Involvement of protein kinase-C in the effect of angiotensin-II on adenosine 3',5'-monophosphate production in lactotroph cells.

0013-7227/91/1294-2231$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 4 Printed in U.S.A.

Involvement of Protein Kinase-C in the Effect of Angiotensin-II on Adenosine 3',5'-Monophosphate Production in Lactotroph Cells V. AUDINOT*, R. RASOLONJANAHARY, P. BERTRAND, M. PRIAM, C. KORDON, AND A. ENJALBERT U.159 INSERM, Centre Paul Broca, 75014 Paris, France

ABSTRACT. When applied to rat anterior pituitary cells, angiotensin-II (All) exerted two opposite effects on adenylate cyclase (AC) activity: a pertussis toxin (PTX)-sensitive inhibition of the enzyme with a maximal effect of —42 ± 2% in crude cell membrane preparations, and, in contrast, a non-PTX-sensitive stimulation of cAMP production (maximal effect = 38 ± 3%) in intact cells. The apparent affinity of both effects was equal to 1.8 nM. The stimulation of cAMP formation parallels the stimulation of PRL release. Under the same conditions, dopamine (DA) inhibited both membrane AC activity and cAMP formation in intact cells by a PTX-sensitive mechanism. After separation of pituitary cell types by sedimentation at unit gravity, the effects of All and DA on intracellular cAMP and membrane AC activity coincided in the same fractions (those enriched in PRL cells). The stimulatory effect of All on cAMP formation was about 5 times weaker than that of peptides positively coupled to AC as vasoactive intestinal peptide in total as well as in PRLenriched cells. Since the All receptor is also coupled to phospholipase-C (PLC) in a non-PTX-sensitive manner, we investigated whether protein kinase-C (PKC) could indirectly account

P

RL RELEASE is regulated by several hypothalamic factors, which exert their action by different effects on second messengers including cAMP, protein kinaseC (PKC), inositol phosphates (IPs), and K+ and Ca2+ channels (for rewiew, see Ref. 1). For instance, vasoactive intestinal peptide (VIP) stimulates, while dopamine (DA) and angiotensin-II (All) inhibit adenylate cyclase (AC) activity (2-4). All and thyreoliberin TRH stimulate, while DA inhibits phospholipase-C (PLC) activity (5-7). DA and TRH can also modulate the activity of ionic channels (8, 9). As a rule, the neurohormone effects on second messengers parallel those on secretion (1012), with the apparent exception of All, which inhibits AC activity in membrane preparations, while it stimulates PRL release (13). All receptors have been characterized in various anterior pituitary cell types (14-16) as well as in a number Received April 22,1991. * To whom all correspondence and requests for reprints should be addressed.

for the positive effect of All on cAMP formation. 12-O-Tetradecanolylphorbol 13-acetate (TPA), a stimulator of PKC was indeed able to increase intracellular cAMP; this effect was not additive with that of All. Conversely, application of the PKC inhibitors H7 [l-(5-isoquinolylsulfonyl)2-methyl-piperazine] and staurosporine or desensitization of PKC by long exposure of the cells to TPA abolished the cAMP response to TPA as well as that to AIL In addition, thyreoliberin, another activator of the PLC pathway, was able to stimulate cAMP formation in a PKC-dependent manner. DA inhibition of intracellular cAMP was not affected by any PKC inhibition. We conclude that in lactotroph cells, 1) the All inhibitory coupling to AC observed in membrane preparations does not exist in intact cells, at least under basal conditions; and 2) the All intracellular cAMP stimulation observed is not accounted for by a direct coupling with AC; it is due to a cross-talk of the PLC pathway mediated by PKC, an effect that might be shared by other PLC-stimulating mediators and may participate in the regulation of PRL release. (Endocrinology 129: 2231-2239, 1991)

of peripheral targets (for review, see Ref. 17). Only one type of All receptor has been described using All analogs, although a possible receptor heterogeneity has been reported in hepatocytes (17,18). The recent use of nonpeptide analogs of All have confirmed the existence of All receptor subtypes (19). All receptor activation of PLC appears to be insensitive to pertussis toxin (PTX), a bacterial toxin that selectively uncouples ai/ao-subunits of G-proteins. The question, however, is not completely settled, since a PTX-sensitive G-protein has been reported to mediate part of the PLC response to All in adrenal fasciculata cells (20). In addition, a PTX-dependent coupling of the All receptor to AC has been described in membrane preparations on all All targets (17). However, intracellular cAMP can be either inhibited, through a PTX-sensitive mechanism, in liver and glomerulosa cells (21, 22), unaffected in vascular smooth muscle cells (23), or stimulated in the case of adrenal medulla cells (24). In view of these discrepancies, it was of interest to

