0013-7227/91/1293-1605$03.00/0 Endocrinology Copyright (p) {991 by The Endocrine Society
Vol. 129, No. 3 Printed in U.S.A.
ai-Adrenergic Receptor Coupling with Phospholipase-C Is Negatively Regulated by Protein Kinase-C in Primary Cultures of Hypothalamic Neurons and Glial Cells* SOPHIA V. DROUVA, ANNIE FAIVRE-BAUMAN, CATHERINE LOUDES, ELIANE LAPLANTE, AND CLAUDE KORDON U. 159INSERM, Unite Dynamique des Systemes Neuroendocriniens, Centre Paul Broca, 75014 Paris, France
of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in young and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alR. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alR, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture. {Endocrinology 129: 1605-1613, 1991)
ABSTRACT. In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a doseand time-dependent manner. The NE effect was mediated by ai-receptor (alR) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alR with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation
"XTORADRENERGIC neurons are widely distributed l l in the mammalian brain (2, 3). Norepinephrine (NE) is known to regulate hypohyseal hormone secretion by affecting the release of several hypothalamic neuropeptides (4-7). Different adrenergic receptors (au a2, ft, /32, and /?3) mediate the physiological effects of epinephrine (E) and NE via GTP-binding proteins (G-proteins), which couple each receptor subtype to a distinct intracellular effector system (8, 9). Upon agonist binding, the «i-adrenergic receptor («1R) has been shown in different tissues to transduce its message by activating phospholipase-C (PLC) and A2 (10-17), increasing calcium influx via receptor-operated channels (18), modulating neuronal currents (19), and regulating intracellular levels of cAMP (8, 20, 21) and cGMP (21). PLC-mediated hydrolysis of phosphoinositides (Pins) leads to the production of inositol 1,4,5-triphosphate Received March 25, 1991. Address all correspondence and requests for reprints to: Dr. S. V. Drouva, Department of Neuroendocrinology, U-159 INSERM, 2 ter rue D'Alesia, F-75014 Paris, France. * A preliminary report of these data was presented at the Seventh ESN meeting (1).
[Ins(l,4,5)P3], which can mobilize calcium from intracellular stores, inositol 1,3,4,5-tetrakis phosphate, which may act to open calcium channels, and diacylglycerol (DAG), which together with calcium can activate a calcium- and phospholipid-dependent protein kinase, protein kinase-C (PKC) (22, 23). The tumor-promoting phorbol esters mimick the effects of endogenously produced DAG in activating PKC (23) and, thus, provide a powerful tool to study the role of PKC in cell function. Activation of PKC results in the inhibition of agonistinduced Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and Ca2+ mobilization in a variety of tissues (24-30). These observations revealed the important role of PKC activity in the feedback regulation of the receptor-operated Ins(l,4,5)P3/Ca2+ signaling pathway (2430). In the present study, using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures (31, 32) we attempted to investigate the possible interactions of adrenergic receptors with enzymatic activities, such as PLC and PKC implicated in membrane coupling mechanisms. Responses of immature neurons sampled after 7 days in vitro (7 Div) before synaptoge-'
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PKC REGULATION OF «1R COUPLING WITH PLC
nesis (33, 34) and those of more developed hypothalamic neurons (14 Div) were compared in view of previous data (33, 34) showing that hypothalamic neurons in serumfree cultures displayed growth cones and a few varicosities after 6 days in culture, whereas mature synapses are only apparent in hypothalamic neurons after 10 days in vitro. Neuronal responses were also compared to those in glial cells at the same developmental stages. Materials and Methods Hypothalami were taken from albino Swiss mice (Iffa-Credo, Labresle, France) on the 16th day of gestation. Primary cultures of hypothalamic cells were prepared as previously described (31, 32). Briefly, the hypothalami were excised under a dissecting microscope and mechanically dissociated in Ham's F-12Dulbecco's Modified Eagle's Medium (F12-DMEM; Gibco, Grand Island, NY; vol/vol) supplemented with 10% fetal calf serum. Cells dispersed from eight hypothalami were resuspended in serum-free medium and plated (~4.5 X 106 cells/ dish) on gelatin/poly-Mysine- and fetal calf serum-coated dishes (Lux Corp., Thousand Oaks, CA) of 60 mm. The cells were grown for different times (7 or 14 days) in serum-free F12-DMEM (vol/vol), supplemented as previously described (5 jug/ml insulin, 100 jug/ml human transferrin, 2 X 10"8 M progesterone, 3 X 10"8 M selenium, 10"4 putrescine, 10"12 M 170estradiol, 1 