Biphasic regulation of Na+-HCO3- cotransporter by angiotensin II type 1A receptor.

Although angiotensin (Ang) II is known to regulate renal proximal transport in a biphasic way, the receptor subtype(s) mediating these Ang II effects remained to be established. To clarify this issue, we compared the effects of Ang II in wild-type mice (WT) and Ang II type 1A receptor-deficient mice (AT(1A) KO). The Na+-HCO3- cotransporter (NBC) activity, analyzed in isolated nonperfused tubules with a fluorescent probe, was stimulated by 10(-10) mol/L Ang II but was inhibited by 10(-6) mol/L Ang II in WT. Although valsartan (AT1 antagonist) blocked both stimulation and inhibition by Ang II, PD 123,319 (AT2 antagonist) did not modify these effects of Ang II. In AT1A KO, in contrast, this biphasic regulation was lost, and only stimulation of NBC activity by 10(-6) mol/L Ang II was observed. This stimulation was blocked by valsartan but not by PD 123,319. More than 10(-8) mol/L Ang II induced a transient increase in cell Ca2+ concentrations in WT, which was again blocked by valsartan but not by PD 123,319. However, up to 10(-5) mol/L Ang II did not increase cell Ca2+ concentrations in AT1A KO. Finally, the addition of arachidonic acid inhibited the NBC activity similarly in WT and AT(1A) KO, suggesting that the inhibitory pathway involving P-450 metabolites is preserved in AT(1A) KO. These results indicate that AT(1A) mediates the biphasic regulation of NBC. Although low-level expression of AT(1B) could be responsible for the stimulation by 10(-6) mol/L Ang II in AT1A KO, no evidence was obtained for AT2 involvement.

Quantifying porphobilinogen deaminase mRNA in microdissected nephron segments by a modified RT-PCR.

BACKGROUND: Quantifying mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR), although widely exercised, is still difficult. METHODS: A modified quantitative RT-PCR in which genomic DNA was used as standard was developed. The quantity of mRNA was expressed as the ratio of the PCR product from cDNA and that from genomic DNA (CG ratio). Nephron distribution of porphobilinogen deaminase (PBGD) mRNA was examined in microdissected nephron segments using the method. The enzyme activity and mRNA quantity of PBGD also were measured in tissue homogenates. RESULTS: Tubular segments expressed substantially more PBGD mRNA than glomeruli (expressed as CG ratios, 1.04 +/- 0.10 in glomeruli, 4.53 +/- 0.32 in PCT, 5.71 +/- 0.25 in PST, 5.13 +/- 0.52 in mTAL, 5.29 +/- 0.20 in cTAL, 4.05 +/- 0.35 in DCT, 2.88 +/- 0.25 in CCD, and 4.90 +/- 0.24 in OMCD). PBGD mRNA level in liver homogenate (3.17 +/- 0.36) was much higher than glomeruli but lower than most of the tubular segments. The enzyme activity in tissue homogenates correlated well with mRNA levels. CONCLUSION: The method reported here is simple and reliable, and especially suitable for quantitating specific mRNA amounts in minute tissue samples such as microdissected nephron segments.