EFFECT OF EXTRACELLULAR HYPERTONICITY AND ALKALOSIS ...

February 27, 2004EUROPEAN JOURNAL OF MEDICAL RESEARCH 71Eur J Med Res (2004) 9: 71-77 © I. Holzapfel Publishers 2004EFFECT OF EXTRACELLULAR HYPERTONICITY AND ALKALOSIS ONENDOTHELIAL-DERIVED EA.hy 926 CELLS IN VITROC. Hirsch 1 and C. Haller 1, 21 Department of Medicine III, University of Heidelberg, Heidelberg, Germany2 Hegau Klinikum, Singen, GermanyAbstract: Endothelial and local metabolic mechanismscontribute in concert to the regulation ofblood flow. In vivo extracellular alkalosis inducesa vasoconstriction, hyperosmolarity a vasodilatation.The interaction between local metabolic andendothelial mechanisms is poorly understood.Therefore we investigated in endothelial-derivedEA.hy926 cells the secretion of endothelial modulatorsof vascular tone under hypertonic stresswith and without alkalosis: hyperosmolality wasgenerated by either the addition of NaHCO 3 (25,50, 100 mM, pH up to > 8) or mannitol (50, 100,200 mM) to the cell culture media. The cells werestudied using automated cell counting, measurementof the activity of the lactate dehydrogenase(LDH) and a bromo-deoxyuridine (BrdU) cell proliferationassay. Endothelin and cGMP, a surrogatemarker for nitric oxide (NO), were measuredwith specific ELISAs.EA.hy 926 cells formed stable monolayers invitro. The secretion of endothelin, but not ofcGMP was inversely correlated with the osmolalityof the incubation media: the endothelin concentrationin the supernatants decreased in bothmannitol- and NaHCO 3 -treated cells in a concentration-dependentmanner (152.4 ± 6.2 pg/ml(control) to 24.4 ± 2.4 pg/ml (200 mM mannitol),res. to 18.2 ± 2.7 pg/ml (100 mM NaHCO 3 ).Neither hypertonic bicarbonate nor mannitol solutionsdecreased the monolayer cell density orcell viability during the 6 hour incubation period.In conclusion, EA.hy926 cells are quite resistantagainst a 6-hour hypertonic/alkaline stress.Hypertonicity decreases the secretion of endothelinand has no effect on cGMP. At each level ofhypertonicity the endothelin concentration wassimilar in the NaHCO 3 and mannitol media arguingagainst a direct role of endothelin in alkalosis-inducedvasoconstriction in vivo. The decreasedsecretion of endothelin during hypertonicitycould contribute to the hyperosmolal vasodilationseen in vivo.Key words: Alkalosis; Hypertonicity; Mannitol;Alkalosis; Endothelin; VE-cadherin*Part of this work was presented at the XIVth WorldCongress of Cardiology 2002 in Sydney, Australia.Abbreviations: BrdU: bromo-deoxyuridine, FCS:fetal calf serum, NO: nitric oxide, cGMP: cyclicguanosine monophosphate, LDH: lactate dehydrogenaseINTRODUCTIONThe regulation of blood flow is mediated byneuro-humoral, myogenic, endothelial and localmetabolic mechanisms. The latter include hydrogenions, potassium, adenosine and hyperosmolalityof the extracellular fluid (Kuschinsky andWahl 1978; Sparks 1999). The local metabolicregulation of blood flow is a negative feedbacksystem: a locally increased metabolic demandstimulates a local vasodilatation through the releaseof vasoactive metabolites from metabolicallyactive cells; the vasodilatory metabolites are thenrapidly dissipated by the increased blood flow.This negative feedback system provides thematching between metabolic demand and bloodflow.In 1980 Furchgott and Zawadzki reported thatthe vasodilatation to acetylcholine is converted toa vasoconstriction by removal of the endothelium(Furchgott and Zawadzki 1980). Since this seminalobservation several endothelial mechanisms ofblood flow regulation have been identified. Thebest characterized endothelial blood flow modulatorsare nitric oxide (NO) (Palmer et al. 1987) andendothelin (Yanagisawa et al. 1988). NO causes avasodilatation by stimulating an intracellularguanylate cyclase (Moncada et al. 1988). ThuscGMP is often used as a surrogate parameter forthe short-lived NO (Jaimes et al. 2001; Mitchell etal. 1991). In contrast, endothelin induces a pronouncedvasoconstriction via activation of phospholipaseC and increase of intracellular calciumlevels (Highsmith et al. 1989; Luscher et al. 1992).Both the metabolic and endothelial regulationof blood flow have been intensely investigated. Incontrast, the interactions between endothelialmodulators and local metabolic factors are lesswell understood. The vascular reactivity can be alteredexperimentally by damaging the vascular endothelium(Haller et al. 1987; Liu et al. 1994;Urabe et al. 1991). However, the experimental removalor damage of the endothelium causes secondaryalterations like tissue edema and possibly col-

