THE CONTROL OF BLOOD PRESSURE DURING EXTERNAL HYPERCAPNIA IN THE RAINBOW TROUT (ONCORHYNCHUS MYKISS)

The Journl of Experimentl Biology, 77 9 (999) Printed in Gret Britin The Compny of Biologists Limited 999 JEB9 77 THE CONTROL OF BLOOD PRESSURE DURING EXTERNAL HYPERCAPNIA IN THE RAINBOW TROUT (ONCORHYNCHUS MYKISS) S. F. PERRY,, R. FRITSCHE, T. M. HOAGLAND, D. W. DUFF AND K. R. OLSON Deprtment of Biology, University of Ottw, Mrie Curie, Ottw, Ontrio, Cnd KN 6N, Deprtment of Zoophysiology, Göteorg University, Medicinregtn 8A, Box 6, Göteorg, Sweden, Indin University School of Medicine, South Bend Center, University of Notre Dme, Notre Dme, IN 66-67, USA nd Deprtment of Biology, Indin University t South Bend, South Bend, IN 66, USA e-mil: sfperry@science.uottw.c Accepted My; pulished on WWW 9 July 999 Adult freshwter rinow trout (Oncorhynchus mykiss) were exposed cutely (pproximtely min) in stepwise mnner to incresing levels of environmentl cron dioxide rnging etween.7 nd 9. mmhg (.. kp). Experiments were performed to exmine, for the first time, the influence of hypercpnic cidosis on spects of crdiovsculr physiology including lood pressure, crdic output nd vsculr resistnce. Fish displyed dose (wter CO prtil pressure) -dependent increses in ventrl ortic ( 9 %) nd dorsl ortic (7 %) lood pressures tht reflected mrked increses in systemic vsculr resistnce (6 78 %); rnchil vsculr resistnce ws unffected y hypercpni. At the highest level of hypercpni (9. mmhg), centrl venous pressure ws significntly elevted y %. Although crdic output remined constnt, hert rte ws significntly lowered y 7 ets min t the two highest levels of hypercpni. To determine whether the crdiovsculr responses to hypercpni were eing lunted y the stepwise increse in externl P CO, seprte group of fish ws exposed directly to single step of hypercpni (wter P CO 8. mmhg). The crdiovsculr responses were similr to those exhiited y the more grdully exposed fish except tht centrl venous pressure did not increse nd the extent of the rdycrdi ws greter ( ets min ). After confirming the effectiveness of yohimine in locking the vsoconstrictory α-drenoreceptors of the systemic vsculture, this ntgonist ws used s tool to Summry ssess the importnce of α-drenoreceptor stimultion in promoting the crdiovsculr responses during hypercpni. Prior tretment of fish with yohimine prevented the incresed lood pressures nd systemic vsculr resistnce during hypercpni ut did not influence the CO -induced rdycrdi. Plsm levels of ctecholmines did not chnge during hypercpni, nd therefore the stimultion of the systemic α- drenoreceptors presumly reflected incresed sympthetic nerve ctivity. To determine whether the crdiovsculr chnges elicited y hypercpni were relted to cidosis-induced hypoxemi, fish were exposed to hypoxi in stepwise mnner (wter P O 6 mmhg). The crdiovsculr responses to hypoxi were mrkedly different from those to hypercpni nd consisted of pronounced increses in systemic nd rnchil vsculr resistnce, ut only t the most severe level of hypoxi; ventrl nd dorsl ortic pressures were unffected. The differences etween the responses to hypercpni nd hypoxi, coupled with the smller reductions in lood oxygen content during hypercpni, support the hypothesis tht the crdiovsculr responses to CO re direct nd re unrelted to hypoxemi. Key words: Oncorhynchus mykiss, hypercpni, lood pressure, systemic resistnce, gill resistnce, hert, ctecholmine, hypoxi, crdic output, ventiltion. Introduction Environmentl hypercpni, n elevtion of mient P CO, is exploited routinely in studies of fish physiology s n nesthetic (Yoshikw et l., 99; Bernier nd Rndll, 998) or s tool to elucidte mechnisms of cid se regultion (Lloyd nd White, 967; Cmeron nd Rndll, 97; Cmeron, 976), ionic lnce (Eddy et l., 979; Perry et l., 987; McKenzie nd Perry, 997), respirtory gs trnsfer (Thoms et l., 99), lood gs trnsport (Eddy, 976; Eddy et l., 977; Perry nd Kinked, 989), metolism (Wlsh et l., 988; Perry et l., 988; Mommsen et l., 988) or ventiltory control (Jnssen nd Rndll, 97). In mny instnces, the physiologicl effects of hypercpni in teleosts re ttriuted to hypoxemi rising indirectly from Bohr nd Root effects. For exmple, the increses in ventiltion during moderte hypercpni in teleost fish hve trditionlly een linked to depression of lood O content rther thn to ny

78 S. F. PERRY AND OTHERS specific effect of CO or cidosis (Rndll, 98; Smith nd Jones, 98). Historiclly, the presence nd/or importnce of CO /H + chemoreceptors in piscine ventiltory control hve een lrgely discounted. In recent yers, however, growing ody of evidence hs ccumulted implicting specific role for CO /H + in the stimultion of rething in elsmornchs nd teleosts (for reviews, see Perry nd Wood, 989; Milsom, 99,). These results revel the presence of centrl nd/or peripherl CO /H + chemoreceptors in fish tht presumly function in conjunction with the well-studied O chemoreceptors (for review, see Fritsche nd Nilsson, 99). In ddition to their effects on ventiltion, O chemoreceptors re lso linked to severl crdiovsculr reflex djustments tht re medited y the neuronl (sympthetic nd prsympthetic nerves) nd humorl (circulting ctecholmines) components of the utonomic nervous system (Fritsche nd Nilsson, 99; Bushnell nd Jones, 99; Frrell, 99; Olson, 998). Although reltively few species hve een exmined, generlised responses to mient hypoxi include n elevtion of lood pressure, incresed systemic vsculr resistnce nd rdycrdi (Stchell, 96; Holeton nd Rndll, 967; Piiper et l., 97; Wood nd Shelton, 98; Fritsche nd Nilsson, 989, 99; Fritsche, 99; Bushnell nd Brill, 99; Mxime et l., 99). Unlike the O chemoreceptors, potentil link etween CO /H + chemoreceptors nd crdiovsculr reflexes hs not een