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Increased responsiveness of the cerebral cortical phosphatidylinositol system to noradrenaline and carbachol in senescent rats
Neuroscience Letters, 107 (1989) 195 199
195
Elsevier Scientific Publishers Ireland Ltd. NSL 06477
Increased responsiveness of the cerebral cortical phosphatidylinositol system to noradrenaline and carbachol in senescent rats Irena Nalepa 2, Annita Pintor l, Stefano Fortuna l, Jerzy Vetulani 2 and Hanna Michalek l JLaboratory of Pharmacology, lstituto Superiore di Sanita, Rome (Italy) and :Department of Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Krakow (Poland) (Received 19 April 1989; Revised version received 3l July 1989; Accepted 31 July 1989)
Key words." Rat; Aging brain; Phosphatidylinositol hydrolysis; cq-Adrenoceptor; Muscarinic Mt receptor; Receptor responsiveness The responsiveness of cerebral cortical ~radrenoceptors and cholinergic muscarinic M~ receptors was assessed in young (3 months) and aged (24 months) male Sprague Dawley rats. The measure of responsiveness was the accumulation of inositol phosphate (IP) formed in [3H]myo-inositol-preloaded cerebral cortical slices in the presence of lithium, following stimulation with various concentrations of noradrenaline (l 300 pM) and carbachol (5 1000 ,uM). In old rats the maximum response to noradrenaline was higher by 80%, and that to carbachol by 33%, indicating an increased responsiveness of the investigated receptors in senescence.
Different neurochemical systems in the brain are modified during aging [6, 7, 17] and it is believed that senescence is connected with a general decline in the function of the central nervous system. Behavioural deficits observed in senescent animals were often associated with changes in neurotransmitter systems, but at least in the cerebral cortex - no decline, but rather small increases in the levels of noradrenaline and acetylcholine were noted (see, e.g. refs. 3, 12). Therefore, the characteristics of various classes of central neurotransmitter receptors, particularly those for catecholamines and acetylcholine, were investigated in senescent animals. In general no agerelated changes in the affinities of the investigated receptors were found, but their densities in many cases declined. Thus for muscarinic cholinergic receptors Pedigo et al. [15] have reported a decline in density in the cerebral cortex of Fischer 344 (F344) rats. We have recently confirmed this finding for F344 rats but found no agerelated changes of this type in Wistar or Sprague-Dawley rats [10, 11, 16]. Several Correspondence." H. Michalek, Lab. of Pharmacology, lstituto Superiore di Sanitfi, Viale Regina Elena, 229, 00161 Roma, Italy. 0304-3940,/89,,'$ 03.50 ~(~ 1989 Elsevier Scientific Publishers Ireland Ltd.
196
studies on fl-adrenoceptors indicate a decline in their density in most brain areas, with the exception of the cortex, in which the results are conflicting [18]. No age-related changes were observed with :~2-adrenoceptors in the Wistar rat [14]. The conflicting data concerning :q-adrenoceptors may be related to strain differences: in SpragueDawley rats no age-related changes in :q-adrenoceptor density were noted [4], while in F344 rats a decline was found [13]. The changes in the density of receptors for neurotransmitters do not necessarily determine the responsiveness of the system regulated by those receptors. A better estimate of functional changes is given by measurement of formation of second messengers. Such studies in old animals were carried out on dopamine and/~'-adrenoceptors by measuring cyclic AMP formation induced by agonists. In most cases a decline in responsiveness of cyclic AMP generating system in some, but not all, brain regions was observed [17, 18]. It was speculated that the declined response might be caused by diminished receptor availability [I 8]. The purpose of the present study was to assess whether there are differences in responsiveness of cortical :q-adrenoceptors and cholinergic muscarinic M i receptors of young and senescent Sprague Dawley rats, using accumulation of inositol phosphate (IP) after challenge with agonists: noradrenaline and carbachol, as the measure of response. Young (3 months old, weighing 250-300 g) and senescent (24 months old, weighing 650-800 g) male Sprague-Dawley rats were purchased from Charles River Italia, Calco (Como). They were kept two in a cage in an animal room under standard conditions (12:12 h DL cycle, free access to standard food and tap water). The experiments were performed in balanced replications. Every day one aged and one young rat were decapitated with a guillotine, the cortex was dissected and cut into 300 × 300 l,m slices using a Mcllwain tissue chopper. One half of the slices was used for stimulation with 5 concentrations of noradrenaline (1.0 300/,tM), the other with 5 concentrations of carbachol (5.0-1000/~M). Protein content was assayed with the method of Lowry et al. [9]. The incubation of slices and the assay of IP were carried out according to Brown et al. [2] with some modifications. The incubation proceeded in three stages: 1. Preincubation, during which the slices obtained from a single rat were incubated at 37~C in 50 ml of O2:CO 2 (95:5) gassed, glucose-containing Krebs-Ringer medium (in mM: NaCI 118, KCI 5, CaC12 1.3, MgSO4 1.2, KH2PO4 i.2, NaHCO3 25, glucose 11.7: pH 7.4) for 1 h, with three changes of the buffer. 