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Inositol 1,4,5-trisphosphate arm of the phosphatidylinositide signal transduction pathway in the rat cerebellum during aging
Neuroscience Letters, 164 (1993) 167 170 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940193/$ 06.00
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Inositol 1,4,5-trisphosphate arm of the phosphatidylinositide signal transduction pathway in the rat cerebellum during aging Orisa J. Igwe*, Li Ning Department (~f Pharmacology, Schools of Pharmacy and Medicine, University Qf Missouri at Kansas City, M3--104. MED. 2411 Holmes Street. Kansas City. MO 64108 2792. USA (Received 2 June 1993; Revised version received 26 July 1993; Accepted 27 September 1993)
Key words." Aging; Phosphatidylinositide; Inositol 1,4,5-trisphosphate; Inositol 1,4,5-trisphosphate receptor: |ntracellular calcium regulation: Signal transduction To determine whether the intracellular calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (InsPd and its receptor (InsP3Rt display age-dependent coordinate regulation, InsP 3 content and [~HlInsPa-binding characteristics were investigated in cerebellar particulate membranes prepared from male Fischer 344 rats at 3, 12 and 25 months of age. Cerebellar lnsP 3 content was significantly increased in 25-month-old rats compared with 3-month-old animals, Cerebellar InsP3R densities were significantly reduced at 12 and 25 months of age but InsP~-binding affinity was significantly decreased only in the 25-month-old animals. The present data strongly suggest that modulation of the phospfioinositide second messenger system may contribute to impaired neuronal responsiveness associated with the aging process in the cerebellum.
As one of the proposed second messengers in the phosphatidylinositide (P1) transduction system, InsP3 links receptor activation with the release of nonmitochondrial stores of calcium [2] by binding to an InsP3specific receptor [8, 29], localized to portions of the endoplasmic reticulum in cerebellar Purkinje neurons [24]. The lnsP3 receptor (InsP3R) has been isolated [9], molecularly cloned [17] and functionally reconstituted into lipid vesicles [7]. Many processes associated with the PI second messenger system and calcium homeostasis have been reported to change with the aging process [1, 11]. Changes in PI turnover-calcium regulation during the aging process may be involved in the development of age-related alterations in neurological functions [13]. Calcium mobilization and phospholipid hydrolysis are intimately interrelated and sometimes complimentary to each other [20], thus, the size of InsP3-sensitive calcium stores appears to depend on InsP 3 concentration [27]. Given the central role of InsP 3 in calcium-signaling through binding to InsP3R [2], it is important to determine whether InsP3 content and InsP3R are coordinately regulated with age. The present study examines the effect of aging on InsP3 content and the binding characteristics of InsP3R in the rat cerebellum, a region with particularly high density of InsP3Rs [24]. *Corresponding author.
