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CALCIUM UPTAKE KINETICS INTO BRUSH-BORDER MEMBRANE VESICLES : HIGHER VMAX IN THE SPONTANEOUSLY HYPERTENSIVE RAT THAN IN NORMOTENSIVE CONTROL U. Hennessen,*

T. Dr:tieke,*

L. Comte,* M. C. Steuf,*

D. A. McCarron,#

and B. Lacour*

* INSERM U90 and D$artement de NCphrologie, HGpital Necker, Paris, France # Division of Nephrology and Hypertension, Oregon Health Sciences University, Portland, Oregon Received

June

5, 1990

Arterial hypertensionin the spontaneouslyhypertensiverat (SHR) is associatedwith an abnormalCa2+homeostasis, comparedwith its normotensivecontrol, the Wistar Kyoto rat (WKY). In particular, epithelial Ca2+transportis perturbed,with intestinal absorption and renal tubular reabsorptionbeingdecreasedin the adolescentand adult SHR. In the presentstudy we examinedCa2+uptakeinto isolatedduodenalbrush-bordermembrane vesicles(BBMV) in 12-14week-old malerats. This uptakecan be separatedin a nonsaturableand a saturablecomponent.The latter follows Michaelis-Menten kinetics. Vmax of this componentwasfound to be significantly higher (~40.05) in SHR than in WKY (0.58s. 19 versus 0.35kO.06nmol/mg protein x 10 set, mean&SD)whereasKmdid not differ. Thus, the defect in the intestinalCa2+absorptionpreviously identified in the SHR of this ageis not due to a decreasein Ca uptakeat the level of the duodenalbrush-border membrane,but is most likely locatedin the baso-lateralmembrane. 0 1990Academic Press,rnc.

An increasingnumberof reportsin animalsandin manindicate that calcium (Ca) plays an important role in arterial hypertension(1). Alterations of Ca metabolismhave been particularly well describedin the SHR comparedwith its normotensivecontrol rat, the WKY (2-5). Moreover, provision of an increaseddietary Ca (2% by weight) to the young SHR prevented the emergenceof hypertension(6,7). During recent years, we (8) and others(9) have found a decreasein intestinalCa absorptionin the mature SHR, even though this finding is still a matter of debate(10). A defect in intestinal Ca transporthas beenobservedin different modelsincluding the whole rat, the isolatedduodenalsegment, andthe isolatedenterocyte (11). However, even the useof isolatedcells did not allow us to ascertainwhether the alteration waslocalized at the brushbordermembraneor the basolateralmembranelevel. Therefore, we studiedCa uptakein isolatedduodenalbrushborder membranevesicles(BBMV) in SHR andWKY rats. MATERIALS

AND METHODS

Animals. Male SHR and WKY were obtained at the ageof 8 weeksfrom IFFA CREDO (Centrede rechercheet ClevagedesOncins; l’Arbesle, France), During 4-6 weeksthey were fed a synthetic diet containingCa 1% , P 0.46% , and Vitamin D2 2200 U/kg food (Centre National de RecherchesZootechniques,la Mini& par Versailles, 0006-291X/90 Copyright All rights

$1.50

0 1990 by Academic Press. Inc. of reproduction in any form reserved.

