Characterization of endothelin I receptor and signal transduction mechanisms in rat medullary interstitial BARRY M. WILKES, ELIZABETH GIRARDI, RICHARD BARNETT,

ANNE S. RUSTON, PETER DIANE HART, MONIQUE AND EDWARD P. NORD

MENTO, VANDER

cells

MOLEN,

Divisions of Nephrology and Hypertension, Departments of Medicine, North Shore University Hospital and the Department of Medicine, Cornell University Medical College, Manhusset 11030; and University Hospital and Northport Veterans Administration Medical Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794

WILKES, BARRY M., ANNE S. RUSTON, PETER MENTO, ELIZABETH GIRARDI, DIANE HART, MONIQUE VANDER MOLEN, RICHARD BARNETT, AND EDWARD P. NORD. Characterization of endothelin 1 receptor and signal transduction mechunismsin

rut meduhy interstitial ceUs.Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F579-F589, 1991.-Previous autoradiographic studies have delineated the renal medulla as the predominant site of renal endothelin (ET) receptors. Accordingly, cultured rat renal medullary interstitial cells (RMICs) were studiedasa target tissuefor ET action. Scatchard analysis revealedpresenceof a singleclassof high-affinity receptor sites (&, 57 & 10 pM; receptor density, 749 t 124 fmol/mg protein). Relative potency order for displacing 1251-ET-1was ET-l > ET-2 > sarafotoxin > big endothelin (human) = big endothelin (porcine). ET-3, unrelated pressor substances,vasodilators, Ca2+channel antagonists, atrial natriuretic factor, GTP, and GppNHp did not inhibit binding. Challengeof monolayerswith ET-l evoked a biphasic elevation in cytosolic free Ca2+concentration ([Ca”]i). Initial transient rise in [Ca2+]i observed in absence of extracellular Ca2+ and accumulation of inositol trisphosphate (IP3) was consistent with activation of phosphatidylinositol-specific phospholipaseC (PI-PLC). Half-maximal activation concentration of ET-l for the processwas 0.5 and 1 nM for [Ca”‘]i and IPB, respectively. The late sustained phase in [Ca2+]i elevation was completely blocked by Ni2+, unperturbed by nimodipine, and accompaniedby influx of Mn2+, indicating presenceof receptor-operated Ca2’ channels. Ca2+ channel openingwasdetected at lo-l6 M ET-l, whereas>10-12 M agonist was required to mobilize Ca2+ from intracellular stores and/or stimulate phosphoinositol hydrolysis, indicating that ET activation of PI-PLC and Ca2+channel opening were independent events. ET-l markedly stimulated prostaglandin Es synthesis in a concentration-dependent manner that paralleled PI-PLC activation and mobilization of [Ca2+]i. In summary, cultured rat RMICs possessET receptors that are linked to PI-PLC, Ca2+channels, and perhapsphospholipaseA2. inositol phosphate;calcium channel; prostaglandin E2 ENDOTHELIN (ET) is a recently identified

novel peptide synthesized by vascular endothelial cells that exhibits potent vasoconstrictor activity (38). Although smooth muscle cells of the vasculature were originally identified as the major target tissue, accumulating evidence indicates that nonmyogenic cells possess ET’ receptors. The recent identification of ET receptors on osteoblasts (32) 0363-6127/91

$1.50

Copyright

has led to the suggestion that ET may play an important role in bone metabolism. In Swiss 3T3 fibroblasts (31) ET has been shown to be a potent mitogen. The observation that the vast majority of ET receptors in the kidney were located in the renal medulla (17) prompted us to explore the nature of the cell type involved and the mechanisms of ET action. Interstitial cells of the renal medulla (16, 21) are the predominant site of renal prostaglandin production. Because these cells are found in close juxtaposition to other components of the renal medulla, it is not difficult to envision a paracrine role for their synthetic products. The present study identifies the rat renal medullary interstitial cell (RMIC) as a target cell for ET action. The results demonstrate that the cultured cells possess a single class of high-affinity receptor sites linked to a phosphatidylinositol-specific phospholipase C (PI-PLC) and possibly a phospholipase A2 (PLAZ). ET also activated receptor-linked Ca2+ channels at concentrations of agonist well below that required to stimulate PI-PLC activity. METHODS

AND

MATERIALS

Cell Culture

RMICs were kindly provided by Dr. T. Ma&k, Department of Physiology, Cornell University Medical College (10). These cells have been previously characterized by other workers (21). Cultures were maintained in a complete growth medium composed of RPM1 1640,10% heat-inactivated newborn calf serum, 15 mM N-2-hydroxyethylpiperazine-N’ -2.ethanesulfonic acid (HEPES), and 25 mM NaHC03 to which 50 IU/pl penicillin and 50 pg/ml streptomycin were added. Cells were grown either on sterile 25.mm diameter glass cover slips (microfluorometry), six-well culture dishes (inositol phosphate and prostaglandin determinations), 35 x lomm plastic Petri dishes (inositol phosphate determinations), or 75.cm3 plastic flasks (receptor binding). Cultures were maintained in a humidified incubator at 37°C in 95% air-5% CO2 (culture medium pH 7.3) and fed at intervals of 48-72 h. Experiments were performed on passages 16-32.

0 1991 the American

Physiological

Society

F579

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F580 ET-1 Radioreceptor

ENDOTHELIN

1 MECHANISMS

Measurements

Tissue preparation. RMICs grown to confluence in 75 cm2 flasks were harvested by addition to the flask of a balanced salt solution composed of the following (in mM): 116 NaCl, 26 NaHC03, 5 KCl, 1 NaH2P04, 5.5 glucose, and 1 ethylene glycol-bis(p-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) brought to pH 7.4 with 5% C02. Harvested cells were suspended in phosphate-buffered saline prior to homogenization. The cell suspension was homogenized with a Polytron PTA 10s (Brinkman Instruments, Westbury, NY) at setting 7 for 15 s. The homogenate was centrifuged at 1,000 g for 20 min (4”C), the resultant supernatant was centrifuged at 44,000 g for 10 min, and the pellet was washed twice in ice-cold buffer. The pellet was resuspended in a buffer composed of (in mM) 50 tris(hydroxymethyl)aminomethane (Tris), 154 NaCl, and 0.5 EGTA (pH 7.4) and was then frozen at -70°C before experimentation. On the day of the binding studies, the tissue was slowly thawed at room temperature and protein content was determined by the method of Lowry (17a), with bovine serum albumin as the standard. Radio&and receptor-binding assay. Binding studies were performed in a total volume of 250 ~1 containing plasma membranes (6.25 pg of membrane protein in 25 ~1 of sucrose buffer) and 225 ~1 of binding buffer composed of the following (in mM): 50 Tris, 154 NaCl, 1 EDTA, 25 MnC12, and 1 N-acetyl-DL-methionine, as well as 2.5% bovine serum albumin of radioimmunoassay (RIA) grade, buffered to pH 7.5. The &3Cetyl-DL-methionine was added to preserve the integrity of the sulfhydryl bridges in ET-l. Assay tubes were incubated at 25°C in a Dubnoff shaking water bath at 60 cycles/ min with varying amounts of ET-1 for the times specified in the legends to Figs. l-3. The binding reaction was terminated by addition of 3 ml of ice-cold Tris-isosaline (pH 7.5; 50 mM Tris in 0.154 M NaCl) to the reaction tube, and bound counts were separated from free by rapid filtration through glass-fiber filters (no. 30, Schleicher & Schuell). The test tube and the trapped membrane fragments were washed three times with 3 ml of ice-cold buffer. The filters were analyzed for 12? by use of a gamma radiation detector (1277 GammaMaster; LKB, Turku, Finland). ET-1 (0.1 PM) caused maximal displacement of the ligand and defined the level of nonspecific binding. Specific binding was determined as the difference between the amount of 1251-labeled ET-1 bound in the absence and presence of 0.1 pM ET-l. Kinetic binding experiments. The rates of association and dissociation ( /z+~ and k+, respectively) of 1251-ET-l (40 PM) to RMIC membranes were measured in separate experiments. In the association experiments the binding reaction was carried out for 2.5 min to 24 h, and the data analyzed as a pseudo-first-order ligand receptor reaction (34). The value of kSI was determined by measuring specific binding 30 min to 25 h after the addition of 0.1 PM unlabeled ET-l to membranes already bound to equilibrium and analyzed as a true first-order reaction. The equilibrium dissociation constant (K#) was calculated by the formula & = k-I/k+I. Equilibrium binding studies. Equilibrium binding stud-

