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Endothelin receptor binding and cellular signal transduction in neurohybrid NG108-15 cells
Neuroscience Vol. 44, No. 1, pp. 215222, Printed in Great Britain
0306-4522/91 $3.00 + 0.00 Pcrgamoo Fkss plc 0 1991IBRO
1991
ENDOTHELIN RECEPTOR BINDING AND CELLULAR SIGNAL TRANSDUCTION IN NEUROHYBRID NG108-15 CELLS T. L. YuE,* P.
NAMBI,
H. L. Wu and G. FEUERSTEIN
Department of Pharmacology, SmithKline Beecham, King of Prussia, PA 19406-0939, U.S.A. Abstrac-Endothelins are a novel group of potent vasoconstrictor peptides originally isolated from cultured porcine endothelial cells. We and others have previously reported the presence of endothelin receptors in the central nervous system, and this study was designed to further characterize endothelin receptors and their transduction mechanism in cultured neurohybrid NGl08-15 cells. Specitic binding of [1251)endothelin-l to NGl08-15 cells reached saturation within 60min at 22°C and was only partially reversible. Scatchard analysis of the saturation binding revealed the presence of one class of high-affinity binding sites with an apparent dissociation constant of 16OpM and a maximal binding capacity of 3.3 x 10’ sites/cell. Unlabeled endothelin analogues competitively inhibited [‘251]endothelin-l binding to NGlOS-15 cells and the apparent dissociation constant values obtained from the competition curves correlated well with the ECX) values obtained for inducing elevation of intracellular free Ca*+ level. Endothelin stimulated phosphoinositide metabolism in a dose-dependent manner with an EC, value of 5.4 nM for inositol trisphosphate formation. The protein kinase C-activator phorbol ester dosedependently inhibited endothelin-induced phosphoinositide turnover and intracellular free Caz+ increase, suggesting the involvement of protein kinase C in the regulation of endothelin-induced responses. Neither endothelin-induced phosphoinositide hydrolysis nor endothelin-induced increase in intracellular free Ca2+ were affected by pertussis toxin. These dam indicate that endothelin receptors are present on NGl08-15 cells and the G protein coupled to endothelin receptor for inducing activation of phospholipase C and increase of free intracellular Ca*+ is insensitive to pertussis toxin.
Endothelins are a novel group of highly potent vasoconstrictor peptides originally isolated from vascular endothelial cell~~**~and subsequently found in the human genome in at least three isoforms, i.e. endothelin-1, endothelin-2 and endothelin-3.20 Recent studies have conkmed that endothelin-1 and endothelin-3 are present in cells other than the endothelium and particularly in neuronal cells. Material that cross-reacted with antibodies to endothelin has been detected in the porcine spinal cord32 as well as in the hypothalamus and posterior pituitary of pig and rat.@ A role for endothelin in the central nervous system has been suggested by studies showing potent cerebrovasculaIj*6~**3’*37 and autonomic33 responses following central administration of the various endothelin peptides. In addition to cerebrovascular
*To whom correspondence should be addressed at: Department of Pharmacology, SmithKline Beecham, P.O. Box 1539, L-510, King of Prussia, PA 19406-0939, USA. Abbreviations:BSA, bovine serum albumin; [Ca’+],, cytosolit free calcium concentration; DMEM, Dulbecco’s modified Eagle medium; EDTA, ethylenediaminetetraacetate; fura 2/AM, penta-acetoxymethyl ester of fura-2; IP,, inositol monophosphate; IP,, inositol bisphosuhate; IPJ, inositol trisphosphate; i&l buffer, Kkbs-Binger Henseleit; PDBu. uhorbol 12.13-dibutvrate: 4a-PDD. 4a -phorbol 12,13_didecanoate;~ PKC, piotein kinase Ci PLC, phospholipase C, PTX, pertussis toxin (Islet Activating Protein); TCA, trichloroacetic acid.
hemodynamic effects, endothelin-1 given intracisternally produced severe apnea which was independent of changes in systemic arterial blood pressure.‘O A discrete brain lesion of ischemic nature following intraparenchymal administration of endothelin- 1 into the striatal region of the rat brain was demonstrated, indicating that endothelin may contribute to the development of cerebral infarcts and neuronal death.” It has also been suggested that endothelin may function as a neuromodulator since it was observed that endothelin stimulated gonadotropin secretion in anterior pituitary cell~.~ Additional support for the potential role of endothelin in modulation of brain function has been drawn from autoradiographic studies showing a discrete, nonvascular pattern of distribution of endothelin-binding sites.‘s*21*uSpecific binding sites for endothelin-1 and endothelin3 in rat brain were also demonstrated recently in our laboratory, which showed a clear heterogenous distribution among various brain regions with a general increase in density in the hindbrain versus the forebrain regions.26 While discrete binding sites in brain tissue seem to be strongly suggested, the cellular origin of this receptor in the nervous system is still unclear.‘*” Moreover, the functional studies of endothelin at the cellular level in neuronal tissue, in particular the secondary messenger system associated with these receptors are rather limited.7Y*36 215
216
T. I.. YUE et al
In previous studies we found that endothelin induced a significant increase in cytosolic free calcium concentration ([Ca* ‘Ii) from both extracellular and intracellular sources in NGl08-15 cells, a hybrid of mouse neuroblastoma/rat glioma cells,4’ but not in NCB-20 cells which are a hybrid of mouse neuroblastoma/Chinese hamster cortical neurons.’ We hypothesized that the discrepancy in response to endothelin between NG108-15 and NCB-20 cells might reflect the fact that NCB-20 cells do not express the endothelin receptors. Further, the endothelin-induced increase in [Ca*+], in NG108-15 cells may be related to effect on phosphoinositide turnover since receptormediated phosphoinositide hydrolysis is obligatory for intracellular calcium release.4 In order to confirm our hypothesis the present study was designed to identify and characterize specific endothelin binding sites in NGl08- 15 and NCB-20 cells and to study the effect of endothelin on phosphoinositide turnover in these cells. EXPERIMENTAL
PROCEDURES
Maierials Endothelin- 1 (ET- 1) (human/porcine), endothelin-2 (ET-2) (human), endothelin-3 (ET-3) (lmman/rat) and big endothelin 22-38 (big ET) (human) were purchased from Peninsula Laboratories (Belmont, CA). [‘251]Endothelin-l (specific activity: 2200 Ci/mmol) and [31Qnyoinositol (specific activity: 17.4 Ci/mmol) were provided by New England Nuclear (Boston, MA) and Amersham (Des Plaines, IL), respectively. Phorbol 12,13_dibutyrate (PDBu) and 4u -phorbol 12,13-didecanoate (4a -PDD) were obtained from Sigma (St. Louis, MO). Pertussis toxin (Islet Activating Protein, PTX) was purchased from List Biological Laboratories (Campbell, CA). Pentaacetoxymethyl ester of fura-2(fura 2/AM) and AG l-X8 anion exchange resin (formate form, 2OWlOO mesh) were from Calbiochem (La Jolla, CA) and Bio-Rad (Richmond, CA), respectively. NGlOS-15 and NCB-20 cells were kindly provided by Dr M. Nirenbera (National Heart. Lung and Blood Institute, NIH). - ~ Cell culture NG108-15 and NCB-20 cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented by 10% (v/v) heat-in&tivated fetal calf serum,- 0.1 mM hypoxanthine. 0.4 uM aminonterin and 16 uM thvmidine in 150-cmZ flasks, in’a humid&d environment oi5% (for NCR20 cells) or 10% (for NGl08-15 cells) CO, at 37°C. After subculturing, the cells were grown to conffuence before harvesting. Binding experiments [1251]Endothelin-l binding experiments were performed on confluent NGlOS-I5 or NCB-20 cells. The culture medium was replaced with serum-free medium 24 h prior to experiment. The cells were harvested by shaking, and washed once with Dulbecco’s phosphate buffered saline containing 0.2% bovine serum albumin (BSA), 5mM glucose and 10 mM MgCl, and resuspended in the same buffer at 2 x IO6 cells/ml. Binding of [i2$ndothelin-1 to the cells was initiated bv adding 1 x lo5 cells/tube in a total volume of 0.25ml containing [‘251]endothelin-l in the absence (total binding) or presence (nonspecific binding) of 100 nM unlabeled endothelin-1. Cells were incubated at 22°C for 60min unless otherwise stated. The cell-associated radioactivity was separated from free l&and by vacuum filtration on GF/C filters presoaked in 0.1% BSA. The filters were
