Modulation of voltage-dependent Ba2+ currents in the guinea-pig gastric antrum by cyclic nucleotide-dependent pathways

Abstract

We have investigated whether the activation of cAMP- and cGMP-dependent pathways modifies the properties of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes using patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Both dibutyryl cAMP (db-cAMP, 0.1-1 mM), a membrane-permeable ester of cAMP, and isoproterenol, a selective beta-stimulant, inhibited I(Ba) in a concentration-dependent manner. Forskolin, but not dideoxy-forskolin, an inactive isomer of forskolin, inhibited the peak amplitude of I(Ba). In the presence of either Rp-cAMP or the PKA (cAMP-dependent protein kinase) inhibitor peptide 5-24 (PKA-IP), neither forskolin nor db-cAMP inhibited I(Ba). After establishing a conventional whole-cell recording, the peak amplitude of I(Ba) gradually decreased when the catalytic subunit of PKA was included in the pipette. The further application of Rp-cAMP reversibly enhanced I(Ba). Sodium nitroprusside (0.1-1 mM) and 8-Br-cGMP (0.1-1 mM) also inhibited I(Ba) in a concentration-dependent manner. The inhibitory effects of forskolin or db-cAMP on I(Ba) were not significantly changed by pretreatment with a cGMP-dependent protein kinase (PKG) inhibitor. Similarly, the inhibitory actions of 8-Br-cGMP on I(Ba) were not modified by PKA-IP. The membrane-permeable cyclic nucleotides db-cAMP and 8-Br-cGMP caused little shift of the voltage dependence of the steady-state inactivation and reactivation curves. Neither of the membrane-permeable cyclic nucleotides db-cAMP or 8-Br-cGMP had additive inhibitory effects on I(Ba). These results indicate that two distinct cyclic nucleotide-dependent pathways are present in the guinea-pig gastric antrum, and that both inhibited I(Ba) in an independent manner.

Figure 1

Effects of db-cAMP (1 mM) on I_Ba using conventional whole-cell recording from an isolated gastric antral myocyte. The upper four traces show (a) inward currents, elicited by voltage steps in control solution ((a) (i)), after the application of db-cAMP ((a) (ii)), following washout of db-cAMP ((a) (iii)) and in the presence of nifedipine ((a) (iv)). The cell capacitance was 51 pF. (b) The time course of inhibition of the peak amplitude of I_Ba by db-cAMP (1 mM) is shown. Time 0 indicates the time when db-cAMP was applied. The inhibition produced by db-cAMP was not reversed by washing with drug-free solution. The application of 10 μM nifedipine inhibited most of the db-cAMP-resistant current. The nifedipine-resistant current was suppressed by Cd²⁺ (100 μM). In each experiment, inward currents were elicited by voltage steps (1 s duration) to +10 mV from a holding potential of −70 mV every 20 s.

Figure 2

Effects of forskolin (30 and 100 nM) on I_Ba in a conventional whole-cell recording. (a) The time course of inhibition of the peak amplitude of I_Ba produced by the application of forskolin (30 and 100 nM) is shown. Time 0 indicates the time when 30 nM forskolin was applied. Inward current were elicited by voltage steps (1 s duration) to +10 mV from a holding potential of −70 mV every 20 s. The cell capacitance was 39 pF. (b) Examples of inward current traces recorded at the indicated points in (a) are shown in ((b) (i)–(iv)). (c) The time course of the current decay I_Ba was identical when the current traces of I_Ba in the absence and presence of forskolin (30 and 100 nM) were superimposed. (d) It shows the relationship between different concentrations of forskolin and the normalized peak amplitude of I_Ba when the peak amplitude of I_Ba was taken as one just before application of each concentration of forskolin; each column indicates the mean of 4–9 observations with +s.d. shown by vertical lines.

Figure 3

Effects of forskolin (100 nM) and isoproterenol (10 and 100 μM) on the peak amplitude of I_Ba using conventional whole-cell recording. (a) Effects of forskolin (100 nM) on the peak amplitude of I_Ba. The current–voltage relationships were obtained in the absence (control) or presence of 100 nM forskolin. The current amplitude was measured as the peak amplitude of I_Ba in each condition. The lines were drawn by eye. The cell capacitance was 43 pF. (b) Effects of isoproterenol on the peak amplitude of I_Ba. The current–voltage relationships were shown in the absence and presence of isoproterenol (10 and 100 μM). Isoproterenol also inhibited the peak amplitude of I_Ba evoked by depolarizing pulses (1 s duration) from −70 mV at levels more positive than −30 mV and the inhibitory effects of isoproterenol on I_Ba showed a voltage- and concentration dependency. The lines were drawn by eye. The cell capacitance was 52 pF.

