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Widespread co-localization of mRNAs encoding the guanylate cyclase-coupled natriuretic peptide receptors in rat tissues
BIOCHEMICAL
Vol. 189, No. 2, 1992 December
AND BIOPHYSICAL
15, 1992
RESEARCH COMMUNICATIONS Pages 61 O-61 6
WIDESPREAD CO-LOCALIZATION OF mRNAS ENCODING THE GUANYLATE CYCLASBCOUPLED NATRIURETIC PEPTIDE RECEPTORS IN RAT TISSUES Teresa Tallerico-Melnyk*+, Cecil C. Yip+, and Valerie M. Watt* * Department of Physiology and+Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5S lA8 Received
October
22,
1992
Natriumtic peptides modulate vasorelaxation, diuresis, and natriurcsis through the stimulation of cGMP production by the guanylate cyclase-coupled natriumtic peptide receptors, GC-A and GC-B. We used reverse transcription-polymerase chain reaction to determine the distribution of mRNA encoding both receptors in rat tissues. GC-A and GC-B transcripts were detected in all peripheral and neural tissuesexamined. Since the atrial natriuretic peptide gene is expressed in all these tissues, our widespread detection of GC-A and GC-B mRNAs now suggests that natriuretic peptides may act as endocrine and paracrine hormones as well as neurotransmitters via both GC-A and GC-B receptors. 0 1992 Academic Press, Inc.
Atrial natriuretic peptide (ANP), secreted primarily by cardiac atria, reduces systemic blood pressure and intravascular volume (reviewed in ref. 1). ANP is the most studied member of a family of related peptides which also includes brain natriuretic peptide or BNP (2) and C-type natriutetic peptide or CNP (3). Both BNP and CNP also have vasorelaxant, diuretic, and natriumtic effects (2,3). Two functionally and structurally distinct types of receptors for natriuretic peptides have been identified (reviewed in ref.4). An -65~kDa clearance receptor is likely to play au important role in regulating plasma ANP levels (5) but does not mediate the known physiologic actions of ANP (reviewed in ref.@. In contrast, two - 130-kDa receptors, guanylate cyclase-A (GC-A) or ANP-A (7,8) and guanylate cyclase-B (GC-B) or ANP-B (9,10), contain an intracellular guanylate cyclase catalytic domain activated by natriuretic peptides. Since many physiological actions of natriumtic peptides such as smooth muscle relaxation and Na+ transport inhibition are associated with elevated cGMP levels, GC-A and GC-B are considered the biologically active natriuretic peptide receptors (reviewed in ref.16). GC-A and GC-B are differentially activated by natriuretic peptides: GC-A is activated by ANP efficiently and by BNP -lo-fold less weLl(9), whereas GC-B is selectively stimulated by CNP (11). Identification of the tissues which express the GC-A and GC-B genes is essential to the understanding of the physiological actions of both the natriuretic peptides and their receptors. Abbreviations: ANP, atrial natriuretic peptide; BNP, brain natriumtic peptide; bp, base pairs; CNP, C-type natriuretic peptide; GC-A, guanylate cyclase-coupled natriuretic peptide receptor-A, GC-B, guanylate cyclase-coupled natriumtic peptide receptor-B; RT-PCR, reverse transcriptionpolymerase chain reaction. 0006-291X/92 Copyright
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Initial studies using Northern blot analyses localized GC-A mRNA to kidney, adrenal, ileum, and adipose tissue (8), and GC-B mRNA to brain, kidney, and lung (9). Based on an extensive study of the tissue distribution of both GC-A and GC-B mRNAs using in situ hybridization in rhesus monkey (12), it was proposed that GC-A is involved in both the peripheral and central control of body fluid homeostasis whereas GC-B’s role is predominantly central. To determine the tissue localization of GC-A and GC-B with a more sensitive technique, we have used reverse transcription-polymerase chain reaction (RT-PCR) to assessthe distribution of GC-A and GC-B mRNAs in a wide variety of rat tissues including regions of the brain and kidney. The presence of both GC-A and GC-B mRNAs in all peripheral and neural tissues examined suggests that there is no qualitative difference in the distribution of GC-A and GC-B in the rat.
