Pflugers Arch - Eur J Physiol (2002) 444:760–770DOI 10.1007/s00424-002-0870-5

O R I G I N A L A R T I C L E

Vesa Paajanen · Matti Vornanen

The induction of an ATP-sensitive K+ current in cardiac myocytesof air- and water-breathing vertebrates

Received: 18 March 2002 / Revised: 15 April 2002 / Accepted: 24 April 2002 / Published online: 8 August 2002� Springer-Verlag 2002

Abstract Opening of ATP-sensitive potassium channels(KATP) is an effective cardioprotective mechanism inmammals. The amplitude of the ATP-sensitive K+ current(IK,ATP) and the opening sensitivity of KATP channels are,however, poorly known in ectotherms. As O2-sensingmechanisms and reactions to O2 deficiency differ inaquatic and terrestrial animals, we hypothesised that theresponse of KATP channels to metabolic inhibition wouldbe different between air- and water-breathers. We there-fore compared IK,ATP in ventricular myocytes of ananoxia-sensitive (Oncorhynchus mykiss) and an anoxia-tolerant fish (Carassius carassius), two amphibians(Xenopus laevis and Rana temporaria) and a terrestrialreptile (Lacerta vivipara) using the whole-cell patch-clamp method. IK,ATP was induced by preventing mito-chondrial and/or glycolytic ATP production and perfusingmyocytes with an ATP-free pipette solution. All specieshad a glibenclamide-sensitive IK,ATP, but the currentamplitude was much greater in air-breathers than inwater-breathers. Furthermore, the IK,ATP in air-breatherswas more sensitive to intracellular ATP depletion than inwater-breathing animals. These findings indicate thatIK,ATP is larger and more easily induced in air- than water-breathers. In all ectotherms, the first response to completemetabolic inhibition was the induction of a large inwardcurrent, the amplitude of which exceeded that of IK,ATP.Thus, the protective effect of the IK,ATP may be physio-logically significant only during partial metabolic block-ade.

Keywords KATP channel · Cardiomyocytes ·Glibenclamide · Fish · Frog · Lizard · Metabolicinhibition · Environmental adaptation

Introduction

Since the detection of a sarcolemmal (SL) ATP-sensitiveK+ current (IK,ATP) by Noma in 1983 [30] numerousstudies have addressed IK,ATP, and the importance of KATPchannels in cardiac protection is generally accepted [9,17, 37]. In normoxia, KATP channels are kept closed bythe high intracellular [ATP] ([ATP]i), but in ischaemiaand hypoxia, opening of KATP channels is induced bydecreased [ATP]i and/or different phosphorylation cas-cades [16, 18, 23, 24]. IK,ATP shortens the duration of theaction potential (AP), thereby limiting Ca2+ influx andsaving energy by reducing cardiac contractility [4, 38,39]. SL KATP channels may, together with mitochondrialKATP channels, also play a role in cardiac preconditioning,a process whereby short periods of ischaemia providecardiac protection against subsequent ischaemic insults orreperfusion injury [5, 13, 41]. The preconditioningresponse has been found in several mammals, birds and,recently, also in fish [14].

The whole-body energy demand in most ectothermicvertebrates is 4–5 times lower than in most endotherms.At the level of the heart, the maximum power output islikewise much lower in most ectotherms than in en-dotherms [7, 11]. This has an advantage under hypoxicconditions because, although the glycolytic capacity forcardiac work does not differ greatly among ectotherms, itcan represent a sizeable proportion of the maximalcapacity for cardiac power output [3, 36].

The respiratory medium (air or water) is another factorthat affects hypoxia/anoxia tolerance. The [O2] in water ismuch lower and more variable than in air, resulting insignificant differences in O2 sensing and cardio-respira-tory regulation between terrestrial, semi-aquatic andaquatic ectotherms [27, 40]. Fish, for example, sensechanges in water [O2] with O2-sensitive receptors in thefirst gill arch, and respond immediately to decreasing O2content with increased ventilation and bradycardia beforeany changes in blood [O2] occur [35]. Air-breathingvertebrates are seldom exposed to environmental hypoxia,with the exception of diving, burrowing and high-altitude