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ies were performed at 25°C. Eight concentrations of 1251ET-l (4-500 PM) were studied in the presence and absence of 0.1 PM unlabeled ET-l, yielding total and nonspecific binding isotherms. Specific binding, calculated as the difference between total and nonspecific binding, was analyzed by use of nonlinear regression curve-fitting analysis of the untransformed data and by linear regression analysis according to Scatchard (29) to yield estimates of receptor density and apparent dissociation constants (K,). The possible existence of multiple classes of receptors was investigated by the comparison of goodness to fit to a one-, two-, or three-component binding equation using the program of Munson and Rodbard (22). Competitive binding studies. Competition for specific binding of 40 pM 1251-ET-1 was studied by incubating for 3 h in the presence of unlabeled compounds (10-13lo-* M). The data were analyzed by nonlinear regression analysis, and the dissociation constants characterizing competing drugs were calculated from the equation defined by Cheng and Prusoff (9) & = I&/(1 + S/K,): where IC50 is the concentration of competing drug causing 50% inhibition of specific binding, S is the concentration of the radioligand, and K, is the affinity of the radioligand for its specific binding sites. Monitoring of Changes in Cytosolic Free Ca2+ Concentration

Changes in cytosolic-free Ca2+ concentration ([Ca”]i) were monitored fluorometrically by use of the acetoxymethyl ester (AM) of the Ca2+-sensitive probe fura(14). For this purpose a previously reported cell suspension method (1, 2, 5) was adapted for microfluorometry. Cells grown on 25-mm diameter glass cover slips were incubated with 5 PM fura-2/AM (in dimethyl sulfoxide) for 15-20 min at 37°C. Preparatory experiments established that intracellular dye concentration had peaked by this time. Loaded monolayers were rinsed three times in assay buffer composed of the following (in mM): 135 NaCl, 5 KCl, 1.5 CaC12, 1 MgC12, 2 NaH2P04, 5 glucose, and 6 HEPES titrated to pH 7.4 at 37°C and bubbled with oxygen. The cover slip with attached monolayer was mounted onto an open Dvorak-Stotler chamber (Nicholson Precision Instruments, Gaithersburg, MD), and cells were continuously bathed in the assay buffer. The temperature of the chamber was maintained at 37°C by wrapping it in coiled plastic tubing through which water at 39°C was rapidly perfused. The chamber was positioned on the stage of a Nikon Diaphot inverted microscope (Nikon, Garden City, NY) equipped with a x40 CF fluor objective. The microscope was coupled to a Delta-Scan dual-excitation spectrofluorometer system (PTI, S. Brunswick, NJ) equipped with a chopper and operated via a desk-top computer (5). Excitation wavelengths were set at 340 and 380 nm (slit widths, 3 nm). A 4OO-nm dichroic mirror and 510-nm barrier filter allowed for collection of emitted light at the appropriate wavelength. Fluoresence intensity was followed as a function of time, and data were printed on a Citizen MSP40 dot-matrix printer. To initiate an experiment, cells were bathed in the

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ENDOTHELIN

1 MECHANISMS

Ca2+-replete assay buffer, or (where required) in a Ca2+free buffer that was devoid of Ca2+ and contained 0.1 mM EGTA. The final osmolarity of all solutions was 300 mosmol/kgHzO. To evoke a change in [Ca2+]i, test compounds were added to the assay buffer (where indicated) in a 1:lOO dilution from a stock solution, and the bathing solution was mixed with a Pasteur pipette. Preliminary experiments establ .ished that the -vehicles and compounds themselves did not alter the fluorescent signal. Ratio 340/380 was converted to [Ca2+]i according to the formula described by Grynkiewicz et al. (14). The minimum and maximum-ratios of fluorescence intensity (Rmin and R,,,) were determined as described by Williams et al. (37) using buffered calcium standards (World Precision Instruments, New Haven, CT). Phosphoinositide

Meling,

Extraction,

and Analysis

Inositol phosphates were measured by incorporation of nyo- [ 3H] inositol into confluent monolayers, perchlorate extraction, and separation by anion-exchange chromatography as previously described (2, 25). Monolayers grown in 35 X- lo-mm plastic Petri dishes or six-well culture dishes were incubated in an inositol-free medium containing 1% newborn calf serum and 0.5 &i/ml myo[3H]inositol (12.8 Ci/mmol) for 24 h. Two hours before experimentation, LiCl (10 mM) was added to the incubation medium. Culture dishes were tran sferred to a shaking water bath (37”C), monolayers were rinsed three times with a balanced salt solution containing LiCl, and agonist or vehicle (water) was added in a 1:lOO dilution. Agonist concentration and time period of incubation are detailed in the legends to Figs. l-7. The reaction was terminated by addition of chilled perchloric acid (final concentration 5%), and all subsequent steps were performed at 4°C. Cells were scraped, sonicated, and after a 300min time interval were neutralized with KOH. Total inositol phosphates were determined as previously described (2). Individual inositol phosphates were analy pzed by application of the perchlorate extract to prewashed (5 mM disodium tetraborate, 60 mM ammonium formate, and 5 mM myo-inositol) 2-ml columns packed with AG l-X2 formate anion exchange resin. Inositol-l-monophosphate (IP), inositol-1,4=bisphosphate (IPZ), and inositol trisphosphate (IP3) were sequentially eluted with 16 ml each bf 0.2, 0.4, and 1.0 M ammonium formate in 0.1 M formic acid. Separation of the different IP3 isomers cannot be accomplished by this technique. Each eluate was assayed by liquid scintillation spectroscopy. Separation and recovery of the various fractions was validated by use of authentic IP standards. Prostaglandin

E2 (PGE2) Determination

PGE2 production by RMICs was measured by means of a standard RIA (5). For this purpose monolayers grown in six-well culture dishes were washed three times and subsequently incubated in 2 ml of modified Robinson’s buffer composed of the following (in mM): 140 NaCl, 4 KCl, 2 CaCIZ, 1 MgSO,, 2.6 Na2HP04, 0.7 KH2P04, and 5.5 glucose buffered to pH 7.4 and containing 2 mg/ml essentially fatty acid-free bovine, serum albumin. Exper-