MATERIALS
AND METHODS
Total RNA from tissues of adult male Wistar rats (Charles River) was extracted in guanidine thiocyanate, purified on CsCl gradients, and extracted with phenol:chloroform:isoamyl alcohol (25:24: 1, ref. 13). Using 20 pg total RNA, single-stranded cDNA was synthesized at 42 C for 1 h with 0.5 pg oligo(dT) adapter (5’-GACTCGAGTCGACATCGAe-3’) and 200 units SuperScript reverse transcriptase (GIBCO BRL) in the recommended buffer (20 p.l). Each PCR contained 15% of the denatured (10 mm at 50C) and neutralized cDNA reaction (ref. 14) and 1 pg of each primer in 100 pl Tq buffer (33.5 mM Tris-HCI pH 8.8,3.35 mM MgC12,8.3 mM (NH4)2SO4,3.4 @I EDTA, 4.95 mM Bmercaptoethanol, 0.75 mM dNTPs, 85 &ml bovine serum albumin). The PCR was initiated at 96 C for 5 min, 60 C for 1.5 min, and 72 C for 1.5 min in a temperature cycler (Ericomp) before enzyme addition (2.5 units Taq DNA polymerase, Boehringer Mannheim), followed by amplification for 25 cycles at 93 C for 1 min, 60 C for 1.5 min, and 72 C for 2 min. Primers (Kronem Systems) used for PCR amplification of GC-A were 5’-GGATGCCTTCAGGAATCTGA-3’ (bases 959-978), and S-TGACACAGCCA’ITAGCTCCT-3’ (complementary to bases 1507-1526) (7); and those used for GC-B were 5’-AGCAACCTCAGTGTGCAACA-3’ (bases 652-671), and 5’-TGAAAGTCGCCAGACTCCAA-3’ (complementary to bases 1270-1289) (9). Negative controls included ribonuclease treatment of RNA prior to reverse transcription as well as amplification in the absence of template, Tq DNA polymerase, or one primer. Ampliied DNA (-5% of total) was size-fractionated by electrophoresis on agarose gels containing ethidium bromide. DNA amplified from whole kidney was cloned into pBluescript (Stratagene) and sequenced by the dideoxy-chain termination method with Sequenase 2.0 (United States Biochemical).
RESULTS To determine the tissue diitribution of guanylate cyclase-coupled natriutetic peptide receptors, we have used RT-PCR with RNA isolated from rat tissues to assessthe localization of GC-A and GC-B transcripts. To ensure that the GC-A PCR product was derived from mRNA and not contaminating genomic DNA, the amplified cDNA encompassed several exons (exons 2 to 6, ref. 15). An analogous region of the GC-B gene was chosen since the stmcture of the GC-B gene is not known, and homologous genes often have similar introonlexon organization (e.g.. ref. 16). Amplified DNA was initially characterized by gel electrophoresis and visualized with ethidium bromide. We detected DNA amplified with GC-A primers as well as GC-B primers in all tissues examined (Fig. 1A). DNA was amplified from cDNAs isolated from whole brain as well as from 611
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m RT-PCR Analysis of the TissueDistribution of GC-A and GC-B Transcripts. Amplified DNA and H&II-digested PhiX174marker DNA were size-fractionatedby electrophoresison 1% agarosegels and visualizedby ethidium bromide staining. A. DNA was amplified from first-strand cDNA synthesizedfrom rat tissuetotal RNA. B. First-strandcDNA synthesizedfrom rat brain total RNA was amplified in a completereaction (lanes11,12); or without Tuq DNA polymerase(lanes 1,2), 5’ primers (lanes3,4), 3’ primers (lanes5,6), or cDNA (lanes7.8); or with cDNA synthesizedfrom ribonuclease-treatedRNA (lanes9.10). GC-A (lanes 1,3,5,7,9,11) or GC-B primer(s) (lanes2,4,6,8,10,12) was present.Lane 13, HaeIII-digested PhiX174 marker DNA.
the cerebral cortex, cerebellum, and brainstem (Fig. 1A). GC-A and GC-B products were also amplified from many peripheral organs including the adrenal, aorta, kidney, liver, lung, pituitary, spleen, and testis, as well as from the three major regions of the kidney: cortex, medulla, and papilla (Fig.lA). The sizes of the amplified products, -575 bp for GC-A and -645 bp for GC-B (Fig. IA), agreed with the sizes predicted on the basis of nucleotide sequence analysis of the isolated GC-A (568 bp, ref.7) and GC-B cDNAs (638 bp, ref.9). This is the first demonstration of co-localization of these two receptors in a wide variety of peripheral and neural tissues. To eliminate the possibility that the RT-PCR amplification had generated artefactual products, cDNAs amplified in complete reactions (Fig.lB, lanes 11,12) were compared to those in incomplete reactions (Fig.lB, lanes l-10). Amplification was enzyme dependent since omission of Tuq DNA polymerase did not yield any PCR products (Fig. lB, lanes 1,2). Amplified GC-A and GC-B DNAs were not the result of misprinting by a single primer since both 5’- and 3’-primers were required (Fig. lB, lanes 3-6). In addition, the RT-PCR amplification was dependent on the presence of the cDNA template since omission of the cDNA or ribonuclease treatment of RNA prior to reverse transcription totally abolished PCR amplification (Fig. lB, lanes 7- 10). Thus, the amplified products could not be attributed to contaminating cDNA or genomic DNA. To prevent the detection of physiologically trival amounts of mRNA that can be detected with PCR (e.g., ref.17,18), we assessed the effect of the number of amplification cycles (5, 10, 15,20,25, 30) on the level of GC-A and GC-B amplified products. We used 25 amplification cycles in subsequent experiments, since at this number of cycles the limited quantities of liver GC-B product were visible and the amplification of the relatively abundant brain products had not plateaued (data not shown). 612
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GC-B GC-A
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&&&, Restriction Enyzme Mapping of RT-PCR AmpliEd Rat Brain GC-A and GC-B. DNA amplified from first-strand cDNA synthesized from tat brain total RNA with GC-A or GC-B primers was digested with EcoRI, SmaI, or StuI. Amplified DNA and HueIII-digested PhiX174 marker DNA were size-fractionated by electrophotesis on a 2% agarose gel and visualized by ethidium bromide staining.
