Neurobiologyof Aging, Vol. 13. pp. 89-92. ~ Pergamon Press plc. 1991. Printed in the U.S.A.
0197-4580/92 $5.00 + .00
Inositol 1,4,5-Trisphosphate Receptor in Developing and Senescent Rat Cerebellum P E T E R P. LI, *l G I A C O M O
G. V E C I L , t
M A R T Y A. G R E E N * A N D J E R R Y J. W A R S H * t $
*Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, "kDepartment of Pharmacology and 4:Institute of Medical Sciences, Universit 3' of Toronto, Toronto, Ontario, Canada, M5T 1R8 R e c e i v e d 6 July 1989; A c c e p t e d 24 M a y 1991 LI, P. P., G. G. VECIL, M. A. GREEN AND J. J. WARSH. lnositol 1,4,5-trisphosphate receptor in developing and senescent rat cerebellum. NEUROBIOL AGING 13(1) 89-92, 1992.--Numerous processes associated with intracellular calcium homeostasis have previously been found to vary with age. To determine whether the binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (InsP3), also displays such variation, [3H]InsP3 binding was investigated in cerebellar or cerebral cortical membranes prepared from rats at different ages from birth up to 24 months of age. In the cerebellum, the InsP3 receptor density was very low during the first week after birth, increased markedly between days 8 and 28 and then reached an apparent plateau between 28 to 56 days of age. The InsP3 receptor binding affinity was comparable at different developmental stages. No age-related differences were found in InsP3 receptor density or affinity in the cerebral cortex of 3-, 6-, 9-, 12-, and 24-month-old rats. In the cerebellum, InsP3 receptor density but not affinity was significantly reduced in 24-month-old compared only to 3-month-old animals. Our data suggest that the changes in InsP3 receptor binding during early development might reflect the growth and maturation of neurons containing these receptors (i.e., Purkinje cells). Furthermore, the age-dependent reduction in InsP3 receptor density, together with the recent report of senescent changes in protein kinase C activity, indicate that disruption of phosphoinositide second messenger system may be of importance to the impairment of neuronal responsiveness and behavioral deficits observed with aging. Inositol 1,4,5-trisphosphate
Cerebellum
Cerebral cortex
THE pivotal role of inositol 1,4,5-trisphosphate (InsP3) as a calcium-mobilizing second messenger for many hormonal, neurotransmitter and growth factor signals has been well documented in a variety of cell types including brain (5). InsP3 releases calcium from intracellular stores by binding to specific InsP 3 receptors that are coupled to calcium channels (9, 13, 23). The putative lnsP 3 receptor was first identified by direct ligand binding with radiolabeled InsP 3 (13, 31, 32). In both rodent and human brain InsP 3 receptors are heterogeneously distributed with the highest concentration occurring in cerebellum (17, 31, 32), where they are found to be selectively localized to Purkinje cell dendrites in the molecular layer (20,31). Recently, the InsP 3 receptor has been purified in rat (30) and mouse (20,21) cerebellum and more recently, the complementary DNAs encoding the InsP 3 receptor have been cloned (11,22). Reconstitution and functional expression studies have confirmed that the receptor protein behaves as an ionotropic receptor containing both recognition sites for lnsP3 and the calcium channel (9,23). Considerable evidence indicates that intracellular free calcium concentration plays a major role in regulating cell growth, differentiation as well as maintaining cell viability (5,7). As well, there is suggestion that phosphoinositide turnover and subsequent production of second messengers, InsP 3 and diacylglycerol, may be important in maintaining intracellular calcium homeostasis (3,24). Many processes associated with the phosphoinositide
Purkinje cell
Postnatal development
Aging
second messenger system are known to change during the course of development and with the aging process. For example, the efficiency of coupling of CNS receptors to phosphoinositide turnover varies during different stages of development (14) and with age (8,25). It has been suggested that protein kinase C (PKC), a calcium-dependent and diacylglycerol-activated enzyme, is specifically involved in the developmental processes of the brain, as the expression of PKC parallels that of brain maturation (15). In addition, perturbation in neuronal calcium homeostasis and deficits in intracellular action of the second messengers, calcium and PKC, have been reported in aged rats (4, 10, 12). Given the central role of InsP 3 in calcium signalling through binding to its receptor (5,24), it was therefore of interest to examine the characteristics of InsP 3 receptors during postnatal development of rat cerebellum, a region in which the ontogenic changes of synapses have been well described (1,2) and which has a particularly high density of InsP 3 receptors (20, 21, 28, 31). To further characterize the effects of aging on InsP 3 receptors, InsP 3 binding was examined in the cerebral and cerebellar cortex of rats 3, 6, 9, 12, or 24 months of age.
