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Endocrinology Vol. 138, No. 11 4591-4598
Copyright © 1997 by The Endocrine Society

ARTICLES

Localization and Differential Expression of Adenylyl Cyclase Messenger Ribonucleic Acids in Rat Adrenal Gland Determined by in Situ Hybridization1

Tiansheng Shen2, Yosuke Suzuki3, Madeleine Poyard, Martin Best-Belpomme, Nicole Defer and Jacques Hanoune

Institut National de la Santé et la Recherche Médicale U-99 Hôpital Henri Mondor, F-94010 Créteil, France

Address all correspondence and requests for reprints to: Jacques Hanoune, Institut National de la Santé et la Recherche Médicale U-99 Hôpital Henri Mondor, F-94010 Créteil, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of adenylyl cyclases (ACs) in the adult rat adrenal gland was examined. In situ hybridization revealed specific patterns of AC messenger RNA (mRNA) distribution. AC1 was limited exclusively to the adrenal medulla. AC5 and AC6 were mainly expressed in the adrenal medulla, with a weak expression in the zona glomerulosa. AC9 was found in all the three regions of the adrenal cortex but not in the adrenal medulla. All these ACs were detected on postnatal day 1 (PN1), and their pattern of expression was unchanged on PN7, PN21, and PN90 (adult). We analyzed the response of these ACs to various physiological conditions known to affect the synthesis of aldosterone and corticosterone in the adrenal cortex. Our study demonstrates a specific increase of AC6 but not AC5 mRNA in the zona glomerulosa of rats given a low sodium diet. AC9 mRNA was increased in all the three cortical zones of rats treated with ACTH. We suggest that AC6 and AC9 play important roles in different pathways associated with the regulation of aldosterone and corticosteroid production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADRENAL gland has a dual origin. The adrenal cortex is derived from mesodermal tissue and comprises the outer zona glomerulosa that secretes aldosterone, and the inner zonae fasciculata-reticularis that secrete glucocorticoids and androgen. The medulla is derived from neuroectodermal cells of sympathic ganglia, and is the source of catecholamines. In both cortex and medulla, the hormonal secretion is under the control of cAMP. This nucleotide is directly involved in the control of glucocorticoid secretion by ACTH and of catecholamine secretion by stimulation of the preganglionic fibers (1, 2, 3). The secretion of aldosterone is mainly regulated by phosphatidyl inositol (4, 5, 6), but cAMP also plays a significant role (7, 8).

To date, nine isoforms of adenylyl cyclase (AC) (the enzyme responsible for the synthesis of cAMP from ATP) have been cloned, sequenced and characterized (9). They can be grouped into four subfamilies. AC1, AC3, and AC8 are activated by the calcium/calmodulin complex, and are rather specific for neural tissues (10, 11, 12, 13). AC2, AC4, and probably AC7 are activated by the ß{gamma} subunits of G proteins (14, 15, 16, 17). AC5 and AC6 are inhibited by submicromolar concentrations of calcium (18, 19). Finally, AC9 is regulated neither by calcium nor by ß{gamma} subunits (20). It is likely that the diversity of these regulatory mechanisms accounts for the tissue-specific pattern of cAMP synthesis, and in particular, for the occurrence of subtle regulation by cross-talk or convergence between independent signaling pathways.

Therefore, the aim of the present study was to elucidate the distribution of various AC isoforms in the rat adrenal gland using the in situ hybridization technique. Our results reveal a discrete distribution of the messenger RNAs (mRNAs) coding for AC1, AC5, AC6, and AC9 in the various zones of the gland. They further demonstrate a good correlation between the amount of AC6 or AC9 and the production of aldosterone and corticosterone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diets
Sprague-Dawley rats were purchased from Janvier (Le Genest St. Isle, France). Male and female rat pups were killed on postnatal days 1 (PN1) and 7 (PN7), and males were killed at postnatal 21 (PN21) and 90 (PN90) days of age for the developmental study. To study the effect of sodium depletion, adult male rats, weighing 150 g, were divided in two groups: the control group that received a synthetic diet containing a normal amount of Na (86 mmol/kg), and a sodium-restricted group that received a synthetic diet containing a low (8.6 mmol/kg) amount of Na for 9 days as described previously (21, 22). For ACTH-treated rats, animals were intramuscularly injected with daily doses of 40 µg ACTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) (Synacthene retard; Ciba-Geigy, Rueil-Malmaison, France) for 10 days before death. All rats were housed under a 12-h light/12-h dark cycle with food and tap water available ad libitum.