Fkure 3, Autoradiograph of Rat Whole Kidney GC-A and GC-B Sequencing Gels. The cDNA sequences obtained correspond to 1480-1505 bp for GC-A (7) and 674-699 bp for GC-B (9).
Conlkrnation that the PCR products identified were indeed amplified from GC-A and GCB cDNAs was obtained by restriction enzyme digestion and nucleotide sequence analyses. The expected restriction fragments, based on the predicted nucleotide sequence encoding rat GC-A (7) and GC-B (9), were obtained from each tissue for the GC-A FCR product digested with EcoFU (341,227 bp), SmaI (568 bp), or StuI (398, 170 bp); as well as for the GC-B PCR product digested with EcoRI (638 bp), SmaI (373, 145, 120 bp), or StuI (352, 286 bp) (e.g., rat brain, Fig.2). Nucleotide sequence analysis of cloned amplified DNAs encoding GC-A and GC-B from kidney (Fig.3), and of uncloned GC-A PCR products from adrenal, brain, kidney, and liver (data not shown), directly confirmed their identity.
DISCUSSION
The widespread co-localization of GC-A and GC-B mRNA in many rat tissues including the brain, kidney, adrenal, aorta, liver, lung, pituitary, spleen, and testis has been demonstrated using RT-PCR. Transcripts for both receptors were also detected in regions of the rat brain including the cerebral cortex, cerebellum, and brainstem as well as in rat renal cortex, medulla, and papilla Restriction enzyme digestion and nucleotide sequence analyses confiied the identity of the amplified products. Our detection of GC-A transcripts in the cerebral cortex, cerebellum, and brainstem (Fig.lA) illustrates the widespread distribution of GC-A in the central nervous system. GC-A mRNA had previously been observed in whole brain of rats using Northern blot analysis and RT613
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PCR (9,19). Using in situ hybridization, however, GC-A mRNA was detected in rhesus monkey cerebellum but not in cerebral cortex (12). This discrepancy may be due to reduced detection sensitivity using in situ hybridization or to species variability of GC-A expression since the distribution of central nervous system ANP receptors exhibit species-related heterogeneity (reviewed in ref.20). Our observed distribution of GC-A mRNA (Fig. 1A) parallels that for rat brain ANP transcripts (21), further supporting a role for ANP acting as a neurotransmitter via GCA in many regions of the brain. Synaptic GC-A may also be activated by BNP, since BNP at lofold higher concentrations than ANP stimulates cGMP production by GC-B (9). The presence of GC-A mRNA in rat kidney provides further evidence of a renal mechanism for natriuretic peptides in controlling fluid and electrolyte homeostasis. Renal GC-A transcripts had previously been observed by Northern blot analysis in human and rat (8,9), as well as by PCR analysis in rat (22), and by in situ hybridization in rhesus monkey (12). Our detection of GC-A mRNA in all kidney regions, confirms the widespread distribution of GC-A mRNA as detected by RT-PCR throughout the kidney nephron (22) and suggests that ANP can control renal tubular function at multiple sites via its second messenger cGMP. In the renal cortex, ANP increases glomerular filtration rate and inhibits renin secretion from juxtaglomerular cells. As well, ANP is believed to inhibit net sodium and water reabsorption in cortical and medullary collecting tubules (reviewed in ref. 1). Our demonstration of GC-A transcripts in rat adrenal is consistent with the previous detection of GC-A mRNA in human adrenal using Northern blot analysis (8) and in rhesus monkey adrenal medulla and cortex using in situ hybridization ( 12). The presence of GC-A receptors with ANP-stimulated guanylate cyclase activity in the adrenal provides a mechanism for the inhibition of aldosterone synthesis by ANP. Although until recently cGMP was unable to account for this inhibition, cGMP analogues have now been shown to inhibit