2. Labelling incubation. Portions of 50/,d of preincubated, density-packed slices (2.7 mg of protein on average) were pipetted into fiat-bottom 3 ml plastic vials containing 200/11 of the buffer, 30/tl of LiC1 solution (final conc. 5 mM) and 20/tl of the purified [3H]myo-inositol solution. The vials were gassed (95% 02:5% CO2), capped and shaken at 37°C for 30 min. During this stage [3H]myo-inositolwas being incorporated into membrane phospholipids, forming phosphatidylinositols (PI, PIP, PIP2). 3. Hydrolytic incubation, at the beginning of which 10/A of agonist solutions: noradrenaline (DL-arterenol, Sigma: 5 concentrations, from 1 to 300/tM) or carbachol
197
(Sigma; 5 concentrations, from 5 to 1000/~M) were added to the medium. The incubation was continued for 45 min and was stopped by addition of 1000/al of a chloroform-methanol (1:2) mixture. To separate phases further 350 pl of chloroform and 350 pl of water were added and the mixture was centrifuged for 10 min at 1000 g. A portion of 1000 pl of the upper aqueous phase was transferred into vials containing 2.5 ml of water and 0.5 ml of 50% (w/v) slurry of Dowex to bind phosphates. After 3 washes with 5 ml portions of 5 mM aqueous myo-inositol solution the phosphates were eluted with 600 pl of a mixture of 1 M ammonium formate and 0.1 M formic acid (1:1). A portion of 500 pl of this eluate was added to 3 ml of Bray's fluid and counted for radioactivity in a Beckman LS 3801 scintillation counter at approx. 36% efficiency. The results are expressed in percentage of the basal IP concentrations. The doseresponse curves for each animal were constructed separately, and average maximum effects and ECs0 values were calculated. The significance of difference of these parameters for young and old rats was assessed with the t-test. In addition, differences in responses between young and old rats were assessed for significance with two-way analysis of variance followed by Dunnett's test. The protein content in the cortex of young and old rats did not differ, being 120 + 6 and 128 + 8 mg/g of tissue, resp. (n = 6 in both cases). The maximum noradrenalineand carbachol-induced accumulation of IP in cerebral cortical slices from old rats was higher than in the preparation from young animals, by 76 and 68%, respectively, while the ECs0 values did not differ between young and old rats in the case of response to noradrenaline (2.16+0.46 and 3.86+1.08 pM), but were significantly lower in old than in young rats in the case of carbachol (31 + 7 vs 91 + 15/IM) (n = 5 in all cases) (Fig. 1). The two-way analysis of variance demonstrated a significant difference both between the age groups and the agonist concentration for both noradrenaline and carbachol. Altogether, the results indicate a greater responsiveness of ~radrenoceptors and cholinergic muscarinic M1 receptors in aged rats. A possible reason for increased second messenger response, i.e. an increased efficiency of translation of the neurohormonal signal, may be either an increase in receptor availability (i.e. an increase in the density of receptors), or increased coupling between the receptor and the second messenger synthesising enzyme, in this case phospholipase C. Our most recent data indicate that in the senescent Sprague-Dawley rats, the strain used for the present study, the density of cortical cholinergic muscarinic receptors does not change with age [11]. The available data for ~l-adrenoceptors do not suggest an increase in their density in senescence either [4]. It seems, therefore, that the observed increased responsiveness of phosphoinositide system upon agonist stimulation is related to increased efficiency of post-receptor mechanisms. It should be mentioned, however, that the receptor binding studies carried out up till now cannot exclude the possibility of age-related redistribution of receptors with their higher proportion on the cell surface. The presently found increased responsiveness of cholinergic muscarinic M1 receptors seems to have behavioural correlates. In our previous paper [16] we have noted that senescent rats are more vulnerable to the toxic action of diisopropyl fluoro-
198
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young
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.
