J Comp Physiol B (2001) 171: 623±634 DOI 10.1007/s003600100213
O R I GI N A L P A P E R
S.F. Perry á C.J. Montpetit á J. McKendry P.R. Desforges á K.M. Gilmour á C.M. Wood K.R. Olson
The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss ) and the marine dog®sh (Squalus acanthias ) Accepted: 28 May 2001 / Published online: 9 August 2001 Ó Springer-Verlag 2001
Abstract The aim of the present study was to evaluate the eects of endothelin-1-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dog®sh (Squalus acanthias). In both species, endothelin-1 (666 pmol kg±1) caused a rapid (within 4 min) reduction (ca. 30±50 mmHg) in arterial blood partial pressure of O2. The eects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly dierent in trout and dog®sh. In trout, arterial CO2 partial pressure was increased transiently by 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dog®sh after injection of endothelin-1 and was increased only slightly (0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml±1) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused
Communicated by L.C.-H. Wang S.F. Perry (&) á C.J. Montpetit á J. McKendry á P.R. Desforges Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5 E-mail:
[email protected] Tel.: +1 613-5625800 ext 6005 Fax: +1-613-5625486 K.M. Gilmour Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 C.M. Wood Department of Biology, McMaster University, 1280 Main Street W., Hamilton, Ontario, Canada L8S 4K1 K.R. Olson Indiana University School of Medicine, South Bend Center, University of Notre Dame, Notre Dame, IN 46556-5607, USA
a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dog®sh) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaected. In dog®sh, arterial blood pressure was lowered by 10 mmHg at 6±10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure re¯ected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing eects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output. Keywords Catecholamines á Cardiac output á Ventilation á Endothelin-1 á Carbonic anhydrase Abbreviations CA carbonic anhydrase á ET-1 endothelin-1 á fH cardiac frequency á fR ventilation (respiration) frequency á GDIFF diusion conductance á HSV cardiac stroke volume á Pa arterial blood pressure á PCA caudal artery pressure á PDA dorsal aortic pressure á PaCO2 á arterial blood á PCO2 á PaO2 arterial PO2 á pHa arterial pH á _ cardiac output á RG branchial vascular Q resistance á RS systemic vascular resistance á Vent. Amp. ventilation amplitude
624
Introduction Endothelin-1 (ET-1) is a 21-amino acid peptide hormone that is produced predominantly, though not exclusively, in vascular endothelial cells (Miyauchi and Masaki 1999). In mammals (Masaki 1993) and lower vertebrates (Poder et al. 1991), ET-1 is a potent vasoconstrictor of systemic blood vessels via its interaction with ETA or ETB receptors (Neylon 1999). Typically, stimulation of vascular smooth muscle ETA/B receptors promotes direct vasoconstriction owing to accumulation of intracellular Ca2+ (Miyauchi and Masaki 1999). Stimulation of endothelial cell ETB receptors, however, indirectly evokes vasodilation as a consequence of localised release of endotheliumderived relaxing factors (EDRFs), in particular nitric oxide (de Nucci et al. 1988). Indeed, it is the activation of ETB receptors that promotes the pronounced vasodilatory response of the pulmonary vasculature to ET-1 in mammals (Muramatsu et al. 1999). In ®sh, the functional equivalent of the pulmonary vascular bed is the microcirculation of the gill. In marked contrast to the mammalian lung, however, ET-1 elicits pronounced vasoconstriction of the branchial circulation (Olson et al. 1991; Sundin and Nilsson 1998; Stenslokken et al. 1999; Hoagland et al. 2000). The blood channels within the gill lamella lack true endothelial cells and thus unlike in the pulmonary circulation, EDRFs cannot contribute to ET-1 induced vasodilatation. The mechanism underlying the branchial vasoconstriction is contraction of lamellar pillar cells (Stenslokken et al. 1999). The resultant narrowing of blood channels leads to a redistribution of lamellar blood ¯ow whereby the peripheral or marginal channels are perfused preferentially at high velocity while the central channels comprising the lamellar vascular sheet receive reduced ¯ow at low velocity (Sundin and Nilsson 1998; Stenslokken et al. 1999). The marginal channels (in particular the inner or basal channels) are less ecient sites of gas transfer (Farrell et al. 1980) and their eciency would be lowered further by the increased velocity of ¯ow (Stenslokken