Activation of the newly discovered cyclostome renin angiotensin system in the river lamprey Lampetra fluviatilis

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1 The Journl of Experimentl Biology 208, Pulished y The Compny of Biologists 5 doi: /je Activtion of the newly discovered cyclostome renin ngiotensin system in the river lmprey Lmpetr fluvitilis J. Anne Brown 1, *, Christopher S. Co 1,, Susn C. Frnkling 1 nd J. Cliff Rnkin 2, 1 School of Biologicl nd Chemicl Sciences, Htherly Lortories, University of Exeter, Prince of Wles Rod, Exeter, EX4 4PS, UK nd 2 Aqutic Biology Reserch Centre, Institute of Biology, University of Southern Denmrk Odense University, Hindsholmvej 11, DK-5300 Kerteminde, Denmrk *Author for correspondence (e-mil: J.A.Brown@exeter.c.uk) Present ddress: The Peninsul Medicl School, St Luke s Cmpus, University of Exeter, Exeter, EX1 2LU, UK Present ddress: Deprtment of Chemicl nd Biologicl Sciences, University of Huddersfield, Queensgte, Huddersfield, HD1 3DH, UK Accepted 27 Octoer 4 This study descries the first investigtions of the physiologicl signls involved in ctivting the newly discovered cyclostome renin ngiotensin system (RAS) nd its role in the river lmprey Lmpetr fluvitilis. Experimentl mnipultion showed tht volume depletion (removl of 40% lood volume) rpidly ctivted the RAS of lmpreys cclimted to wter t 576 mosm kg 1 (21 p.p.t.), significntly incresing plsm ngiotensin concentrtions fter 30 min nd 60 min. In greement with these results, rpid chnge in environmentl slinity (758 mosm kg 1 to freshwter (FW) nd FW to 605 mosm kg 1 ), resulted in rpid decrese nd increse in plsm [ngiotensin], respectively. Intrperitonel (i.p.) injection of FW-cclimted river lmpreys with 1% ody mss y volume of nominlly isosmotic sline (120 mmol l 1 NCl; 233 mosm kg 1 ) resulted in significnt decrese in the plsm ngiotensin concentrtion within 15 min. In contrst, i.p. injection of hyperosmotic sline (4 mol l 1 NCl) t 1% ody mss y Summry volume, which significntly incresed plsm osmollity, hd no significnt effect on plsm [ngiotensin], suggesting tht volume/pressure receptors nd osmoreceptors interct in regulting the lmprey RAS. These results indicte n importnt role for volume/ pressor receptors, s in teleosts, ut with n dditionl osmoreceptor mechnism, such tht circultory [ngiotensin] is determined y interction of volume/ pressure nd osmoreceptors nd their reltive sensitivities. The volume/pressure sensitivity is in keeping with the recent evidence of vsoconstrictor ction of homologous lmprey ngiotensin nd provides evidence tht the fundmentl role of the RAS in mintining volume nd pressure is n ncient function conserved over 500 million yers of verterte evolution. Key words: renin ngiotensin system, river lmprey, Lmpetr fluvitilis, plsm ngiotensin, slinity dpttion, volume regultion. Introduction The renin ngiotensin system (RAS) hs een identified in the mjority of verterte groups, including fish (Koyshi nd Tkei, 1996). In the systemic RAS, circulting ngiotensinogen, synthesised y the liver, is cleved y renin relesed from the kidney to form the inctive decpeptide, ngiotensin I (Ang I). Ang I is further cleved y ngiotensin converting enzyme to produce the physiologiclly ctive octpeptide, ngiotensin II (Ang II) nd further clevge results in formtion of ngiotensin III (Ang III: frgment 2 8 Ang II) nd ngiotensin IV (Ang IV: frgment 3 8 Ang II), oth of which lso show ioctivity, lthough their function remins to e estlished in most vertertes (Koyshi nd Tkei, 1996). In teleost fish there is evidence tht the RAS plys importnt roles in regulting lood pressure, controlling renl nd crdiovsculr function, stimulting interrenl steroidogenesis nd in determining drinking rtes (Brown et l., 1980; Arnold- Reed nd Blment, 1994; Tierney et l., 1995,; Bernier et l., 1999; Bernier nd Perry, 1999; Butler nd Zhng, 1). For decdes, the RAS ws elieved to hve first evolved in ony fishes nd to e sent from oth elsmornchs nd cyclostomes (Nishimur et l., 1970; Nishimur nd Ogw, 1973; Nishimur, 1985; Henderson et l., 1993). However, Ang I hs now een isolted nd sequenced in the elsmornch Trikis scylli (Tkei et l., 1993) nd more recently in two cyclostomes: the se lmprey Petromyzon mrinus (Tkei et l., 4) nd the river lmprey Lmpetr fluvitilis (Rnkin et l., 4). The Ang II component in these lmprey species ws identified s Asn 1,Vl 5 -Ang II, s is found in most, ut not ll teleosts (Hsegw et l., 1984; Khosl et l., 1985; Conlon et l., 1996; Blment et l., 3). Recognition of this similrity enled our first mesurements

