Biochimica et Biophysica Acta, 1055 (1990) 63-68
63
Elsevier BBAMCR 12792
Endothelin isopeptides evoke C a 2 + signaling and oscillations of cytosolic free [Ca 2+] in human mesangial cells Michael S. Simonson 1, T o m o h i r o Osanai 1 and Michael J. D u n n 1,2 Departments of I Medicine, 2 Physiology and Biophysics, School of Medicine, Case Western Reserve University, Division of Nephrology, University Hospitals of Cleveland, Cleveland, OH (U.S.A.)
(Received 24 January 1990) (Revised manuscript received18 June 1990)
Key words: Signaltransduction; Second messengersystem; Calcium; Calcium channel; Endothelium; Glomerular mesangium
Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+ ] ([Ca2+]i) in human giomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [CaZ+]i constitutes a common pathway of signal transduction for the ET peptide family.
Introduction Endothelins (ET) are a family of 21-amino acid peptides ( - 2 . 5 kDa) secreted by endothelial cells in vitro and in vivo [1]. At least three distinct genes encode highly conserved isopeptides designated ET-1, ET-2 and ET-3 (see Refs. 2 - 4 for review). Sarafotoxins, a group of peptides purified from venom of Atractaspis engaddensis, share close sequence homology with ET [5], and it seems likely that the two peptide families share a common evolutionary origin. Although the physiologic and pathophysiologic roles of ET remain to be elucidated, ET exerts profound effects on vascular and nonvascular smooth muscle contraction, mitogenesis and gene expression [2-4]. Binding sites for radiolabeled ET are widely distributed in the body [2,3] and activation of these putative ET receptors could mediate a variety of biological functions.
We and others [6,7] have previously shown that in rat mesangial cells ET-1 evokes a Ca 2÷ signaling system via activation of the phosphoinositide cascade. The increase in cytosolic free [Ca 2÷ ] ([Ca 2÷ ]i) results from phospholipase-C catalyzed formation of inositol 1,4,5-trisphosphate to release Ca 2÷ from intracellular stores and from influx of extracellular Ca 2÷ [6]. In light of the actions of ET-1 in rat mesangial cells, we examined the effects of ET-1 and other ET isopeptides and sarafotoxin S6b on Ca 2÷ signaling in human mesangial cells. We report here that (i) all ET isopeptides and S6b evoke complex patterns of Ca 2÷ signaling; that (ii) ET-1 and ET-2 stimulate oscillations of [Ca 2÷]i previously undescribed in mesangial cells; and that (iii) desensitization of the Ca 2+ signaling induced by ET-1 is independent of protein kinase C activation.
Materials and Methods
Abbreviations: ET, endothelin; S6b, sarafotoxin S6b; [Ca2+ ]i, cytosolic free [Ca2+ ]; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethylester; DTPA, diethylenetriaminepentaacetic acid; KHH, Krebs Henseleit-Hepesbuffer. Correspondence: M,J. Duma, Department of Medicine, University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
Materials. Fura-2 (fura-2 acetoxymethylester) and BAPTA were from Molecular Probes. Ionomycin, Bay K 8644 and nifedipine were from Calbiochem. Ethylene glycol-bis( fl-aminoethyl e t h e r ) - N , N , N ' , N ' - t e t r a a c e t i c acid (EGTA) and D T P A were obtained from Sigma. ET-1 was purchased from Peninsula Laboratories, whereas ET-2, ET-3 and sarafotoxin S6b (S6b), from Peptide Institute, were a generous gift from Dr. Thomas
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
64 Rimley, Glaxo Pharmaceuticals. Stock solutions of ET isopeptides (0.1 mM) in sterile H20 were stored in aliquots at - 4 0 ° C for no more than 3 weeks. Human mesangial cell isolation and culture. Human glomerular mesangial cell strains were prepared and characterized as previously reported [8] by partial digestion of isolated glomerular explants by bacterial collagenase to form 'glomerular cores'. Human kidneys were obtained (approved protocol of Institutional Review Board, University Hospitals) immediately after surgical nephrectomy or from donor, cadaveric kidneys that were judged unsuitable for transplant. Cells were maintained in RPMI 1640 supplemented with 17% fetal bovine serum, 100 U / m l penicillin, 100 # g / m l streptomycin, 5 / ~ g / m l each of insulin and transferrin, and 5 n g / m l sodium selenite at 3 7 ° C in 5% CO2/95% air. Monolayers were subcultured every 7-10 days using t r y p s i n / E D T A , and media in all cultures was renewed every 2-3 days. Four different cell strains (passages 3-15) were used in these experiments. Determinations of [Ca 2 +] i. [Ca2 + ]i in populations of mesangial cells was measured fluorimetrically using fura-2. As reported [6,8], confluent monolayers on 1.4 cm 2 Aclar coverslips (Allied Engineered Plastics) were loaded with 1 laM fura-2 acetoxymethylester for 40 min at 37 ° C followed by a 20 min incubation in fura-2-free RPMI 1640 to allow for intracellular dye cleavage. The cells were kept at 4 ° C in K H H (pH 7.4) until used. Coverslips were mounted in a cuvette with 2 ml K H H maintained at 37 ° C with constant stirring and fluorescence was measured with a University of Pennsylvania Biomedical Instruments Group spectrofluorimeter [9] using an air-driven rotary shutter for alternate excitation at 340 and 380 nm wavelengths with emission set at
A. ,--. 975 ~
oJ o
121
,u,
510 nm. Calibration was made using ionomycin followed by EGTA-Tris, as described [6,8], except that the formula of Grynkyewicz et al. [10] was used to calculate [Ca2÷ ]i assuming the K d of the fura-2-Ca 2÷ interaction to be 224 nM. All agonists were tested independently for autofluorescence. In experiments in which BAPTA was used, 10/~M BAPTA acetoxymethylester was added and coincubated with fura-2 in RPMI 1640 exactly as described above. In some coverslips leakage of fura-2 was assessed by the addition of 100/tM Mn 2÷ followed by rapid chelation with 4 mM DTPA. Results E T isopeptides and S6b cause both transient and sustained increases in [Ca 2 +] i
When added to human mesangial cell monolayers loaded with fura-2, 0.01-0.1/~M of ET-1, ET-2 and S6b caused a biphasic increase in [Ca2+]i consisting of a rapid (2-5 s) transient increase followed by a lower but sustained phase (Fig. 1A, B and D). The peak A[fa2+]i was dose-dependent with ET-1 = ET-2 > S6b >> ET-3 (Fig. 2). 0.1 /~M ET-1 and ET-2 caused a longer sustained phase at higher levels of [Ca2+]i than did equimolar additions of S6b. 0.1 #M ET-3 caused a minimal increase in [Ca2+]i in only one of the four cell strains studied (Fig. 1C). It is interesting to note that in 3 of the 4 cell strains studied, 0.1 ttM ET-1 and ET-2 (but not S6b or ET-3) caused oscillations of [Ca 2+ ]i that appeared immediately following the initial transient increase in [Ca2+]i (Fig. 1A and B). The oscillations were periodic with approx. 30-40 s between peaks and were sustained for only 2-3 min after the addition of ET-1 or ET-2. Oscillations of [Ca2+]i were not observed at lower
I030.