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All EFFECT ON cAMP IN LACTOTROPHS

2232

study more thoroughly the effects of All in adenohypophyseal cells. We attempted to reassess the impact of All receptors on the AC pathway in intact pituitary cells and in lactotroph-enriched populations in a model allowing the concomitant determination of PRL release. We also compared the effects of All with those of neurohormones exhibiting comparable coupling mechanisms: DA for the negative coupling with AC, and TRH for the positive coupling to PLC and its possible, although conflicting, coupling with cAMP production (25, 26). Using intact cells, All inhibition of AC was not observed under our experimental conditions. In contrast, the peptide stimulated intracellular cAMP formation. The same result was observed with TRH. This cAMP stimulation appeared to be secondary to PKC stimulation rather than dependent upon a direct coupling with AC. That mechanism might represent an additional mechanism involved in the regulation of PRL secretion.

Materials and Methods

Endo • 1991 Vol 129 • No 4

antibiotics (penicillin and sreptomycin; 0.05 mg/ml) for 5 days. PTX or 12-O-tetradecanoylphorbol 13-acetate (TPA) was added to the medium 20 h before the experiment. Cell separation by unit gravity sedimentation In some experiments, dispersed cells were sedimented according to their size and density through a discontinuous gradient of BSA (1-4%) at room temperature using a large area cell sorter (LACS), as previously described (28). Cells (120 x 106) were loaded at the top of the gradient and submitted to sedimentation for 30 min. After sedimentation, 20 fractions were collected from the top of the gradient. Aliquots were stored at -20 C until PRL, GH, /3-endorphin, LH, and TSH contents were determined by RIA (29-33). Cell counts and size were estimated with a Coulter counter ZBI coupled to a volume analyzer C1000. The average recovery from the gradient was 70%. Similar cell fractions were then pooled and plated. The distribution of PRL and GH cells after 5 days in culture was evaluated by immunofluorescence studies, using specific antiPRL and anti-GH antibodies. The percent recovery was expressed with respect to the total cell count in the fraction.

Materials

Membrane AC assay

GTP, ATP, cAMP, creatine kinase, creatine phosphate, Dulbecco's Modified Eagle's Medium (DMEM), BSA (fraction V), fetal calf serum, glutamine, and staurosporine were obtained from Boehringer Mannheim (Indianapolis, IN); VIP and GRF from Peninsula (Belmont, CA); Dextran T70 from Pharmacia (Piscataway, NJ); PTX from List (Campbell, CA); DA from Calbiochem (Meudon, France); and culture dishes from Nunc (Roskild, Denmark). All other compounds were purchased from Sigma (St. Louis, MO). Radioactive compounds were obtained from New England Nuclear (Boston, MA).

The culture medium was discarded, and cells were resuspended with the use of a rubber policeman, and broken in a glass-Teflon Potter Elvehjem homogenizer in 3 mM Tris-1 mM EGTA (pH 7.2) containing 3% sucrose, and centrifuged for 3 min at 500 x g. The supernatant was then centrifuged for 20 min at 12,500 x g. The resulting pellet was resuspended in the Tris-EGTA buffer containing 10% sucrose. AC activity was measured by the conversion of [a-32P]ATP to [32P]cAMP, as previously described (3). The final incubation medium (50 nl) contained 50 mM Tris-maleate (pH 7.2), 1.5 mM MgSO4, 1 mM cAMP, 5 mM creatine phosphate, creatine kinase (0.1 mg/ml), 0.15 mM ATP, 0.01 mM GTP, 10 mM theophylline, [3H]cAMP (0.001 MCi; 30.5 Ci/mmol), and [a-32P]ATP (2 MCi; 27 Ci/ mmol). The reaction was initiated by the addition of 10 fx\ of membranes (10-30 ng proteins) and was performed at 30 C for 30 min. The incubation was stopped by the addition of ice-cold buffer [40 mM Tris-maleate (pH 7.2) containing 2.5 mM ATP, 4 mM cAMP, 10 mM CaCl2, and 0.1 N HC1]. Radioactive nucleotides were isolated according to the method of Salomon et al. (34). Recovery of [3H]cAMP varied from 70-80% and was identical in all experimental groups. Protein content was determined according to the method of Lowry et al. (35), and results were expressed as picomoles of cAMP per mg protein/30 min.

