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calmodulin-dependent protein phosphorylation in rat neostriatal slices
Brain Research, 545 (1991) 343-346 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 0006899391246026
343
BRES 24602
Neurotensin effects on calcium/calmodulin-dependent protein phosphorylation in rat neostriatal slices J o h n Kasckow 1, Scott T. Cain I and Charles B. N e m e r o f f 1'2 1Departments of Psychiatry and 2Pharmacology, Duke University Medical Center, Durham, NC 27710 (U.S.A.)
(Accepted 8 January 1991) Key words: Neurotensin; Protein phosphorylation; Calcium; Calmodulin; Neostriatum; Brain slice
Neurotensin (NT) is an endogenous brain tridecapeptide for which high affinity binding sites exist in the central nervous system. We have investigated the effects of NT incubation with rat neostriatal slices on calcium/calmodulin (Ca/CaM)-dependent protein phosphorylation. Slices were incubated with NT (5 or 50 nM) for 3, 10, 16 or 30 min followed by in vitro phosphorylation, electrophoresis and autoradiography. NT significantly altered the phosphorylation of a 62 kDa protein which is likely the fl subunit of the Ca/CaM dependent protein kinase. These changes may reflect the ability of NT to influence calcium mediated signal transduction. Neurotensin (NT) is a tridecapeptide known to exhibit many of the characteristics of a neurotransmitter 6. For example, in the CNS, NT binds to a high affinity, saturable receptor site 1°'12. In whole brain slices, Goedert et al. (1984) demonstrated that there was a significant correlation between the magnitude of NT-stimulated phosphatidylinositol hydrolysis and the number of specific NT binding sites in various brain regions 9. Bozou et al. (1989) reported that, in HT29 cells, NT receptors are linked to calcium mobilization but not to activation of the calcium/phospholipid-dependent protein kinase C 4. Furthermore, Battaini et al. (1986) demonstrated that NT increases the permeability of membrane calcium channels in rat striatai slices2. It is becoming increasingly apparent, therefore, that calcium-mediated processes are critically involved in the intracellular mediation of the NT extracellular signal. The reversible phosphorylation of specific CNS proteins has been implicated in numerous physiologic processes including neurotransmitter release, modulation of ionic channel function and neurotransmitter synthesis 8,17. Specific phosphoprotein substrates which mediate these events are phosphorylated through the actions of a variety of calcium dependent protein kinases including calcium/calmodulin (Ca/CaM)- and calcium/phospholipid-dependent protein kinases. Thus, in view of the evidence implicating calcium mobilization as an important component of NT signal transmission, we have studied the effects of NT on Ca/CaM-dependent protein phosphorylation in rat neostriatal slices.
Male Sprague-Dawley rats were killed by decapitation. The neostriatum was rapidly dissected out and sliced into 300-400/~m sections using a Mcllwain tissue chopper. Slices were placed into artificial cerebrospinal fluid (CSF) consisting of 124 mM NaCI, 2.5 mM KCI, 1.25 mM KHzPO4, 2 mM MgSO4, 2 mM CaC12, 25 mM N a H C O 3, 11 mM glucose, 0.2 mM bacitracin, and 0.1% bovine serum albumin oxygenated with 95% 02/5% CO2. Slices were preincubated for 45 min at 35 °C. They were then incubated in artificial CSF in the presence of NT (5 or 50 nM) or vehicle (Bachem, Inc., Torrance, CA) for 3, 10, 16 or 30 min. For each time and dose of NT treatment there was a corresponding paired control. Following incubation, slices were immediately homogenized in cold 150 mM HEPES, 3.0 mM E G T A , pH 7.1 and rapidly frozen. A portion of the homogenate was used for protein determination 14. The remaining homogenate was then diluted to a concentration of 2.4 mg protein/ml prior to phosphorylation. Aliquots of the diluted homogenate were preincubated at 30 °C for 1 min and 40 s followed by addition of second messenger or vehicle for 20 s. Samples were then phosphorylated for 1 min with 10/~M ATP (containing [y32p]ATP; 4500 Ci/mmole). The final reaction contained 50 mM HEPES, 1 mM E G T A , 7.2 mM MgCI 2 with 0.8 /,M calmodulin/0.5 mM CaCI2, 20/~M 8-bromo-cAMP or vehicle. The reaction was quenched with an SDS-stop solution so that final concentrations were 3% SDS, 1.9% glycerol, 2.25% fl-mercaptoethanol, 0.375 M Tris, pH 6.9.