2 224 J. A. Brown nd others of circulting ngiotensin in cyclostomes, using commercil ntiser with high cross-rectivity to Asn 1,Vl 5 -Ang II nd Vl 5 -Ang III (Rnkin et l., 1). This study showed higher plsm Ang II nd Ang III concentrtions in river lmpreys cclimted to sewter (SW) thn in freshwter (FW)- cclimted lmpreys (Rnkin et l., 1). From this it cn e suggested tht for ndromous lmprey species, migrting etween rivers nd the mrine environment during their life cycle (Hrdisty, 1979), the RAS could e involved in ody fluid homeostsis s hs een identified in teleost fish (Olson, 1992; Koyshi nd Tkei, 1996). There re similrities in the processes responsile for regulting ody fluid volume nd composition of teleosts nd lmpreys with, for exmple, hyper-osmoregultion in FW requiring excretion of lrge mounts of dilute urine to eliminte the high osmotic wter influx (Brown et l., 1980; Evns, 1993). Also, in oth lmpreys nd teleosts, hypoosmoregultion in more sline environments is ssocited with n osmotic wter loss tht is lnced y drinking the surrounding wter, with fluid sorption in the gut, together with drstic reduction in urine output nd renl djustments to excrete high concentrtions of divlent ions (Logn et l., 1980; Evns, 1993; Rnkin, 1997, 2; Brown nd Rnkin 1999). In teleosts nd elsmornchs there is evidence tht the RAS is controlled y volume receptors with, for exmple, relese of renin nd elevted circulting ngiotensin concentrtions during hypovolemi nd/or reduction in lood pressure (Nishimur et l., 1979; Bernier et l., 1999; Anderson et l., 1). However, the physiologicl signls responsile for ctivtion of the lmprey RAS re s yet uncler. We therefore undertook rnge of experimentl mnipultions of river lmpreys followed y mesurement of circulting ngiotensin levels to investigte the ctivtion of the lmprey RAS nd to fcilitte the development of hypotheses relting to its physiologicl role. Specificlly, we hve exmined: (1) whether lood removl ctivtes the lmprey RAS nd hence increses circulting ngiotensin concentrtion; for these studies we used lmpreys held in hyperosmotic environment to ensure tht volume depletion could not e rpidly compensted for y the nturl osmotic influx of wter; (2) the impct of extrcellulr fluid expnsion fter intrperitonel (i.p.) injection of nominlly isosmotic sline; (3) whether chnges in extrcellulr fluid osmollity ccompnying volume expnsion influence the lmprey RAS; (4) how circulting ngiotensin concentrtion is ffected fter rpid chnges in externl slinity from FW to hyperosmotic medium (605 mosm kg 1 ) nd from hyperosmotic medium (758 mosm kg 1 ) to FW. Mterils nd methods Experimentl nimls Adult river lmpreys Lmpetr fluvitilis L. were cught in eel trps in Ringkøing Fjord (c mosm kg 1 ) on the west cost of Jutlnd, Denmrk, during Septemer nd Octoer, t the strt of their migrtion into FW (severl months prior to spwning in the following spring). During this migrtion, river lmpreys metmorphose from silver mrine form through intermedites to yellow form (Rnkin, 1996, 1997). River lmpreys cught in Ringkøing Fjord (c mosm kg 1 ) in the utumn re predominntly silver forms, with mny individuls hving gstrointestinl trcts full of lood nd still cple of hypo-osmoregulting in SW for long periods of time fter cpture (Rnkin, 1997). Cptured river lmpreys were trnsported on ice to the Aqutic Biology Reserch Centre, Kerteminde, Denmrk nd kept in either erted FW (15 mosm kg 1 ; 0.5 p.p.t.) or grdully cclimted to Kerteminde SW (576 mosm kg 1 ; 21 p.p.t.) in closed nd filtered qurium system using Gret Belt surfce SW. Some lmpreys were cclimted to higher slinity of 758 mosm kg 1 (higher slinity wter thn 26 p.p.t. cnnot normlly e otined in Kerteminde). Experimentl studies were crried out during Octoer nd Novemer nd ll lmpreys were held t 10 C under 12 h:12 h light:drk cycle with reduced light chieved y shding most of the tnk surfce with lid. Tnk wter osmollity (vpour pressure osmometry: model 5520, Westcor Inc., Logn, UT, USA or freezing point depression: Osmomt model 030, Gonotech Gmtt, Berlin, Germny), slinity nd temperture (model 30M/50FT, YSI Inc., Yellow Springs, OH, USA) were monitored dily. River lmpreys were held in these conditions for 3 5 weeks prior to experimentl mnipultion. Anesthesi nd lood smpling River lmpreys were nesthetised y immersion in MS222 (3-mino-enzoic cid ester methnesulfonte slt; Sigm, Poole, UK; g l 1, s previously, Brown nd Rnkin, 1999). Once nesthetised, lmpreys were weighed nd plced on their cks in individul Perspex troughs with their heds immersed in erted wter contining nesthetic nd mintined t 10 C; the rest of the ody ws covered with dmp tissue. Respirtory movements were monitored, nd nesthesi djusted to mintin strong ventiltion nd stle lood pressure nd renl function (McVicr nd Rnkin, 1983; Brown nd Rnkin, 1999; Rnkin et l., 4). Blood smples were collected y needle puncture of the cudl vein. Blood for mesurement of circulting ngiotensin concentrtions ws collected into 100 µl of inhiitor solution (0.225 mol l 1 EDTA, 50 Kllikrein IU protinin nd 0.05 mol l 1 1,10- phennthroline; Sigm, UK) in chilled syringes contining irdried mmonium heprin (Sigm, UK). Smples were centrifuged ( g, 2 min t 4 C, Microcentrifuge, Ole Dich, Hvidovre, Denmrk), plsm removed nd trnsferred into fresh tue, nd tues frozen in liquid nitrogen. Hemtocrit (with inhiitor dilution) ws lso determined in heprinised microhemtocrit tues (Mikro Hettich Zentrifugen, Berlin, Germny; g, 2 min) to enle correction for the dilution of mesured plsm ngiotensin concentrtion y the inhiitor solution; the dilution fctor ws determined from hemtocrit nd totl mss of lood smple. Immeditely fter collecting the first lood smple, further