Animal
METHOD
Timed pregnant Wistar rats (Charles River, Montreal) were housed individually, allowed water and food ad lib, and were
1Requests for reprints should be addressed to Dr. P. P. Li, Section of Biochemical Psychiatry, Clarke Institute of Psychiatry. 250 College Street, Toronto, Ontario, Canada, M5T 1R8.
89
tA VECIL, ()REP.N AND ~,~'ARSH
~0
maintained on a 12-hour light/dark cycle. Pups were maintained with their mothers and weaned on day 21. Rats of both sexes were killed by decapitation at 1, 8, 11, 15, 21, 28 and 56 days of age, and the whole brains removed. The cerebella were dissected on ice, frozen and stored at - 7 0 ° C until assay. For animals less than 28 days of age, sufficient brains (ranged from 2 to 12) were pooled to give a total tissue wet weight of between 0.25 to 0.50 g for each binding assay. To examine possible senescent changes in InsP 3 receptor binding, male Wistar rats (Charles River, Montreal) ranging in age from 3 to 24 months were obtained from a colony maintained in our animal care facility. Animals were killed at various ages (3, 6, 9, 12 and 24 months old) and the brains removed, dissected, frozen and stored at - 7 0 ° C until assay.
20
--4t
'- 1 5 ~
/
I
,o r~
*/
~loj
/
:
0.4
Br~.,= 8,23 ~/M
/ m 0.3
KO: 22.7 nM
\
r = - 0.99
o2 z
o 0.1 E 5 113
0 - 2
4
6
8
10
3H4nsP3 B O U N D (nM)
[SH]InsP3 Binding Assay
0
Specific [3H]InsP3 binding was determined as described recently (17, 18, 32) with minor modifications. Briefly, thawed cerebella were homogenized in 40 volumes (w/v) of ice-cold buffer (50 mM Tris, 1 mM EDTA, pH 8.3) using a Polytron at setting 9 for 10 s. The homogenates were then centrifuged at 35,000 × g for 20 rain at 4°C, after which the supematants were decanted and the tissue pellets were resuspended in buffer and used immediately in binding assays. The incubation mixtures contained approximately 0.3~3.5 mg membrane protein, 0.2 nM [3H]InsP3 (47 Ci/mmol; Amersham), InsP 3 (7 different concentrations ranging from 1 to 1000 nM; Sigma), 1 mg/ml bovine serum albumin in a total volume of 1 ml of 50 mM Tris-HC1 buffer (pH 8.3) containing 1 mM EDTA. Triplicate samples (duplicates in 1-day-old pups) were incubated at 4°(2 for 10 min followed by centrifugation at 12,000×g for 10 min (Beckman Microfuge). The pellets were washed with 1 ml ice-cold buffer and subsequently solubilized with 0.5% sodium dodecyl sulphate overnight. The radioactivity in the pellets was quantified by liquid scintillation spectrometry (counting efficiency 50%). Nonspecific binding was defined as the amount of radioactivity bound in the presence of 1 I.~M InsP3. Protein was measured by the method of Sedmak and Grossberg (29) using bovine serum albumin as standard.