Mg/ml arachidonic acid, 0.5 ^g/ra\. docosahexaenoic acid, 10"8 M T3, and 10"7 M corticosterone) (31-34). Cultures were maintained at 37 C in 7% CO2-93% air and 92-95% humidity. The medium was first renewed 5 days after seeding with cytosine arabinoside (10 6 M)-supplemented medium to block the proliferation of glial cells, and then twice a week. In such cultures the proportion of hypothalamic neurons vs glia cells is around 5-6:1 (31, 34); they are refered to as neuronal cultures. Primary cultures of glial hypothalamic cells were initiated from hypothalami of the same developmental stage and grown in medium supplemented with 10% fetal calf serum for the first 4 days. Cells were then incubated for 30 min in versene-EDTA, gently scraped, and replated for 48 h in the serum-supplemented medium, then switched to the serum-free medium (32). These glial cell cultures reveal few oligodendrocytes and a vast majority of astrocytes (32). Measurement of inositol phosphates (IPs) Hypothalamic neurons or glial cells were prelabeled with myo-[3H]inositol (2.5 /iCi/ml; 12.8 Ci/mmol; New England Nuclear, Boston, MA) 72 h before the experiment. On the day of the experiment (7 or 14 Div), the cells were washed and then incubated for 15 min in the regular serum-free culture medium in which 10 mM LiCl was added. The substances to be tested were then administered for the time and at concentrations indicated in Results or in the legends to the figures. Incubations were terminated by elimination of the incubation medium and addition of ice-cold perchloric acid (PCA; 5%). The cells were scraped and transferred to extraction tubes, and PCA-precipitable proteins and lipids were removed by centrifugation (5000 X g for 10 min). The acid-soluble fractions
Endo • 1991 Vol 129 • No 3
were collected and neutralized with 1.5 mM KOH containing 70 mM HEPES. IPs were extracted by chromatography on ion exchange columns (AG 1x8 formate form, Bio-Rad, Richmond, CA). Tritiated myo-inositol was eluted with bidistilled water, and the total [mono (IPi)-, bis (IP2)-, and trisphosphate (IP3)] radioactive phosphorylated IPs were eluted with a 1 M ammonium formate/100 mM formic acid solution, as described previously (35). In some experiments in which the IPs were separated, the elution was as follows: [3H]glyceryl-phosphoryl inositol (60 mM ammonium formate-5 mM sodium tetraborate), IPi (200 mM ammonium formate-100 mM formic acid), IP2 (400 mM ammonium formate-100 mM formic acid), and IP 3 (1 M ammonium formate-100 mM formic acid), as described previously (35). Radioactivity in all fractions was determined by scintillation counting. Results are expressed as counts per min/dish (4.5 x 106 cells) or counts per min/10 ng DNA. Quantification of cellular levels of DAG
DAG was measured according to the procedure of Preiss et al (36), using Amersham's assay reagents system (RP2 200, Arlington Heights, IL). Briefly, the assay is based on the enzymatic conversion of DAG to phosphatidic acid (PA) using DAG kinase (Amersham) and [ T - 3 2 P ] A T P (3 Ci/mmol; Amersham). The [32P]PA reaction product was extracted and separated by Amprep Si minicolumns chromatography (Amersham) using the solvent system consisting of choloroform-methanolacetic acid (65:10:10, vol/vol/vol). The radioactivity attributable to [32P]PA was determined by liquid scintillation counting. The amount of DAG present in the original sample was calculated from the [32P]PA formed, the sample volume, and the specific activity of [T- 3 2 P]ATP. The [32P]PA formed in the experimental samples was compared with the [32P]PA generated by standards containing known amounts of DAG (0-1000 pmol). Results are expressed as picomoles per dish (4.5 X 106 cells). Measurement of membrane Phosphoinositid.es (Pins) The cells were incubated with myo-[3H]inositol for 72 h. Then, the 5% trichloroacetic acid-precipitated Pins were extracted successively in solution I (chloroform-methanol-HCl, 1:1:0.005, vol/vol/vol) and solution II (chloroform-methanolHCl, 1:2:0.005, vol/vol), followed by a further extraction step with a 2:1 (vol/vol) mixture of chloroform and the aqueous phase obtained from chloroform-methanol-HCl (0.1 N; 2:1:1.5, vol/vol/vol), as described previously (37). TLC was performed to separate the Pins; the silica plates (Kieselgel 60, Merck, Rahway, NJ) impregnated in potassium oxalate-methanol (1:1, vol/vol) were developed in chloroform-methanol-NH4OH (4 N; 9:7:2.1, vol/vol/vol) (37). Radioactivity corresponding to bands of Phosphotidylinositol (PI), Phosphotidylinositolmonophosphate (PIP), PIP 2 (same Rf values as those of corresponding standards developed simultaneously) was evaluated by liquid scintillation spectrometry. DNA was assayed according to the method of Hill and Whatley (38), using calf thymus DNA as a standard.