adrenocorticotropininduced aldosterone secretion by lowering CAMP levels via a cGMP-dependent increase in CAMP phosphodiesterase activity (23). We also detected GC-A mRNA in tissues additional to the brain, kidney, and adrenal: peripheral organs including aorta, liver, lung, pituitary, spleen, and testis. This extends the Northern blot analysis that demonstrated GC-A transcripts in rat lung as well as kidney (9). Northern blot analysis of human tissue had also detected GC-A transcripts in two peripheral tissues, ileum and adipose tissue (S), although in contrast to our results, none was detected in human spleen. As well, in situ hybridization studies in rhesus monkey did not identify GC-A receptors in most peripheral organs including aorta, liver, lung, spleen, and testis (12). Our observation of the widespread distribution of GC-A mRNA suggests that ANP acts on multiple organs in the periphery. At these sites, ANP may be acting as a cardiac hormone. Alternatively, ANP may be acting locally since expression of the ANP gene has been demonstrated in all tissues in which we detected GC-A transcripts (24-29). Although a predominantly central role for GC-B (11) had recently been strengthened by in situ hybridization analysis in rhesus monkey where GC-B transcripts were not detected in many peripheral tissues (12), our widespread co-localization of GC-B mRNA with GC-A mRNA suggests that GC-B also has a significant peripheral role (Fig.lA). Consistent with our results, mRNA encoding GC-B was recently detected using RT-PCR in human kidney (30). While this 614
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study was in progress, rat GC-B and a second form of this receptor with a 75 bp deletion were detected by RT-PCR in tissues additional to the brain and kidney and which included adrenal, aorta, heart, intestine, kidney, lung, pituitary, and testis. GC-B mRNA was not detected, however, in rat liver (3 1). Since the proposed ligand for GC-B, CNP (1 l), is undetectable in plasma (32), our demonstration of the widespread localization of GC-B mRNA in peripheral organs raises the possibility that CNP can act as a paracrine hormone. Indeed, rat pituitary, kidney, ileum, and colon were recently shown to contain CNP (32). Alternatively, since CNP mRNA was found predominantly in rat brain (33), another physiological ligand for GC-B may exist. Our detection of GC-A and GC-B mRNA in many rat tissues suggests that the actions of the natriuretic peptides occur at multiple sites. These receptors may act to mediate the hypotensive action of natriuretic peptides in the tissue vasculature. Alternatively, the widespread synthesis of ANP suggests that natriuretic peptides may have a unique paracrine role in each organ. In addition, the presence of GC-A and GC-B transcripts in the cerebral cortex, cerebellum, and brainstem raises the possibility that ANP, BNP, and CNP all act as neurotransmitters in many regions of the brain.
Acknowledgments; We thank J. Chan and S.B. Runciman for technical advice and assistance and J. Chan, C.J. Ingles, M.L. Moule, H. Sonnenberg, and A.T. Veress for critical review of the manuscript. This work was supported by Medical Research Council of Canada grants to CCY and to VMW, and by Heart and Stroke Foundation of Ontario grant to VMW.
REFERENCES 1. i: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Brenner, B.M., Ballermann, B.J., Gunning, M.E., and Zeidel, M.L. (1990) Physiol. Rev. 70, 665-699. Sudoh, T., Kangawa, K., Minamino, N., and Matsuo, H. (1988) Nature 332,78-81. Sudoh, T., Minamino, N., Kangawa, K., and Matsuo, H. (1990) B&hem. Biophys. Res. Commun. 168, 863-870. Garbers, D.L. (1992) Cell 71, l-4. Almeida, F.A., Suzuki, M., Scarborough, R.M., Lewicki, J.A., and Maack, T. (1989) Am. J. Physiol. 256, R469-R475. Maack, T. (1992) Annu. Rev. Physiol. 54, 11-27. Chinkers, M., Garbers, D.L., Chang, M.