Erythropoietin induced transmembrane calcium influx in essential hypertension

L a f e S c i e n c e s , Vol. P r i n t e d ~n t h e U S A

51,

ERYTHROPOIETIN

pp.

161-167

Pergamon

Press

INDUCED TRANSMEMBRANE CALCIUM INFLUX IN ESSENTIAL HYPERTENSION

Martin Tepel, Hubert Waschmowsk! and Walter Zldek Med Umv-Pollkhnak, Unlvers W of Muenster, Albert-Schwe~tzer-Str 33, W-4400 Muenster, Germany

(Recelved

in final

form M a y

7, 1992)

Summary The effects^ of erythropomtm (EPO) on cytosohc free calcmm concentration ([CaZ+]i) in platelets of 20 essentml hypertensive patients (HT) and of 25 normotenstve subjects.)(NT) were anvesngated using the fura2 technique In resting platelets [Ca-~-]. were not slgmficantly higher in H T compared t o N T ( 7 4 3 -+ 7 8 n ~ v s 5 9 ~ _+ 7 0 r i M , mean + SEM) A d d l t m n o f EPO slgmficantly mcreased [Ca-+]l m H T compared to NT (13 8 _+ 5 3 nM vs 0 9 + 1 9 nM, p < 0 01) EPO increased the amount of calcmm in lntracellular_stores This was confirmed independently using thrombm-mduced changes of [Ca z+ ]. m a calcmm-free medium and using chlorotetracychne as a marker of sto{-ed calcmm After preincubaUon with EPO thrombm-mduced changes of [Ca :z+] were significantly lower in H T compared to NT (306 1 _+ 30 0 nM vs 407.7 _+ 35 nM, p < 0 05) In a calcium-free medium after preincubatmn with EPO thrombminduced changes of [Ca z+ ], were significantly lower in H T compared to NT (54.7 _+ 11 8 nM vs 100 9 + 10 5-nM, p < 0 05) indicating lower storage capacity m H T It ~s concluded that elevated response to EPO may provide a powerful tool to evaluate dmgnosls and underlying pathophysmlogical mechamsms in essentml hypertensmn Since the mtroductmn of recombinant erythropoletm (EPO) m the treatment of renal anemia, development or worsening of hypertension emerged to be one of the most important side effects (1-3) Although increased whole blood VlSCOSW may contribute to the development of hypertension, EPO-lnduced changes in viscos W and in blood pressure show a poor correlation (4) On the other hand is generally agreed that abnormalities of cytosohc free calcium ([Ca~'-]l) may influence vascular tone m hypertension (5,6). Impaired calcium metabohsm m hypertension have often been studied in platelets (7-10). Platelets have a calcium dependent contraction couphng mechanism (11,12), and cellular signal transductlon pathways are similar m platelets and vascular smooth muscle cells, which are directly involved in the pathogenesls of hypertension (13,14) To evaluate an increased response of hypertensive cells to EPO, [Ca z+ ]1 was mvesngated m intact platelets