B.
107 - ,u ~ ET-I, O.I,uM
2501 ~
~ ET-2, O.I~M
I
E.
7 0 '-
e E T - 3 . O.I~M
IOI
"
S6b, O.I.aM
'
"
Fig. 1. ET isopeptidesand S6b evoke both transient and sustained[Ca2+ ]i waveforms in fura-2-loadedmesan~alcells. Human mesangialcell monolayerson plasticcoverslipswereloadedwith fura-2 and [Ca2+ ]i was measuredby dual-wavelengthfluorimetry as describedin Materialsand Methods. Representativetracings are shown for the biphasic (A-D) and sustained(E) [Ca2+ ]i waveforms.Similarresults were observed in three to six monolayersin separateexperiments.
65
1,200[
A,
1,000 {
.
~
I
+
t
+
Nifedipine IOj~M
ET-I, O.IJ~M I 0
:576+4uB.
l
0 - - - - ~ ~
0~
0
-II0 -9 -7 -6 log lET Isopeplide] (M) Fig. 2. Dose-dependency of peak A[Ca ~+ ]i. The peak A[Ca 2+ ]i was
calculated and plotted as a function of ET isopeptide concentration. Monophasic waveforms were observed only at 1.0 nM ET-1 and ET-2. Data are mean 5: S.E. for four to seven monolayers in different experiments.
J
I I rain
i
o
1:51 "
NiCl 2 O.5mM
ET-I, O.I .=M
r C. . . - i
150 [ 95L
........... ...... --__~ .,_ Bay K 8644 50/zM
__
2 0 0 [ D.
125 KCl 30 mM A.
Fig. 4. Dihydropyridine insensitivity of ET isopeptide-mediated Ca 2÷ entry. (A) Nifedipine and (B) NiCI 2 were added before addition of 0.1 #M ET-1 (at arrow) to fura-2 loaded mesangial monolayers. (C) Addition of the dihydropyridine agonist Bay K 8644 and of (D) high Kff (30 mM). In the protocols illustrated in A and B, identical results were observed in three to four experiments with 0.1 #M ET-2 (not shown).
,.m.[Co2"] o
ET-I, O.I~M
B.OmM [Co2+]o N 116
0 ,(-L
C. 289
concentrations of ET-1 or ET-2 (10 riM) which also generated a biphasic increase in [Ca2+]i . 1 n M ET-1 and ET-2, but not ET-3 or S6b, caused a modest, sustained increase in [Ca2+]i that contrasted with the clear-cut biphasic response observed at higher concentrations (Fig. 1E).
+ ET*I, 0.1 ,/JM I O
OmM [Ca2+]o
[L ~ EGTA 4ram
~ I I min
.........
~ ET-I, O.I,uM
O.
r 0 mM [Coz+]o / BAPTAi
/
/
I 7L
"--'~ kk + I~ET-I, O.I~M IonomycnrTco 2 Fig. 3. Relative contributions of mobilization of intrace]]u]ar Ca 2+ and influx of extracel]u]ar Ca 2+ to the ET-inducex] changes in [Ca2+]i. (A) Addition of ET-I in Ca2+-containing KHH, (B) in
nominally Ca2+.free KHH, (C) in nominally Ca2+-free KHH preceded by a 30 s preincubation with 4 mM EGTA, and (D) in mesangial cells loaded with BAPTA (as described in Materials and Methods) in Ca2+-free KHH. Tracing are representative of three to five separate monolayers for each protocol. Similar results were observed in all experimental protocols (i.., A-D) after addition of 0.1 #M ET-2 and S6b (not shown).
Relatioe contribution of mobilization of intracellular Ca 2 + vs. extracellular influx As shown in Fig. 3 A - C , when ET-1 was a d d e d in nominally Ca2+-free K H H or in Ca2+-free K H H following addition of E G T A , the transient phase of [Ca2+]i was attenuated and the sustained or plateau phase was abolished. The oscillation could be initiated in Ca 2 +-free m e d i u m but could not be maintained, suggesting that extracellular Ca 2+ is required for m a i n t e n a n c e o f the [Ca2+]i oscillations (Fig. 3B and C). Oscillations following addition of 0.1 # M ET-2, and the sustained phase following addition of 0.1 # M S6b, were similarly blunted in Ca2+-free K H H (data not shown). W e also loaded mesangial cells with 10 # M B A P T A , a high affinity Ca 2+ chelator that does not interfere with the fluorescence determination of [Ca 2+ ]i with fura-2. In mesangial cells loaded with b o t h B A P T A and fura-2, ET-1 (Fig. 3D) and ET-2 (not shown) failed to elevate [Ca2+]i and the subsequent addition of i o n o m y c i n a n d Cab + con-
66 A
117
IET-I, 0.1 ~ M
I ET- l, O.I,uM
TPA (OI,~M} Pcetreotment (20hrs)
.: N 0 L)
162
1ET.I, O,i,ut d
l E T . I , 0 i.~ M t 0
i
,J Imin
agonist, Bay K 8644, and depolarization induced by high [K÷]0 failed to increase [Ca2+]i in mesangial cells (Fig. 4C and D). In three separate experiments addition of [K+]0 after BAy K 8644 also failed to increase [Ca2+]i (data not shown). Thus two lines of evidence suggest that in human mesangial cells ET-stimulated Ca 2+ influx occurs independent of a voltage-gated, dihydropyridine-sensitive Ca 2÷ channel: (i) ET-induced [Ca2+]i waveforms are insensitive to blockade with dihydropyridine channel antagonists and (ii) human mesangial cells fail to express active or otherwise detectable voltage-gated Ca 2÷ channels.
