Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

Abstract

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca(2+)](i) (Ca(2+) transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca(2+) transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca(2+) transients synchronously with each other, but did not often show a close temporal relationship with Ca(2+) transients in USMCs. Nicardipine (1 microm) suppressed Ca(2+) transients in USMCs but not in ICC-LCs. Ca(2+) transients in ICC-LCs were abolished by cyclopiazonic acid (10 microm), ryanodine (50 microm) and caffeine (10 mm) or by removing extracellular Ca(2+), and inhibited by 2-aminoethoxydiphenyl borate (50 microm) and 3-morpholino-sydnonimine (SIN-1; 10 microm), but facilitated by increasing extracellular Ca(2+) or phenylephrine (1-10 microm). These results indicated that Ca(2+) transients in urethral ICC-LCs in situ rely on both Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through non-L-type Ca(2+) channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.

Figure 1. Identification of ICC-LCs in the rabbit urethra Panels

a show fluorescent images of ICC-LCs in the rabbit urethra stained using ACK2 antibody against Kit labelled with Alexa 488. Panels b show micrographs of preparations viewed with Nomarski optics. A, ICC-LC (arrow head) which had a spindle-shaped cell body is shown lying in parallel with a muscle bundle (SMC). B, another ICC-LC having a stellate-shaped cell body is shown located in the connective tissue between the muscle bundles. C, in a different preparation, which had been loaded with fura-2, ICC-LCs identified by immunoreactivity against Kit (a) had higher F₃₄₀ fluorescence than neighbouring smooth muscle cells (SMC, b), whilst having similar F₃₈₀ fluorescence (c).

Figure 2. Spontaneous Ca²⁺ transients recorded from USMCs in the rabbit urethra

Aa, a series of frames at intervals of 0.1 s demonstrating two non-propagating Ca²⁺ transients generated by USMCs within a muscle bundle. b, another series of frames at intervals of 0.1 s demonstrate an intercellular Ca²⁺ wave within the same smooth muscle bundle. B, Ca²⁺ transients initiated in USMC (1) sometimes spread across a muscle bundle to trigger Ca²⁺ transients in USMC (3) and vice versa. On other occasions the Ca²⁺ wave stopped at USMC (2) or did not propagate at all (asterisks). Numbers for traces in B correspond to those in Aa. The first and second frames correspond to images in Ab and Aa, respectively. Dotted arrows indicate the direction of Ca²⁺ wave propagation.

Figure 3. Spontaneous Ca²⁺ transients recorded from ICC-LCs in situ in the rabbit urethra

Aa, a series of frames at interval 1 s demonstrating Ca²⁺ transients recorded from a spindle-shaped ICC-LC. b, the ICC-LC generated four spontaneous Ca²⁺ transients over 90 s. c, averaged Ca²⁺ transients recorded from ICC-LCs had much longer durations than those of the overlaid averaged USMC Ca²⁺ transients. Ba, another series of frames at intervals of 1 s demonstrate Ca²⁺ transients recorded from a pair of stellate-shaped ICC-LCs. b, in the same preparation, ICC-LCs generated four spontaneous Ca²⁺ transients over 90 s. Note that prominently bright ICC-LCs (arrowheads in Aa and Ba) did not exhibit any Ca²⁺ signals.

Figure 4. Analysis of the temporal relationship of Ca²⁺ transients between a pair of ICC-LCs

A, a series of frames at intervals of 1 s demonstrating a pair of ICC-LCs (1 and 2) generating spontaneous Ca²⁺ transients. B, in the same preparation synchronous Ca²⁺ transients were generated by a pair of ICC-LCs. C, a cross-correlogram for this pair of ICC-LCs had a peak near lag period zero. D, a comparison of mean frequency between pairs of ICC-LCs showed a close temporal correlation between ICC-LCs for 17 of the 22 pairs of ICC-LCs investigated (○) but not for the remaining five pairs of ICC-LCs (•).

