Stimulation of Early by Angiotensin II in Glomerulosa Cells: and Protein Kinase

Adrian

J. L. Clark,

Tamas

Balla,

Mark

Gene Expression Bovine Adrenal Roles of Calcium C

Ft. Jones,

and Kevin

J. Catt

Endocrinology and Reproduction Research Branch National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892

sponse to All, suggesting the involvement factors in stimulus-transcription coupling AT, receptor. (Molecular Endocrinology 1898,1992)

The adrenal glomerulosa cell is a major site of action of angiotensin II (All), which binds to AT, receptors to stimulate phosphoinositide hydrolysis and Ca*+ mobilization, and the subsequent production of aldosterone. All also influences adrenal growth and proliferation and promotes thymidine incorporation in adrenocortical cells. In primary cultures of bovine glomerulosa cells, All was found to induce the expression of several early growth response genes (c-fos, c-jun, JunB, and Krox 24). This effect of All was dose-dependent and was blocked by [Sar’,lle’] All and the nonpeptide antagonist DuP 753, indicating that it is mediated by the AT, subtype of the All receptor. ACTH, which elevates CAMP in glomerulosa cells, was a relatively weak inducer of c-fos expression but was as potent as All in stimulating the expression of JunB. ACTH did not further enhance the maximal effect of All on c-fos expression. The role of the All-induced cytoplasmic Ca*+ increase in generating the c-fos response was suggested by the ability of the Ca*+ ionophore ionomycin to induce c-fos expression. However, mobilization of intracellular Ca2+ by the Ca*+ ATPase inhibitor thapsigargin, as well as the stimulation of Ca*+ influx by depolarization with potassium, were less potent stimuli of c-fos expression. Omission of Ca*+ from the extracellular medium, which abolishes the plateau phase of the All-induced Ca*+ signal without affecting the early increase due to Ca*+ mobilization, enhanced the early phase of the All-induced c-fos response, indicating that Ca*+ also has an inhibitory effect on the early gene response. Activation of protein kinase C by phorbol 12-myristate, 13-acetate (PMA) also stimulated c-fos expression, but the combination of PMA and ionomycin did not further increase the c-fos response. Inhibition of protein kinase C by staurosporine, or its depletion by prolonged exposure to PMA, prevented the c-fos response to PMA but only partially inhibited the re-

of other from the 6: 1889-

INTRODUCTION

Angiotensin II (All) plays a critical role in the maintenance of mammalian salt and water balance and acts as a potent vasoconstrictor as well as stimulating the adrenal glomerulosa zone to produce aldosterone. In addition, All and its receptors are widely distributed in many organs including the adrenal, kidney, liver, brain, and gonads, and presumably fulfill a number of as yet poorly defined local functions. In most of its target cells, All interacts with its plasma-membrane receptors to stimulate phospholipase C via a pertussis-toxin insensitive GTP-binding protein and thus promotes the hydrolysis of inositol phospholipids (l-4). The rapid formation of inositol 1,4,5-trisphosphate [lns(l ,4,5)P3] and the consequent mobilization of intracellular calcium are the primary events that initiate aldosterone secretory responses to All in adrenal glomerulosa cells. In addition to its rapid stimulatory actions on intracellular signaling processes and aldosterone secretion, several lines of evidence have indicated that All exerts long-term regulatory effects on adrenal glomerulosa cells. These include stimulation of the expression of its receptor sites in the adrenal glomerulosa zone (5, 6) as well as trophic actions on the glomerulosa cell. Maintenance of rats on low salt intake causes widening of the adrenal glomerulosa layer with cellular hypertrophy and hyperplasia (7). These effects have been attributed to increased activity of the renin-angiotensin system (8). However, the mitogenic effects of All on adrenal cortical cells in vitro are more controversial. Although All has been reported to cause increased [3H]thymidine incorporation in bovine adrenocortical cells (9, lo), an antimitogenic effect of the peptide has been described in glomerulosa cells (11).

0888-8809/92/1889-1898$03 00/O Molecular Endocmology CopyrIght % 1992 by The Endocrine Soctety

1889

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MOL 1890

Vol6No.