Correspondence: S.T. Cain, Duke University Medical Center, Box 3859, Durham, NC 27710, U.S.A.
344 MW x 10 "3
!
205
TABLE I N T incubation with neostriatal slices: effects on Ca~CaM-dependent protein phosphorylation
~116
62 kDa Phosphoprotein
50 kDa Phosphoprotein
5nM
66 ~ " 62
m
50 ~
45 36 29
i
V
Ca/CaM
I
I
V
Ca/CaM
3" 10" 16"
$ 32.7 + 9.14 $ 58.3 + 3.37** 1' 164 ___26.5*
1'33 + 32.9 t 80 + 29.6 1' 148 + 20.5*
50 nM
3" 10" 16"
~"251 + 45.9* 1' 37 +_31 ~ 26.8 + 3.21"
1'42 + 27.8 1' 19 + 28.8 ~ 32.2 + 14.9
Slices were incubated with NT and then phosphorylated in the presence of Ca/CaM. This was followed by electrophoresis and autoradiography as described in the text. The magnitude of Ca/CaM-dependent stimulation following NT incubation and control incubation at each dose and time were compared using Student's t-test (n = 3 independent experiments at each time and dose). Data is expressed as % difference from Ca/CaM stimulation following control incubation as a function of NT dose and incubation time. *P < 0.05; **P < 0.01.
J
Neurotensin Fig. 1. Autoradiographs illustrating Ca/CaM stimulated phosphorylation of rat neostriatal slices following incubation with 50 nM NT for 3 min. Proteins were phosphorylated in vitro and separated on 9-14% polyacrylamide gels as described in the text. Gels were vacuum-dried and autoradiograms prepared. Arrows depict migration of the 62 and 50 kDa phosphoprotein. Other numbers represent electrophoretic migration of known molecular weight standards. The first lane is control/vehicle; the second lane is control/Ca/CaM. The third lane is NT/vehicle and the fourth lane is NT/Ca/CaM. Ca/CaM stimulation of the 62 kDa protein was increased following 50 nM NT incubation for 3 min. C ontrol
Phosphorylated samples were boiled and proteins separated on a 9 - 1 4 % gradient SDS polyacrylamide gel. Gels were fixed and stained in 40% methanol/10% acetic acid containing 0.1% Coomassie blue. They were destained in 20% methanol/10% acetic acid and vacuumdried. Autoradiograms were prepared from the dried gels. Phosphate incorporation into specific proteins was analyzed by microdensitometry and phosphate incorporation quantitated by peak height measurement. A representative autoradiograph illustrating both Ca/ CaM-dependent protein phosphorylation and the effects of prior incubation of neostriatal slices with 50 nM NT for 3 min is provided in Fig. 1. Note that Ca/CaM stimulated the phosphorylation of proteins with mol. wt. of 86, 80, 62, 55, 50 and 45 k D a (lane 1 vs lane 2). Based on molecular weights and sensitivity to Ca/CaM, tentative identifications of certain of these proteins can be made. The 86 and 80 k D a proteins are likely synapsin Ia and Ib 22. The 62 and 50 k D a proteins are likely the fl and a subunits, respectively, of the Ca/CaM-dependent protein kinase 1116'2°. We have noted that c A M P does not
stimulate the phosphorylation of the 62 or 50 k D a proteins (data not shown). It is apparent from the autoradiograph (Fig. 1) that the magnitude of Ca/CaM stimulation of the 62 k D a protein is increased following 50 nM N T incubation for 3 min (see lanes 3 and 4 vs lanes 1 and 2). Quantitation of the time course and dose effects of NT on Ca/CaM-dependent phosphorylation is provided in Table I. After 3 min of exposure to 50 nM NT, Ca/CaM stimulation of the 62 kDa phosphoprotein in the NT-treated group was 251 + 45.9% greater than Ca/CaM-stimulation in the control group (P < 0.05). After 10 min of incubation with NT, Ca/CaM stimulation was still increased (1' 37 + 31%) although the difference was not statistically significant. However, after 16 min of exposure to NT, the amount of Ca/CaM stimulation of the 62 k D a protein was significantly attenuated relative to Ca/CaM stimulation following control incubations [ $ 26.8 + 3.21% (P < 0.02)]. In contrast to the stimulation of Ca/CaM-dependent phosphorylation of the 62 k D a protein we observed following incubation with 50 nM NT, incubation with 5 nM N T for 3 and 10 min resulted in reduced Ca/CaM-dependent 62 kDa phosphorylation [$ 32.7 + 9.14% (P < 0.1) and 58.3 + 3.37% (P < 0.01), respectively]. However, after 16 min of incubation with 5 nM NT, the magnitude of Ca/CaM-dependent stimulation of the 62 k D a protein was increased relative to samples incubated with control buffer [1' 164 + 26.5% (P < 0.05)]. By 30 min, Ca/CaM-stimulation returned to control values with both NT doses. As the 50 k D a phosphoprotein is likely the a subunit of Ca/CaM-dependent protein kinase II, we examined
