Possible role of bradykinin on stimulus-secretion coupling in adrenal chromaffin cells

Possible role of bradykinin on stimulus-secretion coupling in adrenal chromaffin cells

Hitoshi Houchi^a, Mami Azuma^a, Masanori Yoshizumi^b, Toshiaki Tamaki^b, and Kazuo Minakuchi^a

^aDivision of Pharmacy, University Hospital, and ^bDepartment of Pharmacology, The University of Tokushima School of Medicine, Tokushima, Japan

Abstract:Nonapeptide bradykinin is known to be a central nervous system neurotrans-mitter and to play a role in regulation of neuronal function. However, few details are known of the function of its peptide on stimulus-secretion coupling in neuronal cells. In this article, the role of bradykinin on catecholamine biosynthesis, secretion and Ca2+movement in adrenal chromaffin cells as a model for catecholamine-containing neurons are examined. Bradykinin receptors are classified as B1 and B2 receptor subtypes. These receptors are present on the adrenal chromaffin cell membrane. Bradykinin increases the influx of Ca2+ and the turnover of phosphoinositide through the stimulation of bradykinin B2 receptor. The secretion of catecholamine from the cells is initiated by the raise of [Ca2+]i. An increase in [Ca2+]i and production of diacylglycerol stimulate the activation of calcium-dependent protein kinases. These kinases stimulate the activation of tyrosine hydroxylase, a rate-limiting enzyme in the biosynthesis of catecholamine. Otherwise, bradykinin increases Ca2+ efflux from the cells through the stimulation of the bradykinin-B2 receptor. This action may be explained by an extracellular Na+-dependent mechanism, probably through acceleration of Na+/Ca2+ exchange. It is interesting that bradykinin, which stimulates the biosynthesis and secretion of catecholamine in adrenal chromaffin cells, plays a role in the termination of calcium-signal transduction through the stimu-lation of Ca2+ efflux from the cells. J. Med. Invest. 46:1-9, 1999

Keywords:Bradykinin, Catecholamine, Biosynthesis, Secretion, Calcium

INTRODUCTION
The nonapeptide bradykinin has been found to influence several physiological processes including pain generation (1), blood pressure (2) and cardio-vascular regulation (3). It has also been suggested to be a central nervous system neurotransmitter and to play a role in the regulation of neuronal function (4, 5).
Adrenal chromaffin cells and their cancer cell line, pheochromocytoma PC-12 cells, are useful for studying the mechanism of the stimulus-secretion coupling, and are regarded as a model for catecholamine-containing neurons. Physiological stimulations of the cells cause an increase in the levels of intracellular free Ca2+([Ca2+]i) which comes from both the intracellular and extracellular pools. The increase in [Ca2+]i causes exocytosis including the stimulation of catecholamine secretion and activation of its biosynthesis (6, 7).
In this article, the role of bradykinin on catechol-amine biosynthesis, secretion and Ca2+ movement in adrenal chromaffin cells and pheochromocytoma PC-12cells are examined.

General stimulus-secretion coupling in adrenal chromaffin cells
Chromaffin cells synthesize, store and secrete cate-cholamines (dopamine, noradrenaline and adrena-line) in nerve stimulations. The cells have an excitable action in response to acetylcholine or to electrical stimulation of the splanchnic nerve. Following acti-vation of acetylcholine nicotinic receptors, acetyl-choline causes the opening of the receptor-mediated ion channel, allowing the influx of Na+, and to a lesser extent Ca2+ (8, 9). This influx into the chromaffin cells of Na+ results in a mild depolarization of the cell membrane sufficient to activate voltage-dependent Na+ channels (10). The opening of Na+ channels induces the activation of voltage-dependent Ca2+channels (11). The opening of Na+ and Ca2+ channels causes the firing of action potentials and the entry of Ca2+ from extracellular spaces (12). An increase in [Ca2+]i is the trigger for exocytosis of chromaffin granules (secretion of catecholamine) and stimula-tion of biosynthesis of catecholamine. Acetylcholine nicotinic and muscarinic receptors are present in chromaffin cells, and these receptors in most spe-cies stimulate secretion of catecholamine. In bovine adrenal chromaffin cells, however, only nicotinic re-ceptor stimulation evoke secretion of catecholamine. Catecholamine secretion can also be evoked by high K+ which directly activate the voltage-dependent channels. The depolarization induced by high K+ directly opens the Ca2+ channels without the contri-bution of the Na+ channel. The role of bradykinin, a putative neuropeptide, on stimulus-secretion cou-pling including biosynthesis and secretion of cate-cholamine in adrenal chromaffin cells and pheo-chromocytoma PC-12 are discussed below.