washed with 5 x 5 ml of 50 mM Tris buffer @H :‘.?I)WIItaining 10 mM MgCl, (4°C). Specific binding was defined as total binding minus nonspecific binding in the presence of 100 nM unlabeled endothelin-1. All experiments were prrformed in 12 x 75mm glass tubes in triplicate. Measurement of inositol phosphates accumulation Confluent NG108-15 cells were prelabeled wtth [3H]myoinositol (ZpCi/ml in T150 flask) in inositol-free DMEM overnight. The cells were harvested and washed twice with Krebs-Ringer Henseleit (KRH) buffer containing 0.1% BSA and in mM: NaCI. I 18: KCI. 4.6: NaHCO,. 24.9; KH,PG,, 1.0; glucose, 11.1; MgSG,, 1.1 and CaCI,, I:O, pH 7.4. and finally resuspended in KRH buffer containing 1OmM LiCl at I x IO6 cells/ml. An aliquot of t.he cell suspension (0.5 ml) was preincubated for 10 min at 37 c‘ and the reaction was initiated by the addition of agonist, cndothelin and continued for various times as indicated in the figures. The reaction was stopped by addition of 50~1 of 100% trichloroacetic acid (TCA). Samples were maintained on ice for 20 min and then centrifuged. TCA was extracted from the supernatant with 5 x 2m 1 of water-saturated diethylether. Excess ether was removed with nitrogen and the samples were neutralized with 10 ~1 of 0.5 M Tris base. Samples were stored frozen (- 80°C) until the mositol phosphates were separated by column chromatography. Samples prepared as described above were mixed with 4ml of 5mM sodium tetraborate containing 0.5 mM EDTA, and poured onto disposable polystyrene columns (1 x 10cm) containing 1 g of AG 1 x 8 anion exchange resin. [‘H]Inositol mono-, bis- and trisphosphate ([‘H]IP,. [‘H]IP, and [3H]IP,) were sequentially eluted into scintillation vials with 4ml of 0.2, 0.4 and 0.8 M ammonium formate containing 0.1 M formic acid, respectively. 10 ml of scintillation solution (Ready Gel, Beckman) was added to the vials and the radioactivity determined with a counting efficiency of 30%. Measurement of c:vtosolic free calcium concentration mobilization [Ca2+li was measured as described previously.4’ Briefly, the harvested cells were resuspended at 2 x 106/ml in KRH buffer and incubated with 2 MM of fura-2/AM at 37°C for 45min. The cells were centrifuged, resuspended in KRH buffer, and incubated at 37°C for an additional 20 min to allow for complete hydrolysis of entrapped ester. After loading, cells were washed and suspended in KRH buffer at 1 x 106/ml. The fluorescence of fura-2-containing cells was measured with a spectrofluorometer designed by the Biomedical Instruments Group, University of Pennsylvania. The wavelengths were set at 339 nm for excitation and 505 nm for emission. All experiments were performed at 37°C. The [CaZ+li was calculated according to the method of Grynkiewiez et al. Is For the studies in Ca* +-free KRH, fura-2-loaded cells were spun down and resuspended in pre-warmed (37°C) Ca 2+-free KRH buffer. The cells were used immediately. Statistics Data in the text and figures are mean + S.E.M. unless otherwise stated. Statistical significance between two groups was examined through one way analysis of variance and Dunnetts multiple range test. Significant differences were accepted at P < 0.05. RESULTS
[‘251]Endothelin-l binding to NG 108- 15 cells
Binding of [‘251Jendothelin-l to NG108-15 cells was a time-dependent process and reached equilibrium between 30 and 60min at 22°C (Fig. IA). To determine the dissociation of cell bound [‘251jendothelin-l, NG 108-l 5 cells were incubated with [1251]endothelin-1
217
Endotbelin receptor and signal transduction (0.2 nM) at 22°C for 60 min, followed by addition of 250 nM unlabeled endothelin-1, with continued incubation at 22°C. The cell-bound [‘25r]endothelin-l decreased only slightly during subsequent incu~tion at 22°C as shown in Fig. lB, more than 60% of the initial cell bound radioactivity remained after 60 min. Even after 12 h more than 50.1% of the initial cell-bound [‘*~I]endothelin-1 still remained on the cells (data not shown). Specific binding of [~‘~~endothe~n-~ to the cells was a saturable process and represented 60-80% of the total binding when the concentration of [‘251’Jendothelin-l was below 180pM (Fig. 2A). Scatchard analysis of the saturation binding data by Lundon 1 program revealed the presence of a single class of ~~-a~ty binding site (Fig. 2B). The apparent dissociation constant (&) was 160 rt 60 pM and the maximal binding capacity (B_) was 3.34 j, 0.77 x 104 sites/cell (n = 3). When saturation binding experiments were performed at 30°C and 4°C under eq+ibrium binding conditions similar Kd and B_ were obtained (data not shown).
(A)
f:i’~
'*%ET-l
Bound
(PM)
(fmol)
Fig. 2. Saturation kinetics (A) and Scatchard analysis (E) of [I~~endo~e~n-l binding to NGlO8-15 cells. Cells ,were incubated at 22°C for 60 mm witb various concentrations of [‘zsIjendotbelin-l as indicated. Specificbinding was obtained by subtracting nonspecific binding in the presence of IOOnM of unlabeled endotbelin-I from the total binding. The data presented are from a sin8le experiment using triplicate samples and are representative of three experiments with similar results. Unlabeled endothelins competitively inhibited the binding of [‘2~IJendothelin-l to NGIOS-15 cells (Fig. 3). Analysis of the data by Lundon 2 program
ii_.._
0
15
50
45
80
Time (min)
Fig. 1. (A) Specific binding of [l~~ndo~e~n-~ to NG10815cells as a function of time. Confluent NGlO8- 15cells were barvested and cell suspension was prepared as described in Experimental Procedures. Cells (1 x 10s cells/tube) were inc~batcd with 0.2nM [“‘Ijendotbelin-l at 22°C for the times as indicated. Specisc binding was obtained by subtracting nonapecik binding in the presence of IOOnM unlabeled ~do~~n-~ ftom the total binding. Each point is tbe mean of triplicate sampies. (Et) Dissociation of cellbound [‘“&ndothelin-l from NG108-15 ceils. After incubation with 0.2 nM [%)endotbelin-I at 22°C for 60 mm and followed by addition of 250 nM of unlabeled endotbelin-1 (0 mm), the cells were fur&r incubated at 22’%Z.At the indicated times tbe cell-bound radioactivity was determined. Each point ia tbe mean of triplicate samples
01
11
10
s
8
7
6
I
ET (-log M)
Fig. 3. Conflation-de~&nt ~bition of [‘%I& endotbebn-i binding to NGIOt%IS cells by endotbelin-1, endotbelin-2, endotbebn-3 and big endotbelin. NGIOS-15 cells were incubated with 0.21&i [‘*~Ijendotbelin-1 and different concentrations of unlabeled endotbebns as in& cated in the fi8ure for 6Omin at 22°C. The data are expressed as tbe penxntagc of binding observed at each concentration of analogue. Each point is the mean of triplicate detonations from a representative experiment.
T. l_
218
YUE
et
ai.
Time Kd (-log
Fig. 4. Correlation between the apparent dissociation constants (&) of endothelin-I, endothelin-2 and endothelin-3, which are obtained from binding study (Fig. 3) and their corresponding EC% values for inducing [Ca2+limobilization in NG108-15 cells. The ECSO values for inducing [Ca’+J
mobilization are from our previous study.4’ for l-site and 2-site models showed the presence of high and low-a&&y binding sites for endothelin-1 and endothelin-2 when the concentrations of unlabeled ligand were greater than 1 nM. The Kd values of the high-affinity binding sites for endothelin-1 and endothelin-2 calculated from the competition curves were 220 pM and 560 pM, respectively. The Kd values of the low-affinity binding sites for endothelin-1 and endothelin-2 were 120 nM and 140 nM, respectively. The & for endothelin-3 and big endothelin were 62 nM and > 1 PM, respectively. Figure 4 shows the correlation between 1yd values of three endothelins from the present binding study and their ECU values for inducing elevation of [Ca’+]i in NG108-15 cells from our previous study.4’ There was no specific binding of [“‘Ilendothelin-1 to NCB-20 cells at all concentrations tested, as shown in Fig. 5. Endothelin
induced
(min)
M)
turnover
phosphoinositide
in
NG 108- 15 cells Addition of endothelin to cultured NGl08-15 cells prelabeled with [3H]myoinositol caused a 2.5- to
Fig. 6. Time course of endothelin-l-induced formation of [‘HjIP,, rH]IP, and [W]IPIp,in NG108-15 cells. Confluent NG108-15 cells were prelabeled with [3H]myoinositol in inositol-free DMEM overnight. The cells were harvested and suspension was prepared as described in Experimental Procedures. The cells were preincubated at 37°C for 10 min in the presence of 1OmM LiCl before addition of 1WnM
endothelin-1 or saline (control). The reaction was stopped at the indicated times and [%Qnositol phosphates were separated by column chromatography. The basal values of [‘H]IP,, IP, and IP, were 6553 f 380, 620 + 38 and 255 + 3 d.p.m., respectively, and did not change significantly during 20min incubation. Each point is the mean of a triplicate determination from a representative experiment which was repeated three times with similar results.
3-fold increase in the production of IP, (Figs 6 and 7). [3H]IP3 formation was observed immediately after addition of endothelin with the peak response occurring at 0.5-2 min (Fig. 6). Production of IP, and IP, was also increased when compared with controls, however, no obvious peak-response patterns could be identified (Fig. 6). The accumulation of IP, continued to increase, even after 20 min incubation with endothelin. The endothelin-induced production of IP, was directly dependent on the concentration of the agonist, as shown in Fig. 7. The concentration of endothelin-1 required for half-maximal stimulation (i.e. EC,) was 5.4nM and the saturating concentration was approximately 100-200 nM. Under the same condition endothelin-3 caused only a slight increase of IP, production (Fig. 7).
