Adrenergic regulation of translocation of protein kinase C isozymes in rat pinealocytes

Molecular and Cellular Endocrinology 150 (1999) 169 – 178

Adrenergic regulation of translocation of protein kinase C isozymes in rat pinealocytes A.K. Ho, K. Hashimoto, W. Matowe, C.L. Chik * Department of Physiology and Department of Medicine, Faculty of Medicine, Uni6ersity of Alberta, 7 -26 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada Received 3 August 1998; received in revised form 23 October 1998; accepted 23 October 1998

Abstract Protein kinase C (PKC) is involved in the a1-adrenergic-potentiation of b-adrenergic stimulated cyclic nucleotide responses in rat pinealocytes. In the present study, the PKC isozymes expressed in rat pinealocytes and their regulation by norepinephrine (NE) were investigated. Western blot analysis identified PKCa (a classical PKC isozyme), PKCd and o (novel PKC isozymes), and PKCz: (atypical PKC isozymes). NE caused an increase in PKCa, d, and o, but not PKCz, in the particulate fraction. BAPTA-AM, which clamps intracellular Ca2 + , reduced NE mediated translocation of PKCa, d, and o. Subjecting the animals to stimulus deprivation, which altered adrenergic-stimulated cyclic nucleotide responses, had no effect on the expression of PKCa, d, o, and z. Overnight treatment with 4b-phorbol 12-myristate 13-acetate, an activator of PKC, down-regulated PKCa, d, and o, but not PKCz. Our results indicate that all three classes of PKC isozymes (PKCa, d, o, and z are expressed in the rat pineal gland. However, selective activation of these PKC isozymes does not appear to account for the differences in the pineal cAMP and cGMP responses to stimulation. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: PKC isozymes; Norepinephrine; Stimulus deprivation; Cyclic nucleotide; Rat pineal

1. Introduction In rat pinealocytes, the accumulation of cyclic nucleotides is regulated by a dual receptor system involving the synergistic interaction of mechanisms regulated by a1- and b-adrenergic receptors (Klein, 1985; Klein et al., 1988; Chik and Ho, 1989). b-adrenergic activation is an absolute requirement and a1-adrenergic stimulation acts to potentiate both cAMP and cGMP responses. At the post-receptor level, protein kinase C (PKC) plays a central role in the potentiation mechanism (Sugden et al., 1985; Ho et al., 1987, 1988c). This Abbre6iations: DG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; ISO, isoproterenol; LD, 14 h of light/24 h; LL, continuous light for 5 days; NE, norepinephrine; PKC, protein kinase C; PMA, 4b-phorbol 12-myristate 13-acetate. * Corresponding author. Tel.: + 1-403-4927213; fax: + 1-4034928915. E-mail address: [email protected] (C.L. Chik)

conclusion is based on observations that: (i) activation of a1-adrenergic receptors causes translocation of PKC (Sugden et al., 1985; Ho et al., 1988c); (ii) inhibitors of PKC are effective in blocking the norepinephrine (NE)stimulated cAMP and cGMP responses (Ho et al., 1988b,c); and (iii) agents that cause activation of PKC also potentiate the b-adrenergic-stimulated cAMP response and, in most cases, the cGMP response (Sugden et al., 1985; Ho et al., 1988c). In earlier studies, PKC activity was determined by phosphorylation of histone (Sugden et al., 1985; Ho et al., 1988c) and the role of specific PKC isozymes in the potentiation mechanism was not determined. Since treatment with NE causes a large increase in both cAMP and cGMP accumulation while 4b-phorbol 12-myristate 13-acetate (PMA) potentiates predominantly the cAMP response (Sugden et al., 1985; Ho et al., 1988c), it is possible that this difference in cyclic nucleotide responses is secondary to activation of a different subset of PKC isozymes.

0303-7207/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 9 8 ) 0 0 2 2 7 - 5

