sensi i e calcium channels in ascula smoo h muscle and hei possible ole in hypoxic a e ial elaxa ion (02 sensing/hypoxia/cy osolic Ca2+ oscilla ions) A. FRANCO-OBREGON, J. URENA, AND J. LOPEZ-BARNEO* Depa amen o de Fisiologia Medica y Bio isica, Uni e sidad de Se illa, Facul ad de Medicina, A enida Sanchez Pizjuan, 4, E-41009, Se ille, Spain Communica ed by Clay M. A ms ong, Uni e si y o Pennsyl ania Medical Cen e , Philadelphia, PA, Feb ua y 15, 1995 ( ecei ed o e iew Decembe 10, 1994) ABSTRACT We ha e in es iga ed he modi ica ions o cy osolic [Ca2 +] and he ac i i y o Ca2+ channels in eshly dispe sed a e ial myocy es o es whe he lowe ing 02 ension (P02) di ec ly in luences Ca2+ homeos asis in hese cells. Unclamped cells loaded wi h u a-2 AM exhibi oscilla- ions o cy osolic Ca22+ whose equency depends on ex acel- lula Ca2+ in lux. Swi ching om a P02 o 150 o 20 mmHg leads o a e e sible a enua ion o he Ca2' oscilla ions. In ol age-clamped cells, hypoxia e e sibly educes he in lux o Ca2+ h ough ol age-dependen channels, which can accoun o he inhibi ion o he Ca2+ oscilla ions. Low P02 selec i ely inhibi s L- ype Ca2' channel ac i i y, whe eas he cu en media ed by T- ype channels is unal e ed by hypoxia. The e ec o low P02 on he L- ype channels is ma kedly ol age dependen , being mo e appa en wi h mode a e depola iza- ions. These indings demons a e he exis ence o 02- sensi i e, ol age-dependen , Ca2' channels in ascula smoo h muscle ha may c i ically con ibu e o he local egula ion o ci cula ion. Oxygen ension (P02) has been ecognized o decades as an impo an ac o in he local egula ion o ascula one in i o (1), and i is known ha hypoxia causes elaxa ion o sys emic a e ies in i o (2-4). Ne e heless, he mechanisms unde ly- ing his physiological esponse o low P02 emain la gely unknown. Based on s udies done in o gan ba h p epa a ions, i is belie ed ha oxygen in luences ascula esis ance by di ec ly in e e ing wi h he ise o cy osolic Ca2+ equi ed o con ac ion o smoo h muscle cells (4-7); ne e heless, e y li le is unde s ood abou oxygen- egula ed p ocesses in a e- ial myocy es. I has been sugges ed ha dila a ion o he co ona y a e y (8, 9), and pe haps o he a e ies, in esponse o ex eme hypoxia migh be media ed by myocy e hype po- la iza ion in esponse o he opening o ATP- egula ed K+ channels. Howe e , i seems unlikely ha his is he only unde lying mechanism because he sensi i i y o hypoxia in mos a e ies occu s o e a physiologic ange o P02 wi hou a comp omise o ene gy me abolism (3). Since in he pas ew yea s 02- egula ed ion channels ha e been demons a ed o pa icipa e in a numbe o cellula unc ions (10), we hypo h- esized ha ascula one could be also egula ed by di ec modula ion o ol age-ga ed Ca2+ channels by P02. He e, we show he e e sible inhibi ion o he mac oscopic Ca2+ cu en o a e ial myocy es in esponse o hypoxia, which can explain he supp ession o he cy osolic [Ca2+] oscilla ions in he same cells by low P02. These da a demons a e he exis ence o 02- egula ed Ca2+ channels in ascula smoo h muscle and sugges ha hey migh pa icipa e in hypoxic a e ial elax- a ion. METHODS Expe imen s we e pe o med on enzyma ically dispe sed smoo h muscle cells om he celiac and emo al a e ies o adul abbi s. In b ie , a e ies we e emo ed, placed in cold (4°C) Hanks' balanced sal solu ion, and opened longi udi- nally. Thei ou e and inne su aces we e cleaned o he ad en i ia and endo helium, espec i ely. Pieces o a e y (- 1 mm2) we e placed in o 5 ml o Hanks' solu ion o which 7 mg o papain, 5 mg o collagenase (Sigma, ype IA), and 3.5 mg o bo ine se um albumin (Sigma, ac ion V) had