1
0
.M
72
100
144 ‘%El-1
270
1M)
360
(pM)
Fig. 5. Binding of [‘*?Jendothelin-1 to NCB20 cells. NCB20 cells were incubated with 0.2 nM [‘wtbelin-1 at tbe same condition as for NG108-15 cells described in Fig. 1. Each point is the mean of triplicate determination.
Fig. 7. Concentration dependence of cndothelin-1 stimulated production of mlrp, in NGIOS-15 cells. The experimental conditions are as described in kgend to Fig. 6.
Results are expressed as means f S.E.M. of fhree indcpendent experiments performed in triplicate. The basal value of f’H]IP, was 235 + 24 d.p.m. (n = 3).
219
Endothelin receptor and signal transduction
Phorbol
ester
(-log
M)
Fig. 8. Effects of phorbol ester on endothelin-l-induced rIIlIP, accumulation in NGlO&15 cells. Experimental conditions are as described in Experimental Procedures except that indicated concentrations of PDBu or 4u-PDD were added 15 min prior to the addition of IOOnM of endothelin-1 or saline (basal). The data presented are means f S.E.M. of three independent experiments. IOOnM of endothelin-l-induced accumulation of IP, at 20min after stimulation was 52 f 5.3% (n = 3) over the basal level in the absence of phorbol ester.
Effect ofphorbol ester on endothelin-l-induced inositol monophosphate accumulation and elevation of [Ca2 ‘Ii in NG 108-l 5 cells The maximum accumulation of IPr in NG108-15 cells induced by 100 nM of endothelin-1 over the basal was 3025 f 250 d.p.m. (n = 3) at 20 min after stimulation. Pretreatment of NG108-15 cells with the biologically active phorbol ester PDBu for 15 min at 37°C reduced the endothelin-l-induced response in a dose-dependent manner (Fig. 8). The maximum inhibition was 62% when the concentration of PDBu reached 1 PM. The basal accumulation of IP, was not significantly affected by PDBu (Fig. 8). The biologically inactive 4a-PDD had no effect on endothelin-linduced IP, accumulation in NG108-15 cells. Endothelin-1 elevated [Ca2 ‘Ii in NG108-15 cells (Table 1). In the presence of 0.25 and 1 PM PDBu, 100 nM endothelin-l-induced elevation of [Ca2+li in NG108-15 cells was reduced by 13.1 f7.0% (P > 0.05, n = 5) and 26.2 If: 5.5% (P > 0.05, n = 5), respectively. However, the inhibitory effect of PDBu on endothelin-l-induced elevation of [Ca2 ‘Ii increased substantially when the NG108-15 cells were suspended in Ca2+-free KRH buffer. As shown in Table 1, the elevation of [Ca2+li induced by 100 nM of endothelin-1 was reduced by 51 .O f 4.4%
(P < 0.01, n = 4) and 68.8 k 4.1% (P < 0.01, n = 4) in the presence of 0.25 PM and 1 PM PDBu, respectively. Under the same condition, 4a-PDD had no effect on endothelin-l-induced elevation of [Cazfli in NG108-15 cells (data not shown). Preincubation of NGlOS-15 cells with 1 PM PDBu for 20min did not change the maximal binding capacity for endothelin (B_) on the cells, but Kd value was increased by 2835% (n = 2) indicating the reduction of athnity of receptors to [1251]endothelin-1. When the concentration of PDBu was increased to 5 PM, still no difference in B_ was found between PDBu-treated and the control cells, but & value in treated cells was 47% higher than the control. Effect of pertussis toxin on endothelin-l-induced inositol mono- and trisphosphate accumulation and elevation of [Ca2+li in NG 108-15 ceils Endothelin-1 at 100 nM induced increase of IP, and IPr formation by 169.5 + 5.2% and 48.5 + 2.5% (n = 3) at 1 min and 20 min after addition of the agonist, respectively. Pretreatment of NG108-15 cells with 10, 100 or 500 ng/ml of PTX for 16-18 h had no significant effect on endothelin-l-induced IPr and IP, formation (Table 2). Meanwhile, [Ca’ ‘Ii mobilization induced by 100 nM endothelin-1 was not significantly different between control or PTX-treated cells (data not shown). Moreover, PTX did not affect the basal levels of Ca2+.
DISCUSSION
The present study demonstrates the existence of a single class of high a&&y and high density endothelin receptors on NG108-15 cells. The binding of [1251Jendothelin-l to these cells was specific and s&urable with an apparent & of 160pM and B_ of 3.34 x 104 sites/cell as analysed by Lundon 1 program. Unlabeled endothelin analogues such as endothelin-1, 2, 3 and big endothelin displaced [rz51Jendothelin-1 binding in a concentration-dependent manner. Analysis of the competition binding by Lundon 2 program showed that endothelin-1 and endothelin-2 displayed both high and low affinity binding sites with Kd values of 220 pM and 120 nM for endothelin-1 and 560pM and 140 nM for endothelin-2 for high and low afhnity sites, respectively.
Table 1. Effects of phorbol ester on 100 nM endothelin-l-induced elevation of cytosolic free calcium concentration in NG108-15 cells [Ca*+l, (nM)
P
Cells suspended in KBH buffer Endothelin Endothelin + PDBu (0.25 p M) Endothelin + PDBu (1 PM) Endothelin Endothelin + PDBu (0.25 PM) Endothelin + PDBu (1 rM)
406k66 353 f 28 302 f 23 Cells suspended in Ca2+-free KBH 14s*9 13 f 6 46k7
PDBu was added 10 min prior to the addition of endothelin-1; n = 4-5.
> 0.05 > 0.05 < 0.01 < 0.01
220
T. I.. YtiE et al
Table 2. Pertussis toxin did not inhibit endothelin-l-induced phate synthesis in NGl08-15 cells
inositol phos-
IP formation (% over basal) Endothelin- 1 Endothelin-I + PTX (10 ng/ml) Endothelin- 1 + PTX ( 100 ng/ml) Endothelin-1 + PTX (500 ng/ml) Endotbelin- 1 Endothelin- 1 + PTX (10 ng/ml) Endothelin- I+ PTX (100 ng/ml) Endothelin-I + PTX (500 rig/ml)
I’HllP, 49 & 7 42 rt; 6 40 It 5 42 i: 6 [‘H]IP, 170& 15 162 2 16 190 * 19 162 + 14
[‘H]myoinositol-labeled NGl08-15 cells were treated with saline or d&rent concentrations of PTX for 16-18 h and then washed for use. The cells were stimulated with 1OOnM of endothelin-1 for 1 mm (for IP, formation) or 20min (for IP, formation), respectively. The inositol phosphates were separated and measured as described in Experimental Procedures. ‘Ike basal values of [3~]~nositol phosphates were not affected by PTX. Each value is the mean of 3-5 experiments done in triplicate.