Catecholamine biosynthesis regulated by brady-kinin
Tyrosine hydroxylase, which is a rate-limiting enzyme in the biosynthesis of catecholamine, is phos-phorylated and activated by cAMP-dependent pro-tein kinase and calcium-dependent protein kinases (calcium/calmodulin-dependent protein kinase and protein kinase C) (13-16):it is activated on incu-bation of cells with cAMP analogs or forskolin (an activator of adenylate cyclase), phorbol esters (activa-tors of protein kinase C) or compounds that elevate the [Ca2+]i concentration (compounds causing nico-tinic receptor stimulation or K+-induced depolariza-tion or calcium ionophores). Increased activation and phosphorylation of tyrosine hydroxylase on depolar-ization with high K+ or treatments with cholinergic agonists, dibutyryl cAMP, phorbol esters and bioactive neuropeptides (vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide) in adrenal chromaffin cells were reported (17-32).
Fig. 1 compares the activation and phosphorylation of tyrosine hydroxylase produced by bradykinin with the activation and phosphorylation of tyrosine hydroxylase produced by treatment with dibutyryl cAMP, phorbol ester phorbol myristate acetate (PMA) and high K+ in pheochromocytoma PC-12. Concentrations of these compounds were chosen to produce maximal activation and phosphorylation of tyrosine hydroxylase and the additivity of this activation and phosphorylation with that produced by bradykinin was evaluated. The increase in tyrosine hydroxylase activity produced by bradykinin (356%) and dibutyryl cAMP (181%) were additive (544%).Likewise, the enhanced phosphorylation of tyrosine hydroxylase produced by bradykinin (114%) and dibutyryl cAMP (44%) were additive (153%). PMA increased by 19% the activation of tyrosine hy-droxylase produced by bradykinin. Likewise, the phosphorylation of tyrosine hydroxylase was in-creased to a similar degree. High K+ had no effect on the increase in tyrosine hydroxylase activity or phosphorylation produced by bradykinin. These data suggest that the mechanism of activation and phosphorylation of tyrosine hydroxylase produced by bradykinin is similar to that produced by high K+ and to a minor extent by PMA.
In order to evaluate this further, the sites on tyrosine hydroxylase that were phosphorylated by bradykinin were determined. PC-12 cells were incubated with 32P for 1hr to label the cellular ATP stores. After ex-posure of the cells to bradykinin, tyrosine hydroxylase was isolated, subjected to SDS-polyacrylamide gel electrophoresis, eluted from the gels, and digested with trypsin for 12 to 18hr at 37°C. The 32P-labeled phosphopeptides derived from tyrosine hydroxylase were separated by HPLC. Fig. 2 illustrates the HPLC elution pattern of free 32P-labeled peptide peaks that were produced by bradykinin, peptide peak A (reten-tion time, 16min), peak B (19min) and peak C (27min). The phosphorylation of peak A and B was increased markedly when PC-12 cells were treated with brady-kinin, whereas peak C was unaffected. It is known that peak B is phosphorylated by activators of protein kinase C (PMA or 1-oleyl-2-acetylglycerol), peak C is phosphorylated by activators of cAMP-dependent protein kinase (dibutyryl cAMP or forskolin), and peak A is phosphorylated by compounds that in-crease [Ca2+]i concentration and possibly activate cal-cium/calmodulin-dependent protein kinase (high K+ or ionomycin). As bradykinin selectively stimulated the phosphorylation of peaks A and B, the possibility exists that bradykinin may activate tyrosine hydroxylase by an action on both calcium/calmodulin-dependent protein kinase and protein kinase C.