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Vol. 51, No. 2, 1992

METHODS 20 essential hypertensive patients (HT, 14 male, 6 females, mean age 47 +_ 3 years) and 25 normotenslve subjects (NT, 17 male, 8 females, mean age 42 _+ 3 years) were examined A diagnosis of hypertension was based on a blood pressure > 160/90 m m H g or a diastolic (Korotkoff phase V) blood pressure >95 m m H g obtained by conventional s p h y g m o m a n o m e m c methods. In H T diagnosis of essential hypertension was established m 3 measurements m the sitting position after exclusion of secondary forms of hypertension by appropriate chmcal, radlologlcal and laboratory examlnatmns, Le 24h urinary catecholamlnes, metanephrlnes and free cortisol, plasma renin activ W and aldosterone concentration and digital subtraction angiography of renal arteries. No subject had taken annhypertenslve or any other medication for at least 4 weeks before the date of measurements In H T systolic and dlastohc blood pressure was slgmflcantly higher compared to NT~172 +. 5 / 104 +. 2,oamHg vs 128 _+ 3 / 82 +_ 1 mmHg, p
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fluorescence values at 340 and 380 nm excitation was calculated. [Ca 2+ ] was calculated according to the equation reported by Grynkiewlcz et al. (18) [Ca z + ], = K* ( ~ - R m i n ) / ( R m a x R), where Rmin stands for the ratio in calcium free solution (range "1 07 to 1 30), Rmax for the ratio at calcium saturation (range 17 to 22), and K for 224 nmol/l*Fmln2/Fmax2, the latter representing the fluorescence m l m m u m and maximum at 380 nm excitation. Recombinant human erythropoietln in buffer solutmn was provided by Dr T Brune, Behringwerke, Marburg, Germany 50 ~1 EPO at a final concentration of 250 U / m l or an inactive EPO fragment (1-26) (Sigma) in an e~ulmolar concentration were added to 1000 ~1 of stirred platelets, and the change of [ C a Z + ] i 10 minutes after addition of E P O was measured In dose-response-experiments rura2 loaded platelets were incubated with various EPO concentrations from 5 to 250 U / m l for 30 minutes at room temperature prior fluorescence measurements and 0 15 U / m l thrombln (Behringwerke) was added to activate platelets The data out of these experiments were plotted according to Lmeweaver and Burk, and the apparent K D of EPO was calculated using a specific activity of 120,000 U / m g protein and a molecular weight of 34,000 g/Mol (19,20). To discriminate thrombln-mduced calcium reflux from calcium mobihsataon from mtracellular stores 1 mM extracellular calcium_was chelated by addmon of 5 mM E G T A at p H 7 4 (21), and 30 seconds later, changes in [CaZ+]i were induced by addition of 0 15 U / m l thrombln To determine calcmm concentration m lntracellular stores 10 uM chlorotetracychne (Sigma) were added to native washed platelets in the presence of 1 mM external calcium after premcubatlon with 250 U / m l EPO or buffer alone for 30 minutes. The fluorescence of chlorotetracyclIne was measured at 530 nm emission and 390 nm excitation The fluorescence showed an instantaneous increase followed by a slow increase of fluorescence, the latter lS due to fluorescence arising from binding of the calclum-chlorotetracychne complex to tuner surfaces of calcium-sequestering organelles Since there is an equlhbrlum between free and protein-bound calcmm in these organelles, the obtained fluorescence is propomonal to the free calcium concentration in these organelles (21,22) From all measurements mean values and standard error of mean are reported For staust~cal evaluanon of the data Wflcoxon's test was used and two-tailed p values less than 0 05 were considered to be s~gnlflcant RESULTS [Ca2+]1 in resting platelets tended to be higher in H T (74 3 -+ 7 8 nM, mean _+ SEM, n=20) than in NT (59 8 + 7 0 nM, n =25) but the differences between the two groups did not reach statlStlCak slgmflcance Fig 1A shows a representaUve raw trace Qf EPO-lnduced increase of [Ca Z + ] m platelets from H T Using platelets from 17 H T [CaZ + ]. 10 minutes after addmon of 25'0 U / m l EPO was slgmficantly higher compared to the con{rol value 10 minutes after addmon of buffer.alone (97 9 _+ 14 0 nM vs 84 1 + 10.8 nM, p < 0 01, Fig. 1B). Using platelets from 13 NT [Ca Z + ]. was not significantly different 10 minutes after addition of 250 U / m l EPO compared to the~ control value 10 minutes after addition of buffer alone (57 4 + 11 2 nM vs 56.5 _+ 11 0 nM, Fig 1B) The mean increase of [ c a z + ] , 10 minutes after addmon of E P O was significantly higher in platelets of H T compared to N T [13 8 + 5 3 nM vs 0 9 + 1 9 nM, p<0.01) indicating an elevated resaonse of hypertensives to EPO. Addition of buffer or EPO fragment (1-26) did not change [Ci z + ]i in either group To exclude an alteration of calcmm channels not specifically related to the action of EPO, the thrombln-mduced changes of [Ca:Z+] . were studied. A d d m o n of 0 15 U / m l thrombm increased [ c a z + ] , in H T to 244 0 - 25 ~.nM and m N T to 257 4 _+ 24 5 nM. EPO dose-dependently elevated i'hrombin-stimulated [Ca z+ ] The stimulating effect of E P O could be observed at concentrations as low as 5 U / m l (Fig. ~A) The apparent Kr~ was similar in H T (448 _+ 65 pM, n = 4 ) and NT (611 _+ 99 aM, n = 4 ) After preincubataofi"wath 250 U / m l EPO for 30 minutes, thrombin-stimulated [Ca ]i were significantly lower in H T compared to NT (306.1 _+ 30.0 nM vs 407.7 _+ 35.7 nM, p <0.05, Fig. 2B). The effect of a transplasmalemmal calcium influx on [Ca2+]~ may be buffered by a calcmm uptake into cellular stores To evaluate the influence of E P O ~n the amount of stored calcmm the thrombm-mduced increase was measured m an E G T A containing buffer (21). Fig 3 shows typical tracings for H T and NT. Using an E G T A containing buffer thrombin-