The Kd for high affinity site for endothelin-1 compared well with the & obtained from Scatchard analysis. The low affinity site was not detected in the saturation binding because the highest concentration of [‘251jendothelin used was 250 pM. The K,, values for endothelin-3 and big endothelin determined from competition were 62 nM and > 1 PM, respectively, suggesting that endothelin-3 was much less potent than endothelin-I and endothelin-2 whereas big endothelin was the least efficient. These binding data compare very well with the concentration required for stimulation of IP, formation (Fig. 7) and intracellular calcium release (Fig. 4). An increasing body of evidence has confirmed that endothelin is a potent stimulator of phosphoinositide turnover, however most studies have been confined to the cardiovascular system.“*‘7Js*2*,35 A few studies have recently reported that endothelin induced inosito1 phosphate formation in brain tissue,‘,7 perifused pituitary cells” and glial cell~,“*~~but whether endothelin has any effect on cerebellar granule cells is still controversial. ~6 Endothelin-indu~d production of IP, in NG108- 15 cells was briefly reported recently but its properties have not been characterized fully.’ The present study clearly shows that endothelin causes a robust, dose-dependent increase in the breakdown of phosphoinositide in NG108-15 cells. The EC= for endothe~n-I-induct IPs production was 5.4 nM (Fig. 7) which was quite in agreement with the EC, for endothelin-l-induced increase in [Ca”]i (6.7 nM) in these c.ell~.~iA profound increase in IP, production was found less than 10 s after addition of endothelin-I, which was coincident to the onset of the endothelin-l-induced increase in [Ca2+&. The peak increase in IP, appeared at 3WO s which was delayed relative to the peak increase in [Ca* ‘Ii (10-20 s).” The reason for this apparent discrepancy could be due to the fact that the IP, measured in the present study represents multiple isomers including 1,4,5-IP,, 1,3,4IP, and possibly others, while only 1,4,5-IP, was found to be able to stimulate Ca2 + signal4 It has been
reported that in addition to dephosphorylation to form 1,4-IP,, 1.4,5-IP3 can be metabolized via a novel pathway to form 1,3,4,5-IP., which is then transformed to 1,3,4-IP,. The enzyme required for this transformation was found in brain tissue. Therefore, the appearance of 1,3,4-IP, would be delayed relative to 1,4,5-IP,.19 In order to obtain further information on endothelin-induced activation of phospholipase C (PLC), the effects of phorbol ester were studied. Endothelin-iinduced IP, accumulation and elevation of fCaz+fi were attenuated by the biologically active phorbol ester PDBu in a dose-dependent manner. In contrast, neither phosphoinositide turnover nor Ca2+ signal induced by endothelin was affected by the biologically inactive 4a-PDD. In our recent study we have shown that endothelin-indu~d [Ca2+li increase in NGIOS-15 cells involves both infIux of extraceh~ CaZ+ (70%), and intracellular Ca2+ release (30%).4’ The present study demonstrated that PDBu at 1 PM inhibited endothelin- l-induced elevation of [CaZ+1; by 26% in the presence of extracelhtlar Ca* * . However, when the extraceilular Cati was depleted, the per cent inhibition by PDBu increased to 69% which was similar to the inhibition of IP, accumulation (62%) by the same concentration of PDBu (Fig. 8 and Table I), suggesting that the effect of PDBu was mainly via inhibition of pho~hoinositide turnover, which underlies the reduction of in~a~llular Ca*+ release. PDBu up to 1 JIM had no effect on the basal levels of IP, accumulation in NGlO&15 cells (Fig. 8), suggesting that the activated protein kinase C (PKC) did not at&t the basal activity of PLC. Pretreatment of NGIOS-15 cells with I pM PDBu resulted in an increase of & by 28-35% for [‘~~~o~e~n-l with no change in B_. Even when the concentration of PDBu was increased to 5 FM there was still no difference in B,, between control and PDBu-treated cells but the jyd was increased by 45.1%~~These results suggested that PDBu-induced reduction of endothelin receptor affinity in NG108-15 cells could be at
221
Endo~etin nceptor and signal transduction least one of the mechanisms by which PDBu inhibited endothelin-stimulated phosphoinositide turnover as well as [Ca*+]i mobi~tion. Phorbol ester-induced reduction of receptor afhnity has also been found in adrenergic receptors (on rat cardiac myocytes)23 and insulin receptors (on lymphocytes, macrophages and a~~t~).14*16 The ~ptor-rn~ia~ activation of PLC involves a GTP binding protein (G protein) which serves as a coupling unit between the receptor and the effector PLC.12 Whether the G protein coupled to endothelin receptors is sensitive to PTX has not been fully studied. It was reported that G protein responsible for the coupling of endothelin receptor to PLC activation in rabbit aorta smooth muscle cells was not affected by PTX.’ However, it has been demonstrated recently in our laboratory that rat renal mesangial cells possess endothelin receptors which are coupled to phosphoinositidePLC through a PTX-sensitive G protein2’ The present study clearly showed that pretreatment of NG108-15 cells with PTX up to 500ng/ml failed to show any effect on endothelin-induced [Ca2+], mobilization as well as endothelin-stimulated phosphoinositide turnover. Besides, the basal levels of [Ca2 +li, IP, and IP, were not significantly different between the control and PTX-treated cells. These observations suggest that G protein coupled to endothelin receptor
in NG108-15 cells could be PTX-insensitive. It also indicated that the eudothehn receptor-stimulated responses in difierent cell types could be regulated by different G proteins as observed for other receptors.‘2 CONCLUSION The present study has identified the presence of specific endothelin receptors on NG108-15 cells but not on NCB-20 cells. The results confirm our previous hypothesis that NCB-20 cells have no response to endothelin because of the absence of endothelin receptors on this cell line. Endothelin-induced [Ca2 ‘Ii mobilization in NGlOS-15 cells was a receptor-mediated process, and related at least partly to endothelininduced synthesis of inositol phosphates. Phorbol ester inhibited endothelin-induced increase in phosphoinositide turnover as well as in [Ca2 +li, indicating the involvement of PKC in the regulation of endothelin-induced response. Endothelin-indu~d phosphoinositide turnover and [Ca2+li mobilization were not affected by PTX, suggesting that the G-protein coupled to endothelin receptors in these cells was not sensitive to PTX. Since it has been suggested that endothelin plays a crucial role in neuronal injury, our findings may contribute to understanding of the mechanism of endothelin action on neuronal cells.
REFERENCES
1. Ambar I., Kloog Y., Schrartz I., Hazum E. and Sokolovsky M. (1989) Competitive interaction between endothelin and sarafotoxin: binding and phosphoinositides hydrolysis in rat atria and brain. Biachem. biophys. Rex Commun. 158, 195-201. 2. Araki S.-I., Kawahara Y., Kariya K.-I., Sum&o M., Fukuxaki H. and Takai Y. (1989) Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells. Biochem. biophys. Res. Commun. 159, 10724079. 3. Armstead W. M., Murro R., Liffler C. W. and Busija D. W. (1989) Influence of endothelin on piglet cerebral ~~~~~ation. Am. J. Physiol. 257, H707-710. 4. Berridge M. J. and Irvine R. F. (1984) Inositol t&phosphate, a novel second messenger in cellular signal transduction. Nature 312, 315-321. 5. Chuang D.-M. (1986) Carbacho-induced accumulation of inositol-l-phosphate in neurohybridoma NCB-20 cells: effects of lithium and phorbol esters. Biochem. biophys. Res. Commun. 136, 622429. 6. Cintra A., Fuxe K., Anggard E., Tinner B., Staines W. and Agnati L. F. (1989) Increased endothelinlike immunoreactivity in ibotenic acid Iesioned hippocampal formation of the rat brain. Actu physiol. stand. 137, 557458. 7. Crawford M. L. A., Hiley C. R. and Young J. M. (1990) Characteristics of endotbelin-1 and endothelin-3 stimulation
8. 9. 10. 11. 12. 13. 14. 15. 16.
of phosphoinositide breakdown differ between regions of guinea-pig and rat brain. Naunyn-Schmiedeberg’s Arch. Pharmac. 341,268-271. Faraci F. (1989) Effects of endothehn and vasopressin on cerebral blood vessels. Am. J. Physiol. 257, H799-H803. Fu T., Chang W., Ishida N., Saida K., Mitsi Y., Okano Y. and Noxana Y. (1989) Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells. Fe& Eur. biochem. Sots Lett. 257, 351-353. Fuxe K., Andbjer B., Kalia M. and Agnati L. F. (1989a) Centrally administered endo~elin-1 produces apnea in the a-cbloralose-anesthetized male rat. Acta physiol. stand. 137, 157-158. Fuxe K., Cintra A., Andbjer B., Anggard E., Goldstein M. and Agnati L. F. (1989b) Centrally administered endothelin-1 produces lesions in the brain of the male rat. Acta physiol. stand, 137, 155-157. Gilman A. G. (1987) G proteins: transductions of receptor-generated signals. A. Rev. Biochem. 56, 615-649. Goto K., Kasuya Y., Mats&i N., Takuwa Y., K&ham H., Ishikawa T., Kimura S., Yanagisawa M. and Masaki T. (1989) Endothelin activates the dihydropy~dine-~nsitive, voltage-dependent Ca2+ channel in vascular smooth muscle. Proc. nafn. Acad. Sci. USA 86, 391_%3918. Grunberger G. and Gorden P. (1982) Affinity alteration of insulin receptor induced by a phorbol ester. Am. J. Physiol. 243, E3 19E324. Grynkiewin G., Poenie M. and Tsien R. (1985) A new generation of calcium indicators with greatly improved fluorescence properties. J. biol. Chem. 2&I, -3450. Hating H. U., Kirsch D., Obermaier B., Ermel B. and Mach&o F. (1985) Tumor-promoting phorbol esters increase the K,,, of the ATP-binding site of the insulin receptor kinase from rat adipocytes. J. biol. Chem. 261, 3869-3873.
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17. Hirata Y., Yoshimi H., Takata S., Watanake T. X., Kumagai S., Nakajima K. and Sakakibara S. (198X) ~‘cllular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Rio&m biophys. Res. Commun. 154, 868-875. 18. Hoyer D., Waeber C. and Palacios J. M. (1989) [‘Z51]endothelin-I binding sites: autoradiographic studies m the brain and periphery of various species including humans. J. cardiouusc. Pharmac. 13 (Suppl. 5), S162-S165. 19. Irvine R. F., Letcher A. J., Heslop J. P. and Berridge M. J. (1986) The inositol tris/tetrakisphosphate pathwaydemonstration of Ins(1,4,5,)P, 3-kinase activity in animal tissues. Nature 320, 631-634. 20. Inoue A., Yanagisawa M., Kimura S., Kasuya Y., Miyachi T., Goto K. and Masak T. (1989) The human endothelin family-three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Prcjc nam Acud. Sci. USA 86. 2853-2867.
