ARTICLE |
Correspondence to: Anne-Marie O'Carroll, University Research Centre for Neuroendocrinology, U. of Bristol, Marlborough Street, Bristol BS2 8HW, UK. E-mail: A.M.OCarroll@bristol.ac.uk
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Summary |
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Somatostatin (somatotropin-release inhibitory factor, SRIF) exerts multiple inhibitory actions throughout the central nervous system and the periphery by binding to specific membrane-bound SRIF receptors (sstrs) of which five subtypes (sstr15) have now been identified. Individual sstr subtypes have been suggested to mediate selective biological actions of SRIF. Although the adrenal gland is a known target of SRIF action, the sstr subtypes involved in its actions are unclear. This study examined the expression of sstr15 in rat adrenal gland by RT-PCR analysis and in situ hybridization (ISH) histochemistry. Using RT-PCR expression combined with Southern blotting, sstr1, -2, -4, and -5 mRNAs were shown in the adrenal gland. ISH histochemistry revealed strong expression of sstr2 mRNA alone localized to the zona glomerulosa of the adrenal cortex and moderate labeling in scattered cells of the adrenal medulla, indicating a possible role for sstr2 in mediating SRIF physiology in this tissue by altering adrenal aldosterone and catecholamine secretion. These data also point to potential roles for sstr subtypes sstr1, -4, and -5 in the adrenal gland. (J Histochem Cytochem 51:5560, 2003)
Key Words: somatostatin receptors, zona glomerulosa, adrenal medulla, aldosterone secretion
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Introduction |
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SOMATOSTATIN (SRIF) is a hormone/neurotransmitter that was first described as a potent physiological regulator of growth hormone secretion from the anterior pituitary (
The adrenal gland is a known target of SRIF action, where it inhibits angiotensin II-stimulated aldosterone secretion in collagenase-dispersed rat adrenal glomerulosa cells (
Contradictory information regarding individual sstr mRNA distribution in the rat adrenal gland has been reported (
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Materials and Methods |
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RNA Extraction and RT-PCR
Total RNA from rat pituitary and rat adrenal gland was extracted by the RNAzol B method and quantified by UV absorption. Before RT, residual genomic DNA was removed by digestion of with RQ DNase (Promega; Madison, WI). RQ DNase (5 U) and RNasin (10 U; Promega) were added to the RNA samples (2 µg) and digestion was carried out at 37C for 15 min. Reactions were terminated by phenol extraction and the RNA was precipitated with ethanol and resuspended in 15 µl of distilled water. The DNase-treated rat pituitary and adrenal RNAs were reverse transcribed in 20 µl reaction volumes using random hexamer oligonucleotide primers (100 pmol) and Superscript II reverse transcriptase (100 U; BRL, Gaithersburg, MD). Corresponding control samples containing no reverse transcriptase were included for each tissue. After an initial incubation for 10 min at room temperature (RT), the reactions were allowed to proceed at 42C for 45 min and were then heated to 95C for 5 min. cDNA was stored at 20C and 1-µl aliquots were used for PCR.
PCR was carried out using primers for rat sstr1-5 (see
Southern Blotting
Standard protocols (-32P]-ATP and T4 polynucleotide kinase. Membranes were washed at 60C in 1 x SSC/0.1% SDS and exposed to Kodak XAR film.
Preparation of Probes for ISH Histochemistry
The rat sstr constructs used to generate 35S-labeled riboprobes were made as follows. sstr1 (631 bp) and sstr4 (436 bp) probes were generated by PCR using 1 µg of plasmid DNA from a rat cortex cDNA library, and while the sstr-3 probe (656 bp) was generated using
1 µg of rat genomic DNA (Promega). The PCR products were gel-purified and subcloned into pGEM-4Z. The resulting transcripts were complementary to bases 7981429 of rat sstr1, bases 16212277 of rat sstr3, and bases 10281464 of rat sstr4. sstr-2 (789 bp) and sstr5 (800 bp) probes were generated by PCR using
1 µg of plasmid DNA from a rat pituitary cDNA library plasmid DNA. The PCR products were gel-purified and subcloned into pGEM-3Z (Promega). The resulting transcripts were complementary to bases 12142003 of rat sstr2 and bases 8441644 of rat sstr5.
For all five sstr subtypes, sense and antisense riboprobes were generated using T7 and SP6 polymerases with [35S]-UTP and the MAXIscript in vitro transcription kit (Ambion; Austin, TX). The integrity of the probes was checked by restriction enzyme digests and DNA sequencing. The specificity of the probes had been described previously (
ISH Histochemistry (ISHH)
Sections (12 µm) of tissues from adult (180200 g) Wistar rats were thaw-mounted onto polylysine-coated slides and stored at 80C until hybridization. Contralateral adrenals of the same rats used for RT-PCR were used for ISHH. Hybridization was carried out as described previously (1 x 106 cpm) was applied to the sections for 18 hr at 55C. After hybridization the sections were washed four times for 5 min with 4 x SSC at RT, 30 min at 37C in RNase solution (20 µg/ml RNase A in 0.5 M NaCl, 10 mM Tris-HCl, pH 8.0, 0.25 mM EDTA), twice for 5 min in 0.1 x SSC/1 mM DTT at RT, and twice for 30 min at 65C in 0.1 x SSC/1 mM DTT. All slides were quickly rinsed in distilled water, dehydrated in a graded alcohol series, and dried. Slides were exposed to Kodak XAR film for 5 weeks at RT, dipped in Ilford K5 nuclear emulsion to localize the cellular distribution of silver grains, and stored desiccated at 4C for 4 months before development using Kodak D19 at RT. Tissue sections were counterstained with toluidine blue and slides were viewed by light/darkfield microscopy. No specific hybridization was detected with the sense probes.