72 EUROPEAN JOURNAL OF MEDICAL RESEARCHFebruary 27, 2004lateral damage to the vascular smooth musclecells. These problems make the interpretation ofthe results of such experiments difficult.Secondary effects can be excluded by studying theinteractions of modulators of blood flow regulationin vitro. We chose the EA.hy 926 cell line asan endothelial model system in vitro. This cellline was originally established in 1983 by Edgelland coworkers through the hybridization ofhuman umbilical vein endothelial cells with thehuman lung carcinoma cell line A 549 (Edgell etal. 1983). EA.hy926 cells are a well characterizedendothelial cell model system (Bauer et al. 1992;Emeis and Edgell 1988; Li et al. 2002). We studiedthe effects of the metabolic stimuli alkalosisand/or hyperosmolality on the release of endothelinand cGMP from EA.hy 926 cells into the cellculture medium.Since extracellular alkalosis induces a vasoconstrictionand hyperosmolarity a vasodilatation invivo, the hypothesis was tested in vitro that alkalosisis associated with the release of the vasoconstrictorysubstance endothelin and hyperosmolalitywith that of vasodilatory NO.METHODSCELL CULTUREEA.hy 926 cells were grown in Dulbecco´s HAT(hypoxanthine 13,6 mg/l, aminopterine 0,176mg/l, thymidine 3,88 mg/l) medium supplementedwith 10% fetal calf serum (FCS) and 10000IU/ml penicillin and 10000 μg/ml streptomycin.Confluent cell monolayers were passaged at a 1:5ratio. The cells were used up to passage 20 afterthawing from frozen stock (obtained from Dr.Edgell via Prof. Friedel, Darmstadt, Germany).MEASUREMENT OF CELL SIZEConfluent cell monolayers were trypsinized andthe cells were suspended in an electrolyte solution.The cell suspension was injected into a cellcounter (Casy® 1 Model DT, Schärfe SystemGmbH, Reutlingen, Germany). The cells werecounted photoelectrically and the cell counts andcell diameters were plotted.EXPERIMENTAL INCUBATIONSThree groups of confluent cell monolayers fromthe same cell passage were studied in parallel.A: control, EA.hy 926 that were grown in regulargrowth medium as described above.B: cell monolayers incubated with hyperosmolalmedia containing mannitol (50, 100, 200 mM)C: cell monolayers incubated with sodium bicarbonate(25, 50, 100 mM).pH AND OSMOLALITYThe pH of the incubation solutions was measuredby using a pH-meter (WTW Weilheim, Germany)and osmolality was measured using an Osmomat030 (ABBOTT GmbH, Wiesbaden, Germany).ENDOTHELIN AND cGMP MEASUREMENTSThe concentration of endothelin and cGMP wasmeasured in the cell culture supernatant, respectivelycell lysates using commercial ELISA-kits accordingto the manufacturers’ instructions (endothelin:Cayman Chemical Company, Ann Arbor,Michigan, USA; cGMP: Amersham PharmaciaBiotech, Freiburg, Germany). The concentrationmeasurements were carried out photometrically(Modell Spectra, SLT-Labinstruments GmbH,Grödig/Salzburg, Austria), the background ofcell-free incubation control medium was subtractedfrom the readings. Standard curves were establishedfor each experiment in the phenol-red containingincubation media by adding endothelin/cGMPin known-concentrations.VIABILITY TESTSThe activity of LDH was measured in the conditionedsupernatants using standard clinical chemistrytechniques (LDH reagent kit, Rolf GreinerGmbH, Frickenhausen, Germany). The cells werecounted photoelectrically using an automatic cellcounter (Casy® 1 Model DT, Schärfe SystemGmbH, Reutlingen, Germany). The diameter ofEA.hy 926 cells (11 - 26 μm) was determined inpilot experiments.CELL PROLIFERATION ASSAYThe proliferation of EA.hy 926 cells was assessedby measuring the incorporation of bromodeoxyuridine(BrdU) into the cells. The measurementswere carried out using the proliferation ELISA-kit(Roche, Mannheim, Germany) according to themanufacturer´s instructions. The incorporationof the pyrimidine-analogue BrdU instead of thymidinewas measured photometrically after incubationwith a peroxidase-coupled anti-BrdU-antibodyand a subsequent reaction with tetramethylbenzidine.STATISTICSThe results are shown as means ± standard errorof the mean (SEM). Comparative statistics werecarried out with Student’s t-test, P < 0.05 wasconsidered significant. The Bonferroni-Holm correctionwas used for multiple comparisons.RESULTSVIABILITY TESTSFigure 1 shows the distribution of cell size. Thecell size was normally distributed between 11 and26 μm; cells with a diameter between 11 and 26 μmwere counted in the experimental measurements.Table 1 shows the pH and osmolality of the ex-