Tissue preparation and sectioning
The animals were killed by decapitation. The adrenal glands were excised immediately, removed carefully from the surrounding fat tissues, embedded in Tissue-Tek OCT (Miles, Elkhart, IN), and quick-frozen at -20 C. Tissue blocks were stored at -80 C. Ten-micrometer thick cryostat sections were thaw-mounted on gelatin-coated slides and stored in air-tight containers at -20 C.

Probes
Probes specific for each of the AC subtypes were chosen from regions where the sequences are the most divergent. A 459-bp fragment (amino acids 498–650) of the sequence for AC5 was obtained by reverse transcription of 4 µg total RNA from the ventricle of an adult rat, followed by PCR amplification. The PCR product was subcloned into the pCRII plasmid (23). Probes specific for AC6 were obtained by RT-PCR using mRNA from rat brain and specific oligonucleotides: the primers 5'-TGCTGCTGGTCACCGTGCTCAT-3' (sense) and 5'-GGACGCTAAGCAGTAGATCATAGTTGTCAA-3' (antisense), providing amplification of a 493-bp fragment of the rat AC6 (membrane spans 8–11) (23) that was subcloned into the pCRII plasmid. A 739-bp fragment inserted in the SmaI site of the pGEM 3 was derived from mouse AC9 (nucleotides 1–739) (20). A 50-mer: 5'-ATGTTCAGGTCTACTTCAGTAGCCTCAGCCACGGATGTGATGGTATCGAT-3' oligonucleotide was used as a probe for AC1, this being complementary to sequence encoding part of the first putative cytoplasmic loop (24). A 35-mer (5'-TCCCTGAGTTATTAGTGCTGCCACAATGCCACTGT-3') corresponding to positions 857–891 bp of rat aldosterone synthase P-450 (CYP11B2) was constructed to provide specific RNA hybridization for aldosterone synthase P-450 (25). A 35-mer (5'-TGACAGAGTACTCTGTGCTACCATCTCGGATATGA-3') corresponding to positions 863–897 bp of rat 11ß-hydroxylase (CYP11B1) was constructed to provide specific RNA hybridization for 11ß-hydroxylase (26). Before in vitro transcription for generating complementary RNA (cRNA) probes, the pCRII plasmids containing the 459-bp fragment of the rat AC5 and the 493-bp fragment of the rat AC6 were linearized with either XhoI or BamHI; the pGEM 3 plasmid containing the 739-bp fragment of the mouse AC9 was linearized with either EcoRI or AvaII to prepare antisense or sense cRNA probes, respectively. The in vitro transcription was carried out at 40 C for 60 min in a 20-µl reaction mixture containing 1 µg linearized plasmid templates, and Sp6 or T7 RNA polymerase in the presence of [{alpha}-33P]uridine triphosphate (>3000 Ci/mmol, 1 mCi = 37 Mbq; ICN Biomedicals, Costa Mesa, CA). The three oligonucleotides were labeled with [{alpha}-33P]deoxycytidine ATP (>1000 Ci/mmol, 1 mCi = 37 TBq; Amersham, Arlington Heights, IL), and terminal transferase (Promega, Madison, WI) in a 20-µl mixture containing 100 mM cacodylate buffer (pH 6.8), 1 mM CoCl2, and 0.1 mM dithiothreitol, oligonucleotides (9.4 pmol), terminal transferase (40 U), [{alpha}-33P]deoxycytidine ATP for 1 h at 37 C. Reactions were stopped by heating at 70 C for 10 min. The 33P-labeled probes were separated from free nucleotides by spin column (Sephadex G-50 fine) and then concentrated by ethanol precipitation.

