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Somatostatin receptors coupled to the inhibition of adenylyl cyclase in the rat frontoparietal cortex are modulated by α2 adrenoceptors
MOLECULAR BRAIN RESEARCH ELSEVIER
Molecular Brain Research 25 (1994) 143-146
Short Communication
Somatostatin receptors coupled to the inhibition of adenylyl cyclase in the rat frontoparietal cortex are modulated by adrenoceptors Susana Ldpez-Safiudo, Eduardo Arilla * Unidad de Neuroendocrinolog[a Molecular, Departamento de Bioqu[mica y Biolog[a Molecular, Facultad de Medicina, Unit'ersidad de Alcald, E-28871, Alcald de Henares, Madrid, Spain Accepted 8 April 1994
Abstract
The administration of an c~2-adrenoceptor agonist, clonidine, increased the number of somatostatin (SS) receptors and the affinity constant in frontoparietal cortex membranes. In addition, in the clonidine group, the capacity of SS to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity in the frontoparietal cortex was significantly higher than in the control group. Pretreatment with the C~z-adrenoceptor antagonist yohimbine prevented the clonidine-induced changes in SS binding and SS-inhibited AC activity. Yohimbine alone had an opposite effect from clonidine. These experiments provide further evidence that the a2-adrenergic system modulates the rat frontoparietal cortex somatostatinergic system. Key words: Clonidine; Yohimbine; Somatostatin receptor; Adenylyl cyclase; G protein
Several studies have shown anatomical and functional interconnections between ae-adrenergic and somatostatinergic systems. In the cerebral cortex, noradrenergic fibers originating in the locus coeruleus are quite close to somatostatin (SS)-containing terminals [9,11]. It has been reported that noradrenaline (NA) can stimulate SS release and vice-versa in rat cortical slices [2,20]. The somatostatinergic and noradrenergic systems have been implicated in locomotor activity [5,17]. The frontoparietal cortex contain high concentrations of SS as well as of their receptors [6,9]. Some of these receptors are coupled to the adenylyl cyclase (AC) enzyme system via the guanine nucleotide inhibitory protein G i [18]. Clonidine, an a2-adrenergic agonist, and yohimbine, an a2-adrenergic blocking agent, were used to determine whether the o~2-adrenergic system exerts some effect on the specific binding of SS to its receptors and on the ability of SS to inhibit AC activity in the rat frontoparietal cortex. The study also evaluates guanine nucleotide regulatory proteins (G i and G o) through experiments on pertussis toxin (PTX)-catalyzed ADP-
* Corresponding author. Fax: (34) 91-885-45-44. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 01 6 9 - 3 2 8 X ( 9 4 ) 0 0 0 7 8 - S
ribosylation and SS-like immunoreactivity (SSLI) content in the frontoparietal cortex. Synthetic Tyr~I-SS and SS tetradecapeptide were purchased from Universal Biologicals Ltd (Cambridge, UK); carrier free Na125I (IMS 30, 100 m C i / m l ) and rabbit antibody were purchased from the Radiochemical Centre (Amersham, UK). All other chemicals were from Sigma (Madrid, Spain). The animals used in this study were Sprague-Dawley rats (n = 60) weighing between 200 and 250 g. Clonidine (1 m g / k g ) and yohimbine (5 mg/kg) were dissolved in distilled water or saline respectively and were administered intraperitoneally (i.p.) at 16 h (first injection) and 10 h (second injection) prior to assay as previously described [3]. In another experimental group, yohimbine (5 mg/kg, i.p.) was administered 1 h before clonidine (1 mg/kg, i.p.). Control injections were equivalent volumes of distilled water or saline. Rats were sacrificed by decapitation 10 h after the last injection. The brain was rapidly removed and the frontoparietal cortex was dissected over ice. TyrU-SS was radioiodinated by Chloramine-T iodination according to the method of Greenwood [8]. The specific activity of the radioligand was 600 Ci/mmol. SS was extracted from the frontoparietal cortex and measured by a radioimmunoassay method [14], with a
144
S. L6pez-Sahudo, E. Arilla / Molecular Brain Research 25 (1994) 143-146