.
.
.
.
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.--. young zx---~x o k d f
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Fig. 1. The effect of noradrcnalinc and carbachol on inositol phosphate (IP) accumulation in slices from the cerebral cortex of young and old Sprague Dawley rats. Ordinate: the concentration of IP expressed as the percentage of basal value. The basal values were 1068_+ 150 dpm/tube for the young, and 956+ 115 dpm/tube tbr old rats. Abscissa: concentrations of noradrenaline or carbachol. Each point represents the mean + S.E.M. of resulls from 5 animals. Each assay was carried out in triplicate. The two way analysis of variance showed significant differences between old and new rats ( F - 19.23 for noradrenaline and 20.37 l'or carbachol: in both cases d r = l, 40, P < 0.001), and between agonist concentrations ( F = 17.68 and 22.42 li)r noradrenaline and carbachol, respectively, d f = 4 , 40, P<0.001 in both cases). Asterisks denote a singificant difference ( P < 0.05, Dunnett's test) between the results from old and young rats at a given concentration of the agonist.
phosphate, an anticholinesterase agent. Moreover, Pedigo et al. [15] have reported that aged F344 rats are more responsive to pharmacological actions of oxotremorine, despite lower cholinergic muscarinic receptor density. No data on responsiveness of aged rats to behavioural effects of alpha-adrenomimetics are available. Regardless of the molecular mechanism of the observed phenomenon, the increased responsiveness of phosphoinositide system in old age may be regarded as a compensatory mechanism. In the old age the cellular calcium homeostasis is impaired, the brain calcium uptake is reduced and the calcium availability for the cell is decreased [7]. As pointed out by Barritt [1], phosphoinositide hydrolysis may
199
be important for changes in this homeostasis. An increase in responsiveness to agonists would counteract to some extent the decline in calcium availability as increased formation of inositol polyphosphates may facilitate both the mobilisation of the intracelllar calcium stores and external calcium influx [5]. Supported by Progetto Finalizzato Farmaci (Sottoprogetto: Studio di modelli sperimentali di demenze senili). I.N. and J.V. were visiting scientists from the National Council of Research (Consiglio Nazionale delle Ricerche), partially supported by CPBP 06.02. 1 Barritt, G.J., lntracellular free calcium and inositol polyphosphate action as potential targets in the ageing process, Neurobiol. Aging, 8 (1987) 359-361. 2 Brown, E., Kendall, D.A. and Nahorski, S.R., Inositol phospholipid hydrolysis in rat cerebral cortical slices. I. Receptor characterization, J. Neurochem., 42 (1984) 1379-1387. 3 Consolo, S., Wang, J.-X., Fiorentini, F., Vezzani, A. and Ladinsky, H., In vivo and in vitro studies on the regulation of cholinergic neurotransmission in striatum, hippocampus and cortex of aged rats, Brain Res., 374 (1986) 212-218. 4 DeBlasi, A., Cotecchia, S. and Mennini, T., Selective changes of receptor binding in brain regions of aging rats, Life Sci., 31 (1982) 335-340. 5 Exton, J.H., Mechanism of action of calcium-mobilizing agonists: some variations on a young theme, FASEB J., 2 (1988) 267(~2676. 6 Finch, C.E., The regulation of physiological changes during mammalian aging, Quart. Rev. Biol., 51 (1976) 49 83. 7 Gibson, G.E. and Peterson, C., Calcium and the aging nervous system, Neurobiol. Aging, 8 (1987) 329 343. 8 Ixart, G., Barbanel, G., Conte-Devolx, B., Grino, M., Oliver, C. and Assenmacher, I., Evidence for basal and stress-induced release of corticotropin releasing factor in the push-pull cannulated median eminence of conscious free-moving rats, Neurosci. Lett., 74 (1987) 85-89. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 Michalek, H., Fortuna, S. and Pintor, A., Age-related differences in brain choline acetyltransferase, cholinesterases and muscarinic receptor sites in two strains of rats, Neurobiol. Aging, l0 (1989) 143 148. 