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PKC REGULATION OF «1R COUPLING WITH PLC Pharmacological agents The following were used: NE (Sigma, St. Louis, MO), phenoxybenzamine-HCl (Smith, Kline, and French, Philadelphia, PA), D,L-propranolol-HCl (Sigma), prazosin-HCl (Pfizer, Paris, France), yohimbine-HCl (Sigma), PN-200-110 (Sandoz, Hanover, NJ), BAY-5009-nitrendipine [3'-methyl-5'-ethyldihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3-5'-dicarboxylate; generously provided by Prof. F. Hoffmeister (Bayer AG Laboratories, Wuppertal, Germany), w-conotoxin (Sigma), 12O-Tetradecanoylphorbol 13-acetate (TPA), and 4a-phorbol12-13-didecanoate (a-PDD) (Sigma), and pertussis toxin [isletactivating protein (IAP); List Biological Laboratories, Inc., Campbell, CA], Statistical analysis
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mature 14 Div neuronal cultures. As Fig. 2 shows, the pattern of IPs production followed the classical pattern described in other systems (22); IP 3 was immediately increased (within seconds) and further enhanced for 10 min, IP2 progressively increased up to 30 min (maximum time tested), and IPi started to be significantly augmented after 5 min of NE stimulation. In addition, the levels of DAG were significantly increased after 1- and 5-min exposure to NE (10'6 M; Fig. 2B). To characterize the type of adrenergic receptors involved in NE-induced IP production, adrenergic blockers with different specificities for /?-, ax-, and a2-receptor subtypes were administered, either alone or in combination with NE (10 6 M) for 10 min. As Fig. 3 shows, the /3-
Data from several experiments were pooled, and statistical analysis between experimental groups was performed using analysis of variance, followed by post-hoc Scheffe F statistic for multiple comparisons, with an overall two-tailed a value of 0.05.
Results Effect of NE on IPs and DAG formation is mediated by alR in hypothalamic neurons in culture NE applied for 30 min to an enriched preparation of hypothalamic neurons cultivated for 14 days highly stimulated IPs production in a dose-dependent manner, with an EC50 of 10 6 M (Fig. 1). IPs were separated and individually estimated at different times after NE administration (10 6 M) to the
3000
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0 C 10- 9 10" 8 10"7 10- 6 10" 5 10" 4 Norepinephrine concentration (Molar) FIG. 1. Dose-response curve for NE-induced IPs production in 14 DIV hypothalamic neuronal cultures; the time of stimulation was 30 min. Values represent the mean ± SEM from the number of experimental points indicated in parentheses.
C
Imin 5min
FIG. 2. A, Time-dependent effect of NE (106 M) on IP lt IP2, and IP3 production in 14 Div hypothalamic neurons in primary cultures; O, D, and A, Control (C; same in following figures); • , • , and A, stimulated. Values represent the means from three experiments run in triplicate. B, NE (10'6 M)-induced DAG production in 14 Div cultures of hypothalamic neurons. Values represent the mean ± SEM; numbers in parentheses indicate the numbers of experimental points. *, P < 0.01 us. corresponding control group.
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PKC REGULATION OF alR COUPLING WITH PLC
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12000 10000 8000 6000 4000 2000 0
NE+ NE+ Phenox Pru NE+ Oored NE+ Phenox Prat Propran Yohimb
FlG. 3. alR involvement in NE (106 M for 10 min)-evoked IPs formation in 14 Div hypothalamic neurons in primary cultures. Phenox, Phenoxybenzamine (10'6 M); praz, prazosine (106 M); propran, propranolol (10'6 M); clonid, clonidine (10'6 M); yohimb, yohimbine (10'6 M). Values represent the mean ± SEM; numbers in parentheses indicate the number of experimental points. **, P < 0.01 us. corresponding control groups.
