Regulation and Mechanism of L-Type Calcium Channel Activation via V1a Vasopressin Receptor Activation in Cultured Cortical Neurons

Neurobiology of Learning and Memory 76, 388–402 (2001) doi:10.1006/nlme.2001.4020, available online at http://www.idealibrary.com on

Regulation and Mechanism of L-Type Calcium Channel Activation via V1a Vasopressin Receptor Activation in Cultured Cortical Neurons Michael C. Son and Roberta Diaz Brinton Department of Molecular Pharmacology and Toxicology, Pharmaceutical Sciences Center, University of Southern California, Los Angeles, California 90089

We have sought to ellucidate the biochemical mechanisms that underlie the memory enhancing properties of the neural peptide vasopressin. Toward that goal we have investigated vasopressin induction of calcium signaling cascades, long held to be involved in long-term memory function, in neurons derived from the cerebral cortex, a brain region associated with long-term memory. Our previous studies demonstrated that in cultured cortical neurons, V1a vasopressin receptor (V1aR) activation resulted in a sustained rise in intracellular calcium concentration that was dependent on calcium influx (Son & Brinton, 1998). To investigate the

Grateful acknowledgment is given to Richard Thompson for his many years of thoughtful committed mentorship and support. I first became aware of Richard Thompson as a graduate student while studying his brilliant and systematic research on the neurobiology of learning and memory in the mammalian brain. It is hard to imagine that just 20 years ago, the idea of studying the anatomical and neurochemical basis of memory in the mammalian brain was revolutionary, and certainly not without its critics, but the annals of science have proved Thompson’s revolutionary vision correct. Later when recruited to the University of Southern California as part of the Neuroscience initiative, it was Richard Thompson’s presence and support that was the deciding factor in my acceptance of the position. While many have and will write about Richard Thompson’s scientific contributions, I want to gratefully acknowledge the mentorship that both Richard and Judith Thompson have graciously and freely given to many who have crossed their path. When I was a new faculty member at USC, Richard and Judith Thompson graciously welcomed me into the Thompson fold and I have been profoundly grateful ever since. Throughout my tenure at USC, Dr. Thompson has been a thoughtful and conscientious mentor of my development into a successful neuroscientist. Richard graciously gave of his time and resources to mentor so many, happily I am one of them, because he recognized that each stage of professional development benefits from mentorship. Even while Richard embraced the enormous responsibilities of leading the USC Neuroscience Program he made time and energy to mentor individual students and faculty with grace and seemingly inexhaustible energy. Richard was able to see the best in each of us and to nourish (sometimes with great effort on his part!) that best into reality. Richard Thompson’s legacy to science is manifested in his many scientific discoveries and in the numerous successful scientific careers he has fostered. It is a legacy that has already stood the test of time and one that has been full of much joy and of remarkable successes. This work was supported by grants from the National Science Foundation (IBN-9601248) and the Norris Foundation to R.D.B. Address correspondence and reprint requests to Roberta Diaz Brinton, University of Southern California, Pharmaceutical Sciences Center, 1985 Zonal Avenue, Los Angeles, CA 90089. Fax: 323-442-1489. E-mail: [email protected]. 1074-7427/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

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mechanism of V1aR-induced calcium influx, we investigated V1aR activation of the calcium channel subtype(s) in cortical neurons cultured from Sprague–Dawley rat embryonic day 18 fetuses. The results of these analyses demonstrated that the L-type calcium channel blocker nifedipine blocked 250 nM V1 vasopressin receptor agonist (V1 agonist)-induced calcium influx. Intracellular calcium imaging analyses using fura-2AM demonstrated that blockade of L-type calcium channels prevented the 250 nM V1 agonist-induced rise in intracellular calcium concentration. These results indicate that the influx of extracellular calcium via L-type calcium channels is an essential step in the initiation of the V1 agonist-induced rise in intracellular calcium concentration. To determine the mechanism of V1aR activation of Ltype calcium channels, regulatory components of the phosphatidylinositol signaling pathway were investigated. The results of these analyses demonstrated that V1 agonist-induced calcium influx was blocked by both a phospholipase C inhibitor (U-73122) and a protein kinase C inhibitor (bisindolylmaleimide I). Further analysis of V1aR activation of protein kinase C (PKC) demonstrated that V1 agonist induced PKC activity within 1 min of exposure in cultured cortical neurons. These data indicate that in cultured cortical neurons, V1aR activation regulates the influx of extracellular calcium via L-type calcium channel activation through a protein kinaseC-dependent mechanism. The results of these studies provide biochemical mechanisms by which vasopressin could enhance memory function. Those mechanisms include a complex cascade that is initiated by activation of the phosphatidylinositol pathway, activation of protein kinase C, followed by phosphorylation of L-type calcium channels to initiate the influx of extracellular calcium to activate a cascade of calcium-dependent release of intracellular calcium. 䉷 2001 Academic Press