estlished in fish. Indeed, the effects of externl hypercpni on crdiovsculr function hve not yet een investigted in ny teleost. Thus, considering the recent impliction of CO in ventiltory control in fish nd the known dul role of hypoxi in eliciting ventiltory nd crdiovsculr djustments, the present study ws undertken to ssess the crdiovsculr responses of freshwter-dpted rinow trout to environmentl hypercpni nd to investigte the underlying control mechnisms. Mterils nd methods Experimentl nimls Rinow trout [Oncorhynchus mykiss (Wlum)] of either sex, weighing etween 66 nd 79 g (N=6; mss 6±6 g, men ± S.D.), were otined from locl htchery nd held indoors in lrge (volume l) fireglss tnks supplied with flowing, erted well wter. The temperture of the holding nd experimentl tnks vried etween nd C; the photoperiod mtched the sesonl light:drk cycle. Fish were fed dily to stition with commercil trout diet (Purin), ut were not fed for 8 h prior to experimenttion. Surgicl procedures Mesurement of crdiovsculr nd ventiltory vriles Fish were nesthetised in : (w/v) solution of enzocine (ethyl-p-minoenzote) kept t C. After the cesstion of rething movements, the fish were trnsferred to n operting tle nd the gills were irrigted with : solution of enzocine ( C) throughout surgery. A polyethylene cnnul (Cly Adms, PE 6;. mm i.d.,.7 mm o.d.) ws implnted into the uccl cvity to fcilitte mesurement of inspired wter P O, P CO nd ph. To permit mesurement of dorsl ortic lood pressure (P DA ), drug injections nd lood smpling, polyethylene cnnul (Cly Adms, PE 6;.76 mm i.d.,. mm o.d.) ws implnted into the dorsl ort vi percutneous puncture of the roof of the uccl cvity (Olson et l., 997). A smll (pproximtely cm) ventrl incision ws mde to expose the pericrdil cvity, nd the pericrdium ws dissected to expose the ulus rteriosus. To llow mesurement of centrl venous pressure (P VEN ), non-occlusive silicone cnnul (. mm i.d.,.9 mm o.d.) ws implnted into the right horn of the ductus of Cuvier nd secured using cynocrylte glue (Olson et l., 997). The cnnul ws then ttched to length of polyethylene tuing (Cly Adms, PE 9;.86 mm i.d.,.7 mm o.d.) filled with heprinised ( i.u. ml sodium heprin) sline (.9 % NCl, w/v). After clmping the ventriculo-ulr junction, the ulus rteriosus ws cnnulted nd secured using protocol similr to tht used for the ductus (Olson et l., 997). The pressure recorded in the ulus rteriosus is referred to throughout this pper s ventrl ortic pressure (P VA ). Upon removl of the clmp, S or S ultrsonic flow proe (Trnsonics Systems Inc., Ithc, NY, USA) ws plced non-occlusively round the ulus to enle the mesurement of crdic output (V. ). The incision ws closed with sutures nd seled with cynocrylte gel, nd the ulus cnnul nd flow proe cles were secured to the ventrl skin using sutures. Smll ( cm ) rss pltes were stitched to the externl surfce of ech operculum to llow the mesurement of ventiltion mplitude using n impednce converter (Peyrud nd Ferret-Bouin, 96). After surgery, the fish were revived nd llowed to recover for h while constrined in open-ended individul lck tues ( cm dimeter) suspended in holding tnks supplied with erted well wter. Mesurement of lood respirtory vriles Fish were nesthetised (see ove), nd polyethylene cnnul (PE 6) ws inserted into the dorsl ort. The cudl vein ws cnnulted using polyethylene tuing (PE ) filled with heprinised ( i.u. ml sodium heprin) sline (.9 % NCl). Briefly, lterl incision ws mde t the level of the cudl peduncle to expose the heml rch. The cudl vein ws cnnulted percutneously, the wound ws closed, nd the cnnul ws secured to the ody wll using silk ligtures. This llowed n extrcorporel circuit to e initited in which the rteril dorsl ortic lood ws pumped continuously over series of electrodes (ph, P O, P CO ) nd then returned to the fish vi the cudl vein cnnul (Thoms, 99). After surgery, the fish were revived nd llowed to recover for h while constrined in open-ended individul lck tues ( cm dimeter) suspended in holding tnks supplied with erted well wter. The O electrodes (Rdiometer, model E7-) were clirted y pumping (using the peristltic pump of the extrcorporel shunt) zero solution ( % sodium sulphite) or

Crdiovsculr effects of CO in trout 79 ir-sturted wter continuously through the electrode smple comprtments until stle redings were recorded. The CO electrodes (Rdiometer, model E7-) were clirted in similr mnner using mixtures of. % nd. % CO in ir provided y gs-mixing flowmeter. The ph electrode (Metrohm, model 6..) ws clirted using precision uffers. The CO, O nd ph electrodes were clirted prior to ech experiment. Immeditely prior to experimenttion, the extrcorporel shunt ws rinsed for min with solution of sodium heprin ( i.u. ml in sline) to prevent lood from clotting in the tuing nd electrode chmers. Experimentl protocol Mesurement of crdiovsculr nd ventiltory vriles The dorsl ortic, ductus nd ulus cnnule were flushed with heprinised sline ( i.u. ml ) to prevent clotting nd then connected to Argon (-7 A) disposle CDXPRESS pressure trnsducers preclirted ginst sttic column of wter. Anlog lood pressure signls were mesured using Hewlett Pckrd (78A) ptient monitors. Crdic output ws determined y ttching the ultrsonic flow proe to Trnsonic T6 dul-chnnel lood flowmeter. The flow proes were clirted in situ fter ech experiment y pumping (using peristltic pump) sline t known flow rtes into the hert of ded fish immersed in wter; sline nd wter tempertures were mintined t C. Operculr impednce chnges were monitored using custom-uilt impednce converter nd mplifier nd were converted to liner operculr deflections (in cm) fter pproprite clirtion. Wter P CO nd P O were mesured y continuous pumping of inspired wter from the uccl cnnul over