Endo • 1991 Voll29«No3
enhanced at a later stage of neuronal development (14 Div cultures). In addition, basal IPs production was slightly but significantly higher (P = 0.05) in 14 Div cultures than in 7 Div cultures of hypothalamic neurons (Fig. 4). This increase was not due to cell proliferation during that interval of culture, since the numbers of cells per dish were not significantly different at the two stages of neuronal development tested [micrograms of DNA per dish: 7 Div, 17.1 ± 0.5 (n = 7); 14 Div, 16.5 ± 1.15 (n = 8)]. Furthermore, the levels of membrane PI, PIP, and PIP 2 (Pins) were also measured to evaluate differences in the available substrates for the enzyme activities related to generation of Pins and IPs formation during neuronal development; (counts per min/dish: 7 Div, PI, 26,428 ± 2,896; PIP, 4,891 ± 131; PIP2, 2,312 ± 108; 14 Div, PI, 31,651 ± 3,647; PIP, 6,061 ± 246*; PIP2, 3,162 ± 371"; n = 8/group; *, P < 0.05 vs. the corresponding control values). Effect of PKC stimulation on alR-mediated IPs production
12000
C
NE
NE+ Praz
C
NE
NE+ Praz
FIG. 4. Developmental evolution of PLC activity associated with alR in hypothalamic neuronal 7 and 14 Div cultures. NE (10'6 M) was administered for 10 min. Praz, Prazosin (106 M). Values represent the mean ± SEM. **, P < 0.01; *, P < 0.05.
adrenergic blocker propranolol had no effect on either basal (data not shown) or NE-induced IP formation; in addition, the /3-receptor agonist clonidine (10 6 M) did not affect phosphoinositides hydrolysis. In contrast, the a-adrenergic blocker phenoxybenzamine (10 6 M), although devoid of effect by itself, completely blocked the NE (10 6 M)-induced IPs formation. Furthermore, the «i-blocker prazosin (10 6 M), but not the «2-antagonist yohimbine (10'6 M), completely inhibited NE-evoked IPs production (Fig. 3). Developmental evolution of IPs formation in response to NE
To determine whether the IP response to NE is dependent upon neuronal maturation, NE-induced IPs formation was evaluated before (7 days of culture) or after the onset of synaptogenesis (14 days of culture). As shown in Fig. 4, PLC activity was already coupled to the alR at 7 days of culture; the amplitude of the IP response to NE stimulation (10'6 M for 10 min), however, was
To evaluate possible feedback control of PKC on PLC activity, neuronal cultures were exposed to phorbol esters [TPA; 10 (data not shown) or 100 ng/ml] at different times before NE (10'6 M for 10 min) stimulation. Simultaneous administration of TPA with NE slightly but significantly diminished the NE effect on IPs production, while 5-min pretreatment with TPA dramatically inhibited NE-induced IPs formation (Table 1). Administration of TPA to the cultures for 20 min before the stimulus (Table 1 and Fig. 5), completely abolished the effect of the amine in both neuronal maturation stages studied (14 and 7 Div cultures). A biologically inactive phorbol ester, 4a-phorbol 12,13-didecanoate, used as a control at a concentration of 100 ng/ml for 20 min, was ineffective (Fig. 5). Longer pretreatments with TPA (up to 15-20 h), known to effectively deplete PKC (23, 28), had no significant effect on NE-evoked IP production (Table 1). Voltage-dependent Ca2+ channels and G-proteins in basal and NE-induced IPs formation
To examine whether NE-induced PLC activation is dependent on the activity of voltage-dependent Ca2+ channels, different Ca2+ channel blockers were tested in mature 14 Div neuronal cultures, either alone or in combination with the transmitter. As Fig. 6 shows, basal as well as NE (10 min; 10'6 M)evoked IPs production remained unchanged after the administration of any of the specific blockers of calcium voltage-dependent channels (L- or N-type voltage-sensitive calcium channels) (39) to 14 Div hypothalamic cultures. Nitrendipine and PN200-110 (L-type inhibitors) were applied at 10"6 M and co-conotoxin (L- and Ntype inhibitor) at 10"7 M for 10 min, either alone or in
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PKC REGULATION OF a l R COUPLING WITH PLC
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TABLE 1. Effect of PKC activation on NE-induced IP formation in 14 Div neuronal cultures (counts per min/dish) TPA (100 ng/ml) pretreatment to NE (10~6 M) for 10 min stimulation
6
NE (10~ M for 10 min)
Control 3,827 ± 386 (9)
Simultaneous administration
12,410 ± 1,042 (9)a
9,084 ± 386 (5)6
5 min
20 min
6,015 ± 409 (6)°
20 h 10,922 ± 820 (4)c
3,350 ± 250 (6)"
Values represent the mean ± SEM; the number of experimental points is in parentheses. " P < 0.01 us. corresponding control group. 6 P < 0.05 us. corresponding control group. C P = NS.