21. Jones C. R., Hiley’R. C., Pelton J. T. and Mohr M. (1989) Autoradiographic visualization of the binding sites lor [‘Z51]endothelin in rat and human brain. Neurosci. Left. 97. 27&279. 22. Koiaki C., Imai M., Hirata Y., Yanagisawa M. and Masaii T. (1989) Autoradiographic distribution in rat tissues ot binding sites for endothelin: a neuropeptide? Am. J. Physiol. 256, R858-R866. 23. Limas C. J. and Limas C. (1985) Phorbol ester- and diacylglycerol-mediated desensitization of cardiac B-adrenergic receptors. Circulation Res. 57, 44349. 24. MacCumber M. W., Ross C. A. and Snyder S. H. (1990) Endothelin in brain: receptors, mitogenesis and biosynthesis in glial cells. Proc. natn. Acud. Sci. USA 87, 2359-2363. 25. Marsden P. A., Danthuluri N. R., Brenner B. M., Ballermann B. J. and Brock T. A. (1989) Endothelin action on vascular smooth involves inositol trisphosphate and calcium mobilization. Biochem. biophys. Rex Commun. 158,8&93. 26. Nambi P., Pullen M. and Feuerstein G. (1990) Identification of endothelin receptors in various regions of rat brain. Neuropeptides 16, 195-199. 27. Nambi P., Wu S. L., Pullen M., Zabko-Potopovich B. and Albrightson-Winslow C. R. (1989) Identification. characterization and regulation of endothelin (ET) receptor in rat renal mesangial cells in culture (Abst.). 22nd Annual Meeting of the American Society of Nephrology. 28. Resink T. J., Scott-Burden T. and Buhler F. K. (1988) Endothelin stimulates phospholipase in cultured vascular smooth muscle cells. Biochem. biophys. Rex Commun. 157, 136&1368. 29. Robinson M. J. and McCulloch J. (1990) Contractile responses to endothelin in feline cortical vessels in situ. J. cerebr Blood Flow Metub. 10, 285-289.
30. Saito A., Shiba R., Kimura S., Yanagisawa M., Goto K. and Masaki T. (1989) Vasoconstrictor response of large cerebral arteries of cats to endothelin, an endothelium-derived vasoactive peptide. Eur. J. Pharmuc. 162, 353-358. 31. Shigeno T., Mima T., Takokura . K., M., Saito A., Goto K. and Masaki T. (1989) Endothelin-1 acts in . Yanagisawa _ . . .^ cerebral arteries from adventltlal but not from lummal side. J. curdiouasc. Pharmac. 13 (Suppls), 5174-5176. 32. Shirmi O., Kimura S., Yoshizawa T., Uchiyama Y., Sugida Y., Kanazawa I., Yanagisawa M., Goto K. and Masaki T. (1989) Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination. Biochem. biophys. Res. Commun. 162, 340-346. 33. Siren A. L. and Feuerstein G. (1989) Hemodynamic effects of endothelin after systemic and central nervous system administration in the conscious rats. Neuropeptides 14, 231-236. 34. Stojilkovic S. S., Merelli F., Iida T., Krsmanovic L. Z. and Catt K. J. (1990) Endothelin stimulation of cytosolic calcium
and gonadotropin secretion in anterior pituitary cells. Science 248, 1663-1666. 35. Sugiura M., Inagami T., Hare G. M. T. and Johns J. A. (1989) Endothelin action: inhibition by protein kinase C inhibitor and involvement of phosphoinositols. Biochem. biophys. Res. Commun. 158, 170-176. 36. Supattapone S., Simpson A. W. M. and Ashley C. C. (1989) Free calcium rise and mitogenesis in glial cells caused by endothelin. Eiochem. biophys. Res. Commun. 165, 1115-1122. 37. Willette T., Sauermelch C., Ezekiel M., Feuerstein G. and Ohlstein E. (1990) The effect of endothelin (ET-l) on local cortical microvascular perfusion in the rat. Stroke 21, 451458. 38. Yanagisawa M., Inome A., Ishikawa T., Kasuya Y., Kimura S., Goto K. and Masaki T. (1988a) Primary structure, synthesis and biological activity of rat endothelin, an endothelin-derived vasoconstrictor peptide. Proc. natn. Acud. Sci. USA 85, 69646967. 39. Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi
M., Mitsui Y., Yazaki Y., Goto K. and Masaki T. (1988b) Endothelin: a novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 41 l-415. 40. Yoshizawa T., Shinmi O., Graid A., Yanagisawa M., Gibson S. J., Kimura S., Udnyawa Y., Polak J. M., Masaki T. and Kanazawa I. (1990) Endothelin: a novel peptide in the posterior pituitary system. Science 247, 462-464. 41. Yue T. L., Gleason M. M., Lysko P. G. and Feuerstein G. (1990) Effect of endothelins on cytosolic free calcium concentration in neuroblastoma NG108-15 cells. Neuropeprides 17, 7-12. (Accepted 14 February 1991)
0306-4522/91 $3.00 + 0.00 Pcrgamoo Fkss plc 0 1991IBRO
1991
ENDOTHELIN RECEPTOR BINDING AND CELLULAR SIGNAL TRANSDUCTION IN NEUROHYBRID NG108-15 CELLS T. L. YuE,* P.
NAMBI,
H. L. Wu and G. FEUERSTEIN
Department of Pharmacology, SmithKline Beecham, King of Prussia, PA 19406-0939, U.S.A. Abstrac-Endothelins are a novel group of potent vasoconstrictor peptides originally isolated from cultured porcine endothelial cells. We and others have previously reported the presence of endothelin receptors in the central nervous system, and this study was designed to further characterize endothelin receptors and their transduction mechanism in cultured neurohybrid NGl08-15 cells. Specitic binding of [1251)endothelin-l to NGl08-15 cells reached saturation within 60min at 22°C and was only partially reversible. Scatchard analysis of the saturation binding revealed the presence of one class of high-affinity binding sites with an apparent dissociation constant of 16OpM and a maximal binding capacity of 3.3 x 10’ sites/cell. Unlabeled endothelin analogues competitively inhibited [‘251]endothelin-l binding to NGlOS-15 cells and the apparent dissociation constant values obtained from the competition curves correlated well with the ECX) values obtained for inducing elevation of intracellular free Ca*+ level. Endothelin stimulated phosphoinositide metabolism in a dose-dependent manner with an EC, value of 5.4 nM for inositol trisphosphate formation. The protein kinase C-activator phorbol ester dosedependently inhibited endothelin-induced phosphoinositide turnover and intracellular free Caz+ increase, suggesting the involvement of protein kinase C in the regulation of endothelin-induced responses. Neither endothelin-induced phosphoinositide hydrolysis nor endothelin-induced increase in intracellular free Ca2+ were affected by pertussis toxin. These dam indicate that endothelin receptors are present on NGl08-15 cells and the G protein coupled to endothelin receptor for inducing activation of phospholipase C and increase of free intracellular Ca*+ is insensitive to pertussis toxin.
Endothelins are a novel group of highly potent vasoconstrictor peptides originally isolated from vascular endothelial cell~~**~and subsequently found in the human genome in at least three isoforms, i.e. endothelin-1, endothelin-2 and endothelin-3.20 Recent studies have conkmed that endothelin-1 and endothelin-3 are present in cells other than the endothelium and particularly in neuronal cells. Material that cross-reacted with antibodies to endothelin has been detected in the porcine spinal cord32 as well as in the hypothalamus and posterior pituitary of pig and rat.@ A role for endothelin in the central nervous system has been suggested by studies showing potent cerebrovasculaIj*6~**3’*37 and autonomic33 responses following central administration of the various endothelin peptides. In addition to cerebrovascular
*To whom correspondence should be addressed at: Department of Pharmacology, SmithKline Beecham, P.O. Box 1539, L-510, King of Prussia, PA 19406-0939, USA. Abbreviations:BSA, bovine serum albumin; [Ca’+],, cytosolit free calcium concentration; DMEM, Dulbecco’s modified Eagle medium; EDTA, ethylenediaminetetraacetate; fura 2/AM, penta-acetoxymethyl ester of fura-2; IP,, inositol monophosphate; IP,, inositol bisphosuhate; IPJ, inositol trisphosphate; i&l buffer, Kkbs-Binger Henseleit; PDBu. uhorbol 12.13-dibutvrate: 4a-PDD. 4a -phorbol 12,13_didecanoate;~ PKC, piotein kinase Ci PLC, phospholipase C, PTX, pertussis toxin (Islet Activating Protein); TCA, trichloroacetic acid.
