Multiple muscarinic pathways mediate the suppression of voltage-gated Ca2+ channels in mouse intestinal smooth muscle cells

Abstract

Background and purpose: Stimulation of muscarinic receptors in intestinal smooth muscle cells results in suppression of voltage-gated Ca2+ channel currents (I(Ca)). However, little is known about which receptor subtype(s) mediate this effect.

Experimental approach: The effect of carbachol on I(Ca) was studied in single intestinal myocytes from M2 or M3 muscarinic receptor knockout (KO) and wild-type (WT) mice.

Key results: In M2KO cells, carbachol (100 microM) induced a sustained I(Ca) suppression as seen in WT cells. However, this suppression was significantly smaller than that seen in WT cells. Carbachol also suppressed I(Ca) in M3KO cells, but with a phasic time course. In M2/M3-double KO cells, carbachol had no effect on I(Ca). The extent of the suppression in WT cells was greater than the sum of the I(Ca) suppressions in M2KO and M3KO cells, indicating that it is not a simple mixture of M2 and M3 receptor responses. The G(i/o) inhibitor, Pertussis toxin, abolished the I(Ca) suppression in M3KO cells, but not in M2KO cells. In contrast, the G(q/11) inhibitor YM-254890 strongly inhibited only the I(Ca) suppression in M2KO cells. Suppression of I(Ca) in WT cells was markedly reduced by either Pertussis toxin or YM-254890.

Conclusion and implications: In intestinal myocytes, M2 receptors mediate a phasic I(Ca) suppression via G(i/o) proteins, while M3 receptors mediate a sustained I(Ca) suppression via G(q/11) proteins. In addition, another pathway that requires both M2/G(i/o) and M3/G(q/11) systems may be operative in inducing a sustained I(Ca) suppression.

Figure 3

Comparison of the inhibitory effect of carbachol (CCh) on the voltage-gated Ca²⁺ channel currents (I_Ca) in cells from wild-type (WT) and muscarinic receptor knockout (KO) mice. (A) Time courses of the change in the mean percentage suppression of I_Ca[the beginning of CCh (100 µM) application was taken as zero]. Each point indicates mean ± SEM. (B) The mean percentage suppression of I_Ca measured 30 s (four left-hand columns) and 3 min (four right-hand columns) after CCh application. Each column shows the mean with one SEM. In (A and B), the numbers of cells used are presented in parentheses. Asterisks in (B) represent statistically significant differences from the corresponding value in WT cells (P < 0.05). (C) An assumed sustained component of I_Ca suppression mediated by M₂/M₃ pathway. Each point was estimated by subtracting the average percentage suppression curve of I_Ca in both M₂KO and M₃KO cells from that in WT cells shown in (A).

Figure 1

Effects of carbachol (CCh) on the holding current and the voltage-gated Ca²⁺ channel currents (I_Ca) in a single longitudinal smooth muscle cell isolated from the small intestine of a wild-type (WT) mouse. I_Ca was elicited by a 30 ms step pulse to 0 mV from the holding potential of −60 mV at a frequency of 0.1 Hz. CCh (100 µM) was applied extracellularly during application of the depolarizing pulses. (A) CCh induced a cationic inward current (I_CCh) consisting of an initial transient component followed by a sustained component. Every sharp current deflection shows overlapped I_Ca and the capacitive current. (B) Time course of the change in I_Ca amplitude (○) in the cell plotted against time (the beginning of CCh application was taken as zero). Points 1–4 in the graph correspond to actual I_Ca records (1–4) in (C). The amplitude of I_Ca was measured as the difference from the current level (interrupted line) obtained by the depolarizing pulse in the presence of nicardipine (1 µM). In the following figures, measurements and plots of I_Ca amplitude were made in the same way.

Figure 2

Effects of carbachol (CCh) on the voltage-gated Ca²⁺ channel currents (I_Ca) in cells isolated from muscarinic receptor knockout (KO) mice. (A–D) Time courses of the change in I_Ca amplitude in cells from wild-type (WT) (A), M₂KO (B), M₃KO (C) and M₂/M₃-double KO mice (D). Points 1–3 in each graph correspond to actual I_Ca records in insets 1–3.

Figure 4

Effect of guanosine 5′-(γ-thio) triphosphate tetralithium salt (GTP-γ-S) on the voltage-gated Ca²⁺ channel currents (I_Ca) in cells from wild-type (WT) and muscarinic receptor knockout (KO) mice. GTP-γ-S (200 µM) was applied intracellularly via patch pipettes. The mean percentage suppression of I_Ca measured 10 min after the establishment of whole-cell clamp configuration are shown. Each column represents the mean with one SEM. The numbers of cells used are shown in parentheses.

Figure 5

Effect of YM-254890 on BK current (I_BK) in cells isolated from M₂KO mice. CCh (100 µM)- or caffeine (10 mM)-induced Ca²⁺ release events were detected as I_BK at the holding potential of 0 mV in M₂KO cells. Cells were dialysed with a K⁺-rich pipette solution containing 0.3% DMSO (control) or YM-254890 (1–10 µM). (A and B) Typical current traces from a control cell (A) and YM-254890 (3 µM)-dialysed cell (B). (C) Summary of CCh- and caffeine- induced I_BK in the presence or absence of YM-254890. The individual columns indicate the mean with one SEM. The numbers of cells used are shown in parentheses. Asterisks represent significant differences from the corresponding control values (P < 0.05). CCh, carbachol; DMSO, dimethyl sulphoxide; KO, knockout.