END0.1992

In vascular smooth muscle cells, All induces the expression of c-fos (12-I 4) c-jun (15) and c-myc (16), events which represent the initial cellular response to mitogenic stimulation in many tissues and cell lines. The relationships of the various second messenger systems to the activation of these early genes have been analyzed in several cell types. In the present study the effects of All on early gene responses were examined in adrenal glomerulosa cells, and the participation of known second messengers in this stimulus-transcription process was investigated. RESULTS

AND DISCUSSION

All Increases Genes

mRNAs

of Several

Early

Response

In bovine adrenal glomerulosa cells, stimulation with 3 x lo-* M All is known to be maximally effective in eliciting inositol phosphate and cytoplasmic Ca*+ signals, and stimulating aldosterone production (17). As shown in Fig. 1, glomerulosa cells treated with this concentration of All showed increased steady state levels of mRNAs of several early response genes, namely c-fos, c-jun, JunB, and Krox 24. The kinetics of these changes were all quite rapid, with increased levels

12 -

c-jun

c-fos

10 864g

2-

: C 0

0

I

1

I

/

1

Jun

6

/

,

1

I

/

/

I

Krox

24

2 4-

i_ 0

P 0

20

44 60

80 loo 120

d , 0

I 20 40

i , , 1 60 80 loo 120

Time hn)

Fig. 1. All-Induced Changes in Steady State mRNA Levels of Early Response Genes Bovine adrenal glomerulosa cells were cultured for 3-4 days and serum-deprived for 1 day before addition of All (3 x lo-* M). Total cellular RNA was extracted and Northern analysis performed as described in Materials and Metbods. Results are expressed as fold induction relative to baseline values. The results shown are derived from a single experiment by repeated hybridization with the different probes to the same Northern blot. Similar results were obtained in three additional experiments.

11

of c-fos mRNA within 15 min, reaching a peak between 30 and 45 min, and declining rapidly to moderately elevated levels by 90 min. These kinetics are similar to those of the c-fos response to All in smooth muscle (12, 14) and to a range of other stimuli in various cell types. Krox 24 (also known as fgrl or zif 268) is another recently described member of the early response gene family which, unlike c-fos and c-jun, does not form part of the APl protein complex. It encodes an 82-kilodalton zinc finger protein that specifically recognizes a GC-rich region with resemblance to the SPl response element (18, 19). The expression of Krox 24 mRNA in glomerulosa cells was also increased by All with similarly rapid kinetics to c-fos, but remained elevated at 120 min. In contrast, JunB showed a more delayed response and reached a peak between 60 and 90 min, followed by a less marked decline. The kinetics of the c-jun responses were more variable; in some experiments they were similar to those of c-fos (Fig. 1), but in others the response was more delayed (not shown). When glomerulosa cells were incubated with All in the presence of cycloheximide (50-100 PM), a concentration previously shown to be nontoxic to these cells in the short term but sufficient to block effects that involve de nova protein synthesis (20), there was no diminution of the c-fos response (data not shown). Such independence of c-fos expression from protein synthesis is consistent with findings in other cell types stimulated with mitogens (e.g. 21, 22). In further experiments the dose-dependence of the early gene response to All was examined. The c-fos response was detectable at All concentrations as low as lo-” M, and the ECsO for All was approximately 1 O-’ M (Fig. 2). These values are similar to those found for the aldosterone secretory response to All (17) and are compatible with the All receptor binding characteristics observed in these cells (dissociation constant, lo-’ M) (23, 24). The dose-response curves for the All-induced increases in c-jun, JunB, and krox 24 were indistinguishable from that of c-fos (data not shown). To determine whether the effects of All on the early gene responses were mediated via the AT, or the AT2 receptor subtype, we used the subtype-specific nonpeptide All receptor antagonists DuP 753 and PD 123177 (25,26), as well as the nonselective All receptor antagonist [Sar’,lle8]All. As shown in Fig. 3, both [Sar’ ,lle8]All and the AT, receptor-specific antagonist DuP 753 inhibited the effect of All on c-fos expression. The AT2 receptor antagonist PD 123177, applied at a concentration (lo+ M) that is sufficient to displace All binding from rat AT* receptor sites (24,25), was without effect. These data indicate that the stimulatory effects of All on this early response gene is mediated through the AT, receptor subtype. It is interesting to note that stimulation of c-fos mRNA levels required only a short exposure to All. In experiments where the antagonists, [Sar’,lle8]All or DuP 753, were added at selected times after All, we found that both were without effect on the 30-min c-fos response if added longer than 5 min after the agonist (data not shown). Such a transient activa-