Male Fischer 344 rats of three age groups were used, i.e., 3-month-old (young virgins, 208 230 g body wt), 12-month-old (middle-aged retired breeders, 414-462 g body wt) and 25-month-old (senescent retired breeders, 412450 g body wt). All animals were obtained from the National Institute on Aging colonies (Harlan-Sprague Dawley, Indianapolis, IN) and housed in pairs in the animal holding area with a 12-h light-dark cycle. Animals were acclimatized to the holding area for 1 wk before use in these experiments. Water and food were made available ad libitum. On the day of the experiment, animals were weighed and then sacrificed by decapitation and exsanguination. The brains were removed immediately and chilled on ice. All subsequent procedures were conducted at 4°C. Cerebelli were dissected out on an ice-chilled glass plate and a sagittally cut portion from the right lateral side (< I0 mg wet wt) was taken for InsP 3 assay with the rest used for InsP3R characterization. Tissue was processed immediately for InsP3 contents by homogenization (Brinkman Polytron setting 8:8 s) in ice-cold 1 M trichloroacetic acid (TCA) containing 10 mM EDTA to minimize postdecapitation reactions. The homogenate was incubated on ice for 15 rain and then centrifuged at 1000 x g for 10 min at 4°C. The pellet was used to determine tissue protein content after solubilization in 1 M NaOH for 4 h at 37°C. The supernatant was incubated at room temperature for 15 min and InsP3 ex-
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Age (Months) Fig. 1. Effect of aging on content of inositol t,4,5-trisphosphate in rat cerebellum. Tissue lnsP~ in three age groups was extracted and quantified as described in text. Values are mean -2 S.E.M. with n = 8 ~ats/age group with experiments carried out in duplicates. ~.,~ =6.59 (P = 0.006), *P -< 0.05 (ANOVA, Scheffe'sF test of multiple comparisons). tracted with a 3/1 (v/v) mixture of F R E O N and tri-noctylamine. After T C A removal, InsP3 contained in the upper aqueous layer was quantified by using a simple, specific and sensitive radioreceptor assay kit (Dupont N E N , Wilmington, DE). The assay buffer used for the standards and blanks were also carried through the extraction procedure to make corrections for the samples. The characteristics of [3H]InsP3-binding were determined in cerebellar particulate membranes. Tissue was homogenized (Polytron setting 9; 5 s) in 10 vols. of icecold buffer A (50 m M Tris HCI, pH = 8.3, at 25°C, 2.5 m M E D T A and 1 m M dithiothreitolL pelleted by c e n t r i f ugation (39,000 x g for 10 min) and resuspended in the same buffer. Binding assays contained 100/11 ( - t 0 0 - 2 1 0 /,/g protein) of the particulate preparation and 0.2 nM (~10,000 dpm) of [3H]InsP~ (spec. act. = 3 4 4 4 Ci/mmol; Amersham, Arlington, ILl with varying concentrations of unlabeled InsP3 in a total incubation volume of 0.5 ml of buffer A. Assays were incubated in microcentrifuge tubes for 40 rain at 4°C and the reaction stopped by centrifugation at 12,000 x g for 5 rain, followed by aspiration o f the supernatant. Tissue pellets were dissolved overnight in 250/Jl Soluene 350 (Packard Instrument, Meriden, CT), followed by addition of 4 ml Hionic-Fluor
(Packard lnsli-ument) scintillaticm cocktail and the radb oactivity determined by liquid scintillation spectroscop$, Nonspecilic binding was determined ill lhc plCSCllCC ~'~i I/aM unlabeled InsP3 and was generally .... 10% oI" total binding. Receptor density (B ...... ). binding affinity {Kd) and Hill coefficient (nil) were deterntined using an iteratire curve-fitting program L I G A N D 1181, The B ...... values were standardized to tissue protein content determined by the bicinchoninic acid method [26], using bovine serum albumin as the s t a n d a r d Fig. 1 shows that cerebellar InsP 3 contents were increased by ~ 44 and 76% (P -< 0.05) in 12- and 25-monthold rats, respectively, compared with the 3-month-old animals. Accumulation of lnsP~ was used in this study as an index of PIP, turnover. As tissues were processed within a short time (< 2 min) alter animal sacrifice, postdecapitation changes could not account for the differences in lnsP~ content determined in the present study [41. A number of plausible explanations could account for the age-dependent increase in cerebellar lnsP3 content. First, the aging process may attenuate the regulation of cerebellar PI metabolism resulting in altered Ca > homeostasis, as dendritic spike bursts of Purkinje cells are modulated by both Ca e+ and Pt turnover {l 5]. The intracellular Ca ~'~ levels in Purkinje cells of aging cerebellum may increase due to mobilization of Ca 2* from 'leaky' plasma membranes and/or Ca -,+ channels, resulting in both increased tissue InsP~ accumulation and decreased lnsP~-binding (negative feed back). This apparent paradox can be explained on the basis of the activation of phospholipase C (PLC) under situations of increased Ca > levels [16]. Recently, age-dependent increase in InsP~ content in red blood cells was reported [12]. Another possible mechanism for age-dependent increase in intracellular levels of InsP3 may lie in altered regulation of InsP3 3-kinase [25], an lnsP, metabolizing enzyme [5], found in abundance in cerebellar Purkinje cells [30]. Saturable InsP3-binding was found in all cerebellar particulate membranes examined (Fig. 2A). Scatchard were TABLE 1 EFFECTS OF AGING ON INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN RAT CEREBELLUM Values are mean _+S.E.M. of 8 animals/group./'~.2~= 5.82 fP = 0.0097t; *P = 0.02; F~.z~ = 22.62 (P = 0.0001l. **P _<0.001 compared with 3-month old rats (ANOVA, Schefle's F test of multiple comparisons). Age fmonthsl
3 12 25
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B (pmol/mg protein! ......