Anterior pituitary cell culture Anterior pituitaries were dissected rapidly after decapitation of female Wistar rats (175-200 g; from Charles River Breading Laboratories, St. Aubin-les-Elbeuf, France), and the cells were dispersed according to the procedure described by Hopkins and Farquhar (27). Briefly, anterior pituitaries were cut in small pieces (~0.5 mm in diameter) and incubated for 15 min with 0.5% trypsin in DMEM at 37 C. DNase (2 Mg/ml) was then added for 1 min to the medium. After enzymatic digestion, the medium was removed, and tissues were incubated for 5 min in DMEM with trypsin inhibitor (1 ng/ml). The medium was changed, and tissues were first incubated in Ca2+/Mg2+-free medium containing 2 mM EDTA for 5 min, then for 15 min in the same medium containing 1 mM EDTA. Cells were then mechanically dispersed in the Ca2+/Mg2+-free medium. After centrifugation (10 min; 300 X g), cells were counted with a Coulter counter ZBI (Coulter Electronics, Hialeah, FL) and plated in petri dishes (10 cm in diameter) for membrane adenylate cyclase assay (8 X 106 cells/dish) or in six-well plates (3.5 cm diameter) for determination of intracellular cAMP and hormonal release (106 cells/well) and IP (2 x 106 cells/well). Cells were maintained in culture in DMEM supplemented with 10% fetal calf serum [after overnight adsorption on Norit-A charcoal (1%) and dextran (0.1%)], glutamine (2 mM), and

Intracellular cAMP formation and hormonal release determinations The intracellular cAMP formation was determined according to the method of Gannon and Hough (36) adapted to antehypophyseal cells. After removal of the culture medium, cells were incubated for 2 h at 37 C in 1 ml DMEM containing 25 mM HEPES, 10~3 M isobutylmethylxanthine (IBMX), (pH 7.35 ± 0.05), and [3H]adenine (5 MCi/ml; 20.7 Ci/mmol) to perform the incorporation of [3H]adenosine in ATP. The radioactive supernatant was discarded and replaced by 1 ml DMEM sup-



Results

plemented with HEPES, IBMX, and test substances for 15 min at 37 C. The incubation was stopped by removal of the medium and addition of ice-cold trichloroacetic acid (5%) containing 1 mM cAMP and 1 mM ATP. [3H]ATP and [3H]cAMP were isolated according to the method of Salomon et al. (34). Results were expressed as the ratio of ([3H]cAMP/[3H]ATP) X 103 to take into account the tritiated ATP pool in each well. The incubation medium was stored at —20 C until PRL RIA was performed (29).

Effects of AH on AC activity, intracellular cAMP, IP production, and PRL release: comparison with other neurohormones Effects of AH and DA in pituitary cells. As previously shown (40), both All and DA inhibited the AC activity in pituitary membrane preparations, with respective EC50 values of 1.8 ± 0.8 and 170 ± 30 nM. The maximal effect of All (-42 ± 2%) was higher than that of DA (-23 ± 2%; Fig. 1A). In intact cells, DA also inhibited intracellular cAMP formation, with a greater EC50 (80 ± 10 nM) than that observed on AC activity. Maximal inhibition was also higher (—38 ± 3%). In contrast to the membrane preparations, no All inhibitory effect in intact cells was observed; on the contrary, All stimulated cAMP formation with an EC50 of 1.8 ± 0.6 nM and a maximal effect of 38 ± 3% (Fig. IB). Under the same experimental conditions, PRL release was stimulated by All [EC50 = 0.4 ± 0.2 nM; maximal effect (mE) = 176 ± 15%] and inhibited by DA (EC50 = 120 ± 10 nM; mE = -60 ± 8%; Fig. 1C). To determine the possible involvement of PTX-sensitive G-proteins in these modulations of the AC pathway, cells were pretreated for 20 h with PTX. Increasing doses of PTX, with a maximal effect at 100 ng/ml, reversed DA inhibition of membrane AC activity and cAMP formation as well as All inhibition of AC activity measured in membrane preparations (Fig. 2). In contrast, All stimulation of intracellular cAMP formation remained unaffected at any dose of the toxin (Fig. 2B). Under these conditions, DA inhibition of PRL release was reversed, whereas All stimulation remained unaffected (data not shown). In those experiments basal AC activity and basal level of cAMP were not significantly modified by PTX pretreatment, whereas basal PRL release was slightly increased, as previously observed (41).