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Early Gene

Responses

in the Adrenal

I o%+10

Zona Glomerulosa

I I 9 8 All I- log(M)1

1891

I 7

Fig. 2. Dose Dependence

of the c-fos mRNA Response to All After incubation of serum-deprived bovine adrenal cells with All (lo-“‘-3 x lo-* M) for 30 min, Northern analysis was performed on total cellular RNA. Results are expressed as fold stimulation above the nonstimulated basal value. The inset shows a Northern blot for c-fos mRNA. The data are representative of results from three similar experiments.

All 3 x 10’9M Antagmst

-

+ -

+ PD123177 @M

+ Dup753 10 5M

+ iSar’,lleu*lAll IO-‘M

merulosacells. These cells increasetheir aldosterone production in responseto ACTH and elevated levelsof potassium, as well as to All. ACTH is believed to act primarilyvia the CAMP messengersystem, while potassium stimulatesCa2+influx through activation of voltage-sensitiveCa2+channels.In vivo administrationof ACTH (28) and acute stress (29) have been shown to increasec-fos expressionin the adrenalcortex. ACTH also stimulatesthe expression of c-fos in Y-l adrenocortical tumor cells (30) and of c-fos, c-jun, and JunB in adrenal fasciculata cells (31). As shown in Table 1, several stimuli were found to induce early gene responsesin adrenalglomerulosacells, but with marked differences in potency and efficacy. ACTH was a relatively weak stimulusof c-fos, c-jut?,and krox 24 expression as compared to All, but was as potent as All in stimulatingexpressionof JunB. This finding is consistent with a recent report (31) in which ACTH was found to be a potent stimulusof JunB expressionin adrenocortical cells. These data suggest the importance of a CAMP responseelement(CRE)in regulatingJunB expressionin the adrenal, probably more so than in the case of the other early genes examined. Such differences in gene responses to CAMP-mediated stimuli have been observed in other cell types. Thus, stimulationof hemopoietic THP-1 cells with (Bu),cAMP generatesa potent c-fos responsebut little c-jun response,in contrast to the more uniform response produced by phorbol 12myristate 13-acetate(PMA) stimulation(22). This difference has been attributed to the lack of a typical CRE in the c-jun promoter (32) in contrast to c-fos. However, isoproterenol stimulation of neonatal myocardial cells was found to causea significantc-jun response,but no krox 24 response (33). A typical CRE sequence is present in the krox 24 promoter 140 base pairs (bp) upstreamof the transcriptionalinitiationsite but has not yet been shown to be functional (19). The present findings suggest that the krox 24 gene is under dual control in adrenalglomerulosacells,sinceits expression was increasedby both Ca2+-mobilizing(All) and CAMPproducing(ACTH) agonists. Among the physiologicalstimulitested, All was found

Fig. 3. Receptor

Specificity of the c-fos Response to All Glomerulosa cell culture, incubations, and quantification of mRNA were performed as described in the legend to Fig. 1. All (3, x 10M9 M) was added for 30 min together with the antagonists at concentrations of 1 Om7 M for [Sar’,Ile*]All, 1 O-5 M for DuP 753, and 1 Oe6 M for PD 123177. The results shown are representative of two similar experiments and are expressed as fold induction above basal.

requirement was also found for muscarinicreceptors stimulatingc-fos expression(27).

tion

Effects of Other Physiological Gene Responses

Stimuli

on Early

Further experiments were performed to evaluate the agonist specificity of the early gene responsesin glo-

Table 1. Expression jun, JunB, and krox Various Stimuli Agent All ACTH PMA K+ Serum

of the Early Response Genes, c-fos, 24 in Glomerulosa Cells Exposed to

n

c-fos

c-jun

JunB

6 4 3 3 2

7.2 (2.7) 2.2 (0.5) 6.8(4.6) 3.2 (1.0) 7.0

4.1 (2.4) 1.5 (0.6) 3.6 (1.2) 2.4(1.5) 13.1

4.8(2.4) 4.1 (2.7) 5.6(1.4) 4.3(4.1) 15.0

c-

krox 24

5.3 2.2 4.4 2.9 12.2

(2.4) (0.5) (0.8) (1.1)