9.54 _+1.02 36.87 + 2.36 15.89 ; 1.66 22.03 +_ 1.96"* t9.35 ± 2.99* 20.78 +- 1 t0"* .........................................
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1.053 4_0.1)09 1.045 +_0.0t8 1.008 +_0.004
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InsP3 Concentration, [ nM ]
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[3H]lnsP3 Bound (pmol/mg protein)
Fig. 2. Saturation-binding isotherms (A) of [3H]InsP3 in cerebellar particulate membrane preparations derived from 3-, 12- and 25-month-old rats. Each datum point represents mean + S.E.M. of eight independent experiments performed in triplicate. B shows Scatchard (Rosenthalt plot transformations of data in A (see Table I for values).
linear in all cases (Fig. 2B). Similarly, corresponding Hill coefficients (nn) were close to unity (Table I), showing that no cooperativity was involved in InsP_~-binding. Thus, both methods of analysis indicated the presence of a homogenous population of InsP3R in the cerebellum of the three age groups studied. Receptor densities (B.... ) were significantly reduced by - 40 and 44% in 12- and 25month-old rats, respectively (Table I). Although InsP3 bound to cerebellar InsP~R with a high affinity (Ka) in all age groups, the Kd value was significantly decreased by ~103% in the 25-month-old senescent rats only (Table 1) compared with the 3-month-old animals. The present study using Fischer 344 rats gave similar Kd values as those obtained using Wistar rats [14]. Thus, the differences between the present data and the Wistar rat data with respect to age-dependent changes in both Bma x and Kd values could only reflect strain differences. It is likely that the reductions in InsP3R density at 12 and 25 months of age found in the present study is related to growth and maturation of neurons containing these receptors although cerebellar InsP3R density has been shown to reach adult values 28 days postnatal [14]. As cerebellar InsP3R is highly localized in Purkinje cells [10, 24], decrease in cerebellar InsP3R density at 12 and 25 months of age may be related to age-dependent
loss of Purkinje cells [22]. Thus, reduction in InsP3R density, as shown in the present study, would suggest a decrease in the number of lnsP3-activatable Ca -'+ channels in the cerebellum. The present data also suggest that aging causes a transformation of InsP3R from a high-affinity state to a lower-affinity state, a conversion which may play a role in spatio-temporal oscillations of Ca 2+ signals during aging [11]. The heterogeneity of cerebellar InsP~R subtypes, possibly with a range of affinities for InsP3. has recently been shown using recombinant cDNA technology [23]. Speculatively, aging may modulate the expression of one receptor type over the other, resulting in a change in affinity. Curiously, the binding data for all three ages consistently indicated only a single type of receptor. This discrepancy may be due to the use of tritiated ligand which may not be sensitive enough to delineate the affinities of InsP3R subtypes, Furthermore, considering the magnitude of age-dependent change in binding affinity determined in these studies (which is small), the decreased binding affinity may be indicative of the phosphorylation states [6, 28] of different InsP~R subtypes at different ages. Ratios of cerebellar ]nsP 3 content and InsP3R density increased with age. A positive and significant relation-
170
ship (y = 2.28 + 0.2147x, Pearson product moment coefficient, r = 0.691, n = 8 pairs/age group, P -< 0.005), exists between the ages of animals studied and changes in the ratios of InsP3 content and InsP3R density. The agedependent increase in this ratio strongly suggests a reduced ability of InsP3 to mobilize Ca 2+, even in the presence of high InsP 3 content, leading to attenuated Purkinje cell simple spike activity shown during aging [21]. In conclusion, the present findings provide evidence for an age-dependent modulation of phosphoinositidecoupled signal transduction process occurring beyond the receptor recognition sites. The attenuation of this second messenger system may contribute to the diminished neuronal responsiveness to the effects of neurotransmitters with aging [3]. The authors thank C. Nyquist-Battie for critical reading of the manuscript and K. Shaw for secretarial support. This study was supported by grants from the Scientific Education Partnership funded by Marion Merrell Dow Foundation and USPHS NIH AR41606.