sensitivity limit of 10 pg/ml. Dilution curve for the frontoparietal cortex was parallel to the standard curve. The intra- and inter-assay variation coefficients were 6.8% and 8.1%, respectively. Frontoparietal cortex membranes were prepared as described by Reubi et al. [16]. Proteins were assayed by the method of Lowry et al. [12], with BSA as a standard. Specific SS binding was measured according to the modified method of Czernik and Petrack [6]. Membranes (0.15 mg protein/ml) were incubated in 0.25 ml of a medium containing 50 mM Tris-HC1 buffer (pH 7.5), 5 mM MgCI2, 0.2% (w/v) BSA and 0.1 m g / m l bacitracin with 250 pM mSI-TyrH-SS either in the absence or in the presence of 0.01-10 nM unlabelled SS. After 60 rain incubation at 30°C, membrane-bound peptide was isolated by centrifugation at 11,000 × g for 2 min, and radioactivity was determined. Non-specific binding was obtained from the amount of radioactivity bound in the presence of 10 - 7 M SS and represented about 20% of the binding observed in the absence of unlabelled peptide. This non-specific component was subtracted from the total bound radioactivity in order to obtain the corresponding specific binding. The inactivation of 125I-Tyr H-SS in the incubation medium after exposure to membranes was studied by observing the ability of the peptide to rebind to fresh membranes. AC activity was measured as previously reported [10] with minor modifications. Briefly, rat frontoparietal cortex membranes (0.06 m g / m l ) were incubated with 1.5 mM ATP, 5 mM MgSO4, 10 /xM GTP, an ATP-regenerating system (7.5 m g / m l creatine phosphate and 1 m g / m l creatine kinase), 1 mM IBMX, 0.1 mM PMSF, 1 m g / m l bacitracin, 1 mM EDTA, and test substances ( 1 0 - 4 M SS or 10 -5 M forskolin (FK)) in 0.1 ml of 0.025 M triethanolamine/HCl buffer (pH 7.4). After 15 min incubation at 30°C, the reaction was stopped by heating the mixture for 3 rain. After refrigeration, 0.2 ml of an alumina slurry (0.75 g / m l in triethanolamine/HC1 buffer, pH 7.4) was added and the suspension was centrifuged. The supernatant was
Table 1 Effect of clonidine, yohimbine and yohimbine plus clonidine on somatostatin-like-immunoreactive (SSLI) concentration and equilibrium parameters for SS binding to frontoparietal cortex membranes Groups
Control Clonidine Control Yohimbine Control Yohimbine + clonidine
SS receptors
SSLI
Bm,,x
Kd
432 _+ 19 716+39 *** 448 _+ 18 298_+ 12 *** 439 _+34
0.34 + 0.02 0.78_+0.04 *** 0.28 + 0.03 0.12_+0.01 * 0.26 _+0.02
446 _+25
0.31 + 0.04
10.01 _+0.64 9.53_+0.76 9.94 + 0.82 9.21 _+0.36 8.37 _+0.94 9.01 _+0.76
Binding parameters were calculated from Scatchard plots by linear regression. Units for SSLI are ng of SS per mg protein, units for K a are nM and units for Bnl,~X are femtomoles of SS bound per mg of protein. The results are represented as the means+S.E.M, of five separate experiments. Statistical comparison versus control: * P < 0.05, *** P < 0.001.
taken to assay the cyclic AMP (cAMP) by using the method of Gilman [7]. ADP-ribosylation of rat frontoparietal cortex membranes with PTX was carried out after toxin (16 txg/ml) activation, by incubation with membranes (0.8 mg of protein/ml), 1 p~M [32p]NAD+ (30 Ci/mmol), 1 mM ATP, 100/xM GTP, 2.5 mM MgC12, 1 mM EDTA, 10 mM thymidine and an ATP-regenerating system in 100 mM T r i s / H C l buffer, pH 8.0, for 30 min at 30°C [4]. Then, SDS-PAGE was performed and followed by autoradiography. For Scatchard analysis, the stoichiometric binding data were treated with the L I G A N D computer program [13]. For statistical evaluation, Student's t-test was used. Clonidine or yohimbine had no effect on the SSLI content in the frontoparietal cortex as compared with the control group (Table 1). SS inactivation by plasma membranes was similar (8-10%) after 60 min incubation at 30°C in all the experimental groups. The specific binding of 125I-TyrlLSS to rat frontoparietal cortex membranes increased in clonidine-
Table 2 Effect of somatostatin (SS) (10 4 M) and forskolin (FK) (10 -5 M) on brain adenylyl cyclase (AC) activity (pmol c A M P / m i n / m g protein) in control (n = 15), clonidine- (n = 5), yohimbine- (n = 5) and yohimbine plus clonidine-treated rats (n = 5) Clonidine
Basal activity Basal activity + 1 0 4 M S S % SS inhibition of basal activity +10 -5 M F K Fold FK stimulation over basal 10-SMFK+10-4MSS %SSinhibitionofFKstimulation
Yohimbine
Yohimbine plus clonidine
Control
Treated
Control
Treated
Control
Treated
337 265 21 776 2.3 614 21
335 + 12 237 _+11 29 _+ 1 " * * 803 +21 2.4_+ 0.1 516 _+22 36 -+ 1"**
299 232 22 767 2.6 597 22
276 240 13 724 2.6 648 10
298 231 22 711 2.4 557 20
272 199 27 693 2.5 533 23
_+ 15 _+12 + 1 +20 +_ 0.1 +_17 _+ 1
_+ 18 _+13 _+ 2 _+28 + 0.1 +12 + 2
+ 17 +22 + 2" +47 + 0.1 +52 -+ 1"**
+ 9 _+15 + 3 + 16 + 0.1 +15 -+ 2
Experiments were performed as described in the text. Values represent the mean + S.E.M. of the determinations performed. Statistical comparison versus control: * P < 0.05, *** P < 0.001.