11 Michalek, H., Fortuna, S. and Pintor, A., Responsiveness of brain muscarinic acetylcholine receptors in aging, Ann. Ist. Super. Sanitfi., 25 (1989) in press. 12 Moretti, A., Carfagna, N. and Trunzo, F., Effect of aging on monoamines and their metabolites in the rat brain, Neurochem. Res., 12 (1987) 1035-1039, 13 Misra, C.H., Shelat, H.S. and Smith, R.C., Effects of age on adrenergic and dopaminergic receptor binding in rat brain. Life Sci., 27 (1980) 521-526. 14 Nomura, Y., Kitamura, Y., Kawai, M. and Segawa, T., ~2-Adrenoceptor - GTP binding regulatory protein adenylate cyclase system in cerebral cortical membranes of adult and senescent rats, Brain Res., 379 (1986) 118-124. 15 Pedigo, N.W., Jr., Minor, L.D. and Krumrei, T.N., Cholinergic drug effects and brain muscarinic receptor binding in aged rats, Neurobiol. Aging, 5 (1984) 227-233. 16 Pintor, A., Fortuna, S., Volpe, M.T. and Michalek, H., Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate, Life Sci., 42 (1988) 2113-2121. 17 Pradhan, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643-1656. 18 Scarpace, P.J. and Abrass, I.B., Alpha and beta-adrenergic receptor function in brain during senescence, Neurobiol. Aging, 9 (1988) 53 58.
195
Elsevier Scientific Publishers Ireland Ltd. NSL 06477
Increased responsiveness of the cerebral cortical phosphatidylinositol system to noradrenaline and carbachol in senescent rats Irena Nalepa 2, Annita Pintor l, Stefano Fortuna l, Jerzy Vetulani 2 and Hanna Michalek l JLaboratory of Pharmacology, lstituto Superiore di Sanita, Rome (Italy) and :Department of Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Krakow (Poland) (Received 19 April 1989; Revised version received 3l July 1989; Accepted 31 July 1989)
Key words." Rat; Aging brain; Phosphatidylinositol hydrolysis; cq-Adrenoceptor; Muscarinic Mt receptor; Receptor responsiveness The responsiveness of cerebral cortical ~radrenoceptors and cholinergic muscarinic M~ receptors was assessed in young (3 months) and aged (24 months) male Sprague Dawley rats. The measure of responsiveness was the accumulation of inositol phosphate (IP) formed in [3H]myo-inositol-preloaded cerebral cortical slices in the presence of lithium, following stimulation with various concentrations of noradrenaline (l 300 pM) and carbachol (5 1000 ,uM). In old rats the maximum response to noradrenaline was higher by 80%, and that to carbachol by 33%, indicating an increased responsiveness of the investigated receptors in senescence.
Different neurochemical systems in the brain are modified during aging [6, 7, 17] and it is believed that senescence is connected with a general decline in the function of the central nervous system. Behavioural deficits observed in senescent animals were often associated with changes in neurotransmitter systems, but at least in the cerebral cortex - no decline, but rather small increases in the levels of noradrenaline and acetylcholine were noted (see, e.g. refs. 3, 12). Therefore, the characteristics of various classes of central neurotransmitter receptors, particularly those for catecholamines and acetylcholine, were investigated in senescent animals. In general no agerelated changes in the affinities of the investigated receptors were found, but their densities in many cases declined. Thus for muscarinic cholinergic receptors Pedigo et al. [15] have reported a decline in density in the cerebral cortex of Fischer 344 (F344) rats. We have recently confirmed this finding for F344 rats but found no agerelated changes of this type in Wistar or Sprague-Dawley rats [10, 11, 16]. Several Correspondence." H. Michalek, Lab. of Pharmacology, lstituto Superiore di Sanitfi, Viale Regina Elena, 229, 00161 Roma, Italy. 0304-3940,/89,,'$ 03.50 ~(~ 1989 Elsevier Scientific Publishers Ireland Ltd.