1 Barritt, G.J., Intracellular free calcium and inositol polyphosphate action as potential targets in the aging process, Neurobiol. Aging, 8 (1987) 359-361. 2 Berridge, M.J., Inositol trisphosphate and diacylglycerol: two interacting second messengers, Annu, Rev. Biochem., 56 (1987) 159193. 3 Bickford-Wimer, EC., Hoffer, B.J. and Freedman, R., Interaction of norepinephrine with Purkinje cell responses to cerebellar afferent inputs in aged rats, Neurobiol. Aging, 6 0985) 89-94, 4 Bredt, D.S., Mourey, R.J. and Snyder, S.H., A simple, sensitive and specific radioreceptor assay for inositol 1,4,5-trisphosphate in biological tissues, Biochem. Biophys. Res: Commun., 159 (1989) 976-982. 5 Erneux, C. and Takazawa, K., Intracellular control of inositol phosphates by their metabolizing enzymes, Trends Pharmacol. Sci., 12 (1991) 174-176. 6 Ferris, C.D., Huganir, R.L., Bredt, D.S., Cameron, A.M. and Snyder, S.H., lnositot trisphosphate receptor: phosphorylation by protein kinase C and calcium calmodulin-dependent protein kinases in reconstituted lipid vesicles, Proc. Natl. Acad, Sci USA, 88 (t991 ) 2232-2235. 7 Ferris, C.D., Huganir, R.L., Supattapone, S. and Snyder, S.H., Purified inositol 1,4,5-trisphosphate receptor mediates calcium influx in reconstituted lipid vesicles, Nature (London), 342 (1989) 87-89. 8 Ferris, C.D. and Snyder, S., Inositol 1,4,5-trisphosphate-activated calcium channels, Annu. Rev, Physiol., 54 (1992) 469-488). 9 Furuichi, T., Miyawaki, A., Maeda, N., Nakade, S., Nakanishi, S., Nakagawa, T., Yoshikawa, S. and Mikoshiba, K., Structure and function of the inositol 1,4,5-trisphosphate receptor. In N. Kawai et al. (Eds.), Neuroreceptors, Ion Channels and Brain, Elsevier, Amsterdam, The Netherlands, 1992, pp. 103-111. l0 Furuichi, T., Yoshikawa, S., Miyawaki, A., Wad& K., Maeda, N. and Mikashiha, K., Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400, Nature (London), 342 (1989) 32-38.