been added. The issue was hen s o ed in his enzyme solu ion o 1-6 h a 6°C. In p epa a ion o he dissocia ion o smoo h muscle cells, he issue and enzyme solu ion we e placed a 37°C o 15-25 min wi h low s i ing. Upon de ec ing he i s ee cells, he issue was hen ans e ed o esh Hanks' solu ion con aining bo ine se um albumin (10 mg/50 ml) and mechan- ically dissocia ed h ough he i e-polished ip o a glass pipe e. Cells we e pla ed on pieces o poly(L-lysine)-coa ed glass co e slips. Fo expe imen s, a co e slip was placed in a eco ding chambe o -0.2 ml wi h con inuous low o solu- ion. Ex e nal solu ions we e equilib a ed wi h ei he ai (Po2 150 mmHg) o mix u es o N2 and ai o ob ain he desi ed 02 concen a ions. P02 in he chambe was moni o ed wi h an 02-sensing elec ode (11). Cy osolic [Ca2+] was es ima ed in unclamped myocy es loaded wi h u a-2 ace oxyme hyl es e . Expe imen s we e pe o med on an in e ed mic oscope wi h s anda d op ical componen s and equipped o epi luo es- cence and dual-wa eleng h pho ome y (12, 13). Calib a ion o he luo escence signals in e ms o [Ca2+] was pe o med in i o as desc ibed (14). Mac oscopic calcium cu en s we e eco ded in isola ion using he whole-cell con igu a ion o he pa ch-clamp echnique (15, 16) a e blockade o he ol age- dependen K+ channels. Al hough Na+ channels a e p ac i- cally absen in a e ial myocy es, some expe imen s we e also pe o med wi h e odo oxin (0.2 A M) added o he ex e nal solu ion. The holding po en ial was ei he -80 o -70 mV. Ba2+ was used as cha ge ca ie ins ead o Ca2+ o a o he low o cu en h ough he Ca2+ channels, and ATP was added o he in e nal solu ion o p e en he wash-ou o he channels. The composi ion o he eco ding solu ions is gi en in he igu e legends. Capaci y cu en ansien s we e well i ed by single exponen ial unc ions wi h a e age ime con- s an s o 120 ± 45 ,ls (n = 24) and 113 ± 30 As (n = 10) (mean ± SD) o celiac and emo al myocy es, espec i ely. This indica es ha despi e he la ge size o he cells (-100 , m in leng h and 6-8 , m in diame e ), we had a easonably as ol age-clamp and, hus, we could di ec ly moni o ail cu - en s. Vol age-clamp speed was a o ed by ballis ic cha ge o memb ane capaci ance and he use o low- esis ance elec- odes (be ween 1 and 3 MQI) (16). Due o he ela i ely small size o he cu en s, se ies esis ance was no sys ema ically compensa ed. Analog cu en signals we e low-pass il e ed *To whom ep in eques s should be add essed. 4715 The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked "ad e isemen " in acco dance wi h 18 U.S.C. §1734 solely o indica e his ac . P oc. Na l. Acad ScL USA 92 (1995) (cu o equency be ween 3 and 10 kHz), digi ized a a sample in e al o 20 o 50 , s, and s o ed on compu e o analysis. The expe imen s we e conduc ed a oom empe a u e (22- 25°C). RESULTS The e ec o hypoxia on single a e ial smoo h muscle cells was i s s udied by moni o ing he modi ica ions o cy osolic [Ca2+] in esponse o changes in P02 wi h double-wa eleng h mic o luo ime y. Fu a-2-loaded myocy es s imula ed wi h a pu ine gic agonis (ATP) gene a ed hy hmical oscilla ions o cy osolic [Ca2+]. In all myocy es exhibi ing egula Ca2+ oscilla ions ha we e hen subjec ed o he comple e expe i- men al p o ocol (n = 8), exposu e o hypoxia (swi ching om a ba h solu ion equilib a ed wi h a P02 o 150 o one o 20 mmHg) elici ed a ma ked e e sible educ ion in he ampli- ude and equency o he Ca2+ spikes. Hypoxic ea men e en ually esul ed in he comple e supp ession o he Ca2+ oscilla ions. A ep esen a i e example o his cellula esponse o low Po2 is shown in Fig. 1A, which also includes he signal om an 