Rdiometer CO nd O electrodes housed within thermosttted cuvettes nd connected to PHM 7 cid se nlyser (Rdiometer). In few cses, wter ph ws monitored using ph electrode (Metrohm) housed in thermosttted chmer. The nlog outputs from the impednce converter nd cid se nlyser were converted to digitl signls y interfcing with dt-cquisition system (Biopc Systems Inc.) using Acknowledge dt-cquisition softwre (smpling rte set t Hz) nd 86 PC. Crdiovsculr signls (lood pressure nd crdic output) were converted to digitl dt using n A/D converter nd LTech Noteook softwre using second 86 PC. Smpling frequency ws Hz; s running verges were compiled. The timing of dt smpling from the two dt-cquisition systems ws synchronised. Thus, continuous dt recordings were otined for mss-specific crdic output (V. ), hert rte (fh; utomtic rte clcultion from the pulstile crdic output trce), crdic stroke volume (V S ; V. /fh), ventiltion frequency (fg; utomtic rte clcultion from the rw impednce trce), operculr displcement (the difference etween mximum nd minimum impednce vlues), men lood pressures, rnchil vsculr resistnce [R g ; (men P VA minus men P DA )/V. ] nd systemic vsculr resistnce [R s ; (men P DA minus P VEN )/V. ]. In some instnces, mesurements of P VEN were not otined owing to cnnul filure; in these cses, R s ws clculted s men P DA /V.. Four seprte experimentl series were performed. Series I: stepwise increses in externl P CO. When stle seline hd een estlished, experiments commenced with period of normocpni followed y three phses of progressively incresing hypercpni nd recovery. During normocpni, the inflowing wter (>. l min ) ws provided from gs equilirtion column gssed vigorously with ir. After min period of recording, pre-hypercpni lood smple (.6 ml) ws withdrwn from the dorsl ortic cnnul. After centrifugtion ( g), plsm (> µl) ws dded to µl of mmol l sodium sulphite, frozen, kept on dry ice for pproximtely weeks nd then shipped while frozen to the University of Ottw for storge t 8 C until susequent nlysis of ctecholmine levels. Hypercpni ws initited y gssing (gs flow. l min ) the equilirtion column tht provided wter to the fish tues with mixtures of CO in ir rnging etween. nd. %. The mixtures were provided y GF-/MP gs-mixing flowmeter (Cmeron Instrument Compny, Port Arnss, TX, USA). These levels of CO elicited stle levels of inspired P CO of., 6. nd 9. mmhg within min. Additionl lood smples were withdrwn nd processed t the conclusion of ech step of hypercpni. After exposure to the finl step of environmentl hypercpni, fish were returned to normocpnic conditions nd llowed to recover for further min, t which time finl lood smple ws tken. Plsm ctecholmine levels (drenline nd nordrenline) were determined on lumin-extrcted plsm smples (. ml) using high-performnce liquid chromtogrphy (HPLC) with electrochemicl detection (Woodwrd, 98). The extrcted smples were pssed through n Ultrtechsphere ODS-C8 µm column (HPLC Technology Ltd), using ctecholmine nd metnephrine moile phse (Chromosystems, Munich, Germny). Concentrtions were clculted reltive to pproprite stndrds nd with,-dihydroxyenzylmine hydroromide (DHBA) s n internl stndrd in ll determintions. Series II: single increses in externl P CO. Becuse the fish in series I were eing exposed to incrementl increses in externl P CO, there ws possiility tht the crdiovsculr responses to the higher levels of CO (i.e. the second nd third increments) were eing lunted s result of hitution. Therefore, in series II, fish were exposed to single step increse in externl P CO designed to mtch the finl level of hypercpni chieved in series I (9. mmhg). In prctice, however, the ctul P CO reched in the single-step experiment (8.±.8 mmhg) ws slightly lower thn in series I. The experimentl protocol ws identicl to tht of series I in ll other respects except tht lood smples were not withdrwn for plsm ctecholmine nlysis. Series III: the effects of α-drenoceptor lockde on the crdiovsculr responses to hypercpni. The competitive α- drenoceptor ntgonist yohimine (Nichols nd Ruffolo, 99) ws selected for these experiments ecuse previous studies hve estlished it to e relile nd well-tolerted locker of ctecholmine-medited systemic vsoconstriction in rinow

8 S. F. PERRY AND OTHERS trout (Holmgren nd Nilsson, 97; Wood, 976; Wood nd Shelton, 98,). In the present study, detiled preliminry experiments were performed to determine the correct dosge of yohimine nd the durtion of its α-drenoceptor lockde. In these experiments, fish were injected vi the dorsl ortic cnnul with nmol kg drenline ( ml kg ) (L-drenline itrtrte slt; Sigm), nd the effects on crdiovsculr vriles were recorded for min or until vlues hd returned to seline levels. Fish were then given olus intr-rteril injection of yohimine t doses rnging etween nd mgkg. The effects on seline R s were recorded, nd fter new stedy stte hd een reched (usully within min), second injection of drenline ws dministered. The lowest dose of yohimine consistently to lock the drenline-induced rise in R s ws mg kg, nd this dose ws therefore used in ll susequent experiments requiring α-drenoceptor lockde. To ssess the effects of α-drenoceptor lockde on the crdiovsculr responses to hypercpni, fish were pretreted with mg kg of yohimine nd, fter min, sujected to single step increse in wter P CO (to 7.