I I Gibson. G.E. and Peterson, C., Calcium and the aging nervous s~stern, Neurobiol. Aging, 8 (1987) 329 343. 12 Hrusova, H., Strunecka, A., Piacentini, M.R. Accorsi, A. and Magnani, M., Red blood cell age-dependent moditications of inositol 1,4,5-trisphosphate, Mech. Aging Dev., 67 (1993) 13 19. 13 Khachaturian, Z.S., Hypothesis on the regulation of cytosol calcium concentration and the aging brain, Neurobiol. Aging, 8 (1987) 345 346. 14 Li, RR, Vecil, G.G., Green, M.A. and Warsh, J.J., lnositol 1,4,5trisphosphate receptor in developing and senescent rat cerebellum, Neurobiol. Aging, 13 (1991)8992. 15 Llinas, R and Suigimori, M., Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices, J. Physiol., 305 (1980) 197--213. 16 Mignery, G.A., Johnson, RA. and Sudhof, T.C., Mechanism of Ca -,+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar lnsP3 receptor, J. Biol. Chem., 267 (1992) 7450-7455. 17 Mignery, G.A.. Newton, C.L., Archer, B,T. and Sudhof, T.C., Structure and expression of the rat inositol 1,4,5-trisphosphate receptor, J. Biol. Chem., 265 (1990) 12679-12685. 18 Munson, RJ. and Rodbard, D., LIGAND: a versatile computerized approach for the characterization of ligand binding systems, Anat. Biochem., 107 (1980) 220-239. 19 Nahorski, S,R. and Potter, B.V.L., Molecular recognition of inositol polyphosphates by intracellular receptors and metabolic enzymes, Trends Pharmacol. Sci., 10 (1989) 139-,144. 20 Nishizuka, Y., Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 (1992) 607-614. 21 Rogers, J., Silver, M.A., Shoemaker, W.J. and Bloom, F.E., Senescent changes in a neurobiological model system: cerebellar Purkinje cell electrophysiology and correlative anatomy, Neurobiol. Aging, l (1980) 3-12. 22 Rogers, J., Zornetzer, S.F., Bloom, F.E. and Mervis, R.E., Senescent microstructural changes in rat cerebellum, Brain Res., 292 (1984) 23 32, 23 Ross, C.A., Danoff, S.K., Schell, M.J., Snyder, S.H. and Ullrich; A., Three additional inositol 1,4,5-trisphosphate receptors. Molecular cloning and differential localization in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 89 (1992) 4265~1269. 24 Ross, C.A., Meldolesi, J., Milner, T.A., Satoh, T., Supattapone, S. and Snyder, S.H., Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebeltar Purkinje neurons, Nature (London), 339 (1989) 468-470. 25 Sire, S.S., Kim, J.W. and Rhee, S.G., Regulation of o-myo-inositol 1,4,5-trisphosphate 3-kinase by cAMP-dependent kinase and protein kinase C, J. Biol. Chem., 265 (1990) 10367-10372. 26 Smith, EK., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.A., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Chem., 150 (1985) 76-85. 27 Taylor, W.C. and Potter, B.V.L., The size of inositol 1,4,5-trisphosphate sensitive Ca 2+ stores depends on inositol 1,4,5-trisphosphate concentration, Biochem. J., 266 (1990) 189-194. 28 Walaas, S.I., Nairin, A.C. and Greengard, P., PCPP-260, a Purkinje cell-specific cyclic AMP-regulated membrane phosphoprotein of Mt 260,000, J. Neurosci., 6 (1986) 954-961. 29 Worley, P.F., Baraban, J.M., Supattapone, S., Wilson, V.S. and Synder, S.H., Characterization of inositol trisphosphate receptor binding in brain, J. Biol. Chem., 262 (1987) 12132-12136. 30 Yarnada, M., Kakita, A., Mizuguchi, M., Rhee, S.G., Kim, S.U. and lkuta, F., Ultrastructural localization of Ins (1,4,5) P3 3-kinase in rat cerebellar cortex, Brain Res., 578 (1992) 41-48.