-S., Lowe, D.G., Chin, H., Goeddel, D.V., and Schulz, S. (1989) Nature 338,78-83. Lowe, D.G., Chang, M.-S., Hellmiss, R., Chen, E., Singh, S., Garbers, D.L., and Goeddel. D.V. (1989) EMBO J. 8.1377-1384. Schulz, S., Singh, S.; Belle& R.A:, Singh, G., Tubb, D.J., Chin, H., and Garbers, D.L. (1989) Cell 58, 1155-l 162. Chang, M.-S., Lowe, D.G., Lewis, M., Hellmiss, R., Chen, E., and Goeddel, D.V. (1989) Nature 341, 68-72. Koller, K.J., Lowe, D.G., Bennett, G.L., Minamino, N., Kangawa, K., Matsuo, H., and Goeddel, D.V. (1991) Science 252,120-123. Wilcox, J.N., Augustine, A., Goeddel, D.V., and Lowe, D.G. (1991) Mol. Cell. Biol. 11, 3454-3462. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Dunn, J.M., Phillips, R.A., Zhu, X., Becker, A., and Gallie, B.L. (1989) Mol. Cell. Biol. 9, 4596-4604. Yamaguchi, M., Rutledge, L.J., and Garbers, D.L. (1990) J. Biol. Chem. 265,204142042& Shier, P., and Watt, V.M. (1989) J. Biol. Chem. 264, 14605-14608. 615
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17. Chelly, J., Concordet, J.-P., Kaplan, J.-C., and Kahn, A. (1989) Proc. Natl. Acad. Sci. USA 86, 2617-2621. 18. Sarkar, G., and Sommer, S.S. (1989) Science 244,331-334. 19. Kutty, R.K., Fletcher, R.T., Chader, G.J., and Krishna, G. (1992) Biochem. Biophys. Res. Commun. 182, 851-857. 20. Stewart, R.E., Swithers, S.E., Plunkett, L.M., and McCarty, R. (1988) Neurosci. Biobehav. Rev. 12, 151-168. 21. Gardner, D.G., Vlasuk, G.P., Baxter, J.D., Fiddes, J.C., and Lewicki, J.A. (1987) Proc. Natl. Acad. Sci. USA 84,2175-2179. 22. Terada, Y., Moriyama, T., Martin, B.M., Knepper, M.A., and Garcia-Perez, A. (1991) Am. J. Physiol. 261, F1080-F1087. 23. MacFarland, R.T., Zelus, B.D., and Beavo, J.A. (1991) J. Biol. Chem. 266, 136-142. 24. Gardner, D.G., Deschepper, C.F., Ganong, W.F., Hane, S., Fiddes, J., Baxter, J.D., and Lewicki, J. (1986) Proc. Natl. Acad. Sci. USA 83,6697-6701. 25. Morel, G., Chabot, J.-G., Garcia-Caballero, T., Gossard, F., Dihl, F., Belles-Isles, M., and Heisler, S. (1988) Endocrinology 123, 149-158. Ritter, D., Needleman, P., and Greenwald, J.E. (1990) J. Clin. Invest. 87, 208-212. 2 Throsby, M., Lee, D., Huang, W., Yang, Z., Copolov, D.L., and Lim, A.T. (199 1) Endocrinology 129,991-1000. Vollmar, A.M., Friedrich, A., and Schulz, R. (1990) J. Androl. 11,471-475. 2’;. Vesely, D.L., Palmer, P.A., and Giordano, A.T. (1992) Peptides 13, 165-170. 30: Canaan-Kuhl, S., Jamison, R.L., Myers, B.D., and Pratt, R.E. (1992) Endocrinology 130, 550-552. 31. Ohyama, Y., Miyamoto, K., Saito, Y., Minamino, N., Kangawa, K., and Matsuo, H. (1992) Biochem. Biophys. Res. Commun. 183,743-749. 32. Komatsu, Y., Nakao, K., Suga, S.-I., Ogawa, Y., Mukoyama, M., Arai, H., Shirakami, G., Hosoda, K., Nakagawa, O., Hama, N., Kishimoto, I., and Imura, H. (1991) Endocrinology 129,1104-l 106. 33. Kojima, M., Minamino, N., Kangawa, K., and Matsuo, H. (1990) FEBS Lett. 276, 209213.
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WIDESPREAD CO-LOCALIZATION OF mRNAS ENCODING THE GUANYLATE CYCLASBCOUPLED NATRIURETIC PEPTIDE RECEPTORS IN RAT TISSUES Teresa Tallerico-Melnyk*+, Cecil C. Yip+, and Valerie M. Watt* * Department of Physiology and+Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5S lA8 Received
October
22,
1992
Natriumtic peptides modulate vasorelaxation, diuresis, and natriurcsis through the stimulation of cGMP production by the guanylate cyclase-coupled natriumtic peptide receptors, GC-A and GC-B. We used reverse transcription-polymerase chain reaction to determine the distribution of mRNA encoding both receptors in rat tissues. GC-A and GC-B transcripts were detected in all peripheral and neural tissuesexamined. Since the atrial natriuretic peptide gene is expressed in all these tissues, our widespread detection of GC-A and GC-B mRNAs now suggests that natriuretic peptides may act as endocrine and paracrine hormones as well as neurotransmitters via both GC-A and GC-B receptors. 0 1992 Academic Press, Inc.
