We hypothesized that acute volume expansion by saline infusion triggers the release of endothelin-1. Bolus intravenous saline infusion (8 mL/min) in six groups of conscious Wistar rats and spontaneously hypertensive rats did not change mean arterial pressure or heart rate (n = 8 to 12). At 1 min after infusion, the plasma endothelin-1 level was significantly increased in Wistar rats and in spontaneously hypertensive rats by 42% and 61%, respectively (unpaired data). In 12 Wistar rats, the endothelin-1 level increased from 0.68 ± 0.13 to 1.19 ± 0.17 fmol/mL (mean ± SEM, P < .0001, paired data). Thus, acute volume load by rapid saline infusion increases plasma endothelin-1 levels. Vasoconstriction induced by endothelin-1 may counteract enhanced circumferential stretch created by volume expansion. Am J Hypertens 2003;16:1057-1061 @ 2003 American Journal of Hypertension, Lt

Abdel-Sayed, Saad

Brunner, Hans Rudolf

Nussberger, Juerg

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Brief CommunicationsVolume Expansion EnhancesPlasma Endothelin-1Saad Abdel-Sayed, Hans Rudolf Brunner, and Juerg NussbergerWe hypothesized that acute volume expansion by salineinfusion triggers the release of endothelin-1. Bolus intra-venous saline infusion (8 mL/min) in six groups of con-scious Wistar rats and spontaneously hypertensive rats didnot change mean arterial pressure or heart rate (n  8 to12). At 1 min after infusion, the plasma endothelin-1 levelwas significantly increased in Wistar rats and in sponta-neously hypertensive rats by 42% and 61%, respectively(unpaired data). In 12 Wistar rats, the endothelin-1 levelincreased from 0.68  0.13 to 1.19  0.17 fmol/mL(mean  SEM, P  .0001, paired data). Thus, acutevolume load by rapid saline infusion increases plasmaendothelin-1 levels. Vasoconstriction induced by endothe-lin-1 may counteract enhanced circumferential stretch cre-ated by volume expansion. Am J Hypertens 2003;16:1057–1061 © 2003 American Journal of Hypertension,Ltd.KeyWords: Endothelin, enzyme-linked immunoassay,vasoconstriction, hypervolemia, hoop (circumferential)stretch.E ndothelin-1 (ET-1) is a potent vasoconstrictor pep-tide hormone of 21 amino acids. It is predomi-nantly produced by vascular endothelial cells.1Many factors are known to stimulate endothelin release,such as hormones, growth and metabolic factors, hypoxia,and mechanical forces acting on the vascular wall.2Among the mechanical forces, vascular shear stress andcircumferential stretching may play a predominant role inendothelin release.3,4 The circumferential expansion of theblood vessels during the cardiac cycle is also known ashoop stretch.Fyhrquist et al5 showed that in anesthetized Wistar rats,basal and volume-stimulated atrial natriuretic peptide(ANP) release was decreased by pretreatment with endo-thelin antibodies. They suggested that endothelin physio-logically modulates ANP release. Furthermore, thespecific blockade of the endothelin ETA receptors bluntsthe peak ANP response to rapid infusion of saline inconscious Wistar rats.6 Thus, endothelin appears to stim-ulate the ANP response to acute volume overload throughthe ETA receptor.At present, despite good evidence of the involvement ofendothelin in the physiologic ANP response to acute vol-ume load, plasma endothelin concentrations have not beenmeasured under such conditions. Different rat models ofhypertension based on chronic volume overload such asDOCA-salt or 1-kidney, 1-clip hypertension (Goldblatthypertension) (1K1C Goldblatt rats) present increasedplasma ET-1 levels.7–9 The present study investigates theeffect of acute volume load by rapid saline infusion onplasma ET-1 levels in conscious normotensive Wistar andspontaneously hypertensive rats (SHR).MethodsExperimental AnimalsMale Wistar rats and SHR of 8 weeks of age, weighing220 to 246 g, were purchased from Iffa Credo (L’Arbresle,France). Rats were housed in transparent plastic cages in aquiet room at 22°C and maintained on