02-sensing elec ode placed in he icini y o he cell. In hose myocy es ha had a ela i ely high es ing [Ca2+] (abo e 50 nM), he inhibi ion o he oscilla ions by hypoxia was accompanied by a dec ease in es ing cy osolic Ca2+ le els. These obse a ions indica e ha low P02 may exe i s elaxing ac ion by dec easing in acellula [Ca2+], which is he a iable ha de e mines con ac ion in ascula smoo h muscle (17- 20). Al hough la gely due o Ca2+ elease om in acellula s o es (21, 22), Ca2+ oscilla ions in exci able (23) and nonex- ci able (24) cells equi e, and a e acili a ed by, ansmem- b ane Ca;+ in lux. In acco d wi h his idea, depola iza ion o myocy es wi h 60 mM ex e nal K+ elici s an inc ease in he equency o he Ca2+ oscilla ions p eceding a main ained ele a ion o cy osolic [Ca2+] (Fig. 1B). An opposi e e ec (abolishmen o he Ca2+ spikes and dec ease o es ing cy osolic Ca2+) was obse ed a e b ie ly emo ing ex e nal Ca2+ (Fig. 1 C) o blockade o ol age-ga ed Ca2+ channels wi h ni edipine (Fig. 1D). The hypoxic supp ession o Ca2+ oscilla ions could be caused by a ious 02-dependen cellula p ocesses. Re illing o s o es p e iously deple ed wi h ca eine was no p e en ed by hypoxia. Fu he mo e, hypoxia did no a ec he elease o Ca2+ om in e nal s o es e oked by ei he ca eine (10 mM) o no epineph ine (3 ApM). The e o e, one o he ac ions o low P02 migh be o inhibi Ca2+ in lux h ough ol age-dependen channels. This was di ec ly es ed by eco ding he cu en h ough Ca2+ channels in whole-cell pa ch-clamped myocy es. Cu en sweeps gene a ed in esponse o s ep depola iza ions o + 10 mV in a celiac myocy e exposed o no moxic (con ol and eco e y) and hypoxic ex e nal solu ions a e shown in Fig. 2A. The eco dings demons a e an -40% e e sible educ- ion in cu en ampli ude upon exposu e o low P02. The ela ionship be ween cu en ampli ude and P02 is illus a ed in Fig. 2B, whe e he alues o peak cu en (do s) elici ed by depola izing pulses deli e ed a di e en P02 le els a e plo - ed. The g aphs indica e ha he e e sible hypoxic inhibi ion o cu en ampli ude occu s oughly wi h he ime cou se o ba h exchange and ha i is pa icula ly appa en a P02 le els below 70 o 80 mmHg. The ime cou se o he inhibi ion o he calcium cu en by low P02 was simila o ha o ni edipine block (no shown). Fo compa ison, e e sible hypoxic inhi- bi ion o Ca2+ channel ac i i y in a emo al myocy e is illus a ed in Fig. 2C. Simila quali a i e esul s ha e been ob ained in all cells s udied so a (n = 33). I has been shown ha ascula smoo h muscle cells con ain wo majo kine ically A 150 E /oxygen ensiol E 50 (P2) +" ~~~60K+ 200 200 - "i" + 100 0 K n s C .350 u 150 0 cn 2 50 5 o D + N . u 0 >% 1 min 0 Ca2+, 0.5 mM EGTA 0.5 FIM ni edipine 1min FIG. 1. Oscilla ions o cy osolic [Ca2+] in a e ial myocy es and e ec o changes in ambien oxygen ension (Po2). (A) Pa allel eco dings o Po2 in he eco ding chambe and in acellula [Ca2+] in a u a-2-loaded myocy e illus a ing he e e sible inhibi ion o Ca2+ oscilla ions in esponse o hypoxia. No e ha he spikes a e supp essed upon eaching ex eme low Po2 le els (=20 mmHg). A e age es ing cy osolic [Ca2+] was 61 ± 28 nM (mean + SD, n = 22) and oscilla ions we e igge ed wi h a ansien (=30 s) exposu e o 1 mM ATP (26). The a e age equency and ampli ude o he oscilla ions we e 1.6 ± 0.6 spikes pe min and 271 ± 172 nM (n = 8), espec i ely. (B) Re e sible inc ease o he equency o Ca2+ oscilla ions in esponse o memb ane depola iza ion by 60 mM ex e nal K+. Reini ia ion o he oscilla ions a e eco e y o he basal Ca2+ le els was ob ained by a b ie pulse o 1 mM ATP. (C and D) Supp ession o Ca2+ oscilla ions a e b ie emo