INTRODUCTION Vasopressin, the endogenous ligand for V1a vasopressin receptor, is a neuropeptide that is synthesized in a number of sites in the brain, including the paraventricular, supraoptic, and suprachiastmatic nuclei of the hypothalamus, the bed nucleus of the stria terminalis, and the medial amygdala (Buijs, 1987; Caffe, van Leeuwen, & Luiten, 1987; Sofroniew, 1985). Within the nervous system, vasopressin impacts a broad spectrum of functions, including those regulating homeostasis (DeWied, 1988; Kasting, Veale, & Cooper, 1982), the stress response (DeWied, 1988; Jezova, Skultetyova, Tokarev, Bakos, & Vigas, 1995; McEwen & Brinton, 1987), and learning and memory (Brinton, 1998; DeWied, 1988; Jolles, 1987). Our previous studies have demonstrated that at the biochemical level, V1aR activation leads to phosphatidylinositol signaling, which results in regulation of intracellular calcium concentrations in cultured cortical neurons (Son & Brinton, 1998). Calcium is critical to the development of the cellular and structural features of the nervous system (Berridge, 1993; Nishizuka, 1992). One major factor in determining the nature of a neuronal calcium signal is the opening of permeability pathways for calcium in the cell membrane to allow the influx of calcium from the extracellular space. Typically, these have been grouped into voltage-sensitive calcium channels and receptor-operated calcium channels. Our previous findings indicate that such channels may be involved in V1 agonist-induced calcium influx in cultured cortical neurons (Son & Brinton, 1998). There are a growing number of different types of calcium channels detected in neurons