hemodynamic effects, endothelin-1 given intracisternally produced severe apnea which was independent of changes in systemic arterial blood pressure.‘O A discrete brain lesion of ischemic nature following intraparenchymal administration of endothelin- 1 into the striatal region of the rat brain was demonstrated, indicating that endothelin may contribute to the development of cerebral infarcts and neuronal death.” It has also been suggested that endothelin may function as a neuromodulator since it was observed that endothelin stimulated gonadotropin secretion in anterior pituitary cell~.~ Additional support for the potential role of endothelin in modulation of brain function has been drawn from autoradiographic studies showing a discrete, nonvascular pattern of distribution of endothelin-binding sites.‘s*21*uSpecific binding sites for endothelin-1 and endothelin3 in rat brain were also demonstrated recently in our laboratory, which showed a clear heterogenous distribution among various brain regions with a general increase in density in the hindbrain versus the forebrain regions.26 While discrete binding sites in brain tissue seem to be strongly suggested, the cellular origin of this receptor in the nervous system is still unclear.‘*” Moreover, the functional studies of endothelin at the cellular level in neuronal tissue, in particular the secondary messenger system associated with these receptors are rather limited.7Y*36 215
216
T. I.. YUE et al
In previous studies we found that endothelin induced a significant increase in cytosolic free calcium concentration ([Ca* ‘Ii) from both extracellular and intracellular sources in NGl08-15 cells, a hybrid of mouse neuroblastoma/rat glioma cells,4’ but not in NCB-20 cells which are a hybrid of mouse neuroblastoma/Chinese hamster cortical neurons.’ We hypothesized that the discrepancy in response to endothelin between NG108-15 and NCB-20 cells might reflect the fact that NCB-20 cells do not express the endothelin receptors. Further, the endothelin-induced increase in [Ca*+], in NG108-15 cells may be related to effect on phosphoinositide turnover since receptormediated phosphoinositide hydrolysis is obligatory for intracellular calcium release.4 In order to confirm our hypothesis the present study was designed to identify and characterize specific endothelin binding sites in NGl08- 15 and NCB-20 cells and to study the effect of endothelin on phosphoinositide turnover in these cells. EXPERIMENTAL
PROCEDURES
Maierials Endothelin- 1 (ET- 1) (human/porcine), endothelin-2 (ET-2) (human), endothelin-3 (ET-3) (lmman/rat) and big endothelin 22-38 (big ET) (human) were purchased from Peninsula Laboratories (Belmont, CA). [‘251]Endothelin-l (specific activity: 2200 Ci/mmol) and [31Qnyoinositol (specific activity: 17.4 Ci/mmol) were provided by New England Nuclear (Boston, MA) and Amersham (Des Plaines, IL), respectively. Phorbol 12,13_dibutyrate (PDBu) and 4u -phorbol 12,13-didecanoate (4a -PDD) were obtained from Sigma (St. Louis, MO). Pertussis toxin (Islet Activating Protein, PTX) was purchased from List Biological Laboratories (Campbell, CA). Pentaacetoxymethyl ester of fura-2(fura 2/AM) and AG l-X8 anion exchange resin (formate form, 2OWlOO mesh) were from Calbiochem (La Jolla, CA) and Bio-Rad (Richmond, CA), respectively. NGlOS-15 and NCB-20 cells were kindly provided by Dr M. Nirenbera (National Heart. Lung and Blood Institute, NIH). - ~ Cell culture NG108-15 and NCB-20 cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented by 10% (v/v) heat-in&tivated fetal calf serum,- 0.1 mM hypoxanthine. 0.4 uM aminonterin and 16 uM thvmidine in 150-cmZ flasks, in’a humid&d environment oi5% (for NCR20 cells) or 10% (for NGl08-15 cells) CO, at 37°C. After subculturing, the cells were grown to conffuence before harvesting. Binding experiments [1251]Endothelin-l binding experiments were performed on confluent NGlOS-I5 or NCB-20 cells. The culture medium was replaced with serum-free medium 24 h prior to experiment. The cells were harvested by shaking, and washed once with Dulbecco’s phosphate buffered saline containing 0.2% bovine serum albumin (BSA), 5mM glucose and 10 mM MgCl, and resuspended in the same buffer at 2 x IO6 cells/ml. Binding of [i2$ndothelin-1 to the cells was initiated bv adding 1 x lo5 cells/tube in a total volume of 0.25ml containing [‘251]endothelin-l in the absence (total binding) or presence (nonspecific binding) of 100 nM unlabeled endothelin-1. Cells were incubated at 22°C for 60min unless otherwise stated. The cell-associated radioactivity was separated from free l&and by vacuum filtration on GF/C filters presoaked in 0.1% BSA. The filters were
washed with 5 x 5 ml of 50 mM Tris buffer @H :‘.?I)WIItaining 10 mM MgCl, (4°C). Specific binding was defined as total binding minus nonspecific binding in the presence of 100 nM unlabeled endothelin-1. All experiments were prrformed in 12 x 75mm glass tubes in triplicate. Measurement of inositol phosphates accumulation Confluent NG108-15 cells were prelabeled wtth [3H]myoinositol (ZpCi/ml in T150 flask) in inositol-free DMEM overnight. The cells were harvested and washed twice with Krebs-Ringer Henseleit (KRH) buffer containing 0.1% BSA and in mM: NaCI. I 18: KCI. 4.6: NaHCO,. 24.9; KH,PG,, 1.0; glucose, 11.1; MgSG,, 1.1 and CaCI,, I:O, pH 7.4. and finally resuspended in KRH buffer containing 1OmM LiCl at I x IO6 cells/ml. An aliquot of t.he cell suspension (0.5 ml) was preincubated for 10 min at 37 c‘ and the reaction was initiated by the addition of agonist, cndothelin and continued for various times as indicated in the figures. The reaction was stopped by addition of 50~1 of 100% trichloroacetic acid (TCA). Samples were maintained on ice for 20 min and then centrifuged. TCA was extracted from the supernatant with 5 x 2m 1 of water-saturated diethylether. Excess ether was removed with nitrogen and the samples were neutralized with 10 ~1 of 0.5 M Tris base. Samples were stored frozen (- 80°C) until the mositol phosphates were separated by column chromatography. Samples prepared as described above were mixed with 4ml of 5mM sodium tetraborate containing 0.5 mM EDTA, and poured onto disposable polystyrene columns (1 x 10cm) containing 1 g of AG 1 x 8 anion exchange resin. [‘H]Inositol mono-, bis- and trisphosphate ([‘H]IP,. [‘H]IP, and [3H]IP,) were sequentially eluted into scintillation vials with 4ml of 0.2, 0.4 and 0.8 M ammonium formate containing 0.1 M formic acid, respectively. 10 ml of scintillation solution (Ready Gel, Beckman) was added to the vials and the radioactivity determined with a counting efficiency of 30%. Measurement of c:vtosolic free calcium concentration mobilization [Ca2+li was measured as described previously.4’ Briefly, the harvested cells were resuspended at 2 x 106/ml in KRH buffer and incubated with 2 MM of fura-2/AM at 37°C for 45min. The cells were centrifuged, resuspended in KRH buffer, and incubated at 37°C for an additional 20 min to allow for complete hydrolysis of entrapped ester. After loading, cells were washed and suspended in KRH buffer at 1 x 106/ml. The fluorescence of fura-2-containing cells was measured with a spectrofluorometer designed by the Biomedical Instruments Group, University of Pennsylvania. The wavelengths were set at 339 nm for excitation and 505 nm for emission. All experiments were performed at 37°C. The [CaZ+li was calculated according to the method of Grynkiewiez et al. Is For the studies in Ca* +-free KRH, fura-2-loaded cells were spun down and resuspended in pre-warmed (37°C) Ca 2+-free KRH buffer. The cells were used immediately. Statistics Data in the text and figures are mean + S.E.M. unless otherwise stated. Statistical significance between two groups was examined through one way analysis of variance and Dunnetts multiple range test. Significant differences were accepted at P < 0.05. RESULTS
[‘251]Endothelin-l binding to NG 108- 15 cells
Binding of [‘251Jendothelin-l to NG108-15 cells was a time-dependent process and reached equilibrium between 30 and 60min at 22°C (Fig. IA). To determine the dissociation of cell bound [‘251jendothelin-l, NG 108-l 5 cells were incubated with [1251]endothelin-1
217
Endotbelin receptor and signal transduction (0.2 nM) at 22°C for 60 min, followed by addition of 250 nM unlabeled endothelin-1, with continued incubation at 22°C. The cell-bound [‘25r]endothelin-l decreased only slightly during subsequent incu~tion at 22°C as shown in Fig. lB, more than 60% of the initial cell bound radioactivity remained after 60 min. Even after 12 h more than 50.1% of the initial cell-bound [‘*~I]endothelin-1 still remained on the cells (data not shown). Specific binding of [~‘~~endothe~n-~ to the cells was a saturable process and represented 60-80% of the total binding when the concentration of [‘251’Jendothelin-l was below 180pM (Fig. 2A). Scatchard analysis of the saturation binding data by Lundon 1 program revealed the presence of a single class of ~~-a~ty binding site (Fig. 2B). The apparent dissociation constant (&) was 160 rt 60 pM and the maximal binding capacity (B_) was 3.34 j, 0.77 x 104 sites/cell (n = 3). When saturation binding experiments were performed at 30°C and 4°C under eq+ibrium binding conditions similar Kd and B_ were obtained (data not shown).