C
lET-, D
O. LuM lET-I,
O.I.~M
Fig. 5. Adaptive responses and inhibition of ET-induced Ca 2+ signaling by protein kinase C. (A) ET-1 was added to a mesangial monolayers, and at the time indicated (break in tracing) the monolayer was washed five times with 2 ml KHH over a period of 5 rain before the readdition of ET-1. (B) Mesangial monolayers were pretreated for 20 h with 0.1 ~M TPA before addition of ET-1, washing and readdition of the agnnist as described in (A). (C) TPA was added 1 rain prior to the addition of ET-1 as indicated (arrow). (D) After the addition of ET-1, TPA was added (lower trace) to the cuvette; tracings from concurrent controls (upper trace) were superimposed for each of comparison. In protocols (A-C) similar results were observed in two to three independent experiments with ET-1, ET-2 and S6b.
firmed that BAPTA loading did not interfere with the fura-2 detection system. Taken together, these results demonstrate that the transient increase in [Ca2+]i resuits from both mobilization of intracellular Ca 2÷ stores and influx of extracellular Ca 2÷, whereas the sustained phase is dependent on extracellular Ca 2÷ influx. E T isopeptides Ca 2 + channel
activate
a
dihydropyridine-insensitive
The involvement of voltage-gated Ca 2+ channels in ET-induced Ca 2÷ signaling has been reported by several laboratories [11-13]. In contrast, in human mesangial cells nifedipine (10 min preincubation) failed to block either the transient, sustained or oscillatory phase of [Ca2+]i in response to ET-1 (Fig. 4A). Pretreatment of 0.5 mM NiC12, which blocks receptor-gated Ca 2+ channels in numerous cells, attenuated the transient increase in [Ca2÷]i in response to ET-1 (difference in peak [ C a 2 + ] i , n M = - 4 3 0 + 160, n = 3, P < 0.05) and completely prevented the sustained increase (Fig. 4B). In addition, both the dihydropyridine-sensitive channel
Desensitization of Ca 2÷ signaling with E T isopeptides and the effect of protein kinase C on ET-induced Ca 2+ signaling
ET isopeptides induced an adaptive, desensitization response in Ca 2+ signaling. As shown in Table I and Fig. 5A, ET isopeptides blocked the increase in [Ca2+]i induced by a subsequent, equimolar addition of the same ET agonist. Pretreatment with ET-3 and S6b, which stimulated only a modest increase i n [Ca2+]i compared with ET-1 and ET-2 (i.e., Fig. 2), also blunted the increase in [Ca2+]i in response to a subsequent addition of 0.1 /xM ET-1 (Table I). In mesangial cells depleted of protein kinase C by a 20 h pretreatment with 0.1/~M TPA, both the spike and sustained increase in [Ca2+]i were amplified (difference in peak [Ca2+]i, n M = + 320 + 73, n = 4, P < 0.05), but ET-l-induced desensitization persisted (Fig. 5B). These results suggest that protein kinase C did not mediate the desensitization response. Because Ca 2+ signaling by ET-1 was amplified in protein kinase C-depleted cells, we tested the possibility that protein kinase C downregulates ETinduced Ca 2÷ signaling. As shown in Fig. 5C, a 1 min pretreatment with TPA markedly reduced the [Ca2÷], TABLE I Desensitization of ET-induced Ca 2 + signaling by E T isopeptides Human mesangial cell monolayers loaded with fura-2 were treated with the first agonist for 2 rain, then the monolayer was washed two times with 2 ml KHH over a 5 rain period followed by addition of the second agonist as indicated. Data are meanS:S.E, for three to five independent experiments. Addition
A peak [Ca 2+ ]i, nM first addition
second addition
ET-1, 0.1/~M ~ ET-1, 0.1/~M 10 nM ~ 10 nM 1 nM---,1 nM
13685-177 9415:211 1625:76
ET-2, 0.1 #M ~ ET-2, 0.1/~M 10 nM ---,10 nM lnM~lnM
11415:189 3935:36 53 5 : 2 6
41 5 : 2 5 17+ 15 0
ET-3, 0.1 ~M ---,ET-1, 0.1/xM
51 5 : 1 2
682 + 163
1295:27
135 + 25
S6b, 0.1 ~M - , ET-1, 0.1/.tM
8+ 0 0
6
67 waveform in response to ET-1 (difference in peak [Ca2+]i, nM = - 5 5 2 5: 227, n = 3, P < 0.05), consistent with a protein kinase C-mediated negative feedback loop to dampen Ca 2+ signaling. Preincubation with equivalent concentrations of 4a-phorbol had no effect on ET-induced Ca 2+ signaling (data not shown). As shown in Fig. 5D, TPA added after treatment with ET-1 gradually reduced the sustained phase of [Ca2+]i, perhaps reflecting protein kinase C-mediated activation of C a 2+ pumps to extrude cytosolic C a 2+ o r by inhibiting hydrolysis of phosphatidylinositol 4,5P2.