vates certain G-proteins by catalyzing their ADP ribosylation. Pretreatment with the toxin had no effect on basal IPs levels; in contrast, NE-stimulated IPs production was significantly, although not completely, inhibited by IAP administration in cultures before (7 Div) and after (14 Div) synaptogenesis (Fig. 7). PLC activity associated with alR in hypothalamic astroglial cell cultures NE NE+ TPA NE+ TPA 4..PD
NE NE+ TPA NE+ TPA
4aP0
FiG. 5. Feedback control between PKC and PLC activities associated with ttlR of hypothalamic neurons of 7 and 14 Div cultures. TPA and the inactive phorbol ester 4aPDD (100 ng/ml) were applied to the cultures 20 min before NE (10'6 M for 10 min) stimulation. Values represent the mean ± SEM. **, P < 0.01 us. corresponding control group, n, Number of experimental points.
16000 -
Control
NE
Addition of NE (10 6 M) for 30 min to primary cultures enriched in hypothalamic astrocytes (7 or 14 Div as well) but devoid of neurons resulted in increased IPs formation (Fig. 8 and Table 2). The potency of the response to the adrenergic stimulus was greater in 14 Div cultures; however, the basal IP levels were slightly but significantly higher (P = 0.05) in 7 Div cultures (Table 2). Since the number of glial cells in 14 Div is greater than that in 7 Div cultures, the IPs response to adrenergic stimulus was also expressed as counts per min/10 ng DNA (Table 2). NE-induced production of IPs also seems to be mediated by an alR subtype, as in neuronal cultures, since prazosin (10 6 M) completely blocked the NE effect (Fig. 8). In addition, the IP response to NE remained unaffected after administration of the L- or N-type voltage-
Nitrend NE+ PN200 NE+ w-conot NE+ Nitrend PN200 a>-conot
FiG. 6. Interaction of calcium voltage-dependent channels with basal as well as alR-coupled PLC activity in 14 Div hypothalamic cultures. Values represent the mean ± SEM; the numbers in parentheses indicate the number of experimental points. **, P < 0.01 vs. the corresponding control group. C
combination with the amine. To determine whether the alR inducing IPs formation involved an IAP-sensitive G-protein, 14 or 7 Div cultures of hypothalamic neurons were pretreated overnight with pertussis toxin (10 or 100 ng/ml), an agent that inacti-
NE IAP NE+ IAP
C
NE IAP NE+ IAP NE+ IAP IAP
FiG. 7. Involvement of G-proteins in NE (10'6 M for 10 min)-induced IPs formation in 7 and 14 Div neuronal hypothalamic cultures. Values represent the mean ± SEM; the numbers in parentheses indicate the number of experimental points. **, P < 0.01 compared to the corresponding control group.
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PKC REGULATION OF «1R COUPLING WITH PLC
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14 days (6) *•
i
(4)
I
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- | 3000 2000 (6)
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NS
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1 (5) **
15)
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NE NE+ TPA NE+ IAP NE+ Praz TPA IAP
FIG. 8. alR-mediated IPs production in 14 Div hypothalamic glial cultures: interaction with PKC activity and G-proteins. Values represent the mean ± SEM; the numbers in parentheses indicate the number of experimental points. **, P < 0.01 compared to the corresponding control group. TABLE 2. IP response of glial cells to NE stimulation (counts per min/ 10 ng DNA) Age of cultures (Div)
Control
NE (10- M for 30 min)
7 14
1090 ± 118° 395 ± 45
2380 ± 300 (5)c 1722 ± 44 (5)°
Values represent the mean ± SEM; the number of experimental points is in parentheses. 0 P < 0.01 us. the corresponding control group.
