Ranakinin, a Naturally Occurring Tachykinin, Stimulates

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 2 Printed in U.S.A.

Ranakinin, a Naturally Occurring Tachykinin, Stimulates Phospholipase C Activity in the Frog Adrenal Gland* MAGLOIRE K. KODJO, LAURENCE DESRUES, LUISA LAVAGNO†, ALDO FASOLO, J. MICHAEL CONLON, MARIE-CHRISTINE TONON, AND HUBERT VAUDRY European Institute for Peptide Research (IFRMP no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen (M.K.K., L.D., M.-C.T., H.V.), 76821 Mont-Saint-Aignan, France; the Department of Animal Biology, University of Torino (L.L., A.F.), 10123 Torino, Italy; and the Regulatory Peptide Center, Department of Biomedical Science, Creighton University Medical School (J.M.C.), Omaha, Nebraska 68178 ABSTRACT We have previously shown that the frog adrenal gland is innervated by a dense network of fibers containing ranakinin, one of the endogenous tachykinins in the amphibian Rana ridibunda, and we have found that ranakinin stimulates in vitro corticosteroid secretion by frog adrenal tissue. To elucidate the mechanism of action of ranakinin on the frog adrenal gland, we investigated the effect of ranakinin on cAMP formation and polyphosphoinositide metabolism. Incubation of frog adrenal explants with various tachykinins, including ranakinin, substance P, neurokinin A, or neurokinin B, did not produce any significant modification of cAMP concentrations. In contrast, ranakinin induced a time- and dose-dependent stimulation of inositol phosphate formation with a concomitant decrease in mem-

brane polyphosphoinositides. Pretreatment of the tissue slices with the phospholipase C inhibitor U-73122 or with pertussis toxin completely abolished the stimulatory effect of ranakinin on inositol phosphate formation. Prolonged administration of U-73122 to perifused frog adrenal explants markedly attenuated the ranakinin-evoked stimulation of corticosterone and aldosterone secretion. Taken together, these data indicate that in the frog adrenal gland, ranakinin has no effect on the adenylyl cyclase system, but enhances polyphosphoinositide hydrolysis. The stimulatory action of ranakinin on inositol phosphate formation and corticosteroid secretion is mediated through activation of a phospholipase C positively coupled to a pertussis toxin-sensitive G protein. (Endocrinology 139: 505–512, 1998)

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ANAKININ is an undecapeptide that has been isolated from the brain of the European green frog Rana ridibunda (1). Ranakinin belongs to the tachykinin family, which, in mammals, comprises substance P, neurokinin A (NKA), and neurokinin B (NKB; Table 1). Besides ranakinin, two other tachykinins have been identified in Rana ridibunda, i.e. NKB, which has the same amino acid sequence as mammalian NKB (1), and [Leu3,Ile7]NKA (2) (Table 1). Tachykinins are widely distributed in the central and peripheral nervous systems and display a wide range of biological activities (3). In particular, the presence of several tachykinins, including substance P, NKA, and NKB, has been demonstrated in the rat (4), bovine (5), and human (6) adrenal gland. In the frog, the occurrence of a dense network of fibers containing ranakinin-like and [Leu3,Ile7]NKA-like immunoreactivities has been shown in the adrenal parenchyma (7). Concurrently, tachykinins have been found to stimulate corticosteroid secretion in mammals (8, 9). In vivo studies have

shown that tachykinins may also control the growth and differentiation of rat zona glomerulosa cells (10). In amphibians, ranakinin and other tachykinins stimulate corticosterone and aldosterone secretion in vitro (7, 11). Tachykinins exert their effects through activation of at least three types of seven-transmembrane domain receptors that are coupled to various transduction systems (12). Although tachykinin receptors are frequently associated with phospholipase C (13–15), tachykinins can also activate adenylyl cyclase (16, 17) or phospholipase A2 (11, 18) and can modulate several ion conductances (19, 20). The aim of the present work was to determine the signaling pathways involved in the effect of tachykinins on the frog adrenal tissue by studying the action of ranakinin on cAMP formation and phosphoinositide metabolism. We also investigated the role of the adenylyl cyclase and phospholipase C pathways in the stimulatory action of ranakinin on corticosteroid secretion. Materials and Methods