V. Paajanen · M. Vornanen ())Department of Biology, University of Joensuu, P.O. Box 111,80101 Joensuu, Finlande-mail: matti.vornanen@joensuu.fiTel.: +358-13-2513562Fax: +358-13-2513590

residence, and do not have external O2 receptors [40].These animals sense O2 internally by means of the carotidbodies and other O2-sensitive receptors, and show cardio-respiratory responses to hypoxia when it occurs locally intissue cells or when blood [O2] decreases. Given thesefundamental differences in systemic O2 sensing, theremight be also important differences in cellular hypoxiadefence mechanisms between water- and air-breathingectotherms. We hypothesised that air-breathing verte-brates would have a greater risk of acute hypoxic damage,and therefore more powerful cellular defence mechanismsthan water-breathers. To test this hypothesis, we used thewhole-cell patch-clamp technique to measure the ampli-tude and opening sensitivity of KATP channels, the majorcardio-protective mechanism of the vertebrate heart, inventricular myocytes of aquatic, semi-aquatic and terres-trial vertebrates.

Five species were chosen, representing different envi-ronments and respiratory mechanisms. The Europeancommon lizard Lacerta vivipara is a completely terres-trial, air-breathing reptile. Both amphibian species arebimodal breathers capable of lung and cutaneous respi-ration, but the common frog Rana temporaria is semi-aquatic whereas the clawed frog Xenopus laevis iscompletely aquatic. The two water-breathing teleost fishdiffer in their hypoxic tolerance, with the crucian carpCarassius carassius being anoxia tolerant [6], whilst therainbow trout Oncorhynchus mykiss is hypoxia sensitive.Since temperature significantly modifies cardiac functionin fish at the organ [12] and cellular [46] levels, IK,ATPwas studied in both warm- (w.a.) and cold- (c.a.)acclimated fish.

Materials and methods

Animals

Rainbow trout were obtained from a local fish farm and cruciancarp were caught in local ponds near Joensuu in eastern Finland.The fish were held for at least 3 weeks in large tanks (500 or 1000 l)at constant temperature (20 �C and 4 �C for carp; 17 �C and 4 �C

for trout). Aerated groundwater was delivered to the tanks at about0.5 l/min. Trout were fed ad libitum with nutrient pellets (Biomar,Denmark) and w.a. carp with aquarium fish food (Tetra). Coldacclimated carp do not forage [31] and were therefore not fed. Thephotoperiod was a 15 h:9 h, light-dark cycle.

Common frogs and lizards were caught in the Joensuu districtbetween June and August. Clawed frogs were raised in thelaboratory in large tanks (500 l) filled with tap water at constanttemperature (17 �C). Two adult rats (Wistar) and one neonatal (4-day old) rat were used as mammalian references. All procedureswere conducted with the permission of the local committee foranimal experimentation.

Myocyte isolation

Ventricular myocytes were isolated using previously establishedprotocols [44, 45, 48] (Table 1). Teleosts were stunned by a sharpblow to the head, and rats were anaesthetised with diethyl ether. Allanimals were killed by decapitation. A cannula was inserted inaorta (rat, frogs) or through bulbus arteriosus into the ventricle(teleosts). Whole hearts were perfused retrogradely using ahydrostatic pressure head of 80 cm H2O, first with a nominallyCa2+-free solution (see below) and then with digestive enzymesdissolved in the Ca2+-free saline (Table 1). Both solutions wereoxygenated continuously (100% O2), and the enzyme solution wasrecirculated using a peristaltic pump. After digestion, ventricleswere separated and diced with scissors into small species. Singlecells were liberated by agitation of the tissue samples through theopening of a Pasteur-pipette. Lizard hearts were not perfused due totheir small size. Instead, the ventricle was chopped into smallpieces with scissors and incubated in Ca2+-free solution for two 10-min periods. The broken myocytes were removed and treatmentwas continued with enzyme solution under constant stirring at20 �C. Enzyme treatment was continued until sufficient numbers(>20%) of spindle-shaped cells had been obtained. Myocytes werestored 4 �C and used within 12 h of isolation.

Solutions

Ca-free solution contained (in mM): 100 NaCl, 10 KCl, 1.2KH2PO4, 4 MgSO4, 50 taurine, 20 glucose, and 10 HEPES (pH 6.9with KOH). The physiological saline contained the following (inmM): 150 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 glucose, and 10HEPES (pH 7.6 with NaOH). Electrophysiological recordings weremade using pipettes filled with K+-based electrode solutioncontaining (in mM): 140 KCl, 1 MgCl2, 5 EGTA, 10 HEPES and4 MgATP (omitted in ATP-free pipettes) (pH 7.2 with KOH).Tetrodotoxin (TTX, 0.5 �M) and nifedipine (10 �M) were added in