sensitive calcium channel blockers (14 Div; counts per min/dish: control, 1398 ± 190; 10 6 M NE for 30 min, 5221 ± 124; NE plus 10"6 M nitrendipine, 5040 ± 190; NE plus 10"7 M oo-conotoxin, 5178 ± 89; 10"6 M nitrendipine, 1402 ± 68; n = 4/group). As in primary cultures of hypothalamic neurons, pretreatment with the phorbol ester TPA (100 ng/ml) for 20 min completely abolished NE-induced IPs formation (Fig. 8). In contrast to the observation made on neuronal cultures, alR-mediated PLC activation in astroglial cells was not coupled to pertussis toxin-sensitive G-proteins. Administration of IAP (100 ng/ml) for 15 h before the addition of NE did not affect NE-induced IPs formation (Fig. 8). Discussion The «1R has been previously shown to be linked to PLC and PLA-A2 activation in various tissues, including the central nervous system (10-17). In the present work we demonstrated that NE caused an increase in the production of IPs and DAG in hypothalamic neurons and astroglial cells grown in primary culture. The effect was observed in mature neurons (14 Div) as well as in younger cultures (7 Div) before synaptogenesis. The release of these second messengers was mediated by alR, since it was blocked by the selective ai-adrenergic antag-
E n d o • 1991 Vol 129 • No 3
onist prazosin. It has been reported that in the absence of dopaminergic stimulation, Dl receptors did not develop adequately or were not maintained (40). Interestingly, although NE is not present in our hypothalamic neuronal and glial cultures (41), the coupling of alR is already present in immature cultures and further develops in cultures in which synaptogenesis has been achieved. NE-induced IPs formation was more potent in hypothalamic neuronal cultures on 14 Div than on 7 Div. Since the number of neurons per dish is about the same in both 7 and 14 Div cultures, and since glial divisions are blocked by cytosin arabinoside, this could represent a difference in «1R number between younger and mature cultures and/or higher levels of PLC activity linked to «1R. Further work is needed to clarify these points. It is of interest to note, however, that basal levels of IPs are slightly but significantly higher in 14 Div than in 7 Div of hypothalamic neurons. This may suggest higher amounts of PLC activity or available substrate for the enzyme, such as PIP and PIP2. In fact, the levels of these substrates are slightly higher in 14 than 7 Div hypothalamic neuronal cultures. In another in vitro system of striatal neuronal cultures it has been reported that the basal levels of [3H]IP formation increased progressively up to 7 Div and remained constant thereafter (17); however, NE-stimulated [3H]IP production was increased in a linear fashion between 3-14 Div cultures (17). Furthermore, Nicoletti et al. (42) have demonstrated that in rat hippocampal slices the stimulation of Pins hydrolysis by glutamate progressively declined during postnatal development despite the increasing density of glutamate-binding sites. In contrast, the stimulation of Ips formation elicited by NE increased during development. None of the different dihydropyridine-sensitive calcium channel blockers used impeded the generation of IPs induced by NE. This indicates that the calcium voltage-dependent channels of L or N type present on hypothalamic neurons (43) are not implicated in the alR-operated PLC activation. Similar results have been obtained in spinal cord neurons (12) and cardiac myocytes (16). In hypothalamic glial cells, as in neurons, the IP response to NE stimulus was mediated by the alR subtype. As in neurons it was more potent in 14 Div than in 7 Div cultures; surprisingly, however, the reverse situation was recorded concerning the basal levels of IPs. In addition, it is of interest to note, that the basal levels of IPs as well as the absolute values of NE-induced IPs were lower in glial cells; however, the potency of the NE response (increase in IPs over the basal levels) was approximately the same (~3- to 4-fold) in both cell types. Furthermore, as in neurons, NE-induced IPs production
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PKC REGULATION OF «1R COUPLING WITH PLC