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Inositol 1,4,5-trisphosphate arm of the phosphatidylinositide signal transduction pathway in the rat cerebellum during aging Orisa J. Igwe*, Li Ning Department (~f Pharmacology, Schools of Pharmacy and Medicine, University Qf Missouri at Kansas City, M3--104. MED. 2411 Holmes Street. Kansas City. MO 64108 2792. USA (Received 2 June 1993; Revised version received 26 July 1993; Accepted 27 September 1993)
Key words." Aging; Phosphatidylinositide; Inositol 1,4,5-trisphosphate; Inositol 1,4,5-trisphosphate receptor: |ntracellular calcium regulation: Signal transduction To determine whether the intracellular calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (InsPd and its receptor (InsP3Rt display age-dependent coordinate regulation, InsP 3 content and [~HlInsPa-binding characteristics were investigated in cerebellar particulate membranes prepared from male Fischer 344 rats at 3, 12 and 25 months of age. Cerebellar lnsP 3 content was significantly increased in 25-month-old rats compared with 3-month-old animals, Cerebellar InsP3R densities were significantly reduced at 12 and 25 months of age but InsP~-binding affinity was significantly decreased only in the 25-month-old animals. The present data strongly suggest that modulation of the phospfioinositide second messenger system may contribute to impaired neuronal responsiveness associated with the aging process in the cerebellum.
As one of the proposed second messengers in the phosphatidylinositide (P1) transduction system, InsP3 links receptor activation with the release of nonmitochondrial stores of calcium [2] by binding to an InsP3specific receptor [8, 29], localized to portions of the endoplasmic reticulum in cerebellar Purkinje neurons [24]. The lnsP3 receptor (InsP3R) has been isolated [9], molecularly cloned [17] and functionally reconstituted into lipid vesicles [7]. Many processes associated with the PI second messenger system and calcium homeostasis have been reported to change with the aging process [1, 11]. Changes in PI turnover-calcium regulation during the aging process may be involved in the development of age-related alterations in neurological functions [13]. Calcium mobilization and phospholipid hydrolysis are intimately interrelated and sometimes complimentary to each other [20], thus, the size of InsP3-sensitive calcium stores appears to depend on InsP 3 concentration [27]. Given the central role of InsP 3 in calcium-signaling through binding to InsP3R [2], it is important to determine whether InsP3 content and InsP3R are coordinately regulated with age. The present study examines the effect of aging on InsP3 content and the binding characteristics of InsP3R in the rat cerebellum, a region with particularly high density of InsP3Rs [24]. *Corresponding author.
Male Fischer 344 rats of three age groups were used, i.e., 3-month-old (young virgins, 208 230 g body wt), 12-month-old (middle-aged retired breeders, 414-462 g body wt) and 25-month-old (senescent retired breeders, 412450 g body wt). All animals were obtained from the National Institute on Aging colonies (Harlan-Sprague Dawley, Indianapolis, IN) and housed in pairs in the animal holding area with a 12-h light-dark cycle. Animals were acclimatized to the holding area for 1 wk before use in these experiments. Water and food were made available ad libitum. On the day of the experiment, animals were weighed and then sacrificed by decapitation and exsanguination. The brains were removed immediately and chilled on ice. All subsequent procedures were conducted at 4°C. Cerebelli were dissected out on an ice-chilled glass plate and a sagittally cut portion from the right lateral side (< I0 mg wet wt) was taken for InsP 3 assay with the rest used for InsP3R characterization. Tissue was processed immediately for InsP3 contents by homogenization (Brinkman Polytron setting 8:8 s) in ice-cold 1 M trichloroacetic acid (TCA) containing 10 mM EDTA to minimize postdecapitation reactions. The homogenate was incubated on ice for 15 rain and then centrifuged at 1000 x g for 10 min at 4°C. The pellet was used to determine tissue protein content after solubilization in 1 M NaOH for 4 h at 37°C. The supernatant was incubated at room temperature for 15 min and InsP3 ex-
168 200