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The PKC family of enzymes can be subdivided into three classes based on their structures and requirements for activation (Nishizuka, 1988, 1995): (i) classical PKC isozymes (a, b1, b2, and g) which require Ca2 + for their activation and diacylglycerol (DG) to increase their affinity for Ca2 + ; (ii) no6el PKC isozymes (d, o, h, u, and m) which do not require Ca2 + for activation but can be activated by DG; and (iii) atypical PKC isozymes (z and i, l) which do not bind phorbol ester and therefore do not respond to DG or phorbol esters. These isozymes differ in their tissue distribution, subcellular localization, substrate specificity and requirement for activation (Ohno et al., 1987; Nishizuka, 1988; Ono et al., 1988; Sekiguchi et al., 1988; Kiley et al., 1991; Koide et al., 1992; Nishizuka, 1992; Ogita et al., 1992; Ozawa et al., 1993; Nishizuka, 1995). In most cell types, only a subset of the PKC isozymes is expressed (Kiley et al., 1991; Dekker and Parker, 1994). The objective of the present study was to determine the adrenergic regulation of PKC isozymes in rat pinealocytes. First, the PKC isozymes expressed in rat pinealocytes were characterized using Western blot analysis. Second, the NE-mediated translocation of each class of PKC isozymes and the receptor involved were investigated. Third, the effect of stimulus deprivation on the in vivo expression of PKC isozymes and their responses to down regulation after prolonged treatment with PMA were determined.

2. Materials and methods

2.1. Materials Isoproterenol (ISO), NE, PMA, prazosin and propranolol were obtained from Sigma Chemical Corp. (St. Louis, MO). BAPTA-AM and ionomycin were obtained from Calbiochem Corp. (San Diego, CA). Peptide specific antibodies (IgG fraction, rabbit) that recognize PKCa, b, g, d, o, and z were obtained from Gibco/BRL (Gaithersburg, MD). Ac-MBP(4-14), opeptide, z-peptide and fetal calf serum were obtained from Gibco/BRL (Gaithersburg, MD). [g 32P]-ATP, [125I]-cAMP and [125I]-cGMP were obtained from ICN (Costa Mesa, CA). All other chemicals were of the purest grades and were obtained commercially. Antibodies for the radioimmunoassays of cAMP and cGMP were gifts from Dr A. Baukal (NICHHD, NIH, Bethesda, MD).

2.2. Animals Sprague–Dawley rats (male; 150 g) were obtained from the University of Alberta Animal unit. Animals were housed under a lighting regimen providing 14 h of light every 24 h (LD), with lights on at 0600 h. To

achieve stimulus deprivation, rats were subjected to continuous light for 5 days (LL) and animals were killed between 0830 and 0930 h at the end of the treatment period. An identical protocol was used previously in the see-saw signalling studies (Vanecek et al., 1986; Chik and Ho, 1991).

2.3. Preparation and treatment of rat pinealocytes Pinealocytes were prepared from glands by trypsinization as described previously (Buda and Klein, 1978; Ho et al., 1987). The cells were suspended in DMEM containing 10% fetal calf serum and maintained at 37°C for 24 h in a gas mixture of 95% air and 5% CO2 before the experiment. For the determination of PKC translocation, aliquots of cells (2× 105 cells/0.5 ml) were treated with drugs for 6 min and the cells were collected by centrifugation (2 min, 12 000× g). To separate the membrane and particulate fractions, the pinealocytes were permeabilized by resuspension and incubation (7 min, 4°C) in 100 ml of 50 mM digitonin in buffer A (20 mM Tris–HC1, containing 0.5 mM EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulphonyl fluoride, 25 mg/ml each of leupeptin and aprotinin, pH 7.5) as described previously (Ho et al., 1988b, 1996). The samples were centrifuged (1 min, 12 000× g) to separate the cytosolic from the particulate fraction. Aliquots from the same sample were then used for the determination of PKC enzymatic activities or different PKC isozymes by immunoblotting. For the cyclic nucleotide measurement, aliquots of cells (1.5× 104 cells/0.4 ml) were treated with drugs which had been prepared in concentrated solutions in water or dimethylsulfoxide. The final concentration of the latter never exceeded 0.5%. At this concentration, dimethylsulfoxide had no effect on the NE- or ISOstimulated cAMP and cGMP responses. The duration of drug treatment was 15 min for cAMP and cGMP accumulation. At the end of the treatment period, cells were collected by centrifugation (2 min, 10 000×g). The supernatant was aspirated and the tube was placed on solid CO2. The frozen cell pellets were lysed by the addition of 5 mM acetic acid (100 ml) and boiling (5 min). The lysates were stored at − 20°C until analysis.