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such as L-, N-, P-, Q-, R-, and T-type calcium channels (Ishibashi, Rhee, & Akaike, 1997; Randall & Tsien, 1995; Sabatier, Richard, & Dayanithi, 1997; Zhang et al., 1993). Calcium channels play a critical role in many aspects of neuronal function, such as neurotransmitter release, neurite outgrowth, gene expression, and modulation of learning and memory (Bliss & Collingridge, 1993; Ginty, Bading, & Greenberg, 1992; Goelet, Castellucci, Schacher, & Kandel, 1986; Kennedy, 1989). Calcium channel activity is often regulated by phosphorylation/dephosphorylation of its subunits via various kinases. Protein kinase C and calcium/calmodulin-dependent kinases have been implicated as potential mediators of calcium-regulated neuronal adaptive responses, such as long-term potentiation (Bliss & Collingridge, 1993). In sensory neurons, protein kinase C inhibits calcium channel activation, whereas in sympathetic neurons protein kinase C enhances calcium channel activation (Zhu & Ikeda, 1994). Three reasons governed the selection of investigating the mechanism of V1aR activation of L-type calcium channels in cortical neurons. First, our previous work demonstrated that V1aR activation in cultured cortical neurons led to activation of the phosphatidylinositol signal pathway, uptake of calcium from the extracellular medium, and induction of complex intracellular calcium signaling (Son & Brinton, 1998). Furthermore, our findings of V1 agonist-induced calcium influx (Son & Brinton, 1998) indicated V1aR regulation of calcium channels in cultured cortical neurons. Second, presence of multiple calcium channel subtypes has been reported in cortical neurons, including L-type calcium channels (Zhang, Hirano, & Hiraoka, 1995). Second, in immunocytochemical studies, L-type calcium channels were localized primarily to neuronal cell bodies and to the base of proximal dendrites (Ahlijanian, Westenbroek, & Catterall, 1990; Westenbroek, Ahlijanian, & Cattrall, 1990). Last, calcium entry through calcium channels induces gene expression which can lead to structural and functional changes that underlie long-term adaptive responses (Ginty, Bading, & Greenberg, 1992; Goelet, Castellucci, Schacher, & Kandel, 1986). Indeed, L-type calcium channels have been demonstrated to be involved in the mechanism(s) of learning and memory (Murphy, Worley, & Baraban, 1991). Based on these findings, we investigated the mechanism of V1aR regulation of L-type calcium channel activation in cultured cortical neurons. METHODS AND MATERIALS Cell Culture Preparation Cultures of cortical neurons were prepared following the method described by Son and Brinton (1998). Cortices were dissected from the brains of embryonic day 18 (E18, with E0 as breeding day) Sprague–Dawley rat fetuses. The tissue was treated with 0.05% trypsin in Hanks’ Balanced salt solution (50 mM KCl, 3 mM KH2PO4, 80 mM NaCl, 0.9 mM NaH2PO4 ⭈ 7H2O, 10 mM dextrose, 0.3 M Hepes) for 5 min at 37⬚C. Following incubation, trypsin was inactivated with cold phenol red-free Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 mM NaHCO3, 10% fetal bovine serum, 5 U/ml penicillin, 5 ␮g/ml streptomycin, and 10% F12 nutrient medium for 3 min. Tissue was then washed with Hanks’ balanced salt solution (2⫻) and dissociated by repeated passage through a series of fire-polished constricted Pasteur pipettes. Cells were plated at a concentration of 1 ⫻ 106 cells/ml onto polyethylenimine (PEI)-coated (10 ␮g/ml;

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Sigma. St. Louis) 35-mm petri dishes. Cells were grown in 2 ml of phenol red containing Neurobasal Medium (which does not promote glial cell proliferation; Gibco No. 320-1103), B27 medium supplement (Gibco No. 680-7504), 25 ␮M glutamate, 0.5 mM glutamine, 5 U/ml penicillin, and 5 ␮g/ml streptomycin and maintained in a 37⬚C 5% CO2 incubator. Assessment of [3H]IP1 Accumulation After 2 days of incubation, 1 ml of the media was aspirated off and replaced with 0.5 ml of media containing 4 ␮Ci/ml of [3H]myo-inositol (sp act ⫽ 23.45 Ci/mmol). Preliminary studies indicated that 24 h of incubation with [3H]myo-inositol was optimal for incorporation into the cell lipids. Cells were rinsed twice with 1 ml of Krebs Ringer bicarbonate (KRB) buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2 ⭈ 6H2O, 1.2 mM KH2PO4, 26 mM NaHCO3, 10 mM dextrose, 1 mM CaCl2) and then preincubated in 1 ml of KRB for 20 min at 37⬚C. Following the preincubation period, solution was exchanged for KRB ⫹ 10 mM LiCl (inositol phosphatase inhibitor) ⫹ test peptides at 20 min at 37⬚C for the dose response analysis and at varying time points for the time course analysis. Peptides were dissolved in KRB solution (without LiCl) immediately prior to use. The reaction was terminated by the addition of 750 ␮l of ice-cold methanol, and cells were scraped from the petri dishes with a cell scraper, and transferred to test tubes containing 1 ml of chloroform and 0.5 ml of deionized distilled water. An additional 750 ␮l of ice-cold methanol was added to the petri dishes and then transferred to the same test tubes. Chloroform samples were vortexed and then centrifugated (5 min, 2000 rpm). The aqueous phase was transferred to test tubes containing 4 ml of deionized distilled water, vortexed, and centrifuged (5 min, 2000 rpm). Five milliliters of the sample was filtered through 1-ml Dowex columns (Bio-Rad, MO), which had been generated using 1 ml of 1 M ammonium formate/0.1 M formic acid. Columns were washed with 5 ml of distilled deionized water (2⫻), which was discarded, followed by 2.5 ml of 1 M ammonium formate/0.1 M formic acid. This eluate containing the inositol phosphates was collected and 1 ml of the 2.5-ml eluate was counted by scintillation in 5 ml of scintillation fluid. In order to present comparable data cross experiments, [3H]IP1 accumulation data were analyzed by determining the ratio of aqueous cpm/organic cpm and expressed as a percentage of basal accumulation. Calcium Imaging Cortical neurons to be used in calcium imaging studies were cultured at a density of 1 ⫻ 106 cell/ml onto (⫹) poly-L-lysine-coated coverslips and then placed onto coverslip clamp chamber MS-502S (ALA Scientific Instruments; NY) for the calcium imaging analysis. Unless stated otherwise, neurons were used 7 days following seeding. Neurons were briefly washed with Krebs buffer (137 mM NaCl, 5.3 mM KCl, 1.0 mM MgCl2 ⭈ 6H2O, 1.2 mM KH2PO4, 10 mM Hepes, 25 mM dextrose, and 1.5 mM CaCl2) and then loaded with fura-2 acetoxymethyl ester (5 ␮mol/L; Molecular Probes, Inc., OR) by incubating for 45 min at 37⬚C. Excess fura-2 dye was removed by washing with Krebs buffer and incubated for 30 min at 37⬚C for neurons to equilibrate. Five micromolar glutamate was used at the end of each experiment as positive control. Fluorescence measurements of intracellular calcium concentration were performed using InCyt2, Fluorescence Imaging System (Intracellular Imaging, Inc., OH). Neurons were placed on a