Catecholamine secretion regulated by bradykinin
Activation of acetylcholine nicotinic receptors with cholinergic agents or membrane depolarization with high K+ is known to initiate transient rapid catecholamine secretion accompanied by an increase in Ca2+ influx into the cells (6, 33, 34).
The time courses of catecholamine secretion induced by10-6M bradykinin and10-5M ouabain, an Na+/K+ ATPase inhibitor, separately or in combi-nation were carried out in adrenal chromaffin cells. Bradykinin alone induced slight, transient secretion of catecholamine within 2min, followed by slower, sustained secretion for at least 30min. Ouabain alone also induced slight secretion for at least 30 min. The secretion was markedly higher with bradykinin plus ouabain, about 15% of the total intracellular catecholamine being secreted in 30min. Stimulation of catecholamine secretion in either the presence or absence of 10-5M of ouabain was observed with more than10-8M bradykinin and was maximal with10-5M bradykinin. The concentration of ouabain necessary to potentiate the bradykinin-induced catecholamine secretion was 10-6M to 10-4M. Catecholamine secretion stimulated by bradykinin in the presence of ouabain was inhibited by10-6M D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, a bradykinin-B2 receptor antagonist (35), but not by 10-6M Des-Arg9-[Leu8]-bradykinin, a bradykinin-B1 receptor antagonist (3), and was re-duced in medium without Ca2+. These results indicate that the stimulatory effect of bradykinin plus ouabain on catecholamine secretion was mediated via the bradykinin-B2 receptor and dependent on the pres-ence of Ca2+ in the medium. It is further indicated that stimulation of the bradykinin-B2 receptor may stimulate the influx of Ca2+ into the cells.
We examined the 45Ca2+ influx into the cells stimu-lated by bradykinin and ouabain separately or together. Bradykinin or ouabain alone increased 45Ca2+ influx into the cells slightly, whereas bradykinin plus ouabain increased 45Ca2+ influx markedly. The time courses of 45Ca2+ influx were similar to those of catecholamine secretion, indicating that continuous45Ca2+influx is associated with catecholamine secretion.
To determine whether Na+, as well as Ca2+, is involved in the secretion of catecholamine induced by bradykinin and bradykinin plus ouabain, we ex-amined their effects in Na+-depleted sucrose medium. We found that Na+-depleted sucrose medium con-taining Ca2+ itself induced secretion about 5-6% of the total catecholamine by increasing Ca2+ influx into the cells. Therefore, we incubated the cells with bradykinin for30min in Ca2+-free medium in the presence or absence of ouabain and then stimu-lated them for 15min with Ca2+-medium (containing0.55-2.2mM Ca2+) without bradykinin and ouabain. As shown in Fig. 3, prior stimulation of the cells with bradykinin increased the catecholamine secretion induced by each concentration of Ca2+ tested, and this stimulatory effect of bradykinin was greatly poten-tiated by the presence of ouabain. The stimulatory effect of bradykinin plus ouabain on catecholamine secretion in Na+-free sucrose medium was much lower. During the first stimulation of the cells with bradykinin plus ouabain in Ca2+-free medium,22Na+ accumulation in the cells was significantly higher than that with bradykinin or ouabain alone, the levels induced by 10-6M bradykinin, 10-5M ouabain and10-6M bradykinin plus 10-5M ouabain being 25±4,210±24 and 285±31 (nmol/dish in 30min), res-pectively. These results indicated that activation of the bradykinin-B2 receptor and inhibition of the Na+ pump by ouabain both increase the accumulation of Na+ in the cells, resulting in increases in Ca2+ influx, the [Ca2+]i level and catecholamine secretion (36).