Atrial natriuretic peptide (ANP), secreted primarily by cardiac atria, reduces systemic blood pressure and intravascular volume (reviewed in ref. 1). ANP is the most studied member of a family of related peptides which also includes brain natriuretic peptide or BNP (2) and C-type natriutetic peptide or CNP (3). Both BNP and CNP also have vasorelaxant, diuretic, and natriumtic effects (2,3). Two functionally and structurally distinct types of receptors for natriuretic peptides have been identified (reviewed in ref.4). An -65~kDa clearance receptor is likely to play au important role in regulating plasma ANP levels (5) but does not mediate the known physiologic actions of ANP (reviewed in ref.@. In contrast, two - 130-kDa receptors, guanylate cyclase-A (GC-A) or ANP-A (7,8) and guanylate cyclase-B (GC-B) or ANP-B (9,10), contain an intracellular guanylate cyclase catalytic domain activated by natriuretic peptides. Since many physiological actions of natriumtic peptides such as smooth muscle relaxation and Na+ transport inhibition are associated with elevated cGMP levels, GC-A and GC-B are considered the biologically active natriuretic peptide receptors (reviewed in ref.16). GC-A and GC-B are differentially activated by natriuretic peptides: GC-A is activated by ANP efficiently and by BNP -lo-fold less weLl(9), whereas GC-B is selectively stimulated by CNP (11). Identification of the tissues which express the GC-A and GC-B genes is essential to the understanding of the physiological actions of both the natriuretic peptides and their receptors. Abbreviations: ANP, atrial natriuretic peptide; BNP, brain natriumtic peptide; bp, base pairs; CNP, C-type natriuretic peptide; GC-A, guanylate cyclase-coupled natriuretic peptide receptor-A, GC-B, guanylate cyclase-coupled natriumtic peptide receptor-B; RT-PCR, reverse transcriptionpolymerase chain reaction. 0006-291X/92 Copyright
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Initial studies using Northern blot analyses localized GC-A mRNA to kidney, adrenal, ileum, and adipose tissue (8), and GC-B mRNA to brain, kidney, and lung (9). Based on an extensive study of the tissue distribution of both GC-A and GC-B mRNAs using in situ hybridization in rhesus monkey (12), it was proposed that GC-A is involved in both the peripheral and central control of body fluid homeostasis whereas GC-B’s role is predominantly central. To determine the tissue localization of GC-A and GC-B with a more sensitive technique, we have used reverse transcription-polymerase chain reaction (RT-PCR) to assessthe distribution of GC-A and GC-B mRNAs in a wide variety of rat tissues including regions of the brain and kidney. The presence of both GC-A and GC-B mRNAs in all peripheral and neural tissues examined suggests that there is no qualitative difference in the distribution of GC-A and GC-B in the rat.
MATERIALS
AND METHODS
Total RNA from tissues of adult male Wistar rats (Charles River) was extracted in guanidine thiocyanate, purified on CsCl gradients, and extracted with phenol:chloroform:isoamyl alcohol (25:24: 1, ref. 13). Using 20 pg total RNA, single-stranded cDNA was synthesized at 42 C for 1 h with 0.5 pg oligo(dT) adapter (5’-GACTCGAGTCGACATCGAe-3’) and 200 units SuperScript reverse transcriptase (GIBCO BRL) in the recommended buffer (20 p.l). Each PCR contained 15% of the denatured (10 mm at 50C) and neutralized cDNA reaction (ref. 14) and 1 pg of each primer in 100 pl Tq buffer (33.5 mM Tris-HCI pH 8.8,3.35 mM MgC12,8.3 mM (NH4)2SO4,3.4 @I EDTA, 4.95 mM Bmercaptoethanol, 0.75 mM dNTPs, 85 &ml bovine serum albumin). The PCR was initiated at 96 C for 5 min, 60 C for 1.5 min, and 72 C for 1.5 min in a temperature cycler (Ericomp) before enzyme addition (2.5 units Taq DNA polymerase, Boehringer Mannheim), followed by amplification for 25 cycles at 93 C for 1 min, 60 C for 1.5 min, and 72 C for 2 min. Primers (Kronem Systems) used for PCR amplification of GC-A were 5’-GGATGCCTTCAGGAATCTGA-3’ (bases 959-978), and S-TGACACAGCCA’ITAGCTCCT-3’ (complementary to bases 1507-1526) (7); and those used for GC-B were 5’-AGCAACCTCAGTGTGCAACA-3’ (bases 652-671), and 5’-TGAAAGTCGCCAGACTCCAA-3’ (complementary to bases 1270-1289) (9). Negative controls included ribonuclease treatment of RNA prior to reverse transcription as well as amplification in the absence of template, Tq DNA polymerase, or one primer. Ampliied DNA (-5% of total) was size-fractionated by electrophoresis on agarose gels containing ethidium bromide. DNA amplified from whole kidney was cloned into pBluescript (Stratagene) and sequenced by the dideoxy-chain termination method with Sequenase 2.0 (United States Biochemical).
RESULTS To determine the tissue diitribution of guanylate cyclase-coupled natriutetic peptide receptors, we have used RT-PCR with RNA isolated from rat tissues to assessthe localization of GC-A and GC-B transcripts. To ensure that the GC-A PCR product was derived from mRNA and not contaminating genomic DNA, the amplified cDNA encompassed several exons (exons 2 to 6, ref. 15). An analogous region of the GC-B gene was chosen since the stmcture of the GC-B gene is not known, and homologous genes often have similar introonlexon organization (e.g.. ref. 16). Amplified DNA was initially characterized by gel electrophoresis and visualized with ethidium bromide. We detected DNA amplified with GC-A primers as well as GC-B primers in all tissues examined (Fig. 1A). DNA was amplified from cDNAs isolated from whole brain as well as from 611
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m RT-PCR Analysis of the TissueDistribution of GC-A and GC-B Transcripts. Amplified DNA and H&II-digested PhiX174marker DNA were size-fractionatedby electrophoresison 1% agarosegels and visualizedby ethidium bromide staining. A. DNA was amplified from first-strand cDNA synthesizedfrom rat tissuetotal RNA. B. First-strandcDNA synthesizedfrom rat brain total RNA was amplified in a completereaction (lanes11,12); or without Tuq DNA polymerase(lanes 1,2), 5’ primers (lanes3,4), 3’ primers (lanes5,6), or cDNA (lanes7.8); or with cDNA synthesizedfrom ribonuclease-treatedRNA (lanes9.10). GC-A (lanes 1,3,5,7,9,11) or GC-B primer(s) (lanes2,4,6,8,10,12) was present.Lane 13, HaeIII-digested PhiX174 marker DNA.