Table 1 The protocols and enzymes used for the isolation ofventricular myocytes from the rat and the five ectothermicvertebrates (the rainbow trout Oncorhynchus mykiss, the cruciancarp Carassius carassius, the clawed frog Xenopus laevis, the

European common frog Rana temporaria and the Europeancommon lizard Lacerta vivipara) (w.a. warm acclimated, c.a. coldacclimated)

N Body mass(g)

Ca2+-freeperfusion(min)

Collagenase(mg/ml)

Trypsin IX(mg/ml)

BSAa

(mg/ml)Protease III(mg/ml)

Enzymedigestion(min)

R. norvegicus 3 10 0.75b 0.1 20L. vivipara 7 2.6€0.4 10+10d 0.75c 0.5 0.75 20+6d

R. temporaria 6 32.6€5.7 10 0.75c 0.5 0.75 20X. laevis 6 89.2€8.6 10 0.75c 0.5 0.75 20O. mykiss (w.a.) 31 256.3€30.9 10 0.75c 0.5 0.75 20O. mykiss (c.a.) 5 246.2€40.2 10 0.75c 0.5 0.75 20C. carassius (w.a.) 81 44.9€9.5 20 1.5c 1 1 40C. carassius (c.a.) 11 47.9€6.9 20 1.5c 1 1 40

a Fatty-acid free; b collagenase type I (Sigma); c collagenase type 1A (Sigma); d The “chunk” method was used for the European commonlizard due to the small heart size

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the external solution to block Na+ (INa) and L-type Ca2+ currents(ICa,L), respectively. TTX was omitted with experiments on ratmyocytes as INa of the mammalian heart is rather insensitive toTTX.

Protocols

The treatments used to trigger IK,ATP are listed in Table 2. Control(C) experiments were made with oxygenated external solutionsusing either ATP-supplemented (CATP) or ATP-free pipette solu-tions (CATP-free). Chemical anoxia was caused by scavenging thedissolved O2 in the medium with 0.1 mM sodium dithionate(Na2S2O3) added to the external saline just before use. Effects ofanoxia were studied with 4 mM MgATP (AATP) or without ATP(AATP-free) in the pipette. For complete metabolic inhibition (MI),5 mM iodoacetic acid (IAA) and 0.1 mM Na2S2O3 were dissolvedin the physiological solution and pH readjusted to 7.6 with 1 MNaOH. These treatments were also conducted either with 4 mMMgATP (MIATP) or without ATP (MIATP-free) in the pipette.Cromakalim (10 �M; Sigma) and pinacidil (10 �M; ICN) wereused as openers, and glibenclamide (10 �M; Sigma) as a specificblocker of IK,ATP. As the depolarising current overlaps with theoutward IK,ATP, the latter was determined in metabolically inhibited(MIATP, MIATP-free) myocytes after washout of the inhibitors (seeResults).

Electrophysiological recordings

Small aliquots of myocytes were taken for electrophysiologicalrecordings, allowed to settle on the bottom of a small chamber(0.5 ml) and subsequently superfused at a constant 1.4 ml/min withphysiological solution. Temperature in the chamber was maintainedat 17€0.1 �C (TC-10 controller; Dagan, Minneapolis, Minn., USA)for all animals except for c.a. fishes for which it was 10€0.1 �C(this was the lowest temperature that could be used throughout theyear without condensation of water on the optics of the micro-scope). Patch pipettes were pulled from borosilicate glass (Garner,Claremont, Calif., USA) on an appropriate puller (L/M-3P-A, ListMedical, Darmstadt, Germany). The tip resistance of patch pipetteswas 2.88€0.04 MW (n=387). The experiments were conducted inwhole-cell voltage-clamp configuration using an appropriate am-plifier (EPC-9, HEKA, Lambrecht, Germany) and commercial

computer software (Pulse, HEKA). Cell membrane capacitance wasmeasured by integrating the capacitive current induced by smallhyperpolarising steps. The current/voltage (I/V) relations wereconstructed by applying ramp pulses (from 40 to –120 mV;–140 mV/s) every 5 s from the holding potential of –80 mV (Fig. 1).Data were analysed using Pulse (v. 8.53) and SigmaPlot (v. 6.0)programs.

Statistical analyses

Data are given as means€SEM. IK,ATP between two or moretreatments was compared using Student’s t-test for unpairedsamples or one-way ANOVA with Student-Newman-Keuls methodas the post-hoc test. Differences among animal groups were testedusing one-way ANOVA. P<0.05 was considered significant.