Discussion There is accumulating evidence that the biological actions of ET are not limited to the peripheral vasculature but are of considerable importance in most organs including the kidney. Previous experiments suggest that an increase in [Ca2+]i m i g h t be an early signal mediating ET-induced biological actions [2-4,6,7]. The present results in human glomerular mesangial cells demonstrate that ET isopeptides and S6b evoke a dose-dependent, increase in [Ca2+]i . The relative potency for A peak [Ca2+]i was ET-1 = ET-2 >> S6b > ET-3. The [Ca2+]i response to ET-3 was minimal or absent in several of the cell strains studied. It is interesting to note that the duration of the sustained increase of [Ca2+]i in response to ET-1 was shorter in human as compared to rat mesangial cells [6,7]. The agoniststimulated transient increase in [Ca2+]i resulted from both release of intracellular Ca 2+ stores and extracellular Ca 2+ influx, whereas the sustained phase resulted only from extracellular influx (Fig. 3). Previous studies have generated discrepant results regarding the dihydropyridine sensitivity of ET-induced vasoconstriction and nonvascular smooth muscle contraction [6,7,11-14]. Our experiments demonstrate that ET-induced Ca 2+ influx occurs via activation of dihydropyridine-insensitive C a 2+ influx pathway, perhaps occurring through receptor-gated C a 2+ channels. Thus it appears that ET isopeptides activate either voltage-gated or receptor-gated Ca 2+ channels in a cell-specific manner. The [Ca 2 ÷ ]i waveforms generated by ET are of greater amplitude and duration than any agonist yet tested in rat or human mesangial cells (see Ref. 15). It is noteworthy, therefore, that ET-induced C a 2+ signaling undergoes a marked desensitization response (Table I and Fig. 5). Addition of ET either completely blocked or attenuated the C a 2+ signal evoked by the subsequent readdition of an equimolar dose of the same peptide. Even ET-3 and S6b, which stimulated a modest increase in [Ca2+]i compared to ET-1 and ET-2, attenuated the increase in [Ca2+]i stimulated by 0.1 /xM ET-1. The desensitization response was apparently independent of protein kinase C (Fig. 5). It should be noted that some investigators have reported complex, partially irreversi-
ble binding of ET to its receptor (see Refs. 2 and 3), suggesting that ET might be difficult to displace from its receptor using repetitive washings as reported in Fig. 5. Therefore continued receptor occupancy might account for some of the adaptive response observed in these studies, and further experiments are necessary to define the mechanisms involved. Prior activation of C-kinase by phorbol esters markedly inhibited Ca 2+ signaling by ET, suggesting that activation of protein kinase C by ET could serve as a negative feedback signal to dampen ET-induced increments in [Ca2+]i. Both ET-induced desensitization and the downregulation of Ca 2+ signaling by protein kinase C presumably protect glomerular mesangial cells from sustained stimulation by ET. The present experiments also demonstrate that high concentrations of ET-1 and ET-2 stimulate oscillations of [Ca2+]i following the transient phase (Fig. 1A and B). These oscillations could be initiated, but not sustained, in the absence of extracellular Ca 2+. Although the physiological significance of oscillations of [Ca2+]i in non-excitable cells remains unclear [16], to our knowledge oscillations of [Ca2+]i in response to any CaE+-mobilizing agonist in mesangial cells [15] have not been previously observed and deserve further investigation. ET-induced oscillations of [Ca 2+ ]i have also been reported in A10 vascular smooth muscle cells [17]. The most likely explanation for ET-induced oscillations of [Ca2+]i in mesangial cells is that the inositol (1,4,5)P 3insensitive Ca 2+ stores trigger a Ca 2+ wave that propages by a process of CaE+-induced Ca 2+ release coupled to diffusion [18]. In human mesangial cells sufficient Ca 2+ probably passes through gap junctions to trigger a wave of C a 2 ÷ in neighboring cells, thereby recruiting a local population of mesangial cells to function in unison. Similar results have been observed in monolayers of ciliated tracheal epithelium [19] and cardiac trabeculae [20]. Other mechanisms, such as CaE+-mediated inhibition of Ins 1,4,SP3-stimulated Ca 2+ release, could also account for wave propagation and further experiments are necessary to elucidate the mechanisms involved. In conclusion, our results support the concept that the ET family of peptides evoke complex, cellspecific Ca 2+ signaling systems that might mediate some ET-induced biological actions.
Acknowledgements We gratefully acknowledge Julie A. Wolfe for excellent technical assistance. This work was supported by National Institutes of Health Grant HL-22563.
References 1 Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature 332, 411-415.
68 2 Yanagisawa, M. and Masaki, T. (1989) Biochem. Pharmacol. 38, 1877-1883. 3 Vane, J.R., Botting, R. and Masaki, T. (eds.) (1989) J. Cardiovsc. Pharmacol. 13, 1-231. 4 Simonson, M.S. and Durra, M.J. (1990) Hypertension 15 (Suppl. 1), 5-12. 5 Kloog, Y. and Sokokovsky, M. (1989) Trends Pharmacol. Sci. 10, 212-214. 6 Simonson, M.S., Warm, S., Men6, P., Dubyak, G.R., Kester, M., Nakazato, Y., Sedor, J.R. and Dunn, M.J. (1989) J. Clin. Invest. 83, 708-712. 7 Badr, K.F., Murray, J.J., Breyer, M.D., Takahashi, K., Inagami, T. and Harris, R.C. (1989) J. Clin. Invest. 83, 336-342. 8 Simonson, M.S., Men6, P., Dubyak, G.R. and Dunn, M.J. (1988) Am. J. Physiol. 255, 771-780. 9 Scarpa, A. (1979) Methods Enzymol. 56, 301-338. 10 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. 11 Van Renterghem, C., Vigne, P., Barhanin, J., Schmid-Alliana, A.,
12
13 14 15 16 17 18 19 20
Felin, C. and Lazdunski, M. (1988) Biochem. Biophys. Res. Commun. 157, 977-985. Goto, K., Kasuya, Y., Matsuki, N., Takuwa, Y., Kurihara, H., Ishikawa, T., Kimura, S., Yanagisawa, M. and Masaki, T. (1989) Proc. Natl. Acad. Sci..86, 3915-3918. Silberberg, S.D., Poder, T.C., Lacerda, A.E. (1989) FEBS Lett. 247, 68-72. Mitsuhashi, T., Morris, R.C. and Ives, H.E. (1989) J. Clin. Invest. 84, 635-639. Men6, P., Simonson, M.S. and Dunn, M.J. (1989) Am. J. Physiol. 256, 375-386. Rink, T.J. and Hallam, T.J. (1989) Cell Calcium 10, 385-395. Simpson, A.W.M. and Ashley, C.C. (1989) Biochem. Biophys. Res. Commun. 163, 1223-1229. Berridge, M.J. and Irvine, R.F. (1989) Nature 341, 197-205. Sanderson, M.J., Chowm, I. and Dirksen, E.R. (1988) Am. J. Physiol. 254, 63-74. Mulder, B.J.M., De Tombe and Ter Keurs, H.E. (1989) J. Gen. Physiol. 93, 943-961.