+ 16 _+ 3 + 3 +49 + 0.l -+40 + 5
S. Ldpez-Sahudo, E. Arilla / Molecular Brain Research 25 (1994) 143-146
treated rats as compared to control conditions. This increase is due to an increase in the maximal number of SS receptors and in the affinity constant, as revealed by Scatchard plots of the binding data (Table 1). The addition of either 10 -5 M clonidine or yohimbine to the incubation medium changed neither the number nor the affinity of the SS receptors in the m e m b r a n e s of normal rats (data not shown). Pretreatment with yohimbine completely blocked the clonidine-induced changes in the number of SS receptors following clonidine injection (Table 1). The administration of yohimbine alone produced a significant decrease in 1251Tyrtl-SS binding in the frontoparietal cortex m e m branes (Table 1). As shown in Table 2, no significant differences were seen for either the basal or the FK-stimulated AC enzyme activities in the control, clonidine a n d / o r yohimbine groups in frontoparietal cortex membranes. In all experimental groups, SS inhibited the basal and the FK-stimulated A C activities, which is in agreement with other authors [18]. In the clonidine group, however, the capacity of SS to inhibit the basal and FKstimulated AC activity in the frontoparietal cortex was significantly higher than in the control group, whereas the yohimbine had the opposite effect (Table 2). Clonidine and yohimbine had no effect on the PTX-catalyzed ADP-ribosylation of G i a n d / o r G O alpha subunits (data not shown). The SSLI content as well as the binding parameters of SS receptors in the control rats were similar to those previously reported by others [6]. The Scatchard analysis of the stoichiometric data suggests the existence of only one type of SS receptor. This finding agrees with some studies [16] where t25I-Tyrtt-ss as a tracer was also used, but differs from other previously reported data where different SS analogs were used and two SS receptor subtypes distinguished [15]. Recently, five different SS receptor subtypes have been cloned [1]. The changes in 125I-Tyr1~-SS binding were not due to a direct effect of clonidine or yohimbine on SS receptors, because no change was detected in tracer binding following incubation of fresh frontoparietal cortex m e m b r a n e s with 10 -5 M clonidine or yohimbine. Furthermore, az-adrenoceptors seem to mediate the action of clonidine, since the changes induced by clonidine in the SS binding were prevented by pretreatment with the a2-adrenergic blocking agent yohimbine. Adrenergic antagonist or agonist injection of the a 2 type profoundly affects the firing rate of noradrenergic neurons in the locus coeruleus [19]. It is therefore likely that following noradrenergic antagonist or agonist treatment the degree of N A release from noradrenergic terminal in the cortex varies [19]. It is possible that since N A stimulates SS release in the frontoparietal cortex [2], any change in the N A release provoked by az-adrenergic agents leads to a modifica-
145
tion in the SS release in the frontoparietal cortex. The fact that SSLI content does not change bears no relevance to the degree of SS release since the percent of peptide that is released when compared to the actual content is minimal ( < 1%). It is therefore very likely that the rate of SS release varies following noradrenergic agonist or antagonist treatments without any reflection on the SS content. It is tempting to speculate, therefore, that a decrease in the SS release in the frontoparietal cortex leads to sensitization or up-regulation of SS receptors in this brain area and vice-versa. A relatively high concentration of SS was required to produce inhibition of AC. The same concentration has been used by other authors in their studies on SS inhibition of rat brain AC [18]. The SS concentration eliciting the maximal inhibition of AC activity was about 3 log units the necessary to displace ~25I-Tyrtl-SS binding. A possible explanation for this discrepancy may lie in the observation that the PTX-sensitive G proteins can modulate the affinity of SS receptors a n d / o r the receptor coupling to the effector system (AC among others). These results suggest that the modification of the inhibitory activity of SS on AC activity in this study is most likely