Ligand-gated purinergic receptors are differentially expressed in the adult rat vestibular periphery.

To further characterize the pattern of expression of the ligand-gated purinergic P2X receptors in the peripheral vestibular system, we conducted reverse transcription-polymerase chain reaction amplification of P2X1 and P2X2 messenger RNA extracted from adult rat vestibular ganglia (Scarpa's ganglia) and vestibular end organs. Transcripts encoding P2X1 were found in both Scarpa's ganglia and the end organs, but transcripts encoding P2X2 were found only in the vestibular end organs. These results support previous electrophysiological data, and they provide a more complete understanding of the specific role of purinergic (adenosine-5'-triphosphate) transmission in the vestibular periphery.

Adenylyl cyclase isoforms in the vestibular periphery of the rat.

The expression of adenylyl cyclase (AC) isoforms in the adult rat vestibular periphery was investigated using reverse transcription polymerase chain reaction (RT-PCR). AC II, IV and V mRNAs were expressed in both Scarpa's ganglion and vestibular end organs. In addition, in the vestibular end organs, an AC mRNA not previously reported in the rat was identified. The cloned sequence (GenBank accession no. AF184150) represented 95 amino acids with 100% similarity to the human AC VII and 94% to the bovine AC VII. AC VII mRNA also was found in the cerebellum but was undetectable in heart, kidney, liver and Scarpa's ganglion.

Partial cDNAs encoding G-protein alpha subunits in the rat vestibular periphery.

To begin understanding what G-proteins are involved in signal transduction in the vestibular periphery, the expression of Galpha subunits in rat primary afferent neurons (Scarpa's ganglia) and end-organs was studied. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers corresponding to two conserved regions of the Galpha protein coding sequence produced partial cDNAs encoding two distinct forms of Galpha(s) subunit (Galpha(s2) and anove) Galpha(s2) subunit,GenBank accession number AF1841510); and two forms of Galpha(i2) subunits. A novel truncated form of Galpha(i2) (designated Galpha(i2(vest)),Gen Bank accession number AF189020) was detected in the vestibular periphery. Galpha(i2(vest)) was also expressed in rat cerebellum and heart. The possible role of the identified Galpha protein cDNAs in the function of the vestibular periphery is discussed.

Aminopeptidase-A. II. Genomic cloning and characterization of the rat promoter.

Aminopeptidase-A (APA) has a widespread tissue distribution consistent with a role in the metabolism of circulating or locally produced ANG II or CCK-8. APA is also highly expressed in pre-B lymphocytes, but its role in lymphoid cell development is unknown. To begin to understand the basis for cell-specific regulation of APA expression, we sought to clone and characterize the rat gene promoter. Screening of a rat genomic library with a partial rat APA cDNA resulted in isolation of a 12-kb clone found to contain the first exon and >3 kb of 5'-flanking sequence. Primer extension of rat kidney mRNA indicated that the major transcription start site was 312 bp upstream of the translation start codon and 22 bp downstream from a TATA box. Constructs containing portions of the 5'-flanking region placed upstream of a chloramphenicol acetyltransferase reporter gene indicated that expression was cell specific and that high activity could be obtained with constructs containing as little as 110 bp of 5'-flanking region sequence. We further identified an upstream regulatory element between -1063 and -348 that suppressed transcription in a cell-specific manner. This element (termed upstream suppressor of APA, or USA) also suppressed transcription of a heterologous promoter. These results indicate that the organization and regulation of the rat APA is not consistent with it being a housekeeping gene and further suggest that rat APA gene transcription might be regulated through the presence of a novel strong upstream suppressor element.

Aminopeptidase-A. I. CDNA cloning and expression and localization in rat tissues.