Intracellular cAMP determination Preincubation and incubation conditions were the same as those used for the measure of cAMP formation, except that [3H]adenine was omitted. The intracellular cAMP level was determined using a New England Nuclear cAMP RIA kit. Inositol phosphate determination Cells were incubated in the presence of [3H]myo-inositol (5 ^iCi/dish; 16.5 Ci/mmol) for 3 days. The radioactive medium was discarded, and cells were rinced and preincubated for 1 h with DMEM (25 mM HEPES, pH 7.35 ± 0.05). The incubation medium (DMEM-HEPES plus 10 mM LiCl), containing the drugs to be tested was then added. After 15 min, the medium was removed and replaced by 1 ml cold methanol. Cells were scraped and transferred into a glass tube in which 8 ml of a 0.5-mM EDTA solution (pH 7.4) were added. IPs were separated by anion exchange chromatography, as described by Berridge et al. (37). Data analysis The results reported here were obtained at least three times. Representative experiments are shown. Data were submitted to analysis of variance (38). To determine the statistical significance of experiments involving multiple comparisons, Duncan's multiple range test was used. The EC50 values of the doseresponse curves were calculated by the Parker and Waud method (39).

FIG. 1. Dose-dependent effects of All (•) and DA (O) on AC activity (A), intracellular cAMP (B), and PRL release (C). AC activity was measured in membrane preparations after a 30-min incubation. Intracellular cAMP and PRL release were determined in the same wells after a 15-min incubation, as described in Materials and Methods. Results are expressed as the percentage of basal values [166 ± 10 pmol cAMP/mg protein30 min for AC activity, 4.50 ± 0.12 (cAMP/ATP) x 103 for cAMP accumulation, and 420 ± 35 ng/dish for PRL release]. Values are the mean ± SE of six determinations.

2233

•50 0

-10

-9

-8

-7

-6

-5

LOGM



2234 PTX ng/ml 1

,

10

i

formed in heterogenous populations of pituitary cells, this All effect could be diluted by the presence of the other cell types. Thus, studies in separated pituitary cell types were performed. Pituitary cells were separated by sedimentation at unit gravity. The separation profiles of PRL, GH, LH, /3-endorphin, and TSH contents are shown in Fig. 3A. The inset included in Fig. 3A represents the percentage of immunoreactive PRL and GH cells detected in the fractions obtained after sedimentation. To compensate for unequal numbers of cells recovered from each fraction, comparable fractions were pooled. Experiments performed in intact cells are represented in Fig. 3B. The DA (10 6 M) inhibition as well as the All (10'7 M) stimulation of intracellular cAMP formation were both exclusively found in the PRL-enriched fractions (II-IV; Fig. 3B). The effect of All on intracellular cAMP formation was much lower than that elicited by VIP at 10"7 M (fraction III; 40 ± 3% us. 170 ± 10%). The effects of the neurohormones on PRL release paralleled their effects on intracellular cAMP (data not shown). AC activity was determined in membranes from the same fractions. As shown in Fig. 3C, maximal inhibition by All and DA of membrane AC activity occurs in lactotroph-enriched fractions.

100

•60

60

40

20

•20

-40 0

1

10

Endo • 1991 Vol 129 • No 4

100

PTX ng/ml

FiG. 2. Dose-dependent effect of PTX pretreatment on the effects of All (107 M) and DA (106 M) on AC activity (A) andintracellular cAMP (B). Cells were pretreated for 20 h with different doses of PTX or vehicle (1 mM phosphate buffer, pH 7.5). Experimental conditions were the same as those described in Fig. 1. Results are expressed as a percentage of the basal values of each pretreated group. Values are the mean ± SE of six determinations. **, 0.01 < P > 0.001; ***, P < 0.001 (vs. control All inhibition). OOO, P < 0.001 {vs. control DA inhibition).