related to the observed changes of SS receptors. At present, there is no direct evidence that the regulation of the frontoparietal cortex somatostatinergic system by the a2-adrenergic system is physiologically significant. However, this mechanism may provide a means by which the enviroment could modulate the SS action mechanism, and therefore, the sensitivity to SS in a subset of SS-sensitive neurons. The authors thank Carol F. Warren, from the Alcal~i de Henares University Institute of Education Sciences for her editorial help. This study was supported by a Grant (PB 92-0049) from the Direcci6n General de Investigaci6n Cientlfica y T6cnica of Spain. [1] Bell, G.I. and Reisine, T., Molecular biology of somatostatin receptors, Trends Neurol. Sci., 16 (1993) 34-38. [2] Bennett, G.W., Edwardson, J.A., Marcano de Cotte, D., Berelowitz, M., Pimstone, B.L. and Kronheim, S., Release of somatostatin from rat brain synaptosomes, J. Neurochem., 32 (1979) 1127-1130. [3] Blaustein, J.D. and Letcher, B., Noradrenergic regulation of cytosol strogen receptors in female rat hypothalamus: possible role of a2-noradrenergic receptors, Brain Res., 404 (1987) 51-57. [4] Bokoch, G.M., Katada, T., Northup, J.K., Hewlett, E.L. and Gilman, A.G., Identification of the predominant substrate for ADP-ribosylation by islet activating protein, J. Biol. Chem., 258 (1983) 2072-2075. [5] Clineschmidt, B.V., Flataker, L.M., Faison, E. and Holmes, R., An in vivo model for investigating a x- and a2-receptors in the CNS: studies with mianserin, Arch. Int. Pharmacodyn., 242 (1979) 59-76. [6] Czernik, A.J. and Petrack, V., Somatostatin receptor binding in rat cerebral cortex. Characterization using a nonreducible somatostatin analog, J. Biol. Chem., 285 (1983) 5525-5530.
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S. L6pez-Safiudo, E. Arilla / Molecular Brain Research 25 (1994) 143-146
[7] Gilman, A.G., A protein binding assay for adenosine 3',5'-cyclic monophosphate, Proc. Natl. Acad. Sci. USA, 67 (1970) 305 312. [8] Greenwood, F.C., Hunter, W.M. and Glober, J.S., The preparation of t311-1abelled human growth hormone of high specific radioactivity, Biochem. J., 89 (1963) 114-123. [9] H6kfelt, T., Elde, T., Johansson, O., Ljundgahe, A., Schultzberg, M., Fuxe, K., Goldstein, M., Nilsson, G., Pernow, B., Terenius, L., Ganten, D., Jeffwate, S.L., Rehfeld, J. and Said, S., Distribution of peptide-containing neurons, In M.A. Lipton, A. DiMascio and K.F. Killam (Eds.), Psychopharmacology: ,4 Generation of Progress, Raven, New York, 1986, pp. 39-66. [10] Houslay, M.D., Metcalfe, J.C., Warren, G.B., Hesketh, T.R. and Smith, G.A., The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it. Biochim. Biophys. Acta, 436 (1976) 489-494. [11] Levitt, P. and Moore, R.Y., Noradrenaline neuron innervation of the neocortex in the rat, Brain Res., 139 (1978) 219-231. [12] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem,, 193 (1951) 265-275. [13] Munson, P.J. and Rodbard, D., LIGAND: A versatile computerized approach for characterization of ligand binding systems. Anal. Biochem., 107 (1980) 220-239.
[14] Patel, J.C. and Reichlin, S., Somatostatin in hypothalamus, extrahypothalamic brain and peripheral tissues of the rat, Endocrinology, 102 (1978) 523-531. [15] Reubi, J.C.. Evidence tk)r two somatostatin-14 receptor types in rat brain cortex, Neurosci. Lett., 49 (1984) 259-263. [16] Reubi, J.C., Perrin, M.H., Rivier, J.E. and Vale, V., High affinity binding sites for the somatostatin-28 analogue in rat brain, Life Sci., 28 (1981) 2191-2198, [17] Rezek, M., Havlicek, V., Hughes, K.R. and Friesen, H., Behavioural and motor excitation and inhibition induced by the administration of small and large doses of somatostatin into the amygdala, Neuropharmaeology, 16 (1977) 157-162. [18] Schettini, G., Florio, T., Meucci, O., Landolfi, E., Grimaldi, M., Ventra, C. and Marino, A., Somatostatin inhibition of adenylate cyclase activity in different brain areas, Brain Res., 492 (1989) 65-71. [t 9] Starke, K., Gothert, M. and Kilbinger, H., Modulation of neurotransmitter release by presynaptic autoreceptors, Physiol. Rec., 69 (1989) 864-989. [20] Tsujimoto, A. and Tanaka, S., Stimulatory effect of somatostatin on norepinephrine release from rat brain cortex slices, Lifo Sci., 28 (1981) 9(13-910.