63
Elsevier BBAMCR 12792
Endothelin isopeptides evoke C a 2 + signaling and oscillations of cytosolic free [Ca 2+] in human mesangial cells Michael S. Simonson 1, T o m o h i r o Osanai 1 and Michael J. D u n n 1,2 Departments of I Medicine, 2 Physiology and Biophysics, School of Medicine, Case Western Reserve University, Division of Nephrology, University Hospitals of Cleveland, Cleveland, OH (U.S.A.)
(Received 24 January 1990) (Revised manuscript received18 June 1990)
Key words: Signaltransduction; Second messengersystem; Calcium; Calcium channel; Endothelium; Glomerular mesangium
Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+ ] ([Ca2+]i) in human giomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [CaZ+]i constitutes a common pathway of signal transduction for the ET peptide family.
Introduction Endothelins (ET) are a family of 21-amino acid peptides ( - 2 . 5 kDa) secreted by endothelial cells in vitro and in vivo [1]. At least three distinct genes encode highly conserved isopeptides designated ET-1, ET-2 and ET-3 (see Refs. 2 - 4 for review). Sarafotoxins, a group of peptides purified from venom of Atractaspis engaddensis, share close sequence homology with ET [5], and it seems likely that the two peptide families share a common evolutionary origin. Although the physiologic and pathophysiologic roles of ET remain to be elucidated, ET exerts profound effects on vascular and nonvascular smooth muscle contraction, mitogenesis and gene expression [2-4]. Binding sites for radiolabeled ET are widely distributed in the body [2,3] and activation of these putative ET receptors could mediate a variety of biological functions.
We and others [6,7] have previously shown that in rat mesangial cells ET-1 evokes a Ca 2÷ signaling system via activation of the phosphoinositide cascade. The increase in cytosolic free [Ca 2÷ ] ([Ca 2÷ ]i) results from phospholipase-C catalyzed formation of inositol 1,4,5-trisphosphate to release Ca 2÷ from intracellular stores and from influx of extracellular Ca 2÷ [6]. In light of the actions of ET-1 in rat mesangial cells, we examined the effects of ET-1 and other ET isopeptides and sarafotoxin S6b on Ca 2÷ signaling in human mesangial cells. We report here that (i) all ET isopeptides and S6b evoke complex patterns of Ca 2÷ signaling; that (ii) ET-1 and ET-2 stimulate oscillations of [Ca 2÷]i previously undescribed in mesangial cells; and that (iii) desensitization of the Ca 2+ signaling induced by ET-1 is independent of protein kinase C activation.
Materials and Methods
Abbreviations: ET, endothelin; S6b, sarafotoxin S6b; [Ca2+ ]i, cytosolic free [Ca2+ ]; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethylester; DTPA, diethylenetriaminepentaacetic acid; KHH, Krebs Henseleit-Hepesbuffer. Correspondence: M,J. Duma, Department of Medicine, University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
Materials. Fura-2 (fura-2 acetoxymethylester) and BAPTA were from Molecular Probes. Ionomycin, Bay K 8644 and nifedipine were from Calbiochem. Ethylene glycol-bis( fl-aminoethyl e t h e r ) - N , N , N ' , N ' - t e t r a a c e t i c acid (EGTA) and D T P A were obtained from Sigma. ET-1 was purchased from Peninsula Laboratories, whereas ET-2, ET-3 and sarafotoxin S6b (S6b), from Peptide Institute, were a generous gift from Dr. Thomas
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
64 Rimley, Glaxo Pharmaceuticals. Stock solutions of ET isopeptides (0.1 mM) in sterile H20 were stored in aliquots at - 4 0 ° C for no more than 3 weeks. Human mesangial cell isolation and culture. Human glomerular mesangial cell strains were prepared and characterized as previously reported [8] by partial digestion of isolated glomerular explants by bacterial collagenase to form 'glomerular cores'. Human kidneys were obtained (approved protocol of Institutional Review Board, University Hospitals) immediately after surgical nephrectomy or from donor, cadaveric kidneys that were judged unsuitable for transplant. Cells were maintained in RPMI 1640 supplemented with 17% fetal bovine serum, 100 U / m l penicillin, 100 # g / m l streptomycin, 5 / ~ g / m l each of insulin and transferrin, and 5 n g / m l sodium selenite at 3 7 ° C in 5% CO2/95% air. Monolayers were subcultured every 7-10 days using t r y p s i n / E D T A , and media in all cultures was renewed every 2-3 days. Four different cell strains (passages 3-15) were used in these experiments. Determinations of [Ca 2 +] i. [Ca2 + ]i in populations of mesangial cells was measured fluorimetrically using fura-2. As reported [6,8], confluent monolayers on 1.4 cm 2 Aclar coverslips (Allied Engineered Plastics) were loaded with 1 laM fura-2 acetoxymethylester for 40 min at 37 ° C followed by a 20 min incubation in fura-2-free RPMI 1640 to allow for intracellular dye cleavage. The cells were kept at 4 ° C in K H H (pH 7.4) until used. Coverslips were mounted in a cuvette with 2 ml K H H maintained at 37 ° C with constant stirring and fluorescence was measured with a University of Pennsylvania Biomedical Instruments Group spectrofluorimeter [9] using an air-driven rotary shutter for alternate excitation at 340 and 380 nm wavelengths with emission set at
A. ,--. 975 ~
oJ o
121
,u,
510 nm. Calibration was made using ionomycin followed by EGTA-Tris, as described [6,8], except that the formula of Grynkyewicz et al. [10] was used to calculate [Ca2÷ ]i assuming the K d of the fura-2-Ca 2÷ interaction to be 224 nM. All agonists were tested independently for autofluorescence. In experiments in which BAPTA was used, 10/~M BAPTA acetoxymethylester was added and coincubated with fura-2 in RPMI 1640 exactly as described above. In some coverslips leakage of fura-2 was assessed by the addition of 100/tM Mn 2÷ followed by rapid chelation with 4 mM DTPA. Results E T isopeptides and S6b cause both transient and sustained increases in [Ca 2 +] i
When added to human mesangial cell monolayers loaded with fura-2, 0.01-0.1/~M of ET-1, ET-2 and S6b caused a biphasic increase in [Ca2+]i consisting of a rapid (2-5 s) transient increase followed by a lower but sustained phase (Fig. 1A, B and D). The peak A[fa2+]i was dose-dependent with ET-1 = ET-2 > S6b >> ET-3 (Fig. 2). 0.1 /~M ET-1 and ET-2 caused a longer sustained phase at higher levels of [Ca2+]i than did equimolar additions of S6b. 0.1 #M ET-3 caused a minimal increase in [Ca2+]i in only one of the four cell strains studied (Fig. 1C). It is interesting to note that in 3 of the 4 cell strains studied, 0.1 ttM ET-1 and ET-2 (but not S6b or ET-3) caused oscillations of [Ca 2+ ]i that appeared immediately following the initial transient increase in [Ca2+]i (Fig. 1A and B). The oscillations were periodic with approx. 30-40 s between peaks and were sustained for only 2-3 min after the addition of ET-1 or ET-2. Oscillations of [Ca2+]i were not observed at lower
I030.