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Age (Months) Fig. 1. Effect of aging on content of inositol t,4,5-trisphosphate in rat cerebellum. Tissue lnsP~ in three age groups was extracted and quantified as described in text. Values are mean -2 S.E.M. with n = 8 ~ats/age group with experiments carried out in duplicates. ~.,~ =6.59 (P = 0.006), *P -< 0.05 (ANOVA, Scheffe'sF test of multiple comparisons). tracted with a 3/1 (v/v) mixture of F R E O N and tri-noctylamine. After T C A removal, InsP3 contained in the upper aqueous layer was quantified by using a simple, specific and sensitive radioreceptor assay kit (Dupont N E N , Wilmington, DE). The assay buffer used for the standards and blanks were also carried through the extraction procedure to make corrections for the samples. The characteristics of [3H]InsP3-binding were determined in cerebellar particulate membranes. Tissue was homogenized (Polytron setting 9; 5 s) in 10 vols. of icecold buffer A (50 m M Tris HCI, pH = 8.3, at 25°C, 2.5 m M E D T A and 1 m M dithiothreitolL pelleted by c e n t r i f ugation (39,000 x g for 10 min) and resuspended in the same buffer. Binding assays contained 100/11 ( - t 0 0 - 2 1 0 /,/g protein) of the particulate preparation and 0.2 nM (~10,000 dpm) of [3H]InsP~ (spec. act. = 3 4 4 4 Ci/mmol; Amersham, Arlington, ILl with varying concentrations of unlabeled InsP3 in a total incubation volume of 0.5 ml of buffer A. Assays were incubated in microcentrifuge tubes for 40 rain at 4°C and the reaction stopped by centrifugation at 12,000 x g for 5 rain, followed by aspiration o f the supernatant. Tissue pellets were dissolved overnight in 250/Jl Soluene 350 (Packard Instrument, Meriden, CT), followed by addition of 4 ml Hionic-Fluor
(Packard lnsli-ument) scintillaticm cocktail and the radb oactivity determined by liquid scintillation spectroscop$, Nonspecilic binding was determined ill lhc plCSCllCC ~'~i I/aM unlabeled InsP3 and was generally .... 10% oI" total binding. Receptor density (B ...... ). binding affinity {Kd) and Hill coefficient (nil) were deterntined using an iteratire curve-fitting program L I G A N D 1181, The B ...... values were standardized to tissue protein content determined by the bicinchoninic acid method [26], using bovine serum albumin as the s t a n d a r d Fig. 1 shows that cerebellar InsP 3 contents were increased by ~ 44 and 76% (P -< 0.05) in 12- and 25-monthold rats, respectively, compared with the 3-month-old animals. Accumulation of lnsP~ was used in this study as an index of PIP, turnover. As tissues were processed within a short time (< 2 min) alter animal sacrifice, postdecapitation changes could not account for the differences in lnsP~ content determined in the present study [41. A number of plausible explanations could account for the age-dependent increase in cerebellar lnsP3 content. First, the aging process may attenuate the regulation of cerebellar PI metabolism resulting in altered Ca > homeostasis, as dendritic spike bursts of Purkinje cells are modulated by both Ca e+ and Pt turnover {l 5]. The intracellular Ca ~'~ levels in Purkinje cells of aging cerebellum may increase due to mobilization of Ca 2* from 'leaky' plasma membranes and/or Ca -,+ channels, resulting in both increased tissue InsP~ accumulation and decreased lnsP~-binding (negative feed back). This apparent paradox can be explained on the basis of the activation of phospholipase C (PLC) under situations of increased Ca > levels [16]. Recently, age-dependent increase in InsP~ content in red blood cells was reported [12]. Another possible mechanism for age-dependent increase in intracellular levels of InsP3 may lie in altered regulation of InsP3 3-kinase [25], an lnsP, metabolizing enzyme [5], found in abundance in cerebellar Purkinje cells [30]. Saturable InsP3-binding was found in all cerebellar particulate membranes examined (Fig. 2A). Scatchard were TABLE 1 EFFECTS OF AGING ON INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN RAT CEREBELLUM Values are mean _+S.E.M. of 8 animals/group./'~.2~= 5.82 fP = 0.0097t; *P = 0.02; F~.z~ = 22.62 (P = 0.0001l. **P _<0.001 compared with 3-month old rats (ANOVA, Schefle's F test of multiple comparisons). Age fmonthsl
3 12 25
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B (pmol/mg protein! ......