IN

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F581

iments were performed at 37°C in a shaking water bath. After a IO-min basal incubation period, agonist or vehicle was added to the culture well in a 1:lOO dilution from a stock solution. Agonist concentration and time period of incubation are detailed in the legends to Figs. 12-14. For the time-dependent experiments 0.2-ml aliquots of incubation buffer were removed at 0, 2, 5, and 10 min; in the concentration dependence experiments 0.5.ml aliquots were obtained. Aliquots were frozen at -20°C before analysis for PGE,. Duplicate 50.~1 aliquots were subjected to RIA as previously described (5). Statistical

Analysis

Data are presented as mea .ns t SE. Al 1 experiments were performed at least three times, each on a different cell passage. Where appropriate, linear regression analysis or analysis of variance for repeated measurements was applied. The null hypothesis was rejected when P < 0.05. Materials

RPM1 and the penicillin-streptomycin solution were purchased from GIBCO Laboratories (Chagrin Falls, OH), and newborn calf serum was from Sigma Chemical (St. Louis, MO). Endothelin 1 (1-21; human, porcine), endothelin 2 (l-21; human), endothelin 3 (l-21; human, rat), big endothelin-(1-38) (human), big endothelin-(l-39) (porcine), and sarafotoxin S6b were purchased from Peptides International (Louisville, KY). 1251-endothelin 1 (2,200 Ci/mmol) and [3H]PGEz (200 Ci/mmol) were obtained from Du Pant/New England Nuclear (Boston, MA). All other vasoactive compounds used in the radioligand competition experiments were purchased from Sigma. Fura-B/AM was obtained from Molecular IProbes (Eugene, OR), and EGTA was from Fluka Chemical (Ronkonkoma, NY). Myo- [ 3H] inositol (12.8 Ci/mmol) was purchased from Amersham (Arlingtons Height, IL), AG l-X2 (100-200 mesh) formate anion-exchange resin was from Bio-Rad (Richmond, CA), and PGE2 antiserum was ‘from Advanced Magnetics (Boston, MA). All standard chemicals used were purchased at the highest commercial grade available. RESULTS

Radioligand Binding Experiments

Kinetic binding experiments were performed by studying the rates of association of ET-1 (40 PM) with specific receptor sites as a function of time. After the addition of ligand, specific binding increased for 4 h, after which equilibrium was achieved (Fig. lA). Nonspecific binding accounted for ~4% of total counts added. Specific binding remained stable for at least 24 h (Fig. lA ). The rate of dissociation of ligand from its receptor was determined by measuring the decrease in specific binding after the addition of ET-l (0.1 PM) at equilibrium. A time-dependent reduction in specific binding was observed (Fig. 1B) with an apparent k-1 of (2.13 t 0.24) X low3 mine1 (n = 3). Only 30-35% of specific binding was reversible by competition with unlabeled ET-l by 24 h.

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F582

ENDOTHELIN #loo-

1 MECHANISMS *a

A

IN RENAL

INTERSTITIAL

CELLS

*

2 QD b 3 75 86 3

g F:

50

ic

.32

3 .16

w tj

Kd = 80 PM f+, - 581 fmollmg

=.a 5I

E

25/"'v:

0

s 8 Eo

I

I

I

I

I

60

120

180

240

300

20 40 TIME (mid II

80

I

N 1380

I

0 I

1440

TIME (min)

200 SPECIFIC

BOUND

400 (fmoVmg)

600

FIG. 2. Saturation binding of ?-ET-l to RMIC membranes. After addition of various concentrations of ligand (l-500 PM), total and nonspecific binding were measured at equilibrium (5 h) at 25OC. Nonspecific binding was measured in presence of 0.1 PM of unlabeled ET1. Specific binding was calculated as difference between total and nonspecific binding. Points are means of triplicate measurements in a representative experiment (n = 6). I&,, receptor density.

TIME (mid

8 t-

6~

5 0

a5Ob Y

0

1 60

I 120

I 240

I 360 TIME (mid

Y c

I 1320

I 1440

FIG. 1. Kinetics of binding of ‘%I-endothelin 1 (ET-l, 40 PM) to rat medullary interstitial cell (RMIC) membranes. A: time course of formation of ligand receptor complex at 25OC. Inset: pseudo-first-order linear analysis of observed slope (&,J from which rate constant for association (&) is calculated according to the formula k+l = (]Zoh &)/[LR] where k-, is rate constant for dissociation and [LR] is concentration of radioligand. LR, bound ligand concentration at equilibrium. B: time course of dissociation of ‘?-ET-l receptor complex studied at 25°C. Time-dependent reduction in specific binding was studied after addition of 0.1 PM unlabeled ET-l to a preequilibrated (5 h) assay mixture. Inset: linear analaysis of rate constant for dissociation (&). Data points represent mean of triplicate measurements. One of 3 similar experiments is depicted.

The value of k+l calculated from formula k+l = (lz,b, kl)/[LR] was 2.22 x lo8 M-’ lrein-‘, where kbs is the observed slope and [LR] is the concentration of radioligand. & as calculated from the formula Kd = k-l/k+l, was 10 pM. The properties of ET-1 receptors were further characterized in equilibrium binding studies. Scatchard analysis of binding data revealed a single class of highaffinity receptor sites for ET-1 with a K# of 57.0 t 10.0 pM and a density of 749 k 124 fmol/mg protein (n = 6). A typical Scatchard plot is shown in Fig. 2. The specificity of the binding sites was studied in competitive displacement studies (Fig. 3). ET-1 was the most potent inhibitor of binding following ET-2, sarafotoxin, big endothelin (human), and big endothelin (porcine). Endothelin 3 (ET-3) at concentrations as high as 10B8 M did not displace ET-1 from its receptors (Fig. 3). ET-1 binding was not inhibited by low4 M concentrations of unrelated pressor substances (phenylephrine, norepinephrine, angiotensin II, or the thromboxane A2 agonist, U-46619), vasodilators (bradykinin, nitroprusside), Ca2+ channel antagonists (lanthanum, diltiazem), p-blockers (propranolol, 2 x 100~ M), or atria1 natriuretic factor

--

-12

-8

-10

-6

LOG DOSE FIG. 3. Competitive inhibition of specific binding of ‘%ET-1 in RMIC membranes. 12’I-ET-1, 40 pM, and varying concentrations of indicated substances were incubated with RMIC membranes for 5 h, and specific binding was measured. Points represent means of 3 experiments performed in triplicate. ET-2 and ET-3, endothelin 2 and 3, respectively; big ET P and H, big endothelin-( l-39) and (l-38), porcine and human, respectively.