the cerebral cortex, cerebellum, and brainstem (Fig. 1A). GC-A and GC-B products were also amplified from many peripheral organs including the adrenal, aorta, kidney, liver, lung, pituitary, spleen, and testis, as well as from the three major regions of the kidney: cortex, medulla, and papilla (Fig.lA). The sizes of the amplified products, -575 bp for GC-A and -645 bp for GC-B (Fig. IA), agreed with the sizes predicted on the basis of nucleotide sequence analysis of the isolated GC-A (568 bp, ref.7) and GC-B cDNAs (638 bp, ref.9). This is the first demonstration of co-localization of these two receptors in a wide variety of peripheral and neural tissues. To eliminate the possibility that the RT-PCR amplification had generated artefactual products, cDNAs amplified in complete reactions (Fig.lB, lanes 11,12) were compared to those in incomplete reactions (Fig.lB, lanes l-10). Amplification was enzyme dependent since omission of Tuq DNA polymerase did not yield any PCR products (Fig. lB, lanes 1,2). Amplified GC-A and GC-B DNAs were not the result of misprinting by a single primer since both 5’- and 3’-primers were required (Fig. lB, lanes 3-6). In addition, the RT-PCR amplification was dependent on the presence of the cDNA template since omission of the cDNA or ribonuclease treatment of RNA prior to reverse transcription totally abolished PCR amplification (Fig. lB, lanes 7- 10). Thus, the amplified products could not be attributed to contaminating cDNA or genomic DNA. To prevent the detection of physiologically trival amounts of mRNA that can be detected with PCR (e.g., ref.17,18), we assessed the effect of the number of amplification cycles (5, 10, 15,20,25, 30) on the level of GC-A and GC-B amplified products. We used 25 amplification cycles in subsequent experiments, since at this number of cycles the limited quantities of liver GC-B product were visible and the amplification of the relatively abundant brain products had not plateaued (data not shown). 612
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GC-B GC-A
I GATC’ -
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&&&, Restriction Enyzme Mapping of RT-PCR AmpliEd Rat Brain GC-A and GC-B. DNA amplified from first-strand cDNA synthesized from tat brain total RNA with GC-A or GC-B primers was digested with EcoRI, SmaI, or StuI. Amplified DNA and HueIII-digested PhiX174 marker DNA were size-fractionated by electrophotesis on a 2% agarose gel and visualized by ethidium bromide staining.
Fkure 3, Autoradiograph of Rat Whole Kidney GC-A and GC-B Sequencing Gels. The cDNA sequences obtained correspond to 1480-1505 bp for GC-A (7) and 674-699 bp for GC-B (9).
Conlkrnation that the PCR products identified were indeed amplified from GC-A and GCB cDNAs was obtained by restriction enzyme digestion and nucleotide sequence analyses. The expected restriction fragments, based on the predicted nucleotide sequence encoding rat GC-A (7) and GC-B (9), were obtained from each tissue for the GC-A FCR product digested with EcoFU (341,227 bp), SmaI (568 bp), or StuI (398, 170 bp); as well as for the GC-B PCR product digested with EcoRI (638 bp), SmaI (373, 145, 120 bp), or StuI (352, 286 bp) (e.g., rat brain, Fig.2). Nucleotide sequence analysis of cloned amplified DNAs encoding GC-A and GC-B from kidney (Fig.3), and of uncloned GC-A PCR products from adrenal, brain, kidney, and liver (data not shown), directly confirmed their identity.