±. mmhg) tht pproximtely mtched the finl level chieved in series II. All other spects of the protocol were identicl to those in series II. Series IV: stepwise decreses in externl P O. To ssess the potentil contriution of cidosis-induced hypoxemi to the crdiovsculr responses to hypercpni, fish were exposed to stepwise hypoxi. Once stle seline vlue hd een estlished, experiments commenced with period of normoxi followed y three episodes of progressively incresing hypoxi, nd recovery. Hypoxi ws initited y gssing (. l min ) the equilirtion column tht provided wter to the fish tues with mixtures of N in ir rnging etween % nd % N. These mixtures were provided y GF-/MP gs-mixing flowmeter (Cmeron Instrument Compny, Port Arnss, TX, USA). These levels of hypoxi were chosen ecuse they elicited rnge of hypoxemi spnning the reductions in lood O concentrtion chieved during the hypercpni exposures. In prctice, this protocol elicited stle levels of inspired P O of, 9 nd 6 mmhg within min. Blood smples (.6 ml) were withdrwn nd processed s in series I prior to hypoxi, t the conclusion of ech step of hypoxi nd fter return to normoxi. Mesurement of lood respirtory vriles To relte crdiovsculr chnges elicited y externl hypercpni or hypoxi to chnging lood respirtory sttus, rteril lood P O (P O ), P CO (P CO ) nd ph (ph) were monitored in seprte experimentl series during incrementl nd single-step hypercpni (protocols identicl to series I nd II) nd incrementl hypoxi (protocol identicl to series IV). Blood respirtory sttus ws ssessed continuously using n extrcorporel lood shunt (see ove). Crdiovsculr mesurements were not mde during these experiments nor ws ny lood smpled. Sttisticl nlyses nd presenttion of dt Dt re presented s mens ± stndrd error of the men (S.E.M.). The dt were nlysed sttisticlly y prmetric repeted-mesures one-wy nlysis of vrince (ANOVA). When the ANOVA indicted significnt difference, posthoc multiple-comprison test (Bonferroni t-test) ws used to identify dt points tht were significntly different from ech other (ll pirwise multiple comprison used in stepwise experiments) or from single control point (i.e. the finl vlue of the pre-hypercpnic period in single-step experiments). When prmetric test ssumptions were violted, the dt were nlysed using Friedmn s repeted-mesures ANOVA on rnks followed y Dunn s ll pirwise multiple-comprison test or Dunnett s comprison with single control point. In experiments designed to vlidte the effectiveness of yohimine s n α-drenoceptor ntgonist, pre- nd post-drenline injection dt were nlysed using two-tiled pired Student s t-test; differences etween the control nd yohimine-treted fish were determined y ANOVA. All sttisticl tests, including determintions of normlity nd vrince, were performed using commercil softwre (SigmStt version.). The fiducil limit of significnce ws set t %. Results Crdiovsculr nd ventiltory responses to hypercpni Series I: stepwise increses in externl P CO Fish (N=) were exposed to three stepwise increses in mient P CO to chieve (within min) levels of hypercpni of.±., 6.±. nd 9.±. mmhg (Tle ). Wter ph Tle. The effects of stepwise increse in externl P CO on wter/lood gses nd ventiltion vriles in rinow trout Oncorhynchus mykiss Pw CO Pw O P CO P O fg V mp (mmhg) (mmhg) (mmhg) (mmhg) ph (min ) (cm) Pre.66±.6.7±.9,c.8±. 9.6±6.,c 7.8±. 9.6±.9.77±.9,d Step.±.9.±.8,c.99±. 7.±.7 7.76±. 89.±..9±.,c,d Step.99±.9 c 9.9±.9 6.±. c.±. 7.6±. c 87.7±..±.9,c Step 9.±.6 d 9.9±.9 9.±. d.6±. 7.±. d 87.±.8.7±.6,c Post.7±.8.±.,c.±.6.±.7,c 7.8±. 9.±..7±.9 d For ech vrile, vlues tht do not shre common letter re sttisticlly different from ech other (P<.). Pre, Post, pre- nd post-hypercpni recording periods; Pw CO, externl P CO ; Pw O, externl P O ; P CO, rteril P CO ; P O, rteril P O ; fg, gill ventiltion rte; V mp, ventiltory mmhg=. kp.

P Crdiovsculr effects of CO in trout 8 Pw CO (mmhg) 8 6 A c Pre Post d R s (mmhg ml min - kg - ) E,c,d,c,c,,c Pre Post Fig.. The effects of stepwise increses in externl P CO (Pw CO ) (A) on crdiovsculr vriles in rinow trout (Oncorhynchus mykiss) including (B) ventrl ortic lood pressure (P VA; N=8), (C) dorsl ortic lood pressure (P DA; N=), (D) centrl venous lood pressure (P VEN; N=9), (E) systemic vsculr resistnce (R s; N=), (F) gill vsculr resistnce (R g; N=8), (G) crdic output (V. ; N=) nd (H) hert rte (fh; N=). Pre nd Post refer to the pre- nd post-hypercpni recording periods; the numers denote the three discrete steps of incresing hypercpni (., 6. nd 9. mmhg). The dt re presented s mens + S.E.M.; vlues tht do not shre identicl letters re significntly different (P<.). mmhg=. kp. P VA (mmhg) P DA (mmhg) P VEN (mmhg) B C D c Pre Post c Pre Post Pre Post d d R g (mmhg ml min - kg - ) V (ml min - kg - ). fh (min - ) 8 6 F Pre Post G Pre Post H,c,d,c,d,c Pre Post (phw) ws monitored in two experiments nd verged 7.6 during normocpni nd 7.7, 7.6 nd 7.6 during the three steps of hypercpni, respectively. The effects on the mesured nd clculted crdiovsculr vriles re summrised in Fig.. P VA nd P DA were incresed t ll levels of hypercpni in P CO -dependent mnner (Fig. B,C). At the highest level of hypercpni, P VA nd P DA were incresed y 9 % nd %, respectively. P VEN ws incresed y %, ut only during the finl episode of hypercpni (Fig. D). Systemic resistnce ws lso incresed up to 78 % in P CO -dependent fshion, lthough the chnge ws not sttisticlly significnt t the lowest level of hypercpni (Fig. E); R g ws unffected y hypercpni (Fig. F). Although hert rte ws lowered during hypercpni (Fig. H), crdic output ws mintined (Fig. G) ecuse of significnt elevtion of stroke volume ( 6 %) during the ltter two periods of hypercpni (dt not shown). The ventiltory response to incrementl hypercpni ws extremely vrile nd, consequently, ventiltory mplitude (s determined y operculr displcement) ws sttisticlly elevted from pre-hypercpni vlues only during the finl stge of hypercpni (Tle ). Brething frequency ws unltered during hypercpni (Tle ). The effects of hypercpni on plsm levels of ctecholmines re summrised in Tle. The concentrtions of drenline or nordrenline were low nd constnt t ll levels of hypercpni. The totl ctecholmine levels (drenline plus nordrenline) in the plsm never exceeded nmol l. Series II: single increse in externl P CO Exposing rinow trout (N=9) to single increse in externl P CO (Pw CO ), designed to mtch the highest level of hypercpni chieved in step of series I, cused similr