standard rat chowcontaining 0.22% sodium and 0.20% potassium and tapwater ad libitum. The Governmental Animal Care Com-mittee approved the study protocols.Twenty-four hours before the study day, rats wereanesthetized by inhalation of 2% halothane in oxygen. Forarterial blood pressure (BP) measurements and blood sam-pling, polyethylene (PE) catheters were inserted into afemoral artery. Catheters consisted of a 6-cm PE-10 intra-vascular segment that was welded to a 25-cm PE-50tubing. For saline infusions into the femoral vein, similarcatheters and tubings were used but the tip of the intra-vascular segment was replaced by a less rigid 2-cm sili-cone tubing of 0.3-mm internal and 0.6-mm externaldiameter (Ulrich Swiss, St. Gallen, Switzerland). TheReceived June 3, 2003. First decision June 19, 2003. Accepted July 9,2003.From Division of Hypertension and Vascular Medicine, UniversityHospital, Lausanne, Switzerland.This work was supported by the Cardiovascular Research Founda-tion, Lausanne, Switzerland.Address correspondence and reprint requests to Dr. Juerg Nussberger,Hypertension Division, CHUV, CH-1011 Lausanne, Switzerland; e-mail:juerg.nussberger@chuv.hospvd.chAJH 2003; 16:1057–10610895-7061/03/$30.00© 2003 by the American Journal of Hypertension, Ltd.doi:10.1016/S0895-7061(03)01031-8Published by Elsevier Inc.PE-50 tubings of both the intra-arterial and the intravenouscatheters were threaded under the skin and exteriorized atthe back of the neck. The catheterized rats were housedindividually in plastic cages and kept in a quiet place at22°C.Study ProtocolOn the study day, conscious rats were placed in a plastictube for partial immobilization. After a 20-min rest, meanarterial pressure (MAP) and heart rate (HR) were moni-tored using the Notocord computerized system (Paris,France). Then 2 mL of arterial blood was collected forendothelin measurement from control Wistar rats (groupA, n  11) and control SHR (group B, n  8).To four different groups of Wistar rats and SHR, 8 or 10mL of 0.9% saline were rapidly infused in 1 min (SageInstruments, Cambridge, MA). At 1 min after the end ofthe saline infusion, arterial plasma ET-1 was measured,and MAP and HR were registered. In Wistar rats, onegroup (C, n  11) received 8 mL of saline without beingbled for endothelin measurement before the saline infu-sion. Another group (D, n  12) consisting of two lots of7 and 5 Wistar rats received 10 mL of saline after an initial2-mL bleeding for endothelin measurement. In SHR, onegroup (E, n  11) received 10 mL of saline without initialbleeding for endothelin measurement and another group(F, n  9) received 10 mL of saline after an initial 2-mlbleeding.Blood SamplingTwo milliliters of blood were collected from the femoralartery of conscious rats through an indwelling catheter.Blood was immediately transferred into prechilled glasstubes containing tripotassium ethylenediaminetetraacetateto achieve by mixture a final plasma concentration of 7mmol/L. Blood was centrifuged at 4°C for 10 min at 1660g. Plasma (1.2 mL) was stored in polypropylene tubes at20°C.Determination of Dilution FactorHematocrit (Hct) was assessed in heparinized capillarytubes (Baxter Healthcare Corp., Dearfield, IL) after 10 minof centrifugation in a bench-top microcentrifuge (Hettich,L’Abergement, Switzerland). The plasma dilution factor(DF) due to saline infusion was defined as the ratio of therelative plasma volume after infusion (100  HctA) overthe relative plasma volume before infusion (100  HctB):DF  (100  HctA)/(100  HctB).Measurement of ET-1Plasma immunoreactive ET-1 was measured by liquidphase extraction and subsequent ELISA, as previouslydescribed in detail.9 Briefly, endothelin was extractedfrom 1 mL of plasma by acetone-HCl (recovery 55% to75%) and specifically quantitated by a sandwich-typeELISA. The detection limit in plasma was 0.05 fmol/mL.Endothelin concentrations after saline infusion were cor-rected for plasma dilution by multiplying measured levelswith the DF, which varied between 1.04 and 1.26.Statistical AnalysisFor comparison of hemodynamic means, a one-way anal-ysis of variance was performed followed by Tukey’s mul-tiple comparison test. Endothelin concentrations beforeand after saline infusion were evaluated by unpaired orpaired Student t test as appropriate. The significance levelwas P  .05.ResultsEndothelin Responseto Acute Volume LoadFig. 