In situ hybridization
In situ hybridization was performed as described previously (27) with some modifications. Briefly, frozen sections were fixed with 4% paraformaldehyde in PBS, treated with acetic anhydride, delipidated in a graded series of ethanol and chloroform, and preincubated for 5–12 h in a humid chamber at 42 C for the oligonucleotide probes, or at 55 C for cRNA probes, with 40 µl prehybridization buffer (50% formamide, 4x SSC (1x SSC contains 0.15 M NaCl and 0.015 M sodium citrate), 1% sarkosyl, 1x Denhardt’s reagent, 10 mM dithiothreitol, 0.1 M phosphate buffer, pH 7.4, 250 µg/ml denatured salmon sperm DNA). Hybridization was performed at 37 C for the oligonucleotide probes, or at 55 C for cRNA probes, overnight in 40 µl prehybridization buffer containing 10% dextran sulfate and 2.0 x 107 cpm/ml probes. Slides were rinsed in 1x SSC and for cRNA probes, incubated for 30 min at 37 C with 20 µg/ml RNase A and 10 U/ml RNase T1 in 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA. Subsequently, most stringent washes were performed in 0.1x SSC at 42 C for the oligonucleotide probes or at 60 C for cRNA probes. Sections were dehydrated with ethanol, exposed to Bio-Max film (Eastman Kodak, Rochester, NY) for 3–4 days to generate autoradiographic images, then coated with light microscopy 1 emulsion (Amersham), and exposed in the dark at 4 C. After 3 days (for CYP11B1 and CYP11B2) to 2 weeks (for ACs), the slides were developed, fixed, and then counterstained with toluidine blue. Specificity of the AC probes was controlled in two ways. First, Northern blots were performed using either poly(A+) RNAs from rat brain and kidney (28, 29) or total RNAs from rat adrenal gland; each probe labeled a unique band with a size corresponding to its own mRNA (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) (data not shown). Second, adjacent sections were hybridized with labeled oligonucleotide probes with or without a 100-fold excess of the same unlabeled oligonucleotide (AC1), or with either the 33P-labeled antisense cRNA probes or the 33P-labeled sense cRNA probes (the other ACs). Under these conditions, no signal was detected in the control sections.

Quantification of AC expression
To quantify in situ hybridization data, nonoverlapping areas (n = 8) from the different zones of the adrenal cortex were determined on enlarged photographes. In each area, nuclei and silver grains were counted, and data (after deducting the related background fractions) are expressed as autoradiographic grains/10 nuclei. Statistical analyses were performed using the nonpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AC mRNAs expression in the adult rat adrenal gland
To assess the level of expression of the AC isotypes in the adrenal gland and localize their mRNAs in the different structures, in situ hybridizations were performed. Figures 1Go and 2Go show specific signals obtained with probes for AC1, AC5, AC6, and AC9 in the adult rat adrenal gland. In contrast, the expression of AC2, AC3, AC4, AC7, and AC8 is too low, if it exists, to be detected by in situ hybridization. AC1 was expressed exclusively in the medulla and Fig. 2Go shows that the autoradiographic grains representing hybridization to AC1 mRNA accumulated over all chromaffin cells of the medulla. By contrast, for AC9, the autoradiograph revealed a nearly homogenous hybridization signal in the cortex, and no specific signal in the medulla; under the light microscope, accumulation of silver grains was seen over the three zones of the cortex. As for AC5 and AC6, the two closely related calcium-sensitive ACs, they appeared to have the same distribution: in the outer part of the cortex and in the medulla; the light microscopic observation demonstrated an accumulation of silver grains over the medullary cells and a few silver grains over the glomerulosa cells, whereas no grains were detected in the zonae fasciculata/reticularis.



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  		Figure 1. AC expression in rat adult adrenal gland. X-ray autoradiographs illustrating hybridization signals obtained in sections from adult rat adrenal glands. Left, Expression of AC1, AC5, AC6, and AC9 mRNAs in adult rat adrenal glands visualized by hybridization with 33P-labeled oligonucleotide (for AC1) or with antisense cRNA probes (for AC5, AC6, and AC9). Right, Nonspecific labeling on control tissue sections incubated with either 33P-labeled oligonucleotide together with a 100x excess of same unlabeled oligonucleotide (for AC1) or 33P-labeled sense cRNA probes (for AC5, AC6, and AC9). C, cortex; M, medulla. Exposure time: 4 days. All experiments were carried out with at least three different animals and repeated two or three times. Similar results were obtained and typical results are shown.

 


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  		Figure 2. AC mRNA expression in adult rat adrenal gland. Bright-field autoradiographs of emulsions-coated sections labeled with oligonucleotide probe for AC1 (1) or antisense cRNA probes for AC5 (2A and 2B), AC6 (3A and 3B), and AC9 (4A and 4B). Left,Labeling in zona glomerulosa (G) and zona fasciculata (F). Right, Labeling over zona reticularis (R) and medulla. Exposure time: 2 weeks. Magnification: x630.