8 S. F. PERRY AND OTHERS Tle. The effects of stepwise increse in externl P CO on plsm ctecholmine levels in rinow trout Oncorhynchus mykiss [Nor- [Totl Pw CO drenline] [Adrenline] ctecholmines] (mmhg) (nmol l ) (nmol l ) (nmol l ) Pre.66±.6.±..7±..±. Step.±.9.±..8±..±. Step.99±.9 c.±.7.8±..±.7 Step 9.±.6 d.±..±.6.±.9 Post.7±.8.±.8.6±.6.8±. For ech vrile, vlues tht do not shre common letter re sttisticlly different from ech other (P<.). Pre, Post, pre- nd post-hypercpni recording periods; Pw CO, externl P CO. mmhg=. kp. crdiovsculr responses (Fig. ). Two notle differences were tht P VEN did not increse (Fig. D) nd tht fh ws lowered to greter extent (Fig. H). Becuse of the high degree of vrince, pprent increses (pproximtely %) in crdic stroke volume were not sttisticlly significnt (P=.6; dt not shown). Ventiltory mplitude ws incresed, wheres rething frequency ws unchnged y hypercpni (Tle ). In two experiments in which phw ws mesured, it decresed from 7.6 to 7. during hypercpni. Series III: the effects of α-drenoceptor lockde on the crdiovsculr responses to hypercpni Initil experiments were designed to confirm the effectiveness of yohimine s n α-drenoceptor ntgonist in trout. The results of these experiments re depicted in Fig.. The injection of drenline into untreted fish cused expected increses in P VA, P DA, P VEN, R s nd V. ; R g, fh nd V S were unffected. Injection of yohimine ( mg kg ) cused lowering of seline R s (Fig. D) nd olished the pressor nd R s responses to drenline injection. Tretment of fish with yohimine resulted in n elevted V. during normocpni (Fig. G) resulting in n incresed V S (dt not shown) nd eliminted the pressor nd systemic resistnce increses normlly ssocited with hypercpni (Fig. ). However, the rdycrdi elicited y hypercpni ws not ffected y α-drenoceptor lockde (Fig. H). Series IV: stepwise decreses in externl P O Fish (N=7) were exposed to three stepwise decreses in mient P O to chieve (within min) levels of hypoxi of.±.6, 9.±. nd 6.9±.7 mmhg (Tle ). The effects of the stepwise hypoxi on the recorded nd clculted crdiovsculr vriles re illustrted in Fig.. Unlike in the hypercpnic fish, P VA (Fig. B) nd P DA (Fig. C) remined constnt during hypoxi, wheres P VEN ws elevted during the most severe stge of hypoxi (Fig. D). Systemic nd gill vsculr resistnces remined constnt t the two first levels of hypoxi, ut were significntly incresed during the finl episode (Fig. E,F). Crdic output nd fh decresed significntly during the finl stge of hypoxi (Fig. G,H); V S ws unffected (dt not shown). As in the hypercpni series, the ventiltory response to stepwise hypoxi exhiited high degree of vriility, nd ventiltory mplitude ws therefore sttisticlly elevted from Tle. The effects of single increse in externl P CO on wter/lood gses nd ventiltion vriles in rinow trout, Oncorhynchus mykiss Pw CO Pw O P CO P O fg V mp (mmhg) (mmhg) (mmhg) (mmhg) ph (min ) (cm) Pre.6±..±..7±. 8.±8. 7.9±. 87.7±..±. Hypercpni 7.98±.7.8±. 8.±.6.±. 7.9±.7 87.9±..±.7 Post.6±.6 9.8±..7±.7 6.±. 7.88±. 8.±.6.±.7 indictes significnt difference from the pre-hypercpni vlue (P<.). Arevitions re explined in Tle. mmhg=. kp. Tle. The effects of stepwise decrese in externl P O on wter/lood gses nd ventiltion vriles in rinow trout, Oncorhynchus mykiss Pw O Pw CO P CO P O fg V mp (mmhg) (mmhg) (mmhg) (mmhg) ph (min ) (cm) Pre.±..8±.7.±. 9.6±6. 7.9±.,c 96.9±..±.6,c Step.±.6.±.6.7±. 9.8±. 7.9±.,c 9.±6..9±.8,c Step 9.±. c.9±.6.98±.9 6.±.6 c 7.96±.,c 99.±.9.6±.,c Step 6.9±.7 d.7±..9±. 9.6±.8 d 7.87±. 9.9±.9.98±.8 Post.8±..±.7.6±. 7.±.9 7.78±. 9.±..±.6 c For ech vrile, vlues tht do not shre common letter re sttisticlly different from ech other (P<.). Arevitions re defined in Tle. mmhg=. kp.

P VEN (mmhg) P DA (mmhg) V (ml min - kg - ) P VA (mmhg) R g (mmhg ml min - kg - ) Pw CO (mmhg) R s (mmhg ml min - kg - ) Crdiovsculr effects of CO in trout 8 A E 8 6 B F C G Fig.. The effects of single exposure of rinow trout (Oncorhynchus mykiss) to hypercpni on (A) wter cron dioxide tension (Pw CO N=9), (B) ventrl ortic lood pressure (P VA, N=6), (C) dorsl ortic lood pressure (P DA, N=9), (D) centrl venous pressure (P VEN; N=8), (E) systemic vsculr resistnce (R s; N=9), (F) gill vsculr resistnce (R g; N=6), (G) crdic output (V. ; N=9) nd (H) hert rte (fh; N=9). The solid horizontl rs in ech pnel represent the durtion of the exposure to hypercpni. The doule-heded rrows indicte extended periods when sttisticlly significnt differences etween the pre-hypercpnic nd hypercpnic vlues occurred. The dt re presented s mens + S.E.M. An sterisk indictes specific points tht were sttisticlly different from the prehypercpnic vlues. mmhg=. kp. D Time (min). fh (min - ) 8 6 H Time (min) pre-hypoxi vlues only during the finl stge of hypoxi (Tle ). Brething frequency ws unltered during hypoxi (Tle ). The effects of hypoxi on plsm levels of ctecholmines re summrised in Tle. The circulting levels of drenline nd nordrenline remined constnt until the finl stge of hypoxi (Pw O =6 mmhg) ws reched, t which time levels of oth ctecholmines were significntly elevted. Blood respirtory vriles during hypercpni nd hypoxi Exposure of fish to stepwise hypercpni cused predictle Pw CO -dependent chnges in lood respirtory nd cid se sttus, including reduction in ph nd n increse in P CO (Tle ). P O ws incresed t the first level of hypercpni nd therefter remined constnt until the return to normocpni. However, during the single-step hypercpni experiment (series II), P O remined constnt (Tle ). During stepwise hypoxi, P O decresed in ccordnce with the fll in externl P O (Pw O ) (Tle ). Arteril ph ws elevted during the second stge of hypoxi (Pw O =9 mmhg) nd proly reflected the tendency for reduction in P CO during hyperventiltion (leit the chnges were not sttisticlly significnt). Discussion Crdiovsculr responses to environmentl hypercpni The effects of externl hypercpni on ventiltion nd lood cid se lnce re well documented in fish (e.g. Jnssen nd Rndll, 97; Thoms nd Le Ruz, 98; Thoms, 98; Perry et l., 987). The present study, however, is the first to investigte the effects of externl hypercpni on