Erythropozetln and Essential Hypertenszon

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51, No.

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A

B

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p
01

n

s

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FIG 1 9+

Effect of erythropoletm (EPO) on cytosohc free calcium concentration ([Ca- ],) in fura2 loaded platelets A Typical raw traces of EPO-mduced increase (~f [Caz + ]1 in platelets from hypertensives, showing the fluorescence signal at 340 nm (upper cur~e) and 380 nm excltauon (lower curve), the 340/380 nm excitation ratto, and [CaZ+]l from one hypertenswe pattent Note that y-axis is not linear The tracing is representatwe for 17 slmdar experiments B [CaZ+], 10 minutes after addlnon of 250 U / m l EPO or buffer as control (C) in platelets from hypertensives (HT, n= 17) or normotenswes (NT, n--13) Mean _+ SEM and significance levels are indicated ,

k

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NT

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FIG 2 Effect of erythropoletln (EPO) on cytosohc free calcium concentranon ([Ca 2 + ]1) m thrombm-sUmulated platelets A Platelets were incubated with increasing EPO concentrations for 30 minutes and then [Ca z+], was measured 3 minutes after addition of 0 15 U / m l thrombln ( m e a n + SEM,'n=3) B Comparison of the effect of EPO on thrombln-stlmulated [Ca z + ]iln platelets from hypertensives (HT, n=17) and normotenswes (NT, n=13) -Prior to thrombin-stimulatlon platelets were incubated with 250 U / m l EPO or buffer alone (control) for 30 minutes

1992

Vol. 51, NO. 2, 1992

Ca2* (nM)

Erythropoietin and Essential Hypertension

A

Hypertensives

c,2. (nM) 300 -

B Normotens,ves

400 •

Erythropoletln

200

300 Erythropoletln 200 ~

¢

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~

100

100

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50

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Control

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FIG 3 Effect of erythropoletm on calcium storage in platelets from hypertensives (Fig 3A) and from normotensIves (Fig 3B) After incubation of fura2 loaded platelets with 250 U / m l erythropoletm or buffer alone (control) for 30 minutes 1 mM extracellular calcium was chelated by addmon of 5 mM EGTA, pH 7 4, and 30 seconds later platelets were stimulated by 0 15 U/ml thrombln Representanve tracings from 22 similar experiments are shown. Note that y-axis ]s not hnear.