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France). Animals had free access to food and water. They were kept in well-lit rooms with a 12 h light/dark cycle. All experiments were performed on 12-14 week-old rats, which were fasted during the 18h before sacrifice. Preparation of brush-border membrane vesicles (BBMV). Animals underwent a light ethylether anesthesia. Duodenum was excised and rinsed twice with ice-cold saline. Intestine was opened and gently scraped. Mucosa was immediately frozen in liquid nitrogen and stored at -7O’C till the preparation of vesicles. Four rats were used for each vesicle-preparation and vesicles from SHR and WKY were prepared the same day in parallel. We used the method described by Forstner et al (12) with slight modifications. Briefly, duodenal mucosa was suspended in chilled buffer I (20 mM Hepes, 5 mM NajEDT.A, 0.1 mM PMSF at pH 7.4) and homogenized by a Polytron. After centrifugation for 10 min at 3,000 g the supernatant was discarded and the pellet was resuspended in buffer I and recentrifugized for 10 min at 3,000 g. Washing and centrifugation were repeated two times and the pellet was suspended in chilled buffer II (20 mM Hepes, 0.8 mM NaJEDTA, 90 mM NaCl, pH 7.4). The suspension was allowed to rest at 4°C during 30 min and afterwards filtered twice under vacuum. The resulting filtrat was spun for 10 min at 3,000 g. The pellet was resuspended in buffer III (100 mM mannitol, 20 mM HEPES, 0.1 mM PMSF, pH 7.4). After 20 min centrifugation at 24,000 g the pellet was collected and homogenized in a glass-teflon Potter and to a final volume of 20 ml buffer III MgS04 was added (final concentration of 1 mM). The brushborder vesicles were contained in the pellet resulting from a second centrifugation at 24,000 g. Enzyme measurements. Sucrase activity was measured according to the technique described by Dahlquist (13) and Na+,K+-ATPase activity was measured by a slightly modified method of Bergmeyer (14) and defined as the ouabain sensitive hydrolysis of ATP. Protein was measured as described by Lowry et al. (15) using bovine albumin as standard. Uptake studies . Buffer III was used as incubation solution. About 150 pg of BBMV protein were incubated at 2S’C during 10 set in a solution with final Ca2+ concentrations varying between 0.036 and 1.0 mM to which radiolabelled %a was added. Reaction was stopped by the addition of chilled stop solution containing (in mM) 100 mannitol, 20 HEPES, ‘75 MgC12, and 1 EGTA at pH 7.4 and the samples were immediately filtered under vacuum (Millipore filters, type HA, 0.45 pm, 25 mm in diameter). The filter was additionally washed with 7.5 ml stop solution. Radioactivity remaining on the filter was determined by liquid scintillation after dissolving filter in 5 ml of Pica Fluor 30 (Packard). Kinetic constants were derived from each individual experiment. Kinetics of saturable and non-saturable Ca transport were calculated as described by Wilson et al. (16). Vmax (maximal rate of saturable Ca uptake at infinite medium calcium concentration) and Km (medium Ca concentration of half Vmax) were calculated for each preparation by the method of Lineweaver and Burk (17). Statistical analysis was done by Student’s t test. Materials. N-2-hydroxyethylpiperazine N-2 ethanesulfonic acid (HEPES), phenylmethylsulfonyl-fluoride (PMSF), ethyleneglycol-bis-R-aminoethylether NNN’N’tetraacetic acid (EGTA), ethylenediamine-tetraacetic acid &odium salt (Na3EDTA) and other chemicals were from Sigma Chemicals (St Louis, MO). RESULTS We used sucrase activity as a marker for the brush-border membrane and ouabainsensitive Na+,K+-ATPase activity as a marker for the basolateral membrane. Sucrase activity enrichment was lo-fold for both strains and Na+,K+-ATPase activity was 0.6fold diminished in WKY (Table 1). These values correspond to values reported in the literature. (15,16). In BBMV preparations of SHR we were unable to detect duodenal Na+,K+- ATPase activity even when high concentrations of ouabain were used (10 n&I), and we detected only a weak activity in WKY, indicating only minor contamination by baso-1ate:ral membrane. 743

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Table 1 MARKER ACTIVITY

ENZYME

Na+,K+-ATPase ACTIVITY (U/mg protein) WKY SHR

SUCRASE ACTIVITY (U/mg protein) WKY SHR Homogenate

RESEARCH

0.23+0.04

0.22&0.08

0.20f0.03

0.16f0.05

BBMV 2.5tiO.91 2.49M.82 0.09&0.03 0 (ND*) Enzyme marker activities were measured in homogenate and in final duodenal BBMV of SHR and WKY . Values are the results of 6 separate preparations and expressed as mean&SD. * not detectable.

Total Ca2+uptake by isolatedBBMV is shownin Fig. 1. It tendedto be greater in SHR than in WKY but the difference was not statistically significant. Similarly, non-saturable uptake was comparablein both strains(Fig. 1). SaturableCa2+uptakewascalculated as total uptake minusnon-saturableuptake.It was significantly greaterin SHR than in WKY, asillustrated in Fig. 2. When expressedby Michaelis-Menten analysisVmaxwas significantly higher in SHR than in WKY (Table 2). Values for Km did not differ significantly betweenthe two strains. DISCUSSION The novel finding of the presentstudy wasan increasein the Vmaxof BBMV mediated Ca2+transportin the SHR, comparedwith that of the WKY. The apparentaffinity (Km) of this transport systemfor Ca2+wasnot different betweenthe two strains.The duodenumwas usedexclusively for the presentin vitro studiesbecausethis part of the smallintestinerepresentsthe siteof maximal active Ca2+transport(19). This choice restricted the numberof experimentswhich could be done with the samepreparation.