2.4. Immunoblotting of PKC isozymes SDS-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (1970) using 7.5 or 10% acrylamide in the presence of 1 mg/ml SDS (Mini-Protein II gel system, Bio-Rad). Following electrophoresis, gels were equilibrated for 20 min in transfer buffer (48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 20% methanol). Proteins were transferred onto PDVF membranes (1 h, 100 V). The membranes

A.K. Ho et al. / Molecular and Cellular Endocrinology 150 (1999) 169–178

were incubated with a blocking solution containing 5% dried skim milk in 100 mM Tris, pH 7.5, 0.9 g/100 ml NaCl, and 0.1 g/100 ml Tween 20 for a minimum of 1 h. After incubation in the blocking solution, the membranes were incubated overnight at 4°C with diluted specific anti-PKC antisera. The specificities of the antibodies were confirmed by pre-incubation with the immunizing peptides. After overnight incubation, the membranes were washed twice with the blocking solution before incubation with HRP-conjugated anti-rabbit IgG in the blocking solution for 1 h. The membranes were then washed and developed using enhanced chemiluminescence (Amersham International, Little Chalfont, England). Densitometric measurements were performed by scanning the specific immunoblots and the image was analysed using the Sigmagel software (Jandel Inc., St. Rafael, CA).

2.5. In 6itro PKC assay PKC activity was measured in duplicate (Ho et al., 1988c; Yasuda et al., 1990; Ho et al., 1996). The reaction mixture contained 20 mM Tris – HCl, 1.0 mM CaCl2, 20 mM MgCl2, 50 mM Ac-MBP(4-14), o-peptide or z-peptide as substrate, 0.5 mg/ml leupeptin, 0.1 mM ATP (1–5 × 106 cpm of [g-32P]ATP). Phosphatidylserine (280 mg/ml) and PMA (10 mM) were added to some tubes to demonstrate phospholipid-dependent protein kinase activity. The reaction was initiated by adding 1 – 2 mg of pineal protein and the incubation (6 min, 37°C) was stopped by immediate spotting of the reaction mixture onto phosphocellulose discs. The discs were then washed twice with 1% phosphoric acid and three times with distilled water. The radioactivity retained by the filter disc was determined by scintillation counting. PKC activity was calculated from the difference in 32P incorporated into the PKC substrate peptide in the presence and absence of added phospholipids.

171

PKC, cAMP and cGMP measurements and by the paired t-test for the densitometric measurements. Statistical significance was set at P B 0.05.

3. Results

3.1. PKC isozymes expressed in the rat pineal gland The expression of PKC isozymes in the rat pineal gland was compared with that in other tissues including the cortex, cerebellum, hypothalamus, pituitary, retina, lung and the adrenal gland. Western blot analysis showed that all three classes of PKC isozymes were present in the rat pineal gland (Fig. 1): PKCa, a classical Ca2 + -dependent PKC isozyme, PKCd, and o, novel Ca2 + -independent PKC isozymes, and PKCz, an atypical phorbol ester-insensitive PKC isozyme. PKCb and PKCg were not identified in the pineal although they were clearly detectable in retina, cortex, hypothalamus and cerebellum. The specific PKC isozyme band was blocked by pre-incubation with the peptide against which the antibody was raised (data not shown). The immunizing peptide for PKCz blocked both 82 and 75 kDa bands, but the 82 kDa band was also blocked by the immunizing peptide for PKCa, indicating that PKCa was also detected with anti-PKCz as reported previously (Allen et al., 1994). In subsequent studies, the regulation of PKC isozymes from three different classes, PKCa, d, o, and z, by NE was characterized.

2.6. Cyclic nucleotide assays The lysates were centrifuged (10 min, 12 000× g) and samples of the supernatant were used to determine cellular cAMP and cGMP content, using a radioimmunoassay procedure in which samples were acetylated prior to analysis (Harper and Brooker, 1975; Ho et al., 1987).

2.7. Statistical analysis PKC activity, cAMP and cGMP measurements were presented as the mean9SEM from three to four observations done in duplicate. Each experiment was repeated at least three times, with representative results being reported. Statistical comparisons were analysed by ANOVA followed by the Newman Keuls test for the

Fig. 1. Tissue distribution of PKC isozymes. Tissue protein (25 mg) was subjected to 10% SDS-PAGE. PKCa, b, g, d, o, and z were identified by Western blotting using polyclonal antibodies as described under Section 2. Adr, adrenal; Cer, cerebellum; Cor, cortex; Hyp, hypothalamus; Lun, lung; Pin, pineal; Pit, pituitary; Ret, retina. Results are representative of three independent experiments.