Molecular Brain Research 25 (1994) 143-146
Short Communication
Somatostatin receptors coupled to the inhibition of adenylyl cyclase in the rat frontoparietal cortex are modulated by adrenoceptors Susana Ldpez-Safiudo, Eduardo Arilla * Unidad de Neuroendocrinolog[a Molecular, Departamento de Bioqu[mica y Biolog[a Molecular, Facultad de Medicina, Unit'ersidad de Alcald, E-28871, Alcald de Henares, Madrid, Spain Accepted 8 April 1994
Abstract
The administration of an c~2-adrenoceptor agonist, clonidine, increased the number of somatostatin (SS) receptors and the affinity constant in frontoparietal cortex membranes. In addition, in the clonidine group, the capacity of SS to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity in the frontoparietal cortex was significantly higher than in the control group. Pretreatment with the C~z-adrenoceptor antagonist yohimbine prevented the clonidine-induced changes in SS binding and SS-inhibited AC activity. Yohimbine alone had an opposite effect from clonidine. These experiments provide further evidence that the a2-adrenergic system modulates the rat frontoparietal cortex somatostatinergic system. Key words: Clonidine; Yohimbine; Somatostatin receptor; Adenylyl cyclase; G protein
Several studies have shown anatomical and functional interconnections between ae-adrenergic and somatostatinergic systems. In the cerebral cortex, noradrenergic fibers originating in the locus coeruleus are quite close to somatostatin (SS)-containing terminals [9,11]. It has been reported that noradrenaline (NA) can stimulate SS release and vice-versa in rat cortical slices [2,20]. The somatostatinergic and noradrenergic systems have been implicated in locomotor activity [5,17]. The frontoparietal cortex contain high concentrations of SS as well as of their receptors [6,9]. Some of these receptors are coupled to the adenylyl cyclase (AC) enzyme system via the guanine nucleotide inhibitory protein G i [18]. Clonidine, an a2-adrenergic agonist, and yohimbine, an a2-adrenergic blocking agent, were used to determine whether the o~2-adrenergic system exerts some effect on the specific binding of SS to its receptors and on the ability of SS to inhibit AC activity in the rat frontoparietal cortex. The study also evaluates guanine nucleotide regulatory proteins (G i and G o) through experiments on pertussis toxin (PTX)-catalyzed ADP-
* Corresponding author. Fax: (34) 91-885-45-44. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 01 6 9 - 3 2 8 X ( 9 4 ) 0 0 0 7 8 - S
ribosylation and SS-like immunoreactivity (SSLI) content in the frontoparietal cortex. Synthetic Tyr~I-SS and SS tetradecapeptide were purchased from Universal Biologicals Ltd (Cambridge, UK); carrier free Na125I (IMS 30, 100 m C i / m l ) and rabbit antibody were purchased from the Radiochemical Centre (Amersham, UK). All other chemicals were from Sigma (Madrid, Spain). The animals used in this study were Sprague-Dawley rats (n = 60) weighing between 200 and 250 g. Clonidine (1 m g / k g ) and yohimbine (5 mg/kg) were dissolved in distilled water or saline respectively and were administered intraperitoneally (i.p.) at 16 h (first injection) and 10 h (second injection) prior to assay as previously described [3]. In another experimental group, yohimbine (5 mg/kg, i.p.) was administered 1 h before clonidine (1 mg/kg, i.p.). Control injections were equivalent volumes of distilled water or saline. Rats were sacrificed by decapitation 10 h after the last injection. The brain was rapidly removed and the frontoparietal cortex was dissected over ice. TyrU-SS was radioiodinated by Chloramine-T iodination according to the method of Greenwood [8]. The specific activity of the radioligand was 600 Ci/mmol. SS was extracted from the frontoparietal cortex and measured by a radioimmunoassay method [14], with a
144
S. L6pez-Sahudo, E. Arilla / Molecular Brain Research 25 (1994) 143-146