B.
107 - ,u ~ ET-I, O.I,uM
2501 ~
~ ET-2, O.I~M
I
E.
7 0 '-
e E T - 3 . O.I~M
IOI
"
S6b, O.I.aM
'
"
Fig. 1. ET isopeptidesand S6b evoke both transient and sustained[Ca2+ ]i waveforms in fura-2-loadedmesan~alcells. Human mesangialcell monolayerson plasticcoverslipswereloadedwith fura-2 and [Ca2+ ]i was measuredby dual-wavelengthfluorimetry as describedin Materialsand Methods. Representativetracings are shown for the biphasic (A-D) and sustained(E) [Ca2+ ]i waveforms.Similarresults were observed in three to six monolayersin separateexperiments.
65
1,200[
A,
1,000 {
.
~
I
+
t
+
Nifedipine IOj~M
ET-I, O.IJ~M I 0
:576+4uB.
l
0 - - - - ~ ~
0~
0
-II0 -9 -7 -6 log lET Isopeplide] (M) Fig. 2. Dose-dependency of peak A[Ca ~+ ]i. The peak A[Ca 2+ ]i was
calculated and plotted as a function of ET isopeptide concentration. Monophasic waveforms were observed only at 1.0 nM ET-1 and ET-2. Data are mean 5: S.E. for four to seven monolayers in different experiments.
J
I I rain
i
o
1:51 "
NiCl 2 O.5mM
ET-I, O.I .=M
r C. . . - i
150 [ 95L
........... ...... --__~ .,_ Bay K 8644 50/zM
__
2 0 0 [ D.
125 KCl 30 mM A.
Fig. 4. Dihydropyridine insensitivity of ET isopeptide-mediated Ca 2÷ entry. (A) Nifedipine and (B) NiCI 2 were added before addition of 0.1 #M ET-1 (at arrow) to fura-2 loaded mesangial monolayers. (C) Addition of the dihydropyridine agonist Bay K 8644 and of (D) high Kff (30 mM). In the protocols illustrated in A and B, identical results were observed in three to four experiments with 0.1 #M ET-2 (not shown).
,.m.[Co2"] o
ET-I, O.I~M
B.OmM [Co2+]o N 116
0 ,(-L
C. 289
concentrations of ET-1 or ET-2 (10 riM) which also generated a biphasic increase in [Ca2+]i . 1 n M ET-1 and ET-2, but not ET-3 or S6b, caused a modest, sustained increase in [Ca2+]i that contrasted with the clear-cut biphasic response observed at higher concentrations (Fig. 1E).
+ ET*I, 0.1 ,/JM I O
OmM [Ca2+]o
[L ~ EGTA 4ram
~ I I min
.........
~ ET-I, O.I,uM
O.
r 0 mM [Coz+]o / BAPTAi
/
/
I 7L
"--'~ kk + I~ET-I, O.I~M IonomycnrTco 2 Fig. 3. Relative contributions of mobilization of intrace]]u]ar Ca 2+ and influx of extracel]u]ar Ca 2+ to the ET-inducex] changes in [Ca2+]i. (A) Addition of ET-I in Ca2+-containing KHH, (B) in
nominally Ca2+.free KHH, (C) in nominally Ca2+-free KHH preceded by a 30 s preincubation with 4 mM EGTA, and (D) in mesangial cells loaded with BAPTA (as described in Materials and Methods) in Ca2+-free KHH. Tracing are representative of three to five separate monolayers for each protocol. Similar results were observed in all experimental protocols (i.., A-D) after addition of 0.1 #M ET-2 and S6b (not shown).
Relatioe contribution of mobilization of intracellular Ca 2 + vs. extracellular influx As shown in Fig. 3 A - C , when ET-1 was a d d e d in nominally Ca2+-free K H H or in Ca2+-free K H H following addition of E G T A , the transient phase of [Ca2+]i was attenuated and the sustained or plateau phase was abolished. The oscillation could be initiated in Ca 2 +-free m e d i u m but could not be maintained, suggesting that extracellular Ca 2+ is required for m a i n t e n a n c e o f the [Ca2+]i oscillations (Fig. 3B and C). Oscillations following addition of 0.1 # M ET-2, and the sustained phase following addition of 0.1 # M S6b, were similarly blunted in Ca2+-free K H H (data not shown). W e also loaded mesangial cells with 10 # M B A P T A , a high affinity Ca 2+ chelator that does not interfere with the fluorescence determination of [Ca 2+ ]i with fura-2. In mesangial cells loaded with b o t h B A P T A and fura-2, ET-1 (Fig. 3D) and ET-2 (not shown) failed to elevate [Ca2+]i and the subsequent addition of i o n o m y c i n a n d Cab + con-
66 A
117
IET-I, 0.1 ~ M
I ET- l, O.I,uM
TPA (OI,~M} Pcetreotment (20hrs)
.: N 0 L)
162
1ET.I, O,i,ut d
l E T . I , 0 i.~ M t 0
i
,J Imin
agonist, Bay K 8644, and depolarization induced by high [K÷]0 failed to increase [Ca2+]i in mesangial cells (Fig. 4C and D). In three separate experiments addition of [K+]0 after BAy K 8644 also failed to increase [Ca2+]i (data not shown). Thus two lines of evidence suggest that in human mesangial cells ET-stimulated Ca 2+ influx occurs independent of a voltage-gated, dihydropyridine-sensitive Ca 2÷ channel: (i) ET-induced [Ca2+]i waveforms are insensitive to blockade with dihydropyridine channel antagonists and (ii) human mesangial cells fail to express active or otherwise detectable voltage-gated Ca 2÷ channels.
