Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus

Abstract

1. Intracellular recordings made in single bundle strips of a visceral smooth muscle revealed rhythmic spontaneous membrane depolarizations termed slow waves (SWs). These exhibited 'pacemaker' and 'regenerative' components composed of summations of more elementary events termed spontaneous transient depolarizations (STDs). 2. STDs and SWs persisted in the presence of tetrodotoxin, nifedipine and ryanodine, and upon brief exposure to Ca2+-free Cd2+-containing solutions; they were enhanced by ACh and blocked by BAPTA AM, cyclopiazonic acid and caffeine. 3. SWs were also inhibited in heparin-loaded strips. SWs were observed over a wide range of membrane potentials (e.g. -80 to -45 mV) with increased frequencies at more depolarized potentials. 4. Regular spontaneous SW activity in this preparation began after 1-3 h superfusion of the tissue with physiological saline following the dissection procedure. Membrane depolarization applied before the onset of this activity induced bursts of STD-like events (termed the 'initial' response) which, when larger than threshold levels initiated regenerative responses. The combined initial-regenerative waveform was termed the SW-like action potential. 5. Voltage-induced responses exhibited large variable latencies (typical range 0.3-4 s), refractory periods of approximately 11 s and a pharmacology that was indistinguishable from those of STDs and spontaneous SWs. 6. The data indicate that SWs arise through more elementary inositol 1,4,5-trisphosphate (IP3) receptor-induced Ca2+ release events which rhythmically synchronize to trigger regenerative Ca2+ release and induce inward current across the plasmalemma. The finding that action potentials, which were indistinguishable from SWs, could be evoked by depolarization suggests that membrane potential modulates IP3 production. Voltage feedback on intracellular IP3-sensitive Ca2+ release is likely to have a major influence on the generation and propagation of SWs.

Figure 1. STDs

Simultaneous recordings showing STDs (*) in a strip (A) and in a tissue sheet of circular smooth muscle (B) at separations of 1.7 mm near opposite ends of the 2 mm strip and 1.5 mm in the sheet (circumferential orientation). Application of 20 μM BAPTA AM (C) and 16 μM CPA (D) to strips suppressed STD activity. Numbered arrows point to record segments shown in an expanded form on the right and exemplify the suppression of STDs. The two large responses in D (□) are truncated SWs, which appeared shortly after application of CPA. Nifedipine (1 μM) was present throughout. V_m (mV): A, −62; B, −64; C, −55; D, −58. Voltage scale bar in B also applies to A, and that in D also applies to C.

Figure 2. The onset of SWs reveals pacemaker and regenerative SW components

A–C, recordings of membrane potential from strips during the early onset of SW activity. Expanded records on the right correspond to record segments marked by numbered arrows and show baseline STD activity (A only), sub-threshold pacemaker potentials (bars), and SWs each composed of the rising phase of a pacemaker potential (bar) and a larger regenerative response (□). Nifedipine (1 μM) was present throughout. V_m (mV): A, −59; B, −63; C, −62.

Figure 3. SWs persist upon brief exposure to Ca²⁺-free solutions but are inhibited by BAPTA AM and CPA

Aa and b, recordings of SWs from two strips before, during and after exposure to a nominally Ca²⁺-free, 0.3 mM Cd²⁺-containing solution. Ac, recording made in the same strip as Ab in which the Ca²⁺-free, 0.3 mM Cd²⁺ solution also contained 0.1 mM EGTA. Application of BAPTA AM (20 μM; B) and CPA (16 μM; C) as indicated inhibited SWs. The expanded records on the right correspond to record segments marked by numbered arrows. BAPTA AM and CPA sequentially suppressed the regenerative component of SWs (□), then sub-threshold pacemaker potentials (bars) followed by STDs (*). Nifedipine (1 μM) was present throughout. V_m (mV): Aa, −56; Ab and c, −54; B, −51; C, −64. Scale bars in Ac apply to all records in A. Scale bars in C apply to all respective records in B.

Figure 4. ACh induces SWs

A and B, application of 0.1 μM ACh, as indicated, to strips that showed only STD activity sequentially caused an increase in STD activity, the formation of pacemaker potentials and full SWs (□). Numbered arrows indicate expanded regions on the right showing STD activity before ACh and STD/pacemaker potential activity (bars) shortly after application of ACh in each strip. Nifedipine (1 μM) was present throughout. V_m (mV): A, −59; B, −72. Scale bars in B apply to all respective records.

Figure 5. Inhibition of SWs by heparin

Electrical activity recorded from heparin-loaded and control strips (Methods). Recordings were obtained simultaneously from the two strips in the same perfusion chamber. SW activity was present in physiological saline in the control strip (Aa) but not in the heparin-loaded strip (Ba) 85 min after commencement of recordings. Addition of 0.1 μM ACh, as indicated, 3 h after commencement of recordings caused a small increase in SW frequency in the control strip (Ab) and induced the onset of SW activity in the heparin-loaded strip (Bb). Nifedipine (1 μM) was present throughout. V_m (mV): A, −59; B, −51. Scale bars in B apply to all records.

Figure 6. Effect of membrane holding potential on SWs and reversal of the hyperpolarization-induced inhibition by ACh

Spontaneous electrical activity (upper traces) recorded in a strip sequentially polarized to membrane potentials near −63, −69 and −58 mV (A) and in the same strip at sequential potentials near −69, −83, −69 and −47 mV (B). C, electrical activity recorded in a strip held at −77 mV before, during and after application of 0.1 μM ACh. Numbered arrows point to regions shown in an expanded form on the right which show sub-threshold pacemaker potentials (bars) and SWs, each composed of the rising phase of a pacemaker potential (bar) and a larger regenerative response (□). Nifedipine (1 μM) was present throughout. V_m (mV): A and B, −69 mV; C, −56 mV.

