Carbachol-induced oscillations in membrane potential and [Ca2+]i in guinea-pig ileal smooth muscle cells

Abstract

1. Cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential were simultaneously recorded from single smooth muscle cells of guinea-pig ileum, using a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. Carbachol (CCh, 2 microM) produced oscillatory changes in [Ca2+]i and membrane potential which coincided well in time with each other, and peaks of membrane potential oscillations reached a saturated level of around -7 mV. Thapsigargin (1 microM) abolished these effects of 2 microM CCh. La3+ (3 microM) immediately prevented the discharge of spike potentials, but allowed both on-going oscillatory responses to persist for a while. 3. CCh (0.25-0.75 microM) caused membrane potential and [Ca2+]i to oscillate in some 20 % of cells studied. Every membrane potential oscillation was preceded by the discharge of single or multiple spike potentials. The effects of CCh were readily abolished by La3+ (3 microM). 4. In cells exhibiting no oscillatory response to 0.25-0.75 microM CCh, an electrically evoked action potential usually generated changes in [Ca2+]i and membrane potential similar to those following spontaneously evoked action potentials, and sometimes it did so only after [Ca2+]i or InsP3 had been slightly elevated by repeatedly evoking action potentials or by increasing CCh concentration in the bath medium. 5. The results suggest that in ileal smooth muscle cells, the oscillations of [Ca2+]i and membrane potential arising from muscarinic stimulation result from release of Ca2+ from internal stores and that there is a Ca2+-induced potentiation of coincidently elicited cation channel openings. Under weak muscarinic stimulation, Ca2+ entry upon action potential discharge can trigger such a release of stored Ca2+, resulting in synchronous generation of a large rise in [Ca2+]i and a slow, large membrane depolarization.

Figure 1. Oscillations in membrane potential and [Ca²⁺]_i induced by 2 μM CCh in single smooth muscle cells of guinea-pig ileum

Membrane potential (upper traces) and [Ca²⁺]_i (lower traces) were simultaneously measured using a combination of nystatin-perforated whole-cell patch clamp and fura-2 fluorimetry techniques. A and B, records from two different cells. In A, a pair of records indicated by a and b are also shown on a time-extended scale. Note that the peak of membrane potential oscillations does not exceed the zero potential.

Figure 2. Changes in membrane potential and [Ca²⁺]_i produced by 2 μM CCh after treatment with thapsigargin

Thapsigargin (1 μM) was applied 1.5 min before CCh application. A and B, records of CCh-induced changes in membrane potential (upper traces) and [Ca²⁺]_i (lower traces) from two different cells. Note failure of CCh to produce any oscillatory response. See text for details.

Figure 3. Effect of La³⁺ on CCh-induced oscillations in membrane potential and [Ca²⁺]_i

A, records of CCh (2 μM)-induced changes in membrane potential (upper trace) and [Ca²⁺]_i (lower trace) before and after application of 3 μM La³⁺. Application of CCh and La³⁺ is indicated by bars below the [Ca²⁺]_i recording trace. B, records when CCh was used at 0.5 μM. A pair of records indicated by a and b is also shown on a time-extended scale. A and B are from different cells. Note that both on-going oscillations in membrane potential and [Ca²⁺]_i induced by 2 μM CCh persisted for a while after application of La³⁺, whereas those induced by 0.5 μM CCh stopped immediately. See text for details.

Figure 4. Effect of thapsigargin on changes in membrane potential and [Ca²⁺]_i induced by electrically evoked action potentials in the presence of 0.5 μM CCh

A, records of changes in membrane potential (top trace) and [Ca²⁺]_i (bottom trace) induced by the evoked action potentials before and after CCh application and after application of 1 μM thapsigargin in the continued presence of CCh, as indicated by bars below the [Ca²⁺]_i recording trace. Depolarizing current pulses (20 pA in intensity and 100 ms in duration; middle trace) were used to evoke single action potentials. B, time-expanded recordings of traces indicated by a, b and c in A. Note that both membrane depolarization and [Ca²⁺]_i increase subsequent to the evoked action potential in the presence of CCh were selectively and markedly inhibited by thapsigargin.

Figure 5. Effect of La³⁺ on the membrane depolarization and [Ca²⁺]_i rise following electrically evoked action potentials in the presence of 0.5 μM CCh

A, records of changes in membrane potential (top trace) and [Ca²⁺]_i (bottom trace) following the evoked action potentials before and after application of 3 μM La³⁺ in the continued presence of CCh. B, time-expanded recording traces of the membrane potential changes indicated by a and b in A. Note that La³⁺ abolished both the large membrane depolarization and [Ca²⁺]_i rise as well as the evoked action potential and its concomitant [Ca²⁺]_i rise. See text for details.

Figure 6. Effect of increases in CCh concentration and Ca²⁺ influx on changes in membrane potential and [Ca²⁺]_i following an electrically evoked action potential

Depolarizing current pulses (30 pA in intensity and 100 ms in duration; middle traces) were used to evoke action potentials. A, records of evoked action potential-induced changes in membrane potential (top trace) and [Ca²⁺]_i (bottom trace) before (a) and after application of 0.25 μM (b) and then 0.5 μM CCh (c). B, records of membrane potential and [Ca²⁺]_i changes induced by a single evoked action potential (a) and two evoked action potentials with a 100 ms interval (b) before application of 0.5 μM CCh and of those (c and d, respectively) after its application. A and B are from different cells. Note that the increases of CCh concentration from 0.25 to 0.5 μM (A) and of Ca²⁺ influx by repeated action potential discharge (B) enhanced both the membrane depolarization and [Ca²⁺]_i increase induced by a single pulse-evoked action potential. See text for details.

Figure 7. Effects of paired current pulses on membrane potential and [Ca²⁺]_i in the presence of CCh

A pair of current pulses (30 pA in intensity and 100 ms in duration) was applied to evoke two successive action potentials at varied intervals in the presence of 0.5 μM CCh. A, records of changes in membrane potential (top traces) and [Ca²⁺]_i (bottom traces) in response to a pair of current pulses (middle traces) at interpulse intervals of 3.2 (a), 6 (b) and 20 s (c). Note that as the interpulse interval increased, membrane depolarization and [Ca²⁺]_i rise induced by the second evoked action potential increased. B and C, plots of amplitudes of the [Ca²⁺]_i rise (B) and membrane depolarization (C) induced by the second action potential against the interpulse interval. The amplitudes of both variables (ordinates) are expressed as percentages of those induced by the first evoked action potential (P1). Data points with different symbols are derived from four different cells.