al and chela ion o ex e nal Ca2+ (C) o blockade o ol age-ga ed Ca2+ channels wi h ni edipine (D). The applica ion o he di e en es solu ions is indica ed by he ho izon al ba s. The s anda d ex e nal solu ion con ained (in mM) 140 NaCl, 2.7 KCI, 2.5 CaCl2, 1 MgCl2, and 10 Hepes (pH 7.35-7.40). The 60 K+ solu ion con ained 82.7 mM NaCl and 60 mM KCl. Ni edipine (0.5 iM) was added o he ex e nal solu ion. The 0 Ca2+, EGTA solu ion con ained 4 mM MgCI2 and 0.5 mM EGTA. 4716 Pyilg:Fac a P oc. Na l. Acad. Sci. USA 92 (1995) 4717 A hypoxia con ol 2 3 B o 150 C CD - l!0 1 . 200- 0 0 a 0 0 50 100 150 200 ime (s) FIG. 2. Inhibi ion o ol age-ga ed Ca2+ channels by low P02. (A) Mac oscopic calcium cu en s eco ded om a myocy e dispe sed om he celiac a e y du ing 15-ms s ep depola iza ions o + 10 mV om a holding po en ial o -80 mV. Exposu e o hypoxia (swi ching om an ex e nal solu ion equilib a ed wi h P02 - 150 mmHg o ano he wi h P02 - 20 mmHg; eco ds 2 and 3) induces an inhibi ion in cu en ampli ude. Re e sibili y is illus a ed by he eco e y ace. (B) Pa allel ime cou ses o he changes o P02 in he chambe and he educ ion in cu en ampli udc. The eco ds shown in A a e indica ed by he co esponding numbe . Cu en ampli udes we e measu ed immedia ely be o e he end o he depola izing pulses. (C) Re e sible educ ion o calcium cu en ampli ude by low P02 (20 mmHg) in a myocy e dispe sed om he emo al a e y. Depola izing pulses o +10 mV (15 ms) we e applied om a po en ial o -80 mV. No e ha a slow componen o he ail cu en was una ec ed by low P02. The eco ding solu ions con ained (in mM) he ollowing: Ex e nal [140 NaCl, 2.7 KCI, 10 BaCl2, and 10 Hepes (pH 7.4)]. In e nal (solu ion in he pa ch pipe e and inside he cell) [100 CsCl, 25 CsF, 2 MgCl2, 10 Hepes, 10 EGTA, 5 bis(2-aminophenoxy)e hane- N,N,N',N'- e aace a e, and 4 MgATP (pH 7.3)]. dis inc popula ions o Ca2+ channels (L and T ype) (25, 26) ha , as in o he cell ypes (16, 27), can be iden i ied by hei deac i a ion ime cou ses as slow- (T) and as - (L) deac i a - ing channels. In many emo al and celiac myocy es we de ec wo componen s o he ails. The slowly deac i a ing compo- nen o he ail cu en , which is esis an o ni edipine (Fig. 3A) and inac i a ed by a small depola izing p epulse (Fig. 3 B), was una ec ed by hypoxia (Fig. 3 C) in all cells es ed (see also Figs. 2C and 4A). These obse a ions indica e ha he e ec o low P02 is selec i e o he dihyd opy idine-sensi i e, as - deac i a ing (L- ype), channels, whe eas he slow-deac i a ing (T- ype) Ca2+ channel popula ion is una ec ed by changes in P02. A ema kable cha ac e is ic o he egula o y ac ion o oxygen on Ca2+ channel ac i i y is i s s ong ol age depen- dence. Fig. 4A shows a amily o calcium cu en s gene a ed by depola iza ions om -80 mV o he indica ed memb ane po en ials. Cu en aces eco ded in a low P02 solu ion (H) a e compa ed wi h hose ob ained a no mal P02 (C). Reco - e y om hypoxia was almos pe ec and an example is shown a +10 mV ( ace R). Low P02 p oduced an inhibi ion o cu en ampli ude ha was la ge wi h mode a e depola iza- ion. Wi h s onge depola iza ion he e ec o hypoxia was almos negligible. In celiac myocy es, he a e age inhibi ion o cu en ampli ude by hypoxia (Po2 - 20 mmHg) was 43.17% ± 13.8% (mean ± SD, n = 12) o he con ol alue a 0 mV, bu only 2.08% + 7% (n = 8) a +20 mV. In emo al myocy es, hese alues we e 40% + 27% (n = 7) a 0 mV and 10% ± 13% (n = 6) a +20 mV. The ol age dependence o he hypoxic inhibi ion o Ca2+ channel ac i i y is also clea ly e iden in Fig. 4B, whe e we ha e plo ed he a e age cu en - ol age ela- ionship unde no moxic ( illed symbols) and