(10D5 M). The potential modulation of ET-1 binding by guanosine nucleotides was tested by studying the effects of GTP and the stable GTP analogue, GppNHp, on ET1 binding. Neither GTP nor GppNHp caused significant inhibition of ET-1 binding at a concentration of low4 M. Endothelin-Induced

Ca2+ Increments

Evidence exists in other cell types that ET-1 increases [Ca2+]i (30-33). The source of this Ca2’ (i.e., extracellular vs. intracellular stores) appears to be cell specific. To determine whether ET-1 elevates [Ca”‘]i in RMICs, fura2-loaded cells were challenged with the compound, and changes in [ Ca2+]i were monitored microfluorometrically as detailed in METHODS AND MATERIALS. In a Ca2+replete buffer, 10B8 M ET-1 evoked a marked increment in [Ca2+]i from 110 to 850 nM that peaked at -10-15 s and subsequently returned toward steady-state values (Fig. 4A). Mean values for seven similar experiments were 119 & 11 (basal) and 614 t 116 nM (peak). By 5 min [Ca2+]i had returned toward, but not attained,

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ENDOTHELIN

1 MECHANISMS

IN

800600z 3 400ccic> - 200-

800

ET: 1 O-8 M

[Ca2’J

600

0 0

I 100 ET-l

0 mM

I 200

1 300

+ n

ET-l:

lo-‘M

ET-3:

lo-‘M

3 200 Ic c;:.g

100

0

200

400

SECONDS 4. ET-l-elicited Ca2’ mobilization. Monolayers of RMICs were loaded with furaand mounted onto stage of a Nikon Diaphot microscope as detailed under METHODS AND MATERIALS. Cells were bathed in either a Ca2’-replete medium (A and B) containing (in mM) 135 NaCl, 5 KCl, 1.5 CaC12, 1 MgC12, 2 NaH2POd, 5 glucose, and 6 HEPES titrated to pH 7.4, or cells were bathed in a Ca2’-free medium (C) of identical composition to which 0.1 mM EGTA was added. All solutions were bubbled with oxygen and maintained at 37OC. Endothelin 1 (ET or ET-l) or ET-3 was introduced into bathing solution in a 1:X00 dilution from a stock solution to yield a final concentration of agonist (10m8 or lo-’ M) as indicated. Representative experiments are shown where n = 7,4, and 3 for A, B, and C, respectively. FIG.

steady-state values, i.e., 110 (basal) vs. -200 nM (5 min). [Ca2+]i was not routinely measured beyond 5 min. The prolonged elevation in [Ca2’]i suggested that ET-l promotedCa2+ entry from external sources and raised the possibility of a- receptor-mediated Ca2+ channel in RMICs. The ability of ET-l to mobilize Ca2+ from intracellular

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F583

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stores was examined in a Ca2+-free medium. As illustrated in Fig. 4B, lo- 8 M ET-l increased [Ca2+]i from a baseline of 150 to 778 nM in -10-15 s. Mean values for four similar experiments were 128 t 10 (basal) and 584 tl .30 nM (peak). Although peak values were comparable to cells evaluated in Ca2+-replete bu ffer, by ~2 min [Ca”‘]i had returned to the prechallenge value. These results indicate that, in addition to activating receptormediated Ca2+ channels in RMICs, ET-1 mobilized Ca2+ from intracellular stores. The competitive displacement studies shown in Fig. 3 indicate that ET-l, but not ET-3, was recognized by the RMIC endothelin receptor. The functional correlate to the binding assay is illustrated in Fig. 4C. One of three identical tracings is shown. Challenge of cells with ET-3 (low8 M) failed to elicit a Ca2+ transient; subsequent exposure of the same cells to ET-1 (low9 M) yielded the expected Ca2+ increment, demonstrating the integrity of the receptor. Rechallenge of cells (-3 min) with lo-’ M ET-1 failed to yield a second Ca2+ increment, in keeping with homologous desensitization of the receptor by agonist. These data provide additional evidence that in RMICs the ET-l receptor fails to recognize ET-3, thereby establishing a definitive link between binding site and cellular responses. The concentration dependence of the ET-l-induced [ Ca2+]i increment (A[ Ca2+]i), determined both in the presence and absence of Ca2+ in the bathing medium, is depicted in Fig. 5. In the presence of a Ca2+-replete medium, lo-l6 M ET-l evoked a small but perceptible increment in [Ca2+)i, i.e., A[Ca2+]i of 22 t 11 nM. Maximal A[Ca2+]i of 499 t 99 (n = 7) and 521 t 106 nM (n = 5) were observed at 10D8 M and 5 x lo-’ M ET-l, respectively. Higher concentrations of agonist were not tested. Figure 5 also indicates that the half-maximal activation concentration (K&J for the ET-l-evoked Ca2’ response was -10-l’ M. When Ca2+ was deleted from the bathing medium, lo-l3 M ET-l failed to evoke an increase in [Ca2+]i. Challenge with lo-l2 M ET-1 resulted in A[Ca2+]i of 43 t 10 nM (n = 3), and 10B8 M ET-l caused A[ Ca2+]i of 466 t 65 nM (n = 5). Higher concen-

so0 1

so0 1

+Ca2 + -Ca2

[ENDOTHELIN]

+

M

FIG. 5. Concentration dependence of ET-l-evoked Ca2’ increments (A[Ca”‘]i). Fura-2-loaded cells were bathed in Ca2+-replete or Ca2+-free medium (+Ca2+ and -Ca2’, respectively), as detailed in legend to Fig. 4. Final concentrations of ET-l were obtained by adding agonist (suspended in assay buffer) to bathing medium in a 1:lOO dilution from a stock solution. Each data point is from 3-9 replicate experiments. All values were significantly greater than steady-state values (P < 0.05) except lo-l6 M (Ca”’ -replete condition).

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F584

ENDOTHELIN

1 MECHANISMS

IN RENAL

INTERSTITIAL

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trations of agonist were not tested. The K& for this response was ~5 X 10-l’ M. It is recognized that the peak increment in [Ca”‘]i may not have been attained, so the Ko.5 for mobilization of Ca2+ from intracellular stores may be greater than the value reported. In either event it is evident from the data shown in Fig. 5 that I) higher concentrations of agonist are required to mobilize Ca”’ from intracellular stores than from the cell exterior and 2) the threshold for receptor-mediated Ca2+ channel opening is lower by at least three orders of magnitude than that required to mobilize Ca2’ from intracellular stores.

of Inositol

Stimulation

Phosphates

by Endothelin

One mechanism whereby ET-1 might mobilize Ca2’ from intracellular stores is via activation of PI-PLC with consequent hydrolysis of phosphatidylinositol bisphosphate to IPS and diacylglyerol (DAG). The time course of 10B8 M ET-l-induced IP3 accumulation is illustrated in Fig. 6. By 30 s [3H]IP3 increased from 277 t 30 to 466 t 105 counts*min+ 6well-l (cpm/well), and attained peak values of 590 .=f:71 and 591 t 70 cpm/well at 1 and 2 min, respectively (n = 3), representing a 212 and 213% increment in IP3 above basal values at the respective timepoints. A concurrent 315% increase in IP2 and 157% increase in IP was detected over l-2 min (data not shown). By 5 min IP3 values had declined to 485 t 86 cpmJwel1 (n = 3). The concentration curve for ET-levoked IP3 accumulation over 2 min is depicted in Fig. 7. At lo-l1 M agonist concentration, a small increment in IP3 from 158 & 25 to 168 t 21 cpm/well was detected (n = 3). Increasing ET-1 concentration to 5 x lo-’ or low8 M yielded peak values of 398 * 41 and 330 t 58 cpm/well, respectively (n = 3), representing a 252% (5 X loo9 M) and 209% (low8 M) increment in IP, above basal values. The Ko.6 for the process was -lo-’ M. In separate experiments ET-3 (loo8 M) failed to enhance IP3 accumulation (data not shown). These data demonstrate that in RMICs one consequence of ET-1 binding to its specific

-1 OOOs^ ti s 4002 2000, &%OO; -

loo-

1

t Mmrrcs

2

FIG. 6. Time course for ET-l-evoked inositol trisphosphate (IP3) accumulation. Monolayers grown to confluence in 35 x IO-mm plastic Petri dishes were incubated with 0.5 &i/ml nyo- [3H]inositol for 24 h before experimentation. After exposure to 10 mM LiCl for 2 h, cells were challenged with 10e8 M ET-1 (final concentration). Total IP3 accumulation is shown. Details of anion-exchange chromatography appear in METHODS AND MATERIALS. Values are means from 3 separate experiments in which each individual data point was performed in triplicate or quadruplicate. *P < 0.01 vs. basal value.