Results

Capacitive cell sizes and the amplitudes of the back-ground inward rectifier current (IK1) are presented inTable 3. The conductance of IK1 was significantlydifferent (P<0.01) among animals groups, being highestfor w.a. trout (702€47 pS pF–1) and lowest for c.a trout(334€19 pS pF–1). All other values lay between theseextremes. Cold acclimation affected IK1 oppositely in thetwo fish species, reducing IK1 in the trout and increasing itin the carp. Furthermore, cold acclimation increased thecapacitive cell size in trout (but not in carp) and thischange in capacitive cell size accounted for most of thetemperature-related change in conductance observed inc.a. trout. Perfusion of the cells with ATP-free pipettesolution reduced the IK1 in w.a. trout but increased it inc.a. carp and clawed frog.

Table 2 Treatments used to in-duce the ATP-sensitive K+ cur-rent IK,ATP (IAA iodoacetic acid)

Treatment Drug in external saline (mM) ATP in pipette (mM) Abbreviation

Control None 4 CATPControl None none CATP-freeAnoxia 0.1 Na2S2O3 4 AATPAnoxia 0.1 Na2S2O3 none AATP-freeMetabolic inhibition 0.1 Na2S2O3+5 IAA 4 MIATPMetabolic inhibition 0.1 Na2S2O3+5 IAA none MIATP-freeKATP opener 10 �M cromakalim 4 CromKATP opener 10 �M pinacidil 4 Pinac

Table 3 Capacitive cell sizeand conductance of the inwardrectifier K+ current IK1 in pres-ence (CATP) and absence(CATP-free) of ATP in the controlpipette solution

CATP CATP-free

pF IK1 (pS/pF) n pF IK1 (pS/pF) n

L. vivipara 21.89€1.78 582.0€26.6 28 23.32€1.24 641.1€43.2 34R. temporaria 42.08€3.64 387.1€25.9 33 36.87€3.35 432.2€37.5 28X. laevis 48.16€2.02 371.9€24.9* 18 47.24€3.05 522.3€34.0 27O. mykiss (w.a.) 34.88€2.34† 702.3€46.5*† 41 40.84€2.54† 540.0€25.9† 28O. mykiss (c.a.) 56.75€4.68 334.1€18.5 19 51.67€4.11 357.6€19.8 36C. carassius (w.a.) 19.30€1.22 375.9€22.9† 25 22.50€2.07 421.0€39.8† 18C. carassius (c.a.) 18.91€1.46 552.5€36.7* 23 21.41€1.21 727.3€53.0 29

*P<0.05 vs. ATP-free pipette solutions; †P<0.05 vs. c.a. fish

762

Sensitivity of KATP channels for opening

A glibenclamide- (10 �M) sensitive current was inducedby metabolic inhibition, anoxia or intracellular perfusionwith ATP-free pipette solution, in all animal groups, withthe exception of w.a. rainbow trout (Fig. 1, Table 4).Among animal groups there were important differences inthe delay in the onset of the current (3–30 min) and the

severity of the energy depletion required to elicit theresponse. In the lizard, the opening of the KATP channelswas induced rapidly (in 3–7 min) by all treatments thatimpaired cellular ATP balance (Table 4). Surprisingly,long-duration stimulation of the cells with 1-s ramp pulsesat 0.2 Hz was sufficiently strenuous to the cells to inducethe opening of the KATP channels, even when themyocytes were supplied with a continuous flux of ATP

Fig. 1 Induction of the ATP-sensitive K+ current (IK,ATP) inventricular myocytes of five ectothermic vertebrates (the rainbowtrout Oncorhynchus mykiss, the crucian carp Carassius carassius,the clawed frog Xenopus laevis, the European common frog Ranatemporaria and the European common lizard Lacerta vivipara) bydifferent treatments which compromise cellular ATP status (for adetailed description of treatments and their abbreviations seeTable 2). Myocytes were stimulated from a holding potential of

–80 mV with 1-s ramp pulses from 40 to –120 mV (upper leftcorner) at a frequency of 0.2 Hz. Each line in each plot representsone treatment and is the mean from n=3–10 myocytes. Treatmentswere continued as long as a steady-state response was achieved orin the case of complete metabolic inhibition (MIATP-free, MIATP) aslong as the myocyte was able to tolerate the treatment. Differentletters mark different treatments as indicated by the key in the lowerright corner (w.a. warm acclimated, c.a. cold acclimated)

Table 4 Effectiveness of dif-ferent ATP-depletion treatments(for details and abbreviationssee Table 2) in inducing theIK,ATP in ventricular myocytesof the ectothermic vertebrates