Figure 7. Voltage-induced responses

A, voltage responses to passive depolarization, induced by injection of current pulses of variable amplitude, before (a) and during application of 1 μM nifedipine (b). Sub-threshold initial responses (bars) appear to be composed of closely grouped STDs (*). The slow regenerative response (□) and the spike (+) occurred in response to the larger depolarization with only the slow regenerative response recorded during application of 1 μM nifedipine. All traces are from the same continuous recording. B–D, responses induced by application of depolarizing current pulses or consequent to brief pre-hyperpolarizing pulses in nifedipine (1 μM). Arrows indicate sequential responses shown in an expanded form on the right and exemplify the initial depolarizations (bars) and regenerative responses (□). The ‘plateau’ phase of the regenerative response was complex showing large voltage fluctuations. Lower traces indicate current protocols. Traces in B and C are from the same continuous recording. V_m (mV): Aa, −61; Ab, −59; B and C, −62; D, −58. Scale bars in D apply to all respective records in B and C.

Figure 8. Effect of membrane holding potential on voltage-induced responses

A, responses elicited at membrane potentials of −82, −68 and −61 mV. Lower trace shows the current protocol used. B, expanded record segments corresponding to regions of A marked by numbered arrows with traces aligned according to absolute membrane potential and symbols denoting the initial (bar) and regenerative responses (□). Nifedipine (1 μM) was present throughout. V_m, −68 mV.

Figure 9. Effect of repetitive stimulation near threshold or at supramaximal levels at different interpulse intervals

Aa, voltage responses (upper trace) elicited upon cessation of conditioning hyperpolarizations induced by repetitive application of brief current pulses (lower trace). Ab, expanded voltage record segments corresponding to regions of the record in Aa marked by numbered arrows with traces 1–3 exemplifying sub-threshold initial responses and trace 4 a larger initial response and a ‘triggered’ regenerative response (□). Ac, expanded records of the three sub-threshold responses shown in Ab with traces offset for clarity. B, repetitive activation of regenerative responses (upper traces) induced by brief current pulses (lower traces) applied at interpulse intervals of 20, 15 and 10 s (same continuous recording). Arrowheads mark passive depolarizations where there were no regenerative responses and a minimal initial response. Nifedipine (1 μM) was present throughout. V_m (mV): A, −69; B, −59 mV.

Figure 10. The effects of Ca²⁺-free Cd²⁺-, BAPTA AM- and CPA-containing solutions on voltage-induced responses

A, voltage responses (upper traces) to current injection (lower traces) before and during brief exposure to nominally Ca²⁺-free physiological saline containing 0.3 mM CdCl₂. Responses of a strip prior to and during application of 20 μM BAPTA AM (B) and after 13 min exposure to BAPTA (C), and in another strip after 25 min exposure to this same solution but in the absence of nifedipine (D), depolarization now inducing repetitive L-type Ca²⁺ channel-mediated spikes. E, responses of a strip prior to and during application of 16 μM CPA. Dashed lines in a, b and E denote control V_m for each strip. Nifedipine (1 μM) was used throughout except for D. V_m (mV): A, −61; B, −53; C, −50; D, −61; E, −50. Voltage and current scale bars in E apply to all respective records.

Figure 11. Effects of caffeine, ryanodine, ACh and heparin on voltage-induced responses

A and B, caffeine (caff, 1 mM) applied to two strips reversibly and rapidly (both records < 1 min in caffeine) inhibited the initial and regenerative responses, whereas responses persisted after 20 and 25 min exposure to 16 and 20 μM ryanodine (ryan), respectively. Dashed lines denote V_m in control (con) for each strip. C, responses in control, 0.1 and 0.5 μM ACh (records are the mean of 6–9 responses from a continuous recording in the same strip). D and E, voltage-induced responses in paired control (D) and heparin-loaded (E) strips. Nifedipine (1 μM) was present throughout. V_m (mV): A, −60, B, −63, C, −62, D, −59, E, −51. Voltage (upper records) and current (lower records) scale bars in E apply to all respective records. Time bar in E also applies to C and D.

Figure 12. Components proposed to underlie the generation of the SW action potential

The schema presented here shows a positive voltage change (ΔV⁺) across the cell membrane acting at an unknown functional site(s) such as agonist receptors, G proteins or PLC to activate PLC and initiate synthesis of IP₃. IP₃ activates IP₃ receptor-operated Ca²⁺ release channels in the sarcoplasmic reticulum, with the Ca²⁺ activating a Ca²⁺-activated inward current across the plasmalemma, causing further depolarization. Both IP₃ and Ca²⁺ release can activate adjacent Ca²⁺ release channels which above threshold levels can initiate regenerative Ca²⁺ release and a resultant action potential. This process is likely to be terminated by high Ca²⁺ concentrations acting on the low affinity inhibitory site (-) of the IP₃ receptor.

Figure 13. Comparison of spontaneous and voltage-evoked activities

A, recordings from a strip that exhibited SWs (□) at low frequency in which nifedipine-insensitive action potentials (○) were induced by application of hyperpolarizing pulses. B, mean of seven voltage-induced action potentials (○) and eight SWs (□). C, comparison of a sub-threshold pacemaker potential (arrow 1) and a sub-threshold voltage-induced initial response (arrow 2). D, numbered records of C presented in an expanded form. All records were obtained from the same continuous recording. Nifedipine (1 μM) was present throughout. V_m, −53 mV.