Cells were incubated with 30 nM All, 1 nM ACTH, 10 nM PMA, 20 mM K+, or 10% bovine serum. Values are means @SD) of the ratio of the response after 30 min stimulation with agonist relative to the response in control, unstimulated cells, each normalized to the corresponding 28s RNA signal. n, Number of experiments in each case.

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MOL 1892

ENDO.

1992

to be the most potent inducer of all the early response genes analyzed in this study. These findings raise the possibility that each stimulus can generate qualitatively different components of the AP-1 dimer complex. Thus, All could induce the highly stable fos/jun heterodimer, whereas the response to ACTH could involve the less stable fos/JunB heterodimers. The formation of these various complexes could allow for subtle but significant differences in the activation of regulatory processes that follow the immediate early response. Studies in a wide range of cell types suggest that induction of c-fos depends on the interaction of incompletely characterized transcription factors with either the serum response element (located 300 bp upstream of the transcription initiation site) (34) or with an element having homology to the CRE (located 60 bp upstream of the transcription start site) (35). In PC12 pheochromocytoma cells, this downstream element has been shown to bind the CRE binding protein (CREB) and to confer a response to CAMP and/or calcium (35). It has also been shown in those cells that elevation of intracellular calcium results in the phosphorylation and consequent activation of CREB (36). Adrenal glomerulosa cells provide an interesting model to test this proposal, in that they express Ca’+mobilizing All receptors as well as ACTH receptors that stimulate adenylate cyclase and elevate CAMP. Also, All has been shown to enhance the stimulatory action of ACTH on CAMP production (37) and to induce small increases in CAMP production in bovine adrenal glomerulosa cells (38). In our studies, All marginally increased CAMP levels (by only about 25% above basal) during the usual 30-min incubation period, whereas ACTH caused up to a 40-fold increase in CAMP production (not shown). This, and the relatively lower stimulation of c-fos, c-iun, and krox 24 expression by ACTH, indicate that increased CAMP production is not a major factor in the mechanism of the c-fos response to All. This was also indicated by the lack of a detectable effect of 8-bromo-CAMP on c-fos expression (not shown), consistent with findings in adrenocortical cells (31). We also determined whether the maximal effects of ACTH and All on the c-fos response were additive. As shown in Fig. 4, ACTH (lo-’ M) did not further increase the effect of 3 x 1OT8M All, indicating that the two activation pathways for c-fos regulation converge at some point. It is possible that the effects of All on cfos expression are at least partially mediated by the CREB via a Ca2+-dependent phosphorylation as mentioned above (36). It is worth noting that serum (10% fetal bovine serum) was the most potent stimulus of all early response genes studied to date (Table 1). This indicates the functional importance of the induction of factors that interact with the serum response element, since serum did not cause significant inositol phosphate or [Ca’+]i responses in adrenal glomerulosa cells (Balla, T., A. J. Baukal, and K. J. Catt, unpublished observations).

Vo16No.11

c-f OS 12

10

+ ACTH Fig. 4. Stimulation of c-fos mRNA Levels by All and ACTH Glomerulosa cells were incubated for 30 min with ACTH (lo-’ M) and All (3 x lo-* M), alone and in combination. Incubations and quantification of mRNA were performed as described in the legend to Fig. 1. Values are expressed as fold induction above basal and are means + SEM data from six independent experiments.