3 Lmprey renin ngiotensin system 225 smll smple (0.2 ml lood) ws collected into 1 ml syringe coted with mmonium heprin (Sigm, UK). This smple ws used for determintion of hemtocrit (without inhiitor) s efore, nd plsm osmollity (model 5520 Vpor Pressure Osmometer, Westcor Inc.). Plsm smples for ngiotensin nlysis were held t 80 C until trnsport on dry-ice to the University of Exeter, UK, where they were held t 80 C until extrction nd rdioimmunossy of ngiotensins. Experimentl series Blood volume depletion River lmpreys (42 91 g; N=30) cclimted to Kerteminde SW (576 mosm kg 1 ; 21 p.p.t.) were nesthetised in MS222 s descried ove nd n initil lood smple of 3.2% of ody mss ( ml, clculted to reduce lood volume y 40%) ws collected from the cudl vein for nlysis of plsm ngiotensin nd determintion of smple hemtocrit (with inhiitor). A further 0.2 ml ws tken immeditely for mesurement of lood hemtocrit (without inhiitor) nd plsm osmollity. For ech lmprey, lood smples (pproximtely 1 ml) were susequently collected into inhiitor t 30 min (N=10), 60 min (N=11) or 90 min (N=7) fter the initil lood volume depletion (one time point per lmprey) nd used to mesure plsm ngiotensin nd hemtocrit (in presence of inhiitor). Immeditely fter the llocted smpling point further 0.2 ml lood smple ws collected to determine hemtocrit (without inhiitor) nd plsm osmollity. Isosmotic nd hyperosmotic injections In order to determine the effects of volume expnsion on the RAS, with nd without slt loding, FW-cclimted river lmpreys were lightly nesthetised nd injected i.p. with 1% ody mss y volume of either nominlly isosmotic sline or hyperosmotic sline, with control, non-injected lmpreys run in prllel. FW-cclimted river lmpreys ( g) were lightly nesthetised s outlined ove nd weighed. Experimentl lmpreys were i.p.-injected t 1% y volume of ody mss with either nominlly isosmotic sline (120 mmol l 1 NCl; 233 mosm kg 1 ) or hyperosmotic sline (4 mol l 1 NCl). Blood smples were collected from ech lmprey s descried erlier for determintion of plsm ngiotensin, lood hemtocrit nd plsm osmollity. Smples were collected from seprte lmpreys 15 min fter i.p. injection of hyperosmotic sline (N=8) or isosmotic sline (N=5) nd 30 min fter i.p. injection of hyperosmotic sline or isosmotic sline (N=5 in ech group). Control non-injected lmpreys from the sme stock tnks were held under light nesthesi until removl of single lood smple fter 15 min (N=8) or 30 min (N=5). Acute chnges in externl slinity The qurium system used llowed chnges in environmentl slinity to e rpidly chieved, voiding the stress of removing the lmpreys from the wter. This llowed investigtions to determine how the lmprey RAS is ffected during the initil period fter rpid shifts in externl slinity. In the first experiment, the initil FW (14 mosm kg 1 ) ws ltered to hyperosmotic medium (605 mosm kg 1 ); in second experiment hyperosmotic Kerteminde SW (758 mosm kg 1 ) ws rpidly replced y FW to rech 22 mosm kg 1. For the experiment involving trnsfer from FW to hyperosmotic environment, river lmpreys (45 87 g) were held in flow-through FW system (pprox. 500 litres) for 2 weeks. At the strt of the experiment (time zero), the tnk FW volume ws reduced to depth of 10 cm (pprox. 80 litres) nd switched to closed system with filtrtion for the 24 h prior to the strt of the experiment. An initil group of lmpreys (N=8) ws removed, nesthetised nd lood smples collected. Kerteminde SW (slinity 25 p.p.t.) ws pumped into the tnk to increse the slinity from 0.5 p.p.t. to 20 p.p.t. within 6 min nd chieve pek slinity of 21 p.p.t. (605 mosm kg 1 ) within 20 min. Su-groups of river lmpreys (N=8 per group) were lood smpled t 1, 2, 4, 8 or 24 h from the strt of SW ddition in order to determine plsm osmollity, hemtocrit nd plsm ngiotensin concentrtion, s descried in the lood volume depletion experiments. For the experiment involving trnsfer from hyperosmotic environment to FW, river lmpreys (44 87 g) were held in closed nd filtered system of erted Kerteminde SW (26 p.p.t., 758 mosm kg 1 t 10 C) for 3 weeks. 24 h efore strting the experiment, the SW ws reduced to depth of pproximtely 10 cm. At the strt of the experiment group of lmpreys (N=10) ws removed from the experimentl tnk, nesthetised, nd lood smples collected s in other experiments. Slinity ws lowered y rpid ddition of FW to rech 1 p.p.t. (23 mosm kg 1 ) within 30 min nd 0.6 p.p.t. t 1 h. Su-groups of lmpreys (N=8 per group) were lood smpled t 2 h, 4 h, 8 h nd 24 h fter the strt of FW ddition. Extrction nd rdioimmunossy of plsm ngiotensin Plsm ngiotensin ws extrcted ccording to the method descried y Bernier et l. (1999). Briefly, 100 µl smples of river lmprey plsm held on ice t 4 C were ech mixed with 100 µl cidic cetone (cetone:wter:1 mol l 1 HCl, rtio 40:5:1) nd vortexed vigorously for 1 min. The mixture ws centrifuged t g for 10 min t 4 C. The superntnt ws collected nd the pellet re-soluilised nd re-extrcted, s efore. Superntnts were comined, freeze dried under vcuum t 45 C (Edwrds EF4 Modulyo freeze dryer, Edwrds High Vcuum, Crwley, UK) nd stored t 80 C until rdioimmunossy (RIA). Prior to RIA, the extrcted residues were resuspended in phosphte-uffered sline (PBS, 400 µl t 0.01 mol l 1, ph 7.4, contining 0.25% (w/v) ovine serum lumin frction V RIA grde nd 0.25% (v/v) Triton X-100; Sigm, UK). Triplicte smples of these plsm extrcts nd [Asn 1,Vl 5 ]-Ang II stndrds (100 µl) were incuted overnight t 4 C with 100 µl ( 8000 c.p.m.) 125 I-[Ile 5 ]-Ang II (74 Tq mmol 1, 0 Ci mmol 1 ; Amershm, UK) nd 100 µl of ngiotensin

