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. 2005 May;125(5):443-54.
doi: 10.1085/jgp.200409205.

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat

King-Ho Cheung  1 , George P H Leung, Matthew C T Leung, Winnie W C Shum, Wen-Liang Zhou, Patrick Y D Wong
Affiliations

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat

King-Ho Cheung et al. J Gen Physiol. 2005 May.

Abstract

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.

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Figures

F<sc>igure</sc> 1.
Figure 1.
Isolation of basal cells from rat epididymis using Dynabeads. Schematic diagram showing isolation of basal cells from the rat epididymis (A). After enzymatic separation, cells were incubated with TRPC3 antibody-tagged Dynabeads, which captured (bind) basal cells (open arrows) but not principal cells (closed arrows) (B). After separation of the basal cell/Dynabeads complex with a magnet, principal cells were plated down on Petri dish and stained with hematoxylin (C). Basal cells were freed from the Dynabeads and stained with anti–COX-1 antibody (D).
F<sc>igure</sc> 2.
Figure 2.
Effect of BAPTA-AM. Cultured rat epididymal epithelia (area 0.4 cm2) stimulated with LBK (100 nM, top), or with secretin (100 nM, bottom). In each panel, the epithelium on the left shows the control response to LBK or secretin. The other two epithelia were pretreated with BAPTA-AM (0.05 or 0.1 mM, apical) for 1 h before addition of the hormone. Histogram shows a summary of the results. Each column represents the mean ± SEM of six experiments. †, P < 0.01, compared with control.
F<sc>igure</sc> 3.
Figure 3.
Identification and localization of TRPC and COX-1 in the rat epididymis. (A) RT-PCR and Western blot analysis of TRPC and COX-1 transcripts and proteins. RT-PCR revealed only TRPC1, 3, and 6 transcripts but not TRPC2, 4, and 5 transcripts (not depicted). The PCR products for TRPC1, 3, and 6 were more intense in the caudal than in the proximal region of the epididymis, whereas that of COX-1 was more uniformly distributed along the epididymis. The PCR product for S16 serves as the internal standard. In parallel with the RNA expression, Western blots also show the expression of TRPC1, 3, and 6 proteins in the rat epididymis. (B) Immunohistochemical localization of TRPC1, TRPC3, and TRPC6 proteins in the rat epididymis using polyclonal rabbit anti-TRPC1, anti-TRPC3, and anti-TRPC6 antibodies (1:100 dilution). Higher magnifications are shown in insets. Negative controls were obtained by incubation with antigen-preabsorbed antibodies. Phase contrast images of the corresponding stained sections are shown below. (C) Consecutive sections of the rat cauda epididymidis showing positive immunoreactivity (fluorescence) for COX-1 and TRPC3, respectively. Both proteins were restricted to basal cells in the epithelium but not to the other cell types. The two sections were overlaid to demonstrate coexistence of TRPC3 and COX-1 in the basal cells. Phase contrast image is shown to reveal the relationship of the cells. Basal cells are indicated by arrows.
F<sc>igure</sc> 4.
Figure 4.
Effect of SKF96365 Isc responses to (A) LBK (100 nM) or (B) secretin (100 nM) in cultured rat epididymal epithelia (area 0.4 cm2) in the presence of varying concentrations of SKF96365. Inset shows the concentration–inhibition curves of SKF96365 on LBK (closed circles) and secretin (open circles). Each point shows the mean ± SEM of six experiments. †, P < 0.01, compared with control.
F<sc>igure</sc> 5.
Figure 5.
Effect of antisense oligonucleotide to TRPC3. Two matched cultured rat epididymal epithelia (area 0.4 cm2) from the same batch of cells stimulated with LBK (100 nM) followed by secretin (100 nM). The epithelium on the left was pretreated with sense and on the right antisense to TRPC3. Histogram shows a summary of the results. Each column shows the mean ± SEM of six different experiments. †, P < 0.01, compared with control. The down-regulation of TRPC3 protein after antisense treatment is revealed by Western blot analysis (inset). TRPC1, TRPC6, and COX-1 expressions are not affected by TRPC3 antisense oligonucleotide.
F<sc>igure</sc> 6.
Figure 6.