C
lET-, D
O. LuM lET-I,
O.I.~M
Fig. 5. Adaptive responses and inhibition of ET-induced Ca 2+ signaling by protein kinase C. (A) ET-1 was added to a mesangial monolayers, and at the time indicated (break in tracing) the monolayer was washed five times with 2 ml KHH over a period of 5 rain before the readdition of ET-1. (B) Mesangial monolayers were pretreated for 20 h with 0.1 ~M TPA before addition of ET-1, washing and readdition of the agnnist as described in (A). (C) TPA was added 1 rain prior to the addition of ET-1 as indicated (arrow). (D) After the addition of ET-1, TPA was added (lower trace) to the cuvette; tracings from concurrent controls (upper trace) were superimposed for each of comparison. In protocols (A-C) similar results were observed in two to three independent experiments with ET-1, ET-2 and S6b.
firmed that BAPTA loading did not interfere with the fura-2 detection system. Taken together, these results demonstrate that the transient increase in [Ca2+]i resuits from both mobilization of intracellular Ca 2÷ stores and influx of extracellular Ca 2÷, whereas the sustained phase is dependent on extracellular Ca 2÷ influx. E T isopeptides Ca 2 + channel
activate
a
dihydropyridine-insensitive
The involvement of voltage-gated Ca 2+ channels in ET-induced Ca 2÷ signaling has been reported by several laboratories [11-13]. In contrast, in human mesangial cells nifedipine (10 min preincubation) failed to block either the transient, sustained or oscillatory phase of [Ca2+]i in response to ET-1 (Fig. 4A). Pretreatment of 0.5 mM NiC12, which blocks receptor-gated Ca 2+ channels in numerous cells, attenuated the transient increase in [Ca2÷]i in response to ET-1 (difference in peak [ C a 2 + ] i , n M = - 4 3 0 + 160, n = 3, P < 0.05) and completely prevented the sustained increase (Fig. 4B). In addition, both the dihydropyridine-sensitive channel
Desensitization of Ca 2÷ signaling with E T isopeptides and the effect of protein kinase C on ET-induced Ca 2+ signaling
ET isopeptides induced an adaptive, desensitization response in Ca 2+ signaling. As shown in Table I and Fig. 5A, ET isopeptides blocked the increase in [Ca2+]i induced by a subsequent, equimolar addition of the same ET agonist. Pretreatment with ET-3 and S6b, which stimulated only a modest increase i n [Ca2+]i compared with ET-1 and ET-2 (i.e., Fig. 2), also blunted the increase in [Ca2+]i in response to a subsequent addition of 0.1 /xM ET-1 (Table I). In mesangial cells depleted of protein kinase C by a 20 h pretreatment with 0.1/~M TPA, both the spike and sustained increase in [Ca2+]i were amplified (difference in peak [Ca2+]i, n M = + 320 + 73, n = 4, P < 0.05), but ET-l-induced desensitization persisted (Fig. 5B). These results suggest that protein kinase C did not mediate the desensitization response. Because Ca 2+ signaling by ET-1 was amplified in protein kinase C-depleted cells, we tested the possibility that protein kinase C downregulates ETinduced Ca 2÷ signaling. As shown in Fig. 5C, a 1 min pretreatment with TPA markedly reduced the [Ca2÷], TABLE I Desensitization of ET-induced Ca 2 + signaling by E T isopeptides Human mesangial cell monolayers loaded with fura-2 were treated with the first agonist for 2 rain, then the monolayer was washed two times with 2 ml KHH over a 5 rain period followed by addition of the second agonist as indicated. Data are meanS:S.E, for three to five independent experiments. Addition
A peak [Ca 2+ ]i, nM first addition
second addition
ET-1, 0.1/~M ~ ET-1, 0.1/~M 10 nM ~ 10 nM 1 nM---,1 nM
13685-177 9415:211 1625:76
ET-2, 0.1 #M ~ ET-2, 0.1/~M 10 nM ---,10 nM lnM~lnM
11415:189 3935:36 53 5 : 2 6
41 5 : 2 5 17+ 15 0
ET-3, 0.1 ~M ---,ET-1, 0.1/xM
51 5 : 1 2
682 + 163
1295:27
135 + 25
S6b, 0.1 ~M - , ET-1, 0.1/.tM
8+ 0 0
6
67 waveform in response to ET-1 (difference in peak [Ca2+]i, nM = - 5 5 2 5: 227, n = 3, P < 0.05), consistent with a protein kinase C-mediated negative feedback loop to dampen Ca 2+ signaling. Preincubation with equivalent concentrations of 4a-phorbol had no effect on ET-induced Ca 2+ signaling (data not shown). As shown in Fig. 5D, TPA added after treatment with ET-1 gradually reduced the sustained phase of [Ca2+]i, perhaps reflecting protein kinase C-mediated activation of C a 2+ pumps to extrude cytosolic C a 2+ o r by inhibiting hydrolysis of phosphatidylinositol 4,5P2.