4 226 J. A. Brown nd others ntiserum ( diluted in PBS). The heterologous ntiserum ws initilly rised ginst mmmlin [Asp 1,Ile 5 ]- Ang II (Ymguchi, 1981). Serilly diluted extrcted lmprey plsm rn prllel to the stndrd curve of [Asn 1,Vl 5 ]-Ang II, the ntive Ang II sequence in lmpreys (Fig. 1). The ntiserum used shows <0.5% cross-rectivity with Ang I from mmmls, teleosts nd elsmornchs ut high cross-rectivity with Ang II from mmmls (100%), teleosts (63 85%), elsmornchs (74%) nd lmpreys (63%) (Bernier et l., 1999; Gry Anderson, Gtty Mrine Lortory, University of St Andrews, UK, personl communiction). However, the ntiserum, in common with most commercil Ang II ntiser, lso shows high cross-rectivity ( 90%) with mmmlin Ang III nd Ang IV (G. Anderson, personl communiction). Therefore, our RIA mesurements of ngiotensin levels would incorporte Ang II, Ang III nd Ang IV. The following dy, seprtion of free nd ound ngiotensins ws chieved y ddition of 100 µl of solid phse second ntiody-coted cellulose suspension (nti-rit IgG serum; Sc-Cel IDS, Boldon, UK) nd incution t room temperture for 30 min. Distilled wter (1 ml) ws then dded nd tues centrifuged (2500 g, 4 min, 4 C). Superntnts were spirted nd rdioctivity of the pellets contining ound Ang II ws determined (Pckrd Cor Auto-gmm, B5002, Reding, UK). The ngiotensin content of smples of river lmprey plsm ws determined using softwre pckge (RIASMART, Biosoft, Cmridge, UK). Finl vlues of plsm ngiotensin concentrtion were corrected for the clculted dilution of plsm smples y the inhiitor mix. This dilution ws determined y clcultion of the plsm volume in ech smple sed on the mesured hemtocrit nd the grvimetric determintion of the lood volume collected. Estimted recovery of ngiotensin through the extrction procedure nd RIA ws 78.5±5.7% for [Asn 1,Vl 5 ]-Ang II nd 105.7±3.7% for [Vl 5 ]-Ang III (N=10 in oth cses). Intrssy vriility ws 10.1% nd 7.2% for Ang II t 9.8 pmol l 1 nd 78.1 pmol l 1, respectively, nd 7.3% nd 10.6% for Ang III t identicl concentrtions (N=10 in ll cses). The minimum detectle level of plsm ngiotensin concentrtion ws 11.7 pmol l 1. Sttisticl nlyses All dt re presented s mens or percentges of mens ± stndrd error (S.E.M.). All sttisticl nlyses used SPSS version 10.0 for Windows. All dt were initilly tested y the Kolmogorov Smirnov test to determine whether they were normlly distriuted. When dt were normlly distriuted or normlity ws chieved y trnsformtions, further nlyses used nlysis of vrince (ANOVA), followed y post-hoc multiple comprison tests (Tukey s HSD when dt showed homogeneous vrinces fter Levene s tests, nd Gmes Howell when vrinces differed significntly). When trnsformtions filed to chieve normlity, dt were nlysed y non-prmetric ANOVA for independent smples nd Mnn Whitney U-tests. Plsm ngiotensin dt fter trnsfer of lmpreys from FW to higher environmentl slinities showed significnt differences etween groups (ANOVA) tht could not e locted y post-hoc multiple comprison tests; dt were therefore nlysed y liner contrsts compring ngiotensin concentrtions t ech time point fter exposure to the higher environmentl slinity with ngiotensin concentrtions in FW-cclimted lmpreys t time 0 h. Significnt differences etween pre-lood volume depletion nd post-lood volume depletion dt for plsm ngiotensins, osmolrity nd hemtocrit were estlished using pired t- tests. Sttisticl differences etween mens were considered significnt t P<0.05. Results Blood volume depletion The removl of n estimted 40% of the lood volume of river lmpreys cclimted to 576 mosm kg 1 (21 p.p.t.) resulted in significnt decrese in hemtocrit (Fig. 2A). Blood volume depletion did not significntly ffect the plsm osmollity of these lmpreys (Fig. 2B). A time course investigtion of the chnges in plsm ngiotensin concentrtion fter lood volume depletion showed tht the plsm concentrtion of ngiotensin hd more thn douled fter 30 min (Fig. 2C; P<0.01). Plsm ngiotensin concentrtion remined significntly elevted in lmpreys smpled 60 min fter lood volume depletion (P<0.001) with 62% increse compred to sl levels (Fig. 2C). After 90 min, there ws no significnt difference in plsm ngiotensin concentrtion compred to tht of sl smples tken to chieve the imposed lood volume depletion (Fig. 2C). Isosmotic nd hyperosmotic sline injection The hemtocrit of non-injected lmpreys remined stle under the light nesthesi (Fig. 3A). Injection with either of the sline solutions resulted in pronounced reduction in hemtocrit with the men vlues in non-injected controls of 125 I-inding (%) Lmprey plsm dilution Asn 1 Vl 5 -Ang II stndrds [Angiotensin] (fmol per tue) Fig. 1. Prllelism of the [Asn 1,Vl 5 ]-Ang II rdioimmunossy stndrd curve (shown s mens ± S.E.M., N=6) nd seril dilution of n extrct of pooled plsm from the river lmprey Lmpetr fluvitilis. For detils of the ssy, see Mterils nd methods.