The Roles of Elevated Cytoplasmic Ca2+ and Protein Kinase C in c-fos Responses

In many cell types, increasesin cytoplasmic Ca*+ have been shown to be sufficient for the activation of c-fos expression. All is known to increase [Ca2+li by both mobilizingCa2+from intracellularstores and enhancing Ca*+influx through the plasmamembrane(cf. 39). All also increasesdiacylglycerol levels in bovine glomerulosacells (40) and activates protein kinaseC (41). The dual activation of Ca*‘-calmodulin-dependentkinases and protein kinase C has been implicated in many agonist-stimulatedcellular responses,includingthe aldosterone secretory responseof glomerulosacells to All (4). We therefore examined the participation of these Ca*+ signalingevents in the stimulation of the c-fos response.Omissionof Ca*+from the extracellular medium has been shown to abolishthe Ca’+ influx phase of the All-induced Ca*+ signal without significantly affecting the initial Ca*+ mobilization. When bovine glomerulosacells were stimulated with All under these nominallyCa*+-freeconditions,the plateauphaseof the [Ca”], increasewas abolished(Fig. 5A), but the c-fos

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Early Gene Responses in the Adrenal Zona Glomerulosa

however,

Time (set) (3)*

(6)*

1.3t P-3

----

I.0 L---\-t

its effect was significantly

less than that of

All (Fig. 5). Interestingly, the effect of ionomycin was also enhanced if Ca2+ was omitted from the incubation medium (not shown). To find whether the source of the [Ca’+], increase is relevant to the efficiency of stimulation of c-fos mRNA, we examined the effect of thapsigargin (TG), an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase, on c-fos mRNA and compared its effect to that of 20 mM potassium. By inhibiting the ER Ca2+ ATPase, TG causes a gradual increase in [Ca*+], due to passive leakage of Ca”+ from its intracellular stores. TG also enhances Ca2+ entry indirectly, probably secondary to emptying of the agonist-sensitive Ca2+ stores (42). Elevation of extracellular potassium, however, increases Ca*+ influx by activating voltage-gated Ca2+ channels in the plasma membrane, without emptying the intracellular Ca2+ stores. As shown in Fig. 6 and Table 1, respectively, both TG and potassium ions stimulated c-fos mRNA levels, but their effects were quite weak compared to that of All. Comparison of the [Ca’+], increases evoked by these stimuli (Fig. 7) suggests that the c-fos response is proportional to the change in [Ca”],. In addition, the more sustained [Ca”], increases observed in both TG- and potassium-stimulated cells could exert a negative effect (as discussed above) and may thus contribute to the smaller c-fos response. Although increases in [Ca”], caused a c-fos re-

Time (min) Fig. 5. Effects of Removal of Extracellular Calcium on the AllStimulated c-fos Response A, Cytoplasmic Ca” changes evoked by All (3 x 1 O-’ M) in the presence (a) and absence (b) of extracellular Ca*+. B, A representative Northern blot of the c-fos response after 30 min exposure to All in normal and nominally Ca2+-free medium. C, Time course of c-fos induction by All in the absence (O- - 0) and presence (O-O) of extracellular calcium. Data from three experiments are expressed as percent of the maximal response in the presence of Ca’+ (12.8 rt 4.0-fold over basal, SEM). Also shown are the ratios of the c-fos responses observed in the absence and presence of Ca*+, with the numbers of observations in parentheses (*, significantly different from 1, P < 0.05).

c-f OS 12

t

T

“t

I

response to All was slightly but consistently enhanced during the first 30 min of stimulation (Fig. 5C). This

result not only indicated that the initial Ca2+mobilization was sufficient to trigger the c-fos response but also suggested

that sustained

[Ca”],

elevation

may have an

inhibitory effect on the expression of c-fos mRNA. The c-fos response developed more rapidly and appeared to decline earlier without reaching higher levels than in the presence of Ca2+(Fig. 5C). Pretreatment of cells for 10 min with 1 mM EGTA in Ca2+-free medium attenuated

but did not abolish the All-induced c-fos response (data not shown), indicating that the initial [Ca”], increase (which is reduced due to depletion of intracellular Ca*+ pools under these conditions) is important in the initiation of the early gene response. Consistent with this conclusion, ionomycin was able to increase

the Ca2+ ionophore

c-fos mRNA levels;

C

All

lono

TG

PMA

PMA t Ion0

Fig. 6. The Effects of Ca”-Mobilizing Stimuli and Activation of Protein Kinase C on c-fos mRNA Levels Glomerulosa cells were cultured and c-fos mRNA quantitated as described in the legend to Fig. 1. The concentrations used were: All, 3 x 1 O-’ M; ionomycin (lono), 1 Om6M; TG, 1O-’ M; and PMA, lo-’ M. Means + SEM from six separate experiments are shown, expressed as fold induction above basal.

