Institute of Chemical Physics and *Institute of Biochemistry, University of Tartu, Jakobi 2, EE-2400 Tartu, Estonia
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1 Vol. 45, No. 4, July 1998 Pages ACTIVATION OF camp SYNTHESIS IN RAT BRAIN CORTICAL MEMBRANES BY RUBIDIUM AND CESIUM IONS Katri Rosenthal, Jaanus Lember, *Ello Karelson and Jaak Jfirv Institute of Chemical Physics and *Institute of Biochemistry, University of Tartu, Jakobi 2, EE-2400 Tartu, Estonia Received April 8, 1998 SUMMARY: Rubidium and cesium chlorides accelerated camp synthesis in rat brain cortex membranes, while other alkali metal chlorides had no influence on the rate of this process. The effect was dose-dependent and yielded above 2-fold activation of adenylate cyclase. It has been shown that Rb + and Cs + influenced directly the catalytic subunit of the enzyme and did not substit+ute Mg 2 in formation of the metal-atp complex in this reaction. The stimulatory effect of Rb was additive to the activation of adenylate cyclase by the half-maximal (0.31.tM) as well as by the saturating (10 ~tm) forskolin concentrations, pointing to the fact that these effectors activate different isoforms of the enzyme in rat brain cortex. Key words: camp synthesis activation, adenylate cyclase isoforms, rubidium chloride, cesium chloride, rat brain cortical membranes INTRODUCTION Formation of cyclic 3",5"-AMP (camp) from ATP is catalysed by adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), E.C ]. This reaction is an important component of the intracellular signal transduction pathway which uses camp as the second messenger. This signalling system involves besides adenylate cyclase also some receptor and a guanine nucleotide binding protein (G- protein). The latter components are directly involved in modulation of the adenylate cyclase activity. As general, Gs-proteins activate the catalytic core of this enzyme in most cell types [1,2,3]. Adenylate eyclases form a large multi-gene family of proteins, having 12 transmembrane helices. The intracellular regions of these helices carry the catalytic site as well as regulatory sites [1 ]. Mammalian cells have been shown to synthesise up to nine types of adenylate cyclase isoforms [1,4,5]. Many of these isoforms can be activated by a natural diterpene forskolin [6,7]. This activation occurs through forskolin binding with the intraeellular hydrophobic pocket on the catalytic core of the enzyme [1,4]. Some of the isoforms (e.g. I and VIII) could be directly stimulated by manganese and calcium ions and the presence of specific binding sites for these divalent cations was assumed on these proteins [1,4, 6,8,9] /98/1 oo o5.oo/o Copyright by Academic Press Australia. 745 All rights of reproduction in any form reserved.
2 In this paper we report about selective activation of adenylate cyclase activity in rat brain cortex by monovalent cations of cesium and rubidium. As this effect of Rb and Cs was additive to the activation of camp synthesis by forskolin, it is possible that these factors affect selectively different types of adenylate cyclase in rat brain cortex, where at least three isoforms (I, V and VIII) have been discovered [9,10]. MATERIALS AND METHODS ATP, GTP, bacitracin, theophylline, EGTA, bovine serum albumin and HEPES were supplied by Sigma, camp was obtained from Fluka, [3H]cAMP (specific radioactivity 59 Ci/mmol) from Amersham. Other reagents were of the highest grade commercially available. Membranes of rat brain cortex were fractionated and prepared for the experiments as described previously [11]. The pre-cooled cortexes were homogenised in ice-cold 8 mm HEPES-Na buffer (ratio of buffer to cortical substance 6:1). The homogenate was diluted 10 times in the same buffer, stirred 30 minutes on ice and centrifuged 6 minutes at 1500g. The supematant was removed and the pellets were re-suspended in ice-cold buffer A (20 mm HEPES-Na, 1.5 mm theophylline, 8.25 mm MgCI2, 0.75 mm EGTA, 7.5 mm KCI, 100 mm NaC1, ph 7.4) to final protein concentration mg/ml. Protein concentration in the samples was determined by the method of Lowry et al. [ 12]. Adenylate cyclase was assayed at 0.05 mg/ml of membrane protein in buffer A, containing 0.1 mg/ml bacitraein, 0.05% bovine serum albumin, 1.0 mm ATP and 0.01 mm GTP at 30~ for 15 rain. The reaction was stopped by addition of 0.05 ml 100 mm EDTA (ph 7.4) and boiling the samples during 3 min in a water bath. Precipitate was removed by centrifugation and aliquots of the supernatant were used for determination of camp contents by Brown's method, using endogenous camp binding protein from bovine adrenal cortex [13]. Basal level of specific activity of