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stage of an inverted microscope (MT-2, Olympus) equipped with epifluorescence optics (20X, Nikon). Fluorescence was excited at wavelengths of 340 and 380 nm alternatively using a rotating four-wheel filter changer. To minimize the background noise of the fura2 signal, successive values (16 sample images, 8 background images) were averaged (⬃13 images/min). 45

Ca2⫹ Uptake

Cultures were similar in age and cellular density to those used for [3H]IP1 accumulation. Cultures were washed in Krebs buffer for 20 min at 37⬚C. Following this wash, V1 agonist ⫹ 1.0 ␮Ci 45Ca2+ per 2 ml were simultaneously added to the cultures (sp act of 45 Ca2+, 30.7 mCi/mg). Following the incubation period with V1 agonist, the treatment solution was decanted and cultures were washed two times with 2 ml of Krebs buffer. After decanting the last Krebs wash, 45Ca2+ uptake was terminated by the addition of 1 ml of 7% ice-cold trichloroacetic acid to each culture dish and incubated for 45 min at 4⬚C. Trichloroacetic acid extracts were removed and placed into scintillation vials for counting and 1 ml of NaOH was added to the cultures to solubilize protein for analysis of protein content by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951). Protein Isolation Cells were plated at a concentration of 1 ⫻ 106 cells/ml onto polyethylenimine-coated (10 ␮g/ml; Sigma. St. Louis) 100-mm petri dishes in Neurobasal media and maintained in a 37⬚C 5% CO2 incubator. After 7 days, cells were treated with and without 250 nM V1 agonist for 1 min medium was discarded, and cells were washed with cold PBS solution, and then 2 ml of lysis buffer (20 mM Tris–HCl, pH 7.4, 2 mM EDTA, 0.5 mM EGTA, 0.25 M sucrose, 50 mg/ml PMSF) was added. Cells were scraped and transferred to ultracentrifuge tube (Beckman, CA). Ultracentrifuge tubes were balanced, put into a Ti-70 rotor (Beckman, CA), and then centrifuged at 100,000g for 1 h in a Beckman L870 Ultracentrifuge. Then supernatants that contained the cytosolic proteins were collected. The pellets which contained the membrane proteins were dissolved with 1 ml of lysis buffer and 0.2% Triton X-100 and centrifuged at 100,000g for 1 h, and then the supernatant (membrane protein) was combined with cytosolic proteins to collect the total proteins. Protein Kinase C Assay The PepTag Assay for Non-Radioactive Detection of Protein Kinase C from Promega was used. Protein kinase C was diluted to 2.5 ␮g/ml in protein kinase C buffer and protein kinase C activator (5⫻ solution) was sonicated using a probe sonicator for 20–30 s until it was warm. For protein kinase C assay, reaction mixtures were prepared containing 5 ␮l of PepTag Protein Kinase C Reaction 5⫻ Buffer, 2 ␮g of PepTag C1 Peptide, 5 ␮l of Protein Kinase C Activator 5⫻ Solution, 1 ␮l of Peptide Protection Solution, 5 ␮l of protein sample, and 4 ␮l of deionized water. Four microliters of 2.5 ␮g/ml of protein kinase C was substituted for the sample proteins for a protein kinase C positive control. For a protein kinase C negative control, sample protein was replaced with deionized water. At time 0, reaction mixtures were removed from the ice and incubated in a 30⬚C water