CATP AATP MIATP CATP-free AATP-free MIATP-free

L. vivipara + + + + + +R. temporaria – + + + + +X. laevis – – – – – +C. carassius (w.a) – + ? + + +C. carassius (c.a.) – – ? + + ?O. mykiss (c.a.) – – – – + +O. mykiss (w.a) – – – – – –

+ IK,ATP induced; – IK,ATP not induced; ? IK,ATP not seen, but induction of IK,ATP cannot be excluded ascarp myocytes did not tolerate prolonged metabolic inhibition

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Fig. 2A, B Amplitude of IK,ATPin the ventricular myocytes offive ectothermic vertebrates and4-day old rat (Rattus norvegi-cus). A Representative record-ings of whole cell current/voltage (I/V) relations after theopening of ATP-sensitivechannels was induced. B Mean(€SEM) conductance of theoutward current, measured be-tween –40 and 0 mV, in thesame species. The results arepooled data from differenttreatments that induced theopening of KATP channels: n isthe number of myocytes exam-ined. Dissimilar letters indicatesignificant differences betweenspecies

Fig. 3 The linear current in-duced in metabolically com-promised myocytes wasblocked partially by 10 �Mglibenclamide. Upper row:representative experiments froma ventricular myocyte of thelizard L. vivipara, in whichIK,ATP was induced by perfusingthe cell with 4 mM ATP inpipette solution (CATP; left) orATP-free anoxia (AATP-free;right). Lower row: I/V relationsconstructed at the times corre-sponding to the letters in theupper row

764

from the pipette (CATP). In the common frog, alltreatments except ATP-supplemented control (CATP)induced IK,ATP. In w.a. carp, anoxia or metabolic inhibi-tion were required to induce IK,ATP. Provision of ATPthrough the pipette could not prevent the opening of theKATP channels in these animals when cellular ATPproduction was compromised (AATP, MIATP). In the restof the animals, supplementation of ATP through thepipette prevented the opening of KATP channels both inanoxic (AATP) and metabolically inhibited myocytes(MIATP). w.a. Rainbow trout were resistant to all attemptsto induce a glibenclamide-sensitive current and this wasstriking exception among the animals studied. Neitherprolonged anoxia (39 min) (AATP-free; n=14) nor complete

metabolic inhibition (8 min) (MIATP-free; n=4) inducedIK,ATP. The same treatments triggered the IK,ATP in c.a.rainbow trout in less than 8 and 4 min, respectively.

IK,ATP amplitude

The density and conductance of IK,ATP were higher in theterrestrial (neonatal rat, European common lizard andcommon frog) than in the aquatic species (clawed frog,crucian carp and rainbow trout) (P<0.001, Figs. 1 and 2).The amplitude of the current did not differ significantlybetween the treatments used to induce IK,ATP, with theexception of that in the common frog. Therefore, once the

Fig. 4 Openers of ATP-sensitive K+ channels, cromakalim (Crom,10 �M) and pinacidil (Pinac, 10 �M), are ineffective in ventricularmyocytes of ectothermic vertebrates. Myocytes were stimulated

with voltage ramp pulses (see Fig. 1) at 0.2 Hz and exposed to theopeners for 3–20 min. Comparisons are with control conditions.Means€SEM; n=3–9 myocytes

765

channels were opened, the density of the current wasapproximately the same regardless of how it was induced.However, the amplitude of IK,ATP for the common frogwas reduced approximately by half (P<0.027) when ATPwas provided through the pipette.

The induced current had a linear I/V relationship andwas inhibited partly by 10 �M glibenclamide (Fig. 3). Theextent of glibenclamide inhibition of IK,ATP depended onthe treatment used to induce IK,ATP and was maximally74€6.8% (range 56.2–83.5%) for the five ectotherms.Prolonged exposure to anoxia or metabolic inhibitionmade the current more resistant to the blocker as theaverage inhibition was only 46€9.4% (P<0.05%)

KATP channel openers

Specific activators of mammalian KATP channels, 10 �Mcromakalim and 10 �M pinacidil, did not induce IK,ATP inany of the ectothermic vertebrates (Fig. 4) even thoughmyocytes were exposed to the drugs for up to 20 min.Higher concentrations of the openers were tested in somepreparations without detecting IK,ATP. Instead, 50 and100 �M of both pinacidil and cromakalim partially andreversibly inhibited the background inward rectifier IK1.