345 whether NT-induced changes in the phosphorylation of this protein paralleled those observed in the 62 kDa protein. Consistent with NT-induced alterations in 62 kDa phosphorylation, the 50 kDa protein exhibited significant increases in Ca/CaM-stimulated phosphorylation following a 16-min incubation with 5 nM NT [ t 148 + 20.5% P < 0.05. (See Table I)]. In addition, with 50 nM NT there was a trend in 50 kDa phosphorylation that paralleled that of the 62 kDa protein at 3 min ( 1' 42 + 27.8%), 10 min ( 1' 19 + 28.8%) and 16 min ( ~ 32.2 + 14.9%). We have described here our findings that incubation with 50 and 5 nM NT altered the post-hoe calciumdependent phosphorylation of specific proteins. Although the relative role of NT-modulated protein kinase and protein phosphatase activities cannot be definitively concluded from these post-hoc phosphporylation experiments, it should be mentioned that the phosphorylation assay conditions employed in these experiments reflect primarily the rate of phosphate incorporation (i.e. protein kinase activity) 23'24. The effective concentrations of NT are within the ranges of NT binding to both low (K d = 2.85 nM) and high affinity (K d = 0.1-0.3 nM) sites in rat brain membranes 12, as well as within the range of the EC5o for the excitatory electrophysiological action of NT in the ventral tegmental area (35 nM) 21. In addition to the ventral tegmental area, NT exerts an excitatory effect on frontal cortical ~, substantia nigra TM, and periaqueductal gray neurons 3. It will be of interest to determine if NT effects on calcium-dependent phosphorylation mediate the electrophysiological actions of the peptide. Based on molecular weight, stimulation of phosphorylation by Ca/CaM, and insensitivity to cAMP, we have suggested that the 62 and 50 kDa proteins are the fl and a subunits, resp., of Ca/CaM-dependent protein kinase II. Autophosphorylation of Ca/CaM kinase II converts the enzyme to a calcium-independent state, thereby
altering the rate of substrate phosphorylation. In vitro autophosphorylation of the Ca/CaM kinase II has also been reported to be associated with translocation of the enzyme from a cytoskeletal form to a soluble form 19'2°. Our results may, therefore, reflect the ability of NT to both alter the activity and induce translocation of the Ca/CaM kinase II. The Ca/CaM-dependent protein kinase II is known to regulate neurotransmitter release at the synapse 13 and, furthermore, NT has been shown to modulate dopamine release in cat, rabbit and rodent striatal slices s'7'~5. Thus, altered phosphorylation of the 62 kDa and/or 50 kDa proteins may be one of the mechanisms through which NT exerts its effects on dopamine release. It is noteworthy that the time course of the phosphorylation response to NT was reversed at the high relative to the low dose. It is not clear whether this reflects a time shift in the dose response curve or, alternatively, is an indication that the two doses of NT differentially influence cellular responsiveness, perhaps by acting through the high (5 nM) and low affinity (50 nM) NT receptor. In this regard, it is interesting that, depending on concentration, NT exerts either an excitatory (high concentration) or inhibitory (low concentration) effect on muscle contraction in the guinea pig ileum ~1. We are currently attempting to verify the identity of the 62 and 50 kDa proteins as subunits of CaM kinase II and delineate the relative roles of NT-sensitive kinases and/or phosphatases in the observed effects. Functional studies will focus on evaluating NT dose response effects on both protein phosphorylation and dopamine release in neostriatal tissue.