Data Analysis Saturation analyses of isotopic dilution [3H]lnsP3 binding data and estimates of binding parameters (KD and Bmax) were performed using the EBDA (Elsevier-Biosoft, Cambridge) computer program (19). Data were analyzed by one-way analysis of variance followed by Student's t-test. Mean differences with p values
0197-4580/92 $5.00 + .00
Inositol 1,4,5-Trisphosphate Receptor in Developing and Senescent Rat Cerebellum P E T E R P. LI, *l G I A C O M O
G. V E C I L , t
M A R T Y A. G R E E N * A N D J E R R Y J. W A R S H * t $
*Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, "kDepartment of Pharmacology and 4:Institute of Medical Sciences, Universit 3' of Toronto, Toronto, Ontario, Canada, M5T 1R8 R e c e i v e d 6 July 1989; A c c e p t e d 24 M a y 1991 LI, P. P., G. G. VECIL, M. A. GREEN AND J. J. WARSH. lnositol 1,4,5-trisphosphate receptor in developing and senescent rat cerebellum. NEUROBIOL AGING 13(1) 89-92, 1992.--Numerous processes associated with intracellular calcium homeostasis have previously been found to vary with age. To determine whether the binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (InsP3), also displays such variation, [3H]InsP3 binding was investigated in cerebellar or cerebral cortical membranes prepared from rats at different ages from birth up to 24 months of age. In the cerebellum, the InsP3 receptor density was very low during the first week after birth, increased markedly between days 8 and 28 and then reached an apparent plateau between 28 to 56 days of age. The InsP3 receptor binding affinity was comparable at different developmental stages. No age-related differences were found in InsP3 receptor density or affinity in the cerebral cortex of 3-, 6-, 9-, 12-, and 24-month-old rats. In the cerebellum, InsP3 receptor density but not affinity was significantly reduced in 24-month-old compared only to 3-month-old animals. Our data suggest that the changes in InsP3 receptor binding during early development might reflect the growth and maturation of neurons containing these receptors (i.e., Purkinje cells). Furthermore, the age-dependent reduction in InsP3 receptor density, together with the recent report of senescent changes in protein kinase C activity, indicate that disruption of phosphoinositide second messenger system may be of importance to the impairment of neuronal responsiveness and behavioral deficits observed with aging. Inositol 1,4,5-trisphosphate
Cerebellum
Cerebral cortex
THE pivotal role of inositol 1,4,5-trisphosphate (InsP3) as a calcium-mobilizing second messenger for many hormonal, neurotransmitter and growth factor signals has been well documented in a variety of cell types including brain (5). InsP3 releases calcium from intracellular stores by binding to specific InsP 3 receptors that are coupled to calcium channels (9, 13, 23). The putative lnsP 3 receptor was first identified by direct ligand binding with radiolabeled InsP 3 (13, 31, 32). In both rodent and human brain InsP 3 receptors are heterogeneously distributed with the highest concentration occurring in cerebellum (17, 31, 32), where they are found to be selectively localized to Purkinje cell dendrites in the molecular layer (20,31). Recently, the InsP 3 receptor has been purified in rat (30) and mouse (20,21) cerebellum and more recently, the complementary DNAs encoding the InsP 3 receptor have been cloned (11,22). Reconstitution and functional expression studies have confirmed that the receptor protein behaves as an ionotropic receptor containing both recognition sites for lnsP3 and the calcium channel (9,23). Considerable evidence indicates that intracellular free calcium concentration plays a major role in regulating cell growth, differentiation as well as maintaining cell viability (5,7). As well, there is suggestion that phosphoinositide turnover and subsequent production of second messengers, InsP 3 and diacylglycerol, may be important in maintaining intracellular calcium homeostasis (3,24). Many processes associated with the phosphoinositide
Purkinje cell
Postnatal development
Aging
second messenger system are known to change during the course of development and with the aging process. For example, the efficiency of coupling of CNS receptors to phosphoinositide turnover varies during different stages of development (14) and with age (8,25). It has been suggested that protein kinase C (PKC), a calcium-dependent and diacylglycerol-activated enzyme, is specifically involved in the developmental processes of the brain, as the expression of PKC parallels that of brain maturation (15). In addition, perturbation in neuronal calcium homeostasis and deficits in intracellular action of the second messengers, calcium and PKC, have been reported in aged rats (4, 10, 12). Given the central role of InsP 3 in calcium signalling through binding to its receptor (5,24), it was therefore of interest to examine the characteristics of InsP 3 receptors during postnatal development of rat cerebellum, a region in which the ontogenic changes of synapses have been well described (1,2) and which has a particularly high density of InsP 3 receptors (20, 21, 28, 31). To further characterize the effects of aging on InsP 3 receptors, InsP 3 binding was examined in the cerebral and cerebellar cortex of rats 3, 6, 9, 12, or 24 months of age.