Compared to other AC-stimulating responses, such as the one elicited by VIP, the All effect on intracellular cAMP was moderate (Table 1). Effects of AH and DA in pituitary cells separated by unit gravity sedimentation. As the experiments were per-

Effects of All, TRH, and VIP in pituitary cells. The maximal effects of All, TRH, and VIP on PRL release, AC activity measured in membrane preparations, intracellular cAMP formation, and IP production are shown in Table 1. The three peptides stimulated PRL release with different potencies. TRH stimulated intracellular cAMP formation to the same extent as All, but did not inhibit AC activity. TRH stimulation of IP production was higher than the one elicited by AIL VIP stimulated both AC activity and intracellular cAMP formation, but it did not affect IP production. The technique using [3H]adenine as a precursor of cAMP permits the determination of cAMP formation, but not of cAMP level. Effects on cAMP level were measured using a cAMP RIA kit (Table 2). All and TRH stimulated the intracellular cAMP level, but to a lesser

TABLE 1. Effects of All, TRH, and VIP on PRL release, AC activity, intracellular cAMP, and IP production

Basal % of basal All (lO"7 M) TRH (10-7 M) VIP (10-6 M)

PRL release (ng/ml)

AC activity (pmol cAMP/mg protein • 30 min)

cAMP formation (cAMP/ATP x 103)

IP production (dpm)

450 ± 40

150 ± 5

3.2 ± 0.2

272 ± 19

175 ± 10 300 ± 20 200 ± 16

-42 ± 6 8±6 165 ± 15

39 ± 5 34 ± 5 150 ± 10

224 ± 15 360 ± 25 10 ± 5

Experimental conditions were the same as those described in Fig. 1. IP production was determined after a 15-min incubation, as described in Materials and Methods. Results are expressed as a percentage of the indicated basal values. Values are the mean ± SE of triplicate determinations.


All EFFECT ON cAMP IN LACTOTROPHS 20 22 45 50 75 70 35 3S 25 25 22 20 20 15 PRL 10 10 10 15 25 30 40 50 40 35 40 X 25 20 GH

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extent than VIP. The ratio of stimulations induced by All and TRH us. the VIP effect was similar to the one obtained with the [3H]adenine method. Role of PKC in the AH stimulation of intracellular cAMP formation, IP production and PR release. This All stimulatory effect on the AC pathway in intact cells could either be a direct positive coupling of the All receptor to AC or the consequence of another transduction mechanism. As the All receptor was linked to the PLC system, the eventual impact of PKC on this parameter was investigated. As shown in Fig. 4, the PKC activator TPA at 10"6 M induced a stimulation of intracellular cAMP formation of 37 ± 5%, similar to the one elicited by All at 10"7 M. Both effects were prevented in the presence of the PKC inhibitors H7 [l-(5-isoquinolylsulfonyl)2methyl-piperazine; 10*7 M] and staurosporine (10 7 M). Under these conditions, DA (10 6 M) inhibition of intracellular cAMP formation was not modified. Another means of inactivating PKC is to desensitize the enzyme by a previous exposure (20 h) of the cells to TPA (10 6 M). Under these conditions the TPA stimulation of intracellular cAMP formation was abolished as well as the All stimulation in both pituitary cells (Fig. 5A) and the PRL cell-enriched fractions (data not shown). In addition, the effect of TRH, whose receptor is also coupled to the PLC pathway, was tested. At 10'7 M, TRH was able to stimulate cAMP formation with the same amplitude as All and TPA, and this effect vanished

FIG. 3. A, Distribution of pituitary cell types after separation: profiles of PRL (O), GH (•), LH (•), TSH (•), and 0-endorphin (A) contents and immunoreactivity of PRL and GH cells. The percentages in unfractionated cells were: PRL, 57%; and GH, 23%. Hormonal contents were measured by RIA. Immunoreactivity of PRL and GH cells was determined as described in Materials and Methods. B, Effects in enriched cell type fractions of 107 M All (•) and 10'6 M DA (O) on intracellular cAMP. Intracellular cAMP was measured as described in Materials and Methods. C, Effects in membranes of enriched cell type fractions of 10 7 M All (•) and 10 6 M DA (O) on AC activity. AC activity was determined as described in Materials and Methods. Results are expresed as a percentage of the basal values of corresponding fractions. Values are the mean ± SE of triplicate determinations.