We would like to thank Ward Virts and Sharon Rhoden for preparation of this manuscript. Research was supported by a National Alliance for Research on Schizophrenia and Depression (NARSAD) Award to Scott T. Cain, NSF BNS-8910032 and NIMH MH-39415.
1 Audinat, E., Hermel, J.-M. and Crepel, E., Neurotensininduced excitation of neuroris of the rat's frontal cortex studied intraceUularly in vitro, Exp. Brain Res., 78 (1989) 358-368. 2 Battaini, E, Govoni, S., DiGiovine, S. and Trabucchi, M., Neuroteusin effects on dopamine release and calcium transport in rat striatum: interactions with diphenylalkylamine calcium antagonists, Naunyn-Schmiedeberg's Arch. Pharmacol., 332 (1986) 267-270. 3 Behbehani, M.M., Shipley, M. and McLean, J.H., Effect of neurotensin on neurons in the periaqueductal gray: an in vitro study, J. Neurosci., 7 (1987) 2035-2040. 4 Bozou, J.C., Rochet, N., Magnaldo, I., Vincent, J.P. and Kitabgi, P., Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT 29 cells. Involvement of a G protein, Biochem. J., 264 (1989) 871-878. 5 de Quidt, M.E. and Emson, P.C., Neurotensin facilitates dopamine release in vitro from rat striatal slices, Brain Research, 274 (1983) 376-380.
6 Ervin, G.N. and Nemeroff, C.B., Interactions of neurotensin with dopamine-containing neurons in the central nervous system, Prog. Neuropsychopharmacol. Biol. Psychiatry, 12 (1988) $53-$69. 7 Faggin, B.M., Zubieta, J.K., Rezvani, A.H. and Cubeddu, L.X., Neurotensin-induced dopamine release in vivo and in vitro from substantia nigra and nucleus caudate, J. Pharmacol. Exp. Ther., 264 (1990) 817-825. 8 Gispen, W.H. and Routtenberg, A. (Eds.), Phosphoproteins in Neuronal Function. Progress in Brain Research, Vol. 69, Elsevier, Amsterdam, 1986. 9 Goedert, M., Pinnock, R.D., Downes, C.P., Mantyh, P.W. and Emson, P.C., Neurotensin stimulates inositol phospholipid hydrolysis in rat brain slices, Brain Research, 323 (1984) 193-197. 10 Goedert, M., Pittaway, K., Williams, B.J. and Emson, P.C., Specific binding of tritiated neurotensin to rat brain membranes: characterization and regional distribution, Brain Research, 304 (1984) 71-81.