 
AC mRNA expression in the developing rat adrenal gland
In situ hybridizations were performed at different stages of the developing rat adrenal gland: PN1, PN7, PN21, and adult (PN90) (Fig. 3Go). On PN1, the expression of AC types 1, 5, 6, and 9 mRNAs was already detected in the adrenal gland. The pattern of their expression remained relatively unchanged throughout. The levels of the mRNAs for AC1, AC5, and AC6 at PN1 were not markedly modified during postnatal development (data not shown). In contrast, the levels of AC9 mRNA on PN1 and PN7 (71.8 ± 5.6 and 77.9 ± 8.3 grains/10 cells, respectively), as compared with that (33.5 ± 4 grains/10 cells) in the adult rats, are significantly higher (P < 0.01).



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  		Figure 3. AC mRNA expression in developing rat adrenal gland. X-ray autoradiographs illustrating hybridization signals obtained in sections from adrenal glands of PN1, PN7, PN21, and PN90 animals. No radiolabeling is present in control sections (data not shown). Probes were used as in Fig. 1Go. Exposure time: 4 days.

 
AC mRNAs expression in adrenal gland of dietary sodium-deficient rats
The specificity of steroid biosynthesis to different zones of the adrenal cortex is due to the presence of two highly homologous cytochromes: CYP11B2, which is responsible for the synthesis of aldosterone in the zona glomerulosa, and CYP11B1, which is responsible for the synthesis of corticosterone in the zonae fasciculata-reticularis (6, 22, 30). Dietary sodium restriction is known to induce the proliferation of the glomerulosa cells and to increase the mRNA level of CYP11B2. In the experiment shown in Fig. 4Go and in agreement with previous findings (21, 22), the abundance of CYP11B2 mRNA was markedly increased in rats receiving a low sodium diet for 9 days, whereas no change was found for CYP11B1 mRNA. The important feature observed was that the hybridization signal increased also for AC6, but not for AC5 and AC9, in the outer zona glomerulosa. Additionally, we found no change in the relative abundance of AC1, AC5, and AC6 mRNAs in the adrenal medulla.



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  		Figure 4. AC mRNA expression in rats fed 9 days of a low sodium diet. Rats were fed for 9 days with normal diet (Normal) or with low sodium diet (Hypo-Na+). In situ hybridizations were performed as previously described using CYP11B1 (B1)-, CYP11B2 (B2)-, AC1-, AC5-, AC6-, or AC9-labeled probes. No specific labeling has been found in control sections (data not shown). Exposure time: 3–4 days.

 
Figure 5Go shows that in rats maintained under Na-depleted conditions, the zona glomerulosa cells containing the CYP11B2 mRNA proliferated, and the thickness became about 3-fold greater than that of the control rats. Under these conditions, the mRNA expression for AC6 also increased in the proliferating glomerulosa cells; no significant change in AC5 mRNA expression was observed (Fig. 6Go). To quantify AC5 and AC6 mRNA expression, autoradiographic grains and nuclei were counted in measured areas of enlarged photographs; the mean number of silver grains/10 cells (± SEM) was: 12 ± 1.3 and 40 ± 6 for AC6 in normal and low sodium-diet rats, respectively (significantly different at P < 0.01). This represents a 3-fold increase in the level of expression per cell. In contrast, there was no difference in AC5 expression, it was 8 ± 0.8 and 7.7 ± 0.5 grains/10 cells for AC5 in normal and low sodium-diet rats, respectively. According to Ogishima et al. (22), the increased level of CYP11B2 in the rat adrenal cortex associated with Na depletion could be attributed to an increased population of the cells expressing the enzyme. In our experiments, the increase in the AC6 mRNA expression might be due to both the increased cell population expressing AC6 and the increased level of expression in the individual cells.



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  		Figure 5. Dark-field autoradiographs of low sodium-diet rat adrenal glands. Same legend as Fig. 4Go. CYP11B1 (B1), CYP11B2 (B2), AC6, and AC5 mRNAs expression in adrenal glands of rats fed with either a normal (Normal) or a low sodium diet (Hypo-Na+). G, zona glomerulosa; F, zona fasciculata. Exposure time: 3 days for CYP11B1 and CYP11B2, 2 weeks for AC6 and AC5. Magnification: x100.

 


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  		Figure 6. mRNA expression of AC5 (A and B) and AC6 (C and D) in zona glomerulosa and fasciculata of adrenal gland of rats fed with normal diet (A and C) or low sodium diet (B and D). Exposure time: 2 weeks. Magnification: x630. Quantifications of AC6 and AC5 expression were performed on measured areas in which silver grains and nuclei were counted.