sensitivity limit of 10 pg/ml. Dilution curve for the frontoparietal cortex was parallel to the standard curve. The intra- and inter-assay variation coefficients were 6.8% and 8.1%, respectively. Frontoparietal cortex membranes were prepared as described by Reubi et al. [16]. Proteins were assayed by the method of Lowry et al. [12], with BSA as a standard. Specific SS binding was measured according to the modified method of Czernik and Petrack [6]. Membranes (0.15 mg protein/ml) were incubated in 0.25 ml of a medium containing 50 mM Tris-HC1 buffer (pH 7.5), 5 mM MgCI2, 0.2% (w/v) BSA and 0.1 m g / m l bacitracin with 250 pM mSI-TyrH-SS either in the absence or in the presence of 0.01-10 nM unlabelled SS. After 60 rain incubation at 30°C, membrane-bound peptide was isolated by centrifugation at 11,000 × g for 2 min, and radioactivity was determined. Non-specific binding was obtained from the amount of radioactivity bound in the presence of 10 - 7 M SS and represented about 20% of the binding observed in the absence of unlabelled peptide. This non-specific component was subtracted from the total bound radioactivity in order to obtain the corresponding specific binding. The inactivation of 125I-Tyr H-SS in the incubation medium after exposure to membranes was studied by observing the ability of the peptide to rebind to fresh membranes. AC activity was measured as previously reported [10] with minor modifications. Briefly, rat frontoparietal cortex membranes (0.06 m g / m l ) were incubated with 1.5 mM ATP, 5 mM MgSO4, 10 /xM GTP, an ATP-regenerating system (7.5 m g / m l creatine phosphate and 1 m g / m l creatine kinase), 1 mM IBMX, 0.1 mM PMSF, 1 m g / m l bacitracin, 1 mM EDTA, and test substances ( 1 0 - 4 M SS or 10 -5 M forskolin (FK)) in 0.1 ml of 0.025 M triethanolamine/HCl buffer (pH 7.4). After 15 min incubation at 30°C, the reaction was stopped by heating the mixture for 3 rain. After refrigeration, 0.2 ml of an alumina slurry (0.75 g / m l in triethanolamine/HC1 buffer, pH 7.4) was added and the suspension was centrifuged. The supernatant was
Table 1 Effect of clonidine, yohimbine and yohimbine plus clonidine on somatostatin-like-immunoreactive (SSLI) concentration and equilibrium parameters for SS binding to frontoparietal cortex membranes Groups
Control Clonidine Control Yohimbine Control Yohimbine + clonidine
SS receptors
SSLI
Bm,,x
Kd
432 _+ 19 716+39 *** 448 _+ 18 298_+ 12 *** 439 _+34
0.34 + 0.02 0.78_+0.04 *** 0.28 + 0.03 0.12_+0.01 * 0.26 _+0.02
446 _+25
0.31 + 0.04
10.01 _+0.64 9.53_+0.76 9.94 + 0.82 9.21 _+0.36 8.37 _+0.94 9.01 _+0.76
Binding parameters were calculated from Scatchard plots by linear regression. Units for SSLI are ng of SS per mg protein, units for K a are nM and units for Bnl,~X are femtomoles of SS bound per mg of protein. The results are represented as the means+S.E.M, of five separate experiments. Statistical comparison versus control: * P < 0.05, *** P < 0.001.
taken to assay the cyclic AMP (cAMP) by using the method of Gilman [7]. ADP-ribosylation of rat frontoparietal cortex membranes with PTX was carried out after toxin (16 txg/ml) activation, by incubation with membranes (0.8 mg of protein/ml), 1 p~M [32p]NAD+ (30 Ci/mmol), 1 mM ATP, 100/xM GTP, 2.5 mM MgC12, 1 mM EDTA, 10 mM thymidine and an ATP-regenerating system in 100 mM T r i s / H C l buffer, pH 8.0, for 30 min at 30°C [4]. Then, SDS-PAGE was performed and followed by autoradiography. For Scatchard analysis, the stoichiometric binding data were treated with the L I G A N D computer program [13]. For statistical evaluation, Student's t-test was used. Clonidine or yohimbine had no effect on the SSLI content in the frontoparietal cortex as compared with the control group (Table 1). SS inactivation by plasma membranes was similar (8-10%) after 60 min incubation at 30°C in all the experimental groups. The specific binding of 125I-TyrlLSS to rat frontoparietal cortex membranes increased in clonidine-
Table 2 Effect of somatostatin (SS) (10 4 M) and forskolin (FK) (10 -5 M) on brain adenylyl cyclase (AC) activity (pmol c A M P / m i n / m g protein) in control (n = 15), clonidine- (n = 5), yohimbine- (n = 5) and yohimbine plus clonidine-treated rats (n = 5) Clonidine
Basal activity Basal activity + 1 0 4 M S S % SS inhibition of basal activity +10 -5 M F K Fold FK stimulation over basal 10-SMFK+10-4MSS %SSinhibitionofFKstimulation
Yohimbine
Yohimbine plus clonidine
Control
Treated
Control
Treated
Control
Treated
337 265 21 776 2.3 614 21
335 + 12 237 _+11 29 _+ 1 " * * 803 +21 2.4_+ 0.1 516 _+22 36 -+ 1"**
299 232 22 767 2.6 597 22
276 240 13 724 2.6 648 10
298 231 22 711 2.4 557 20
272 199 27 693 2.5 533 23
_+ 15 _+12 + 1 +20 +_ 0.1 +_17 _+ 1
_+ 18 _+13 _+ 2 _+28 + 0.1 +12 + 2
+ 17 +22 + 2" +47 + 0.1 +52 -+ 1"**
+ 9 _+15 + 3 + 16 + 0.1 +15 -+ 2
Experiments were performed as described in the text. Values represent the mean + S.E.M. of the determinations performed. Statistical comparison versus control: * P < 0.05, *** P < 0.001.