et al. 1999). Therefore, re-routing of blood away from the central vascular sheet and the resultant loss of functional surface area would be expected to lower gas transfer eciency and cause a lowering of arterial PO2 (PaO2) and an increase of arterial PCO2 (PaCO2). The situation is somewhat more complex, however, because the branchial vasoconstriction associated with ET-1 injection is accompanied by a profound decrease in _ Stenslokken et al. 1999; Hoagland cardiac output (Q; et al. 2000). Consequently, the transit or residence time of the remaining blood ¯owing within the central lamellar channels (Stenslokken et al. 1999) is increased. An increased transit time would extend the period for gas equilibration across the gill epithelium and this potentially could increase gas transfer eciency (i.e. raise PaO2 and lower PaCO2). Thus, a
_ could counteract the eects of reduced decrease in Q functional surface area on arterial blood gases (Stenslokken et al. 1999). Therefore, the goal of this study was to assess the impact of these con¯icting eects on arterial blood gas tensions. This was accomplished by continuous measurements of PaO2, PaCO2 and arterial pH (pHa) before and after intravascular injections of ET-1 in rainbow trout (Oncorhynchus mykiss) or Paci®c spiny dog®sh (Squalus acanthias). These two species were selected for comparison because of their presumed dierent strategies for CO2 excretion. The trout relies exclusively on red blood cell (RBC) carbonic anhydrase (CA) to dehydrate plasma HCO3±, whereas the dog®sh is thought to utilise both extracellular and RBC CA in branchial CO2 transfer (Gilmour 1997; Tufts and Perry 1998; Gilmour et al. 2001). Thus, it was reasoned that the presence of extracellular CA in dog®sh would limit the impact on CO2 excretion of diusion-limitations and transit time changes imposed by ET-1. Moreover, it was hypothesised that the addition of bovine CA to trout plasma in vivo would enable rapid extracellular dehydration of HCO3± and thus abolish the inter-speci®c dierences in the PaCO2 response to ET-1.
Materials and methods Experimental animals Paci®c spiny dog®sh (S. acanthias) were collected by net during trawls by local ®shermen or angled by hook and line and transported to holding facilities at Bam®eld Marine Station (BMS; Bam®eld, Vancouver Island, British Columbia). The dog®sh were kept under natural photoperiod in a 75,000-l opaque circular tank provided with aerated full-strength seawater (30±32 mg ml±1) at 12°C and they were fed twice weekly with herring. In the present study a total of 18 dog®sh (average mass=1489131 g) were used within 4 weeks of their capture. Rainbow trout (O. mykiss) of both sexes were obtained from Linwood Acres Trout Farm. All ®sh were kept in large ®breglass tanks supplied with ¯owing, aerated and dechlorinated, city of Ottawa tap water: [Na+]=0.15 mmol l±1; [Cl±]=0.15 mmol l±1; [K+]=0.02 mmol l±1; [Ca2+]=0.40 mmol l±1; [HCO3±]=0.41 mmol l±1; pH=7.5±8.0; temperature=13°C) under a constant 12 h light-12 h dark photoperiod. Trout were fed to satiation on alternate days with commercial trout pellets until 24 h prior to experimentation. All ®sh were allowed at least 2 weeks to acclimate to the holding conditions before any experiments were performed. Two groups of ®sh were used; for experiments involving the recording of cardiovascular and blood respiratory variables, ®sh (n=40) weighing between 500 g and 800 g were used (average mass=63421 g). Smaller ®sh were used for experiments designed to examine the eects of ET-1 on plasma catecholamine levels (average mass=28712 g; n=16). Surgical procedures In vivo experiments Dog®sh were immersed in an aerated anaesthetic solution of benzocaine (ethyl-p-aminobenzoate; 0.1 g l±1) and transferred to an operating table where the gills were irrigated continuously with the same anaesthetic solution. Bi-directional cannulation of the coeliac
625 artery was required to establish an extracorporeal arterial blood circulation (see below). After making a ventral incision and externalising much of the viscera, the coeliac artery was cannulated in the orthograde and retrograde directions using polyethylene tubing (Clay-Adams PE 50). The viscera were re-inserted, the wound sutured, and the cannulae were secured ®rmly to the ventral musculature. A lateral incision was made in the caudal peduncle to expose and cannulate (PE 50; Clay-Adams) both the caudal vein and the caudal artery in the anterograde direction (Axelsson and Fritsche 1994). While the arterial cannula allowed caudal artery blood pressure (PCA) measurements, the caudal vein cannula