196
studies on fl-adrenoceptors indicate a decline in their density in most brain areas, with the exception of the cortex, in which the results are conflicting [18]. No age-related changes were observed with :~2-adrenoceptors in the Wistar rat [14]. The conflicting data concerning :q-adrenoceptors may be related to strain differences: in SpragueDawley rats no age-related changes in :q-adrenoceptor density were noted [4], while in F344 rats a decline was found [13]. The changes in the density of receptors for neurotransmitters do not necessarily determine the responsiveness of the system regulated by those receptors. A better estimate of functional changes is given by measurement of formation of second messengers. Such studies in old animals were carried out on dopamine and/~'-adrenoceptors by measuring cyclic AMP formation induced by agonists. In most cases a decline in responsiveness of cyclic AMP generating system in some, but not all, brain regions was observed [17, 18]. It was speculated that the declined response might be caused by diminished receptor availability [I 8]. The purpose of the present study was to assess whether there are differences in responsiveness of cortical :q-adrenoceptors and cholinergic muscarinic M i receptors of young and senescent Sprague Dawley rats, using accumulation of inositol phosphate (IP) after challenge with agonists: noradrenaline and carbachol, as the measure of response. Young (3 months old, weighing 250-300 g) and senescent (24 months old, weighing 650-800 g) male Sprague-Dawley rats were purchased from Charles River Italia, Calco (Como). They were kept two in a cage in an animal room under standard conditions (12:12 h DL cycle, free access to standard food and tap water). The experiments were performed in balanced replications. Every day one aged and one young rat were decapitated with a guillotine, the cortex was dissected and cut into 300 × 300 l,m slices using a Mcllwain tissue chopper. One half of the slices was used for stimulation with 5 concentrations of noradrenaline (1.0 300/,tM), the other with 5 concentrations of carbachol (5.0-1000/~M). Protein content was assayed with the method of Lowry et al. [9]. The incubation of slices and the assay of IP were carried out according to Brown et al. [2] with some modifications. The incubation proceeded in three stages: 1. Preincubation, during which the slices obtained from a single rat were incubated at 37~C in 50 ml of O2:CO 2 (95:5) gassed, glucose-containing Krebs-Ringer medium (in mM: NaCI 118, KCI 5, CaC12 1.3, MgSO4 1.2, KH2PO4 i.2, NaHCO3 25, glucose 11.7: pH 7.4) for 1 h, with three changes of the buffer. 2. Labelling incubation. Portions of 50/,d of preincubated, density-packed slices (2.7 mg of protein on average) were pipetted into fiat-bottom 3 ml plastic vials containing 200/11 of the buffer, 30/tl of LiC1 solution (final conc. 5 mM) and 20/tl of the purified [3H]myo-inositol solution. The vials were gassed (95% 02:5% CO2), capped and shaken at 37°C for 30 min. During this stage [3H]myo-inositolwas being incorporated into membrane phospholipids, forming phosphatidylinositols (PI, PIP, PIP2). 3. Hydrolytic incubation, at the beginning of which 10/A of agonist solutions: noradrenaline (DL-arterenol, Sigma: 5 concentrations, from 1 to 300/tM) or carbachol
197
(Sigma; 5 concentrations, from 5 to 1000/~M) were added to the medium. The incubation was continued for 45 min and was stopped by addition of 1000/al of a chloroform-methanol (1:2) mixture. To separate phases further 350 pl of chloroform and 350 pl of water were added and the mixture was centrifuged for 10 min at 1000 g. A portion of 1000 pl of the upper aqueous phase was transferred into vials containing 2.5 ml of water and 0.5 ml of 50% (w/v) slurry of Dowex to bind phosphates. After 3 washes with 5 ml portions of 5 mM aqueous myo-inositol solution the phosphates were eluted with 600 pl of a mixture of 1 M ammonium formate and 0.1 M formic acid (1:1). A portion of 500 pl of this eluate was added to 3 ml of Bray's fluid and counted for radioactivity in a Beckman LS 3801 scintillation counter at approx. 