P P 8 S. F. PERRY AND OTHERS P VA (mmhg) DA (mmhg) A B R g (mmhg ml min - kg - ) V (ml min - kg - ). E F Fig.. The effects of intr-rteril injections of drenline (Adr; nmol kg ) in rinow trout (Oncorhynchus mykiss) efore (filled columns) nd fter (open columns) tretment with yohimine ( mg kg ) on (A) ventrl ortic lood pressure (P VA, N=), (B) dorsl ortic lood pressure (P DA, N=7), (C) centrl venous pressure (P VEN; N=), (D) systemic vsculr resistnce (R s; N=7), (E) gill vsculr resistnce (R g; N=), (F) crdic output (V. ; N=7), (G) hert rte (fh; N=7) nd (H) crdic stroke volume (V S; N=7). The dt re presented s mens + S.E.M. An sterisk indictes sttisticlly significnt differences rising from drenline tretment; doule dgger indictes sttisticlly significnt differences from the pre-yohimine vlue (P<.). Pre, efore drenline tretment. mmhg=. kp. P VEN (mmhg) R s (mmhg ml min - kg - ) C D V S (ml kg - ) fh (min - ) 8 6..... G H crdiovsculr physiology in ny teleost. The results clerly demonstrted tht exposing trout to hypercpni cuses mrked crdiovsculr djustments consisting of elevted lood pressures nd rdycrdi. The origin of the increse in rteril lood pressures (P VA nd P DA ) ws lrge elevtion of systemic vsculr resistnce. In teleosts, systemic vsculr resistnce is controlled predominntly y the dul ctions of constrictory α- nd diltory β-drenoceptors (Nilsson, 98). In most species tht hve een exmined, the α-constrictory response predomintes, nd thus n increse in sympthetic nerve ctivity to the systemic vsculture or n elevtion of circulting ctecholmine levels generlly cuses n increse in systemic resistnce (Wood nd Shelton, 98,). In the present study, the increse in systemic resistnce during hypercpni ws clerly cused y the stimultion of α- drenoceptors. This ws estlished y compring the responses of untreted fish with those pre-treted with the α- drenoceptor ntgonist yohimine. Previous studies in fish hve demonstrted tht yohimine is le to olish drenergic increses in systemic resistnce in vivo (Wood nd Shelton, 98) nd in perfused trunk preprtions (Wood, 976) s well s eliminting drenergic increses in vsculr smooth muscle tension in vitro (Holmgren nd Nilsson, 97). Other conventionl α-lockers, such s phentolmine (Smith, 978) or phenoxyenzmine (Stevens et l., 97; Wood, 976; Xu nd Olson, 99), were not used in the present study ecuse of the difficulty of chieving complete lockde (phentolmine) or the requirement for multiple injections spnning severl hours (phenoxyenzmine). The

P Crdiovsculr effects of CO in trout 8 Fig.. The effects of single step increse in wter P CO (Pw CO ) (A) in rinow trout (Oncorhynchus mykiss) efore (filled columns) nd fter (open columns) tretment with yohimine ( mg kg ) on (B) ventrl ortic lood pressure (P VA, N=), (C) dorsl ortic lood pressure (P DA, N=8), (D) centrl venous pressure (P VEN; N=7), (E) systemic vsculr resistnce (R s; N=7), (F) gill vsculr resistnce (R g; N=), (G) crdic output (V. ; N=8) nd (H) hert rte (fh; N=7). The dt re presented s mens + S.E.M. An sterisk indictes sttisticlly significnt difference from the pre-hypercpni (Pre) vlue; doule dgger indictes sttisticlly significnt differences from the untreted (no yohimine) vlue (P<.). Hyp, hypercpni; Post, post-hypercpni. mmhg=. kp. Pw CO (mmhg) P VA (mmhg) P DA (mmhg) P VEN (mmhg) A 8 6 Pre HypPost B Pre Hyp Post C Pre Hyp Post D Pre Hyp Post Pre HypPost Pre Hyp Post Pre Hyp Post Pre Hyp Post R s (mmhg ml min - kg - ) fh (min - ) V (ml min - kg - ) R g (mmhg ml min - kg - ). 8 6 E Pre Hyp Post Pre Hyp Post F Pre Hyp Post Pre Hyp Post G Pre Hyp Post Pre Hyp Post H Pre Hyp Post Pre Hyp Post effectiveness of yohimine s n α-ntgonist ws verified in detiled series of preliminry experiments. Circulting ctecholmine levels versus sympthetic nerve ctivity Plsm ctecholmine levels did not increse t ny level of hypercpni (Tle ). Thus, the stimultion of systemic α-drenoceptors nd resultnt increse in R s during hypercpni were presumly cused exclusively y n increse in sympthetic nerve ctivity. Although previous studies hve demonstrted n elevtion of circulting ctecholmine levels during hypercpni in trout (Perry et l., 987, 989; Kinked et l., 99; Thoms et l., 99; Perry nd Gilmour, 996), the response is highly vrile nd pprently dependent upon the severity of hypercpni imposed on the fish. Severl uthors hve reported stle plsm ctecholmine levels in trout exposed to mild or moderte hypercpni (Kinked nd Perry, 99; Julio et l., 998). The widely different ctecholmine secretory responses to hypercpni in trout my reflect the extent of hypoxemi elicited y the respirtory cidosis (see Discussion in Julio et l., 998). In comprison with other studies, however, it is surprising tht plsm ctecholmine levels were not elevted t the most severe level of hypercpni (Pw CO =9 mmhg). This pprent lunting of the ctecholmine secretory response to hypercpni my reflect the fct tht the trout in this fcility re chroniclly cclimted to reltively high levels of Pw CO in the normocpnic holding wter (e.g. pproximtely.7 mmhg during pre-hypercpni mesurements).