Figure 6

Effects of Pertussis toxin (PTX) and YM-254890 on the CCh-induced sustained I_Ca suppression in M₂KO cells. (A) Time courses of the change in the mean percentage suppressions of I_Ca mediated by CCh (100 µM) in PTX-untreated (control) and -treated cells (the beginning of CCh application was taken as zero). (C) Time courses of the change in the mean percentage suppressions of I_Ca in 0.3% DMSO-dialysed (control) and YM-254890 (3 µM)-dialysed cells. Each point indicates mean ± SEM. The numbers of used cells are shown in parentheses. (B and D) Summary of the mean percentage suppression of I_Ca measured 3 min after the CCh application in control, PTX-treated (B) or YM-254890-dialysed cells (D). Each column shows the mean with one SEM. The asterisk in (D) represents a significant difference from the corresponding control value (P < 0.05). PTX had no effect on the sustained I_Ca suppression, but YM-254890 markedly diminished it. CCh, carbachol; DMSO, dimethyl sulphoxide; I_Ca, voltage-gated Ca²⁺ channel currents; KO, knockout.

Figure 7

Effects of PTX and YM-254890 on the CCh-induced phasic I_Ca suppression in M₃KO cells. (A and C) Time courses of the change in the mean percentage suppression of I_Ca induced by CCh (100 µM) in PTX-treated (A) and YM-254890 (3 µM)-dialysed (C) M₃KO cells plotted in the same way as in Figure 6. (B and D) Summary of the mean percentage suppression of I_Ca measured 30 s after CCh application in PTX-treated (B) and YM-254890-dialysed (D) cells. Asterisk in (B) represents significant difference from the corresponding control value (P < 0.05). PTX abolished the phasic I_Ca suppression in M₃KO cells, but YM-254890 had no effect on this response. CCh, carbachol; I_Ca, voltage-gated Ca²⁺ channel currents; KO, knockout; PTX, Pertussis toxin.

Figure 8

Effects of PTX and YM-254890 on the CCh-induced I_Ca suppression in WT cells. (A and C) Time courses of the change in the mean percentage suppression of I_Ca induced by CCh (100 µM) in PTX-treated (A) and YM-254890 (3 µM)-dialysed (C) WT cells. For comparison, the averaged I_Ca suppression curves against time after CCh application in PTX-untreated M₂KO cells (Figure 6) and YM-254890-untreated M₃KO cells (Figure 7) were superimposed in (A and C) respectively. (B and D) Summary of the mean percentage suppression of I_Ca measured 30 s (left-hand columns) and 3 min (right-hand columns) after CCh application in PTX-treated (B) and YM-254890-dialysed (D) WT cells. Asterisks represent significant differences from the corresponding control value (P < 0.05). CCh, carbachol; I_Ca, voltage-gated Ca²⁺ channel currents; KO, knockout; PTX, Pertussis toxin; WT, wild-type.

Figure 9

Effects of EGTA on the CCh-induced I_Ca suppressions in M₂KO, M₃KO and WT cells. (A, C and E) Time courses of the change in the mean percentage suppression of I_Ca induced by CCh (100 µM) in 0.05 mM EGTA (control)- and 20 mM EGTA-dialysed cells from M₂KO (A), M₃KO (C) and WT (E) mice. In (E), the sum of the average I_Ca suppression curve in M₂KO (A) and M₃KO (C) control cells is also shown. (B, D and F) Summary of the mean percentage suppression of I_Ca measured 30 s or 3 min after CCh application in M₂KO (B), M₃KO (D) and WT (F) cells. The asterisk in (F) represents a significant difference (P < 0.05) from the corresponding control value. CCh, carbachol; I_Ca, voltage-gated Ca²⁺ channel currents; KO, knockout; WT, wild-type.

Figure 10

Assignment of muscarinic receptor subtypes and G proteins mediating the suppressions of L-type voltage-gated Ca²⁺ channels in intestinal smooth muscle cells. M₂ muscarinic receptors, via G_i/o proteins, induce a phasic I_Ca suppression, while M₃ receptors, via G_q/11 proteins, induced a transient I_Ca suppression due to Ca²⁺ release from intracellular stores and a sustained I_Ca suppression. In addition, M₂ and M₃ receptors can cooperatively suppress I_Ca. The M₂/M₃-mediated pathway involves both G_i/o and G_q/11 proteins and a Ca²⁺-dependent process to induce a sustained I_Ca suppression. The present results are consistent with two possible models for the M₂/M₃-mediated pathway; one is a M₂/M₃-synergistic model in which the M₃/G_q/11-mediated sustained suppression is potentiated by the M₂/G_i/o-mediated, Ca²⁺-dependent signal (A), and the other one is a M₂/M₃-complex model in which both the M₂ and M₃ receptor subtypes and the respective G proteins form a complex to operate a Ca²⁺-dependent signal and induce a sustained I_Ca suppression (B), as suggested for the activation mechanisms of muscarinic cationic channels (Sakamoto et al., 2007). I_Ca, voltage-gated Ca²⁺ channel currents.