(A)
f:i’~
'*%ET-l
Bound
(PM)
(fmol)
Fig. 2. Saturation kinetics (A) and Scatchard analysis (E) of [I~~endo~e~n-l binding to NGlO8-15 cells. Cells ,were incubated at 22°C for 60 mm witb various concentrations of [‘zsIjendotbelin-l as indicated. Specificbinding was obtained by subtracting nonspecific binding in the presence of IOOnM of unlabeled endotbelin-I from the total binding. The data presented are from a sin8le experiment using triplicate samples and are representative of three experiments with similar results. Unlabeled endothelins competitively inhibited the binding of [‘2~IJendothelin-l to NGIOS-15 cells (Fig. 3). Analysis of the data by Lundon 2 program
ii_.._
0
15
50
45
80
Time (min)
Fig. 1. (A) Specific binding of [l~~ndo~e~n-~ to NG10815cells as a function of time. Confluent NGlO8- 15cells were barvested and cell suspension was prepared as described in Experimental Procedures. Cells (1 x 10s cells/tube) were inc~batcd with 0.2nM [“‘Ijendotbelin-l at 22°C for the times as indicated. Specisc binding was obtained by subtracting nonapecik binding in the presence of IOOnM unlabeled ~do~~n-~ ftom the total binding. Each point is tbe mean of triplicate sampies. (Et) Dissociation of cellbound [‘“&ndothelin-l from NG108-15 ceils. After incubation with 0.2 nM [%)endotbelin-I at 22°C for 60 mm and followed by addition of 250 nM of unlabeled endotbelin-1 (0 mm), the cells were fur&r incubated at 22’%Z.At the indicated times tbe cell-bound radioactivity was determined. Each point ia tbe mean of triplicate samples
01
11
10
s
8
7
6
I
ET (-log M)
Fig. 3. Conflation-de~&nt ~bition of [‘%I& endotbebn-i binding to NGIOt%IS cells by endotbelin-1, endotbelin-2, endotbebn-3 and big endotbelin. NGIOS-15 cells were incubated with 0.21&i [‘*~Ijendotbelin-1 and different concentrations of unlabeled endotbebns as in& cated in the fi8ure for 6Omin at 22°C. The data are expressed as tbe penxntagc of binding observed at each concentration of analogue. Each point is the mean of triplicate detonations from a representative experiment.
T. l_
218
YUE
et
ai.
Time Kd (-log
Fig. 4. Correlation between the apparent dissociation constants (&) of endothelin-I, endothelin-2 and endothelin-3, which are obtained from binding study (Fig. 3) and their corresponding EC% values for inducing [Ca2+limobilization in NG108-15 cells. The ECSO values for inducing [Ca’+J
mobilization are from our previous study.4’ for l-site and 2-site models showed the presence of high and low-a&&y binding sites for endothelin-1 and endothelin-2 when the concentrations of unlabeled ligand were greater than 1 nM. The Kd values of the high-affinity binding sites for endothelin-1 and endothelin-2 calculated from the competition curves were 220 pM and 560 pM, respectively. The Kd values of the low-affinity binding sites for endothelin-1 and endothelin-2 were 120 nM and 140 nM, respectively. The & for endothelin-3 and big endothelin were 62 nM and > 1 PM, respectively. Figure 4 shows the correlation between 1yd values of three endothelins from the present binding study and their ECU values for inducing elevation of [Ca’+]i in NG108-15 cells from our previous study.4’ There was no specific binding of [“‘Ilendothelin-1 to NCB-20 cells at all concentrations tested, as shown in Fig. 5. Endothelin
induced
(min)
M)
turnover
phosphoinositide
in
NG 108- 15 cells Addition of endothelin to cultured NGl08-15 cells prelabeled with [3H]myoinositol caused a 2.5- to
Fig. 6. Time course of endothelin-l-induced formation of [‘HjIP,, rH]IP, and [W]IPIp,in NG108-15 cells. Confluent NG108-15 cells were prelabeled with [3H]myoinositol in inositol-free DMEM overnight. The cells were harvested and suspension was prepared as described in Experimental Procedures. The cells were preincubated at 37°C for 10 min in the presence of 1OmM LiCl before addition of 1WnM
endothelin-1 or saline (control). The reaction was stopped at the indicated times and [%Qnositol phosphates were separated by column chromatography. The basal values of [‘H]IP,, IP, and IP, were 6553 f 380, 620 + 38 and 255 + 3 d.p.m., respectively, and did not change significantly during 20min incubation. Each point is the mean of a triplicate determination from a representative experiment which was repeated three times with similar results.
3-fold increase in the production of IP, (Figs 6 and 7). [3H]IP3 formation was observed immediately after addition of endothelin with the peak response occurring at 0.5-2 min (Fig. 6). Production of IP, and IP, was also increased when compared with controls, however, no obvious peak-response patterns could be identified (Fig. 6). The accumulation of IP, continued to increase, even after 20 min incubation with endothelin. The endothelin-induced production of IP, was directly dependent on the concentration of the agonist, as shown in Fig. 7. The concentration of endothelin-1 required for half-maximal stimulation (i.e. EC,) was 5.4nM and the saturating concentration was approximately 100-200 nM. Under the same condition endothelin-3 caused only a slight increase of IP, production (Fig. 7).
1
0
.M
72
100
144 ‘%El-1
270
1M)
360
(pM)
Fig. 5. Binding of [‘*?Jendothelin-1 to NCB20 cells. NCB20 cells were incubated with 0.2 nM [‘wtbelin-1 at tbe same condition as for NG108-15 cells described in Fig. 1. Each point is the mean of triplicate determination.
Fig. 7. Concentration dependence of cndothelin-1 stimulated production of mlrp, in NGIOS-15 cells. The experimental conditions are as described in kgend to Fig. 6.
Results are expressed as means f S.E.M. of fhree indcpendent experiments performed in triplicate. The basal value of f’H]IP, was 235 + 24 d.p.m. (n = 3).
219
Endothelin receptor and signal transduction
Phorbol
ester
(-log
M)
Fig. 8. Effects of phorbol ester on endothelin-l-induced rIIlIP, accumulation in NGlO&15 cells. Experimental conditions are as described in Experimental Procedures except that indicated concentrations of PDBu or 4u-PDD were added 15 min prior to the addition of IOOnM of endothelin-1 or saline (basal). The data presented are means f S.E.M. of three independent experiments. IOOnM of endothelin-l-induced accumulation of IP, at 20min after stimulation was 52 f 5.3% (n = 3) over the basal level in the absence of phorbol ester.
Effect ofphorbol ester on endothelin-l-induced inositol monophosphate accumulation and elevation of [Ca2 ‘Ii in NG 108-l 5 cells The maximum accumulation of IPr in NG108-15 cells induced by 100 nM of endothelin-1 over the basal was 3025 f 250 d.p.m. (n = 3) at 20 min after stimulation. Pretreatment of NG108-15 cells with the biologically active phorbol ester PDBu for 15 min at 37°C reduced the endothelin-l-induced response in a dose-dependent manner (Fig. 8). The maximum inhibition was 62% when the concentration of PDBu reached 1 PM. The basal accumulation of IP, was not significantly affected by PDBu (Fig. 8). The biologically inactive 4a-PDD had no effect on endothelin-linduced IP, accumulation in NG108-15 cells. Endothelin-1 elevated [Ca2 ‘Ii in NG108-15 cells (Table 1). In the presence of 0.25 and 1 PM PDBu, 100 nM endothelin-l-induced elevation of [Ca2+li in NG108-15 cells was reduced by 13.1 f7.0% (P > 0.05, n = 5) and 26.2 If: 5.5% (P > 0.05, n = 5), respectively. However, the inhibitory effect of PDBu on endothelin-l-induced elevation of [Ca2 ‘Ii increased substantially when the NG108-15 cells were suspended in Ca2+-free KRH buffer. As shown in Table 1, the elevation of [Ca2+li induced by 100 nM of endothelin-1 was reduced by 51 .O f 4.4%
(P < 0.01, n = 4) and 68.8 k 4.1% (P < 0.01, n = 4) in the presence of 0.25 PM and 1 PM PDBu, respectively. Under the same condition, 4a-PDD had no effect on endothelin-l-induced elevation of [Cazfli in NG108-15 cells (data not shown). Preincubation of NGlOS-15 cells with 1 PM PDBu for 20min did not change the maximal binding capacity for endothelin (B_) on the cells, but Kd value was increased by 2835% (n = 2) indicating the reduction of athnity of receptors to [1251]endothelin-1. When the concentration of PDBu was increased to 5 PM, still no difference in B_ was found between PDBu-treated and the control cells, but & value in treated cells was 47% higher than the control. Effect of pertussis toxin on endothelin-l-induced inositol mono- and trisphosphate accumulation and elevation of [Ca2+li in NG 108-15 ceils Endothelin-1 at 100 nM induced increase of IP, and IPr formation by 169.5 + 5.2% and 48.5 + 2.5% (n = 3) at 1 min and 20 min after addition of the agonist, respectively. Pretreatment of NG108-15 cells with 10, 100 or 500 ng/ml of PTX for 16-18 h had no significant effect on endothelin-l-induced IPr and IP, formation (Table 2). Meanwhile, [Ca’ ‘Ii mobilization induced by 100 nM endothelin-1 was not significantly different between control or PTX-treated cells (data not shown). Moreover, PTX did not affect the basal levels of Ca2+.