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MOL ENDO. 1992 1894

Vo16No.11

TG 30 nM

0

+

300

1 min Control

PMA Pretreated

KCI 20 mM +

300 200 100 0

All 30 nM c

Fig. 7. Cytoplasmic Ca*+ Responses of Glomerulosa Cells to Thapsigargin (1 O-’ M), Elevated Extracellular K+ (20 mM), and All (3 x lo-’ M) Cells were prepared and cultured, and cytoplasmic Cd’ measured with Fura- as described in Materials and Methods. The traces shown are representative of numerous replicates in different cell preparations.

sponse, they did not fully mimic the effect of the Ca’+mobilizing agonist All. We therefore determined whether activation of protein kinase C, alone or in combination with increased [Ca’+],, was able to increase c-fos mRNA levels. As shown in Table 1, and Figs. 6 and 8, PMA (10e7 M) was a potent stimulus of c-fos expression in adrenal glomerulosa cells. However, this effect of PMA varied between experiments, being usually less than that of All (see Fig. 6) but sometimes almost equivalent to the All-induced c-fos response (Table 1 and Fig. 8). The effect of PMA on c-fos expression was not further enhanced by simultaneous treatment with ionomycin (Fig. 6). The role of protein kinase C in the stimulatory action of All was studied by inhibiting the enzyme with staurosporine (10m7 M) or after down-regulation by prolonged exposure to PMA (lo-’ M). As shown in Fig. 8, All was still able to evoke about 60% of the c-fos response under these conditions. At the same time, the abolition of the PMA-induced c-fos response indicated the complete blockade or loss of protein kinase C (Fig. 8). It should be noted that relatively large doses of staurosporine were used in these studies, based on preliminary experiments to determine the dose necessary to prevent the stimulatory effect of PMA on aldosterone production. However, nonspecific

Pretreatment: Stimulus:

ST

PMA

-All3x10-sM

ST -PMAlo-‘M

PMA

ST

J

ACTH WgM

Fig. 8. Effects of Staurosporine and Depletion of Protein Kinase C on the c-fos Response to Stimulation with All, PMA, and ACTH Staurosporine (ST, 1 O-’ M) was added 10 min and PMA (IO-’ M) 16 h before addition of stimuli for 30 min. A, Representative Northern blots (overexposed to better show the ACTH response). B, Responses expressed as percent of the increases caused by the respective stimuli (12.2 + 4.0-, 11.8 + 3.9-, and 4.4 + 25fold increases over basal for All, PMA, and ACTH, respectively. n = 3, SEM).

effects of staurosporine are unlikely to be responsible for inhibition of the All-induced c-fos response, since the minor effect of ACTH on c-fos was not significantly affected. Taken together, these data suggest that activation of protein kinase C participates in the stimulatory action of All on c-fos expression, but that additional factors must account for the full effect of the peptide. The early gene responses to All and other physiological stimuli of the adrenal glomerulosa may reflect the initial events in the long-term effects of these agonists on adrenal function, even though these changes alone do not necessarily lead to mitogenesis. It is well known that enucleated adrenals can regenerate the cortex from the capsular glomerulosa layer, possibly from progenitor cells present in this layer (43). Many studies have suggested that All plays a trophic role in the glomerulosa layer, especially during sodium deficiency (7). All has been shown to increase [3H]thymidine incorporation in bovine adrenocortical cells (9, 10) and also in bovine glomerulosa cells (Balla, T., Y. Tien, and K. J. Catt, unpublished observations). Similarly, K’ ions are known to be important for the maintenance of