5 Lmprey renin ngiotensin system 227 Pre-volume depletion (time 0) A Post-volume depletion Non-injected Isosmotic Hyperosmotic A Hemtocrit (%) *** *** ** Hemtocrit (%) Plsm osmollity (mosm kg 1 ) Plsm [ngiotensin] (pmol l 1 ) B C ** *** 30 min 60 min 90 min Fig. 2. Blood volume depletion in river lmpreys cclimted to Kerteminde sewter t 576 mosm kg 1 (21 p.p.t.). The first lood smple (pre-volume depletion; open rs) ws tken to chieve 40% decrese in lood volume nd second smple (post-volume depletion; htched rs) ws collected fter 30 min (N=10), 60 min (N=11) or 90 min (N=7). Dt for (A) hemtocrit (%), (B) plsm osmollity (mosm kg 1 ) nd (C) plsm ngiotensin concentrtions (pmol l 1 ) re shown (**P<0.01; ***P<0.001, pired t-tests). Plsm osmollity (mosm kg 1 ) Plsm [ngiotensin] (pmol l 1 ) B C c,, 15 min 30 min Time fter intrperitonel injection Fig. 3. (A) Hemtocrit (%), (B) plsm osmollity (mosm kg 1 ) nd (C) plsm ngiotensin concentrtions (pmol l 1 ) of river lmpreys cclimted to freshwter t 15 mosm kg 1. Control lmpreys (noninjected; lck rs) were held under light nesthesi for 15 min (N=8) nd 30 min (N=4) in the sence of further mnipultion. Experimentl lmpreys were lood smpled t either 15 min or 30 min fter n i.p. injection of 1% ody mss with either isosmotic sline (white rs; 120 mmol l 1 NCl; 233 mosm kg 1 ; N=5 t ech time point) or hyperosmotic sline (cross-htched rs; 4 mol l 1 NCl; N=5 t 30 min; N=8 t 30 min). Different letters ove error rs signify groups tht differ significntly (ANOVA nd post-hoc multiple comprison tests). 38.7% nd 41.8% t the two time points declining to men vlues of 20 26% in the four injected groups (Fig. 3A). The reduction in hemtocrit ws similr fter injection of isoosmotic nd hyperosmotic sline nd not significntly ffected over the two smpling time points. Injection of hyperosmotic sline significntly incresed plsm osmollity (Fig. 3B). The nominlly isosmotic sline (233 mosm kg 1 ) proved to e slightly hypo-osmotic in these lmpreys, ut not sufficiently so s to result in ny chnge in plsm osmollity (Fig. 3B). The two experimentl groups showed no differences over the two time points, ut noninjected lmpreys showed slight (3.8%) decrese in plsm osmollity etween the 15 min nd 30 min smpling points. Injection with the nominlly isoosmotic sline resulted in significnt depression in the concentrtion of plsm ngiotensin t 15 min (P<0.01; Fig. 3C), ut plsm ngiotensin concentrtion of lmpreys injected with hyperosmotic sline ws not significntly different from tht c,

6 228 J. A. Brown nd others of the non-injected controls oth fter 15 min nd 30 min (Fig. 3C). Acute chnges in externl slinity The rpid increse in environmentl slinity, rising from 14 to 605 mosm kg 1 within 20 min, resulted in significnt increse in plsm osmollity within 1 h (Fig. 4A). Blood hemtocrit ws not significntly ltered y exposure to high slinities (Fig. 4B). Plsm ngiotensin concentrtions followed visily similr pttern to the rising plsm osmollity (Fig. 4C) ut there ws lrge vriility in plsm ngiotensin concentrtion t ll time points, prticulrly t time 0 h. Sttisticl nlysis y ANOVA indicted significnt chnge fter exposure to higher environmentl slinity, ut multiple comprison procedures did not locte significnt difference etween groups. However, liner contrsts of ngiotensin concentrtions fter exposure to higher slinity, compred to control vlues in FW-cclimted lmpreys, indicted significnt rise fter 4 h (P=0.022), 8 h (P=0.005) nd 24 h (P=0.004). A rpid decrese in environmentl slinity, from 758 to 22 mosm kg 1, significntly lowered the lood hemtocrit nd plsm osmollity in river lmpreys within 4 h (Fig. 5A,B; P<0.05 nd P<0.001, respectively). Plsm osmollity remined depressed t 8 h nd 24 h ut lood hemtocrit ws restored. Plsm ngiotensin concentrtions dropped stedily fter the cute chnge in environmentl slinity, ut only reched significntly lower vlue fter 24 h (Fig. 5C). Discussion Our studies were imed principlly t investigting the signls involved in regulting the recently discovered lmprey RAS. Our recent studies of river lmpreys showed lmost douling of plsm ngiotensin concentrtion in river lmpreys cclimted to SW of 28 p.p.t. slinity (845 mosm kg 1 ) compred to the plsm ngiotensin concentrtion of lmpreys cclimted to FW (Rnkin et l., 1). In the present studies, however, lthough the men plsm concentrtion of immunorective ngiotensin ws slightly higher in lmpreys cclimted to SW of 758 mosm kg 1 thn in FW-cclimted lmpreys, the difference ws not sttisticlly significnt. This seems likely to reflect t lest prtilly the lower osmotic chllenge fced y the lmpreys in the present studies, ut differences in ssy techniques etween the two studies lso complicte direct comprisons. Our previous nlyses included HPLC seprtion of Ang II, III nd IV, prior to RIA, ut in the present study the lrge numer of smples precluded routine seprtion. Although the ntiser used in present nd previous studies differed, this is of less importnce s oth showed high cross-rectivity with Ang II, Ang III nd Ang IV nd <0.5% cross-rectivity with Ang I. Thus, in ech cse RIA would hve mesured the mjor ctive ngiotensin frgments (Butler nd Oudit, 1995; Koyshi nd Tkei, 1996). Our previous HPLC seprtion resulted in comined (Ang II plus Ang III) vlue of ~ pmol l 1 in FW-cclimted lmpreys nd