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bath for 2 min. Then sample or protein kinase C was added and incubated at 30⬚C for 30 min. The reaction was stopped by placing the tubes in a boiling water bath or on a 95⬚C heating block for 10 min. Cytosolic and membrane protein samples were loaded onto 0.8% agarose gels in 50 mM Tris–HCl, pH 8.0 and then run at 100 V for 15 to 20 min to separated the phosphorylated and nonphosphorylated PepTag Peptides. To visualize the separation, ultraviolet light was used to view the bands and then photographs were taken for the qualitative assay. To quantitate kinase activity, negatively charged phosphorylated bands from the gel were excised, keeping the total volume at approximately 250 ␮l. Excised bands were placed into a 1.5-ml graduated microcentrifuge tube and heated at 95⬚C until the gel slice was melted. The hot agarose (125 ␮l) was transferred to a tube containing 75 ␮l of Gel Solubilization Solution (that had been warmed to room temperature and mixed well), 100 ␮l of glacial acetic acid, and 200 ␮l of distilled water. Once in the acidified Gel Solubilization Solution, the agarose should remain liquid for several hours. The samples were vortexed and then 500 ␮l of the solution was transferred to a 0.5-ml cuvette. The absorbance was read at 570 nm. The spectrophotometer was zeroed with liquefied agarose without PepTag Peptide. Data Analysis [3H]IP1 accumulation data are presented as mean percentages of basal ⫾SEM, as determined by the ratio of aqueous cpm/organic cpm. The 45Ca2+ uptake data are presented as mean percentages of basal ⫾SEM. Statistical analysis was performed by a Student’s t test or by a one-way analysis of variance (ANOVA) followed by Newman–Keuls posthoc analysis.

RESULTS The L-type calcium channel blocker nifedipine was used to investigate whether V1 agonist-induced calcium entry in cortical neurons was via the L-type calcium channel. The results of these experiments demonstrated that 250 nM V1 agonist induced calcium influx (141.9% ⫾ 6.2, p ⬍ .05), whereas pretreatment with 20 ␮M nifedipine blocked V1 agonist-induced calcium influx (99.0% ⫾ 7.2) in cultured cortical neurons (Fig. 1). To confirm the selectivity of nifedipine, (⫺)S-BayK 8644, an L-type channel activator, was used. Ten micromolar (⫺)S-BayK 8644 induced calcium influx (142.3% ⫾ 7.0, p ⬍ .05), whereas 20 ␮M nifedipine effectively blocked 10 ␮M (⫺)S-BayK-8644-induced calcium influx (96.2% ⫾ 4.6) in cultured cortical neurons (Fig. 1). Calcium imaging studies demonstrated that 20 ␮M nifedipine blocked the V1 agonist-induced rise in intracellular calcium concentration (Fig. 2). Calcium channels can be activated either directly by a ligand or indirectly by intermediate components of the signaling cascade, such as a retrograde signal. U-73122 is a phospholipase C inhibitor that blocks the production of inositol phosphates, whereas BIS I is a protein kinase C inhibitor that allows continuous production of inositol phosphates by inhibiting the negative feedback mechanism of the phosphatidylinositol signaling pathway. U-73122 and BIS I were used to determine the mechanism of V1 agonist-induced calcium

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FIG. 1. The L-type calcium channel antagonist nifedipine blocked V1 agonist-induced uptake of extracellular Ca2+ in cortical neurons. Cultures of cortical neurons were pretreated with 20 ␮M nifedipine for 20 min, followed by 5-s exposure to 250 nM V1 agonist or 10 ␮M BayK 8644. Values represent means ⫾SEM from one experiment and are representative of three separate analyses with 7–8 cultures per condition per experiment. ** p ⬍ .01.