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Table 1 Effect of NE, PMA and ionomycin on translocation of PKC activity in rat pinealocytesa Treatment

Control NE (10 mM) PMA (0.1 mM) Ionomycin (10 mM)

PKC activity (pmol/6 min per 106 cells) Cytosolic fraction

Particulate fraction

1734 938 1516983 1194 967b 1496 936b

2029 28 373 936b 6339 32b 316 9 27b

a

Rat pinealocytes were treated for 6 min with NE, PMA or ionomycin. The cytosolic and particulate fractions were separated and solubilized, and PKC activity was measured using Ac-MBP(4-14) as the substrate. Each value represents the mean9 SEM, n = 3, based on duplicate measurements. b Significantly different from control.

3.2. NE-, PMA- and ionomycin-mediated translocation of PKC acti6ity The effects of NE, PMA (an activator of PKC) and ionomycin (a Ca2 + ionophore) on cytosolic and particulate PKC activity in rat pinealocytes were determined using Ac-MBP(4-14) as the substrate. Treatment of rat pinealocytes with NE (10 mM) caused a significant increase in PKC activity in the particulate fraction (Table 1) as observed previously when histone was used as the substrate (Ho et al., 1987, 1988c). Treatment with PMA or ionomycin also caused a significant redistribution of PKC activity from the cytosolic to the particulate fraction (Table 1).

Fig. 2. Effect of NE, PMA and ionomycin on translocation of PKCa, d, o, and z. Pinealocytes (2 ×105 cells/0.5 ml) were cultured for 24 h, and treated with NE(10 mM), PMA (0.1 mM) or ionomycin (ION, 10 mM) for 6 min. Cytosolic and particulate fractions were prepared and these fractions were analysed by Western blotting using polyclonal antibodies against PKCa, d, o, and z as described in Section 2. The Western blot shown is representative of three independent experiments.

3.4. Characterisation of the effect of NE on PKC isozymes In cells treated with NE, PKCa in the particulate fraction was increased after 3 min (Fig. 3, Table 2). This increase was sustained for 10 min but by 30 min,

3.3. Effects of NE, ionomycin and PMA on translocation of PKC isozymes In unstimulated cells, all four PKC isozymes were localized in both cytosolic and particulate fractions (Fig. 2). Treatment with NE (10 mM) resulted in translocation of PKCa, d, and o from the cytosolic fraction to the particulate fraction. However, NE had no effect on the distribution of PKCz (the 75kDa lower band) (Fig. 2). In cells treated with PMA (0.1 mM), there was a redistribution of PKCa, d, and o from the cytosolic to the particulate fraction with a near complete translocation of PKCd and o to the particulate fraction (Fig. 2). In contrast, PMA only had a minimal effect on PKCz (Fig. 2). Treatment with ionomycin (10 mM) resulted in a small increase in PKCa in the particulate fraction without causing a redistribution of PKCo, d or z from the cytosolic to the particulate fraction (Fig. 2).

Fig. 3. NE-mediated translocation of PKCa, d, o, and z. Pinealocytes (2 ×105 cells/0.5 ml) were cultured for 24 h and treated either with NE (10 mM) for the indicated time or NE(1 or 10 mM) for 6 min. The effects of these treatments on PKCa, d, o, and z in the particulate fraction were analysed by Western blotting using polyclonal antibodies as described in Section 2. The Western blot shown is representative of three independent experiments.

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Table 2 NE-mediated changes in PKCa, d, o, and z in the particulate fractiona Treatment

Experiment 1 NE (10 mM)-3 min NE (10 mM)-10 min NE (10 mM)-30 min Experiment 2 NE (1 mM)-6 min NE (10 mM)-6 min

PKCa PKCd (% of Control)

PKCo

PKCz

1879 10b 162 911b

1409 110b 1609 11b

1869 12b 1769 8b

104 98 1069 11

110 98

1489 9b

1039 5

101 910

149 9 8b 162 9 7b

1529 9b 1839 12b

1399 7b 1559 11b

99 96 98 96

Rat pinealocytes were treated with NE (10 mM) for the indicated time in experiment 1 or two different concentrations of NE for 6 min. The effects of these treatments on PKCa, d, o, and z in the particulate fraction were analysed by Western blotting using polyclonal antibodies as described in Section 2. The results were quantified by densitometry and expressed as % of control. Each value represents the mean9 SEM (n =3). b Significantly different from control. a

PKCa in the particulate fraction had returned to the basal level. The NE-mediated increase in PKCa in the particulate fraction was dependent on concentration (Fig. 3, Table 2). NE had a similar effect on PKCd and o (Fig. 3, Table 2). In contrast, NE did not have an effect on PKCz in the particulate fraction (Fig. 3). Previously it has been shown that NE stimulates PKC activity through activation of a1-adrenergic receptors (Sugden et al., 1985; Ho et al., 1988c). To determine the receptor involved in the activation of PKC isozymes, the effect of NE was determined in the presence of propranolol, a b-adrenergic antagonist and prazosin, an a1-adrenergic antagonist. While prazosin was effective in inhibiting the NE-mediated increase in PKCa, d and o in the particulate fraction, propranolol did not have an effect (Fig. 4). Confirming the involvement of a1-adrenergic receptors, phenylephrine, an a1adrenergic agonist, was effective in increasing PKCa, d and o in the particulate fraction, while ISO, a b-adrenergic agonist, had no effect (Fig. 5).