Animal
METHOD
Timed pregnant Wistar rats (Charles River, Montreal) were housed individually, allowed water and food ad lib, and were
1Requests for reprints should be addressed to Dr. P. P. Li, Section of Biochemical Psychiatry, Clarke Institute of Psychiatry. 250 College Street, Toronto, Ontario, Canada, M5T 1R8.
89
tA VECIL, ()REP.N AND ~,~'ARSH
~0
maintained on a 12-hour light/dark cycle. Pups were maintained with their mothers and weaned on day 21. Rats of both sexes were killed by decapitation at 1, 8, 11, 15, 21, 28 and 56 days of age, and the whole brains removed. The cerebella were dissected on ice, frozen and stored at - 7 0 ° C until assay. For animals less than 28 days of age, sufficient brains (ranged from 2 to 12) were pooled to give a total tissue wet weight of between 0.25 to 0.50 g for each binding assay. To examine possible senescent changes in InsP 3 receptor binding, male Wistar rats (Charles River, Montreal) ranging in age from 3 to 24 months were obtained from a colony maintained in our animal care facility. Animals were killed at various ages (3, 6, 9, 12 and 24 months old) and the brains removed, dissected, frozen and stored at - 7 0 ° C until assay.
20
--4t
'- 1 5 ~
/
I
,o r~
*/
~loj
/
:
0.4
Br~.,= 8,23 ~/M
/ m 0.3
KO: 22.7 nM
\
r = - 0.99
o2 z
o 0.1 E 5 113
0 - 2
4
6
8
10
3H4nsP3 B O U N D (nM)
[SH]InsP3 Binding Assay
0
Specific [3H]InsP3 binding was determined as described recently (17, 18, 32) with minor modifications. Briefly, thawed cerebella were homogenized in 40 volumes (w/v) of ice-cold buffer (50 mM Tris, 1 mM EDTA, pH 8.3) using a Polytron at setting 9 for 10 s. The homogenates were then centrifuged at 35,000 × g for 20 rain at 4°C, after which the supematants were decanted and the tissue pellets were resuspended in buffer and used immediately in binding assays. The incubation mixtures contained approximately 0.3~3.5 mg membrane protein, 0.2 nM [3H]InsP3 (47 Ci/mmol; Amersham), InsP 3 (7 different concentrations ranging from 1 to 1000 nM; Sigma), 1 mg/ml bovine serum albumin in a total volume of 1 ml of 50 mM Tris-HC1 buffer (pH 8.3) containing 1 mM EDTA. Triplicate samples (duplicates in 1-day-old pups) were incubated at 4°(2 for 10 min followed by centrifugation at 12,000×g for 10 min (Beckman Microfuge). The pellets were washed with 1 ml ice-cold buffer and subsequently solubilized with 0.5% sodium dodecyl sulphate overnight. The radioactivity in the pellets was quantified by liquid scintillation spectrometry (counting efficiency 50%). Nonspecific binding was defined as the amount of radioactivity bound in the presence of 1 I.~M InsP3. Protein was measured by the method of Sedmak and Grossberg (29) using bovine serum albumin as standard.
Data Analysis Saturation analyses of isotopic dilution [3H]lnsP3 binding data and estimates of binding parameters (KD and Bmax) were performed using the EBDA (Elsevier-Biosoft, Cambridge) computer program (19). Data were analyzed by one-way analysis of variance followed by Student's t-test. Mean differences with p values