346 11 Huidoboro-Toro, J.P. and Zhu, Y.-X., Neurotensin receptors on
the ileum of the guinea-pig: evidence for the coexistence of inhibitory and excitatory receptors, Eur. J. Pharmacol., 102 (1984) 237-250. 12 Kitabgi, P., Checler, E, Mazella, J. and Vincent, J.P., Pharmacology and biochemistry of neurotensin receptors, Rev. Clin. Bas. Pharmacol., 5 (1985) 397-486. 13 Llinas, R., McGuiness, T.L., Leonard, C.S., Sugimori, M. and Greengard, P., Intraterminal injection of calcium/calmodulin dependent protein kinase II alters neurotransmitter release at the giant squid synapse, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3035-3039. 14 Lowry, O.H., Rosebrough, M.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 15 Markstein, R. and Emson, P., Effect of neurotensin and its fragments neurotensin (1-6) and neurotensin (8-13) on dopamine release from cat striatum, Eur. J. Pharmacol., 152 (1988) 147-152. 16 Miller, S.G. and Kennedy, M.B., Distinct forebrain and cerebellar isozymes of type II Ca2÷/calmodulin-dependent protein kinase associate differently with the postsynaptic density fraction, J. Biol. Chem., 260 (1985) 9039-9046. 17 Nestler, E.J. and Greengard, P., Protein Phosphorylation in the Nervous System, John Wiley and Sons, New York, 1984. 18 Pinnock, R.D., Neurotensin depolarizes substantia nigra dopa-
mine neurons, Brain Research, 338 (1985) 151-154. 19 Saitoh, T. and Schwartz, J.H., Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme in Aplysia neurons, J. Cell Biol., 100 (1985) 835-842. 20 Schulman, H., The multifunctional Ca2+/calmodulin dependent protein kinase. In P. Greengard and G.A. Robinson (Eds.), Second Messenger and Phosphoprotein Research, Vol. 22, Raven, New York, 1988. 21 Seutin, V., Massotte, L. and Dresse, A., Electrophysiological effects of neurotensin on dopaminergic neurons of the ventral tegmental area of the rat in vitro, Neuropharmacology, 28 (1989) 949-954. 22 Sudhoff, T.C., Czernik, A.J., Kao, H.T., Takei, K., Johnston, P.A., Horiuchi, A., Kanazir, S.D., Wagner, M.A., Perin, M.S., de Camilli, P. and Greengard, E, Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins, Science, 245 (1989) 1474-1480. 23 Wiegant, V.M., Zwiers, H., Schotman, P. and Gispen, W.H., Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: some methodological aspects, Neurochem. Res., 3 (1978) 443-453. 24 Zwiers, H., Wiegant, V.M., Schotman, P. and Gispen, W.H., ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: structure-activity, Neurochem. Res., 3 (1978) 455-463.
343
BRES 24602
Neurotensin effects on calcium/calmodulin-dependent protein phosphorylation in rat neostriatal slices J o h n Kasckow 1, Scott T. Cain I and Charles B. N e m e r o f f 1'2 1Departments of Psychiatry and 2Pharmacology, Duke University Medical Center, Durham, NC 27710 (U.S.A.)
(Accepted 8 January 1991) Key words: Neurotensin; Protein phosphorylation; Calcium; Calmodulin; Neostriatum; Brain slice
Neurotensin (NT) is an endogenous brain tridecapeptide for which high affinity binding sites exist in the central nervous system. We have investigated the effects of NT incubation with rat neostriatal slices on calcium/calmodulin (Ca/CaM)-dependent protein phosphorylation. Slices were incubated with NT (5 or 50 nM) for 3, 10, 16 or 30 min followed by in vitro phosphorylation, electrophoresis and autoradiography. NT significantly altered the phosphorylation of a 62 kDa protein which is likely the fl subunit of the Ca/CaM dependent protein kinase. These changes may reflect the ability of NT to influence calcium mediated signal transduction. Neurotensin (NT) is a tridecapeptide known to exhibit many of the characteristics of a neurotransmitter 6. For example, in the CNS, NT binds to a high affinity, saturable receptor site 1°'12. In whole brain slices, Goedert et al. (1984) demonstrated that there was a significant correlation between the magnitude of NT-stimulated phosphatidylinositol hydrolysis and the number of specific NT binding sites in various brain regions 9. Bozou et al. (1989) reported that, in HT29 cells, NT receptors are linked to calcium mobilization but not to activation of the calcium/phospholipid-dependent protein kinase C 4. Furthermore, Battaini et al. (1986) demonstrated that NT increases the permeability of membrane calcium channels in rat striatai slices2. It is becoming increasingly apparent, therefore, that calcium-mediated processes are critically involved in the intracellular mediation of the NT extracellular signal. The reversible phosphorylation of specific CNS proteins has been implicated in numerous physiologic processes including neurotransmitter release, modulation of ionic channel function and neurotransmitter synthesis 8,17. Specific phosphoprotein substrates which mediate these events are phosphorylated through the actions of a variety of calcium dependent protein kinases including calcium/calmodulin (Ca/CaM)- and calcium/phospholipid-dependent protein kinases. Thus, in view of the evidence implicating calcium mobilization as an important component of NT signal transmission, we have studied the effects of NT on Ca/CaM-dependent protein phosphorylation in rat neostriatal slices.