74 EUROPEAN JOURNAL OF MEDICAL RESEARCHFebruary 27, 2004Fig. 3. cGMP was measured as a surrogatemarker for NO. There was aminor, though significant, increaseof cGMP with 25 mM sodium bicarbonateand there was a trend towardslower cGMP with increasing bicarbonateor mannitol concentrations(mean ± SEM; n=12; *p < 0,05).Fig. 4. The photoelectric cell countsshowed no difference of the cell densitywith hypertonic and/or alkalineincubation media. (mean ± SEM;n = 17; *p < 0,05).Fig. 5. The cell proliferation rate wasassessed using the BrdU method. Theabsorbance values correlate with theamount of DNA synthesis, i.e. cellproliferation. Markedly hypertonicincubation media caused a significantreduction of the BrdU incorporationwhich was similar in the mannitoland in the bicarbonate media (mean± SEM; n = 18; *p < 0,05).cell damage despite the preserved cell counts. Thisis consistent with the moderately increased activityof LDH in the experimental incubation solutionscompared with control. However, massivecell death/major cell damage was ruled out, as mechanicalcell lysis yielded a much higher LDH activity(Fig. 6) than the one measured during thehyperosmolar incubations.In conclusion, the present results show thatEA.hy 926 cells are remarkably resilient againsthypertonic stress regardless whether this was inducedby the inert substance mannitol or by alkalinesodium bicarbonate. Moderate hypertonicitywith or without alkalosis had no discernible effecton the release of endothelin or cGMP from thecells in vitro. Massive hypertonic stress as exertedby 200 mM mannitol or 100 mM sodium bicarbonatereduces the concentration of endothelin, butnot of cGMP in the cell culture media. Togetherwith the largely preserved cell viability this heter-

February 27, 2004 EUROPEAN JOURNAL OF MEDICAL RESEARCH75Fig. 6. The mechanical lysis of thecells resulted in a major increase ofthe LDH activity in the cell culturesupernatants indicating the loss ofcell membrane integrity. In contrast,incubation with hypertonic bicarbonateor mannitol media caused only aminor increase in the LDH activityin the cell culture supernatantswhich was not different between thebicarbonate and mannitol media.This suggests that most cells had retainedtheir viability, since the cellmembranes during the hypertonic incubationsremained largely intact.(mean ± SEM; n=6; *p < 0,05).ogeneous effect on endothelin and cGMP arguesagainst major cell death as a cause of the reducedendothelin concentration.DISCUSSIONThe results of the cell viability and cell proliferationanalysis indicate that EA.hy 926 cells survivemoderate hypertonic stress over six hours. Neitheralkaline nor neutral hypertonicity has a significanteffect on cGMP concentration, only the maximalconcentration of sodium bicarbonate or mannitolinduced a significant decrease of the endothelinconcentration in the cell culture supernatant.There was no significant difference between the effectsof sodium bicarbonate or mannitol. Thus extracellularalkalosis had no special effect in additionto the effect of hypertonicity. This resultargues against a major role of endothelin in the alkalosis-inducedvasoconstriction in vivo. Conversely,the decreased endothelin concentration atthe highest level of hyperosmolality could contributeto the hyperosmolal vasodilatation in vivo.The present in vitro results cannot be directlyextrapolated to the in vivo situation. The endothelialcell culture model is an artificial systemunder static conditions, whereas in vivo the shearstress exerted by the flowing blood is an importantstimulus for the endothelial regulation ofblood flow (Morawietz et al. 2000; Ranjan et al.1995). Moreover, in vivo the endothelial cellmonolayer is at the interface between blood andvascular smooth muscle. The endothelial cellmonolayer is polarized and endothelial modulatorsof blood flow are secreted vectorially towardsthe smooth muscle cells (Wagner et al. 1992).Therefore the present results of