36% efficiency. The results are expressed in percentage of the basal IP concentrations. The doseresponse curves for each animal were constructed separately, and average maximum effects and ECs0 values were calculated. The significance of difference of these parameters for young and old rats was assessed with the t-test. In addition, differences in responses between young and old rats were assessed for significance with two-way analysis of variance followed by Dunnett's test. The protein content in the cortex of young and old rats did not differ, being 120 + 6 and 128 + 8 mg/g of tissue, resp. (n = 6 in both cases). The maximum noradrenalineand carbachol-induced accumulation of IP in cerebral cortical slices from old rats was higher than in the preparation from young animals, by 76 and 68%, respectively, while the ECs0 values did not differ between young and old rats in the case of response to noradrenaline (2.16+0.46 and 3.86+1.08 pM), but were significantly lower in old than in young rats in the case of carbachol (31 + 7 vs 91 + 15/IM) (n = 5 in all cases) (Fig. 1). The two-way analysis of variance demonstrated a significant difference both between the age groups and the agonist concentration for both noradrenaline and carbachol. Altogether, the results indicate a greater responsiveness of ~radrenoceptors and cholinergic muscarinic M1 receptors in aged rats. A possible reason for increased second messenger response, i.e. an increased efficiency of translation of the neurohormonal signal, may be either an increase in receptor availability (i.e. an increase in the density of receptors), or increased coupling between the receptor and the second messenger synthesising enzyme, in this case phospholipase C. Our most recent data indicate that in the senescent Sprague-Dawley rats, the strain used for the present study, the density of cortical cholinergic muscarinic receptors does not change with age [11]. The available data for ~l-adrenoceptors do not suggest an increase in their density in senescence either [4]. It seems, therefore, that the observed increased responsiveness of phosphoinositide system upon agonist stimulation is related to increased efficiency of post-receptor mechanisms. It should be mentioned, however, that the receptor binding studies carried out up till now cannot exclude the possibility of age-related redistribution of receptors with their higher proportion on the cell surface. The presently found increased responsiveness of cholinergic muscarinic M1 receptors seems to have behavioural correlates. In our previous paper [16] we have noted that senescent rats are more vulnerable to the toxic action of diisopropyl fluoro-
198
6(10 s
4-00-
,f/
200-
e--.
young
zx---zx o l d cq 2,.
0
~ .....
.
.
.
.
.
7 . . . . . . . . . T5 4-
6
- :og ( L - n o r a d r e n a / / n e ) . M -Q
400 J
Z
/ / / ~
300 i
oo]
.
i
'
.
t
z 2 " ' ~
!00 i
0
,,,-'"
~/~
r
6
. . . .
T .....
5 ~'o~(C~rbachol:
.--. young zx---~x o k d f
4-
7
3
M
Fig. 1. The effect of noradrcnalinc and carbachol on inositol phosphate (IP) accumulation in slices from the cerebral cortex of young and old Sprague Dawley rats. Ordinate: the concentration of IP expressed as the percentage of basal value. The basal values were 1068_+ 150 dpm/tube for the young, and 956+ 115 dpm/tube tbr old rats. Abscissa: concentrations of noradrenaline or carbachol. Each point represents the mean + S.E.M. of resulls from 5 animals. Each assay was carried out in triplicate. The two way analysis of variance showed significant differences between old and new rats ( F - 19.23 for noradrenaline and 20.37 l'or carbachol: in both cases d r = l, 40, P < 0.001), and between agonist concentrations ( F = 17.68 and 22.42 li)r noradrenaline and carbachol, respectively, d f = 4 , 40, P<0.001 in both cases). Asterisks denote a singificant difference ( P < 0.05, Dunnett's test) between the results from old and young rats at a given concentration of the agonist.