1.0

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0

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0.6

0.6

[Ca2*] incubation

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Figure 1. Total Ca2+uptake by duodenal BBMV of WKY and SHR .Total Ca*+ uptake as describedin Materials and Methods by duodenal BBMV of WKY (A) and SHR (B) was measuredas with increasingCa*+concentration (0.036;0.069;0.1; 0.13; 0.49;0.94 mM) in the incubation solution. Presentedare means of 6 separateexperiments in duplicate for eachstrain of rats; calculated non-saturableuptake is presentedby (- -). 744

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1 0

solution

2. Saturable Ca2+ uptake by duodenal BBMV of WKY and SHR. Saturable uptake of Ca into duodenal BBMV of WKY a and SHR (+) was calculated by reducing total Ca uptake by non-saturable uptake. Presented are means of 6 separate experiments in duplicate for each group of rats.

BBMV were essentiallypure, closed,and right-side out, as shownby electron microscopy. Sucraseactivity enrichmentof isolatedBBMV correspondedto valuesfound by other groups(18,19). Moreover determinationsof Na+,K+-ATPase activity indicated only minor contaminationby basolateralmembranes(BLM). All experimentsof BBMV Ca2+uptake were done at the time point of 10 set after addition of Ca2+ in order to ensurethat the uptakemeasuredwasstill in its initial linear phase.In contrast to other authorswho chosetime points at 3 set (16) or 7 set (21), our experimentsshowedlinear uptake up to 30 set (datanot shown).We chose10 set for technicalreasons.Working at this time interval enabledusto separateclearly saturablefrom non-saturabletransportof Ca2+into duodenalBBMV. Non-saturable“uptake” correspondsto diffusive transportof Ca2+into the vesicular spaceand non-specificbinding to the outer surfaceof vesicles. The existenceof a saturableuptakeimpliesa mediatedtransportsystemwhich we shall call transporter. Not much is known aboutthe mechanismsunderlying mediatedCa2+ transport. Experiments exploring the interaction of strontium andmagnesiumwith Ca2+ transport appearto indicate a mobilecarrier ( 16).

-KINETIC

CONSTANTS

Table 2 OF THE SATURABLE

Vmax -

(nmol/mg WKY

protein

0.35kO.06

Caz+ UPTAKE

IN BBMV

Km x 10 s)

(nmol/mg

protein)

0.06f0.02

SHR 0.58f0.19*" 0.11+0.06 Values are expressed as means?SD.Vmaxand Km were calculated described in Materials and Methods . ** ~~0.05.