1 demonstrates the effect of saline infusion in Wistarrats (left panel) and SHR (right panel) for unpaired andpaired data (upper and lower panel, respectively).In normotensive Wistar rats, plasma ET-1 was 42%higher after saline infusion (8 mL) at 1.26 0.13 fmol/mL(group C) as compared to control rats at 0.89  0.09fmol/mL (group A) (P  .05). Fig. 1 also demonstratesindividual increases in plasma ET-1 after saline infusion(10 mL) in the 12 rats where ET-1 was measured bothbefore and after saline infusion (group D). Plasma ET-1increased from 0.68  0.13 to 1.19  0.17 fmol/mL (P .0001). The average increase was 84%. This increase inET-1 after 10 mL of saline was greater than the enhance-ment found after infusion of 8 mL of saline (P  .05,unpaired t test, rank order comparisons for 8-mL infu-sions).In the SHR, plasma ET-1 was 61% higher after salineinfusion at 1.05 0.14 fmol/mL (group E) as compared tocontrol SHR at 0.65  0.03 (group B) (P  .05). Fig. 1(lower right panel) also depicts individual changes inplasma ET-1 after saline infusion in the nine SHR whoseET-1 was measured both before and after saline infusion(group F). Mean plasma ET-1 concentration increasedfrom 0.64 0.03 to 0.81 0.03 fmol/mL (P .02, pairedt test). The average increase was 28%. Among the nineSHR of this group F, six increased and two decreasedET-1 levels in response to saline infusion, whereas one ratkept ET-1 unchanged.BP and Heart Rate ResponseTable 1 shows effects of blood sampling and saline infu-sion on BP and heart rate in Wistar rats and SHR. After theinitial 2-mL bleeding, Wistar rats tended to increase heartrate and fairly maintain BP, whereas SHR had reducedheart rate (P  .05) and their BP decreased (P  .05).Saline infusion reversed these hemodynamic changes to-ward baseline. Immediately after the initial blood sam-pling, BP in SHR was considerably lower than in unbledcontrol SHR (P  .01). No such difference was found inWistar rats.1058 AJH–December 2003–VOL. 16, NO. 12ENDOTHELIN IN VOLUME-LOADED RATSDiscussionAcute volume load by rapid infusion of saline increasesplasma ET-1 levels in Wistar rats and in SHR. In Wistarrats, a net volume enhancement of 8 mL led to similar finalET-1 levels of 1.2 fmol/mL in two differently testedgroups of rats (C without initial bleeding, D with 2 mL ofinitial bleeding). Nevertheless, the 10-mL saline infusionafter an initial 2-mL bleeding increased the endogenousET-1 by 84% (paired data, 12/12 rats increased plasmaET-1), ie, more than the 8-mL saline infusion withoutblood sampling at baseline (42%, unpaired data). Thisenhanced ET-1 response may be due to various reasons.The baseline 2-mL hemorrhage certainly stimulated renalrenin secretion and hence increased angiotensin II levels,which are known to trigger ET-1 release.10 Kowano et al11FIG. 1. (Upper left panel) Intravenous 1-min infusions of 8 mL of physiologic saline in Wistar rats enhances plasma immunoreactiveendothelin-1 by 42% (*P  .05, unpaired t test). (Upper right panel) Intravenous 1-min infusions of 10 mL of physiologic saline in SHRenhances plasma endothelin-1 by 61% (*P .05, unpaired t test). (Lower left panel) Plasma endothelin-1 levels in 12 normotensive Wistarrats before and after 1-min intravenous infusions of 10 mL of physiologic saline: endothelin-1 levels were increased in all rats at the end ofthe infusion (P  .0001). The lowest five levels represent a complete second lot of Wistar rats. (Lower right panel) Plasma endothelin-1levels in nine SHR before and after 1-min intravenous infusions of 10mL of physiologic saline: endothelin-1 levels were significantly increasedat the end of the infusion despite the lack of response in three rats (P  .02). Mean  SEM, n  8 to 12, *P  .05, ***P  .0001.Table 1. Blood pressure and heart rate in Wistar rats and SHR (mean  SD, n  8 to 12)Rat SpeciesBlood Pressure (mm Hg) Heart Rate (min1)BaselineBloodSampled*SalineInfused BaselineBloodSampled*SalineInfusedWistarNot bled 119  3 119  3 117  2 446  8 446  8 428  8Bled 121  3 113  4 108  3 448  4 475  5 437  3SHRNot bled 154  2 156  1 153  1 398  5 387  7 401  8Bled 152  8 118  10†‡ 137  6 410  9 332  34† 365  19SHR  spontaneously hypertensive rats.* Ten l of blood for hematocrit measurement in all rats; two ml of blood for endothelin-1 measurement in “bled” rats.† P  .05 versus baseline, ‡ P  .01 versus not bled.1059AJH–December 2003–VOL. 16, NO. 12 ENDOTHELIN IN VOLUME-LOADED RATShave measured concomitantly increased plasma renin ac-tivity and ET-1 concentrations in anesthetized Wistar ratsafter withdrawing a similar volume of blood (7 mL/kg). Inour conscious rats, 10 mL of saline was infused after a2-min period of hypovolemia. The enhanced ET-1 re-sponse may, therefore, be due to both the 2-mL hemor-rhage at baseline and to the larger volume of saline infused(10 v 8 mL).Contrasting with previous results,9 baseline ET-1 levelsin Wistar rats were not higher than in SHR. Very low ET-1concentrations in one lot of rats enhanced the scatter ofET-1 levels in Wistar rats, whereas SHR had consistentlylow plasma ET-1. In SHR, all infusions consisted of 10mL of saline. The infusions increased the low endogenousplasma ET-1 levels by merely 28% when 2 mL of bloodwere collected before saline infusion, and by 61% withoutprior blood sampling. In six of nine SHR, saline infusionactually increased plasma ET-1, whereas two SHR re-sponded with a slight decrease in ET-1 and in one SHR,the ET-1 level remained unchanged.Our findings in conscious rats are consistent with themodulatory effect of endothelin on ANP release after rapidsaline infusion, as previously demonstrated by the use ofendothelin antibodies5 or ETA receptor blockers.6 Therapid volume expansion by saline infusion triggers a com-pensatory release of ANP that is lost in the presence ofendothelin antibodies or ETA receptor blockers.5,6 Circu-lating endothelin may directly stimulate atrial myocytes tosecrete ANP or indirectly by vasoconstriction and volumecentralization. It was therefore likely that saline infusionwould stimulate endothelin release. Our measurements inWistar rats and SHR verify this hypothesis. We used thesame infusion technique as did Fyhrquist et al5 to obtainconclusive results. Baertschi et al6 had also observed anET-1-mediated increase in plasma ANP after rapid salineinfusion (6.6 mL/min) and even to slow infusions (3.3mL/8 min). It would be of interest to confirm our findingthat circulating ET-1 also increased after the more physi-ologic slow infusions. Nevertheless, previous work in ratsalso suggested7–9 that higher plasma ET-1 levels prevailedin rat models in which BP predominantly depended onenhanced circulating volume (DOCA-salt, 1K1C). Lowerplasma ET-1 levels were found in SHR,9 where enhancedvasoconstriction rather than volume expansion maintainshigh BP. Clinical studies are now awaited to confirm ourfindings in humans.Interestingly, our SHR responded to a 2-mL bloodwithdrawal with a decrease in mean arterial pressure of 40mm Hg. Unlike Wistar rats, SHR were unable to maintainBP by a rapid baroreflex increase in heart rate. One minuteafter blood sampling, heart rate of SHR was actuallydecreased. Albeit SHR have a reduced baroreflex sensitiv-ity, it also appears that SHR as a vasoconstrictor model ofhypertension may respond more drastically to a 2-mLblood loss. The circulating volume before blood samplingmay be reduced in SHR and therefore a relatively largeblood loss is sensed. Furthermore, an already maximallyconstricted vasculature may have little functional reserveto respond