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Early Gene Responses in the Adrenal Zona Glomerulosa

adrenal ceils in culture and for the full activity of the 18hydroxylase/dehydrogenase enzymes (see Ref. 44). The trophic effect of ACTH on the zona fasciculatareticularis is well established; conversely, hypophysectomy causes atrophy of the glomerulosa layer as well as the fasciculata-reticularis zone, although the function of the former is much less impaired. In the zona glomerulosa, ACTH only transiently increases aldosterone secretion and decreases All receptor number. It also causes enzymatic and morphological changes consistent with transformation of glomerulosa cells toward fasciculata-like cells (44) and therefore seems to determine the pattern of differentiation. An analysis of the effects of the different secretagogues on the early gene responses in the glomerulosa layer is thus an important step toward defining their roles in these long-term effects. In this respect it is important to note that our glomerulosa cell preparation is not significantly contaminated with fasciculata cells, which are removed by the combination of tissue selection and percoll separation employed in the cell preparation protocol. In addition, Northern blot analysis of mRNA prepared from these glomerulosa cells (Fig. 9) did not reveal a significant

1895

amount of 17a-hydroxylase mRNA as compared to that present in the adrenal fasciculata zone. It is also important to recognize that the early gene responses to the physiological stimuli did not closely match their effects on aldosterone secretion; this is a clear indication that the second messengers generated during agonist stimulation are not equally potent in evoking secretory and transcriptional responses. It is likely that under physiological conditions, the Ca2+ signal generated by Ca2’-mobilizing hormones, together with the CAMP levels and as yet poorly defined serumactivated factors, are integrated into an activation pattern that optimally regulates cell growth and/or differentiation. The adrenal glomerulosa cell and its morphological responses in viva should be a useful model to further explore the interactions between the several regulatory factors and their messengers at the transcriptional level. In summary, All rapidly stimulates the expression of several early response genes in bovine adrenal glomerulosa cells. This effect is mediated through cell-surface All receptors of the AT, subtype. A transient elevation of [Ca’+], is important for initiating this action of All, but our data suggest that more prolonged calcium elevations may diminish the c-fos mRNA response. The physiologicalsignificanceand mechanismof this effect are not yet clear, but Ca2+might decreasethe stability of the c-fos mRNA. ACTH, acting via CAMP, also stimulates early gene responsesand in the case of c-fos, the combinedstimulationof All and ACTH appearsto be convergent. This finding is compatiblewith the proposal that the effects of calcium and CAMP on c-fos might be mediated through phosphorylation of the CREB protein (45). Activation of protein kinase C is a potent stimulusof the c-fos responsebut isonly partially responsiblefor the effects of All, suggestingthat additional mechanism(s)play a role in All-induced stimulustranscription coupling. The recently described effects of All on tyrosine phosphorylationin hepatoma(46)and mesangialcells(47) could representone such potential mechanism,a possibility that merits further investigation

MATERIALS Preparation

Fig. 9. Northern Blot Analysis of mRNA Prepared from Bovine Adrenal Glomerulosa Cells (Bov. zglom. cells) and Bovine Adrenal Cortex (Bov. zf.) Messenger RNA was prepared by poly(A)+ selection, and samples of 1 pg [and also 2 fig (2X) glomerulosa cell mRNA] were loaded on agarose gels. The blots were hybridized with the full-length cDNA for rat 17ol-hydroxylase mRNA for 16 h at 42 C in the presence of dextran sulfate. Blots were subsequently washed twice for 15 min in 2x SSC, 0.1% SDS and then twice in 1 x SSC, 0.1% SDS for 15 min at room temperature.

AND METHODS and Incubation

of Adrenal

Glomerulosa

Cells

Bovine adrenal glomerulosa cells were prepared and cultured as described previously (48) except that metopyrone (5 PM) was,present during the culture period. Metopyrone has been shown to preserve the steroid hydroxylation capacity of adrenal glomerulosa cells (49) and is therefore routinely added to our primary cultures. Its inhibitory effect on 1 1-hydroxylase is reversible and is removed by a l-h washout period. We observed no significant difference in the c-fos response to All and ACTH whether or not metopyrone was present during the culture period. Cells were kept without serum for 1 day before experiments, which were performed on the third or fourth day of culture. Cells cultured on six-well plates (4 x lo6 cells per well) or 1 O-cm diameter culture plates (12 x 1 O6 cells per plate) were stimulated at 37 C in modified medium 199/Earl’s salt solution (K+, 3.6 mM, Ca 2f , 1.2 mM) containing 0.2% BSA for