indicted tht plsm Ang III ws present t similr concentrtions to plsm Ang II (Rnkin et l., 1). This contrsts with the pprently low level of Ang III found fter RIA of HPLC-seprted trout plsm (Bernier et l., 1999), using the sme ntiody tht we employed in the present studies. Given tht in the present studies we mesured concentrtions of pproximtely pmol l 1 totl immunorective ngiotensin in plsm smples collected from FW-cclimted lmpreys, it is prole tht significnt mount of Ang IV is lso present in lmprey plsm. Light nesthesi for short periods (up to 30 min) hs no pprent effect on plsm ngiotensin levels (see Fig. 3C). In previous studies, trnsfer of river lmpreys from FW to 503 mosm kg 1 suggested possile link etween the circulting Ang II concentrtion nd rising plsm osmollity (Rnkin et l., 1). The present studies therefore exmined Hemtocrit (%) Plsm osmollity (mosm kg 1 ) Plsm [ngiotensin] (pmol l 1 ) 350 A B C * ** Time (h) Fig. 4. Effects of rpid increse in environmentl slinity (FW to 21 p.p.t.; 605 mosm kg 1 ) on (A) plsm osmollity (mosm kg 1 ), (B) lood hemtocrit (%) nd (C) plsm ngiotensin concentrtion (pmol l 1 ) of river lmpreys (N=8 t ech time point: 0 h, 2 h, 4 h, 8 h nd 24 h fter trnsfer). Groups with different letters differ significntly (ANOVA nd post-hoc multiple comprison tests). Asterisks signify groups tht differ significntly from time 0 h (*P<0.05, **P<0.01, ANOVA followed y liner contrst nlyses). **

7 Lmprey renin ngiotensin system 229 more fully the chnges in plsm osmollity nd plsm ngiotensin concentrtions fter rpidly incresing externl slinity from FW to 605 mosm kg 1 nd lso fter the reverse mnoeuvre, rpidly decresing slinity from 758 mosm kg 1 to FW. Lmpreys exposed to these rpid chnges in environmentl slinity showed good osmoregultion over the next 24 h, s shown previously (Rnkin, 2). After n cute rise in externl slinity, there ws no significnt chnge in lood hemtocrit, indicting limited, if ny, volume depletion, ut there ws smll though significnt rise in plsm osmollity tht ws ssocited with similr pttern of chnge in plsm ngiotensin concentrtion, with significnt rise fter 4 h, 8 h nd 24 h suggested y the liner contrst nlyses of dt t ech time point compred to the initil level in FW. In greement with these trends, trnsfer from hyperosmotic sline environment to FW, resulted in stedy decline in plsm ngiotensin concentrtion longside declining plsm osmollity. However, our dt indicte tht reltionships re complex since the lrgest (nd significnt) decline in plsm ngiotensin concentrtion occurred etween 8 h nd 24 h when plsm osmollity ws stle. Movements etween different environmentl slinities re highly likely to ffect extrcellulr fluid volume s well s plsm osmollity, s in teleosts (Olson, 1992). We therefore exmined the impct of oth hypovolemi nd volume expnsion. A severe hemorrhge of 40% of nominl lood volume of 8% of ody mss ws chosen to test for stimultion of the RAS in SW-cclimted lmpreys. Another lmprey species (Petromyzon mrinus) ws estimted to hve lood volume of 8.5% of ody mss, sed on the volume of distriution of Evn s Blue (Thorson, 1959). This technique is inccurte in teleost fish (Olson, 1992), ut in two Petromyzon mrinus otined from Ringkøing Fjord, Denmrk, the more ccurte technique involving re-injection of red lood cells lelled with 51 Cr (Olson, 1992), gve vlues of 8.3% nd 8.1% of ody mss (J. C. Rnkin, unpulished oservtions). SW-cclimted lmpreys re fced with the need to continully drink their environmentl medium in order to regulte ody fluid volume, ut nesthesi hs een found to lock drinking (Rnkin, 1997). Therefore, in the hypovolemi experiments the lmpreys would not hve een le to increse drinking in order to restore lood volume. Bsed on expected drinking rtes in SW-cclimted lmpreys, n inhiition of drinking would hve resulted in further volume loss of 4.8 ml h 1 kg 1 ody mss tht could not e sored from the gut to replce rnchil nd renl losses (Rnkin, 2). Within the 90 min experimentl period fter the imposed lood volume depletion this would hve resulted in further 4.5% hypovolemi. Hypovolemi, induced y removl of n estimted 40% of the lood volume, in river lmpreys held in hyperosmotic medi, would potentilly cuse n immedite drop in lood pressure, s in teleost fish (e.g. Nishimur et l., 1979). This could cuse decrese in kidney perfusion pressure, relesing renin. However, s yet, there is no evidence for renin-relesing grnulr epithelioid cells in the lmprey kidney or Hemtocrit (%) Plsm osmollity (mosm kg 1 ) Plsm [ngiotensin] (pmol l 1 ) 350 A , 45 B 800 C Time (h) Fig. 5. Effects of rpid decrese in environmentl slinity (from 26 p.p.t., 758 mosm kg 1 to FW, 22 mosm kg 1 ) on river lmpreys. Blood smples were collected from group of lmpreys prior to trnsfer nd further groups t 2 h, 4 h, 8 h nd 24 h fter trnsfer. Dt for (A) plsm osmollity (mosm kg 1 ; N=8 t ech time point), (B) hemtocrit (%; N=10 t 0h, 8 t ll other time points) nd (C) plsm ngiotensin concentrtion (pmol l 1 ; N=9 t 0 h, N=8 t 2 h nd 24 h, N=7 t 4 h nd 8 h) re shown). Groups with different letters ove error rs differed significntly (ANOVA nd post-hoc multiple comprison tests). mesurement of plsm renin concentrtion in cyclostomes (Henderson et l., 1993; Koyshi nd Tkei, 1996). Nevertheless, the more thn twofold increse in plsm ngiotensin concentrtion oserved 30 min fter hypovolemi suggests tht pressure/volume receptors ply n importnt role in ctivting the lmprey RAS, s hs een reported in oth teleosts nd elsmornchs (Biley nd Rndll, 1981; Nishimur et l., 1979; Nishimur nd Biley, 1982; Glli nd Phillips, 1996; Bernier et l., 1999). Hypotension following hemorrhge will result in fluid influx to the lood from the interstitil fluid due to the oncotic pressure of plsm proteins. In lmpreys, which lck spleen (nd secondry circultion, s is found in teleost fish), reductions in hemtocrit my