Male Sprague-Dawley rats were killed by decapitation. The neostriatum was rapidly dissected out and sliced into 300-400/~m sections using a Mcllwain tissue chopper. Slices were placed into artificial cerebrospinal fluid (CSF) consisting of 124 mM NaCI, 2.5 mM KCI, 1.25 mM KHzPO4, 2 mM MgSO4, 2 mM CaC12, 25 mM N a H C O 3, 11 mM glucose, 0.2 mM bacitracin, and 0.1% bovine serum albumin oxygenated with 95% 02/5% CO2. Slices were preincubated for 45 min at 35 °C. They were then incubated in artificial CSF in the presence of NT (5 or 50 nM) or vehicle (Bachem, Inc., Torrance, CA) for 3, 10, 16 or 30 min. For each time and dose of NT treatment there was a corresponding paired control. Following incubation, slices were immediately homogenized in cold 150 mM HEPES, 3.0 mM E G T A , pH 7.1 and rapidly frozen. A portion of the homogenate was used for protein determination 14. The remaining homogenate was then diluted to a concentration of 2.4 mg protein/ml prior to phosphorylation. Aliquots of the diluted homogenate were preincubated at 30 °C for 1 min and 40 s followed by addition of second messenger or vehicle for 20 s. Samples were then phosphorylated for 1 min with 10/~M ATP (containing [y32p]ATP; 4500 Ci/mmole). The final reaction contained 50 mM HEPES, 1 mM E G T A , 7.2 mM MgCI 2 with 0.8 /,M calmodulin/0.5 mM CaCI2, 20/~M 8-bromo-cAMP or vehicle. The reaction was quenched with an SDS-stop solution so that final concentrations were 3% SDS, 1.9% glycerol, 2.25% fl-mercaptoethanol, 0.375 M Tris, pH 6.9.
Correspondence: S.T. Cain, Duke University Medical Center, Box 3859, Durham, NC 27710, U.S.A.
344 MW x 10 "3
!
205
TABLE I N T incubation with neostriatal slices: effects on Ca~CaM-dependent protein phosphorylation
~116
62 kDa Phosphoprotein
50 kDa Phosphoprotein
5nM
66 ~ " 62
m
50 ~
45 36 29
i
V
Ca/CaM
I
I
V
Ca/CaM
3" 10" 16"
$ 32.7 + 9.14 $ 58.3 + 3.37** 1' 164 ___26.5*
1'33 + 32.9 t 80 + 29.6 1' 148 + 20.5*
50 nM
3" 10" 16"
~"251 + 45.9* 1' 37 +_31 ~ 26.8 + 3.21"
1'42 + 27.8 1' 19 + 28.8 ~ 32.2 + 14.9
Slices were incubated with NT and then phosphorylated in the presence of Ca/CaM. This was followed by electrophoresis and autoradiography as described in the text. The magnitude of Ca/CaM-dependent stimulation following NT incubation and control incubation at each dose and time were compared using Student's t-test (n = 3 independent experiments at each time and dose). Data is expressed as % difference from Ca/CaM stimulation following control incubation as a function of NT dose and incubation time. *P < 0.05; **P < 0.01.