Received August 18, 1997. Address all correspondence and requests for reprints to: Dr. H. Vaudry, European Institute for Peptide Research (IFRMP n°23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. Email: [email protected]. * This work was supported by grants from INSERM (U-413), DRET (Grant 92– 099), EU Human Capital and Mobility (Grant ERBCHRXCT 92– 0017), French-Italian exchange programs (GALILEE 94022 and CNRINSERM), and the Conseil Re´gional de Haute-Normandie. † Recipient of a fellowship from the EU Erasmus program.

Animals Adult male frogs, Rana ridibunda (40 –50 g body weight), originating from Albania, were obtained from a commercial source (Coue´tard, St. Hilaire de Riez, France). The animals were maintained in glass tanks supplied with a trickle of tap water in a temperature-controlled room (8 6 1 C) under an established photoperiod of 12 h of light/day (lights on from 0600 –1800 h). Animal treatment was performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

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RANAKININ-INDUCED PHOSPHOINOSITIDE HYDROLYSIS

TABLE 1. Primary structure of the main tachykinins identified in the adrenal tissue Ref. no.

Substance Pa Ranakininb NKAa [Leu3, Ile7] NKAb NKBa

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2 His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 His-Lys-Leu-Asp-Ser-Phe-Ile-Gly-Leu-Met-NH2 Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2

5 7 5 7 5

The common carboxyl-terminal amino acid sequence is indicated in bold letters. The variable residue within this sequence is indicated in italic letters. a Peptides identified in the adrenal gland of mammals. b Peptides identified in the adrenal gland of amphibians.

Reagents and test substances Ranakinin and substance P were synthesized by the solid phase method and purified by reversed phase HPLC as previously described (1, 21). Mammalian NKA and NKB were purchased from Novabiochem (Lauselsingen, Switzerland). Leibovitz L15 culture medium, HEPES, forskolin, pertussis toxin (PTX), 3-isobutyl-1-methylxanthine, kanamycin, and the antibiotic-antimycotic solution were purchased from Sigma Chemical Co. (St. Louis, MO). BSA (fraction V) was obtained from Boehringer Mannheim (Indianapolis, IN). Corticosterone and aldosterone were purchased from Merck (Darmstadt, Germany). Myo-[3H]inositol (100 Ci/mmol), the cAMP RIA kit, [1,2,6,7-3H]corticosterone (84 Ci/mmol), and [1,2,6,7-3H]aldosterone (82 Ci/mmol) were obtained from Amersham International (Les Ulis, France). 1-(6-[(17b-3-Methoxyestra-1,3,5-(10)-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U73122) and its inactive analog U-73343 were obtained from Biomol (Plymouth Meeting, PA). N-[2-(p-Bromocinnamyl-amino)ethyl]-5-isoquinoline-sulfonamide (H-89) was purchased from ICN Pharmaceuticals (Orsay, France).

Tissue preparation Frogs were killed by decapitation, and the kidneys were quickly removed. The adrenal glands were dissected free of renal parenchyma and sliced into six to eight pieces. For cAMP measurement, the adrenal slices (six per tube) were immersed in Ringer’s buffer consisting of 112 mm NaCl, 2 mm KCl, 2 mm CaCl2, 15 mm NaHCO3, 15 mm HEPES, 2 mg/ml glucose, and 0.3 mg/ml BSA (pH 7.4) and gassed with a 95% O2-5% CO2 mixture. For myo-[3H]inositol labeling, the tissue explants were placed in petri dishes containing L15 medium adjusted to Rana ridibunda osmolality (L15-water 5 1:0.4) supplemented with 0.4 mm CaCl2, 15 mm HEPES, and 1% of the kanamycin and antibiotic-antimycotic solutions (fL15; pH 7.4).