DISCUSSION
The widespread co-localization of GC-A and GC-B mRNA in many rat tissues including the brain, kidney, adrenal, aorta, liver, lung, pituitary, spleen, and testis has been demonstrated using RT-PCR. Transcripts for both receptors were also detected in regions of the rat brain including the cerebral cortex, cerebellum, and brainstem as well as in rat renal cortex, medulla, and papilla Restriction enzyme digestion and nucleotide sequence analyses confiied the identity of the amplified products. Our detection of GC-A transcripts in the cerebral cortex, cerebellum, and brainstem (Fig.lA) illustrates the widespread distribution of GC-A in the central nervous system. GC-A mRNA had previously been observed in whole brain of rats using Northern blot analysis and RT613
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PCR (9,19). Using in situ hybridization, however, GC-A mRNA was detected in rhesus monkey cerebellum but not in cerebral cortex (12). This discrepancy may be due to reduced detection sensitivity using in situ hybridization or to species variability of GC-A expression since the distribution of central nervous system ANP receptors exhibit species-related heterogeneity (reviewed in ref.20). Our observed distribution of GC-A mRNA (Fig. 1A) parallels that for rat brain ANP transcripts (21), further supporting a role for ANP acting as a neurotransmitter via GCA in many regions of the brain. Synaptic GC-A may also be activated by BNP, since BNP at lofold higher concentrations than ANP stimulates cGMP production by GC-B (9). The presence of GC-A mRNA in rat kidney provides further evidence of a renal mechanism for natriuretic peptides in controlling fluid and electrolyte homeostasis. Renal GC-A transcripts had previously been observed by Northern blot analysis in human and rat (8,9), as well as by PCR analysis in rat (22), and by in situ hybridization in rhesus monkey (12). Our detection of GC-A mRNA in all kidney regions, confirms the widespread distribution of GC-A mRNA as detected by RT-PCR throughout the kidney nephron (22) and suggests that ANP can control renal tubular function at multiple sites via its second messenger cGMP. In the renal cortex, ANP increases glomerular filtration rate and inhibits renin secretion from juxtaglomerular cells. As well, ANP is believed to inhibit net sodium and water reabsorption in cortical and medullary collecting tubules (reviewed in ref. 1). Our demonstration of GC-A transcripts in rat adrenal is consistent with the previous detection of GC-A mRNA in human adrenal using Northern blot analysis (8) and in rhesus monkey adrenal medulla and cortex using in situ hybridization ( 12). The presence of GC-A receptors with ANP-stimulated guanylate cyclase activity in the adrenal provides a mechanism for the inhibition of aldosterone synthesis by ANP. Although until recently cGMP was unable to account for this inhibition, cGMP analogues have now been shown to inhibit adrenocorticotropininduced aldosterone secretion by lowering CAMP levels via a cGMP-dependent increase in CAMP phosphodiesterase activity (23). We also detected GC-A mRNA in tissues additional to the brain, kidney, and adrenal: peripheral organs including aorta, liver, lung, pituitary, spleen, and testis. This extends the Northern blot analysis that demonstrated GC-A transcripts in rat lung as well as kidney (9). Northern blot analysis of human tissue had also detected GC-A transcripts in two peripheral tissues, ileum and adipose tissue (S), although in contrast to our results, none was detected in human spleen. As well, in situ hybridization studies in rhesus monkey did not identify GC-A receptors in most peripheral organs including aorta, liver, lung, spleen, and testis (12). Our observation of the widespread distribution of GC-A mRNA suggests that ANP acts on multiple organs in the periphery. At these sites, ANP may be acting as a cardiac hormone. Alternatively, ANP may be acting locally since expression of the ANP gene has been demonstrated in all tissues in which we detected GC-A transcripts (24-29). Although a predominantly central role for GC-B (11) had recently been strengthened by in situ hybridization analysis in rhesus monkey where GC-B transcripts were not detected in many peripheral tissues (12), our widespread co-localization of GC-B mRNA with GC-A mRNA suggests that GC-B also has a significant peripheral role (Fig.lA). Consistent with our results, mRNA encoding GC-B was recently detected using RT-PCR in human kidney (30). While this 614
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study was in progress, rat GC-B and a second form of this receptor with a 75 bp deletion were detected by RT-PCR in tissues additional to the brain and kidney and which included adrenal, aorta, heart, intestine, kidney, lung, pituitary, and testis. GC-B mRNA was not detected, however, in rat liver (3 1). Since the proposed ligand for GC-B, CNP (1 l), is undetectable in plasma (32), our demonstration of the widespread localization of GC-B mRNA in peripheral organs raises the possibility that CNP can act as a paracrine hormone. Indeed, rat pituitary, kidney, ileum, and colon were recently shown to contain CNP (32). Alternatively, since CNP mRNA was found predominantly in rat brain (33), another physiological ligand for GC-B may exist. Our detection of GC-A and GC-B mRNA in many rat tissues suggests that the actions of the natriuretic peptides occur at multiple sites. These receptors may act to mediate the hypotensive action of natriuretic peptides in the tissue vasculature. Alternatively, the widespread synthesis of ANP suggests that natriuretic peptides may have a unique paracrine role in each organ. In addition, the presence of GC-A and GC-B transcripts in the cerebral cortex, cerebellum, and brainstem raises the possibility that ANP, BNP, and CNP all act as neurotransmitters in many regions of the brain.
Acknowledgments; We thank J. Chan and S.B. Runciman for technical advice and assistance and J. Chan, C.J. Ingles, M.L. Moule, H. Sonnenberg, and A.T. Veress for critical review of the manuscript. This work was supported by Medical Research Council of Canada grants to CCY and to VMW, and by Heart and Stroke Foundation of Ontario grant to VMW.
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