Discussion There is accumulating evidence that the biological actions of ET are not limited to the peripheral vasculature but are of considerable importance in most organs including the kidney. Previous experiments suggest that an increase in [Ca2+]i m i g h t be an early signal mediating ET-induced biological actions [2-4,6,7]. The present results in human glomerular mesangial cells demonstrate that ET isopeptides and S6b evoke a dose-dependent, increase in [Ca2+]i . The relative potency for A peak [Ca2+]i was ET-1 = ET-2 >> S6b > ET-3. The [Ca2+]i response to ET-3 was minimal or absent in several of the cell strains studied. It is interesting to note that the duration of the sustained increase of [Ca2+]i in response to ET-1 was shorter in human as compared to rat mesangial cells [6,7]. The agoniststimulated transient increase in [Ca2+]i resulted from both release of intracellular Ca 2+ stores and extracellular Ca 2+ influx, whereas the sustained phase resulted only from extracellular influx (Fig. 3). Previous studies have generated discrepant results regarding the dihydropyridine sensitivity of ET-induced vasoconstriction and nonvascular smooth muscle contraction [6,7,11-14]. Our experiments demonstrate that ET-induced Ca 2+ influx occurs via activation of dihydropyridine-insensitive C a 2+ influx pathway, perhaps occurring through receptor-gated C a 2+ channels. Thus it appears that ET isopeptides activate either voltage-gated or receptor-gated Ca 2+ channels in a cell-specific manner. The [Ca 2 ÷ ]i waveforms generated by ET are of greater amplitude and duration than any agonist yet tested in rat or human mesangial cells (see Ref. 15). It is noteworthy, therefore, that ET-induced C a 2+ signaling undergoes a marked desensitization response (Table I and Fig. 5). Addition of ET either completely blocked or attenuated the C a 2+ signal evoked by the subsequent readdition of an equimolar dose of the same peptide. Even ET-3 and S6b, which stimulated a modest increase in [Ca2+]i compared to ET-1 and ET-2, attenuated the increase in [Ca2+]i stimulated by 0.1 /xM ET-1. The desensitization response was apparently independent of protein kinase C (Fig. 5). It should be noted that some investigators have reported complex, partially irreversi-
ble binding of ET to its receptor (see Refs. 2 and 3), suggesting that ET might be difficult to displace from its receptor using repetitive washings as reported in Fig. 5. Therefore continued receptor occupancy might account for some of the adaptive response observed in these studies, and further experiments are necessary to define the mechanisms involved. Prior activation of C-kinase by phorbol esters markedly inhibited Ca 2+ signaling by ET, suggesting that activation of protein kinase C by ET could serve as a negative feedback signal to dampen ET-induced increments in [Ca2+]i. Both ET-induced desensitization and the downregulation of Ca 2+ signaling by protein kinase C presumably protect glomerular mesangial cells from sustained stimulation by ET. The present experiments also demonstrate that high concentrations of ET-1 and ET-2 stimulate oscillations of [Ca2+]i following the transient phase (Fig. 1A and B). These oscillations could be initiated, but not sustained, in the absence of extracellular Ca 2+. Although the physiological significance of oscillations of [Ca2+]i in non-excitable cells remains unclear [16], to our knowledge oscillations of [Ca2+]i in response to any CaE+-mobilizing agonist in mesangial cells [15] have not been previously observed and deserve further investigation. ET-induced oscillations of [Ca 2+ ]i have also been reported in A10 vascular smooth muscle cells [17]. The most likely explanation for ET-induced oscillations of [Ca2+]i in mesangial cells is that the inositol (1,4,5)P 3insensitive Ca 2+ stores trigger a Ca 2+ wave that propages by a process of CaE+-induced Ca 2+ release coupled to diffusion [18]. In human mesangial cells sufficient Ca 2+ probably passes through gap junctions to trigger a wave of C a 2 ÷ in neighboring cells, thereby recruiting a local population of mesangial cells to function in unison. Similar results have been observed in monolayers of ciliated tracheal epithelium [19] and cardiac trabeculae [20]. Other mechanisms, such as CaE+-mediated inhibition of Ins 1,4,SP3-stimulated Ca 2+ release, could also account for wave propagation and further experiments are necessary to elucidate the mechanisms involved. In conclusion, our results support the concept that the ET family of peptides evoke complex, cellspecific Ca 2+ signaling systems that might mediate some ET-induced biological actions.
Acknowledgements We gratefully acknowledge Julie A. Wolfe for excellent technical assistance. This work was supported by National Institutes of Health Grant HL-22563.
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68 2 Yanagisawa, M. and Masaki, T. (1989) Biochem. Pharmacol. 38, 1877-1883. 3 Vane, J.R., Botting, R. and Masaki, T. (eds.) (1989) J. Cardiovsc. Pharmacol. 13, 1-231. 4 Simonson, M.S. and Durra, M.J. (1990) Hypertension 15 (Suppl. 1), 5-12. 5 Kloog, Y. and Sokokovsky, M. (1989) Trends Pharmacol. Sci. 10, 212-214. 6 Simonson, M.S., Warm, S., Men6, P., Dubyak, G.R., Kester, M., Nakazato, Y., Sedor, J.R. and Dunn, M.J. (1989) J. Clin. Invest. 83, 708-712. 7 Badr, K.F., Murray, J.J., Breyer, M.D., Takahashi, K., Inagami, T. and Harris, R.C. (1989) J. Clin. Invest. 83, 336-342. 8 Simonson, M.S., Men6, P., Dubyak, G.R. and Dunn, M.J. (1988) Am. J. Physiol. 255, 771-780. 9 Scarpa, A. (1979) Methods Enzymol. 56, 301-338. 10 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. 11 Van Renterghem, C., Vigne, P., Barhanin, J., Schmid-Alliana, A.,
12
13 14 15 16 17 18 19 20
Felin, C. and Lazdunski, M. (1988) Biochem. Biophys. Res. Commun. 157, 977-985. Goto, K., Kasuya, Y., Matsuki, N., Takuwa, Y., Kurihara, H., Ishikawa, T., Kimura, S., Yanagisawa, M. and Masaki, T. (1989) Proc. Natl. Acad. Sci..86, 3915-3918. Silberberg, S.D., Poder, T.C., Lacerda, A.E. (1989) FEBS Lett. 247, 68-72. Mitsuhashi, T., Morris, R.C. and Ives, H.E. (1989) J. Clin. Invest. 84, 635-639. Men6, P., Simonson, M.S. and Dunn, M.J. (1989) Am. J. Physiol. 256, 375-386. Rink, T.J. and Hallam, T.J. (1989) Cell Calcium 10, 385-395. Simpson, A.W.M. and Ashley, C.C. (1989) Biochem. Biophys. Res. Commun. 163, 1223-1229. Berridge, M.J. and Irvine, R.F. (1989) Nature 341, 197-205. Sanderson, M.J., Chowm, I. and Dirksen, E.R. (1988) Am. J. Physiol. 254, 63-74. Mulder, B.J.M., De Tombe and Ter Keurs, H.E. (1989) J. Gen. Physiol. 93, 943-961.
