Modulators of internal Ca2+ stores and the spontaneous electrical and contractile activity of the guinea-pig renal pelvis

Abstract

1. The role of internal Ca(2+) stores in the generation of the rhythmic electrical and contractile activity in the guinea-pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques. 2. Ryanodine (30 microM) transiently increased contraction amplitude, while caffeine (0.5 - 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2-aminoethoxy-diphenylborate (2-APB 60 microM), xestospongin C (1 microM), U73122 (5 microM) and neomycin (4 mM), blockers of IP(3)-dependent release from Ca(2+) stores. 3. 60 mM K(+) saline-evoked contractions were reduced by caffeine (1 mM), U73122 (5 microM) and neomycin (4 mM), but little affected by ryanodine or 2-APB (60 microM). 4. Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6+/-1.0 min(-1)) in small urothelium-denuded strips of proximal renal pelvis. 5. Action potential discharge was blocked in 75 and 35% of cells by 2-APB (60 microM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed. 6. Cyclopiazonic acid (CPA 10 microM for 10 - 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure. 7. We conclude that the action potential in caffeine-sensitive cells and the shoulder component of caffeine-insensitive action potential arise from the entry of Ca(2+) through Ca(2+) channels. The inhibitory actions of modulators of internal Ca(2+) release were partially explained by a blockade of Ca(2+) entry.

Figure 1

Effects of modulators of Ca²⁺ mobilization from ryanodine-sensitive and IP₃-dependent stores on the spontaneous contractile activity of the guinea-pig renal pelvis. Spontaneous contractions of the proximal renal pelvis in the absence and presence of caffeine (0.5 and 1 mM, Ai,ii), ryanodine (30 μM, B), 2-APB (60 μM, Ci), neomycin (4 mM, Cii) or xestospongin C (1 μM, Ciii).

Figure 2

Comparison of the effects of modulators of ryanodine-sensitive or IP₃-dependent Ca²⁺ mobilization on the positive inotropic and chronotropic effects of high K⁺ saline (60 mM). Contractions to K⁺ saline (60 mM for 5 min) were evoked in the absence and presence of caffeine (1 mM, A), ryanodine (30 μM, B), 2-APB (60 μM, C), U73122 (5 μM, D) or neomycin (4 mM, E). Calibration bars apply to all traces.

Figure 3

Typical examples of electrical discharge recorded in the urothelium-denuded proximal renal pelvis with a single (A) or a pair (B) of intracellular microelectrodes. Most commonly recorded action potentials were ‘intermediate' in appearance (Ai,ii, Bi) with quiescent diastolic potentials, however ‘ureteric-like' or ‘driven' action potentials were also recorded (Aiii, Bii,iii). Both intermediate and driven action potentials displayed sub-threshold potential transients (Aiii, Bii). (B) Recordings from pairs of intracellular microelectrodes (100 – 200 μm apart) revealed that electrical responses propagated in both the axial (Bi,ii) and transverse (Biii) direction. Recordings could also occur simultaneously, suggesting that the electrodes were placed in the same smooth muscle bundle (Biii). Peak negative membrane potential between electrical events was −48 (Ai), −43 (Aii), −50 (Aiii), −43 (Bi), −40 (Bii) and −48 (Biii) mV, respectively. Time and voltage calibration bars apply to all traces.

Figure 4

Effects of caffeine on the spontaneous electrical activity recorded in the guinea-pig renal pelvis. Application of caffeine (0.5 mM) produced a substantial block of the spontaneous action potentials (Ai,ii) in 35% of cells (6 of 17 cells) and only a reduction in both the maximum rate of rise and the plateau phase in the remaining 65% of cells (11 of 17) (Bi,ii). Raising the caffeine concentration (1 mM) abolished electrical discharge in the caffeine-sensitive cells (Aiii) and further decreased the plateau of the caffeine-insensitive cells (Biii). The effects of caffeine were readily reversible after 5 min wash out (Aiv, Biv). Membrane potentials in control saline were −42 (A) and −40 (B) mV, respectively. Calibration bars apply to all traces.

Figure 5

Caffeine-induced blockade of electrical activity in the renal pelvis was associated with cessation of transient increases of [Ca²⁺]_i. Membrane potential recordings (A) were made simultaneously with relative changes in [Ca²⁺]_i in two areas (Bi,ii) of the renal pelvis adjacent to the intracellular microelectrode. Spontaneous action potential and increases in [Ca²⁺]_i were recorded synchronously. [Ca²⁺]_i transients consisted of a rapid rising phase and a slower decay phase. The application of caffeine (1 mM for min) induced a cessation of both action potential discharge and the transient increase in [Ca²⁺]_i. Time calibration bar applies to all traces.

Figure 6

Effects of blocking IP₃-dependent Ca²⁺ release with 2-APB. Application of 2-APB (60 μM) caused a time-dependent reduction of the frequency and plateau component of the action potentials (A,B) recorded in the renal pelvis. In 75% of cells (six of eight cells), action potential discharge was completely abolished after 6 min exposure to 2-APB (C). The effects of 2-APB were partially reversible (D) upon washout. The membrane potential in control saline (A) was −48 mV. Calibration bars apply to all traces.

Figure 7

Effects of preventing IP₃ formation with the antibiotic, neomycin, on the electrical discharge in the proximal renal pelvis. Application of neomycin (1 mM) caused a time-dependent blockade of the action potential discharge in both caffeine-sensitive (Ai,ii) and -insensitive (Bi,ii) cells. Action potential discharge was completely restored upon wash-out (Aiii, Biii). The membrane potential in control saline was −45 (A) and −43 (B) mV, respectively. Time and voltage calibration bars apply to all traces.

Figure 8

Effects of blocking the sarcoplasmic reticulum CaATPase with cyclopiazonic acid (CPA). (A) Contraction amplitude and frequency transiently increased and slowly declined with time in the presence of CPA (10 μM for >120 min). (B – C) Application of CPA (10 μM for 10 min) gradually increased the duration of the recorded action potential in both caffeine-sensitive (Bi,ii) and -insensitive (Ci,ii) cells. This effect of CPA was reversed upon wash-out (Biii, Ciii). The membrane potential in control saline was −48 (B) and −42 (C) mV, respectively. Time and voltage calibration bars apply to all traces.