An inward current induced by metabolic inhibition

Although IK,ATP was present in all species, the firstresponse that appeared under metabolic inhibition (MIATP,MIATP-free) was not IK,ATP but an large inward current that

merged within 1 min from the inflow of the inhibitors(Fig. 5). The depolarising current was smaller and themyocytes tolerated the ramp pulses better when the cells

Fig. 5 Complete metabolic inhibition (MIATP-free) induces a largeinward current in ventricular myocytes of ectothermic vertebrates.Myocytes were stimulated with voltage ramp pulses (see Fig. 1) at

0.2 Hz and subjected to metabolic inhibition (0.1 mMNa2S2O3+5 mM iodoacetic acid) for 1–3 min. Means€SEM; n=8–15 myocytes

Fig. 6 The depolarising inward current and IK,ATP can occursimultaneously but are independent entities in the ventricularmyocytes of ectothermic vertebrates. A representative experimentfrom the clawed frog X. laevis. The myocyte was stimulated byvoltage ramp pulses (see Fig. 1) at 0.2 Hz and currents induced bycomplete metabolic (MIATP-free). Under control conditions (A) atypical I/V relation dominated by the background inward rectifier(IK1) was recorded. Soon after (<1 min) metabolic inhibition (B) thedepolarising inward current appeared without any sign of IK,ATP.Later (7 min, C) an outwards current appeared, suggestinginduction of IK,ATP. The outward current was blocked by 10 �Mglibenclamide (D). When all the drugs were washed out (E), thedepolarising current disappeared but IK,ATP remained. Similarresults were obtained in all ectothermic vertebrates

766

were perfused with ATP-supplemented pipette solution(MIATP) (results not shown). In the current-clamp mode,strong depolarisation of the SL was evident, but the cellstolerated it well for up to 20 min providing they were notstimulated to contract (results not shown). The depolar-ising inward current and IK,ATP were completely inde-pendent (Fig. 6). For example, when the myocytes of theclawed frog were inhibited metabolically (MIATP-free), thedepolarising current was the only response that appearedduring the first 7 min. When the inhibitors were washedout, the depolarising current disappeared and only thenormal-sized background inward rectifier, IK1, was pres-ent. In contrast, if the treatment was repeated for a longertime (>7 min), a glibenclamide-sensitive outward currentalso appeared, and upon washout the depolarising currentagain disappeared, whereas a prominent IK,ATP remained.Even this maximally developed IK,ATP was unable tooverride the large depolarising current, which appearedon reapplication of the metabolic inhibitors.

Discussion

The ATP-sensitive K+ current

We have shown here that in cardiac myocytes from awide range of ectothermic vertebrates, a prominent IK,ATPcan be expressed under energy limiting conditions.However, there were equally prominent differences inthe amplitude of IK,ATP and in the opening sensitivity ofthe KATP channels among air- and water-breathingspecies. Generally, IK,ATP was much larger in terrestrialand semi-aquatic vertebrates (rat, European commonlizard and common frog) than in the aquatic species(clawed frog, carp and trout). The density of IK,ATP washighest in ventricular myocytes of the neonatal rat, andthe values of the present study agree well with reportedIK,ATP densities [48]. Likewise, the amplitude of the IK,ATPmeasured here for the common frog was comparable tothat in another frog, Rana esculenta [32]. To ourknowledge the present measurements of the density ofIK,ATP are the first for any lizard species, crucian carp orrainbow trout. The present results indicate that the IK,ATPis much larger in terrestrial lizard than in either of the twofish species.

The resting membrane potential (RMP) of the cardiacmyocytes is maintained by IK1, while repolarisation of theAP is effected by IK1, various delayed rectifier K+ currents(IK) and a transient outward current (Ito) [20, 42]. Thehypoxic protection of the heart by KATP channels is basedon their ability to increase K+ efflux and thereby shortenthe duration of the AP and prevent depolarisation of themembrane. Clearly, the larger IK,ATP in terrestrial verte-brates provides better means of regulating the duration ofAP and RMP than in aquatic species. This finding isconsistent with the hypothesis that terrestrial species aremore dependent on local, cellular protection mechanismsthan aquatic animals that can sense environmental [O2]and therefore quickly alleviate hypoxic insults by sys-

temic regulation of the ventilation pattern or by improv-ing O2 extraction from water by adjusting ventilation-circulation synchrony with the bradycardic reflex [35]. Ifextraction of O2 from water fails, the aquatic vertebratesobtain only limited protection from the opening of theKATP channels. It should be remembered, however, thatectothermic vertebrates obtain the major part of theircardiac energy from carbohydrates, which they canexploit with the aid of their relatively powerful anaerobicmetabolism [10].

