Role of Ca2+ entry and Ca2+ stores in atypical smooth muscle cell autorhythmicity in the mouse renal pelvis

Abstract

Background and purpose: Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs.

Experimental approach: The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal-like cells (ICC-LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo-4.

Results: In 1 microM nifedipine, high frequency (10-30 min(-1)) Ca2+ transients and STDs were recorded in ASMCs, while ICC-LCs displayed low frequency (1-3 min(-1)) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2-aminoethoxy-diphenylborate (2-APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine-sensitive Ca2+ release blocked ICC-LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl(-) with isethionate.

Conclusions: ASMCs generated Ca2+ transients and cation-selective STDs via mechanisms involving Ca2+ release from IP3-dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine-sensitive stores.

Figure 1

Typical recordings of the effects of nifedipine on the spontaneous electrical and Ca²⁺ signals in the mouse renal pelvis. (A) In some preparations, regenerative action potentials recorded in typical smooth muscle cells (TSMCs) (Aai) were completely blocked in 1 μM nifedipine (Aaii); dashed line represents 0 mV. In other preparations (Abi–ii), although the time course and amplitude of TSMC action potential were significantly reduced in 1 μM nifedipine; their frequency of discharge often increased. (Bi–ii) High frequency spontaneous transient depolarizations (STDs) were recorded in approximately 50% of impalements and either little affected or more evident (Abii) in 1 μM nifedipine. (Ci–ii) Long plateau action potentials, which did not trigger muscle contraction were occasionally recorded (at frequency of 1–3 min⁻¹) and little affected by 1 μM nifedipine. (Di–ii) Ca²⁺ waves in TSMC layer were either completely blocked or partially reduced (Di) in 1 μM nifedipine. Ca²⁺ transients were recorded in spindle-shaped ASMCs (E) and fusiform interstitial cells of Cajal-like cells (ICC-LCs) (F) distinguished by their distinctive discharge frequency and time course were little affected by 1 μM nifedipine. (G) Greyscale fluorescence micrographs of cells displaying Ca²⁺ transients in TSMC layer (Gi) in the absence of nifedipine and ASMCs (Gii) and ICC-LCs (Giii) in 1 μM nifedipine. Calibration bars represent 30 μm.

Figure 2

Effects of blocking Ca²⁺ entry and Ca²⁺ uptake into internal stores on the spontaneous electrical and Ca²⁺ signals in the renal pelvis bathed in 1 μM nifedipine. Ca²⁺ free PSS (a, bi–ii) and 10 μM CPA (c, di–ii) both blocked residual action potential and STD (a, c) discharge and depolarized the membrane some 10–20 mV. Blockade of Ca²⁺ transients in ASMCs and interstitial cell of Cajal-like cells (ICC-LCs) by Ca²⁺ free PSS (b) was associated with a reduction of the basal [Ca²⁺]_i of 0.2 F_t/F₀, while CPA (10 μM) blockade of Ca²⁺ transients was associated with a rise in basal [Ca²⁺]_I of 0.3–0.4 F_t/F₀ (d).

Figure 3

La³⁺ (ai–ii) and Gd³⁺ (bi–ii), blockers of pacemaker currents in intestinal ICCs had little effect on the frequency or time course of spontaneous transient depolarizations recorded in the renal pelvis.

Figure 4

Effect of monovalent ion replacement and Cl⁻ channel blocker DIDS on spontaneous transient depolarizations (STDs) generation in the renal pelvis bathed in 1 μM nifedipine. (ai–ii) Residual action potentials (^*) were abolished and STD amplitudes reduced when 93% of the external Na⁺ was replaced by N-methyl-D-glucamine (for 3–4 min). STD amplitudes were not significantly affected when 93% of the Cl⁻ concentration was replaced by isethionate (for 3–4 min) (bi–ii) or in the presence of 100 μM DIDS (ci–ii). Residual action potentials (^*) were blocked in DIDS (bii). (aiii, biii, ciii) The effects of all treatments were reversible upon washout.

Figure 5

Effect of blocking IP₃ receptors with 2-aminoethoxy-diphenylborate (2-APB) on spontaneous transient depolarization (STD) discharge (A) and Ca²⁺ transients in ASMCs (C) and interstitial cells of Cajal-like cells (ICC-LCs) (D) in the renal pelvis bathed in 1 μM nifedipine. 2-APB (60 μM) blocked STD discharge in a manner associated with a membrane depolarization of 20 mV. (Bi–iii) Sections of trace indicated by a–c in (A) displayed on an expanded time base for better comparison. STDs remain blocked even when the membrane was repolarized upon injection of a constant hyperpolarizing current (Bii). Ca²⁺ transients in ASMCs (C) and ICC-LCs (D) were blocked in 50 μM 2-APB.

Figure 6

(A) Effects of blockade of PLC with neomycin (4 mM for 5 min) (A, B) or U73122 (30 μM for 5 min) (A, C) on spontaneous transient depolarization amplitude and discharge frequency were associated with a membrane depolarization of 4–20 mV. (Bi–iii) Sections of trace indicated by a–c in (A) displayed on an expanded time base while (Ci–iii) illustrates the sections of trace denoted by (Ad–f).

Figure 7

(A) Blockade of ryanodine-sensitive Ca²⁺ release produced a concentration-dependent reduction in the synchronistic behaviour of spontaneous transient depolarizations (STD) discharge resulting in a reduction in STD amplitude and ½ width, but an increase in their frequency. (Bi–iii) Sections of trace indicated by a–c in A were displayed on an expanded time base. Superimposing STDs recorded in control PSS (Ci), 30 μM (Cii) or 100 μM (Ciii) ryanodine reveals changes in time course and synchronicity in ryanodine.

Figure 8

Effects of ryanodine (100 μM) on Ca²⁺ transients recorded in single ASMCs (ai–ii) and interstitial cells of Cajal-like cells (ICC-LCs) (bi–ii) of the renal pelvis bathed in 1 μM nifedipine. In comparison to ICC-LCs (bii), Ca²⁺ transients in ASMCs (aii) were only partially reduced even after 30–60 min exposure to ryanodine.

Figure 9

Caffeine (1 mM) rapidly blocked residual action potential discharge (^*) in all tissues bathed in 1 μM nifedipine and hyperpolarized the membrane some 10–20 mV. Spontaneous transient depolarization amplitudes were either reduced (in 50% of preparations) (Ai) or increased in a manner associated with a decrease in their discharge frequency (Aii). (Bi–iii) Sections of trace indicated by a–c in (Aii) displayed on an expanded time base. Caffeine (1 mM 2–4 min) either blocked completely (Ci) or reduced the frequency of discharge of Ca²⁺ transients in ASMCs (Cii) and interstitial cells of Cajal-like cells (ICC-LCs) (D).

Figure 10

(A) Hyperpolarizing effects of caffeine (1 mM for 3–5 min) were prevented by the K_ATP channel blocker gliblenclamide (10 μM). (Bi–vi) Sections of trace indicated by a–f in (A) displayed on an expanded time base for better comparison. It can be seen that membrane hyperpolarization evoked by passing a constant current did not mimic the effects of caffeine (1 mM for 3–5 min) on spontaneous transient depolarisation (STD) amplitude (Bi, iii). (Bii) Repolarizing the membrane to control potentials in the presence of 1 mM caffeine revealed the presence of small amplitude STDs that were not apparent at the more negative potentials. Exposure to 10 μM glibenclamide reduced the hyperpolarizing action of caffeine but did not reduce the caffeine-evoked inhibition of STD discharge (A, Biv-vi).