Calcium efflux from chromaffin cells regulated by bradykinin
After stimulation of catecholamine secretion from adrenal chromaffin cells induced by various secreta-gogues, the increase in [Ca2+]i should rapidly return to the resting level to enable response to a subse-quent stimulation (37-43). In this part, the mecha-nism of decrease in elevated [Ca2+]i and the effect of bradykinin on Ca2+ efflux from adrenal chromaffin cells are discussed.
Fig. 4 shows the effluxes of 45Ca2+ from adrenal chromaffin cells in culture induced by various con-centrations of bradykinin. The stimulatory effect of bradykinin on 45Ca2+ efflux was dose-dependent at concentrations of 10-9-10-6M bradykinin. The efflux of 45Ca2+ increased to a peak value within about 1min after bradykinin addition. The peak value with10-6M bradykinin was 8.2±0.7% of the total 45Ca2+ in the cells. After the peak, the efflux decreased rapidly for the next 5min. The effects of the bradykinin-receptor antagonists Des-Arg9-[Leu8]-bradykinin (B1-receptor antagonist (3)) and D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin (B2-receptor antagonist (35)) on the submaximal 45Ca2+ efflux from the cells induced by10-7M bradykinin were evaluated. This efflux was inhibited 81% by10-6M D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but was not inhibited by Des-Arg9-[Leu8]-bradykinin. This result suggests that the 45Ca2+ efflux induced by bradykinin was mediated through the bradykinin B2-receptor.
To determine whether bradykinin-stimulated 45Ca2+ efflux is mediated by activation of Ca2+ channels, we examined whether it was inhibited by Ca2+ channel blockers. Nifedipine, an organic voltage-dependent Ca2+ channel blocker, had no effect on 45Ca2+ efflux from the cells induced by bradykinin (Fig. 5). Nor did the inorganic Ca2+ channel blockers Co2+ and Cd2+, which inhibit voltage-dependent and receptor-operated Ca2+ channels, inhibit bradykinin-stimulated 45Ca2+ efflux (Fig. 5). Thus stimulation of 45Ca2+ efflux by bradykinin is probably not due to increased Ca2+ flux through Ca2+channels.
The stimulations of bradykinin, histamine and muscarinic acetylcholine receptors are reported to induce breakdown of phosphatidyl inositol 4,5-bisphosphate (PIP2) in bovine adrenal chromaffin cells (44, 45). The breakdown products of PIP2 (inositol1,4,5-trisphosphate (IP3) and diacylglycerol) increase the [Ca2+]i level and stimulate protein kinase C (24, 46, 47). The effects of bradykinin, histamine, acetylcholine and PMA (an activator of protein kinase C) on 45Ca2+ efflux from the cells were examined. Bradykinin (10-6M) and histamine (10-5M) increased the 45Ca2+ efflux to about 460% and 350% of the control level, respectively. Acetylcholine (10-4M) increased 45Ca2+ efflux from the cells slightly, but significantly, whereas PMA (10-6M) had no effect on 45Ca2+ efflux. These results suggest that bradykinin-stimulated 45Ca2+ efflux from the cells is related to the forma-tion of IP3, but not to activation of protein kinase C.
To determine whether the increased 45Ca2+ efflux induced by bradykinin is dependent on the elevation of [Ca2+]i level, the effects of bradykinin, histamine and acetylcholine on [Ca2+]i concentration were ex-amined. Bradykinin (10-6M) and histamine (10-5M) increased the [Ca2+]i to approximately 430nM and360nM from 100nM. Acetylcholine (10-4M) increased it to about 890nM. However, acetylcholine-stimulated 45Ca2+ efflux from the cells was less than bradykinin- or histamine-stimulated 45Ca2+ efflux (Fig. 5). Therefore, bradykinin-stimulated 45Ca2+ efflux from the cells may not be dependent on the elevation of the intracellular [Ca2+]i level in the cells.
It has been established that bradykinin increases cyclic GMP (cGMP) in adrenal chromaffin cells through the activation of guanylate cyclase (48).Therefore, the role of cAMP and cGMP on Ca2+ efflux from cultured bovine adrenal chromaffin cells was evaluated to determine whether these intracellular messengers are involved in regulation of the Ca2+efflux mechanism. The effects of dibutyryl cAMP(10-3M), forskolin (10-6M:an activator of adenylate cyclase (18)), dibutyryl cGMP (10-3M) and nitro-prusside (10-3M:an activator of guanylate cyclase(49, 50)) on 45Ca2+ efflux from cultured bovine adrenal chromaffin cells preloaded with 45Ca2+ were exam-ined. All these agents stimulated efflux of 45Ca2+ from the cells. These stimulations of 45Ca2+ efflux were clearly observed at concentrations of more than2x10-4M of dibutyryl cAMP and dibutyryl cGMP, 10-7M of forskolin and 10-4M of nitroprusside. Since these agents did not affect the [Ca2+]i level measured using the Ca2+ indicator fura-2 (control98±8;10-3M dibutyryl cAMP103±10;10-6M forskolin101±9;10-3M dibutyryl cGMP 97±9;10-3M nitroprusside102±11nM, respectively), this stimulation of Ca2+efflux was not the result of increase in the [Ca2+]i level. These results suggest that both cAMP and cGMP are involved in stimulation of Ca2+ efflux from the cells, through these nucleotide-dependent protein kinases (51).
To determine whether the increased 45Ca2+ efflux induced by bradykinin is Na+-dependent, we carried out a series of experiments in the absence of extracellular Na+. As shown in Fig. 6, complete replacement of Na+ by sucrose significantly blocked the enhanced 45Ca2+ efflux from the cells induced by bradykinin. Amiloride, an inhibitor of the Na+/Ca2+ exchanger (52), also sig-nificantly inhibited bradykinin-stimulated 45Ca2+efflux from the cells. Therefore, the effect of bradykinin in stimulating Ca2+ efflux across the plasma membrane may be mediated in part by a Na+/Ca2+ exchange mechanism (53).
Bradykinin-stimulated 45Ca2+ efflux from the cells may be regulated by nitric oxide (NO)/ cGMP path-way because 1) Bradykinin increases the formation of cGMP (48), 2) an increase in cGMP formation stimulates the calcium efflux from adrenal chromaffin cells (51), and 3) the efflux of calcium is regulated by NO production (54). However, we have no data on whether bradykinin stimulates NO production in adrenal chromaffin cells.

CONCLUSION
The role of bradykinin on stimulus-secretion coupling in adrenal chromaffin cells is illus-trated in Fig7. Bradykinin receptors are clas-sified as B1 and B2 receptor subtypes. These receptors are present on the adrenal chromaffin cell membrane. Bradykinin increases the influx of calcium and the activity of phospholipase C through the stimulation of bradykinin B2 receptor on the cell membrane. Diacylglycerol is produced concurrently with IP3 on break-down of PIP2 by phospholipase C and is thought to activate protein kinase C by increasing the affinity of the enzyme for calcium (46). In the pathway of catecholamine biosynthesis, an in-crease in [Ca2+]i and production of diacylglycerol stimulate the activation of calcium-dependent protein kinases (calcium/calmodulin-dependent protein kinase and protein kinase C). These kinases phosphorylate and acti-vate tyrosine hydroxylase, a rate-limiting enzyme in the bio-synthesis of catecholamine. The catecholamine which is formated by tyrosine hydroxylase is stored in chromaffin granules. In the pathway of catecholamine secretion, the stimulation of the bradykinin-B2 receptor elevates the accu-mulation of Na+ in the cells. An increase in intracellular Na+ concentration leads to Ca2+ influx into the cells through the depolarization of the cell membrane and/or the reverse mode of Na+/Ca2+ exchange mechanism. The secre-tion of catecholamine from the cells is initiated by the raise of [Ca2+]i. However, the detailed mechanism of the calcium-induced catecholamine secretion pathway remains unclear.
Physiological stimulation of adrenal chromaffin cells cause an increase in [Ca2+]i, leading to initiation of stimulus-secretion coupling. How-ever, this increased [Ca2+]i should be restored to a physiological level for response to subsequent stimu-lation. In the pathway of Ca2+ efflux, bradykinin in-creases Ca2+ efflux from the cells through the stimu-lation of the bradykinin-B2 receptor. Bradykinin-induced Ca2+ efflux may be explained by an extra-cellular Na+-dependent mechanism, probably through acceleration of Na+/Ca2+ exchange. It is interesting that bradykinin, which stimulates the biosynthesis and secretion of catecholamine in adrenal chromaffin cells, plays a role in the termination of Ca2+-signal transduction through the stimulation of Ca2+ efflux from the cells.

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Received for publication November 30, 1998 ; accepted December 15, 1998.

Address correspondence and reprint requests to Hitoshi Houchi, Ph. D., Division of Pharmacy, University Hospital, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima770-8503, Japan and Fax:+81-88-633-7472.