A quantitative analysis of purinoceptor expression in the bladders of patients with symptomatic outlet obstruction.

OBJECTIVE: To compare the expression of the seven known P2X receptors in human bladder from male patients with detrusor instability caused by symptomatic bladder outlet obstruction with that from control bladders, using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. PATIENTS AND METHODS: Real-time quantitative RT-PCR provides a system for detecting and analysing RNA. Bladder biopsies were obtained from nine patients undergoing prostate surgery and control biopsies were obtained from eight age-matched men undergoing routine bladder endoscopy studies, and who were asymptomatic. Total RNA was extracted from each sample and 10 ng of this used for individual PCR reactions. The expression levels of the seven P2X genes in the total RNA were then determined. RESULTS: In the control bladder, P2X1 was by far the predominant purinergic receptor at the RNA level, the remainder consistently present in the order P2X1 >> P2X4 > P2X2 > P2X7 > P2X5 >> P2X3 = P2X6 = 0. Calponin, a smooth muscle-specific protein, was used as a marker for smooth muscle content. In bladder from symptomatic patients, the P2X1/calponin ratio was greater than that in controls (P = 0.016). There appeared to be no difference in P2X2, but P2X4, P2X5, and P2X7 were all greater in the symptomatic bladder than in the controls, although these differences were not significant. CONCLUSION: P2X1 is the predominant purinoceptor subtype in the human male bladder, consistent with pharmacological evidence. The amount of P2X1 receptor per smooth muscle cell is greater in the obstructed than in control bladder, suggesting an increase in purinergic function in the unstable bladder arising from bladder outlet obstruction.

A quantitative analysis of purinoceptor expression in human fetal and adult bladders.

PURPOSE: In adults there is evidence that adenosine triphosphate acting at P2X receptors functions as a co-transmitter at vesical smooth muscle. The contractile mechanisms of human fetal bladder have been studied to a limited extent and it remains undetermined whether P2X receptors contribute. We compared the expression of the 7 known P2X receptors in fetal and adult human bladders using a quantitative polymerase chain reaction (PCR) based method. MATERIALS AND METHODS: Real-time quantitative reverse transcriptase-PCR provides a system for the detection and analysis of RNA. Four complete cadaver fetal bladders were obtained at 16 weeks to full-term gestation and divided into a total of 12 segments. Adult bladder samples were obtained from 4 patients requiring bladder biopsy. Total RNA was extracted from each sample and 10 ng. were used for individual PCR reactions. An ABI 7700 machine (PE Applied Biosystems, California) determined expression levels of the 7 P2X genes in total RNA. RESULTS: In adult bladders P2X1 was by far the predominant purinergic receptor at the messenger RNA level. The remaining purinergic receptors were consistently present in the order P2X1 >> P2X4 > P2X7 >> P2X5 > P2X2 >> P2X3 = P2X6 = 0. In fetal bladders the expression of P2X1 transcripts was much lower than in adult bladders, and P2X4 and P2X7 were also present. The rank order of the P2X transcript level was P2X1 = P2X4 > P2X7 >> P2X5 >> P2X2 >> P2X3 = P2X6 = 0. With increasing gestation the P2X receptor transcript level (expression) shifted from the dome to the body of the bladder. CONCLUSIONS: P2X1 is the predominant purinoceptor subtype in adult human bladders, consistent with pharmacological evidence. The fetal expression of all P2X receptor transcripts is much lower than in adults, suggesting that purinergic transmission is of less importance. However, there are also several marked developmental changes in purinoceptor expression in the bladder, in that P2X4 is expressed in developing bladders at relatively high levels. There is also a marked developmental change in the regional distribution of purinoceptors. These changes are likely to reflect the changing role of purinergic transmission in the control of bladder motility during fetal maturation.

Molecular cloning, genomic characterization and expression of novel human alpha1A-adrenoceptor isoforms.

We have isolated and characterized from human prostate novel splice variants of the human alpha1A-adrenoceptor, several of which generate truncated products and one isoform, alpha(1A-4), which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the alpha(1A-4) exon is located between those encoding the alpha(1A-1) and alpha(1A-3) variants. CHO-K1 cells stably expressing alpha(1A-4) showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that alpha(1A-4) is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Expression of human dopamine beta-hydroxylase in Drosophila Schneider 2 cells.

Human dopamine beta-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The Km of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.