3.5. Effect of BAPTA-AM on the NE-mediated changes in PKC isozymes and cyclic nucleotide responses To determine the role of Ca2 + in the regulation of PKC isozymes, pinealocytes were pre-treated with BAPTA-AM which clamps intracellular Ca2 + for 30 min. Pre-treatment with BAPTA-AM (1 mM) largely blocked the NE-induced increase in PKCa, d, and o in the particulate fraction while having no effect on PKCz (Fig. 6). Interestingly, in the unstimulated cells, treatment with BAPTA-AM alone significantly reduced PKCa in the particulate fraction but had no effect on PKCd, o, or z (Fig. 6). Treatment with BAPTA-AM

Fig. 4. Effect of adrenergic antagonists on NE-mediated translocation of PKCa, d, and o. Pinealocytes (2× 105 cells/0.5 ml) were cultured for 24 h and treated with NE (10 mM) in the presence of propranolol (Pro, 1 mM) or prazosin (Prz, 1 mM) for 6 min. The effects of these treatments on PKCa, d, and o in the particulate fraction were analysed by Western blotting using polyclonal antibodies as described in Section 2. The Western blot shown is representative of three independent experiments.

also had no effect on basal or ISO-stimulated cAMP and cGMP accumulation (Table 3). In contrast, while BAPTA-AM was effective in reducing the NE- and ISO+ inomycin-stimulated cAMP and cGMP accumulation, it had no effect on the PMA-potentiation of ISO-stimulated cAMP and cGMP accumulation (Table 3).

3.6. Effect of stimulus depri6ation on the potentiation of the cyclic nucleotide response, expression of PKC isozymes and PKC acti6ity Similar expressions of PKCa, d, o, and z were found in pineal homogenates prepared from animals subjected to LL or LD lighting conditions (Fig. 7). There was

Fig. 5. Effect of adrenergic agonists on translocation of PKCa, d, and o. Pinealocytes (2 × 105 cells/0.5 ml) were cultured for 24 h and treated with NE (10 mM), ISO (1 mM) or phenylephrine (PE, 1 mM) for 6 min. The effects of these treatments on PKCa, d, and o, in the particulate fraction were analysed by Western blotting using polyclonal antibodies as described in Section 2. The Western blot shown is representative of three independent experiments.

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174

Fig. 6. Ca2 + -dependency of NE-mediated translocation of PKCa, d, o, and z. Pinealocytes (2 ×105 cells/0.5 ml) were cultured for 24 h and treated with BAPTA-AM (1 mM) for 30 min prior to treatment with NE (10 mM) for 6 min. The effects of these treatments on PKCa, d, o, and z in the particulate fraction were analysed by Western blotting using polyclonal antibodies as described in Section 2. The Western blot shown is representative of three independent experiments.

also no difference in the PKC activity of LL and LD pineal homogenates determined using Ac-MBP(4-14), o-peptide or z-peptide as substrates (Table 4). Furthermore, the NE-induced redistribution of PKC activity from the cytosolic to the particulate fraction was also similar in pinealocytes prepared from rats subjected to LL or LD lighting conditions (Table 5). In contrast, when the effect of stimulus deprivation on NE-, ISO-, ISO and PMA-, and ISO and ionomycin-stimulated cyclic nucleotide responses was determined, LL treatment caused a greater agonist-stimulated cAMP response and a smaller cGMP response (Tables 5 and 6) as described previously (Vanecek et al., 1986; Chik and Ho, 1991). These observations indicate that while stimTable 3 Effect of BAPTA-AM on ISO, NE, ISO+PMA and ISO+ionomycin-stimulated cyclic nucleotide accumulationa Treatment

cAMP pmol/105 cells

cGMP

−BAPTA

+BAPTA

−BAPTA

Control 40 9 5 ISO (1 mM) 384 931 +PMA (0.1 3321 9205 mM) +Ionomycin 2325 9 185 (10 mM) NE (10 mM) 2530 9250