• ^ •

Control H7 Staurosporine

TABLE 2. Effects of All, TRH, and VIP on intracellular cAMP level cAMP level (pmol cAMP/well) Basal All (10-7 M) TRH (10"7 M) VIP (10"6 M)

3.5 ± 0.1 10 ± 0.9° 9.5 ± 0.8° 51 ±3°

Intracellular cAMP was determined by RIA, as described in Materials and Methods. Results are expressed as picomoles of cAMP per well/15 min. Values are the mean ± SE of triplicate determinations. 0 P < 0.001 vs. basal.

All

DA 7

FIG. 4. Effects of H7 (10 M) and staurosporine (107 M) on the effects of All (10-7 M), TPA (106 M), and DA (106 M) on intracellular cAMP. Intracellular cAMP was determined as described in Materials and Methods. Results are expressed as a percentage of the basal values of each group: 4.6 ± 0.3 (cAMP/ATP) X 103 for control, 4 ± 0.4 (cAMP/ ATP) X 103 for H7, and 4.2 ± 0.3 (cAMP/ATP) X 103 for staurosporine. Values are the mean ± SE of six determinations. ***, P < 0.001 {vs. control). O, P > 0.05; OOO, P < 0.001 {vs. H7 control). • • • , P < 0.001 {vs. staurosporine control).



2236 40

OUntraaled |BTPA2Wi |

-r

5"

Control (dpm/well)

1

wo

•40

NS

All

TPA

TRH

I

300

X

200

I 100 o

»

o •100

DA

T

400

i

TABLE 3. Effect of a 20-h pretreatment with TPA (10"6 M) on TPA (10~6 M), All (10'7 M), and TRH (10~6 M) effects on IP production

20

-20

g

Endo • 1991 Vol 129 • No 4

All

TPA

TRH

DA

FIG. 5. Effect of a 20-h pretreatment with TPA (106 M) on the effects of All (107 M), TPA (106 M), TRH (107 M), and DA (106 M) on intracellular cAMP (A) and PRL release (B). Intracellular cAMP and PRL release were determined as described in Materials and Methods. Results are expressed as the percentage of basal values of each group: 4 ± 0.4 (cAMP/ATP) x 103 and 420 ± 30 ng/ml for cAMP and PRL in the control group, and 3.7 ± 0.3 (cAMP/ATP) x 103 and 300 ± 20 ng/ml for cAMP and PRL after TPA pretreatment. Values are the mean ± SE of triplicate determinations. **, 0.01 < P > 0.001; ***, P < 0.001 us. untreated value.

after TPA pretreatment (Table 1 and Fig. 5A). As it was the case using the PKC inhibitors, DA inhibition of intracellular cAMP formation was not affected by PKC desensitization (Fig. 5A). The PRL release determined in these experiments is shown in Fig. 5B. The long term treatment with TPA almost completely suppressed TPAinduced PRL release and partially but significantly reduced All- and TRH-induced PRL release. In contrast, TPA pretreatment had no effect on the DA inhibition of PRL release. A 20-h exposure to TPA reduced the basal level of IP production by 35%. In nontreated cells, TPA inhibited IP production by 60%, but this effect completely disappeared after the TPA pretreatment. In contrast, the All and TRH stimulations were potentiated after TPA pretreatment (Table 3). Discussion The purpose of this work was to reassess the impact of the All receptor on the AC pathway in anterior pituitary cells in culture. Previous reports have indicated that the receptor is coupled to PLC (5, 40); in addition, the peptide is able to inhibit AC activity when tested in pituitary membrane preparations (4). The latter effect, however, is paradoxical in view of the capacity of the

Basal TPA (10~6 M) All (10"7 M) TRH (10~7 M)

272 ± 107 ± 883 ± 980 ±

19 2 (-60%) 9 (+225%) 45 (+260%)

20-h TPA (dpm/well) 173 ± 176 ± 1053 ± 1020 ±

11 2 (+1%) 2 (+509%) 45 (+490%)

IP production was measured after a 15-min incubation, as described in Materials and Methods. Results are expressed as disintegrations per min/well. Values are the mean ± SE of four determinations.