+ 16 _+ 3 + 3 +49 + 0.l -+40 + 5
S. Ldpez-Sahudo, E. Arilla / Molecular Brain Research 25 (1994) 143-146
treated rats as compared to control conditions. This increase is due to an increase in the maximal number of SS receptors and in the affinity constant, as revealed by Scatchard plots of the binding data (Table 1). The addition of either 10 -5 M clonidine or yohimbine to the incubation medium changed neither the number nor the affinity of the SS receptors in the m e m b r a n e s of normal rats (data not shown). Pretreatment with yohimbine completely blocked the clonidine-induced changes in the number of SS receptors following clonidine injection (Table 1). The administration of yohimbine alone produced a significant decrease in 1251Tyrtl-SS binding in the frontoparietal cortex m e m branes (Table 1). As shown in Table 2, no significant differences were seen for either the basal or the FK-stimulated AC enzyme activities in the control, clonidine a n d / o r yohimbine groups in frontoparietal cortex membranes. In all experimental groups, SS inhibited the basal and the FK-stimulated A C activities, which is in agreement with other authors [18]. In the clonidine group, however, the capacity of SS to inhibit the basal and FKstimulated AC activity in the frontoparietal cortex was significantly higher than in the control group, whereas the yohimbine had the opposite effect (Table 2). Clonidine and yohimbine had no effect on the PTX-catalyzed ADP-ribosylation of G i a n d / o r G O alpha subunits (data not shown). The SSLI content as well as the binding parameters of SS receptors in the control rats were similar to those previously reported by others [6]. The Scatchard analysis of the stoichiometric data suggests the existence of only one type of SS receptor. This finding agrees with some studies [16] where t25I-Tyrtt-ss as a tracer was also used, but differs from other previously reported data where different SS analogs were used and two SS receptor subtypes distinguished [15]. Recently, five different SS receptor subtypes have been cloned [1]. The changes in 125I-Tyr1~-SS binding were not due to a direct effect of clonidine or yohimbine on SS receptors, because no change was detected in tracer binding following incubation of fresh frontoparietal cortex m e m b r a n e s with 10 -5 M clonidine or yohimbine. Furthermore, az-adrenoceptors seem to mediate the action of clonidine, since the changes induced by clonidine in the SS binding were prevented by pretreatment with the a2-adrenergic blocking agent yohimbine. Adrenergic antagonist or agonist injection of the a 2 type profoundly affects the firing rate of noradrenergic neurons in the locus coeruleus [19]. It is therefore likely that following noradrenergic antagonist or agonist treatment the degree of N A release from noradrenergic terminal in the cortex varies [19]. It is possible that since N A stimulates SS release in the frontoparietal cortex [2], any change in the N A release provoked by az-adrenergic agents leads to a modifica-
145
tion in the SS release in the frontoparietal cortex. The fact that SSLI content does not change bears no relevance to the degree of SS release since the percent of peptide that is released when compared to the actual content is minimal ( < 1%). It is therefore very likely that the rate of SS release varies following noradrenergic agonist or antagonist treatments without any reflection on the SS content. It is tempting to speculate, therefore, that a decrease in the SS release in the frontoparietal cortex leads to sensitization or up-regulation of SS receptors in this brain area and vice-versa. A relatively high concentration of SS was required to produce inhibition of AC. The same concentration has been used by other authors in their studies on SS inhibition of rat brain AC [18]. The SS concentration eliciting the maximal inhibition of AC activity was about 3 log units the necessary to displace ~25I-Tyrtl-SS binding. A possible explanation for this discrepancy may lie in the observation that the PTX-sensitive G proteins can modulate the affinity of SS receptors a n d / o r the receptor coupling to the effector system (AC among others). These results suggest that the modification of the inhibitory activity of SS on AC