P P 86 S. F. PERRY AND OTHERS Prior lockde of α-drenoceptors eliminted the pressor responses to hypercpni ut did not eliminte the rdycrdi. This indictes tht the rdycrdi ws direct consequence of the environmentl hypercpni nd not secondry rosttic reflex response. The origin of the rdycrdi ws not investigted in the present study, ut presumly involved n increse in prsympthetic (cholinergic) vgl tone (Frrell nd Jones, 99), which is the predominnt mechnism of controlling hert rte in most teleosts. Despite the significnt reductions in fh, crdic output ws mintined during hypercpni owing to simultneous increses in crdic stroke volume. In the present study, the fish were sujected to extensive crdiovsculr surgery. Previous studies hve shown tht similr surgery (Gmperl et l., 99) nd, in prticulr, opening of the pericrdium (Frrell et l., 988) cn influence crdic performnce. Thus, we cnnot exclude the possiility tht the crdic responses to hypercpni were underestimted in the present study. A modest (.8 mmhg), ut significnt, increse in P VEN ws oserved during the finl stge of stepwise exposure to hypercpni (Fig. ), wheres P VEN ws not significntly ffected y single exposure to nerly the sme Pw CO (lthough it lso ppered to increse slightly). The reson for this discrepncy is not cler, ut it my e explined y the different tretment regimes tht resulted in slightly more severe nd gretly prolonged hypercpni during stepwise tretment. Nevertheless, the increse in P VEN in the stepwise hypercpni experiments is consistent with previous oservtions of sympthetic control of venous tone in trout (Zhng et l., 998). Pw O (mmhg) 6 8 A c d Pre Post R s (mmhg ml min - kg - ) E Pre Post Fig.. The effects of stepwise decreses in externl P O (Pw O ) (A) on selected crdiovsculr vriles in rinow trout (Oncorhynchus mykiss) including (B) ventrl ortic lood pressure (P VA; N=6), (C) dorsl ortic lood pressure (P DA; N=6), (D) centrl venous lood pressure (P VEN; N=), (E) systemic vsculr resistnce (R s; N=6), (F) gill vsculr resistnce (R g; N=6), (G) crdic output (V. ; N=6) nd (H) hert rte (fh; N=6). Pre nd Post refer to the pre- nd posthypoxi recording periods; the numers denote the three discrete steps of decresing hypoxi (, 9 nd 6 mmhg). The dt re presented s mens + S.E.M.; vlues tht do not shre identicl letters re significntly different (P<.). mmhg=. kp. P VA (mmhg) P DA (mmhg) P VEN (mmhg) B C D Pre Post Pre Post,c,c,c Pre Post R g (mmhg ml min - kg - ) V (ml min - kg - ). fh (min - ) 8 6 F G H Pre Post,, Pre Post,, Pre Post

Crdiovsculr effects of CO in trout 87 Hemogloin O -sturtion (%) 8 6 6 8 P O (mmhg) Fig. 6. An in vitro oxygen equilirium curve for rinow trout lood (dt from Montpetit nd Perry, 998) showing the expected pproximte reductions in hemogloin oxygen-sturtion ssocited with the flling rteril lood P O (P O ) during the stepwise hypoxi experiments (series IV). The horizontl length of ech shded ox represents the men P O vlue ± S.E.M. prior to hypoxi nd during the three stges of incresing hypoxi. mmhg=. kp. Does hypoxemi contriute to the crdiovsculr effects of environmentl hypercpni? As result of Bohr nd Root effects, environmentl hypercpni cuses reduction in rteril lood O content. In trout, chnges in lood O content re elieved to ply n importnt role in the control of ventiltion during hypoxi nd hypercpni (Rndll, 98; Smith nd Jones, 98). Thus, we investigted the possile contriution of hypoxemi in promoting the crdiovsculr responses to elevted mient CO. The results clerly exclude significnt role for hypoxemi in mediting these responses to hypercpni in rinow trout. First, the crdiovsculr responses to Tle. The effects of stepwise decrese in externl P O on plsm ctecholmine levels in rinow trout Oncorhynchus mykiss [Nor- [Totl Pw O drenline] [Adrenline] ctecholmines] (mmhg) (nmol l ) (nmol l ) (nmol l ) Pre.±..±..±.7.±.8 Step.±.6.±..7±..±. Step 9.±..±..±..±. Step 6.9±.7 7.7±..±.7.±7. Post.8±..±..7±..±.7 indictes significnt difference from the pre-hypoxi vlue (P<.). Pre, Post, pre- nd post-hypercpni recording periods; Pw O, externl P O. mmhg=. kp. hypercpni were mrkedly different from the responses to hypoxi. Unlike in the hypercpnic fish, P DA nd P VA remined constnt t ll levels of hypoxi. Even when systemic resistnce ws incresed t the most severe level of hypoxi, rteril lood pressures did not rise, presumly s result of concomitnt decreses in fh nd V.. Furthermore, during hypoxi, R s nd R g oth incresed, wheres R g ws unffected during hypercpni. Second, nd perhps more importntly, the expected reduction in lood O content (pproximtely %; see Fig. 6) during the second stge of hypoxi (Pw O =9 mmhg) ws lmost certinly greter thn the degree of hypoxemi experienced t ny level of hypercpni. Although not mesured in the present study, previous experiments utilising similr levels of mient hypercpni hve reported reductions in lood O content of pproximtely % (Perry nd Gilmour, 996; Julio et l., 998). Thus, given the sence of ny crdiovsculr djustments during the second stge of hypoxi (predicted reduction in lood O content %), it is difficult to envisge contriuting role of hypoxemi during exposure to CO. Indeed, even t the most severe level of hypercpni, lood O content ws proly reduced y only %. Finlly, dorsl nd ventrl ortic pressor responses were initited t low levels of hypercpni (e.g. Pw CO =. mmhg). Such levels of hypercpni would hve negligile effects on lood O content considering the minor chnges in lood ph (from 7.8 to 7.76; Tle ). Externl versus internl CO /H + receptors nd possile direct ctions of CO /H + The presence of externl nd/or internl gill oxygen receptors is well estlished (Bmford, 97; Smith nd Jones, 978; Fritsche nd Nilsson, 989, 99; Burleson nd Smtresk, 99,). Stimultion of these receptors initites hyperventiltion s well s t lest two crdiovsculr reflexes, rdycrdi (Wood nd Shelton, 98; Smith nd Dvie, 98; Fritsche nd Nilsson, 989; Fritsche, 99; Sundin, 99) nd hypertension (Holeton nd Rndll, 967; Sunders nd Sutterlin, 97; Wood nd Shelton, 98; Fritsche nd Nilsson, 99). The exct mechnism(s) y which CO exerts its effects on ventiltion nd crdiovsculr function is not known, ut the results of the present study suggest the involvement of externl CO receptors linked to the utonomic nervous system. It seems most likely tht the primry crdiovsculr response (incresed systemic resistnce) ws initited y externl CO receptors ecuse increses in dorsl ortic pressure hve not een oserved in fish experiencing endogenous (hyperoxic) hypercpni (e.g. white sucker Ctostomus commersoni, Wilkes et l., 98; rinow trout, S. F. Perry, unpulished oservtions). CO nd H + could lso ffect the vsculr resistnce y direct ction on the lood vessels. However, this seems unlikely s the sole explntion given (i) the rpidity of the lood pressure response, (ii) the quick recovery of the response fter hypercpni nd (iii) the lockde of the response y yohimine. Furthermore, recent results otined from perfused trunk preprtion (J. McKendry nd S. F.