permitted injections and/or repeated blood sampling. All cannulae were ®lled with heparinised (100 IU ml±1 ammonium heparin; Sigma Chemical, St. Louis, Mo.) dog®sh saline (500 mmol l±1 NaCl). In addition, the pericardial cavity was exposed with a ventral midline incision and the pericardium was dissected to expose the conus _ a 3S or arteriosus. To enable measurement of cardiac output (Q), 4S ultrasonic ¯ow probe (Transonic Systems, Ithaca, N.Y.) was placed non-occlusively around the conus. Lubricating jelly was used with the perivascular ¯owprobe as an acoustic couplant. Silk sutures were used to close the ventral and caudal peduncle incisions, and to anchor the cardiac output probe lead and the cannulae to the skin. In order to assess ventilatory amplitude and frequency, catheters (Clay-Adams PE 160) were inserted into the spiracular cavities and sutured to the head. After surgery, dog®sh were placed into individual ¯ow-through opaque acrylic or wooden boxes and left to recover for approximately 24 h before experimentation. Rainbow trout were anaesthetised in a solution of benzocaine (ethyl-p-aminobenzoate; 0.1 g l±1) cooled to 10°C. After cessation of breathing movements, the ®sh were transferred to an operating table and the gills were irrigated with the same anaesthetic solution throughout the surgery. For experiments assessing the eects of ET-1 on plasma catecholamine levels, a single indwelling polyethylene cannula (Clay-Adams PE 50) was inserted into the dorsal aorta according to the basic method of Soivio et al. (1975). For continuous measurements of blood respiratory variables using an extracorporeal arterial blood loop, a lateral incision (2 cm in length) was made at the level of the caudal peduncle approximately 4 mm below the lateral line. The caudal vein and artery were cannulated in the anterograde and retrograde directions, (ClayAdams PE 50 polyethylene tubing) using standard surgical procedures (Axelsson and Fritsche 1994). The incision was sutured using a running stitch and both cannulae were then secured to the body wall with silk ligatures. A small (1 cm) ventral incision was made to expose the pericardial cavity and the pericardium was dissected _ a 3S to expose the bulbus arteriosus. To enable measurement of Q, or 4S ultrasonic ¯ow probe was placed non-occlusively around the bulbus (see above). Small (1 cm2) brass plates were stitched to the external surface of each operculum to allow the measurement of ventilation amplitude using an impedance converter (Peyraud and Ferret-Bouin 1960). After surgery, ®sh were placed into individual opaque acrylic boxes supplied with ¯owing, aerated water where they were allowed to recover for approximately 24 h before experimentation.
In situ perfusion experiments A pithed perfused head preparation of dog®sh, externally irrigated with seawater and internally perfused with saline at 12± 14°C was employed, following the methods of Part et al. (1998). Irrigation rate (via external recirculation of aerated seawater entering the spiracles) was set to 2.0 l kg±1 min±1 and perfusion rate via the cardiac pump was set to 20 ml kg±1 min±1 in accord with values measured in vivo (Table 1). Perfusate PCO2 was set to 2.3 mmHg, and perfusate trimethylamine oxide concentration was set to 85 mmol l±1. All other experimental details, including composition of the perfusion saline, were identical to those described by Part et al. (1998). A three-way valve on the in¯ow cannula, after the Windkessel and close to the point of ventral aortic cannulation, served for injections of agonists and mea-
Table 1 Selected respiratory variables and circulating catecholamine concentrations in untreated (pre-injected) rainbow trout (Oncorhynchus mykiss) or dog®sh (Squalus acanthias). Values shown are means 1 SEM; n in parentheses (pHa arterial pH, fR respiration frequency, PaO2 arterial partial pressure of O2, PaCO2 arterial partial pressure of CO2) Rainbow trout PaO2 (mmHg) 106.03.4 (28) 2.690.16 (28) PaCO2 (mmHg) pHa 7.840.02 (28) a Vent. Amp. (cm or mmHg) 0.350.03 (14) fR (min±1) 73.42.4 (14) 2.851.38 (8) [Noradrenaline] (nmol l±1) ±1 [Adrenaline] (nmol l ) 0.950.24 (8)
Dog®sh 126.47.7* (11) 1.050.07* (11) 7.890.04 (11) 1.50.2 (11) 33.10.9* (11) 1.180.28 (9) 1.870.21 (9)
a In trout, ventilation amplitude (Vent. Amp.) was assessed by monitoring opercular impedance changes; in dog®sh, Vent. Amp. was assessed by monitoring pressure changes in the spiracle. Owing to the dierent methods of measurement, Vent. Amp. values for dog®sh and trout were not compared statistically *Indicates a statistically signi®cant dierence (P