adenylate cyclase in rat cortical membranes was at an average pmol of camp/min per mg of protein. The effect of salt was measured as a difference in the amount of camp, produced by the adenylate eyclase in the absence and in the presence of different concentrations of RbC1 or CsCI in the reaction medium. In experiments where the effect of salts on the forskolin-stimulated adenylate cyclase activity was studied, the diterpene, solved in the reaction buffer, was added to the assay medium before the salt solution. Statistical analysis of the experimental data was carried out by using a PC version of a STATGRAPHICS package (versions 2.0 and 2.6). RESULTS Rubidium and cesium ions accelerated the synthesis of camp in the rat brain cortex membranes in dose-dependent manner (Fig.I) and the effect was characterised by the EC50 values M and M, respectively. In both cases the maximal activation reached up to 2 times relatively the basal rate of camp synthesis. Potassium ions (Fig. 1.), as well as other alkali metal ions (Li Na data not shown) had no similar influence on camp 746
3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL, I,]. ~ n 0 ' o.o o11 0:2 0:3 0.4 o:s 0.6 [salt], M FIGURE 1. Effect of CsCI ( V ), RbCI ( [] ) and KC! ( o ) on the basal rate of camp synthesis in rat brain cortex membranes, assayed in the presence of 0.1 mg/ml bacitracin, 0.05 % bovine serum albumin, 1.0 mm ATP and 0.01 mm GTP and protein concedntration 0.05 mg/ml. synthesis. Thus, the stimulatory effect of Rb + and Cs + is specific and probably possesses similar mechanism in both cases. In general, this acceleration mechanism can involve: i- the influence of Rb + and Cs + ions on adenylate cyelase activity via modulation of muscarinic receptor, ii- the influence of these cations on G-proteins, which mediate the regulatory signals from the receptor(s) to the enzyme, or iii- direct effect of Rb + and C + ions on the adenylate eyclase catalytic core. The following experiments were designed to differentiate between these possibilities. Firstly, it has been found that blocking of muscarinic receptor by 1 mm atropine, which completely abolished the inhibitory effect of 100 mm carbaehol on the camp synthesis, had no effect on the activation of the same process by 0.1 M CsCI. Thus, the activation phenomenon, observed in the case of CsCI, did not occur via the muscarinic receptor. Secondly, separate experiments were carried out in the presence and absence of 0.01 mm GTP. It has been found that the omission of GTP in the reaction mixture had no effect on activation of the basal adenylate cyclase activity by RbCI (Fig,2, curve A). This finding excluded the possibility of 747
4 ,,, [] Jr C ze31f B A Oi o.o o'.1 o12 =,, o.s o [RbCI], M FIGURE 2. Acceleration of camp synthesis by RbCI in the absence ( A ) and in the presence of 30 ~tm ( B ) and 10 mm ( C ) forskolin. Experiments were made in the absence of GTP (empty symbols) and in the presence of 0.01 mm GTP in the assay buffer. activation of camp synthesis via G-proteins. Consequently, the acceleration phenomenon discovered can be related to the direct influence of Rb + and Cs + ions to the adenylate eyclase catalytic activity. It is generally accepted that the physiological substrate of the adenylate cyclase-catalysed reaction is Mg-ATP complex. Therefore the standard assay medium contained 8.25 mm of MgC12 that was sufficient for saturation of the available ATP molecules. To analyse whether rubidium ions can substitute Mg 2+ ions in the reaction of camp synthesis, the experiments were made in the absence of MgCI2 in the assay mixture. It can be seen in Fig. 3 that the omission of Mg 2+ ions in the reaction mixture remarkably reduced the rate of camp synthesis in the presence as well as in the absence of RbC1. Thus, the acceleration of camp synthesis by Rb + ions was indeed related to the adenylate cyclase catalysed reaction, requiring the presence of Mg 2+ ions. On the other hand, it can also be concluded that even at high concentration Rb + ions cannot substitute Mg 2+ ions in the complex with ATP. In the following experiments the acceleration of camp synthesis by RbC1 was studied in the presence of forskolin, a well-known specific adenylate cyclase activator, acting directly at the 748