Analysis
Five adjacent sections from adrenals from four animals were used for ISH with the five sstr riboprobes. Cells positive for sstr mRNA were identified by clusters of silver grains over cells.
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Results |
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RT-PCR Expression of sstr Subtypes in Adult Adrenal Gland
RT-PCR, using subtype-specific primers, was used to characterize the expression of mRNA encoding sstr15 in the rat adrenal gland. The expression of sstr15 in the rat pituitary was included as a positive control. Expression of all five sstr subtype mRNAs was seen in the pituitary (Fig 1A) as previously reported (
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Southern Blotting
Southern blotting hybridizaton of the RT-PCR products with internal sstr subtype-specific probes was performed to increase the detection limit of the PCR products compared to ethidium bromide staining and to introduce an additional subtype specificity check. Expression of all five sstr subtype mRNAs was seen in the pituitary (Fig 1C, Lane 1). Strong sstr2 mRNA expression was identified in the adrenal gland and weaker signals for sstr1, -4, and -5 mRNAs were observed after Southern blotting of RT-PCR products (Fig 1C, Lane 2). mRNAs for sstr3 were not detectable in the adrenal gland.
Localization of sstr Subtypes in Adult Adrenal Gland
The localization and distribution of sstr15 mRNAs in the rat adrenal gland was also studied by ISHH. 35S-Labeled antisense and sense riboprobes were used for this study. Distribution of sstr15 mRNA expression in classical SRIF target tissues, i.e., brain, pituitary, kidney, and stomach, was examined in parallel to the adrenal as positive controls and expression patterns were as described in the literature (data not shown). Strong hybridization of the sstr2 antisense probe was seen in the cortical region of the adrenal gland (Fig 2), whereas the mRNAs for sstr1, -3, -4, and -5 were not detectable over nonspecific background (not shown). sstr2 labeling was also observed in isolated cells scattered throughout the adrenal medulla (Fig 2). High-power micrographs show sstr2 mRNA distribution localized to the zona glomerulosa of the cortex, where intense labeling was observed (Fig 3A and Fig 3B), but no labeling was seen in the zona fasciculata, zona reticularis, or adrenal capsula.
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Discussion |
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SRIF is a potent physiological regulator of endocrine secretion and exerts its biological effects via five receptor subtypes (sstr15). The presence of high-affinity binding sites for SRIF has been demonstrated in homogenates of rat adrenal zona glomerulosa (
Typically, individual target cells for SRIF express multiple sstr subtypes, suggesting that the effects of SRIF may represent the composite activation of more than one sstr. However, there is evidence that individual sstr subtypes may mediate selective biological actions of SRIF. Recent studies suggest that most if not all of the antihormonal actions of SRIF are mediated via either sstr2 and/or sstr5 (
Conflicting information regarding individual sstr mRNA distribution in the rat adrenal gland has been reported in studies employing methodologies that have different detection sensitivities, e.g., RT-PCR, RNase protection assays, and Northern blotting analyses, and therefore the receptor subtypes involved in the actions of SRIF in this tissue are unclear. Specific expression of sstr-2 mRNA has been described in rat adrenal in one study (
In the present study, RT-PCR in combination with Southern blotting demonstrated that mRNA for sstr1, -2, -4, and -5 is expressed in the rat adrenal gland, suggesting a possible role for these subtypes in mediating SRIF physiology in this tissue. By ISH histochemistry, which determines the exact cellular localization of receptor mRNAs, expression of sstr2 mRNA alone could be demonstrated in the adrenal gland. No expression of sstr1, -3, -4, or -5 mRNAs could be detected above nonspecific background. High levels of sstr2 mRNA were found in the adrenal cortex, with expression localized to the cells of the zona glomerulosa, the exclusive site within the adrenal gland for the synthesis and secretion of aldosterone. sstr2 labeling was also observed in isolated cells scattered throughout the adrenal medulla. These observations are consistent with the known effects of SRIF on the adrenal gland and suggest a specific role for the sstr2 subtype in aldosterone production and in the regulation of catecholamine secretion. Based on the RT-PCR results from this study, however, it is likely that sstr1, -4, and -5 mRNAs, also present in the adrenal gland, are in a position to contribute to SRIF effects in this tissue. The lack of detection of these sstr subtypes by ISH histochemistry can be accounted for by the presence of their mRNAs at levels below the detection limit of this technique in the adrenal gland but which are detected with more sensitive methods such as RT-PCR.
In conclusion, these results demonstrate expression of sstr1, -2, -4, and -5 mRNAs in the rat adrenal gland. sstr2 mRNA expression in the zona glomerulosa and adrenal medulla of the rat adrenal gland indicates a possible role for this sstr subtype in altering adrenal aldosterone and catecholamine secretion. The roles of sstr1, -4, and -5 in regulating adrenal function have yet to be defined and may be clarified by IHC studies using receptor subtype-specific antibodies.
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Acknowledgments |
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The author is grateful for the financial support of the Wellcome Trust.
Received for publication July 10, 2002; accepted August 14, 2002.
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