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Characterization of transepithelial potential oscillations in the Drosophila Malpighian tubule

Edward M. Blumenthal

Department of Biology and NSF Center for Biological Timing, PO Box 400328, University of Virginia, Charlottesville, VA 22904–4328, USA

View larger version (12K): [in a new window]   Fig. 1. Representative recording from a Malpighian tubule showing transepithelial potential (TEP) oscillations. At the beginning of the trace, the electrode is in the bath. The negative deflection reflects the potential across the basolateral membrane of a principal cell as the electrode enters the cell; as the electrode is advanced into the lumen of the tubule, the recorded potential becomes positive. At the end of the trace, the electrode is withdrawn into the bath again.

View larger version (10K): [in a new window]   Fig. 2. Effect of Ba2+ on principal cell basolateral membrane potential. The electrode is positioned inside a principal cell. Application of 3mmoll-1 BaCl2 for the duration indicated by the bar results in a hyperpolarization of Vbl and the appearance of voltage oscillations. Similar results were seen in two additional tubules.

View larger version (18K): [in a new window]   Fig. 3. Dependence of TEP oscillations on [Cl-]p. Eight tubules were exposed to varying concentrations of Cl- (in mmoll-1; indicated at top of panels) in the peritubular bath; seven of these tubules exhibited TEP oscillations. In one tubule (A), the oscillations disappeared at 55.5mmoll-1 Cl- and reappeared as [Cl-]p was reduced further. In the six other tubules, the amplitude of the oscillations decreased as [Cl-]p decreased (B). (C) Averaged data from the latter six tubules only. Values are means ± S.E.M.

View larger version (16K): [in a new window]   Fig. 4. Effect of DPC on tubule electrophysiology. Application of 250µmoll-1 DPC (bar) rapidly depolarizes the TEP, which recovers upon washout of the drug. Similar results were seen in four additional tubules (see Table1).

View larger version (25K): [in a new window]   Fig. 5. Dependence of TEP oscillations on intracellular calcium. Traces from six control (A) and six BAPTA-AM loaded (B) tubules are shown. (C) Quantification of the effect of BAPTA on the coefficient of variation for the 12 traces shown in A and B. See text for details of methods. Values are means ± S.E.M., *P<0.0001 (paired t-test).

View larger version (18K): [in a new window]   Fig. 6. Lack of dependence of TEP oscillations on peritubular calcium. In the trace shown, the tubule is bathed in a normal, calcium-containing saline. At the arrow, the bathing solution is switched to a nominally calcium-free saline. Similar results were obtained from seven other tubules.

View larger version (35K): [in a new window]   Fig. 7. Expression of genes encoding intracellular calcium-release channels in the tubule. The gel shows the result of RT-PCR from whole-fly mRNA (lanes 2,6) and tubule-specific mRNA (lanes 3,4,7,8) using primers specific to the IP3 receptor (IP3R) (lanes 2–4) and the ryanodine receptor (RyR) (lanes 6–8). In lanes 4 and 8, reverse transcriptase was omitted from the cDNA synthesis reaction to control for possible genomic DNA contamination. The PCR reactions give products of the expected sizes: 547base pairs (bp) for RyR, compared to 678bp from genomic DNA, due to the inclusion of two short introns (Adams et al., 2000) and 843bp for IP3R (Sinha and Hasan, 1999). Because the 5' IP3R primer spans an exon–intron junction, genomic DNA should not give any PCR product. Lane 1: 1kb ladder (Stratagene, La Jolla, CA, USA); Lane 5: 100bp ladder (Life Technologies).

View larger version (17K): [in a new window]   Fig. 8. Effect of leucokinin on TEP oscillations. (A) Addition of leucokinin IV (10µmoll-1) rapidly depolarizes the TEP. After drug washout, the TEP recovers amplitude while the oscillations remain inhibited. (B) Average results from six tubules show long-lasting inhibition of oscillations after leucokinin washout. Values are means ± S.E.M., *P<0.01 (paired t-test). Inhibition of oscillations was measured beginning 60s after washout following an application of 10µmoll-1 leucokinin lasting 25–140s.

View larger version (24K): [in a new window]   Fig. 9. CAP2b does not alter the oscillations. (A) Trace from a tubule exposed to 100nmoll-1 CAP2b starting at the arrow. Note the lack of any immediate effect of the drug on the TEP. Similar results were seen in five additional tubules. (B) Graph showing no effect of CAP2b on TEP variability. The TEP coefficient of variation was measured before drug application and during exposure to 100nmoll-1 CAP2b lasting 5.5–11min. Values are means ± S.E.M., N=6 tubules, P>0.14 (paired t-test).