 
AC mRNAs expression in the adrenal gland of rats treated with ACTH
The effects of ACTH administration (with a daily dose of 40 µg im for 10 days) on AC mRNAs expression are shown in Figs. 7Go and 8Go. In agreement with previous reports (30, 31), ACTH induced the proliferation of zonae fasciculata-reticularis cells, as evidenced by an increase of the adrenal gland weight by about 3-fold (50.7 mg ± 1.2 as compared with normal adrenal gland weight: 15.5 mg ± 0.44, which is significantly different at P < 0.01). It specifically induced the expression of CYP11B1 mRNA, whereas the mRNA level of CYP11B2 in the zona glomerulosa was significantly decreased. Concerning the expression of different AC isoforms, the major finding was that AC9 expression was markedly increased in all the proliferating cortical cells, whereas no significant change was observed for the other isoforms. The increase in AC9 mRNA is clearly seen in Fig. 8Go. Quantification of the silver grains over the three zones of the cortex, as described above, showed a 2-fold expression of AC9 per cell in ACTH-treated rats compared with normal animals (The mean value of silver grains per 10 cells ± SEM was: 34 ± 2.7 and 68 ± 4.9, which is significantly different at P < 0.001).



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  		Figure 7. X-ray autoradiographs illustrating hybridization signals obtained in sections from adrenal glands of normal and ACTH-treated rats. Probes were used as in Fig. 4Go. No radiolabeling can be seen in control sections (data not shown). Exposure time: 3–4 days.

 


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  		Figure 8. Dark-field autoradiographs of ACTH-treated rat adrenal glands. Same legend as Fig. 7Go. CYP11B1 (B1), CYP11B2 (B2), and AC9 mRNAs expression in adrenal cortex of normal and ACTH-treated rats. Exposure time: 3 days for CYP11B1 and CYP11B2, 2 weeks for AC9. Magnification: x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study clearly establishes the localization of AC isoforms in the adult rat adrenal gland at the light microscopic level, using the in situ hybridization technique. AC1 is exclusively expressed in the medulla, AC5 and AC6 are expressed in the zona glomerulosa and medulla, whereas the newly described type AC9 is expressed in the three zones of the cortex and is specific for the zonae fasciculata-reticularis. Our results suggest that discrete roles may be fulfilled by these AC subtypes.

The adrenal gland is of neuroendocrine origin. AC1 is considered to be expressed exclusively by neurons or endocrine cells with neuron-like characteristics, and its presence in the adrenal medulla might reflect their embryological origin. On the basis of Northern blot analysis, Xia et al. (32) also detected the expression of AC1 in the bovine adrenal medulla. Manolopoulos et al. (33) detected a strong expression of AC4 and AC6 as well as a weak expression of AC5 in rat whole adrenal tissue homogenates by RT-PCR. We also found AC5 and AC6 to be expressed in the rat adrenal gland, but we were unable to detect AC4 mRNA expression. This discrepancy is probably due to the different methods and probes used. For AC2 and AC3, our results are in agreement with theirs, because no specific signal was found for those two subtypes in the adult rat adrenal gland.

Developmental changes in the expression of different AC subtypes have been reported in the brain, heart, and kidney (28, 34, 35), suggesting that various AC subtypes have specific roles for different tissues in their development. In the rat adrenal gland however, we found that AC types 1, 5, 6, and 9 are already expressed in the neonate, with no evident change during the developmental period examined except for AC9, which has a relatively higher expression in the first week after birth. The biological significance of this age-related reduction in AC9 mRNA in the developing rat adrenals remains to be established.

Of additional interest is the hormonal regulation involved in ACs expression under different physiological conditions (Table 1Go). When rats were given a low sodium diet for 9 days, the thickness of zona glomerulosa increased several-fold as compared with that in rats fed normal diet. The expression of AC6, but not AC5 mRNA, in this zone was increased significantly. These cells also expressed AC9, but the intensity was not different from that in normal rats. As AC5 and AC6 are two closely related cyclase isoforms belonging to the same subfamily, the lack of induction in AC5 mRNA level demonstrates a high degree of specificity in their regulation. We have no clear understanding of the basis for the difference in sodium induction of these highly similar genes within zona glomerulosa cells. Interestingly, previous studies have shown that the expression of AC5 and AC6 is differentially regulated in the rat heart during ontogenic development, with AC5 mRNA increasing after birth until adulthood, but no significant change in AC6 mRNA level (34, 35). It is tempting to speculate that these differences in gene regulation reflect differences in promotor elements that regulate the expression of these two AC types. Unfortunately, at present, with the exception of AC3 (36), the promotor regions of the AC genes are not known.