FIG. 7. Concentration dependence of ET-l-evoked IP, accumulation. Confluent ‘monolayers grown in six-well culture dishes were incubated with 0.5 &i/ml myo-[3H]inositol for 24 h before experimentation. Cells incubated in presence of 10 mM LiCl for 2 h were challenged with indicated concentrations of ET-l. Total IP3 accumulation over 2 min is shown. Details of anion-exchange chromatography appear in METHODS AND MATERIALS. VdUeS are means from 3 separate experiments in which each individual data point was performed in triplicate or quadruplicate. *P < 0.01 vs. basal value.

receptor is activati .on of the phosphoinositol tion pathway. Activation of Receptor-Operated by Endothelin

transduc-

Ca2’ Channels

The sustained elevation above baseline of [Ca”‘]i observed in RMICs challenged with ET-1 in the presence of extracellular Ca2+ (Figs. 4A and 6) might indicate the existence of receptor-activated Ca2+ channels. To further investigate this possibility, cells were challenged with low8 M ET-l, and once the plateau phase had been attained (following the initial transient increment) lOa M Ni2+ (an inorganic Ca2+ channel inhibitor) or low5 M nimodipine (an inhibitor of voltage-sensitive Ca2+ channels) was introduced into the bathing solution. As illustrated in Fig. 8A (one of five similar tracings), 10e3 M Ni2+ abruptly lowered plateau phase [Ca”“]i to the prechallenge value. In contrast, nimodipine (lo-’ M) was without effect (Fig. 8B). Addition of 10m3 M Ni2+ to nimodipine-challenged cells returned [ Ca2+]i to the prechallenge value. These data are consistent with the presence of a receptor-linked Ca2+ channel in RMICs. It might be argued that the phase of sustained [Ca2+]i elevation could be explained by blockade of a Ca2’ exit step, rather than stimulation of Ca2’ entry by the agonist. To eliminate this possibility, cells bathed in a Ca2+replete medium were exposed to log3 M Ni2+ before challenge with ET-l (Fig. 9). Addition of Ni2+ to the bathing solution lowered resting [Ca”‘]i by ~50 nM. Furthermore, challenge of cells with 10V8 M ET-1 resulted in the expected early transient increment in [Ca2+]i. However, in contrast to cells bathed in a Ca2+replete medium without Ni2+ (Fig. 4A), [Ca2+]i returned to basal values within 5 min, with no plateau phase of elevated [Ca2”]i as seen in a Ca2+-free medium (Fig. 4C). In additional experiments cells were challenged with 10 -15 M ET-l, a concentration of agonist that has no effect on PI hydrolysis (Fig. 8), or on release of Ca2+ from intracellular stores (Fig. 5). As depicted in Fig. 10 (top), lo-l5 M ET-1 evoked a small but sustained eleva-

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1 MECHANISMS

800

IN RENAL

INTERSTITIAL

F585

CELLS

ET: 1 O-8 M [Ca*+ J: 1.5 mM

600

ET: 1O-l5 M Ni 2+

[Ca2+ J: 1.5 mM

Ni *+ 4

SECONDS

10. Endothelin-evoked sustained elevation in [Ca”‘]i. Assay conditions are detailed in legend to Fig. 4. Cells, bathed in a Ca2’replete solution, were challenged with 10-l’ M ET-1 (COPand bottom). Ni2+, Joe3 M, was added to bathing solution; n = 4 experiments. FIG.

I

B

600

ET: 1OS8 M

I

[Ca*+J: Nimodipine

400

Ni

1

1.5 mM

2+

ET: 10” M

c

i 200

i 0

f

I

I

1

I

0

100

200

300

400

SECONDS

u-l--

FIG. 8. Effect of Ni+2 and nimodipine on ET-l-induced sustained elevation of [Ca2’]i. Fura-Z-loaded cells were bathed in the Ca2+-replete solution detailed in legend to Fig. 4 and METHODS AND 'MATERIALS. Cells were challenged with 10e8 M ET-l (final concentration). A: Ni2+ ( low3 M final concentration) was added from a stock solution (in H20). B: nimodipine (10’” M final concentration) was added from a stock solution (in ethanol). Subsequently, lOBa M Ni2+ was added. One of five similar experiments is shown.

ET:lO"M [Ca*+J:

1.5 mM

0

lb0

2b0

3bo

ET: loaM

40

1

I$J:“*+5 uz 3% LL-

360nm 0

I

0

100

I

1

200

300

SECONDS 11. Quenching of intracellular fura- by Mn2+. Fura-2-loaded cells were bathed in Ca2+-replete solution delineated in legend for Fig. 4 and METHODS AND MATERIALS. Fluorescence tracings were followed at 340-nm and 360-nm excitation wavelength, with 510-nm emission wavelength. Cells were challenged with 1Om8M ET-l in absence (A) or presence (B) of 10D4 M Mn2’. Representative tracings from 4 experiments are shown. Note that units of ordinate are arbitrary. FIG.

0; 0

I loo

I 200

I 300

I 4Qo

SECONDS 9. Exposure of cells to Ni2+ before challenge with ET-l. Assay condition8 are detailed in legend to Fig. 1. Cell8 were bathed in a Ca2+replete solution. Ni2+ (10” M final concentration) was added to bathing solution where shown, followed by ET-l (lOa M); n = 4 experiments. FIG.

tion in[Ca2+]i. No transient Ca2’ increment was observed. Ni2+, 10B3 M, returned plateau [Ca”‘] value to baseline (Fig. 10, bottom). These findings provide strong additional evidence for the existence of receptor-operated Ca2+ channels in RMICs. Further evidence for receptor-operated Ca2’ channels is provided by the experiments shown in Fig. 11 and is dependent on the fact that the 3600nm excitation wavelength is insensitive to changes in [Ca2+]i but is sensitive to quenching of intracellular fura- by compounds known to have a high affinity for the fluoroprobe, notably Mn2+

(14). Note that the units for Fig. 11 are arbitrary fluorescence units. In a Ca2+-replete solution low8 M ET-1 evoked a rise in the 3400nm (Ca2+-sensitive) wavelength but induced no change in the 360-nm (Ca2+-insensitive) wavelength as expected (Fig. HA). Preincubation of cells with 10B4 M Mn2+ (Fig. 11B) followed by subsequent challenge with 10m8M ET-1 resulted in a smaller increment in the 340-nm (Ca2+-sensitive) component and decrement in the 360~nm (Ca2’ insensitive) tracing, consistent with entry of Mn2+ into the cells and quenching of the fluoroprobe. Because Mn2+ is known to enter cells via Ca2+ channels, (28), these data confirm the existence of ET-l-linked Ca2+ channels in RMICs,