J
Neurotensin Fig. 1. Autoradiographs illustrating Ca/CaM stimulated phosphorylation of rat neostriatal slices following incubation with 50 nM NT for 3 min. Proteins were phosphorylated in vitro and separated on 9-14% polyacrylamide gels as described in the text. Gels were vacuum-dried and autoradiograms prepared. Arrows depict migration of the 62 and 50 kDa phosphoprotein. Other numbers represent electrophoretic migration of known molecular weight standards. The first lane is control/vehicle; the second lane is control/Ca/CaM. The third lane is NT/vehicle and the fourth lane is NT/Ca/CaM. Ca/CaM stimulation of the 62 kDa protein was increased following 50 nM NT incubation for 3 min. C ontrol
Phosphorylated samples were boiled and proteins separated on a 9 - 1 4 % gradient SDS polyacrylamide gel. Gels were fixed and stained in 40% methanol/10% acetic acid containing 0.1% Coomassie blue. They were destained in 20% methanol/10% acetic acid and vacuumdried. Autoradiograms were prepared from the dried gels. Phosphate incorporation into specific proteins was analyzed by microdensitometry and phosphate incorporation quantitated by peak height measurement. A representative autoradiograph illustrating both Ca/ CaM-dependent protein phosphorylation and the effects of prior incubation of neostriatal slices with 50 nM NT for 3 min is provided in Fig. 1. Note that Ca/CaM stimulated the phosphorylation of proteins with mol. wt. of 86, 80, 62, 55, 50 and 45 k D a (lane 1 vs lane 2). Based on molecular weights and sensitivity to Ca/CaM, tentative identifications of certain of these proteins can be made. The 86 and 80 k D a proteins are likely synapsin Ia and Ib 22. The 62 and 50 k D a proteins are likely the fl and a subunits, respectively, of the Ca/CaM-dependent protein kinase 1116'2°. We have noted that c A M P does not
stimulate the phosphorylation of the 62 or 50 k D a proteins (data not shown). It is apparent from the autoradiograph (Fig. 1) that the magnitude of Ca/CaM stimulation of the 62 k D a protein is increased following 50 nM N T incubation for 3 min (see lanes 3 and 4 vs lanes 1 and 2). Quantitation of the time course and dose effects of NT on Ca/CaM-dependent phosphorylation is provided in Table I. After 3 min of exposure to 50 nM NT, Ca/CaM stimulation of the 62 kDa phosphoprotein in the NT-treated group was 251 + 45.9% greater than Ca/CaM-stimulation in the control group (P < 0.05). After 10 min of incubation with NT, Ca/CaM stimulation was still increased (1' 37 + 31%) although the difference was not statistically significant. However, after 16 min of exposure to NT, the amount of Ca/CaM stimulation of the 62 k D a protein was significantly attenuated relative to Ca/CaM stimulation following control incubations [ $ 26.8 + 3.21% (P < 0.02)]. In contrast to the stimulation of Ca/CaM-dependent phosphorylation of the 62 k D a protein we observed following incubation with 50 nM NT, incubation with 5 nM N T for 3 and 10 min resulted in reduced Ca/CaM-dependent 62 kDa phosphorylation [$ 32.7 + 9.14% (P < 0.1) and 58.3 + 3.37% (P < 0.01), respectively]. However, after 16 min of incubation with 5 nM NT, the magnitude of Ca/CaM-dependent stimulation of the 62 k D a protein was increased relative to samples incubated with control buffer [1' 164 + 26.5% (P < 0.05)]. By 30 min, Ca/CaM-stimulation returned to control values with both NT doses. As the 50 k D a phosphoprotein is likely the a subunit of Ca/CaM-dependent protein kinase II, we examined
345 whether NT-induced changes in the phosphorylation of this protein paralleled those observed in the 62 kDa protein. Consistent with NT-induced alterations in 62 kDa phosphorylation, the 50 kDa protein exhibited significant increases in Ca/CaM-stimulated phosphorylation following a 16-min incubation with 5 nM NT [ t 148 + 20.5% P < 0.05. (See Table I)]. In addition, with 50 nM NT there was a trend in 50 kDa phosphorylation that paralleled that of the 62 kDa protein at 3 min ( 1' 42 + 27.8%), 10 min ( 1' 19 + 28.8%) and 16 min ( ~ 32.2 + 14.9%). We have described here our findings that incubation with 50 and 5 nM NT altered the post-hoe calciumdependent phosphorylation of specific proteins. Although