to vasoconstrictor hormones such as norepi-nephrine, angiotensin II, or ET-1.We did not measure circumferential stretch (hoopstretch) in the present experiments. But our results arecompatible with the release of ET-1 in response to aug-mented circumferential stretch due to volume expansion.Lauth et al12 reported upregulated preproendothelin-1mRNA together with enhanced vascular endothelial ET-1content in response to increased intraluminal pressure inrabbit carotid arteries. Ziegler et al4 found in an artificialdevice for shear and stretch modeling that shear stresstransiently increased ET-1 mRNA. The hoop stretch con-cept would make sense. Stretched endothelial cells releaseET-1 to induce vasoconstriction, which protects endothe-lial cells against further stretching. High and low circulat-ing levels of ET-1 in different rat models of hypertensioncould be easily explained by expanded volume or predom-inant vasoconstriction.In conclusion, rapid infusion of saline increases ET-1concentrations in the circulating blood of Wistar rats andSHR. The volume-induced stretching of endothelial cellsmay cause a release of the protecting vasoconstrictorET-1.AcknowledgmentWe thank Catherine Amustutz for excellent technicalassistance.References1. Shimada K, Matsushita Y, Wakabayashi K, Takahashi M, Matsub-ara A, Iijima Y, Tanzawa K: Cloning and functional expression ofhuman endothelin converting enzyme cDNA. Biochem Biophys ResCommun 1995;207:807–812.2. Tamirisa P, Frishman WH, Kumar A: Endothelin and endothelinantagonism: roles in cardiovascular health and disease. Am Heart J1995;130:601–610.3. Harrison VJ, Ziegler T, Bouzoure`ne K, Suciu A, Silacci P, Hayoz D:Endothelin-1 and endothelin-converting enzyme-1 gene regulationby shear stress and flow-induced pressure. J Cardiovasc Pharmacol1998;31(Suppl 1):S38–S41.4. Ziegler T, Bouzoure`ne K, Harrison VJ, Brunner HR, Hayoz D:Influence of oscillatory and unidirectional flow environments on theexpression of endothelin and nitric oxide synthetase in culturedendothelial cells. Throm Vasc Biol 1998;18:686–692.5. Fyhrquist F, Sirvio M-L, Helin K, Sajnmaa O, Metsarinne K,Paakkari I, Asko J, Tikkanen I: Endothelin antiserum decreasesvolume stimulated and basal plasma concentration of atrial natri-uretic peptide. Circulation 1993;88:1172–1176.6. Baertschi AJ, Pedrazzini T, Aubert JF, Roatti A, Pence RA: Role ofendothelin receptor subtypes in volume-stimulated ANF secretion.Am J Physiol Heart Circ Physiol 2000;278:H493–H499.7. Larivie`re R, Thibault G, Schiffrin EL: Increased endothelin-1 con-tent in blood vessels of deoxycorticosterone acetate-salt hyperten-sive but not in spontaneously hypertensive rats. Hypertension 1993;21:294–300.8. Li J-S, Knafo L, Turgeon A, Garcia R, Schiffrin EL: Effect ofendothelin antagonism on blood pressure and vascular structure inrenovascular hypertensive rats. Am J Physiol 1966;271 (Heart CircPhysiol 40):H88–H93.1060 AJH–December 2003–VOL. 16, NO. 12ENDOTHELIN IN VOLUME-LOADED RATS9. Abdel-Sayed S, Nussberger J, Aubert JF, Gohlke P, Brunner HR,Brakch N: Measurement of endothelin-1 in experimental hyperten-sion and in healthy subjects. Am J Hypertens 2003;16:515–521.10. Scott-Burden T, Resink TJ, Hahn AWA, Vanhoutte PM: Inductionof endothelin secretion by angiotensin II: effects on growth andsynthetic activity of vascular smooth muscle cells. J CardiovascPharmacol 1991;17(Suppl 7):S96–S100.11. Kawano Y, Yoshimi H, Matsuoka H, Omae T: Changes in plasmaendothelin-1 concentration during blood volume depletion and ex-pansion: role of the cardiopulmonary baroreflex. Hypertens Res1995;18:43–46.12. Lauth M, Berger M-M, Cattaruzza M, Hecker M: Elevated perfusionpressure up regulates endothelin-1 and endothelin B receptor expres-sion in the rabbit carotid artery. Hypertension 2000;35:648–654.1061AJH–December 2003–VOL. 16, NO. 12 ENDOTHELIN IN VOLUME-LOADED RATS

Volume expansion enhances plasma endothelin-1

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