5 251 s e- (~,.=.., 2.0-[ 1.5- Oj ~ ~> ~ Iv" [MgCI2], mm FIGURE 3. Effect of MgCI2 on camp synthesis in the absenee of RbCI (empty bars) and presence of 0.5 M RbC1 (filled bars). The basal rate of camp synthesis was assayed in the presence of 8.3 mm MgClz in the reaction medium. catalytic core of the enzyme, Under the present experimental conditions the forskolin-elicited increase in camp level was dose-dependent with the EC50 value of 0.3 ~tm and the maximal acceleration was reached at forskolin concentrations above 10 ~tm. It has been found that in the presence of these forskolin concentrations RbC1 still activated the camp synthesis (Fig.2, B and C), while the overall increase in the reaction rate was additive in the presence of these two effectors. Moreover, the EC50 values for RbCI, measured in the absence and presence of forskolin, remained unchanged. Thus, the effects induced by Rb + ions and forskolin were independent, pointing to the fact that part of the cortical enzyme should be stimulated only by Rb + ions, while another part should be activated only by forskolin. It is also important that both of these effects were not depending on the presence or absence of GTP in the reaction medium (Fig. 2). DISCUSSION There are three isoforms of adenylate cyclase still found in mammalian brain cortical membranes (types I, V and VIII) [9,10]. Binding studies with [125I]forskolin have revealed that these isoforms revealed indeed different affinity for this diterpene and the most effective binding was observed in the case of the isoform I [7,16]. This means that the latter type of the enzyme 749
6 might be preferentially activated by the diterpene. At the same time Rb + and Cs + ions might be involved in activation of the isoforms V or/and VIII, located in the membranes of the same brain region. Previous studies on membranes from different brain regions have revealed the possibility of direct activation of some adenylate cyclase isoforms (e.g. I, III,V, VI and VIII) by divalent cations of calcium and manganese [8,10,14]. Although the mechanism of this stimulatory effect is not clear yet, it has been suggested that there should exist metal-binding regulatory site(s) on the catalytic core of the enzyme [1,4]. The present study has extended this list of the regulatory metal ions by adding two monovalent ions, Rb + and Cs +. The effect of these ions was selective, as other alkali metal ions (Li +, N + and K +) had no influence on camp synthesis. Most probably such selectivity can be related to some physico-chemical properties of these ions, recognised by the putative binding site. It can be assumed that size of the ions, characterised by values of the hydrated radii from 3.4 A for Li + to 2.3 A for Cs +, or their hydration energies, ranging from 519 kj/mol for Li + to 264 kj/mol for Cs + [15], should be considered above all. It is interesting to mention that the intensity of hydration of ions should influence their hydrophobic/hydrophilic properties, which in turn may govern interaction of these ions with membrane-bound proteins. Some indirect support to the idea of the presence of ion-selective sites of different binding properties on individual forms of adenylate cyclase can be obtained from data about different sensitivity of these isoforms to changes in intracellular Ca 2 concentration in transfected intact cells [9,14]. However, under the common assay conditions the activities of these isoforms cannot be determined separately. Thus, the possibility of separation of activities of adenylate cyclase isoforms, provided by selective effects of forskolin and Rb + (Cs +) ions, may be of great practical value for investigation into the role and distribution of adenylate cyclase isoforms in various tissues. REFERENCES 1. Zhang, G., Liu, Y., Ruoho, A.E. and Hurley, J.H. (1997) Nature, 386, Gilman, A.G. (1984) Cell36, Miller, R. T. (1991) Kidn. lntern. 39, Houslay, M.D. and Milligan, G. (1997) Trends in Biol. Sci., 22, Hanoune, J., Pouille, Y., Tzavara, E., Shen, T., Lipskaya, L., Miyamoto, N., Suzuki. Y. and Defer, N. (1997)Mol. and Cell Endocrinol., 128, Seamon, K.B., Padgett, W. and Daly, J.W. (1981) Proc.Nat.Acad.Sci.USA, 78,
7 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 7. Sutkowski, E.M, Tang,W.-J., Broome,C.W., Robins, J.D. and Seamon, K.B.(1994) Biochemistry 33, Cooper, D.M.F., Mons, N. and Fagan,K. (1994) Cell.Sign. 6, Taussig,R. and Gilman, A.G. (1995)J.Biol.Chem. 270, Hellevuo, K., Hoffman, P.L. and Tabakoff B. (1996) J. Neurochemistry 67, J~irv, J., Toomela, T. and Karelson, E. (1993) Biochem. Mol. Biol. lntern. 30, Lowry, O.H., Rosenbrough, N.J., Farr A.I. and Randall R.J. (1951) J.Biol.Chem. 193, Brown, B.L., Ekins, R.P. and Albano, J.D.M. (1972)Adv, Cycl.Nucl.Res. 2, Choi, E.J., Wong, S.T., Hinds, T.J and Storm D.R. (1992)J.Biol.Chem. 267, Cosson, A.C. (1988) Advanced Inorganic Chemistry, 5-th Ed., Wiley-Interscience Publisher, p Seamon, K.B., Vaillancourt, R., Edwards, M. and Daly, J.W. (1984) Proc. Nat. Acad Sci. USA, 81,
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