Aminopeptidase-A (APA) is an ectoenzyme that selectively hydrolyzes acidic residues from the amino terminus of oligopeptides, including biologically active [Asp(1)]ANG II and [Asp(1)]CCK-8. We sought to characterize rat APA by cDNA cloning and expression and to determine its tissue distribution by in situ hybridization and immunohistochemistry. Sequence analysis of overlapping cDNA clones isolated from rat kidney cDNA libraries indicated that the full-length cDNA encoded a 945-amino acid protein with a predicted molecular mass of 108 kDa; the size was confirmed by in vitro translation of a full-length cDNA construct. Transient transfection of the full-length cDNA construct in mammalian cells yielded a protein approximately 140 kDa in size, a size that agrees with the immunoblots of APA from rat tissue and is consistent with APA being known as a glycosylated protein. Tissue APA activity and mRNA expression were highest in the kidney and ileum. Localization of APA by in situ hybridization and immunohistochemistry indicated that, with the exception of the kidney and ileum, where APA was localized to the luminal brush border of proximal tubules and enterocytes, respectively, APA was associated with either capillaries or the lining of sinusoids. Areas known to be physiological targets for ANG II, including glomeruli, the zona glomerulosa, and anterior pituitary, had high levels of APA. The localization pattern suggests that APA may subserve multiple functions, i.e., a generalized role in peptide scavenging and perhaps a more specific role in metabolism of circulating or locally produced ANG II or CCK-8.

Evidence for three additional P2X2 purinoceptor isoforms produced by alternative splicing in the adult rat vestibular end-organs.

P2X2 receptors are ligand-gated ion channels that are activated by extracellular ATP. To characterize the expression of P2X2 purinoceptor in the adult rat vestibular periphery, reverse transcription-polymerase chain reaction (RT-PCR) was used. No transcript for P2X2 receptor was found in the vestibular primary afferent neurons (Scarpa's ganglia); however, partial cDNAs encoding four splice variants of the P2X2 receptor were isolated from vestibular end-organs. In all four cDNAs, the deletions were of different lengths but started at the same position on the P2X2 gene (Val-370 codon) located toward the intracellular carboxyl terminus. One of these receptor isoforms was identical in sequence to the recently published P2X2(b) receptor (Simon et al., 1997, Mol. Pharmacol. 52, 237-248) (also known as P2X2-2, in the nomenclature of Brändle et al., 1997, FEBS Lett. 404, 294-298). The remaining three novel splice variants of the P2X2 receptor were designated P2X2(e), P2X2(f) and P2X2(g) (GenBank accession numbers AF028603, AF028604 and AF028605, respectively). The functional significance of these three splice variants remains to be determined. Pituitary and cerebellum were used as survey tissues and only the P2X2(b) receptor cDNA was found.

Differential expression of alpha2-7, alpha9 and beta2-4 nicotinic acetylcholine receptor subunit mRNA in the vestibular end-organs and Scarpa's ganglia of the rat.

To further characterize the pattern of expression of the nicotinic acetylcholine receptor (nAChR) subunits in the peripheral vestibular system, we conducted RT-PCR of all known mammalian nAChR alpha and beta subunits in mRNA extracted from adult rat vestibular primary afferent neurons (Scarpa's ganglia) and vestibular end-organs. Transcripts encoding the alpha2-7 and beta2-4 nAChR subunits were found in the vestibular ganglia, while alpha3, alpha5-7, alpha9 and beta2-4 nAChR subunits were expressed in the vestibular end-organs. These results support previous electrophysiological, immunocytochemical and molecular biological data, and also provide a more complete understanding of the role of nAChRs in the neurochemical transmission subserving the efferent-afferent interaction in the vestibular periphery.

Expression of aminopeptidase A, an angiotensinase, in glomerular mesangial cells.