Figure 4

Effects of forskolin on I_Ba in the presence of PKA inhibitors (Rp-cAMP and PKA-IP). The PKA inhibitor Rp-cAMP (100 μM) potentiated the amplitude of I_Ba and abolished the inhibitory effect of 100 nM forskolin. (a) Inward current were again elicited by voltage steps (1 s duration) to +10 mV from a holding potential of −70 mV every 20 s. Typical current traces are shown in (b), at the points ((i)–(iii)) indicated in (a). The cell capacitance was 36 pF. The change in the peak amplitude of I_Ba as a function of time caused by 100 nM forskolin when PKA-IP (1 μM) was included in the pipette solution is shown in (c). Time 0 indicates the time when a conventional whole-cell configuration was established. The cell capacitance was 34 pF.

Figure 5

Effects of dialysing cells with the catalytic subunit of PKA on I_Ba. The cell capacitance was 49 pF. (a) The time-dependent changes observed in I_Ba using patch pipette, which contained the catalytic subunit of PKA (125 U ml⁻¹), are shown. The ordinate scale shows the peak amplitude of I_Ba evoked by a depolarization pulse (1 s duration) from a holding potential of −70 mV every 20 s. The abscissa scale indicates the time after formation of a conventional whole-cell recording. (b) Sample traces shows I_Ba recorded at the indicated points ((i)–(iii)) in (a).

Figure 6

Effects of 8-Br-cGMP on I_Ba using conventional whole-cell recording. (a) The time course of changes in the peak amplitude of I_Ba was shown before and after application of 8-Br-cGMP (0.1 and 1 mM). Time 0 indicates the time when 0.1 mM 8-Br-cGMP was applied. The cell capacitance was 42 pF. (b) Similarly, it shows that the peak amplitude of I_Ba was little changed by the application of 8-Br-cGMP (0.1 and 1 mM) in the presence of Rp-8-pCPT-cGMP. Time 0 indicates the time when 10 μM Rp-8-pCPT-cGMP was applied. The cell capacitance was 59 pF. (c) The effects are summarized. It can be seen that the selective inhibitor of PKG only inhibited the effects of cGMP analogues, and that the selective inhibitor of PKA only inhibited the effects of cAMP, with no cross interaction being detected. Each column shows the mean of 3–5 observations with +s.d. shown by vertical lines. Asterisks indicate a statistically significant difference, demonstrated using a paired t-test (^**P<0.01).

Figure 7

Effects of db-cAMP (0.1 and 1 mM) on I_Ba using conventional whole-cell recording. (a) The time course of changes in the peak amplitude of I_Ba before and after application of db-cAMP (0.1 and 1 mM) when PKA-IP (1 μM) was included in the pipette solution. Time 0 indicates the time when 0.1 mM db-cAMP was applied. The cell capacitance was 36 pF. (b) Similarly, it shows that the peak amplitude of I_Ba was little changed by the application of db-cAMP (0.1 and 1 mM) in the presence of Rp-8-pCPT-cGMP. Time 0 indicates the time when 10 μM Rp-8-pCPT-cGMP was applied. The cell capacitance was 42 pF. (c) The effects are summarized. It can be seen that the selective inhibitor of PKA only inhibited the effects of cAMP analogues and that the selective inhibitor of PKG only inhibited the effects of cGMP, with no cross interaction being detected. Each column shows the mean of 3–6 observations with +s.d. shown by vertical lines. Asterisks indicate a statistically significant difference, demonstrated using a paired t-test (^*P<0.05, ^**P<0.01).

Figure 8

Effects of cyclic nucleotides (db-cAMP and 8-Br-cGMP) on the voltage-dependent activation and inactivation of I_Ba in guinea-pig antrum. Whole-cell recording, pipette solution Cs⁺-TEA⁺ solution containing 5 mM EGTA and the bath solution 10 mM Ba²⁺ containing 135 mM TEA⁺. Steady-state inactivation curves, obtained in the absence (control) and presence of cyclic nucleotides, were fitted to the Boltzmann equation. Peak current values were used. The steady-state inactivation curve was obtained using the double-pulse protocol (see Methods). The current measured during the test pulse is plotted against membrane potential and expressed as relative amplitude. Activation curves were obtained from the current–voltage relationships, fitting to the Boltzmann equation (see Methods). (a) The steady-state inactivation curves in the absence or presence of db-cAMP (1 mM) were drawn using the following values: (control), I_max=1.0, V_half=−38, k=7.0 and C=0.04; (db-cAMP, 1 mM), I_max=1.0, V_half=−39, k=7.0 and C=0.04. Each symbol indicates the mean of six observations with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol. (b) The steady-state inactivation curves in the absence or presence of 8-Br-cGMP (1 mM) were drawn using the following values: (control), I_max=1.0, V_half=−36, k=8.0 and C=0.06; (8-Br-cGMP, 1 mM), I_max=1.0, V_half=−37, k=7.8 and C=0.06. Each symbol indicates the mean of six observations with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol.