STIMULATION OF CATECHOLAMINE RELEASE FROM ISOLATED ADRENAL GLANDS BY SOME AMINO ACIDS

Transcription

1 STIMULATION OF CATECHOLAMINE RELEASE FROM ISOLATED ADRENAL GLANDS BY SOME AMINO ACIDS Takashige NISHIKAWA*, Katsuya MORITA, Kenji KINJO and Akira TSUJIMOTO** Department of Pharmacology, Hiroshima University School of Dentistry, Hiroshima 734, Japan Accepted December 7, 1981 Abstract-The effects of several amino acids on isolated perfused adrenal glands of dogs were investigated. Methionine (5 x X 10-2 M), glutamic acid (5X10-5_5X10-2 M), glutamine (10-4_5X10-2 M), serine (5 x x 10_2 M), cysteine (5X x 10-2 M), arginine ( X10-2 M), and threonine (5X10-3_5X10-2 M) consistently increased the rate of release of catecholamine (CA) from the glands for about 8 min. Preinfusion with hexamethonium (C6, 10-4 M) plus atropine (5x10-5 M) reduced the effects of methionine and glutamic acid slightly, and it completely inhibited the effect of acetylcholine (ACh, M). Preinfusion of physostigmine did not significantly influence the effects of methionine, glutamic acid, cysteine, and serine. Ca2+ free perfusion fluid depressed or abolished the effects of amino acids. They had no effect on the release of CA from isolated chromaffin granules. It seems likely that amino acids depolarize chromaffin cells in the adrenals and release CA from its storage sites. The effects of these amino acids on adrenal medulla cannot involve release of ACh from splanchnic nerve endings. Mellinkoff et al. (1, 2) first suggested that amino acids might stimulate release of insulin. Since then there have been many reports that infusion of certain amino acids stimulates in vivo or in vitro insulin release in animals and man. Knopf et al. (3) reported that infusion of a mixture of essential amino acids stimulated secretion of growth hormone in humans. Curtis et al. (4) showed that electrophoretically administered (-)-glutamic acid and related acidic amino acids depolarized the neurones in the central nervous system. Brimblecombe and Sutton (5) reported that some amino acid esters, *Present address: Department of Pharmacology School of Dentistry, Kagoshima University, Usuki, Kagoshima 890, Japan **To whom reprint requests should be addressed., such as ethyl -13-demethylaminopropionate methyliodide and methyl-r-dimethylamino butyrate methiodide, stimulated the superior cervical ganglions of cats. On the other hand, Gatgounis and Hester (6) showed that various neutral amino acids, such as leucine, serine, cysteine, methionine, and threonine, produced positive inotropic and pressor responses when administered intravenously and intraaortically in vago tomized open-chest dogs. They suggested that these amino acids might be active circulatory stimulants and that there might be a close interaction between the adrenal system and the stimulant effects of these amino acids. In the present work, the effects of several amino acids on isolated perfused adrenal glands of dogs were

2 investigated. MATERIALS AND METHODS Dogs of either sex weighing 5-13 kg were anaesthetized with sodium pentobarbital (30 mg/kg, i.v.). Both adrenals were exposed through a midline abdominal incision and removed. All detectable side branches entering the adrenal vein were ligated and then the adrenals were cannulated as described by Robinson (7). Retrograde perfusion was carried out at a pressure of 60 cm of water at 30±1 C with modified Krebs-Ringer phosphate solution (ph 7.3) of the following composition in mm: NaCI 137.5, KCI 5.5, CaC12 1.8, MgSO4 1.38, KH2PO4 1.38, Na2HP , and dextrose 8.5 (Tsujimoto and Nishikawa) (8, 9). This solution was equilibrated with 95% 02-5% C02 and perfused at a constant rate in each experiment, ranging from 0.7 to 2.5 ml/min. Glands were perfused for 40 min before any treatment to allow stabilization of output. Amino acids and drugs dissolved in Krebs Ringer phosphate solution were administered by continuous infusion by switching a valve on the tubing leading to the glands. When the Ca2+ concentration was altered in the perfusing medium, the Na+ concentration was correspondingly altered to maintain isotonicity. The ph value of the perfusing medium was always confirmed to be at ph 7.3 before the start of infusion. In some experiments, paired adrenals from the same animal were used as control and compound treated glands to minimize individual variation. When the effects of repeated exposure of adrenals to amino acids were studied, glands were perfused for 20 min with perfusion fluid without any compound before the next control collection. Samples were usually collected 4 drops (0.12 ml) at a time every 30 sec in glass tubes for CA assay. CA output was measured by the tri hydroxyindole fluorometric method of Euler and Lishajko (10) with epinephrine as a standard. In some experiments, epinephrine and norepinephrine output were determined differentially. Fluorescence was determined in a Hitachi type 204 spectrophotofluorometer at activation -fluorescence wavelengths of 405 and 515 nm. Chromaffin granules were prepared by the method of Borowitz (11, 12) except that the final preparation was suspended in 0.32 M sucrose. Granules containing 10 ug CA per mg of protein were incubated in 2.5 ml of 0.32 M sucrose at 30 C and allowed to stand for 20 min. After incubation the mixture was centrifuged at 30,000xg for 20 min, and the CA contents of the super natant and precipitate were determined. For this, the precipitate was extracted with 0.5 ml of 0.4 N HC104. The compounds used in this study were (-)-methionine (Sigma), (-)-glutamic acid (Sigma), (-)-glutamine (Nakarai Chemicals Ltd.), (-)-serine (Nakarai Chemicals Ltd.), (-)-cysteine (Merck), (-)-arginine (Nakarai Chemicals Ltd.), (-) -threonine (Nakarai Chemicals Ltd.), acetylcholine chloride (Daiichi Seiyaku Co., Ltd.), physostigmine salicylate (Merck), atropine sulfate (Sigma), hexamethonium bromide (Sigma), epin ephrine (Merck), and norepinephrine (Merck). The final molar concentrations of amino acids and other compounds are expressed in terms of the free base and the base, respectively. RESULTS Stimulation of adrenal CA release by methionine: The mean spontaneous rate of secretion of CA from 86 adrenal glands was 0.40 ag total CA/min (range iig): the mean concentrations being 0.38 ug/ml. The ranges were fairly wide, although the flow rate through the glands was relatively constant in each experiment. Addition of 5X10-3 M methionine to the perfusion fluid

3 for 10 min resulted in increased liberation of CA from 30 sec after the addition of the amino acid. Methionine significantly increased the proportion of norepinephrine in the effluent. CA release reached a maximum 2-3 min after the addition and then returned to control value within 8 min (Fig. 1). Release of CA by amino acids: Perfusion of isolated glands with methionine (5x10-5_ 5 x10-2 M), glutamic acid (5x10-5-5X10-2 M), glutamine (10-4-5x10-2 M), serine (5x10-4-5x10_2 M), cysteine (5x10-4 5x10-2 M), arginine (10-3-5x10-2 M), or threonine (5x x 10-2 M) for 6 min consistently increased the rate of release of CA from the glands from sec after the start of perfusion of amino acids. CA release reached a maximum 2-4 min after the start of perfusion, and then usually returned to the control value within 5-9 min. Figure 2 shows the dose-responses to these amino acids. Next the effects of a nonamino organic acid, glutaric acid, and various concentrations of Na+ on the amine release were determined. Addition of NaCI (10-3 2x10-2 M) or glutaric acid (10-3-2x10-2 M) into the perfusing fluid for 6 min hardly had any effect on the spontaneous release of CA. From these results, it seems that this release is due to the effect of the amino acids in general. The effects of methionine and glutamic acids at concentrations of 5x10-5_ 5 x 10-2 M were significantly greater (P<0.05 by the Student's t-test) than those of other amino acids at equivalent concentrations. When the amino acids were introduced for successive 6 min periods at 26 min intervals, the second and third responses were 50.1 ±7.3% and 21.3±6.8% of the first response, respectively, for 10-3 M methionine (12 glands), and 47.3±8.5% and 24.8±12.3%, respectively, for 10-3 M glutamic acid (7 glands). Effects of atropine plus hexamethonium (C6) and physostigmine on adrenal CA release: Paired glands from the same animal were perfused with either with drugs or without drugs for 8 min and then stimulated Fig. 1. Releases of epinephrine and norepinephrine induced by methionine. Methionine (5 x 10-3 M) was perfused through the adrenal glands conti nuously for 10 min. Samples of perfusates were collected every 15 sec and analyzed for CA (epinephrine and norepinephrine). Points are means±s.e.m. of values in 10 experiments. E-epinephrine, NE=norepinephrine. Fig. 2. Dose-response curves of CA release by amino acids. Amino acids were perfused through the adrenal glands continuously for 6 min. Samples were collected every 30 sec for the control period and during application of amino acids. Points are means±s.e.m. of values in 4 to 9 experiments for maximum increase in CA output. Values are those of the first response to the amino acids. Met= methionine, Glu=glutamic acid, Gln=glutamine, Ser=serine, Cys=cysteine, Arg=arginine, Thr= threonine.

4 with amino acids. Preperfusion of atropine (5X10-5 M) Plus C6 (10-4 M) slightly reduced the effects of methionine (5 X 10-3 M) and glutamic acid (5 X 10-3 M) on CA release. Preinfusion of atropine plus C6 did not inhibit the response to cysteine sig nificantly, but completely inhibited the response to ACh (Fig. 3). Atropine, C6, and atropine plus C6 caused a slight initial increase in the spontaneous release of CA. Previous perfusion of physostigmine (10-5 M) potentiated the secretory response to ACh (2x10-5 M) significantly, but not that to methionine (5 X 10-3 M), glutamic acid (5X10-3 M), cysteine (5X10-3 M), or serine (5X10-3 M) (Fig. 4). Physostigmine also caused a slight initial increase in spontaneous release of CA. Effects of amino acids on adrenal CA output in Ca 21 free or high Ca2+ Ringer solution: Paired glands from the same animals were perfused with 3.6, 1.8, or 0 mm Ca2+ Fig. 4.%Effect; of pretreatment with physostigmine on CA release by amino acids. Infusion of physostigmine (10-5 M) was started 5 min before treatment with amino acids and was present until the end of the experiments. Ab breviations are as in Fig. 3. Values are the mean increases±s.e.m. *Significantly different from the control at P<0.01. Fig. 3. Effect of pretreatment with atropine plus C6 on CA release by amino acids or ACh. Infusion of atropine (5x10-5 M) plus C6 (10-4 M) was started 8 min before application of amino acids and was present until the end of the experi ments. All amino acids were infused for 6 min. Samples of perfusates were collected for 6 min before and during treatment with amino acids and assayed for CA. Values are the mean increases +S.E.M. Numbers in parentheses are the numbers of experiments. *Significantly different from the corresponding control at P<0.05. **Significantly different from the control at P< Fig. 5. Effects of Ca2--free or high-ca2 Ringer solution on CA release by amino acids. Adrenals were perfused for 15 min with 0 mm or 3.6 mm Ca2 `-modified Krebs-Ringer phosphate solution. Values are mean increases±s.e.m. of values in 4 to 9 experiments. Abbreviations are as in Fig. 3. *Significantly different from the control (1.8 mm Ca2+ modified Krebs-Ringer phosphate solution), P<0.05. **Significantly different from the control at P<0.001.

5 Ringer solution, respectively, for 15 min and then with amino acids. The perfusion fluid of high Ca2l concentration (3.6 mm) sig nificantly increased the effects of methionine (5 x 10-3 M), glutamic acid (5 x 10-3 M), glutamine (5 X 10-3 M), and cysteine (5x10-3 M), but not serine (5 x 10-3 M), compared to the effects with 1.8 m M Ca2+. However, Ca2+ free perfusion solution depressed or abolished the effects of these amino acids (Fig. 5). Effects of amino acids on isolated chrom affin granules: When isolated chromaffin granules in 0.32 M sucrose were incubated at 30 C with methionine (10-3 M), glutamic acid (10-3 M), glutamine (5x10-3 M), or cysteine (5X10-3 M), the amounts of CA released into medium did not differ sig nificantly from that of the controls (data not shown). DISCUSSION The present experiments showed that the addition of rather large concentrations of various amino acids into the perfusing medium increased the rate of release of CA from isolated dog adrenal glands. Perfusion with atropine plus C6 did not conspicuously affect amino acids-induced CA release, although it completely inhibited the response to ACh. This suggests that amino acids do not act on nicotinic or muscarinic receptors. Further, it is unlikely that amino acids release ACh from splanchnic nerve terminals in the adrenal medulla, since perfusion with physostigmine did not potentiate the effects of amino acids. It seems somewhat difficult to assume the existence of the specific receptors for a variety of amino acids, some known to be neurotransmitters while others (e.q. serine or threonine) have not been shown as trans mitters. The amino acid concentrations needed for CA release are slightly high in view of the fact that the plasma free amino acid concentrations are generally not higher than 10-a M, while infusion of various amino acids in known to stimulate insulin release in vivo and in vitro in animals and man. Knopf et al. (3) reported that infusion of a mixture of essential amino acids stimulated secretion of growth hormone in man. Gatgounis and Hester (6) showed that various amino acids, such as leucine, serine, cysteine, methionine, and threonine produced positive inotropic and pressor responses when admin istered intravenously or intraaortically in vagotomized non-adrenalectomized dogs. When these findings are taken into con sideration, rather large concentrations of various amino acids may stimulate CA release from the adrenals by a non-specific progress which seems to involve direct depolarization of the chromaffin cells. Anyhow, the effects of amino acids other than ;r-aminobutyric acid (GABA) on CA release from isolated organs have not been reported by others. Recently, we reported that methionine, cysteine, and serine induced the release of CA from isolated perfused dog hearts (13). Sangiah et al. (14) reported that GABA caused release of CA from isolated perfused bovine adrenal glands and that the secretory response was dependent on Cat{. The effects of amino acids on the secretion of CA also required the presence of Ca2+ since in the absence of Ca2+, amino acids had little or no effect on spontaneous CA release. In addition, the secretory response of the adrenal medulla to amino acids varied directly with the Ca2+ concentration of the perfusion medium (Fig. 4). These observations suggest that amino acids act by enhancing the entry of Ca 21 into the cells rather than by liberating CA from storage sites in the chromaffin granules. In support of the idea that amino acids do not displace CA from storage sites, we observed that amino acids did not increase the release of amines from isolated chromaffin

6 granules. Curtis et al. (4) reported that glutamate and related acidic amino acids electro phoretically administered depolarized the neurones in the central nervous system. The ionic basis of this depolarization and con ductance change has been claimed to be an increased permeability to Na+ into brain slices (15-18). Ferrendelli et al. (19, 20) reported that glutamate and glycine increased the concentrations of cyclic AMP and cyclic GMP in brain slices. Rasmussen (21) and Berridge (22) have proposed a general hypothesis of cell secretion in which the stimulus promotes Ca 21 uptake and simul taneously increases the production of cyclic nucleotides which in turn may regulate intracellular Ca 21 inactivation and mobili zation, and the stimulus may also activate a kinase system which is involved in the transport of the vesicle to the releasing sites. From these circumstances, it seems likely that amino acids depolarize chromaffin cells of the adrenal medulla through an increased permeability to Na+ followed by an enhance ment of Ca2+ entry and this releases CA from its storage sites. Acknowledgements: This study was sup ported in part by a Grant-in-Aid for Scientific Research, No from the Ministry of Education, Science and Culture, Japan. We thank Mrs. M. Mimura for skillful technical assistance. REFERENCES 1) Mellinkoff, S.M., Jenden, D.J. and Frankland, M.: Postprandial serum amino acid levels in viral hepatitis. Archs intern. Med. 94, (1954) 2) Mellinkoff, S.M., Boyle, D., Frankland, M. and Greipel, M.: The effect of amino acid adminis tration upon the blood sugar concentration. Stanford med. Bull. 13, (1955) 3) Knopf, R.F., Conn, J.W., Floyed, J.C., Jr., Fajans, S.S., Rull, J.A., Guntsche, E.M. and Thiffault, C.A.: The normal endocrine response to ingestion of protein and infusion of amino acids: sequential secretion of insulin and growth hormone. Trans. Ass. Am. Physns 79, (1966) 4) Curtis, D.L., Phillis, J.W. and Watkins, J.C.: The chemical excitation of spinal neurones by certain acidic amino acids. J. Physiol. 150, (1960) 5) Brimblecombe, R.W. and Sutton, J.V.: The ganglion-stimulating effects of some amino acid esters. Brit. J. Pharmacol. 34, (1968) 6) Gatgounis, J. and Hester, W.: Inotropic activity of a series of amino acids. Proc. Soc. exp. Biol. Med. 116, (1964) 7) Robinson, R.L.: Stimulation of the release of catecholamines from isolated adrenal glands by tyramine. J. Pharmacol. exp. Ther. 151, (1966) 8) Tsujimoto, A. and Nishikawa, T.: Comparison of the effects of nicotine on catecholamine release from isolated adrenal glands of dogs and monkeys. Europ. J. Pharmacol. 29, (1974) 9) Tsujimoto, A. and Nishikawa, T.: Further evidence for nicotinic and muscarinic receptors and their interaction in dog adrenal medulla. Europ. J. Pharmacol. 34, (1975) 10) Euler, U.S.V. and Lishajko, F.: Improved technique for the fluorometric estimation of catecholamines. Acta physiol. scand. 51, (1961) 11) Borowitz, J.L.: Calcium binding by subcellular fractions of bovine adrenal medulla. J. cell Physiol. 69, (1967) 12) Borowitz, J.L.: Effect of acetylcholine on the subcellular distribution of 45Ca in bovine adrenal medulla. Biochem. Pharmacol. 18, (1969) 13) Nishikawa, T. and Tsujimoto, A.: The positive inotropic and chronotropic actions of some amino acids and their effects in mobilizing noradrenaline. Europ. J. Pharmacol. 37, (1976) 14) Sangiah, S., Borowitz, J.L. and Yim, G.K.W.: Actions of GABA, picrotoxin and bicuculline on adrenal medulla. Europ. J. Pharmacol. 27, (1974) 15) Gibson, I.M. and Mcllwain, H.: Continuous recording of changes in membrane potential in mammalian cerebral tissues in vitro; recovery, after depolarization by added substances. J. Physiol. 176, (1965) 16) Bradfor, H.F. and Mcllwain, Y.: Ionic basis for the depolarization of cerebral tissues by excitatory amino acids. J. Neurochem. 13, (1966)

7 17) Harvey, J.R. and Mcllwain, H.: Excitatory acidic amino acids and the cation content and sodium ion flux of isolated tissues from the brain. Biochem. J. 108, (1968) 18) Harvey, J.R. and Mcllwain, H.: Electrical phenomena and isolated tissues from the brain. In Handbook of Neurochemistry, Edited by Lajtha. A., Vol. 2, p , Plenum Press, New York (1969) 19) Ferrendelli, J.A., Chang, M.M. and Kinscherf, D.A.: Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids. J. Neurochem. 22, (1974) 20) Ferrendelli, J.A., Kinscherf, D.A. and Chang, M.M.: Comparison of the effect of biogenic amines on cyclic GMP and cyclic AMP levels in mouse cerebellum in vitro. Brain Res. 84, (1975) 21) Rasmussen, H.: Cell communication, calcium ion, and cyclic adenosine monophosphate. Science 170, (1970) 22) Berridge, M.J.: The interaction of cyclic nucleotides and calcium in the control of cel lular activity. In Advances in Cyclic Nucleotide Research, Edited by Drumond, G.I., Greengard, P. and Robinson, G.A., Vol. 5, p , Raven Press, New York (1975)