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  		Table 1. Distribution and regulation of AC isoforms in rat adrenal glands

 
The physiological significance of an increase in AC6 mRNA level associated with sodium restriction is not clear. Previous reports showed that the most evident change with a low sodium diet is the increased amount of CYP11B2 mRNA and protein associated with an elevated plasma aldosterone concentration (21, 22). Although it is generally accepted that angiotensin II is the major mediator of aldosterone synthesis in the adrenal cortex, this hypothesis has been challenged recently. Angiotensin II had no stimulatory effect on the promotor activity of 5'-flanking regions of CYP11B2 in transient transfection experiments using mouse adrenocortical Y1 cells (8). On the other hand, a putative cAMP-responsive element, TGACGTGA, was found in the rat CYP11B2 gene (7). The promotor of this gene was shown to be under the control of cAMP, because dibutyryl cAMP could stimulate the promotor activity of this gene (37). We therefore postulate that the increased expression of AC6 mRNA, and in turn the resultant rise in intracellular cAMP level, could partially participate in regulating the transcription of CYP11B2 gene by stimulating its promotor activity. The fact that AC6 is inhibited by submicromolar concentration of calcium and that an excess of cytosolic Ca2+ exerts a negative effect on aldosterone biosynthesis (38, 39), lend support to our hypothesis.

In the rat, corticosterone (the primary glucocorticoid in this animal species) is mainly produced in the zonae fasciculata-reticularis, and the major factor regulating its biosynthesis is ACTH. ACTH exerts both acute and chronic actions to promote adrenocortical steroidogenesis. Within minutes after ACTH administration, cholesterol is metabolized to corticosterone by a series of reactions that involve the participation of several constitutively active cytochrome P-450 mixed-function oxidases including 11ß-hydroxylase (3). ACTH has also a long-term effect on the adrenal cortex to stimulate the synthesis of steroidogenic enzymes at the transcriptional level as part of its trophic effects (2). As a result, ACTH administration elicits a dramatic growth of the adrenal gland (40). All those actions are mediated by activation of the AC system, which leads to the cAMP signaling cascade (1). In the present study, AC9 is the only subtype detected in zonae fasciculata-reticularis. After ACTH treatment, we found a massive adrenal growth, mainly due to the increased width of the zonae fasciculata-reticularis cells that express CYP11B1 mRNA. This was paralleled by an increase in AC9 mRNA level per cell. Because no significant change was observed for the other AC subtypes, our results not only demonstrate again the importance of the AC system in the action of ACTH on adrenal growth and steroidogenesis in the rat, but also provide new information about the specific regulation of AC isoforms by ACTH. Additionally, it should be noted that Ca2+ ions are essential for ACTH to stimulate in vitro AC activity, in naive as well as in ACTH-treated rat adrenal glands (data not shown). Because AC9 is not regulated by Ca2+ (20), it is probably not the site of cross-talk or convergence between the Ca2+ and cAMP signaling pathways in the adrenocortical cells.

In summary, the present study reveals that AC types 1, 5, 6, and 9 mRNAs are expressed in the rat adrenal gland with different distribution patterns. This pattern of expression remains constant throughout development (from PN1 to adult). In rats fed a low sodium diet, the expression of AC6 increases significantly in the zona glomerulosa, in parallel with the increased expression of CYP11B2. In rats treated with ACTH, the expression of AC9 increases also in the proliferating zonae fasciculata-reticularis. We suggest that AC6 and AC9 play important roles in regulating the production of aldosterone and corticosterone.


   Acknowledgments
 
We are grateful to Dr. Robert Barouki, Dr. Yannick Laperche, and Dr. John Laycock for critical reading of the manuscript. We are also thankful to Edith Grandvilliers and Lydie Rosario for expert secretarial assistance.