8 230 J. A. Brown nd others quntittively reflect such hemodilution. If so, the resultnt flls in hemtocrit (y 36.7%, 36.3% nd 40.3% fter 30, 60 nd 90 min, respectively) indicte tht the intention of reducing lood volume y 40%, hd indeed een chieved. The initil trnsient reduction in lood volume (prior to compenstory fluid influx) cn e predicted to hve triggered rpid ctivtion of the RAS nd plsm ngiotensin concentrtion could well hve peked efore the 30 min smple ws tken. The demonstrtion of pressure/volume-sensitive regultion in the lmprey RAS is perhps indictive of the most ncient role of the RAS in lood pressure regultion, which hs remined fundmentl feture throughout verterte evolution. The most immedite effect of elevted levels of circulting ngiotensin fter lood volume depletion is likely to e vsoconstriction, which would serve to restore lood pressure. The men circulting concentrtion of ngiotensin tht we mesured t 30 min fter hypovolemi ws c. 1 nmol l 1 (1054.5±203.5 pmol l 1, N=10). Injection of 1 nmol kg 1 ody mss ngiotensin II produced significnt (6 mmhg) increse in dorsl ortic lood pressure in the river lmprey (Rnkin et l., 4). Although this would hve rpidly produced concentrtion of out 12 nmol l 1 if distriuted only in the lood volume, pressure effects were oserved with lower doses nd initil ngiotensin concentrtions following lood volume depletion could well hve een much higher thn 1 nmol l 1. In the present study, plsm ngiotensin concentrtions recovered within ~1.5 h of lood volume depletion. Our mesurements of the declining concentrtions of plsm ngiotensin fter n infusion of Ang II hve shown tht the hlf-life of ngiotensin in lmpreys is pproximtely 16 min (J. A. Brown, S. C. Frnkling, C. S. Co nd J. C. Rnkin, unpulished dt) nd thus if RAS ctivtion fter volume depletion is short-lived event, sl levels could e chieved within 90 min of volume depletion. Pressure effects would e very rpidly chieved (Rnkin et l., 4) nd restortion of lood pressure would e likely to inhiit further ngiotensin formtion. A further prt of the recovery, t lest in FWcclimted lmpreys, could involve reduction in urine flow rtes initited y ngiotensin, s hs een shown to occur in teleosts (Brown nd Blment, 1997; Brown et l., 0). Studies re required to descrie fully the renl ctions of the lmprey RAS, ut Asn 1,Vl 5 -Ang II t or 10 9 mol min 1 kg 1 ody mss hs een shown to reduce urine flow rtes of FW-cclimted Lmpetr fluvitilis nd Petromyzon mrinus (J. A. Brown, C. S. Co nd J. C. Rnkin, unpulished dt). However, in the lood volume depletion experiments, lmpreys were cclimted to hyperosmotic SW nd urine flows would lredy hve een miniml (McVicr nd Rnkin, 1983; Brown nd Rnkin, 1999). In ddition to exmining the effects of lood volume depletion, we explored the effect of i.p. injection of nominlly isosmotic sline on plsm ngiotensin concentrtions with the im of inducing extrcellulr fluid expnsion, with noninjected FW lmpreys s controls. Anesthesi in the control lmpreys resulted in slight reduction in plsm osmollity etween the 15 nd 30 min smple times, perhps reflecting some wter retention. This could e the result of slight decrese in lood pressure tht would influence individul nephron filtrtion rtes (McVicr nd Rnkin, 1985) nd hence reduce urine flow (Brown nd Rnkin, 1999). This rgument is questionle, however, s lmpreys given pproximtely isosmotic sline (ctully slightly hyposmotic), showed no evidence of ny chnge in plsm osmollity. The i.p. injection of isosmotic sline resulted in significnt decline in plsm ngiotensins compred to the level in non-injected FWcclimted river lmpreys t 15 min fter the injection, nd supports our hypothesis tht volume/pressure receptors re importnt in regulting the lmprey RAS. Similr findings were reported in the Austrlin lungfish Neocertodus forsteri, in which plsm renin ctivity ws significntly lower fter injection of isosmotic sline, lthough plsm ngiotensin concentrtion ws not mesured in this study (Blir-West et l., 1977). Our results suggest tht the i.p. injection of pproximtely isosmotic sline led to expnsion of the extrcellulr fluid volume, lowering lood hemtocrit y 35% (t 15 min), nd hence inhiited the RAS, wheres stimultion of the lmprey RAS occurred fter volume depletion. Although significnt reduction in plsm ngiotensin concentrtion ws induced y i.p. injection of nominlly isosmotic sline, this did not occur fter injection of lmpreys with similr volume of hyperosmotic sline. It is rgule tht i.p. injection of hyperosmotic sline might stimulte initil withdrwl of extrcellulr fluid into the peritonel cvity nd tht reduced plsm volume my ccount for the sence of ny reduction in plsm ngiotensin concentrtion. However, there ws no evidence of ny increse in hemtocrit tht would hve ccompnied fluid redistriution into the peritonel cvity. After oth isosmotic nd hyperosmotic sline injections, hemtocrit ws drmticlly reduced compred to the hemtocrit of non-injected lmpreys nd did not differ significntly etween lmpreys injected with isosmotic sline nd hyperosmotic sline. Therefore, it would pper tht oth groups of lmpreys were exposed to volume expnsion. However, declining plsm ngiotensin concentrtion ws not seen in lmpreys i.p.