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The present results which have identified enhanced mediated Ca2+ uptake by duodenal BBMVs in the SHR have to be interpreted in the light of our previous findings in isolated enterocytes (10,22) which indicated a decreased whole cell Cap+ uptake in the 12- 14 week-old male SHR compared with the WKY of same age and gender. It is conceivable, that the Ca2+ penetrates more rapidly into the cell because of abnormal brush border membrane (BBM) structure (23). Alternatively, a greater number of Ca2+ channels could be open at the site of the BBM level in response to an impaired Caz+ extrusion at the site of the BLM, leading to an increase in cytosolic free Ca2+ and thereby an activation of the Ca2+-ATPase via the Ca2+-calmodulin complex. For technical reasons , the determination of enterocyte cytosolic free Ca2+ is still difficult. In preliminary experiments, it has been estimated to be approximately 130 nmol/l in the intestinal villus epithelial cell (24). In the SHR or WKY enterocyte, a precise measurement of cytosolic free Ca 2+ has not yet been achieved. On the other hand, determinations in erythrocytes and platelets from hypertensive subjects and animals (2526) have revealed an increase of free cytosolic Ca 2+ which could be due to alterations of the kinetic properties of the plasma membrane Ca 2+-ATPase pump (27). In enterocytes this might also be a plausible explanation, though it has not been explored yet. Finally, it is also conceivable that the Ca2+ ion, immediately after its entry into the SHR’s enterocyte, is bound or transported into structures from which it is less accessible than in the WKY for outward transport across the BLM. Taken together, our present and previous findings point to a perturbation of Caz+ extrusion at the level of the baso-lateral membrane. ACKNOWLEDGMENTS This work was supported by grants-in-aid from the ComitC des Salines, France, and the National Dairy Council, United States. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Young, E.W., Bukoski, R.D., McCarron, D.A. (1988) Proc. Sot. Exp. Biol. Med. 198, 141-146 McCarron, D.A. (1989) Kidney Intemat. 35717-736 Resnick, L.M. (1989) Am. J. Hypertens. 2, 179S-18% Drtieke, T., Bourgouin, P., Lacour, B. (1990) Miner. Electrolyte Metab. 16,6-l 1 Bourgouin, P., Lucas, P., Roullet, C., Pointillart, A., Thomasset, M., Brami, M., Comte, L., Lacour, B., Garabedian, M., McCarron, D.A., Drtieke, T. (1990) Am. J. Physiol. (in press) McCarron, D.A., Lucas, P., Shneidman, J., Lacour, B., Drtieke, T. (1985) J. Clin. Invest. 76, 1147-l 154 Hatton, D.C., Scrogin, K.E., Metz, J.A., McCarron, D.A.( 1989) Hypertension 13, 622-629 Lucas, P.A., Brown, R.C., Driieke, T., Lacour, B. Metz, G. McCarron, A.D.(1986) J. Clin. Invest. 78, 221-227 Schedl, H.P., Wilson, H.D., (1988) Hypertension [Dallas] 12, 310-316 Bindels, R.J.M., Van den Brock, L.A.M., Jongen, M.J.M., Hackeng, W.H.L., Lowick, C.W., van OS, C.H. (1987) Pfliigers Arch. 408,395-400 Roullet, C., Young, E.W., Roullet, J.-B., Lacour, B., Drtieke, T., McCarron, D.A. (1989) Am. J. Physiol. 257, F574-F579 746

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Forstner, G.G., Sabesin, S.M., Isselbacher, K.J. (1968) Biochem. J. 106, 381390 Dahlquist, A. (1964) Analyt. Biochem. 7, 18-25 Penefsky, K., Bruist, M.D. (1974) in Methods of Enzymatic Analysis, Vol II, 324-328, Ed. by Bergmeyer Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J.Biol. Chem. 193,256-275 Wilson, H.D., Schedl, H.P., Christensen, K. (1989) Am. J. Physiol. 26, F446F453 Ldneweaver, H., Burk, D. (1934) J. Am. Chem. Sot. 56, 658-666 Gishan, F.K., Wilson, F.A.(1985) Am. J. Physiol. 248, G87-G92 Kabata, H., Inui, K.-I., Itokawa, Y. (1989) Nutr. Res. 9, 791-799 Kimberg, D., Scachter, D., Schenker, H. (1961) Am. J. Physiol. 200, 12561262 Gishan, F.K., Arab, N., Nylander, W., (1989) Gastroenterology 96, 122-129 Lucas, P.A. Roullet, C.M., Duchambon, P., Lacour, B., Dang, P., McCarron, D.A., Driieke T. (1989) Am. J. Hypertens. 2,86-92 Driieke, T., Hennessen, U., Nabarra, B., Ben Nasr, L., Dang, P., Thomasset, hl., Lacour, B., Coudrier, E., McCarron, D.A. (1990) Kidney Intemat. (in press) Emmer, E., Rood, R.P., Wesolek, J.H., Cohen, M-E., Braithewaite, R.S., Sharp, G.W.G., Murer, H., Donowitz, M. (1989) J. Memb. Biol. 108, 207-215 Brushi, G., Brushi, M.E., Caroppo, M., Orlandini, G., Pavarani, C., Cavatorta, A. (1985) Clin. Sci. 68, 179-184 Resnick, L.M., Gupta, R.K., Bhargava, K.K., Laragh, J.H. (1990) Am. J. Hypertens. 3,59A Vincenzi, F.F., Lindner, A., Hinds, T.R. (1990) Am. J. Hypertens. 3, 52A

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Calcium uptake kinetics into brush-border membrane vesicles: higher Vmax in the spontaneously hypertensive rat than in normotensive control.

Arterial hypertension in the spontaneously hypertensive rat (SHR) is associated with an abnormal Ca2+ homeostasis, compared with its normotensive cont...
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