FIG. 2. Blockade of V1 agonist-induced rise in intracellular calcium concentration by the L-type calcium channel antagonist nifedipine in cultured cortical neurons. Cultures of 7-day-old neurons were loaded with fura2 for 30 min (A) and then exposed to 250 nM V1 agonist (B), or pretreated with 20 ␮M nifedipine for 30 min (C), or exposed to 250 nM V1 agonist and 20 ␮M nifedipine (D).

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FIG. 3. Inhibition of protein kinase C by bisindolylmaleimide I and inhibtion of phospholipase C by U73122 both abolish V1 agonist-induced calcium channel activation. Neuronal cultures were pretreated with 5 ␮M BIS I for 20 min and then exposed to 250 nM V1 agonist and 5 ␮M BIS I simultaneously for 5 s. Another set of cultures were pretreated with 1 ␮M U-73122 for 20 min followed by exposure to 250 nM V1 agonist and 1 ␮M U-73122 for 5 s. Values are means ⫾SEM from one experiment and are representative of data obtained in two separate analyses with 7–8 cultures per condition per experiment. ** p ⬍ .01. * p ⬍ .05.

channel activation in cortical neurons by inhibiting various steps along the phosphatidylinositol signal pathway. Results from 45Ca2+ uptake experiments demonstrated that 5 ␮M BIS I and 1 ␮M U-73122 both blocked V1 agonist-induced calcium influx, 90.2% ⫾ 3.5 and 97.8% ⫾ 3.9, respectively (Fig. 3). In addition, the results of the experiments to confirm the efficiency of BIS I and U-73122 showed that 5 ␮M BIS I and 250 nM V1 agonist allowed greater accumulation of inositol phosphates (206.94% ⫾ 13.91, p ⬍ .001; Fig. 4) compared to 250 nM V1 agonist alone (176.30% ⫾ 15.07, p ⬍ .001; Fig. 4), whereas 1 ␮M U-73122 effectively blocked the accumulation of inositol phosphates (83.7% ⫾ 4.5; Fig. 4). These results indicated that V1 agonist regulation of L-type calcium channels required activation of the phosphatidylinositol signaling pathway. One of the well-known components of the phosphatidylinositol signaling pathway is activation of protein kinase C, which can subsequently phosphorylate a variety of proteins. To further investigate the role of protein kinase C in L-type channel regulation, experiments to determine whether V1a vasopressin receptor activation led to protein kinase C activation in cortical neurons were performed. The results of these studies demonstrated that within 1 min. of exposure to 250 nM V1 agonist induction of protein kinase C activity occurred (Fig. 5A). Quantitative analysis indicated a 54.5% increase in the phosphorylation activity of protein kinase C in cortical neurons treated with 250 nM V1 agonist (3.4 U/ml; Fig. 5B) compared to control neurons (2.2 U/ml; Fig. 5B). Last, the effect of protein kinase C inhibition on the V1 agonist-induced rise in intracellular calcium concentration was investigated using intracellular calcium imaging. The results of these determinations showed that blockade of protein kinase C activity inhibited the

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FIG. 4. Antagonism of protein kinase C with bisindolylmaleimide I does not inhibit V1 agonist-induced [3H]IP1, whereas antagonism of phospholipase C with U-73122 does inhibit V1 agonist-induced [3H]IP1. Cultures were pretreated with 5 ␮M BIS I for 20 min and then exposed to 250 nM V1 agonist and 5 ␮M BIS I for 60 min. In a separate set of cultures, neurons were pretreated with 1 ␮M U-73122 for 20 min followed by exposure to 250 nM V1 agonist and 1 ␮M U-73122 for 60 min. Values are means ⫾SEM from one experiment and are representative of data obtained in two separate analyses with 7–8 cultures per condition per experiment. *** p ⬍ .001.