Fluoreecence (arbltrlr

unltl)

~oo

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FGTA Ionomycin It

200

100

0

t Chlorotetracychne

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FIG 4 Effect of erythropoletin on calcium storage in platelets. After incubation with 250 U / m l erythropoietin or buffer alone (control) for 30 minutes 10 ~M chlorotetracychne were added to washed platelets and the fluorescence recorded At the indicated time periods E G T A (5 mM) and lonomycln (1 ~M) were added Representatxve tracings from 3 similar experiments are shown

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reduced changes of [Ca 2+ ]1 In platelets were significantly higher after prelncubaUon with 250 U / m l E P O for 30 minutes compared to control (HT" 85.4 -+ 22 7 nM vs 31.0 + 12 6 nM, p < 0 01; N T 118 1 -+ 11 0 nM vs 17 3 -+ 2 6 nM, p < 0 01) indicating increased calcium storage after E P O treatment In addition, the rise of stored calcium after prelncubatlon with E P O was significantly lower in platelets of H T compared to N T (54.7 -+ 11 8 nM vs 100 9 -+ 10 5 nM, p < 0 05) Chlorotetracycline was used as a marker of intracellular stored calcium (21,22), and the slow phase increase of fluorescence after addmon of chlorotetracychne to washed platelets was considered to reflect calcmm content in storage sites As shown m F~g 4 the increased amount of calcium in mtracellular stores after E P O treatment could be released by addmon of the ionophor lonomycln DISCUSSION EPO significantly increased [Ca2+]l in H T compared to NT. Slmdar effects of EPO on + [Ca ]t m platelets from spontaneously hypertensive rats were recently reported (23) Unspecffm effects seem unlikely, since E P O acts dose-dependently and since both buffer and EPO fragment (1-26) were not effective In addmon, platelet actwanon by other substances like thrombm showed similar effects m H T and NT These findings suggest that E P O interacts with a specific receptor on the platelet surface membrane E P O receptors are known to exist on several non-erythrold cell types, such as endothehal cells (24) and placenta (25) Using dose-response experiments an approximate KD-value could be calculated, and data need to be confirmed by further binding studies However, since the apparent KD-values were slmdar in hypertenswes and normotensives, a difference m receptor affmlf~ to E P O could be excluded The apparent KD-values of 448 pM for H T and 611 pM for N T m the present study are m agreement with those reported for erythrold cells (800 pM) or placenta (900 pM) (25) To evaluate whether EPO-mduced increase of [Ca2+]. was due to increased calcium influx or release from internal stores, the EPO-lnduced cha~ges of the calcmm content in internal stores were lnvest~gated using two different methods Thrombln-sUmulatton of platelets in an EGTA-contamlng buffer and chlorotetracychne were used to measure mtracellular stored calcmm Both methods showed that EPO increased the amount of stored calc.mm Since firstly the amount of stored calcmm increased under EPO, and secondly, [Ca- + ]1 was increased in hypertensives, it was concluded that E P O may act by increasing the calclum reflux Moreover, after mcubauon with EPO, thrombm-lnduced changes in [Ca2+], were sigmficantly lower m H T compared to NT. Using an EGTA-contalnmg buffer st was-shown that th~s difference could be atmbuted to a dlmlmshed calcmm storage in hypertenswe platelets m response to EPO In this regard, it is ~mportant to notice that the resting [CaL+]~ tended to be higher m H T compared to ~ T , although the differences were not stat~stlcally s~gnlficant Earher studms of restmg [Ca~+], in platelets have gwen contradmtory results Elevated (8,9) and identical (26,27) [CaZ+]l were reported m hypertenswes compared to normotenslves The different responses to EPO in H T and NT can be interpreted in several ways First, the response to EPO can be a biochemical marker of essential hypertension Since diagnosis of essential hypertension is based on the exclusion of secondary forms of hypertension, the response of the easily available blood platelet to a specific agomst may improve the diagnostic dilemma Similar tests based on plasmalemmal 1on transport systems were pubhshed earher (28,29) Second, the exaggerated E P O response of hypertensive cells may not be causally related to hypertension Rather ~t may be hnked indirectly, as it is discussed for the effect of lnsuhn and insuhn resistance in hypertensives (30,31). Third, the EPO-mduced increase of cytosohc calcmm may directly play a role in the development of hypertension This view is supported by recent studies, showing direct vasopressor effects of EPO on renal resistance arteries only in the presence of external calcium (32)

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ACKNOWLEDGEMENTS This work was supported by Deutsche Forschungsgemeinschaft (grant Zi 315/2-1). 1 2 3 4. 5. 6. 7 8 9 10 11 12. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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