cAMP measurement Adrenal explants were preincubated for 20 min in Ringer’s buffer containing 1024 m 3-isobutyl-1-methylxanthine to inhibit phosphodiesterase activity. The tissue explants were then incubated for 10 min with either tachykinins (substance P, NKA, NKB, and ranakinin; 1025 m of each) or forskolin (1025 m). The dose of tachykinins used has been previously shown to significantly stimulate corticosteroid secretion (7). The reaction was stopped by removing the incubation medium and adding 5% (wt/vol) perchloric acid at 4 C. The tissues were homogenized in a glass Potter, and the homogenate was centrifuged (13,000 3 g; 5 min). The pellet was used to determine the protein content. The supernatant was collected, diluted with the same volume of potassium bicarbonate (KHCO3; 1 m), and centrifuged (13,000 3 g; 5 min). The cAMP content was determined by RIA following the procedure recommended in the cAMP RIA kit. The sensitivity threshold of the assay was 15 fmol/tube. The results are expressed as the amount of cAMP per mg protein.

Labeling of adrenal tissue with myo-[3H]inositol The effect of ranakinin on polyphosphoinositide metabolism was investigated as previously described (22). Briefly, phosphoinositides and inositol phosphates were labeled by incubating adrenal explants in

FIG. 1. Effect of tachykinins on cAMP production by frog adrenal tissue. The adrenal slices were incubated for 10 min in the absence (C 5 control) or presence of 1025 M ranakinin (RK), 1025 M substance P (SP), 1025 M NKA, 1025 M NKB, or 1025 M forskolin (FSK). Data are the mean 6 SEM values from three independent experiments performed in triplicate. Statistical difference between basal and stimulated cAMP concentrations was assessed by one-factor ANOVA. ns, Not significantly different from the control; ***, P , 0.001 vs. control.

fL15 medium with myo-[3H]inositol (50 mCi/ml) for 18 h at 24 C. The tissue was sampled at random (12 slices/tube) and washed six times with Ringer’s buffer containing 1023 m inositol. The tissue slices were preincubated in Ringer’s buffer containing 10 mm LiCl for 20 min and then incubated with ranakinin in the presence of LiCl for periods ranging from 10 sec to 2 min. The reaction was stopped by removing the incubation medium and adding 1 ml ice-cold 20% trichloroacetic acid. The tissue was homogenized in a glass Potter homogenizer, and the homogenate was centrifuged at 13,000 3 g for 10 min. The supernatant that contained the inositol phosphates (IPs) was stored at 220 C until analysis by anion exchange chromatography. Membrane phosphoinositides were extracted from the pellet by 200 ml chloroform-methanol (2:1, vol/vol). After centrifugation (13,000 3 g, 10 min), the organic phase containing the phosphoinositides was stored at 220 C until analysis by high performance TLC (HPTLC). The protein concentration was determined in the remaining pellet by the method of Lowry.

Analysis of 3H-inositol phosphates [3H]IPs contained in the aqueous phase were separated by anion exchange chromatography using a formate form of AG1-X8 resin (100 – 200 mesh; Bio-Rad Laboratories, Richmond, CA). Free [3H]inositol and inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3) species were sequentially eluted with distilled water and solutions of 0.2, 0.45, and 0.8 m ammonium formate in formic acid (0.1 m), respectively. Quantitative analysis of the chromatogram was performed using a flow scintillation


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FIG. 2. Time course of ranakinin-induced inositol phosphates formation (A) and phosphoinositide metabolism (B) in myo-[3H]inositol-prelabeled frog adrenal tissue. After a 20-min preincubation in Ringer’s buffer supplemented with 10 mM LiCl, the adrenal slices were incubated in the presence of 1025 M ranakinin for the times indicated. Data are the mean 6 SEM values from at least three independent determinations.

detector (Radiomatic Flo-One Beta A-500, Packard, Meridian, CT). Results are expressed as counts per min/mg protein.