The KATP channels provide cardiac protection duringhypoxia and ischaemia by virtue of the direct couplingbetween channel opening and myocyte energy balance.The various procedures employed to impair cellular ATPproduction and availability (CATP-free) suggest that theKATP channels are more easily opened in the hearts ofterrestrial than aquatic vertebrates. In accordance withearlier studies, a few minutes of anoxia were sufficient toinduce IK,ATP in rat cardiac myocytes [21, 38, 39, 48, 49].In the completely terrestrial European common lizard, alltreatments – including the control (CATP) – inducedIK,ATP, while in amphibians and fish partial or completemetabolic blockade was needed. These results suggestthat in terrestrial air-breathing ectotherms, IK,ATP is notonly larger but is also more easily induced. This might berelated to the high risks of acute hypoxia to which theterrestrial vertebrates are exposed in the absence ofexternal O2 sensing.

With respect to the two amphibians, IK,ATP in the semi-aquatic common frog was much more easily induced thanin the aquatic clawed frog. It should be also noted that innorthern latitudes the common frog is exposed regularlyto winter hypoxia, unlike the tropical clawed frog. Fromthe two fish species the anoxia-tolerant crucian carpshowed greater sensitivity for the opening of KATPchannels than the hypoxia-sensitive rainbow trout. Thesefindings are in accordance with “hypoxia risk-hypothe-sis”, in that the crucian carp, in its natural environment ofsmall seasonally anoxic ponds, is exposed regularly tohypoxia and/or anoxia, while rainbow trout prefer streamsand other bodies of water with a high O2 content.Interestingly, the isolated ventricular myocytes of thecrucian carp heart did not tolerate metabolic inhibition aswell as those of the rainbow trout, which is in completedisagreement with the relative hypoxia/anoxia toleranceof the species. The reason for this difference is obscurebut might be associated with the anoxic depolarisation ofthe membrane potential (see below).

In ventricular myocytes of rainbow trout acclimated to17 �C, no IK,ATP was found under any experimentalconditions. This could be due to the absence of KATPchannels or failure of channel opening. It is unlikely thatanoxia (AATP-free) or complete metabolic inhibition(MIATP-free) did not deplete cellular ATP levels to thethreshold value of the KATP channel opening as theyinduced the depolarising inward current. Thus, it is morelikely that KATP channels are not expressed in the w.a.trout. The very large IK1 with a clearly discernibleoutwardly directed component at the plateau voltage

767

range of the cardiac AP (between –50 and –10 mV)(Fig. 4) may alleviate the need for the repolarising IK,ATPin the w.a. trout. The density of the IK1 decreases,however, strongly in anoxia and metabolic inhibition andtherefore reduces the repolarising power in energy-limitedcardiac myocytes. A similar reduction of IK1 by IAApoisoning has been described in ventricular myocytes ofthe neonatal rat [48]. On the other hand, in hypoxia, thew.a. trout can activate the adenosine-sensitive K+ current,IK,Ado, which is not expressed in its c.a. species mates [1].Thus, the hypoxic defence mechanisms seem to betemperature dependent and might involve different K+

channels in w.a. (IK,Ado) and c.a. (IK,ATP) rainbow trout.The existence of KATP channels in ventricular myo-

cytes of goldfish (C. auratus), a close relative of thecrucian carp, has been implied on the basis of single-channel recordings and prolongation of AP by glibencla-mide [15]. The IK,ATP of the goldfish heart is expressedmainly in c.a. fish and it was insensitive to 2 mM ATP.Surprisingly, the channels were open under normoxicconditions [15]. The identity of the current is, however,uncertain as it was recorded under Mg2+-free conditions,which are known to remove the rectification of thebackground inward rectifier, IK1 [8]. Indeed, the presentfinding that the density of the IK1 is higher in c.a. than inw.a. carp is consistent with the higher open-state prob-ability of the K+ current recorded in the c.a. goldfish [15].Furthermore, if the KATP channels were open in normoxia,a clearly discernable outward K+ current should appear inthe whole-cell recordings. Such a current has not beenfound in either crucian carp or rainbow trout cardiacmyocytes ([45, 46], present study).