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MOL 1896

END0.1992

Vo16No.11

the times indicated. Incubations were terminated by removal of the medium followed by rapid freezing of the cells on dry ice. RNA was then extracted from 1 O-l 5 million cells for each determination as described below. RNA Preparation

and Analysis

RNA .was prepared by solubilizing cells in 4 M guanidinium isothiocyanate and centrifugation over a cesium chloride cushion, essentially as described (50). RNA pellets were resuspended in Tris/HCI (10 mM, pH 7.5), EDTA (1 mM) (TE buffer), precipitated once with ethanol, and resuspended in TE buffer. RNA was quantitated by measurement of the absorbance at 260 nm. Northern blots were performed by electrophoresis of lo- or 20-pg quantities of RNA through 1% agarose, 17% formaldehyde gels in 3-(N-morpholino)propanesulfonic acid buffer and transfer to Nytran membranes (Schleicher & Schuell, Keene, NH). After fixation by baking, membranes were prehybridized and hybridized as described (51) at 42 C for 16 h with the appropriate “P-labeled DNA probes (see below). The final wash of filters was in 0.2x SSC (1 x SSC = 0.15 M NaCI, 0.015 M Na citrate, pH 7.0), 0.2% sodium dodecyl sulfate (SDS) at 55 C. Filters were exposed to Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) at -70 C with intensifying screens for 3-24 h. Subsequenfiy, filters were scanned with an Ambis radioanalytic scanning system (San Diego, CA) for 60 min for direct quantification of 32P. The DNA probes used were the inserts from plasmid clones for v-fos (Clontech Labs., Palo Alto, CA), c-jun, JunB, krox 24 (from Dr. R. Bravo, Brystol Myers, Princeton, NJ), and a synthetic oligonucleotide specific for the 28s RNA species (5AAAACGATCAGAGTAGTGGTATTTCACCG-3). The 17cu-hydroxylase probe was a full-length cDNA for the rat enzyme (52). Each probe (25 ng) was labeled with [3*P]dCTP (6000 Ci/ mmol, New England Nuclear, Boston, MA) by the oligo-priming method (53), and unincorporated nucleotide was separated by passage over a Sephadex G-50 column. Specific activities obtained were between 2-5 x 10’ cpm/mg. The oligonucleotide was end-labeled with [32P]ATP (ICN, Cleveland, OH) and T4 polynucleotide kinase (Promega, Madison, WI) and the unincorporated label removed as above. Measurement

of Cytoplasmic

Ca*+ Concentration

Bovine adrenal glomerulosa cells cultured in six-well plates (see above) were removed from the plates by treatment with Ca*‘- and Mg2+-free medium in the presence of 1 mM EDTA. Cells were loaded with 0.5 KM Fura-2/AM, and fluorescence was monitored with a PTI Deltascan dual-wavelength spectrophotometer (South Brunswick, NJ) as described previously (39). Materials All and [Sar’,lle*]All were purchased from Peninsula Laboratories (Belmont, CA). The nonpeptide All antagonists, DuP 753 and PD 123177, were provided by Dr. P. C. Wang (DuPont, Wilmington, DE). TG was a sift from Dr. S. g Christensen (Roval Danish School of Pharmacv. Cooenhaaen. Denmark). iat&ials used for cell isolation and ir&uba&& were described elsewhere (17). Statistics

and Data Analysis

Since actin mRNA showed significant changes upon All stimulation, 28s rRNA was used as a reference to check uniformity of RNA loading on gels. Results are expressed as the fold induction above basal, nonstimulated values. Statistical comparisons were made using Student’s t test or analysis of variance.

Acknowledgments We are grateful to Dr. L. M. Mertz for performing the Northern blot analysis of the mRNA samples with the 17n-hydroxylase probe, and to Dr. M. L. Dufau (NIH, Bethesda, MD) and Dr. R. Bravo (Bristol Myers-Squibb Pharmaceutical Research Institute) for providing the 17a-hydroxylase and the c-jun, JunB, and Krox 24 DNA probes, respectively.

Received July 7, 1992. Revision received August Accepted August 25, 1992. Address requests for reprints to: Dr. Kevin J. Catt, Institute of Child Health and Human Development, nology and Reproduction Research Branch, National of Health, Building 10, Bl/L-400, 9000 Rockville thesda, Maryland 20892.

20, 1992. National EndocriInstitutes Pike, Be-

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