(Bertold, Elancourt, France). Results are expressed as counts per min/mg protein.

Analysis of [3H]phosphoinositides

Perifusion experiments

The phosphoinositide extracts were dried under nitrogen and reconstituted in 10 ml chloroform-methanol (2:1, vol/vol). [3H]Phosphoinositides were separated by HPTLC on precoated Silica Gel 60 F254 plates (Merck, Paris, France) using the solvent system chloroform-methanolammonia-water (45:35:2:8, vol/vol/vol/vol). To calibrate the HPTLC plates, reference standards (PI, lyso PI, [3H]PIP2, and a mixture of PS, PI, PIP, and PIP2) were chromatographed under the same conditions and visualized by counting the radioactivity or by exposure to iodine vapor (23). The radioactivity corresponding to the various 3H-labeled phosphoinositides was determined in an automatic HPTLC linear analyzer

The adrenal explants were rinsed three times with Ringer’s solution and layered between several beds of Bio-Gel P-2 (200 – 400 mesh; Bio-Rad Laboratories) in perifusion chambers (equivalent of eight adrenal glands per chamber) as previously described (24). The tissues were continuously supplied either with Ringer’s solution alone or with test substances freshly dissolved in Ringer’s solution at a constant flow rate (200 ml/min) and temperature (24 C). The experimental procedure commenced after a stabilization period of 2 h. The perifusate effluent from each column was collected at 5-min intervals, and the fractions were stored frozen until assay.

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FIG. 3. Effects of graded concentrations of ranakinin on inositol phosphates formation (A) and phosphoinositide metabolism (B) in myo-[3H]inositol-prelabeled frog adrenal tissue. After a 20-min preincubation in Ringer’s buffer supplemented with 10 mM LiCl, the adrenal explants were incubated for 20 sec in the presence of ranakinin (1029-1025 M). Data are the mean 6 SEM values from at least three independent determinations.

Corticosteroid RIAs Corticosterone and aldosterone concentrations were determined directly in 100-ml aliquots of each perifusion fraction without prior extraction using specific RIAs (25). The working range of the assays was 20 –5000 pg for corticosterone and 10 –2000 pg for aldosterone. None of the test substances showed any interference in the corticosterone and aldosterone assays. For both assays, the intra- and interassay coefficients of variation were less than 4% and 10%, respectively.

Results Effects of ranakinin on cAMP formation

Incubation of frog adrenal slices with ranakinin (1025 m; 10 min) did not induce any significant modification of the cAMP concentration (Fig. 1). Similarly, the other tachykinins

tested, i.e. substance P, NKA, and NKB (1025 m; 10 min) were totally devoid of effect on cAMP formation in the frog adrenal gland. Under the same conditions, forskolin (1025 m; 10 min) induced a 6-fold rise in cAMP content (P , 0.001; Fig. 1). Effects of ranakinin on polyphosphoinositide metabolism and IPs formation

After an 18-h incubation of frog adrenal tissue with myo[3H]inositol, the incorporation of [3H]inositol into membrane phospholipids had reached equilibrium (22). Under these conditions, exposure of frog adrenal explants to ranakinin (1025 m) produced an abrupt increase in IPs formation, fol-


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hibitor H-89 (1025 m) or the phospholipase C inhibitor U-73122 (1026 m). In control conditions, ranakinin induced a 2-fold increase in corticosteroid secretion (Fig. 5, A and B). Prolonged infusion of H-89 (1025 m) did not affect the secretory response of adrenal explants to ranakinin (data not shown). In contrast, during prolonged infusion of U-73122, the stimulatory effect of ranakinin on corticosterone and aldosterone secretion was reduced by 70% (P , 0.01) and 46% (P , 0.05), respectively (Fig. 5, C and D), whereas the inactive analog U-73343 did not affect the ranakinin-evoked stimulation of corticosteroid secretion (Fig. 5, E and F). Discussion