the relative role of NT-modulated protein kinase and protein phosphatase activities cannot be definitively concluded from these post-hoc phosphporylation experiments, it should be mentioned that the phosphorylation assay conditions employed in these experiments reflect primarily the rate of phosphate incorporation (i.e. protein kinase activity) 23'24. The effective concentrations of NT are within the ranges of NT binding to both low (K d = 2.85 nM) and high affinity (K d = 0.1-0.3 nM) sites in rat brain membranes 12, as well as within the range of the EC5o for the excitatory electrophysiological action of NT in the ventral tegmental area (35 nM) 21. In addition to the ventral tegmental area, NT exerts an excitatory effect on frontal cortical ~, substantia nigra TM, and periaqueductal gray neurons 3. It will be of interest to determine if NT effects on calcium-dependent phosphorylation mediate the electrophysiological actions of the peptide. Based on molecular weight, stimulation of phosphorylation by Ca/CaM, and insensitivity to cAMP, we have suggested that the 62 and 50 kDa proteins are the fl and a subunits, resp., of Ca/CaM-dependent protein kinase II. Autophosphorylation of Ca/CaM kinase II converts the enzyme to a calcium-independent state, thereby
altering the rate of substrate phosphorylation. In vitro autophosphorylation of the Ca/CaM kinase II has also been reported to be associated with translocation of the enzyme from a cytoskeletal form to a soluble form 19'2°. Our results may, therefore, reflect the ability of NT to both alter the activity and induce translocation of the Ca/CaM kinase II. The Ca/CaM-dependent protein kinase II is known to regulate neurotransmitter release at the synapse 13 and, furthermore, NT has been shown to modulate dopamine release in cat, rabbit and rodent striatal slices s'7'~5. Thus, altered phosphorylation of the 62 kDa and/or 50 kDa proteins may be one of the mechanisms through which NT exerts its effects on dopamine release. It is noteworthy that the time course of the phosphorylation response to NT was reversed at the high relative to the low dose. It is not clear whether this reflects a time shift in the dose response curve or, alternatively, is an indication that the two doses of NT differentially influence cellular responsiveness, perhaps by acting through the high (5 nM) and low affinity (50 nM) NT receptor. In this regard, it is interesting that, depending on concentration, NT exerts either an excitatory (high concentration) or inhibitory (low concentration) effect on muscle contraction in the guinea pig ileum ~1. We are currently attempting to verify the identity of the 62 and 50 kDa proteins as subunits of CaM kinase II and delineate the relative roles of NT-sensitive kinases and/or phosphatases in the observed effects. Functional studies will focus on evaluating NT dose response effects on both protein phosphorylation and dopamine release in neostriatal tissue.
We would like to thank Ward Virts and Sharon Rhoden for preparation of this manuscript. Research was supported by a National Alliance for Research on Schizophrenia and Depression (NARSAD) Award to Scott T. Cain, NSF BNS-8910032 and NIMH MH-39415.
1 Audinat, E., Hermel, J.-M. and Crepel, E., Neurotensininduced excitation of neuroris of the rat's frontal cortex studied intraceUularly in vitro, Exp. Brain Res., 78 (1989) 358-368. 2 Battaini, E, Govoni, S., DiGiovine, S. and Trabucchi, M., Neuroteusin effects on dopamine release and calcium transport in rat striatum: interactions with diphenylalkylamine calcium antagonists, Naunyn-Schmiedeberg's Arch. Pharmacol., 332 (1986) 267-270. 3 Behbehani, M.M., Shipley, M. and McLean, J.H., Effect of neurotensin on neurons in the periaqueductal gray: an in vitro study, J. Neurosci., 7 (1987) 2035-2040. 4 Bozou, J.C., Rochet, N., Magnaldo, I., Vincent, J.P. and Kitabgi, P., Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT 29 cells. Involvement of a G protein, Biochem. J., 264 (1989) 871-878. 5 de Quidt, M.E. and Emson, P.C., Neurotensin facilitates dopamine release in vitro from rat striatal slices, Brain Research, 274 (1983) 376-380.
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