55 9 6 4289 26 3632 9257

10 9 3 359 4 65 9 8

992 30 9 4 579 7

559955b

5989 45

49 9 9b

628945b

6789 55

45 9 8b

+BAPTA

Fig. 7. Effect of stimulus deprivation on the expression of PKCa, d, o, and z. Animals were subjected to 14 h light/10 h dark (LD) or 5 days of constant light (LL). Pineal protein (25 mg) prepared from these animals was subjected to 7.5% SDS-PAGE and PKC isozymes were identified by Western blotting using polyclonal antibodies as described in Section 2. The Western blot shown is representative of three independent experiments.

ulus deprivation is effective in modulating cyclic nucleotide responses, it has no effect on the expression of PKC isozymes.

3.7. Effect of o6ernight treatment with PMA on PKC isozymes in rat pinealocytes Treatment with PMA for 24 h resulted in a marked reduction in PKCa and o, and a small reduction in PKCd (Fig. 8). In contrast, this treatment had no effect on PKCz, the atypical PKC isozyme (Fig. 8). The effect of selective down-regulation of PKC isozymes in adrenergic-stimulated cyclic nucleotide responses was investigated in rat pinealocytes subjected to acute (added simultaneously) or prolonged (pre-treated for 24 h) treatment with PMA. As shown in Table 7, simultaneous addition of PMA enhanced the ISO-, ISO and ionomycin- or ISO and phenylephrine-stimulated cAMP and cGMP responses, with PMA causing an 8-fold increase in the ISO-stimulated cAMP accumulation. In cells pre-treated with PMA for 24 h, PMA only caused a 4-fold increase in the ISO-stimulated cAMP accumulation (Table 7). Pre-treatment with PMA also reduced the ISO and phenylephrine- or ISO and ionomycin-stimulated cAMP and cGMP responses Table 4 Effect of stimulus deprivation on PKC activity measured in rat pineal homogenates using different peptide substratesa Treatment

LD glands LL glands

PKC activity (pmol/min per mg protein) Ac-MBP(414)

o-peptide

z-peptide

109 916 111 94

3024 9135 29189156

778 9 38 745 9 62

a

Pinealocytes (2×105 cells/0.5 ml) were incubated in DMEM with 10% fetal bovine serum and stimulated by different treatments for 15 min in the presence or absence of BAPTA-AM (1 mM). Each value represents the mean 9 SEM, n= 3, based on duplicate measurements. b Significantly different from treatment without BAPTA-AM.

a Pineal homogenates were prepared from rats subjected to LD or LL lighting conditions and their PKC activity was determined using Ac-MBP(4-14), o-peptide or z-peptide as the substrate. Each value represents the mean 9SEM, n= 3, based on duplicate measurements.

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Table 5 Effect of stimulus deprivation on NE stimulated translocation of PKC and cyclic nucleotide accumulationa

Table 6 Effects of stimulus deprivation on ISO-, ISO+PMA- and ISO+ionomycin-stimulated cyclic nucleotide accumulationa

Treatment

Treatment

Control-LD Control-LL NE (10 mM)-LD NE(10 mM)LL

PKC activity pmol/6 min per 106 cells

cAMP cGMP pmol/105 cells

Cytosolic

Particulate

1625 955 1454 973 1516 983

213 9 30 2209 25 3849 40

50 9 5 459 4 22359 65

15549113

3849 40

30509 105b

109 2 11 9 3 560950 609 5b

a

Dissociated pinealocytes (2×105 cells/0.5 ml) prepared from rats subjected to LL or LD lighting conditions were stimulated with NE for 6 min. Cytosolic and particulate fractions were prepared and solubilized. PKC activity of each fraction was measured using AcMBP(4-14) as substrate. For the cyclic nucleotide measurements, LL and LD pinealocytes were stimulated with NE for 15 min. Each value represents the mean 9SEM, n=3, based on duplicate measurements. b Significantly different from result in LD cells.

when compared with either acute treatment or no treatment with PMA. These results, apart from supporting the involvement of PKC in the potentiation mechanism mediated by a1-adrenergic receptors and intracellular Ca2 + -elevating agents, also suggest that the PKC isozymes involved appear to be down-regulated by 24 h treatment with PMA.