   Footnotes
 
1 This work was supported by the INSERM and the University Paris-Val de Marne. Back

2 Recipient of a fellowship from the People’s Republic of China. Back

3 Recipient of a fellowship from the Fondation de la Recherche Médicale. Back

Received April 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schimmer BP 1995 The 1994 Upjohn Award Lecture. Molecular and genetic approaches to the study of signal transduction in the adrenal cortex. Can J Physiol Pharmacol 73:1097–1107[Medline]
  2. Hanukoglu I, Feuchtwanger R, Hanukoglu A 1990 Mechanism of corticotropin and cAMP induction of mitochondrial cytochrome P450 system enzymes in adrenal cortex cells. J Biol Chem 265:20602–20608[Abstract/Free Full Text]
  3. Simpson ER, Waterman MR 1992 Regulation of expression of adrenocortical enzymes. In: James VHT, ed. The adrenal gland, 2nd ed. Raven Press, New York, pp 191–207
  4. Miller WL 1988 Molecular biology of steroid hormone synthesis. Endocr Rev 9:295–318[Abstract/Free Full Text]
  5. Aguilera G 1993 Factors controlling steroid biosynthesis in the zona glomerulosa of the adrenal. J Steroid Biochem Mol Biol 45:147–151[CrossRef][Medline]
  6. Oertle M, Müller J 1993 Two types of cytochrome P-45011b in rat adrenals: separate regulation of gene expression. Mol Cell Endocrinol 91:201–209[CrossRef][Medline]
  7. Mukai K, Imai M, Shimada H, Okada Y, Ogishima T, Ishimura Y 1991 Structural differences in 5'-flanking regions of rat cytochrome P-45011b genes. Biochem Biophys Res Commun 180:1187–1193[CrossRef][Medline]
  8. Mukai K, Imai M, Shimada H, Ishimura Y 1993 Isolation and characterization of rat CYP11B genes involved in late steps of mineralo-and glococorticoid syntheses. J Biol Chem 268:9130–9137[Abstract/Free Full Text]
  9. Hanoune J, Pouille Y, Tzavara E, Shen T, Lipskaya L, Miyamoto N, Suzuki Y, Defer N 1997 Adenylyl cyclases: structure, regulation and function in an enzyme superfamily. Mol Cell Endocrinol 128:179–194[CrossRef][Medline]
  10. Tang W-J, Krupinski J, Gilman AG 1991 Expression and characterization of calmodulin-activated (type I) adenylylcyclase. J Biol Chem 266:8595–8603[Abstract/Free Full Text]
  11. Defer N, Marinx O, Stengel D, Danisova A, Iourgenko V, Matsuoka I, Caput D, Hanoune J 1994 Molecular cloning of the human type VIII adenylyl cyclase. FEBS Lett 351:109–113[CrossRef][Medline]
  12. Choi E-J, Xia Z, Storm DR 1992 Stimulation of the type 3 olfactory adenylyl cyclase by calcium and calmodulin. Biochemistry 31:6492–6498[CrossRef][Medline]
  13. Cali JJ, Zwaagstra JC, Mons N, Cooper DMF, Krupinski J 1994 Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. J Biol Chem 269:12190–12195[Abstract/Free Full Text]
  14. Taussig R, Quarmby LM, Gilman AG 1993 Regulation of purified type 1 and 2 adenylyl cyclases by G protein ß{gamma} subunits. J Biol Chem 268:9–12[Abstract/Free Full Text]
  15. Tang W-J, Gilman AG 1991 Type specific regulation of adenylyl cyclase by G protein ß{gamma}-subunits. Science 254:1500–1503[Abstract/Free Full Text]
  16. Gao B, Gilman AG 1991 Cloning and expression of a widely distributed (type IV) adenylyl cyclase. Proc Natl Acad Sci USA 88:10178–10182[Abstract/Free Full Text]
  17. Feinstein PG, Schrader KA, Bakalyar HA, Tang W-J, Krupinski J, Gilman AG, Reed RR 1991 Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenlylyl cyclase from rat brain. Proc Natl Acad Sci USA 88:10173–10177[Abstract/Free Full Text]
  18. Katsushika S, Chen L, Kawabe JI, Nilakantan R, Halnon NJ, Homcy CJ, Ishikawa Y 1992 Cloning and characterization of a sixth adenylyl cyclase isoforms: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family. Proc Natl Acad Sci USA 89:8774–8778[Abstract/Free Full Text]
  19. Ishikawa Y, Katsushika S, Chen L, Halnon NJ, Kawabe J-I, Homcy CJ 1992 Isolation and characterization of a novel cardiac adenylyl cyclase cDNA. J Biol Chem 267:13553–13557[Abstract/Free Full Text]
  20. Premont R, Matsuoka I, Mattei MG, Pouille Y, Defer N, Hanoune J 1996 Identification and characterization of a novel and widely-expressed isoform of adenylyl cyclase. J Biol Chem 271:13900–13907[Abstract/Free Full Text]