V1 agonist-induced rise in intracellular calcium concentration in cultured cortical neurons (Fig. 6). DISCUSSION The purpose of this study was to identify the calcium channel involved in V1 agonistinduced calcium influx and to determine the mechanism underlying the activation of this calcium channel. The results of the present study demonstrate that V1 agonist-induced calcium influx occurred through an L-type calcium channel mechanism. Furthermore, we found that the protein kinase C arm of the V1 receptor activated a phosphatidylinositol signaling pathway mediating the L-type calcium channel activation in cultured cortical neurons. The results of the present study add additional insights into understanding of the mechanisms underlying V1 receptor-induced rises in intracellular calcium. Moreover, these data provide the first demonstration of V1 vasopressin receptor activation of L-type calcium channels in cortical neurons and are the first to demonstrate that regulation of these calcium channels by V1 vasopressin receptor activation is a protein kinase-Cdependent process. L-type calcium channels are abundantly expressed in neurons (Ahlijanian et al., 1990; Giffin, Solomon, Burhalter, & Nerbonne, 1991; Nakazawa & Murphy, 1999; Westenbroek et al., 1990), have a long-open time (Ishibashi et al., 1997; Zhang et al., 1993), and have been implicated in learning and memory models such as long-term potentiation and

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FIG. 5. V1 agonist induction of protein kinase C activation. Proteins were collected from 7-day-old cortical neurons and then protein kinase C activity was assayed. Peak activation of protein kinase C occurred following 1 min exposure to 250 nM V1 agonist. Exogenous protein kinase C was used in positive control experiment. (A) The lower band below the loading well shows protein kinase C activity, phosphorylation of PepTag peptide. The equal fluorescent levels of the upper band of nonphosphorylated PepTag peptide demonstrate the quantitative control for the amount of PepTag peptide used for each condition (B) Quantitative determination of protein kinase C activity by cytofluorometry. Ctrl, control; V1, 250 nM V1 agonist; Po. Ctrl, positive control.

neuronal plasticity (Deyo, Straube, & Disterhoft, 1989; Murphy et al., 1991; Yamada et al., 1996). In pituitary adenoma cells, vasopressin increased calcium currents through Ltype calcium channels (Mollard, Vacher, Rogawski, & Dufy, 1988). Moreover, the abundance of L-type calcium channels in neuronal cells, including cortical neurons, suggested an important role of L-type calcium channels in vasopressin-induced calcium influx (Zhang et al., 1995). The results of the current calcium imaging analysis and calcium uptake experiments using nifedipine demonstrated V1 agonist induction of calcium regulation via L-type calcium channels in cultured cortical neurons. Thus far, data from the current study and those of previous studies (Son & Brinton,

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FIG. 6. Inhibition of V1 agonist-induced intracellular calcium increase by protein kinase C inhibition in cultured cortical neurons. (A) Seven-day-old neurons were loaded with fura-2AM (A) and then exposed to 250 nM V1 agonist (B). Neurons shown in C were pretreated with 5 ␮M bisindolylmaleimide I (protein kinase C inhibitor) for 30 min (C) and then exposed to 250 nM V1 agonist with 5 ␮M bisindolylmaleimide I (D).

1998) continue to indicate that V1 agonist-induced calcium signaling in cultured cortical neurons occurs via the “calcium-induced calcium release” model, in which blockade of calcium influx from the extracellular compartment prevents a rise in intracellular calcium despite V1 agonist-induced generation of IP3. In addition to changes in the level of intracellular calcium, the route of calcium entry and its intracellular localization give rise to activation of specific biochemical signaling pathways that mediate particular biological responses. By controlling the route of calcium entry, neuronal calcium channels regulate a wide variety of cellular functions, including spike patterning, neurotransmitter release, and gene transcription (Bito, Deisseroth, & Tsien, 1997; Hernandez-Lopez, Bargas, Surmeier, Reyes, & Galarraga, 1997; Holliday, Adams, Sejnowski, & Spitzer, 1991; Mintz et al., 1995; Wheeler, Sather, Randall, & Tsien, 1994). Influx of calcium during cortical plasticity development underlies the signal transduction events leading to long-term alterations of synaptic efficacy (Geiger & Singer, 1986; Murphy et al., 1991; Singer, 1985). It is now becoming increasingly apparent that the influx of calcium through voltagesensitive calcium channels can be modulated by receptor-mediated events. A number of cell surface receptors are coupled either directly to calcium channels (Fasolato, Innocenti, & Pozzan, 1994) or through the intermediate G-proteins (Hescheler & Schultz, 1993; Strubing et al., 1997). Modulation of calcium channels by both phosphorylation/dephosphorylation and G-protein-dependent regulation has been widely described (Netzer, Pflimlin, & Trube,