the endothelialmodulators determined in cell culture supernatantscannot be directly translated into a potentialeffect on blood flow. However, the present invitro results can be subjected to appropriate testingin vivo.There is no generally accepted permanent endothelialcell culture model. The multiple problemsassociated with (primary) cell culture of freshlyisolated cells include the trauma of cell isolationand changes of the cellular phenotype in cultureand/or cell passaging in vitro. Therefore we studiedthe EA.hy 926 cell line, which is a well characterizedand widely accepted endothelial cell culturemodel. It has often been used as an endothelialcell culture model to study a variety of biologicalquestions including angiogenesis (Bauer et al.1992; Ribatti et al. 1999), coagulation (Edgell et al.1983; Emeis and Edgell 1988) and expression ofendothelin (Saijonmaa et al. 1991) and prostacyclin(Suggs et al. 1986).An important endothelial cell feature of EA.hy926 cells is the production and secretion of von-Willebrand-factor-related antigen (Edgell et al.1983). EA.hy 926 cells also express the endothelium-specificvascular endothelial cadherin (VE-cadherin(Rabiet et al. 1996) own data, not shown)and its cytoskeletal link ß-catenin and take up Di-Ac-LDL (Unger et al. 2002, own data, not shown).These features attest that EA.hy 926 cells have retaineda number of differentiated endothelial cellcharacteristics. This cell line is therefore a usefulmodel system to generate hypotheses in vitrowhich can be tested in vivo.Interactions between metabolic and endothelialmechanisms of blood flow regulation have beenreported (Green et al. 1996; Haller et al. 1987;Lavi et al. 2003). The effect of extracellular alkalosisand hyperosmolality on endothelial mechanismsof blood flow regulation is not well defined.Alkalosis causes a vasoconstriction (Yasue et al.1981) which has been associated with endothelin(Zuccarello et al. 2000). However, in the newbornlung a vasodilatory response to alkalosis has alsobeen reported (Balasubramanyan et al. 2000). Thisparadoxical dilatory response in the pulmonarycirculation has been related to NO-dependent(Mizuno et al. 2002; Vander Heyden et al. 2001)and NO-independent mechanisms (Fineman et al.1993; Vander Heyden et al. 2001).The effect of NO on the vascular reactivity tohyperosmolality also appears to be heterogeneous.Thus NO-dependent (Steenbergen and Bohlen1993) and NO-independent (Morcos et al. 1997)mechanisms have been described as potential contributorsto the hyperosmolal vasodilatation.The results of experimental manipulations ofthe endothelium in vivo are difficult to interpret

76 EUROPEAN JOURNAL OF MEDICAL RESEARCHFebruary 27, 2004because of secondary physiological changes. Thiswas the reason for the present in vitro experiments.On the other hand it is clear that the invitro findings cannot be directly translated intothe in vivo, let alone clinical situation. Vascularresponses may be different in vessels in situ andafter isolation in an organ bath (Gordon et al.2003). Therefore the in vitro cell culture approachcan be useful to generate hypotheses with theunderstanding that the results need to be verifiedin vivo.To our knowledge this is the first study on theinteraction between extracellular alkalosis/hyperosmolalityand endothelial vasoactive factors invitro. The results show an inverse correlationbetween hyperosmolality and endothelin secretedinto the incubation media. There was no differencebetween alkaline and neutral hyperosmolarity.Neither alkalosis nor hypertonicity induced asignificant change of the concentration of cGMPin the tissue culture supernatants after lysis of thecells. cGMP was used as a surrogate marker forNO (Mitchell et al. 1991). EA.hy 926 cells haveNO synthase (Li et al. 2002) and generate NO(Swiatkowska et al. 2000).In summary, despite the inherent limitations ofthe in vitro approach the results of the present experimentsprovide evidence for an inverse correlationbetween extracellular hypertonicity and endothelin,but not nitric oxide in EA.hy 926 cellsin vitro. They also suggest that under static invitro conditions hypertonicity is a more importantfactor in this interaction than alkalosis, sincealkalosis had no additional effects that could notbe ascribed to hyperosmolality alone. 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