LBK activates a nonselective current in basal cells. (A) Whole-cell current recorded before and during exposure to 200 nM LBK in an isolated basal cell clamped at −60 mV dialyzed with K+-based pipette solution and superfused with normal PSS. (B) Incubation of principal cells with LBK (without basal cells) evoked no current at −60 mV. (C) Pretreatment with SKF96365 (20 μM) or (D) Gd3+ (0.1 mM) prevented the LBK-evoked current in basal cells. (E) Summary of the current mean amplitudes in A–D: control, current measured before stimulation; +LBK (peak), current measured at the peak of LBK response; +SKF (Pre) or +GdCl3 (Pre), current measured after pretreatment with SKF96365 or Gd3+. Number of cells shown in brackets. †, P < 0.01, compared with control. (F) Current–voltage relationship of basal cells before and during exposure to LBK (200 nM), then to Gd3+ (0.1 mM). The bathing solution was low-Ca2+ PSS, and internal solution was Cs+-based solution containing 100 nM [Ca2+]i. Data points in B and D are currents taken at 10-s intervals at −60 mV from principal cells and basal cells, respectively. Dotted line indicates zero current level.
F<sc>igure</sc> 7.
Figure 7.
Measurement of intracellular Ca2+, [Ca2+]i. [Ca2+]i measurement in isolated basal cells (squares) or principal cells (upright triangles) using Fluo3 as probe. Addition of Ca2+ (2.5 mM) to the cells did not significantly increase [Ca2+]i. Basal cells, but not principal cells, responded to LBK (100 nM) by an increase in [Ca2+]i, which was blockable by Gd3+ and prevented by pretreatment with SKF96365 (20 μM) (inverted triangles). Each point shows the mean ± SEM of five experiments.
F<sc>igure</sc> 8.
Figure 8.
Measurement of CFTR-Cl channel activity. Whole-cell recording in isolated principal cells cultured alone or with basal cells. (A) Time profile showing whole-cell currents from principal cells held at −70 mV. Data points are from currents at −70 mV obtained at 10-s intervals. (a) When principal cells were cultured without basal cells (PC alone), LBK (200 nM) evoked no current; (c) subsequent addition of PGE2 (100 nM) developed a current that was abolished by the Cl− channel blocker DPC (1 mM). When principal cells were cultured with basal cells (PC + BC), LBK (200 nM) developed a current that was attenuated by (b) addition of the cation channel blocker SKF96365 (10–20 μM), or by (d) the Cl− channel blocker DPC (1 mM). (e) SKF96365 (20 μM) added simultaneously with LBK or piroxicam pretreatment (10 μM) prevented LBK to activate the currents. (f) Summary of results from A. Each column shows the mean current amplitudes taken before or after drug addition. The number shows the number of cells studied. *, P < 0.05, when compared with unstimulated control. (B) Tracings showing current responses to a series of 500-ms voltage steps between −100 and +60 mV from principal cells in the absence (left) or presence of basal cells (right). Corresponding I–V plots are also shown. Note the linear I–V curves and the time independence at each potential level. Dotted line represents zero current level.
F<sc>igure</sc> 9.
Figure 9.
Measurement of PGE2 release. Prostaglandin E2 (PGE2) release from isolated basal or principal cells. Significant release of PGE2 was observed in basal cells but not in principal cells stimulated with LBK (0.1 or 1 μM). PGE2 release from basal cells was significantly reduced by pretreatment of the cells with BAPTA-AM (0.1 mM, 1 h) or SKF96365 (10 μM, 15 min). Each column represents the mean ± SEM of six experiments. *, P < 0.05, compared with unstimulated control; †, P < 0.01, compared with stimulated control without pretreatment with BAPTA-AM or SKF96365.
F<sc>igure</sc> 10.
Figure 10.
Reconstitution of epithelia from cells isolated from rat cauda epididymis. (A) Short-circuit current (Isc) recording in epithelia reconstituted from isolated principal cells (105 cells/ml) or (B) from a mixture of principal cells (0.7 × 105 cells/ml) and basal cells (0.3 × 105 cells/ml). Epithelia reconstituted from principal cells only responded to LBK (100 nM) poorly although they responded to PGE2 (500 nM) with a significant increase in current. Epithelia reconstituted from principal cells plus basal cells responded to PGE2 as in principal cell only–derived epithelia but responded to LBK with a marked increase in current. This LBK response in PC+BC epithelia was reduced and abolished by pretreatment with SKF96365 (10 μM) and piroxicam (10 μM), respectively. Each tracing is representative of six experiments.
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