hypoxic (open symbols) condi ions ob ained wi h measu emen s om ou cells. DISCUSSION Ou esul s indica e ha exposu e o low P02 leads o inhibi- ion o Ca2+ in lux h ough L- ype Ca2+ channels in a e ial myocy es. This is selec i e since T- ype Ca2+ channels appea o be una ec ed. The e ec o 02 ension on he Ca2+ channels is as , is comple ely e e sible, and occu s wi h P02 le els wi hin he physiological ange. This phenomenon explains he dec ease o cy osolic Ca2+ upon exposu e o low P02 and, hus, i is concei able ha i con ibu es o he hypoxic elaxa ion o sys emic a e ies. In ag eemen wi h p e ious wo k by o he au ho s (28, 29), we ha e no de ec ed so a any modula o y e ec o P02 on he mac oscopic K+ cu en s o sys emic myocy es. Pha macological s udies done in isola ed pe used hea s ha e sugges ed ha hypoxic asodila a ion o he co - ona y a e ies could be a consequence o he dec ease o in acellula ATP and he subsequen hype pola iza ion caused by he opening o ATP-sensi i e (KATP) K+ channels (8, 9). Ac i a ion o KATP channels in esponse o hypoxia mos likely equi es main ained exposu e o ex eme low P02 (8, 30). Thus, al hough possibly impo an , his p ocess may ac on a slowe , mo e p o ac ed, ime scale han he acu e esponse o Ca2+ channels desc ibed he e, occu ing o e a ull ange o P02 alues and su ely c i ical o an immedia e a e ial elax- a ion in esponse o hypoxia. The Ca2+ channels selec i ely egula ed by P02 a e o he dihid opy idyne-sensi i e, L- ype, which a e b oadly dis ib- u ed in ascula smoo h muscle and a e known o be ac i a ed by no epineph ine and o he asoac i e agen s (20, 26, 31). In mesen e ic a e y myocy es, he memb ane po en ial- o ce 4 eco e y 00 msA 5 ms C hypoxia 5 ms Physiology: F anco e al. 1 P oc. Na l. Acad. Sci. USA 92 (1995) I | < HP=-50 HP=-80 150 pA 5 ms I HP=-80 mV HP=-50 mV 5 ms FIG. 3. Sepa a ion o as - and slowly deac i a ing componen s in he Ca2+ ail cu en s and selec i e inhibi ion o he as componen by low P02. (A) Supe posi ion o cu en aces eco ded in a celiac myocy e du ing 15-ms depola iza ions o + 10 mV om -80 mV in he con ol solu ion and a e addi ion o 0.2 u M ni edipine (uppe panel). The ail cu en s in he wo expe imen al condi ions a e shown a a expanded ime scale in he middle and bo om panels. As in o he cell ypes (16, 27), he ail cu en s ha e as and slow componen s ha mos likely ep esen he L- ype ( as -deac i a ing) and T- ype (slow-deac i a ing) Ca2+ channels desc ibed in a e ial smoo h muscle cells (25, 26). Ni edipine almos comple ely abolished he as componen o he ail bu le unal e ed he slow componen . This is shown by he simila ampli ude o single exponen ial unc ions ha in he wo expe imen al condi ions we e i ed o he slow componen o he ail. The exponen ial unc ions we e ex apola ed o he onse o epola iza ion (indica ed by downwa d a ows). (B) Supe posi ion o cu en aces ob ained om a emo al myocy e du ing 10-ms depola iza ions o + 10 mV om he indica ed holding po en ials (HP) (uppe aces). The middle and bo om panels show he exponen ial unc ions i ed o he slow..componen o he ail a he wo holding po en ials. No e ha a small main ained depola iza ion ( om -80 o -50 mV) leads o >80% educ ion o he slow componen in he ail, indica ing inac i a ion o he T- ype Ca2+ channels. The as componen o he ail was almos unal e ed. (C) Supe posi ion o cu en aces in a celiac myocy e gene a ed du ing 15-ms s ep depola iza ions o + 10 mV om -80 mV in a myocy e exposed o no moxic (C, con ol, P02 - 150 mmHg) and hypoxic (H, Po2 - 20 mmHg) solu ions (uppe panel). As in A, he eco dings in he middle and bo om panels show ha he slow componen o