9.54 _+1.02 36.87 + 2.36 15.89 ; 1.66 22.03 +_ 1.96"* t9.35 ± 2.99* 20.78 +- 1 t0"* .........................................
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1.053 4_0.1)09 1.045 +_0.0t8 1.008 +_0.004
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[3H]lnsP3 Bound (pmol/mg protein)
Fig. 2. Saturation-binding isotherms (A) of [3H]InsP3 in cerebellar particulate membrane preparations derived from 3-, 12- and 25-month-old rats. Each datum point represents mean + S.E.M. of eight independent experiments performed in triplicate. B shows Scatchard (Rosenthalt plot transformations of data in A (see Table I for values).
linear in all cases (Fig. 2B). Similarly, corresponding Hill coefficients (nn) were close to unity (Table I), showing that no cooperativity was involved in InsP_~-binding. Thus, both methods of analysis indicated the presence of a homogenous population of InsP3R in the cerebellum of the three age groups studied. Receptor densities (B.... ) were significantly reduced by - 40 and 44% in 12- and 25month-old rats, respectively (Table I). Although InsP3 bound to cerebellar InsP~R with a high affinity (Ka) in all age groups, the Kd value was significantly decreased by ~103% in the 25-month-old senescent rats only (Table 1) compared with the 3-month-old animals. The present study using Fischer 344 rats gave similar Kd values as those obtained using Wistar rats [14]. Thus, the differences between the present data and the Wistar rat data with respect to age-dependent changes in both Bma x and Kd values could only reflect strain differences. It is likely that the reductions in InsP3R density at 12 and 25 months of age found in the present study is related to growth and maturation of neurons containing these receptors although cerebellar InsP3R density has been shown to reach adult values 28 days postnatal [14]. As cerebellar InsP3R is highly localized in Purkinje cells [10, 24], decrease in cerebellar InsP3R density at 12 and 25 months of age may be related to age-dependent
loss of Purkinje cells [22]. Thus, reduction in InsP3R density, as shown in the present study, would suggest a decrease in the number of lnsP3-activatable Ca -'+ channels in the cerebellum. The present data also suggest that aging causes a transformation of InsP3R from a high-affinity state to a lower-affinity state, a conversion which may play a role in spatio-temporal oscillations of Ca 2+ signals during aging [11]. The heterogeneity of cerebellar InsP~R subtypes, possibly with a range of affinities for InsP3. has recently been shown using recombinant cDNA technology [23]. Speculatively, aging may modulate the expression of one receptor type over the other, resulting in a change in affinity. Curiously, the binding data for all three ages consistently indicated only a single type of receptor. This discrepancy may be due to the use of tritiated ligand which may not be sensitive enough to delineate the affinities of InsP3R subtypes, Furthermore, considering the magnitude of age-dependent change in binding affinity determined in these studies (which is small), the decreased binding affinity may be indicative of the phosphorylation states [6, 28] of different InsP~R subtypes at different ages. Ratios of cerebellar ]nsP 3 content and InsP3R density increased with age. A positive and significant relation-
170
ship (y = 2.28 + 0.2147x, Pearson product moment coefficient, r = 0.691, n = 8 pairs/age group, P -< 0.005), exists between the ages of animals studied and changes in the ratios of InsP3 content and InsP3R density. The agedependent increase in this ratio strongly suggests a reduced ability of InsP3 to mobilize Ca 2+, even in the presence of high InsP 3 content, leading to attenuated Purkinje cell simple spike activity shown during aging [21]. In conclusion, the present findings provide evidence for an age-dependent modulation of phosphoinositidecoupled signal transduction process occurring beyond the receptor recognition sites. The attenuation of this second messenger system may contribute to the diminished neuronal responsiveness to the effects of neurotransmitters with aging [3]. The authors thank C. Nyquist-Battie for critical reading of the manuscript and K. Shaw for secretarial support. This study was supported by grants from the Scientific Education Partnership funded by Marion Merrell Dow Foundation and USPHS NIH AR41606.
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