peptide to stimulate PRL secretion (13). We first confirmed the inhibitory effect of All on membrane AC under our experimental conditions. The effect was comparable to that obtained with DA, although of somewhat greater amplitude. AC inhibition obtained with All as well as with DA was completely reversed upon pretreatment with PTX, confirming that a PTX-sensitive Gprotein is involved in coupling both receptors to the enzyme (40). In intact cells, however, no corresponding inhibition of cAMP accumulation by All was observed. On the contrary, the peptide stimulated cAMP formation with an EC50 in the nM range, close to those obtained for All stimulation of IP production and PRL release (13, 40). As All induced PRL release, the All stimulation of cAMP formation was not affected by PTX under conditions where both effects of DA were reversed (42). Thus, the All stimulation of cAMP described in this report may reflect an additional transduction mechanism involved in PRL secretion. The differential PTX sensitivity of the opposite All modulations of the AC pathway suggests that different G-proteins mediate these two All effects. Coupling of All receptors to the same transduction mechanism by both PTX-sensitive and unsensitive G-proteins has also been described in the case of PLC in adrenal fasciculata cells (20). All stimulation of basal intracellular cAMP has been reported in All peripheral targets, as in adrenal medulla cells (24). Potentiation of cAMP stimulation was also found in anterior pituitary corticotrophs and adrenocortical cells (43, 44). However, All stimulation of intracellular cAMP was not observed in all target cells. In fact, no effect was found in vascular smooth muscle cells (23), whereas basal or stimulated cAMP levels were inhibited by All in glomerulosa cells and hepatocytes (21, 22). The discrepancy between AH actions on cAMP in membrane or in intact cells could theoretically be accounted for by heterogeneity of the pituitary cell population used. In that case, inhibition of membrane AC and intracellular accumulation of cAMP would occur in distinct cell types. Alternately, the membrane effect may result from direct coupling of the All receptor with AC in lactotrophs, while the intracellular effect could rely



on indirect paracrine interactions involving other pituitary cell types (45). Further characterization of the All effect on intracellular cAMP formation, performed in pituitary cell populations enriched by sedimentation, indicated that All stimulation of cAMP formation closely paralelled the DA inhibition of cAMP formation in the lactotroph-enriched fractions. Both All and DA inhibitions of membrane AC activity were also located in the PRL-enriched fractions. This distribution of the All effect correlated with All binding in lactotrophs (15). Other All-binding sites have been described in corticotrophs and, to a lesser extent, in thyrotrophs (16). Corticotrophs represent a very small proportion of anterior pituitary cells; their distribution partially coincides with the lactotroph distribution, as shown in the hormonal profiles of the separated fractions. Thus, no conclusion on a possible All effect on intracellular cAMP formation in corticotrophs can be drawn from our data. Under those conditions, cellular heterogeneity is unlikely to account for the paradoxical effects of All, which can, instead, be assumed to take place in the same cell population, but to involve different mechanisms. Even in lactotroph-enriched fractions, the All effect on cAMP formation was moderate for a direct coupling to AC. Cross-talks between PLC and AC transduction pathways have been described in several reports (for review, see Ref. 46). An influence of PKC on intracellular cAMP formation could, thus, be suspected, inasmuch as similar effects have already been reported in other tissues (47, 48). Under our experimental conditions, activation of PKC by TPA stimulated PRL release, as previously described (49), but was also able to induce stimulation of cAMP formation comparable to that elicited by AIL Conversely, All-induced cAMP accumulation was suppressed by concomitant treatment of the cells with the PKC inhibitors H7 and staurosporin. As the specificity of the PKC inhibitors is limited (50), although we showed they counteracted the TPA effect, another means to suppress PKC was used. In fact, several reports have shown that pretreatment of cells with TPA induced desensitization of PKC (51-53). The effectiveness of PKC desensitization by pretreatment with TPA was shown by the fact that subsequent administration of TPA induced neither cAMP accumulation nor PRL release. Under these conditions, All was no longer able to affect cAMP production; its efficacy on PRL release was also diminished. These data, thus, validate the hypothesis that All triggers cAMP accumulation in intact cells only indirectly, via an initial activation of PKC. This is further substantiated by the absence of additivity of the effects of TPA and All on cAMP accumulation (data not shown). Comparable effects of All were also described in corticotrophs (43); in that case, however, the action of All could only be seen on a CRF-stimulated cAMP level.