  21. Shibata H, Ogishima T, Mitani F, Suzuki H, Murakami M, Saruta T, Ishimura Y 1991 Regulation of aldosterone synthase cytochrome P-450 in rat adrenals by angiotensin II and potassium. Endocrinology 128:2534–2539[Abstract/Free Full Text]
  22. Ogishima T, Suzuki H, Hata J, Mitani F, Ishimura Y 1992 Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011b in rat adrenal cortex: histochemical basis for the functional zonation. Endocrinology 130:2971–2977[Abstract/Free Full Text]
  23. Premont RT, Chen J, Ma H-W, Ponnapalli M, Iyengar R 1992 Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases. Proc Natl Acad Sci USA 89:9809–9813[Abstract/Free Full Text]
  24. Mons N, Yoshimura M, Cooper DMF 1993 Discrete expression of Ca2+/Calmodulin-sensitive and Ca2+-insensitive adenylyl cyclases in the rat brain. Synapse 14:51–59[CrossRef][Medline]
  25. Imai M, Shimada H, Okada Y, Matsushima-Hibiya Y, Ogishima T, Ishimura Y 1990 Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex. FEBS Lett 263:299–302[CrossRef][Medline]
  26. Nonaka Y, Matsukawa N, Morohashi K-I, Omura T, Ogihara T, Teraoka H, Okamoto M 1989 Molecular cloning and sequence analysis of cDNA encoding rat adrenal cytochrome P-45011b. FEBS Lett 255:21–26[CrossRef][Medline]
  27. Matsuoka I, Giuili G, Poyard M, Stengel D, Parma J, Guellaen G, Hanoune J 1992 Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution. J Neurosci 12:3350–3360[Abstract]
  28. Matsuoka I, Suzuki Y, Defer N, Nakashini H, Hanoune J 1997 Differential expression of type I, II, and V adenylyl cyclase gene in the postnatal developing rat brain. J Neurochem 68:498–506[Medline]
  29. Shen T, Suzuki Y, Poyard M, Miyamoto N, Defer N, Hanoune J 1997 Expression of adenylyl cyclase mRNAs in the adult, developing and Brattleboro rat kidney. Am J Physiol 273:C323–C330
  30. Lauber M, Bôni-Schnetzler M, Mûeller J 1990 Potassium raises cytochrome P-450 11b mRNA level in zona glomerulosa of rat adrenals. Mol Cell Endocrinol 72:159–166[CrossRef][Medline]
  31. Feuillan PP, Aguilera G 1996 Regulation of aldosterone in the 7-day-old rat. Endocrinology 137:3992–3998[Abstract]
  32. Xia Z, Choi E-J, Wang F, Blazynski C, Storm DR 1993 Type I calmoduline-sensitive adenylyl cyclase is neural specific. J Neurochem 60:305–311[CrossRef][Medline]
  33. Manolopoulos VG, Liu J, Vnsworth BR, Lelkes PI 1995 Adenylyl cyclase isoforms are differentially expressed in primary cultures of endothelial cells and whole tissue homogenates from various rat tissues. Biochem Biophys Res Commun 208:323–331[CrossRef][Medline]
  34. Espinasse I, Iourgenko V, Defer N, Samson F, Hanoune J, Mercadier JJ 1994 Type V, but not type VI adenylyl cyclase mRNA accumulates in the heart during ontogenic development. Correlation with adenylyl cyclase activity. J Mol Cell Cardiol 27:1789–1795
  35. Tobise K, Ishikawa Y, Holmer SR, Im M-J, Newell JB, Yoshie H, Fujita M, Susannie EE, Homcy CJ 1994 Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle. Circ Res 74:596–603[Abstract/Free Full Text]
  36. Wang MM, Tsai RYL, Schrader KA, Reed RR 1993 Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression. Mol Cell Biol 13:5805–5813[Abstract/Free Full Text]
  37. Mukai K, Imai M, Shimada H, Okada Y, Ogishima T, Ishimura Y 1991 Structural differences in 5'-flanking regions of rat cytochrome P-450aldo and P-450(11) beta genes. Biochem Biophys Res Commun 180:1187–1193
  38. Python CP, Laban OP, Rossier MF, Vallotton MB, Capponi AM 1995 The site of action of calcium in the activation of steroidogenesis: studies in calcium-clamped bovine adrenal zona glomerulosa cells. Biochem J 305:569–576
  39. Barrett PQ, Ertel EA, Smith MM, Nee JJ 1995 Voltage-gated calcium currents have two opposing effects on the secretion of aldosterone. Am J Physiol 268:C985–C992
  40. Masui H, Garren LD 1970 On the mechanism of action of adrenocorticotropic hormone. J Biol Chem 245:2627–2632[Abstract/Free Full Text]



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