4. Discussion Although the involvement of PKC in the regulation of pineal cyclic nucleotide accumulation has been clearly established (Sugden et al., 1985; Ho et al., 1988c), the specific isozyme(s) involved remain unknown. In the present study we characterized the PKC isozymes expressed in the rat pineal gland and their

Control ISO (1 mM) +PMA(0.1 mM) +Ionomycin(10 mM)

cGMP

LD

LD

LL

LL

35 910 29 98 892 692 323 926 603 931b 37 9 6 26 92b 3042928 4312 9226b 66 9 9 429 5b 2216 9198 2813 9271b 511 9 37 139 9 13b

a Pinealocytes (1.5×104 cells/0.4 ml) were incubated in DMEM with 10% fetal bovine serum and stimulated by different treatments for 15 min. Each value represents the mean9SEM of determinations done in duplicate on three samples of cells. b Significantly different from result in LD cells.

regulation by the endogenous neurotransmitter, NE. Using Western blot analysis, we found that all three classes of PKC isozymes are expressed in the rat pineal gland: PKCa, a classical PKC isozyme, PKCd, and o, novel PKC isozymes and PKCz, an atypical PKC isozyme. The finding of novel and atypical PKC isozymes in the rat pineal gland extends the previous study that showed the presence of type II and type III isozymes (PKCa and b) in these cells (Yoshida et al., 1988). In agreement with the previous study, we demonstrated the presence of PKCa and the absence of PKCg isozyme. However, the presence of PKCb in the pineal could not be confirmed even though the expression of this isozyme is clearly detectable in other tissues, including the cortex, in the same immunoblot. It is possible that this discrepancy is related to the difference Table 7 Effect of acute treatment and overnight treatment with PMA on the potentiation of the ISO-stimulated cyclic nucleotide responses by phenylephrine (PE), and ionomycin (ION)a Treatment

−PMA +PMA(acute) cAMP (pmol/105 cells)

+PMA(24 h)

Control ISO (1 mM) +PE (10 mM) +ION (10 mM)

45 910 50 98 360 9 26 2820 9131b 2318 9178 35139 151b 2416 9198 36509 145b cGMP (pmol/105 cells) 8 92 10 92 40 9 6 66 9 9b 450 950 635 931b 525 947 673929b

30 92 1410 966c 1233 9116c 1336 978c

Control ISO (1 mM) +PE (10 mM) +ION (10 mM) Fig. 8. Effect of treatment with PMA for 24 h on the expression of PKCa, d, o, and z. Pinealocytes (2 × 105 cells/0.5 ml) were cultured for 24 h in the presence or absence of PMA (0.1 mM). Tissue samples prepared from control (C) and PMA-treated (P) cells were analysed by Western blotting using polyclonal antibodies as described in Section 2. The Western blot shown is representative of three independent experiments.

cAMP pmol/105 cells

7 92 30 96c 326921c 412 933c

a Pinealocytes (4×105 cells/5 ml) were cultured for 24 h in the presence (+) or absence (−) of PMA (0.1 mM). After 24 h, cells (l.5×104 cells/0.4 ml) were stimulated by different treatments for 15 min. Each value represents the mean 9 SEM of determinations done in duplicate on three samples of cells. b Significantly different from treatment without PMA. c Significantly different from acute treatment with PMA.