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It should be noted that under PKC desensitization, DA inhibition of cAMP formation and PRL release remained unaffected. TRH, a potent stimulator of PRL release, has been described to stimulate PLC (6), as does AIL Several studies reported conflicting effects of TRH on the AC pathway. In the GH3 PRL-secreting cell line, a stimulatory effect of TRH in membrane preparations and on intracellular cAMP was found by Gautvik et al. (25), whereas others have shown no effect on the AC pathway (26). It was, thus, of interest to test the effect of TRH in our experiments. This peptide did not exert any effect on membrane preparations, but induced a stimulation of intracellular cAMP formation similar to those elicited by All and TPA; this stimulation also disappeared after TPA pretreatment. This suggested that a cross-talk of PKC on the AC pathway could be shared by receptors positively linked to PLC in anterior pituitary cells. PKC interaction with the AC pathway in various systems has been shown to take place either at the level of the receptor, the G-protein, or the AC catalytic subunit itself (54-57). TPA pretreatment did not desensitize the All or TRH receptors, as demonstrated by their remaining stimulatory effects on IP production. Interestingly, acute TPA exerted an inhibition on basal IP production, which disappeared after TPA pretreatment, suggesting that, as in other systems, PKC exerted a negative feedback on PLC (58-60). After the TPA pretreatment, All and TRH stimulated IP production even more than under basal conditions, possibly because the negative feedback of PKC was switched off. In conclusion, the All paradoxical inhibitory coupling to AC found in membrane preparations is not observed in intact cells under basal conditions. In contrast, we have shown a stimulatory effect on intracellular cAMP formation, which indirectly results from the primary coupling of the All receptor to the PLC pathway and is mediated by PKC. This cellular cross-talk is also implicated in the TRH stimulation of intracellular cAMP formation. PKC seems to contribute, although only in part, to the hormonal actions of All and TRH, since the amplitude of PRL release is decreased, but not suppressed, after PKC desensitization. The relevancy of the All negative coupling to AC in intact cells could not be evaluated under the present experimental conditions and remains to be clarified. References 1. Lamberts SWJ, MacLeod RM 1990 Regulation of prolactin secretion at the level of the lactotrophs. Physiol Rev 70:279-317 2. Robberecht P, Coy DH, Waelbroeck M, Heiman ML, De Neef P, Lambert M, Christophe J 1979 VIP activation of rat anterior pituitary adenylate cyclase. FEBS Lett 103:229-233 3. Enjalbert A, Bockaert J 1983 Pharmacological characterization of the D2 dopamine receptor negatively coupled with adenylate cy-


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Involvement of protein kinase C in the regulation of cortisol production by guinea pig adrenocortical cells.

Effect of adenosine on melanogenesis in b16 cells and zebrafish.

The effect of alkylating agents on the activity of adenosine 3',5'-monophosphate-dependent protein kinase in Walker carcinoma cells.

The effect of timolol on isoprenaline induced cyclic adenosine monophosphate production in human isolated lymphocytes [proceedings].

Effect of protein kinase inhibitors on ACTH-stimulated aldosterone production in rat zona glomerulosa cells.

Effect of adenosine and adenosine analogues on cyclic AMP accumulation in cultured mesangial cells and isolated glomeruli of the rat.

In vitro study of the effect of adenosine on frog adrenocortical cells.

Effects of dopamine on voltage-dependent potassium currents in identified rat lactotroph cells.

Influence of lactotroph cell density on prolactin secretion in rats.

Involvement of cyclic adenosine monophosphate in the interleukin 4 inhibitory effect on interleukin 2-induced lymphokine-activated killer generation.

The effect of the unfolded protein response on the production of recombinant proteins in plants.

Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

Conditional knockout of the RNA-binding protein HuR in CD4⁺ T cells reveals a gene dosage effect on cytokine production.

Involvement of prostaglandins in the inhibition of growth hormone production in cultured pituitary cells by insulin.

Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors.

The effect of a selective alpha 2-adrenergic receptor antagonist (midaglizole) on the cyclic 3',5'-adenosine monophosphate production in human mononuclear cells.

Effect of protein deficiency on suppressor cells.

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Effect of exogenous copper on lipid peroxidation in rat hepatocytes. Possible involvement of protein kinase C.

Effect of the salts of deep ocean water on the production of cordycepin and adenosine of Cordyceps militaris-fermented product.

Effect of DEAE-dextran on protein synthesis in HeLa cells.

Effect of thyroliberin on the concentration of adenosine 3':5'-phosphate and on the activity of adenosine 3':5'-phosphate-dependent protein kinase in prolactin-producing cells in culture.

Effect of HCG on the interstitial cells and androgen production in the immature rat testis.

Effect of azithromycin on the LPS-induced production and secretion of phospholipase A2 in lung cells.