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1994). Another important mechanism regulating calcium channel function is phosphorylation by Ca2+/phospholipid-dependent protein kinase C. In addition to the presence in high concentrations of protein kinase C in neuronal tissue (Tanaka & Saito, 1992), a pivotal role in controlling cellular functions, and involvement in the modulation of signal transduction (Nishizuka, 1992), protein kinase C has been implicated in the modulation of calcium channel functions where protein kinase C phosphorylates G-protein and/or calcium channel subunits (Shistik et al., 1999; Swartz, 1993). Several studies have reported L-type calcium channel modulation by protein kinase C activation (Bourinet et al, 1994; Bourinet, Fournier, Lory, Charnet, & Nargeot, 1992; Singer-Lahat et al., 1992; Yang & Tsien, 1993). Moreover, protein kinase C has been demonstrated to mediate the enhancement of L-type calcium channels by vasopressin (Zhang et al., 1995). In cortical neurons, our results indicate that V1 agonist-induced calcium influx was mediated by downstream components of the phosphatidylinositol signaling cascade. More specifically, the application of protein kinase C inhibitor effectively blocked the V1 agonist-induced calcium influx and increase in intracellular calcium concentration, which may suggest a possible phosphorylation of G-proteins and/or calcium channel subunits by protein kinase C in modulation of L-type calcium channels in cultured cortical neurons. Recent evidence of G-protein ␣- and ␤-subunit modification by protein kinase C also suggested that the G-protein could be a “programmable messenger” that after interaction with protein kinase C would interact weakly with calcium channels (Chen & Penington, 1996; Puri, Gerhardstein, Zhao, Ladner, & Hosey, 1997). In rat sympathetic neurons, the activation of protein kinase C antagonizes G-protein-mediated inhibition of calcium channels by shifting calcium channels from the “reluctant” state to the “willing” state (Zhu & Ikeda, 1994). Recent findings suggest that the N-terminus of L-type calcium channel ␣1C subunit acts as an inhibitory gate, and its removal enhances channel activation where protein kinase C may increase the current by attenuating the inhibitory action of the N-terminus (Shistik et al., 1999; Shistik, Ivanina, Blumenstein, & Dascal, 1998). Thus, protein kinase C may phosphorylate a site at ␣1C which interacts directly or allosterically, resulting in weakening the inhibitory gating effect of the Nterminus, or protein kinase C may phosphorylate an unknown auxiliary protein that may obstruct the inhibitory gating effect of the N-terminus. We are currently pursuing identification of the protein kinase C isoform(s) that regulates vasopressin-induced calcium channel activity. For future studies, we will determine the direct or indirect effect of protein kinase C on calcium channels by identifying intermediate proteins that are phosphorylated by protein kinase C in cultured cortical neurons. In conclusion, our studies indicate that the V1 agonist induces calcium influx via L-type calcium channel activation and that V1a vasopressin receptor activation of the L-type calcium channel is mediated by protein kinase C in cultured cortical neurons. These findings provide increasing evidence that vasopressin enhancement of memory function is via activation of a phosphatidylinositol/calcium channel signaling cascade. REFERENCES Ahlijanian, M. K., Westenbroek, R. E., & Catterall, W. A. (1990). Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina. Neuron, 4, 819–832. Berridge, M. J. (1993). Inositol trisphosphate and calcium signaling. Nature, 361, 315–325.

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