phosphate, an anticholinesterase agent. Moreover, Pedigo et al. [15] have reported that aged F344 rats are more responsive to pharmacological actions of oxotremorine, despite lower cholinergic muscarinic receptor density. No data on responsiveness of aged rats to behavioural effects of alpha-adrenomimetics are available. Regardless of the molecular mechanism of the observed phenomenon, the increased responsiveness of phosphoinositide system in old age may be regarded as a compensatory mechanism. In the old age the cellular calcium homeostasis is impaired, the brain calcium uptake is reduced and the calcium availability for the cell is decreased [7]. As pointed out by Barritt [1], phosphoinositide hydrolysis may
199
be important for changes in this homeostasis. An increase in responsiveness to agonists would counteract to some extent the decline in calcium availability as increased formation of inositol polyphosphates may facilitate both the mobilisation of the intracelllar calcium stores and external calcium influx [5]. Supported by Progetto Finalizzato Farmaci (Sottoprogetto: Studio di modelli sperimentali di demenze senili). I.N. and J.V. were visiting scientists from the National Council of Research (Consiglio Nazionale delle Ricerche), partially supported by CPBP 06.02. 1 Barritt, G.J., lntracellular free calcium and inositol polyphosphate action as potential targets in the ageing process, Neurobiol. Aging, 8 (1987) 359-361. 2 Brown, E., Kendall, D.A. and Nahorski, S.R., Inositol phospholipid hydrolysis in rat cerebral cortical slices. I. Receptor characterization, J. Neurochem., 42 (1984) 1379-1387. 3 Consolo, S., Wang, J.-X., Fiorentini, F., Vezzani, A. and Ladinsky, H., In vivo and in vitro studies on the regulation of cholinergic neurotransmission in striatum, hippocampus and cortex of aged rats, Brain Res., 374 (1986) 212-218. 4 DeBlasi, A., Cotecchia, S. and Mennini, T., Selective changes of receptor binding in brain regions of aging rats, Life Sci., 31 (1982) 335-340. 5 Exton, J.H., Mechanism of action of calcium-mobilizing agonists: some variations on a young theme, FASEB J., 2 (1988) 267(~2676. 6 Finch, C.E., The regulation of physiological changes during mammalian aging, Quart. Rev. Biol., 51 (1976) 49 83. 7 Gibson, G.E. and Peterson, C., Calcium and the aging nervous system, Neurobiol. Aging, 8 (1987) 329 343. 8 Ixart, G., Barbanel, G., Conte-Devolx, B., Grino, M., Oliver, C. and Assenmacher, I., Evidence for basal and stress-induced release of corticotropin releasing factor in the push-pull cannulated median eminence of conscious free-moving rats, Neurosci. Lett., 74 (1987) 85-89. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 Michalek, H., Fortuna, S. and Pintor, A., Age-related differences in brain choline acetyltransferase, cholinesterases and muscarinic receptor sites in two strains of rats, Neurobiol. Aging, l0 (1989) 143 148. 11 Michalek, H., Fortuna, S. and Pintor, A., Responsiveness of brain muscarinic acetylcholine receptors in aging, Ann. Ist. Super. Sanitfi., 25 (1989) in press. 12 Moretti, A., Carfagna, N. and Trunzo, F., Effect of aging on monoamines and their metabolites in the rat brain, Neurochem. Res., 12 (1987) 1035-1039, 13 Misra, C.H., Shelat, H.S. and Smith, R.C., Effects of age on adrenergic and dopaminergic receptor binding in rat brain. Life Sci., 27 (1980) 521-526. 14 Nomura, Y., Kitamura, Y., Kawai, M. and Segawa, T., ~2-Adrenoceptor - GTP binding regulatory protein adenylate cyclase system in cerebral cortical membranes of adult and senescent rats, Brain Res., 379 (1986) 118-124. 15 Pedigo, N.W., Jr., Minor, L.D. and Krumrei, T.N., Cholinergic drug effects and brain muscarinic receptor binding in aged rats, Neurobiol. Aging, 5 (1984) 227-233. 16 Pintor, A., Fortuna, S., Volpe, M.T. and Michalek, H., Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate, Life Sci., 42 (1988) 2113-2121. 17 Pradhan, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643-1656. 18 Scarpace, P.J. and Abrass, I.B., Alpha and beta-adrenergic receptor function in brain during senescence, Neurobiol. Aging, 9 (1988) 53 58.