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F586 Stimulation

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1 MECHANISMS

of PGE2 by Endothelin

Hydrolysis of phosphoinositides would not only mobilize Ca2+ from intracellular stores (via IP3 formation), but the parallel formation of DAG would be predicted to stimulate protein kinase C (PKC). PKC, in turn, has been shown to stimulate calcium-sensitive PLA2, a key enzyme involved in the formation of PGE2 from arachidonic acid (23). It is recognized, however, that PGE2 formation may be a consequence of other intracellular events, notably direct activation of PLA2 by the ET receptor. The time course of PGE2 accumulation by RMICs challenged with 10e8 M ET-1 is depicted in Fig. 12. Two minutes postchallenge, PGE2 accumulation had increased from 3.32 t 0.52 to 16.18 * 3.28 ng/mg protein (n = 6, P < 0.01) and to 41.80 t 6.84 ng/mg protein at 5 min (n = 6, P < 0.001) By 10 min a &fold increment of PGE2 above basal values had occurred: 3.32 t 0.52 (basal) vs. 49.70 t 7.97 (10 min) ng/mg protein (n = 6, P < 0.001). Time points beyond 10 min were not examined. The concentration dependence of the ET-l-evoked PGE2 response at 10 min is illustrated in Fig. 13. ET-1 at lo-l4 and lo-l2 M evoked small increments in PGE2 accumulation above basal values: 3.03 t 0.65 (basal), 3.82 t 0.06

0

10 Ml&ES

FIG. 12. Time course for ET-l-evoked prostaglandin Ez (PGEJ accumulation. Monolayers grown to confluence in six-well culture dishes were washed three times and preequilibrated in 2 ml assay buffer (see METHODS AND MATERIALS). Aliquots of assay buffer were removed before (time 0) and at indicated time points after addition of 10V8 M ET-l. PGE2 was assayed by RIA as detailed in METHODS AND MATERIALS. For each data point, n = 6. *P < 0.01 vs. basal value.

t

I

0

1o-w

&12

[ENDOTHLLIN]

lb

RENAL

INTERSTITIAL

CELLS

( lo-l4 M), and 4.40 t 0.49 ng*mg protein-‘. 10 min-’ (lo-l2 M) (n = 6, not significant). ET-l, 10-l’ M, significantly increased PGE2 from 3.03 t 0.65 to 8.28 t 0.85 ng*mg protein-’ 10 min-’ (n = 6, P < 0.05) with further increments in PGE2 measured at higher concentrations of ET-l (Fig. 13). The similar concentration dependence of ET-l-evoked PGE2 accumulation (Fig. 13), IP3 generation, and Ca2+ release from internal stores (Fig. 5) should be noted. Further evidence that the RMIC endothelin receptor fails to recognize ET-3 is provided by the data illustrated in Fig. 14. Whereas lo-’ M ET-1 significantly increased PGE2 from a basal value of 3.20 t 0.50 to 38.21 t 4.62 ng* mg protein-‘. 10 min-’ (n = 6, P < 0.05), 10m8 M ET3 was without effect at 4.21 t 1.55 ng=mg protein-’ 10 min-l (n = 6, not significant vs. basal). l

l

DISCUSSION

These results demonstrate that cultured rat RMICs possess a single class of high-affinity receptor sites for ET-1 (Fig. 2) that are coupled to a PI-PLC (Figs. 4-7) and to receptor-operated Ca2+ channels (Figs. 8-11). In addition, PGE2 accumulation was markedly stimulated (Figs. 12-14), raising the possibility that the ET receptor is linked to a PLA2. Autoradiographic studies of rat kidney slices by use of 1251-ET have recently identified the renal medulla as the predominant site within the kidney where ET receptors are located (17). Radioligand binding studies performed on crude membrane fragments prepared from rat renal papilla confirmed this observation (19). Neither study, however, pinpointed the specific cell type involved. The current investigation determines the medullary interstitial cell as one such cell type. Preliminary data from these laboratories indicate that the epithelial cells lining the collecting duct, i.e., inner medullary collecting duct cells, may be a second target for ET action (8). The affinity of the RMIC ET receptor for substrate is remarkable in comparison to other renal and nonrenal ET receptors identified. The & of 57 t 10 pM (Fig. 2) is -12-fold greater than that reported for a crude membrane preparation of renal medulla, i.e., 662 t 151 pM (19). In fact, the Kd of the RMIC ET receptor is 14- to

*

lit-8

M

13. Concentration dependence of ET-l-evoked PGEZ accumulation. Confluent monolayers grown in six-well culture dishes were preequilibrated as described in legend to Fig. 12 and METHODS AND MATERIALS. Aliquots of assay buffer were removed after lo-min incubation with concentrations of ET-l. RIA was performed as detailed in METHODS AND MATERIALS. For each data point, n = 6. *P < 0.05 vs. basal value. FIG.

IN

CONT

CT-1

ET-3

14. Specificity of ET-l-evoked PGE2 accumulation. Washed, confluent monolayers were preequilibrated as described in legend to Fig. 12 and METHODS AND MATERIALS. Aliquots of assay buffer were removed following lo-min incubation with vehicle (control, CONT), 10s8 M ET-l or ET-3. PGE, was assayed by RIA as detailed in METHODS AND MATERIALS. For each data point, n = 6. *P < 0.01 vs. basal value. FIG.

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-

1 MECHANISMS

24-fold greater than that reported for mesangial cell receptors defined in freshly isolated crude glomerular membrane fragments (1,309 t 112 PM) or from cultured mesangial cells (760 PM), respectively (4,19). Indeed the & of the RMIC receptor compares extremely favorably with that of ET receptors in other tissues, e.g., 300 pM for vascular smooth muscle cells (33), 220 pM for osteoblastic cells (32), 180 pM for Swiss 3T3 fibroblasts (31), and 36 pM for human placenta (35). The RMIC ET receptor also differs importantly from that of the mesangial cell in that ET-3, even at high concentrations, was unable to displace 1251-ET-1 from the receptor (Fig. 3). The inability of ET-3 to evoke a Ca2+ transient (Fig. 4C), accelerate IP3 accumulation, or stimulate PGE2 production (Fig. 14) provides functional confirmation for the radioligand binding studies (Fig. 3). Taken together, these data indicate that the RMIC ET-1 receptor displays distinct characteristics, particularly a remarkable affinity for ET-1 but little if any affinity for ET-3, which is similar to the 73-kDa ET receptor in cultured rat smooth muscle cells (20). The fluoroprobe studies demonstrate ‘a biphasic pattern of ET-l-induced Ca2+ mobilization (Figs. 4 and 8). The initial transient increment in [Ca2+]i is consistent with activation of a PI-PLC, whereas the sustained plateau phase suggests the presence of receptor-operated Ca2+ channels. Similar biphasic responses have been observed in vascular smooth muscle cells (33), mesangial cells (30), osteoblasts (32), and Swiss 3T3 fibroblasts (31). In the current study these two events could be mechanistically and even perhaps temporally separated. With regard to activation of PI-PLC, ET-1 2 lo-l2 M was required to evoke a transient increment in [Ca2+]i in the absence of extracellular Ca2+ (Fig. 5) or an increment in IP3 (Fig. 7). In contrast, lo-l6 M ET-l (Figs. 5 and 10) evoked a sustained elevation in [Ca2+]i without an initial Ca2+ transient, providing compelling evidence that activation of receptor-operated Ca2+ channels in this cell type is not linked to PI hydrolysis (15). Furthermore, these findings argue against the concept that efflux of Ca2+ from intracellular stores is a necessary prerequisite for the operation of plasma membrane Ca2’ channels (24). Opening of the Ca2+ channel at lo-l5 M ET-l (Fig. 10) was a rapid event, unlike slower responses observed at low agonist concentration in preparations such as osteoblasts (32) and mesa&al cells (30). It might be argued that detection of IP3 accumulation by column chromatography may not be ideally sensitive. Although this contention is undoubtedly relevant, it is unlikely that the assay erred by five orders of magnitude, because Ca2+ channel activation could be detected at lo-l6 M ET1 (Fig. 5), whereas lo-l1 M ET-1 evoked only small but statistically insignificant increments in IP3 accumulation (Fig. 7). Furthermore, 10-13-10-16 M ET failed to elevate [ Ca2+]i in the absence of extracellular Ca2+ (Fig. 5). In any event, the mechanism promoting ET-receptor-operated Ca2+ channel activation is currently not known. Additional studies will be required to elucidate this crucial point. Challenge of RMICs with ET-1 also resulted in a timeand concentration-dependent accumulation of PGE2 (Figs. 12-14). The observation that the concentration

