Contribution of reverse Na+-Ca2+ exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra

Abstract

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+-Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mM, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB-R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mM) and noradrenaline (10 microM) induced transient Ca2+-activated chloride currents (I(ClCa)) at -60 mV due to release of Ca2+ from ryanodine- and inositol trisphosphate (IP3)-sensitive Ca2+ stores, respectively, but these responses were not blocked by KB-R7943 or SEA0400 suggesting that neither drug blocked Ca2+-activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration-dependent fashion. Finally, single cell RT-PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+-Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

Figure 1. Effect of low [Na⁺]_o on spontaneous Ca²⁺ oscillations in ICC

A is a representative recording from an isolated ICC showing that reduction of [Na⁺]_o from 130 to 13 mm doubles the frequency of spontaneous Ca²⁺ oscillations in ICC. B–D are summary bar charts showing mean frequency and amplitude of Ca²⁺ oscillations as well as the basal Ca²⁺ levels in 130 mm[Na⁺]_o (open bars) and 13 mm[Na⁺]_o (filled bars). Error bars refer to standard errors of the mean in each example.

Figure 2. Effect of KB-R7943 and SEA0400 on spontaneous Ca²⁺ oscillations in ICC

The reverse NCX inhibitor KB-R7943 abolished Ca²⁺ oscillations and lowered basal Ca²⁺ levels in urethral ICC (A). B–D, summary bar charts showing mean frequency and amplitude of Ca²⁺ oscillations and basal Ca²⁺ levels in the absence (open bars) and presence (filled bars) of KB-R7943. The effect of SEA0400 was not as dramatic as KB-R7943 but it also greatly reduced the frequency of Ca²⁺ oscillations and lowered basal Ca²⁺ levels in ICC as shown in the representative example in E and summary data plotted in F–H.

Figure 3. Effect of SEA0400 on Ca²⁺ oscillations induced by [Na⁺]_o

A and B, representative recordings which show the effect of 13 mm[Na⁺]_o on spontaneous Ca²⁺ oscillations in the absence (A) and presence (B) of SEA0400, respectively. C, summary bar chart which plots the mean frequency of Ca²⁺ oscillations under control conditions, in the presence of low [Na⁺]_o, in the presence of SEA0400 and in the presence of SEA0400 and low [Na⁺]_o together.

Figure 4. Effect of low [Na⁺]_o on spontaneous Ca²⁺ oscillations in ICC

A, representative recording from an isolated ICC showing that reduction of [Na⁺]_o from 130 to 13 mm increases the frequency of STICs in ICC. B and C, summary bar charts showing the effect of 13 mm[Na⁺]_o on mean frequency and amplitude of STICs.

Figure 5. Effect of 0 mm[K⁺]_o on spontaneous Ca²⁺ oscillations in ICC

Representative trace showing that removal of K⁺ from the bath solution to inhibit the Na⁺–K⁺ pump causes an increase in the frequency of spontaneous Ca²⁺ oscillations in ICC.

Figure 6. Effect of KB-R7943 and SEA0400 on STICs in ICC

KB-R7943 (5 μm) and SEA0400 (1 μm) abolished STICs in ICC at −60 mV but not at −100 mV. Representative examples of the effects of KB-R7943 at each potential are shown in A and C. Summary data plotting mean STIC amplitude and frequency at −60 and −100 mV in the absence and presence of KB-R7943 are plotted in B and D, respectively. Typical records showing the effects of SEA0400 at −60 and −100 mV are shown in E and G. F and H are summary bar charts which show the effect of SEA0400 on the amplitude and frequency of STICs at each potential.

Figure 7. Effect of KB-R7943 and SEA0400 on caffeine- and noradrenaline-induced inward currents in ICC

Application of caffeine (10 mm) to ICC voltage clamped at −60 mV resulted in transient inward currents. These responses were not affected by KB-R7943 as indicated by the representative example shown in A and summary data plotting the peak amplitude of the caffeine-evoked current before, during and following washout of KB-R7943 in B. Noradrenaline (10 μm) also evoked a transient inward current in ICC. These responses were also not significantly affected by KB-R9743 (C and D). E and F show that SEA0400 did also not significantly inhibit caffeine responses. Noradrenaline-induced currents were slightly reduced in amplitude as shown in the example in G and summary data in H.

Figure 8. Effect of SEA0400 on spontaneous myogenic tone of rabbit urethra smooth muscle

A and B, representative examples showing the effects of 1 and 10 μm SEA0400 on spontaneous myogenic tone in rabbit urethra smooth muscle. C, summary bar chart plotting the mean amplitude of the relaxations produced by SEA0400.

Figure 9. Expression of NCX in the rabbit urethra

A representative gel displaying amplification products from RNA derived from rabbit brain, urethra and urethra SMC and ICC using gene-specific primers for NCX1, NCX2 and NCX3.