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in the PKCb antiserum used. However, we have obtained similar results with anti-PKCb1 and antiPKCb2 from two other commercial sources (Transduction laboratory and Santa Cruz) (our unpublished observation). In our previous studies, we have shown that PKC activity in rat pinealocytes can be translocated from the cytoplasmic fraction to the particulate fraction by NE, PMA and intracellular Ca2 + -elevating agents (Ho et al., 1988c, 1996). In the present study, we have characterized the PKC isozymes that are translocated by different mechanisms. As expected, PMA is effective in translocating the classical (a) and novel (d and o) PKC isozymes while ionomycin, an intracellular Ca2 + -elevating agent, is effective in translocating the Ca2 + -dependent PKCa. The finding that NE causes intracellular Ca2 + elevation (Ho et al., 1996) and translocates both classical and novel PKC isozymes suggests that NE is utilising different signalling pathways, probably generation of DG and elevation of intracellular Ca2 + , in activating different PKC isozymes. In rat pinealocytes, PKC is involved in the a1-adrenergic potentiation of b-adrenergic stimulated cyclic nucleotide responses and the NE-mediated translocation of PKC is through activation of a1-adrenergic receptors (Sugden et al., 1985; Ho et al., 1988c). Of the four isozymes examined, PKCa, d, and o, but not PKCz, are translocated by NE indicating that PKCz is unlikely to be directly involved in the NE-stimulated cyclic nucleotide responses. The NE-mediated translocation of PKCa, d, and o is mediated through activation of a1-adrenergic receptors since it is mimicked by phenylephrine and inhibited by prazosin, an a1-adrenergic antagonist. Although NE is effective in inducing the translocation of PKCa, d, and o, the temporal profiles of PKC isozyme translocation are not identical. Only the NE-induced increase in PKCd in the particulate fraction is sustained for 30 min. Since the a1-adrenergic mediated potentiation of cyclic nucleotide responses has been shown to be sustained for 30 min (Vanecek et al., 1985), this raises the possibility that PKCd is the isozyme involved in this response. A requirement of Ca2 + for the translocation of classical PKC isozymes is confirmed. Clamping intracellular Ca2 + by BAPTA-AM reduces the NE-mediated translocation of PKCa, a classical PKC isozyme that requires Ca2 + for its activation (Sekiguchi et al., 1988). Not only does BAPTA-AM reduce the NE-mediated increase in PKCa, it also reduces PKCa in the particulate fraction in unstimulated cells. This indicates that the distribution of PKCa in rat pinealocytes is highly sensitive to intracellular Ca2 + concentration even under basal conditions. The effect of BAPTA-AM on PKC appears isozyme-specific since it is not seen with PKCd, o or z, even when the same sample was used for the immunoblot.

Interestingly, BAPTA-AM treatment also blocks the NE-stimulated redistribution of PKCd and o, even though these isozymes are not Ca2 + -dependent and elevation of intracellular Ca2 + by ionomycin has no effect on their distribution. One possible explanation for this observation is that treatment with BAPTA-AM also inhibits the receptor-mediated activation of phospholipase C and hence prevents generation of DG. Indeed we have previously shown that the NE activation of phospholipase C in rat pinealocytes is blocked by EGTA, a Ca2 + chelating agent (Ho et al., 1988a). BAPTA-AM, however, has no effect on basal or NEstimulated distribution of PKCz, confirming that this isozyme does not require Ca2 + or DG for its activation. Many proteins are subjected to photoneural regulation in the rat pineal gland (Stehle et al., 1993; Baler and Klein, 1995). Our observation shows that neither the in vitro PKC activity nor the expression of PKC isozymes is affected by stimulus deprivation. These results are consistent with the observation that PMA remains effective in potentiating the b-adrenergic (Vanecek et al., 1986) or VIP-stimulated cAMP response after stimulus deprivation (Chik and Ho, 1991). Furthermore, our results also indicate that the smaller cGMP response observed after stimulus deprivation (Vanecek et al., 1986; Chik and Ho, 1991) is not related to changes in the expression of PKC isozymes. In the present study, we also found a difference in the sensitivity of PKC isozymes to PMA. Treatment with PMA causes a near complete translocation of PKCd and o from the cytosolic to the particulate fraction. While PMA also causes translocation of PKCa, it has no effect on PKCz, an isozyme not activated by DG or phorbol esters (Ohno et al., 1987; Ways et al., 1992) and therefore not down-regulated by treatment with PMA. This difference in sensitivity of PKC isozymes to PMA is also reflected in their responses to prolonged treatment with PMA. Overnight treatment with PMA causes down regulation of PKCa, d, and o but has little effect on PKCz. As indicated in the introduction, one possible explanation for the difference in cyclic nucleotide responses to potentiation by a1-adrenergic agonists and different activators of PKC is that a different set of PKC isozymes may be activated by specific activators of PKC. However, our results on PKCa, d, o, and z fail to support this hypothesis. For example, while activation of a1-adrenergic receptors and treatment with PMA have similar effects on the four isozymes, the former is effective in potentiating both cAMP and cGMP responses, while the latter is effective in potentiating only the cAMP response. In contrast, while ionomycin potentiates both cAMP and cGMP responses, its effect on the PKC isozymes is different from that of NE. Nonetheless, since we have only examined the regula-

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tion of four specific PKC isozymes, it is possible that other PKC isozymes expressed in the pineal are involved in the different potentiating effects on cAMP and cGMP accumulation.

Acknowledgements A.K. Ho is a scholar of the Alberta Heritage Foundation for Medical Research. This work was supported by grants from the Medical Research Council of Canada and the University of Alberta Hospital Foundation. We would like to express our deep appreciation to Dr A. Baukal for his gift of anti-cAMP and anti-cGMP sera and Dr S. Barton for her helpful discussion.

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