DISCUSSION
The present study demonstrates the existence of a single class of high a&&y and high density endothelin receptors on NG108-15 cells. The binding of [1251Jendothelin-l to these cells was specific and s&urable with an apparent & of 160pM and B_ of 3.34 x 104 sites/cell as analysed by Lundon 1 program. Unlabeled endothelin analogues such as endothelin-1, 2, 3 and big endothelin displaced [rz51Jendothelin-1 binding in a concentration-dependent manner. Analysis of the competition binding by Lundon 2 program showed that endothelin-1 and endothelin-2 displayed both high and low affinity binding sites with Kd values of 220 pM and 120 nM for endothelin-1 and 560pM and 140 nM for endothelin-2 for high and low afhnity sites, respectively.
Table 1. Effects of phorbol ester on 100 nM endothelin-l-induced elevation of cytosolic free calcium concentration in NG108-15 cells [Ca*+l, (nM)
P
Cells suspended in KBH buffer Endothelin Endothelin + PDBu (0.25 p M) Endothelin + PDBu (1 PM) Endothelin Endothelin + PDBu (0.25 PM) Endothelin + PDBu (1 rM)
406k66 353 f 28 302 f 23 Cells suspended in Ca2+-free KBH 14s*9 13 f 6 46k7
PDBu was added 10 min prior to the addition of endothelin-1; n = 4-5.
> 0.05 > 0.05 < 0.01 < 0.01
220
T. I.. YtiE et al
Table 2. Pertussis toxin did not inhibit endothelin-l-induced phate synthesis in NGl08-15 cells
inositol phos-
IP formation (% over basal) Endothelin- 1 Endothelin-I + PTX (10 ng/ml) Endothelin- 1 + PTX ( 100 ng/ml) Endothelin-1 + PTX (500 ng/ml) Endotbelin- 1 Endothelin- 1 + PTX (10 ng/ml) Endothelin- I+ PTX (100 ng/ml) Endothelin-I + PTX (500 rig/ml)
I’HllP, 49 & 7 42 rt; 6 40 It 5 42 i: 6 [‘H]IP, 170& 15 162 2 16 190 * 19 162 + 14
[‘H]myoinositol-labeled NGl08-15 cells were treated with saline or d&rent concentrations of PTX for 16-18 h and then washed for use. The cells were stimulated with 1OOnM of endothelin-1 for 1 mm (for IP, formation) or 20min (for IP, formation), respectively. The inositol phosphates were separated and measured as described in Experimental Procedures. ‘Ike basal values of [3~]~nositol phosphates were not affected by PTX. Each value is the mean of 3-5 experiments done in triplicate.
The Kd for high affinity site for endothelin-1 compared well with the & obtained from Scatchard analysis. The low affinity site was not detected in the saturation binding because the highest concentration of [‘251jendothelin used was 250 pM. The K,, values for endothelin-3 and big endothelin determined from competition were 62 nM and > 1 PM, respectively, suggesting that endothelin-3 was much less potent than endothelin-I and endothelin-2 whereas big endothelin was the least efficient. These binding data compare very well with the concentration required for stimulation of IP, formation (Fig. 7) and intracellular calcium release (Fig. 4). An increasing body of evidence has confirmed that endothelin is a potent stimulator of phosphoinositide turnover, however most studies have been confined to the cardiovascular system.“*‘7Js*2*,35 A few studies have recently reported that endothelin induced inosito1 phosphate formation in brain tissue,‘,7 perifused pituitary cells” and glial cell~,“*~~but whether endothelin has any effect on cerebellar granule cells is still controversial. ~6 Endothelin-indu~d production of IP, in NG108- 15 cells was briefly reported recently but its properties have not been characterized fully.’ The present study clearly shows that endothelin causes a robust, dose-dependent increase in the breakdown of phosphoinositide in NG108-15 cells. The EC= for endothe~n-I-induct IPs production was 5.4 nM (Fig. 7) which was quite in agreement with the EC, for endothelin-l-induced increase in [Ca”]i (6.7 nM) in these c.ell~.~iA profound increase in IP, production was found less than 10 s after addition of endothelin-I, which was coincident to the onset of the endothelin-l-induced increase in [Ca2+&. The peak increase in IP, appeared at 3WO s which was delayed relative to the peak increase in [Ca* ‘Ii (10-20 s).” The reason for this apparent discrepancy could be due to the fact that the IP, measured in the present study represents multiple isomers including 1,4,5-IP,, 1,3,4IP, and possibly others, while only 1,4,5-IP, was found to be able to stimulate Ca2 + signal4 It has been
reported that in addition to dephosphorylation to form 1,4-IP,, 1.4,5-IP3 can be metabolized via a novel pathway to form 1,3,4,5-IP., which is then transformed to 1,3,4-IP,. The enzyme required for this transformation was found in brain tissue. Therefore, the appearance of 1,3,4-IP, would be delayed relative to 1,4,5-IP,.19 In order to obtain further information on endothelin-induced activation of phospholipase C (PLC), the effects of phorbol ester were studied. Endothelin-iinduced IP, accumulation and elevation of fCaz+fi were attenuated by the biologically active phorbol ester PDBu in a dose-dependent manner. In contrast, neither phosphoinositide turnover nor Ca2+ signal induced by endothelin was affected by the biologically inactive 4a-PDD. In our recent study we have shown that endothelin-indu~d [Ca2+li increase in NGIOS-15 cells involves both infIux of extraceh~ CaZ+ (70%), and intracellular Ca2+ release (30%).4’ The present study demonstrated that PDBu at 1 PM inhibited endothelin- l-induced elevation of [CaZ+1; by 26% in the presence of extracelhtlar Ca* * . However, when the extraceilular Cati was depleted, the per cent inhibition by PDBu increased to 69% which was similar to the inhibition of IP, accumulation (62%) by the same concentration of PDBu (Fig. 8 and Table I), suggesting that the effect of PDBu was mainly via inhibition of pho~hoinositide turnover, which underlies the reduction of in~a~llular Ca*+ release. PDBu up to 1 JIM had no effect on the basal levels of IP, accumulation in NGlO&15 cells (Fig. 8), suggesting that the activated protein kinase C (PKC) did not at&t the basal activity of PLC. Pretreatment of NGIOS-15 cells with I pM PDBu resulted in an increase of & by 28-35% for [‘~~~o~e~n-l with no change in B_. Even when the concentration of PDBu was increased to 5 FM there was still no difference in B,, between control and PDBu-treated cells but the jyd was increased by 45.1%~~These results suggested that PDBu-induced reduction of endothelin receptor affinity in NG108-15 cells could be at
221
Endo~etin nceptor and signal transduction least one of the mechanisms by which PDBu inhibited endothelin-stimulated phosphoinositide turnover as well as [Ca*+]i mobi~tion. Phorbol ester-induced reduction of receptor afhnity has also been found in adrenergic receptors (on rat cardiac myocytes)23 and insulin receptors (on lymphocytes, macrophages and a~~t~).14*16 The ~ptor-rn~ia~ activation of PLC involves a GTP binding protein (G protein) which serves as a coupling unit between the receptor and the effector PLC.12 Whether the G protein coupled to endothelin receptors is sensitive to PTX has not been fully studied. It was reported that G protein responsible for the coupling of endothelin receptor to PLC activation in rabbit aorta smooth muscle cells was not affected by PTX.’ However, it has been demonstrated recently in our laboratory that rat renal mesangial cells possess endothelin receptors which are coupled to phosphoinositidePLC through a PTX-sensitive G protein2’ The present study clearly showed that pretreatment of NG108-15 cells with PTX up to 500ng/ml failed to show any effect on endothelin-induced [Ca2+], mobilization as well as endothelin-stimulated phosphoinositide turnover. Besides, the basal levels of [Ca2 +li, IP, and IP, were not significantly different between the control and PTX-treated cells. These observations suggest that G protein coupled to endothelin receptor
in NG108-15 cells could be PTX-insensitive. It also indicated that the eudothehn receptor-stimulated responses in difierent cell types could be regulated by different G proteins as observed for other receptors.‘2 CONCLUSION The present study has identified the presence of specific endothelin receptors on NG108-15 cells but not on NCB-20 cells. The results confirm our previous hypothesis that NCB-20 cells have no response to endothelin because of the absence of endothelin receptors on this cell line. Endothelin-induced [Ca2 ‘Ii mobilization in NGlOS-15 cells was a receptor-mediated process, and related at least partly to endothelininduced synthesis of inositol phosphates. Phorbol ester inhibited endothelin-induced increase in phosphoinositide turnover as well as in [Ca2 +li, indicating the involvement of PKC in the regulation of endothelin-induced response. Endothelin-indu~d phosphoinositide turnover and [Ca2+li mobilization were not affected by PTX, suggesting that the G-protein coupled to endothelin receptors in these cells was not sensitive to PTX. Since it has been suggested that endothelin plays a crucial role in neuronal injury, our findings may contribute to understanding of the mechanism of endothelin action on neuronal cells.
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