activity in this study is most likely related to the observed changes of SS receptors. At present, there is no direct evidence that the regulation of the frontoparietal cortex somatostatinergic system by the a2-adrenergic system is physiologically significant. However, this mechanism may provide a means by which the enviroment could modulate the SS action mechanism, and therefore, the sensitivity to SS in a subset of SS-sensitive neurons. The authors thank Carol F. Warren, from the Alcal~i de Henares University Institute of Education Sciences for her editorial help. This study was supported by a Grant (PB 92-0049) from the Direcci6n General de Investigaci6n Cientlfica y T6cnica of Spain. [1] Bell, G.I. and Reisine, T., Molecular biology of somatostatin receptors, Trends Neurol. Sci., 16 (1993) 34-38. [2] Bennett, G.W., Edwardson, J.A., Marcano de Cotte, D., Berelowitz, M., Pimstone, B.L. and Kronheim, S., Release of somatostatin from rat brain synaptosomes, J. Neurochem., 32 (1979) 1127-1130. [3] Blaustein, J.D. and Letcher, B., Noradrenergic regulation of cytosol strogen receptors in female rat hypothalamus: possible role of a2-noradrenergic receptors, Brain Res., 404 (1987) 51-57. [4] Bokoch, G.M., Katada, T., Northup, J.K., Hewlett, E.L. and Gilman, A.G., Identification of the predominant substrate for ADP-ribosylation by islet activating protein, J. Biol. Chem., 258 (1983) 2072-2075. [5] Clineschmidt, B.V., Flataker, L.M., Faison, E. and Holmes, R., An in vivo model for investigating a x- and a2-receptors in the CNS: studies with mianserin, Arch. Int. Pharmacodyn., 242 (1979) 59-76. [6] Czernik, A.J. and Petrack, V., Somatostatin receptor binding in rat cerebral cortex. Characterization using a nonreducible somatostatin analog, J. Biol. Chem., 285 (1983) 5525-5530.
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[7] Gilman, A.G., A protein binding assay for adenosine 3',5'-cyclic monophosphate, Proc. Natl. Acad. Sci. USA, 67 (1970) 305 312. [8] Greenwood, F.C., Hunter, W.M. and Glober, J.S., The preparation of t311-1abelled human growth hormone of high specific radioactivity, Biochem. J., 89 (1963) 114-123. [9] H6kfelt, T., Elde, T., Johansson, O., Ljundgahe, A., Schultzberg, M., Fuxe, K., Goldstein, M., Nilsson, G., Pernow, B., Terenius, L., Ganten, D., Jeffwate, S.L., Rehfeld, J. and Said, S., Distribution of peptide-containing neurons, In M.A. Lipton, A. DiMascio and K.F. Killam (Eds.), Psychopharmacology: ,4 Generation of Progress, Raven, New York, 1986, pp. 39-66. [10] Houslay, M.D., Metcalfe, J.C., Warren, G.B., Hesketh, T.R. and Smith, G.A., The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it. Biochim. Biophys. Acta, 436 (1976) 489-494. [11] Levitt, P. and Moore, R.Y., Noradrenaline neuron innervation of the neocortex in the rat, Brain Res., 139 (1978) 219-231. [12] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem,, 193 (1951) 265-275. [13] Munson, P.J. and Rodbard, D., LIGAND: A versatile computerized approach for characterization of ligand binding systems. Anal. Biochem., 107 (1980) 220-239.
[14] Patel, J.C. and Reichlin, S., Somatostatin in hypothalamus, extrahypothalamic brain and peripheral tissues of the rat, Endocrinology, 102 (1978) 523-531. [15] Reubi, J.C.. Evidence tk)r two somatostatin-14 receptor types in rat brain cortex, Neurosci. Lett., 49 (1984) 259-263. [16] Reubi, J.C., Perrin, M.H., Rivier, J.E. and Vale, V., High affinity binding sites for the somatostatin-28 analogue in rat brain, Life Sci., 28 (1981) 2191-2198, [17] Rezek, M., Havlicek, V., Hughes, K.R. and Friesen, H., Behavioural and motor excitation and inhibition induced by the administration of small and large doses of somatostatin into the amygdala, Neuropharmaeology, 16 (1977) 157-162. [18] Schettini, G., Florio, T., Meucci, O., Landolfi, E., Grimaldi, M., Ventra, C. and Marino, A., Somatostatin inhibition of adenylate cyclase activity in different brain areas, Brain Res., 492 (1989) 65-71. [t 9] Starke, K., Gothert, M. and Kilbinger, H., Modulation of neurotransmitter release by presynaptic autoreceptors, Physiol. Rec., 69 (1989) 864-989. [20] Tsujimoto, A. and Tanaka, S., Stimulatory effect of somatostatin on norepinephrine release from rat brain cortex slices, Lifo Sci., 28 (1981) 9(13-910.