Glomerular mesangial cells are known to express angiotensin II type 1 receptors and contract in response to circulating and/or locally produced angiotensin II. In addition, stimulation of mesangial cell matrix protein synthesis by elevated levels of angiotensin II is known to contribute to the development of glomerulosclerosis. Previously, we reported that mesangial cells were positively immunostained with antiserum directed against aminopeptidase A, the principal angiotensinase in the metabolism of angiotensin II. Here we demonstrate directly that aminopeptidase A is expressed in mesangial cells cultured from rat kidney. First, cultured mesangial cells had measurable aminopeptidase A enzymatic activity. Second, immunoblots for aminopeptidase A were positive for isolated glomeruli and mesangial cells, although two bands were seen for mesangial cells (approximately 138 and 144 kD), and only the larger band was seen for isolated glomeruli and kidney. Third, Northern blot hybridizations of total RNA from mesangial cells or kidney were positive and labeled similarly sized bands. Fourth, reverse transcription-polymerase chain reaction amplification of mesangial cell total RNA yielded a partial cDNA of the expected size that was confirmed by sequencing to be identical to rat kidney aminopeptidase A. These results indicate that aminopeptidase A is expressed within mesangial cells. These results further suggest that metabolism of angiotensin II by aminopeptidase A could play a protective role in minimizing the adverse effects of angiotensin II stimulation of mesangial cells.

Localization of angiotensin II type 1 receptor subtype mRNA in rat kidney.

The physiological effects of angiotensin II (ANG II) on the kidney are mediated primarily by the ANG II type 1 (AT1) receptor. Two highly similar AT1 receptor subtypes have been identified in the rat by molecular cloning techniques, namely AT1A and AT1B. The intrarenal localization of the AT1A and AT1B receptor subtypes has not been studied by hybridization methods with subtype-specific receptor probes. Using radiolabeled probes from the 3' noncoding region of the AT1A and AT1B cDNAs, we localized AT1 mRNA in rat kidney by in situ hybridization. Specificity of the 3' noncoding region probes was tested by Northern blot and solution hybridization methods. AT1A mRNA levels were highest in the liver, kidney, and adrenal. In contrast, AT1B mRNA levels were highest in the adrenal and pituitary and low in kidney. Autoradiographic localization of 125I-[Sar1,Ile8]ANG II binding indicated that the highest levels of AT1 receptors were found in glomeruli and vascular elements. In situ hybridization with a nonselective AT1 receptor riboprobe indicated that the highest levels of AT1 mRNA were in the outer medullary vasa recta and cortical glomeruli with additional diffuse labeling of the cortex and outer medulla, consistent with labeling of tubular elements. In contrast, in situ hybridization with the AT1 subtype selective probes revealed that AT1A receptor mRNA was primarily localized to the vasa recta and diffusely to the outer stripe of the outer medulla and the renal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization.

Glutamyl aminopeptidase [aminopeptidase A (EAP), EC 3.4.11.7] is an ectoenzyme that selectively hydrolyzes acidic amino acid residues from the amino terminus of oligopeptides. EAP activity is highest within the kidney and small intestine. The murine pre-B cell BP-1/6C3 and the human kidney glycoprotein gp160 differentiation antigens have been reported to have biochemical properties indistinguishable from EAP. It is not known, however, if rat kidney EAP is a homologue of these antigens or molecularly distinct. Using the reverse transcription-polymerase chain reaction method with oligonucleotide primers based on the BP-1/6C3 nucleotide sequence, we isolated a 450-bp partial cDNA from rat kidney poly(A)+ RNA. The partial cDNA encoded a predicted protein that was 92% and 86% identical to the murine BP-1/6C3 and human gp160 antigens, respectively; the amino acid sequence within the zinc-binding domain was completely conserved. Purification of EAP from rat kidney and microsequence analysis of a tryptic digest peptide fragment (18-mer) indicated that the fragment was highly similar to a region within the BP-1/6C3 and gp160 proteins. Northern blot hybridization and immunoblot analyses were also consistent with labeling of products the same size as reported for the BP-1/6C3 and gp160 antigens. There was a good correlation between the cellular distribution of EAP mRNA and EAP immunoreactivity, with proximal tubules and glomerular mesangial cells having the highest densities. These results indicate that rat kidney EAP is a species homologue of the murine BP-1/6C3 and human gp160 antigens. Furthermore, on the basis of its cellular localization, rat kidney EAP is likely to be involved in degradation of oligopeptides within the glomerulus and the glomerular filtrate. Since cells that express EAP also express receptors for angiotensin II, an intrarenal vasoactive hormone that is a substrate for EAP, these results further suggest that EAP may play a role in modulating the activity of intrarenal angiotensin II.