FIG. 4. Effect of the phospholipase C inhibitor (U-73122) and PTX on ranakinin-induced inositol phosphates production by myo-[3H]inositol-prelabeled frog adrenal tissue. The adrenal explants were preincubated with U-73122 (1026 M; 20 min) or PTX (200 ng/ml; 18 h) and then exposed to ranakinin (RK; 1025 M) for 20 sec in the presence of U-73122 or PTX. Data are the mean 6 SEM values from four independent determinations. Statistical difference between experimental values was assessed by one-factor ANOVA. ***, P , 0.001.

lowed by a rapid decay toward baseline. IP3 production increased by 100% (P , 0.01) above the control value within 20 sec after the beginning of the incubation period (Fig. 2A). IP2 increased by 67% (P , 0.05) within the first 20 sec, whereas IP1 increased by 54% (P , 0.05) after 30 sec (Fig. 2A). Concurrently, ranakinin caused a rapid and transient decrease in PIP2 (254%; P , 0.05) and PIP (246%; P , 0.05); the maximum effect was observed 30 sec after the onset of incubation with the peptide (Fig. 2B). In contrast, the PI concentration was not significantly affected (Fig. 2B). Administration of graded concentrations of ranakinin (10291025 m) resulted in a dose-dependent stimulation of IP3, IP2, and IP1 production (Fig. 3A) and a concomitant decrease in PIP2 and PIP concentrations (Fig. 3B). In contrast, whatever the dose of ranakinin, the concentration of PI was not significantly affected (Fig. 3B). Preincubation of the adrenal slices with the phospholipase C inhibitor U-73122 (1026 m; 20 min) did not affect the basal concentration of IPs, but completely abolished the stimulatory effect of ranakinin on IPs formation (Fig. 4). Similarly, incubation of the adrenal tissue with PTX (200 ng/ml; 18 h) had no effect on the basal IPs concentration, but totally abrogated the response to ranakinin (Fig. 4). Effects of H-89 and U-73112 on corticosteroid secretion

It has been shown previously that administration of repeated pulses of tachykinins to frog adrenal explants causes a marked attenuation of the secretory response (7). Given this desensitization phenomenon, a single pulse of ranakinin (1025 m; 20 min) was administered to each perifusion chamber in the absence or presence of the protein kinase A in-

We have previously demonstrated that the stimulatory effect of ranakinin on the frog adrenal gland is mediated through an NK-1-like receptor subtype (26). Studies using Chinese hamster ovary (CHO) cells transfected with the NK-1 receptor have shown that tachykinins can increase both phosphatidylinositol hydrolysis and cAMP formation (16, 27). In bovine adrenocortical cells, substance P acting through an NK-1 receptor type stimulates cortisol secretion and causes a concomitant increase in the intracellular cAMP concentration (8). However, it was found that inhibitors of cAMP-dependent protein kinase do not reduce the steroidogenic response to substance P, indicating that the corticotropic activity of the peptide cannot be accounted for by its stimulatory effect on adenylyl cyclase (8). The present study showed that in the frog adrenal gland, ranakinin and other tachykinins did not stimulate cAMP formation. In addition, the PKA inhibitor H-89 did not affect ranakinin-evoked corticosteroidogenesis. Taken together, these data indicate that the stimulatory effect of ranakinin in the frog adrenal gland cannot be ascribed to activation of the adenylyl cyclaseprotein kinase A pathway. Ranakinin induced a dose-dependent increase in IP3, IP2, and IP1 formation with a concomitant decrease in PIP2 and PIP contents in the frog adrenal gland. The doses of ranakinin required to stimulate phosphatidylinositol breakdown were in the same range as those causing an increase in corticosteroid secretion (7). Time-course studies conducted with various cellular models have previously shown that tachykinins induce a brief stimulation of IPs production (28, 29). Similarly, we have found that the effect of ranakinin on phosphoinositide metabolism in the frog adrenal gland was immediate and transient, suggesting the occurrence of a desensitization phenomenon. In support of this hypothesis, it has been previously shown that the administration of repeated pulses of tachykinins to perifused frog adrenal slices causes a decrease in the stimulatory effect of the peptide on corticosteroid secretion (7). It has also been found that sequential administration of ranakinin in the vicinity of cultured adrenochromaffin cells resulted in a gradual attenuation of the amplitude of the calcium transients (30). Downregulation of NK-1 receptors by substance P has already been documented in other in vitro models, including smooth muscle strips (31), parotid acinar cells (28, 32), and striatal neurons (33). Exposure of frog adrenal explants to the phospholipase C inhibitor U-73122 totally suppressed the stimulatory effect of