did not implicate activation of L- or N-type calcium voltage-sensitive channels. The tumor-promoting phorbol ester TPA has been used in this study to determine a possible relationship between PKC activation and NE-stimulated PI hydrolysis in cultures of hypothalamic and astroglial cells. Active phorbol esters bind to and stimulate PKC (22, 23). In addition, the phorbol ester-binding protein copurifies with PKC (23). The phorbol ester TPA inhibited NE-stimulated production of IPs in young and mature hypothalamic neurons and astroglial cell cultures, as has been found in several other systems to inhibit neurotransmitter-induced PI hydrolysis (24-30, 44, 45). The inhibitory effect of TPA on agonist-evoked IPs formation was immediate and stereospecific, since the stereoisomer phorbol a-PDD had no effect on basal or NE-stimulated IPs levels. Long term TPA treatment, which downregulates PKG (23, 28), was ineffective. These results indicate that PKC may play an important role in the regulation of alR coupling to the phosphoinositide phosphodiesterase; they further suggest that an individual receptor might be capable of modulating its function through the activation of PKC. DAG, the other product of Pins hydrolysis, should thus act through C-kinase as an endogenous inhibitor of hormone-stimulated Pins metabolism. In the present work we did not present data concerning homologous desensitization of agonist-induced Pins hydrolysis. However, it has been shown that exposure of neuronal cultures to carbachol for extended periods resulted, as in the case of TPA, in a loss of the muscarinic stimulated PI response in parallel with a loss of muscarinic recognition sites (27, 44). The inhibition of Pins hydrolysis by PKC may be due to C-kinase-mediated phosphorylation events (46-50): PKC implicated phosphorylation of PLC or of G-proteins coupling the receptor to the PLC has been proposed as a potential mechanism for the inhibitory effects of phorbol esters on receptor-mediated IPs formation (48, 49). Evidence obtained with a vas deferens smooth muscle cell line suggest that phorbol ester causes phosphorylation of an «1R ligand-binding subunit in parallel with a decrease in binding affinity for the agonist (29), thereby uncoupling the receptor from PLC activation (29, 46, 47). Receptor down-regulation may be also achieved by PKC-induced phosphorylation of one of the different enzymes implicated in Pins metabolism (28, 50). Phosphorylation, for example, of membrane protein B50 (50) reduces the amount of substrate available for agonistinduced Pins hydrolysis by affecting PIP kinase activity. We found that pretreatment with phorbol ester had no effect on unstimulated IPs levels in hypothalamic neurons and astrocytes; this may indirectly indicate that the
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synthesis of Pins remained unaffected. However, direct measurements of the [3H]inositol incorporation into Pins of hypothalamic neurons and astrocytes were not performed. Further work is needed to identify the exact mechanism(s) by which different species of PKC (51) might be involved in the phosphorylation of a specific substrate(s) implicated in the physiological feedback regulation of the PI cycle. Preliminary results (Drouva, S. V., unpublished) indicate that, in fact, more than one form of PKC with a differential activity dependence on phospholipids (Arachidonic Acid, phosphatidylserine, oleic acid, etc.) and calcium are present in hypothalamic neuronal and glial cells of 7 and 14 Div cultures. In the present studies a substantial component of the al-adrenergic IPs response in neuronal cultures was not inhibited by the concentrations of pertussis toxin used. Several explanations could account for this observation. It is possible that there is some substrate unresponsive to the actions of the toxin, or alternatively, the alR may be linked to PLC through more than one GTP-binding protein. The component of the phosphatidylinositol response that is resistent to pertussis toxin may be analogous to the PI response in astrocytes; no effect of IAP pretreatment was recorded in primary cultures of hypothalamic astroglial cells. While studies have described agonist-induced phosphoinositides hydrolysis as exclusively pertussis toxin sensitive or pertussis toxin insensitive in different tissues (8, 52-54), the ai-adrenergic IP response in the hypothalamic neurons (but not in astroglial cells) has elements of both, as has been also shown in cardiac myocytes (16) and spinal cord neurons (12). Indeed, in 7 Div as well as 14 Div neuronal cultures we have reported the presence of different species of Gproteins (aO, ai, and as) by both ADP ribosylation and Western blotting experiments (55). In conclusion, since Pins hydrolysis and PKC stimulation have been implicated in several cellular processes, including secretion, ionic permeability, cell growth and division, pH, and volume regulation (22, 23), and because the astrocytes appear to be involved in the modulation of neuronal excitability, pH, and cell growth (56), the properties of the regulatory feedback mechanism(s) described between alR-PLC-PKC may be particularly relevant to these functions during the developmental stages of hypothalamic cells.
Acknowledgments Thanks are given to Dr. A. Tixier-Vidal for her valuable comments, and to C. Rogers for her except secretarial help.
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