IN

RENAL

INTERSTITIAL

CELLS

F587

dependence for ET-l-stimulated PGE2 production (Fig. 13) closely paralleled ET-l-augmented IP3 generation (Fig. 7) raised the possibility, but did not prove, that prostaglandin synthesis was linked to activation of the PI cascade, with release of arachidonic acid from DAG via DAG-lipase (18). It is equally feasible that a PLA2 could account for the eicosanoid production. In this regard, agonist-mediated stimulation of PLA2 might occur secondary to activation of PLC (23) changes in [Ca”+] i (13) or as a direct consequence of receptor-linked PLA2 (7) Given these complexities, including the possibility tht;t several mechanisms may act cooperatively, it is difficult to estimate with certainty the precise processes regulating arachidonic acid metabolism in these cells. Interestingly, in vascular smooth muscle cells ET-mediated activation of PLA2 has been implicated. Failure to block arachidonic acid generation by treatment of cells with neomycin a PLC inhibitor, pointed toward the existence of a ” PLA2 receptor-linked process (26) . In agreement with these findings, pretreatment of cells with phorbol esters inhibited phosphoinositide hydrolysis but potentiated ET-stimulated arachidonic acid release (27). [Ca2+]; was, however, not directly measured in the latter study. The contribution of alterations in [Ca2+]i and/or PKC modulation of PGE2 synthesis in RMICs is currently under examination. It might be contended that some discrepancy exists between the radioreceptor ligand binding studies and other biochemical parameters assayed. This, however, is probably not the case. Rather, careful scrutiny of concentration response data may provide additional insights into endothelin actions. Saturation binding studies indicate that occupancy of ~1% of receptors is required to activate receptor-operated Ca2+ channels (Figs. 2 and 5). It is feasible that a small number of higher-affinity sites might exist, but there was no suggestion of a higher affinity binding site when Scatchard plots were analyzed by the LIGAND program. Stimulation of PI-PLC, on the other hand, requires occupancy of -50% or more of receptors to evoke a response (Figs. 2, 5, 7, and 13). These data suggest that a heirarchy of postreceptor signaling events might occur with increasing concentrations of endothelin. Given the strikingly different experimental conditions dictated by each specific assay, some variability in responses is hardly surprising. It is interesting to speculate on the physiological role of ET in RMIC function, especially in light of its remarkable ability to stimulate PG& production. There is substantial evidence to indicate that the RMIC is a major site of renal PGE2 production (3,6,21,39). Interestingly, in rabbit RMIC, PGE2 synthesis was impressively enhanced by angiotensin II, a potent vasoconstrictor agent (39). Bradykinin, arginine vasopressin (AVP), and hyperosmolarity as agonists were all several times weaker. PGE2, in turn, has been shown to antagonize the hydrosmotic effect of AVP (12). From an anatomic standpoint, RMICs have been shown to straddle the vasa recta and inner medullary collecting duct, a known site of AVP action (5). Taken together, the current findings raise the possibility that ET-l-evoked PGE2 synthesis may play a role in inner medullary collecting duct water transport. Whether the very strong reaction product of ET-l-like

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F588

ENDOTHELIN

1 MECHANISMS

immunoreactivity in the renal medulla and papilla (11, 36) represents synthesis of the peptide by RMICs and or other cells of the medulla remains an intriguing but currently untested hypothesis. The expert word processing support of Sherry1 Krulik is greatly appreciated. This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants DK-36351 and DK-41585 to E. P. Nord, a Veterans Administration Merit Review Award to R. Barnett, and grants from the American Heart Association, New York affiliate, to B. M. Wilkes and P. Mento. The spectrofluorometer was purchased with a challenge grant from the State University of New York, Stony Brook, Research Foundation to E. P. Nord. Address for reprint requests: E. P. Nord, Division of Nephrology, Dept. of Medicine, School of Medicine, HSC T-15 Rm. 020, SUNY, Stony Brook, NY 11794. Received 27 April 1990; accepted in final form 20 November 1990. REFERENCES 1. ABOOLIAN, A., AND E. P. NORD. Bradykinin increases cytosolic free [Ca”] in proximal tubule cells. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F486-F493,1988. 2. ABOOLIAN, A., M. VANDER MOLEN, AND E. P. NORD. Differential effects of phorbol esters on PGE2 and bradykinin induced-elevation of [ Ca*+]i in MDCK cells. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F1135-F1143,1989. 3, AUSIELLO, D. A., AND R. M. ZUSMAN. The role of calcium in the stimulation of prostaglandin synthesis by vasopressin in rabbit renal-medullary interstitial cells in tissue culture. B&hem. J. 220: 139-145,1984. 4. BADR, K. F., K. A. MUNGER, M. SUGIURA, R. M. SNAJDAR, M. SCHWARTZBERG, AND T. INAGAMI. High and low affinity binding sites for endothelin on cultured rat glomerular mesangial cells. Biochem. Biophys. Res. Commun. 161: 776-781,1989. 5. BARNEW, R., P. A. ORTIZ, S. BLAUFOX, S. SINGER, E. P. NORD, AND L. RAMSAMMY. Atria1 natriuretic factor alters phospholipid metabolism in mesangial cells. Am. J. Physiol. 258 (Cell Physiol. 27): C37-C45,1990. 6. BECK, T. R., A. HASSID,

M. J. DUNN. The effect of arginine vasopressin and its analogs on the synthesis of prostaglandin ES by rat renal medullary interstitial cells in culture. J. Pharmacol. Exp. Ther. 215: 15-19,198O. 7. BURCH, R. M., A. LUINI, AND J. A~ELROD. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to cri-adrenergic stimulation in FRTLS thyroid cells. Proc. Natl. Acad. Sci. USA 83: 7201-7205,1986. 8. CASSALS, M., B. M. WILKES, D. HART, M. VANDER MOLEN, R. BARNETT, AND E. P. NORD. Mechanisms of endothelin action in inner medullary collecting duct (IMCD) cells (Abstract). J. Am. AND

Sot. Nephrol. 1: 467, 1990. 9. CHENG, Y. C., AND W. H. PRUSOFF.