88 S. F. PERRY AND OTHERS Perry, unpulished dt) hve demonstrted tht incresing P CO (up to mmhg) does not cuse vsoconstriction of the systemic vsculture in rinow trout. The hyperventiltory response during hypercpni proly resulted from the specific effects of CO /H + on stimulting internl centrl or peripherl CO /H + chemoreceptors (Heisler et l., 988; Grhm et l., 99; Wood et l., 99; Kinked nd Perry, 99; Wood nd Munger, 99; Perry nd Gilmour, 996) s well s the indirect stimulting influence of hypercpnic hypoxemi (Smith nd Jones, 98). Further, we cnnot exclude the possile contriution of externl CO /H + receptors. Although the role of circulting ctecholmines in promoting hyperventiltory responses in fish is under dete (Rndll nd Tylor, 99; Perry et l., 99), the results of the present study (hyperventiltion in the sence of elevted ctecholmine levels) reinforce the view tht incresed levels of plsm ctecholmines re not prerequisite for hyperventiltion to occur (Kinked nd Perry, 99; Perry nd Gilmour, 996). Are the crdiovsculr responses to hypercpni physiologiclly significnt? During hypercpni, P O my increse (see Tle ) or remin constnt (see Tle ; Eddy et l., 977) despite lowering of venous P O (Pv O ) (Thoms et l., 99). This pprent enhncement of gs trnsfer my reflect the comintion of ventiltory nd crdiovsculr djustments. It hs een suggested tht hypertension enhnces rnchil gs trnsfer vi lmellr recruitment. The increse in ventrl ortic pressure is elieved to cuse lmellr recruitment y inititing perfusion of the more distl lmelle on the filments s well s y ltering the flow pttern within ech lmell (Booth, 978, 979,; Frrell et l., 979; Soivio nd Tuurl, 98). Further experiments re required to elucidte the physiologicl significnce (if ny) of the incresed systemic vsculr resistnce during hypercpni. In prticulr, it would e informtive to compre gs trnsfer during hypercpni in untreted fish (lrge increse in R s ) with tht in fish treted with α-drenoceptor ntgonists (no increse in R s ). Trout generlly inhit well-erted wters tht re likely to exhiit low nd constnt levels of CO. However, externl hypercpni could potentilly occur in lrge odies of wter suject to therml strtifiction or in smller lkes or rivers fed y cronte- or icronte-rich wter derived from underground springs. In fish frms, holding trout under crowded conditions in spring-fed ponds cn led to externl hypercpni. Regrdless of whether trout experience elevted CO levels in the nturl environment, their responsiveness to externl hypercpni (s for hypoxi) my reflect phylogenetic linege in which the selective dvntge conferred y the ility to respond to elevted CO nd/or reduced O levels ws significnt. A comprison of the crdiovsculr responses to hypercpni nd hypoxi The two common responses shred y trout exposed to hypercpni with trout nd other teleosts exposed to hypoxi re n elevtion of systemic vsculr resistnce nd rdycrdi. However, it is importnt to point out tht the crdiovsculr responses of teleosts to hypoxi re highly vrile. For exmple, R s responses to hypoxi in teleosts rnge from little or no chnge (Peyreud-Witzenegger nd Soulier, 989; Axelsson nd Fritsche, 99; Bushnell nd Brill, 99; Gmperl et l., 99) to lrge increses (Fritsche nd Nilsson, 989, 99; Axelsson nd Frrell, 99; Sundin, 99). The mechnisms underlying the influence of hypoxi on R s hve een exmined thoroughly in the Atlntic cod Gdus morhu (Fritsche nd Nilsson, 99; Axelsson nd Fritsche, 99). Essentilly, the net effect of hypoxi on R s in cod reflects the stimultion of systemic α-drenoreceptors y sympthetic nerves nd y circulting ctecholmines tht is opposed y unidentified vsodiltor sustnces; generlly, the vsoconstrictor effect predomintes. Thus, reflex stimultion of systemic α-drenoceptors ppers to e common response to oth hypercpni nd hypoxi. The degree of rdycrdi experienced y fish during hypoxi is lso highly vrile nd my depend upon the severity of the hypoxi nd the rpidity with which it is imposed. A similr sitution my exist in fish exposed to hypercpni ecuse the reduction in fh in the present study ws proportionl to the degree of P CO elevtion nd ws gretest in fish rpidly exposed to hypercpni (single step experiment). In summry, hypercpni elicits profound crdiovsculr nd ventiltory djustments in rinow trout. Although the physiologicl significnce of the crdiovsculr chnges remins uncler, it is nevertheless pprent from this nd previous (Heisler et l., 988; Wood et l., 99; Grhm et l., 99; Kinked nd Perry, 99; Wood nd Munger, 99; Perry nd Gilmour, 996) studies tht CO /H + is emerging s n importnt modultor of crdiorespirtory function in fishes. This reserch ws supported y NSERC Reserch nd Equipment grnts to S.F.P. nd y NSF grnts (grnt No. IBN 976) to K.O. We re grteful to Drs Serge Thoms nd Mrk Powell for their contriutions to erlier versions of this work. We lso thnk Dr Peter Bushnell for his vlule dvice nd ssistnce throughout this study. References Axelsson, M. nd Frrell, A. P. (99). Coronry lood flow in vivo in the coho slmon (Oncorhynchus kisutch). Am. J. Physiol. 6, R96 R97. Axelsson, M. nd Fritsche, R. (99). 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