Native KATP channels are heteromultimers of thesulphonylurea receptor and Kir proteins [2, 19, 30, 34,43], whereby the former mediates the effects of inhibitorsand openers on the KATP channel. Unexpectedly, cro-makalim (10 �M) and pinacidil (10 �M) did not inducethe opening of the KATP channels in any of the ectother-mic vertebrates, suggesting the possibility that high-affinity binding sites for pinacidil and cromakalim mightbe absent in the sulphonylurea receptors of these species.This is not, however, supported by the finding that IK,ATPis induced by 1 �M cromakalim in cardiac myocytes ofthe frog R. esculenta [33]. We tested also 50 and 100 �Mcromakalim and pinacidil, but even these high openerconcentrations did not induce IK,ATP in the teleosts or inthe clawed frog. The effect of openers in mammals isreduced strongly by low temperatures [26] and theirineffectiveness in ectotherms might therefore be due toconformational changes at low experimental tempera-tures, which are not unusual for the animals studied herebut are lower than those typically used with mammaliancells and for the earlier study with the frog.

Binding studies have demonstrated the presence of[3H]glibenclamide binding sites in amphibian, reptilian,avian and mammalian hearts, but not in fish hearts [28].In the present experiments, glibenclamide strongly inhib-ited IK,ATP in carp and trout ventricular myocytes, thusindicating the presence of glibenclamide receptors also in

fish hearts. Glibenclamide’s inhibition of the IK,ATP wasless under conditions in which myocyte ATP productionwas prevented completely. The mechanism for thereduced potency of glibenclamide in severely metaboli-cally compromised cells remains unclear, but a similareffect has been observed in ventricular myocytes of theneonatal rat heart [48].

The inward current induced by metabolic inhibition

Although the present study was designed to investigatethe amplitude and opening sensitivity of KATP channels inectothermic vertebrates, the first current induced byanoxia and metabolic inhibition was a large inwardcurrent, the magnitude of which far exceeded that of theIK,ATP. The depolarizing current was not due to the loss ofthe gigaohm seal, as it was non-linear, reversible andpartially blocked by 2 mM Ni2+. This current has beenrecorded previously in anoxic ventricular myocytes of thecrucian carp heart [45], and the present study extendsthese findings to a range of ectothermic vertebrates. Inview of its sensitivity to 2 mM Ni2+, the current has beenascribed to the activity of Na+/Ca2+ exchanger (NCX)[45]. The current, however, rectifies strongly in theinward direction at positive voltages (no outward current),a property not consistent with the voltage dependence ofNCX unless metabolic inhibition were to block selective-ly the reverse mode of NCX. Although there is someevidence that hypoxia and anoxia inhibit preferentiallythe reverse mode of NCX in guinea-pig cardiac myocytes[29], it is unlikely that the current is generated by theNCX since 5 mM EGTA, present in the pipette solution,should have buffered free intracellular Ca2+ to levels thatwould have prevented the forwards mode (inward current)of the NCX. We have not yet examined the contributionof non-specific cation channels, some of which areactivated by metabolic inhibition, to the depolarisingcurrent of ectothermic vertebrates [22, 25, 47]. Thesecurrents have, however, linear I/V relations with aprominent outward component at positive voltages andthus differ clearly from the current recorded here inectotherms. The complex voltage dependence of thecurrent suggests that it might include more than onecurrent component, but this issue cannot be solved on thebasis of the current knowledge. The physiological con-sequences and significance of this strong depolarisingcurrent remain to be elucidated.

That the depolarising current readily overwhelmed theIK,ATP in anoxia (AATP) is further indicated by theprolongation of AP and depolarisation of RMP in cruciancarp ventricular myocytes [45]. In other studies inmammals in which whole cell currents were recordedfor long time in anoxia [21, 38, 39] or glycolyticinhibition [48, 49], no such depolarising current wasobserved. Thus, the net response of ectothermic hearts tocomplete metabolic inhibition, consisting of SL depolar-isation and AP prolongation, is completely opposite to theshortening of AP and hyperpolarisation of RMP in

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mammals [4, 39]. Therefore, it seems that the relativelysmall IK,ATP of ectothermic vertebrates can providecardioprotection only when energy production of cardiacmyocytes is maintained partially.

The present experiments suggest that the magnitude ofIK,ATP and the opening sensitivity of the KATP channels arespecies specific and vary depending on the mode ofrespiration and environmental conditions to which theanimal has adapted, i.e. that they correlate with the risk ofacute metabolic failure of cardiac myocytes. Clarificationof the coupling between the metabolic state of thecardiomyocyte and the opening of the KATP channels inectothermic vertebrates would cast light on the evolu-tionary presses that have resulted in the development ofthe powerful IK,ATP in endotherms.

Acknowledgements This study was supported by the Academy ofFinland (project 53481). We would like to thank the Kontiolahtifish farm for donating the trout

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