-injected with hyperosmotic sline. The injection of hyperosmotic sline significntly rised plsm osmollity within 15 min nd this suggests tht osmoreceptors my either directly ctivte the lmprey RAS, or inhiit the impct of volume/pressure receptors. These results re in greement with the longer-term increse in plsm ngiotensin concentrtion tht ccompnied the rise in plsm osmollity fter exposure to incresed environmentl slinity. In mmmls, renin hs een suggested to e the mjor rtelimiting component of the RAS nd complex rry of mechnisms involving renl nerve stimultion, roreceptors nd the mcul dens interct to control renin relese (Koyshi nd Tkei, 1996; Nishimur, 4; Peti-Peterdi et l., 4). In non-mmmlin vertertes, the control of renin relese is still poorly understood (Henderson et l., 1993; Koyshi nd Tkei, 1996; Nishimur, 4) nd few studies

9 Lmprey renin ngiotensin system 231 hve investigted the impct of extrcellulr osmollity on renin relese. In the fowl, infusion of hypertonic sline into the kidney (vi the renl portl system) nd intrvenous injection of hypertonic sline depress plsm renin relese nd plsm ngiotensin, respectively (Nishimur nd Biley, 1982; Koyshi nd Tkei, 1996). This inhiition of renin relese y high osmollity grees with the results in mmmlin studies, suggesting n inverse reltionship etween plsm sodium/osmollity nd the numer of renin cells, renin relese nd plsm renin ctivity (Koyshi nd Tkei, 1996; Peti- Peterdi et l., 4). Our results show n opposite pttern in lmpreys, since high plsm osmollities were ssocited with high plsm ngiotensin concentrtions in oth experiments involving chnges in externl osmollity. Furthermore, while plsm ngiotensin concentrtion declined fter injection of lmpreys with nominlly isosmotic sline, similr chnges did not occur fter the hyperosmotic sline injection. Stimultion of the RAS fter injection of hyperosmotic sline ws lso reported for the eel (reviewed y Koyshi nd Tkei, 1996), so this my e feture of lower vertertes. However, we cnnot ssume tht renin ws relesed in either the study of eels or in the present study of lmpreys, since plsm renin ctivity ws not determined in either study. Even if plsm renin ctivity ws known, this my not e the only rte-limiting step in the lmprey RAS. In mmmls, secretion of ngiotensinogen hs rte-limiting effect on mximl formtion of plsm Ang I nd ultimtely, the production of Ang II (Klett nd Grnger, 1). Furthermore, our recent studies of the rinow trout exposed to n osmotic stress hve shown incresed heptic ngiotensinogen mrna (R. K. Pley, J. G. Aust, S. J. Aves nd J. A. Brown, unpulished oservtions; Aust, 2). This suggests tht regultion of heptic ngiotensinogen secretion plys significnt role in producing the reported elevtions in circulting ngiotensin levels nd dpttion of teleost fish to hyperosmotic medi. However, s yet we hve no informtion on the regultion of lmprey ngiotensinogen secretion. In conclusion, our results indicte tht volume receptors exert control on the lmprey RAS, ut these receptors my e modulted y sodium/chloride/osmo-sensitive receptors nd circulting ngiotensin levels will e determined y the interction of the puttive volume nd osmo/slt receptors nd their reltive sensitivities. However, the predictle chnges in plsm volume nd electrolytes when ndromous lmpreys migrte etween FW nd SW would ct s complementry signls in ctivting the RAS in hyperosmotic environments nd inhiition of the RAS in FW. While feeding on fish during the mrine phse of their life cycle, lmpreys my fce imposed chnges on extrcellulr fluid volume nd osmolrity tht impct on the functioning of the RAS. For exmple, river lmpreys cught in Ringkøing Fjord, Denmrk, just s they egin their upstrem migrtion, hve een found with intestines distended with lood representing up to 17% of their ody mss (Rnkin, 1997). This gut distension when feeding on teleosts my result in rpid isosmotic volume lod, potentil inhiitory signl to the RAS. In contrst, se lmpreys (Petromyzon mrinus) my meet simultneous volume nd hyperosmotic chllenges when feeding on mrine shrks tht hve ody fluids roughly isosmotic to sewter (Wilkie et l., 4). Although undoutedly difficult to chieve, investigtions exploring the potentil regultory role of endocrine systems such s the RAS during the prsitic feeding of lmpreys, would e extremely vlule. The uthors re grteful to Professor Yoshio Tkei for the ngiotensin ntiserum used in these studies, Lone Morgen for technicl ssistnce in Denmrk nd Dr Dve Hodgson (University of Exeter) for sttisticl dvice. This study ws supported y grnt from the Nturl Environment Reserch Council (NERC GR3/12190). References Anderson, W. G., Cerr, M. C., Wells, A., Tierney, M. L., Tot, B., Tkei, Y. nd Hzon, N. (1). Angiotensin nd ngiotensin receptors in crtilginous fishes. Comp. Biochem. 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