Relation between the inhibition constant [Ki] and the concentration of inhibitor which causes fifty percent inhibition [Km] of an enzyme reaction. Biochem. Pharmacol. 22: 3099-3109,1973. 10. FONTOURA, B. M. A., D. R. NUSSENZVEIG, K. M. PELTON, AND T. MAACK. Atrial natriuretic factor receptors in cultured renomedullary interstitial cells. Am. J. Physiol. 258 (Cell Physiol. 27): C692-C699,1990. 11. FRIED, T. A., K. WALKER, AND M. A. AYON. Immunohistochemical and auto radiographic localization of endothelin in the rat kidney (Abstract). J. Am. Sot. Nephrol. 1: 415,199O. 12. GRANTHAM, J. J., AND J. ORLOFF. Effect of prostaglandin E1 on the permeability response of the isolated collecting tubule to vasopressin, CAMP and theophylline. J. Clin. Invest. 47: 1154-1161, 1968.

13. GRONICH, J. H., J. V. BONVENTRE, AND R. A. NEMENOFF. Identification and characterization of a hormonally regulated form of phospholipase A2 in rat renal mesangial cells. J. Biol. Chem. 263: 16645-16651,1988. 14. GRYNKI ‘EWICZ, G., M. POENIE, AND R. Y. TSIEN. A new generation of Ca*+ indicators with greatly improved fluorescence properties.

IN RENAL J. Biol. 15. IRVINE,

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Chem. 260: 3440-3450,1985. R. F., AND R. M. MOOR.

Micro-injection of inositol l,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca*+. Biochem. J. 240: 917-920,1986. 16. KAISSLING, B., AND W. KRIZ. Structural analysis of the rabbit kidney. In: Advances in Anatomy, Embryology and Cell Biology, edited by A. Brodal, W. Wild, G. Tondury, and E. Wolff. New York: Springer-Verlag, 1990, vol. 56, p. 51-64. 17. KOHZUKI, M., C. I. JOHNSTON, S. Y. CHAI, D. J. CASLEY, AND F. A. 0. MENDELSOHN. Localization of endothelin receptors in rat kidney. Eur. J. Pharmacol. 160: 193-1941989. 17a.LowRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem.

193: 265-275,195l.

18. MAJERUS, P. W., T. M. CONNOLLY, H. DECKMYN, T. S. Ross, T. E. BROSS, H. ISHII, V. S. BANSAL, AND D. B. WILSON. The metabolism of phosphoinositide-derived messenger molecules. Science Wash. DC 234: 1519-1526,1986. 19. MARTIN, E. R., P. A. MARSDEN, BALLERMAN. Identification and

B. M. BRENNER, AND B. J. characterization of endothelin binding sites in rat renal papillary and glomerular membranes. Biochem. Biophys. Res. Commun. 162: 130-137,1989. on MARTIN, E. R., M. B. BRENNER, AND B. J. BALLERMANN. LU. Heterogeneity of cell surface endothelin receptors. J. Biol. Chem.. 265: 14044-14049,199o. 21. MUIRHEAD, E. E., G. GERMAIN, B. E. LEACH, J. A. PITCOCK, P. STEPHENSON, B. BROOKS, W. L. BROSIUS, E. G. DANIELS, AND J. G. HINMAN. Production of renomedullary prostaglandins by re-

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Anal. Biochem. 107: 220-239,198O. PARKER, J., C. W. DANIEL, AND

D. RODBARD. LIGAND: A versatile computerized approach for characterization of ligand-binding systems.

M. WAITE. Evidence of protein kinase involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis. J. Biol. Chem. 262: 5385-5393,

1987. 24. PUTNEY, J. W., H. TAKEMURA, A. R. HUGHES, D. A. HORSTMAN, AND 0. THASTRUP. How do inositol phosphates regulate calcium signaling? FASEB J. 3: 1899-1905, 1989. 25. RAMSAMMY, L. S., C. JOSEPOVITZ, AND G. J. KALOYANIDES. Gen-

tamycin inhibits agonist stimulation of the phosphoinositol cascade in primary cultures of rabbit proximal tubular cells and in rat renal cortex. J. Pharmacol. Exp. Ther. 247: 989-995,1988. 26. RESINK, T.J.,T. SCOTT-BURDEN,AND F.R. BUHLER.

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 158: 279-286, 1989. 27. REYNOLDS, E. E., L. L. S. MOK, AND S. KUROKAWA. Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells. B&hem. Biophys. Res. Commun. 160: 868-873,1989. 28. SAGE, S. O., J. E. MERRITT, T. J. HALLAM,

AND T. J. RINK. Receptor-mediated calcium entry in FURA- loaded human platelets stimulated with ADP and thrombin. Biochem. J. 258: 923-926,

1989. 29. SCATCHARD, G. The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci. 51: 660-672,1949. 30. SIMONSON, M. S., S. WANN, P. MENE, G. R. DUBYAK, M. KESTER, Y. NAKAZATO, J. R. SEDOR, AND M. DUNN. Endothelin stimulates

phospholipase C, Na’/H+ exchange, c-fos expression and mitogenesis in rat mesangial cells. J. Clin. Invest. 83: 708-712, 1989. 31. TAKUWA, N., Y. TAKUWA, M. YANAGISAWA, K. YAMASHITA, AND T. MASAKI. A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J. Biol. Chem. 264: 7856-7861,1989. 32. TAKUWA, Y., T. OHUR, N. TAKUWA,

AND K. YAMASHITA. Endothelin-1 activates phospholipase C and mobilizes Ca*+ from extra and intracellular pools in osteoblastic cells. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E797-E803, 1989. 33. TAKUWA, Y., Y. KASUYA, N. TAKUWA, M. KUDO, M. YANAGISAWA, K. GOTO, T. KASAKI, AND K. YAMASHITA. Endothelin receptor is coupled to phospholipase C via a pertussis toxin-insensitive guanine regulatory protein in vascular smooth mus_ nucleotide-binding -- -de cells. J. Clin. Invest. 85: 653-658, 1990. 34. WEILAND, G. A., AND P. B. MOLINOFF. Quantitative analysis of

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drug-receptor interactions. I. Determination of kinetic and equilibrium properties. Life Sci. 29: 313-330, 1981. 35. WILKES, B. M., P. F. MENTO, A. M. HOLLANDER, M. E. MAITA, S. SUNG, AND E. P. GIRARDI. Endothelin receptors in human placenta: relationship to vascular resistance and thromboxane release. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E864-E870, 1990. 36. WILKES, B. M., S. MYRON, P. F. MENTO, C. M. MACICA, E. P. GIRARDI, E. BOSS, AND E. P. NORD. Localization of endothelin-llike immunoreactivity in rat kidneys. Am. J. Physiol. (Renal Fluid Electrolyte PhysioZ.). In press 37. WILLIAMS, D. A., K. E. FOGARTY, R. Y. TSIEN, AND F. S. FAY.

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CELLS

F589

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