he ail cu en s was una ec ed by low P02 and ha he educ ion o cu en ampli ude is due o selec i e inhibi ion o he as componen o he ail cu en . ela ion almos pe ec ly ma ches wi h he ol age dependence o Ca2+ channel open p obabili y (Popen) (20). Thus, is has A VM (mV) H C 0 -" - :; ~ ~ -_-------" +1 0 / H been sugges ed ha his channel ype is a majo egula o o smoo h muscle ension and hence o a e ial one (20, 28, 31). memb ane po en ial (mV) B -40 -20 ' 0 ' 0 20 40 60 . . .. * -100- -200- 3 CD c 'D 0 a C> I FIG. 4. Vol age dependence o he inhibi o y e ec o low P02 on he Ca2+ channels. (A) Cu en aces eco ded du ing 15-ms s ep depola iza ions om -80 mV o he indica ed memb ane po en ials. Sweeps eco ded om he same celiac myocy e in no moxic (C, P02 - 150 mmHg) and hypoxic (H, P02 - 20 mmHg) solu ions a e supe imposed. No e ha he e ec o low P02 is la ge wi h mode a e depola iza ions. (B) A e age cu en - ol age ela ion in no moxic ( illed symbols) and hypoxic (open symbols) solu ions. Cu en ampli udes we e measu ed in ou celiac myocy es be o e he end o 15-ms depola izing pulses. Ve ical ba s a e he s anda d e o o he mean. A B C Icon ol -slow ail i con ol I ni edipine - B- b ms )- 5 ms 4718 Pyilg:Fac a P oc. Na l. Acad Sci. USA 92 (1995) 4719 In e es ingly, he inhibi ion o he calcium cu en by low Po2 is mo e p onounced a po en ials be ween -30 and 0 mV (see Fig. 4), which is he ange a which he popen- ol age ela ion- ship o he channels is e y s eep (20, 31). The e o e, in pa ially depola ized myocy es low P02 would be expec ed o ha e a majo in luence on channel Popen. In good ag eemen wi h his idea, ea ly s udies had al eady shown ha he sensi i i y o sys emic a e ies o oxygen is mo e ob ious when es ing is ca ied ou on p econ ac ed samples using low o mode a e concen a ions o agonis (5). In conclusion, we ha e ound in a e ial smoo h muscle a ype o Ca2+ channel modula ion ha may pa icipa e in hypoxic a e ial dila a ion. The p ope ies o he oxygen- sensi i e Ca2+ channels make hem well sui ed o ha ing a majo physiological ole in he as adap a ion o egional a e ial esis ance o he deg ee o blood oxygena ion. The exis ence o oxygen-sensi i e K+ channels in a ious issues has been p e iously desc ibed (10) bu oxygen-sensi i e Ca2+ channels a e wi hou p eceden in he li e a u e. Besides celiac and emo al myocy es ( ep esen a i e examples o isce al and skele al muscle a e ies), we ha e obse ed a simila egula- o y ac ion o P02 in muscle cells dispe sed om a and abbi mesen e ic a e ies as well as om he main unk o he abbi pulmona y a e y. This sugges s ha 02- egula ed Ca2+ chan- nels a e pe haps dis ibu ed h oughou he ci cula o y sys em and in di e en species. This channel ype migh be in ol ed in some ca dioci cula o y dis u bances such as hype ension. This wo k was suppo ed by g an s om he Di ecci6n Gene al de In es igaci6n Cien ica y T6cnica (DGICYT) o he Spanish Minis y o Science and Educa ion and he Eu opean Communi y (DGXII). A.F.-O. is an In e na ional Human F on ie s Science P og am Pos - doc o al Fellow. 1. Spa ks, H. V. (1980) in Vascula Smoo h Muscle, Handbook o Physiology, The Ca dio ascula Sys em, eds. Boh , D. F., Somlyo, A. P. & Spa ks, H. V. (Am. Physiol. Soc., Be hesda), Vol. 3, pp. 475-513. 2. Hells and, P., Johansson, B. & No be g, K. (1977) Ac a Physiol. Scand. 100, 69-83. 3. De a , R. (1980) Am. J. Physiol. 238, H761-H769. 4. Ma io , J. F. & Ma shall, J. (1990) J. Physiol. (London) 422, 1-13. 5. Chang, A. E. & De a , R. (1980)Am. J. Physiol. 238, H716-H728. 6. an B eemen, C. & Saida, K. (1989) Annu. 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