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ranakinin on polyphosphoinositide metabolism. Similarly, preincubation of the adrenal tissue with PTX abolished the effect of ranakinin on phosphatidylinositol breakdown. These data indicate that the NK-1-like receptor present in the frog adrenal gland is coupled to phospholipase C through a PTX-sensitive G protein. In agreement with these observations, recent studies have shown that the NK-1 receptors expressed in the rat submaxillary gland and in CHO-transfected cells are positively coupled to phospholipase C through Gq and/or G11 proteins (29, 34). The fact that U-73122 significantly attenuated the stimulatory effect of ranakinin on corticosterone and aldosterone secretion revealed that the corticotropic activity of the peptide could be accounted for at least in part by activation of phospholipase C. Recent studies conducted in CHO cells stably transfected with the NK-1 receptor complementary DNA have shown that substance P stimulates both inositol phosphate production and formation of arachidonic acid metabolites (18). In agreement with these findings, it has been previously shown that in amphibians, the cyclooxygenase inhibitors indomethacin and acetyl salicilic acid markedly attenuate the stimulatory effect of tachykinins on corticosteroid secretion (7, 11). These data strongly suggest that in the frog adrenal gland, the corticotropic activity of the peptide can be ascribed to activation of both phospholipase C and phospholipase A2. In conclusion, the present study has demonstrated that in the frog adrenal gland, ranakinin, a recently discovered amphibian tachykinin, stimulates phospholipase C activity through a PTX-sensitive G protein. In contrast, ranakinin had no effect on the adenylyl cyclase/protein kinase A pathway. The stimulatory effect of ranakinin on corticosteroid secretion is attributable to its stimulatory effect on both phospholipase C and phospholipase A2.

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Acknowledgments The authors thank Mrs. Huguette Lemonnier for expert technical assistance, and Catherine Blonde for typing the manuscript.

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FIG. 5. Effect of ranakinin (RK) alone or during prolonged infusion of U-73122 or U-73343 on corticosteroid secretion from perifused frog adrenal explants. A and B, Control experiments showing the effect of RK (1025 M; 20 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of RK during infusion of the phospholipase C inhibitor U-73122 (1026 M; 180 min) on corticosterone (C) and aldosterone (D) secretion. E and F, Effect of RK during infusion of U-73343, an inactive analog of U-73122, on corticosterone (E) and aldosterone (F) secretion. The pulses of RK were given 80 min after the onset of U-73122 or U-73343 administration. The profiles represent the mean secretion pattern of three independent perifusion experiments. Each point is the mean corticosteroid production (expressed as a percentage of spontaneous steroid output) of two consecutive fractions collected during 5 min. The spontaneous level of steroid release (100% of the basal level) was calculated as the mean of eight consecutive fractions (40 min) collected before administration of the secretagogues (E). The mean basal levels of corticosterone and aldosterone were 35.4 6 4.7 and 18.2 6 1.7 pg/minzadrenal gland.

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