Figure 5. Analysis of the temporal relationship of Ca²⁺ transients between ICC-LCs and USMCs

Aa, a series of frames at intervals of 0.2 s demonstrating USMC Ca²⁺ transients originating from an ICC-LC. B, in the same preparation, synchronous Ca²⁺ transients were generated by an ICC-LC (1) and USMC (2). C, a cross-correlogram for ICC-LCs and USMCs showed a peak near lag period zero. D, in a different preparation, a series of frames at intervals of 0.2 s demonstrating USMC Ca²⁺ transient and subsequent Ca²⁺ transient in a pair of ICC-LCs. E, Ca²⁺ transients in ICC-LCs (1) occurred independently from those of USMCs (2). Scale bars also apply to traces in B. Numbers for traces in B and E correspond to those in A and D. F, a comparison of mean frequency between ICC-LCs and USMCs demonstrates a close temporal correlation between the two types of cells in five preparations but not in the remaining 16 preparations.

Figure 6. Effects of nicardipine on spontaneous Ca²⁺ transients recorded from USMCs and ICC-LCs in the urethra

Aa, spontaneous Ca²⁺ transients recorded form USMCs of the rabbit urethra were strongly suppressed by nicardipine (1 μm) (Ab). Ba, in another preparation, ICC-LC Ca²⁺ transients generated spontaneous Ca²⁺ transients which were not inhibited by nicardipine (1 μm) (Bb). C, summary of the effects of nicardipine on ICC-LC Ca²⁺ transients. Nicardipine (1 μm) did not significantly change amplitude (a), frequency (b) or half-width (c) of ICC-LC Ca²⁺ transients.

Figure 7. Role of intracellular Ca²⁺ stores in generating spontaneous Ca²⁺ transients in ICC-LCs and USMCs of the urethra

CPA (10 μm) abolished spontaneous Ca²⁺ transients recorded from ICC-LC (A) and USMC (B). A and B were recorded from different preparations. Ca, in other preparations, CPA reduced the frequency of spontaneous Ca²⁺ transients recorded from USMCs. b, in the same preparations which had been treated with CPA for 45 min, spontaneous Ca²⁺ transients occurred but with a markedly reduced frequency and amplitude.

Figure 8. Role of extracellular Ca²⁺ in generating spontaneous Ca²⁺ transients in ICC-LCs of the urethra

A, in a urethral smooth muscle preparation, ICC-LC generated spontaneous Ca²⁺ transients. Increasing [Ca²⁺]_o from 2.5 mm to 5 mm accelerated ICC-LC Ca²⁺ transients and increased basal Ca²⁺ concentration. B, in a different preparation, ICC-LC generated spontaneous Ca²⁺ transients. Switching from normal PSS to nominally Ca²⁺ free solution rapidly abolished ICC-LC Ca²⁺ transients.

Figure 9. Effects of ryanodine, caffeine and 2-APB on spontaneous Ca²⁺ transients in urethral ICC-LCs

A, ryanodine (50 μm) prevented the generation of spontaneous Ca²⁺ transients recorded in ICC-LCs. B, in another preparation, caffeine (10 mm) initially increased the frequency, and then abolished the discharge of spontaneous Ca²⁺ transients recorded in ICC-LC. C, in a different preparation, 2-APB (50 μm) greatly suppressed ICC-LC spontaneous Ca²⁺ transients.

Figure 10. Role of nitrergic and α-adrenergic stimulation in the modulation of spontaneous Ca²⁺ transients recorded from the urethral ICC-LCs

SIN-1 (10 μm;A) reduced the amplitude (Ba) of spontaneous Ca²⁺ transients recorded from ICC-LC, but did not significantly alter either their frequency (Bb) or half-width (Bc). Ca, in another preparation, bath-applied phenylephrine (1 μm) increased the frequency of spontaneous Ca²⁺ transients recorded from ICC-LC and raised basal Ca²⁺ levels. b, a higher concentration of phenylephrine (10 μm) further accelerated ICC-LC Ca²⁺ transients which summed to create a sustained rise in the basal Ca²⁺ concentration.