The effect of cyclopiazonic acid on excitation-contraction coupling in guinea-pig ureteric smooth muscle: role of the sarcoplasmic reticulum

Abstract

1. We have investigated the effect of cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase on excitation-contraction (EC) coupling in guinea-pig ureter, by measuring membrane currents, action potentials, intracellular [Ca2+] and force. 2. CPA (20 micrometers) significantly enhanced the amplitude and duration of phasic contractions of ureteric smooth muscle associated with action potentials. This was accompanied by an increase in the duration of the intracellular Ca2+ transient in intact tissue and single cells but not their amplitude. However, CPA also slowed the rate of rise, and fall, of the force 1|1|Phiand1Phi Ca2+ transients. 3. Membrane potential recordings showed that CPA produced a small depolarization and a large increase in the duration of the plateau phase of the action potential. 4. Patch-clamp studies showed marked inhibition of outward potassium current in the presence of CPA and an inhibition of spontaneous transient outward currents (STOCs). CPA had no effect on inward Ca2+ current. 5. These data suggest that the SR plays a major role in modulating the excitability of the ureter, particularly via curtailing the action potential duration. This in turn will shorten the Ca2+ transient and decrease force. This negative action on developed force predominates over any small role it may play in initiating force in the guinea-pig ureter.

Figure 1. The effects of cyclopiazonic acid (CPA) on force and calcium

A, the effect of CPA on the evoked phasic contractions and Ca²⁺ transients of the guinea pig ureter. B, phasic contractions and Ca²⁺ transients recorded in control conditions (i) and after 1.5 min (ii) and 5 min (iii) in CPA. C, the relationship between the amplitude of force and the duration of the Ca²⁺ transient. D, the phase plane diagram showing the force-Ca²⁺ relationship recorded during the development of the individual phasic contractions (see text).

Figure 2. The effect of CPA on Ca²⁺ in single cells

A, changes in intracellular [Ca²⁺] in a single ureteric cell, evoked by brief exposure (1 s) to high-K⁺ solution before and after addition of CPA. B, normalized Ca²⁺ transients in the absence and the presence of CPA, showing the monoexponential fittings to the decaying phase of the Ca²⁺ transients.

Figure 3. The effect of CPA on kinetics of the Ca²⁺ transients and force

A, superimposed records of the normalized Ca²⁺ transients and phasic contractions evoked by electrical field stimulation. B, rates of contraction and relaxation of the normalized phasic contraction recorded in the absence and the presence of CPA, showing the slower rates of both rise and fall produced by CPA. C, superimposed traces of the rising phase of the Ca²⁺ transients recorded in the presence and the absence of CPA.

Figure 4. The effect of CPA on electrical activity

A, simultaneous recording of the force (top trace), Ca²⁺ transients (middle trace) and action potentials (E_m; bottom trace) obtained in the absence and the presence of CPA. B, the relationship between the amplitude of force, the duration of the Ca²⁺ transient measured at its 90 % peak level (t_90%) and the duration of the action potential measured at its 50 % level (t_½)(n = 6).

Figure 5. The effect of CPA outward currents of uretereal myocytes

Clamping protocol: from a holding potential of −80 mV, membrane potential was clamped from −60 to +60 mV. A, control record; B, 5 min in CPA; C, 10 min after removal of CPA from the bath. The pipette solution had normal K⁺ concentration.

Figure 6. The effect of CPA on the initial transient outward current and total membrane currents

The effect of CPA on the K⁺ outward current (A) and spontaneous transient outward currents (STOCs; C and D), recorded from single ureteric cells under voltage-clamp conditions. In A, the pipette solution contained 1 mM EGTA and the cell was depolarized from a holding potential of −80 mV to 0 mV. In C and D the pipette solution contained 0.1 mM EGTA, and the cell was depolarized from −80 mV to −20 mV. B, the current-voltage relationship recorded in the absence and the presence of CPA.

Figure 7. The effect of CPA and CPA plus TEA on the outward K⁺ currents

From a holding potential of −80 mV, the membrane potential was clamped from −60 to +10 mV. A, control record; B, 5 min in CPA; C, 5 min after addition of TEA (5 mM) to the bath; D shows superimposed specimen records from A, B and C recorded during the depolarization to 0 mV. The pipette solution had normal K⁺ concentration.

Figure 8. The effect of CPA on the inward Ca²⁺ currents in the absence (A) and presence (B) of BayK (1 μm) and on the Ba²⁺ current (C)

From holding potential of −80 mV, the membrane potential was clamped from −60 to +60 mV. Pipette solution contained 140 mM Cs⁺. In A and B the bath media contained 2 mM Ca²⁺ and in C it contained 2 mM Ba²⁺. D-F, current voltage plots of inward current peaks vs. membrane potential recorded in 2 mM Ca²⁺ with Bay K and 2 mM Ba²⁺, respectively (in all plots □ are from control currents and • are current values recorded 5 min after addition of 20 μm CPA).

Figure 9. The effects of CPA and CPA plus TEA on the parameters of the action potential

The bottom record shows action potentials (E_m). Simultaneous recordings of Ca²⁺ transient (middle traces) and force (top traces) are also shown.