Alpha2-adrenoceptors couple to inhibition of R-type calcium currents in myenteric neurons

Abstract

Alpha2-adrenoceptors inhibit Ca2+ influx through voltage-gated Ca2+ channels throughout the nervous system and Ca2+ channel function is modulated following activation of some G-protein coupled receptors. We studied the specific Ca2+ channel inhibited following alpha2-adrenoceptor activation in guinea-pig small intestinal myenteric neurons. Ca2+ currents (I(Ca2+)) were studied using whole-cell patch-clamp techniques. Changes in intracellular Ca2+ (delta[Ca2+]i) in nerve cell bodies and varicosities were studied using digital imaging where Ca2+ influx was evoked by KCl (60 mmol L(-1)) depolarization. The alpha2-adrenoceptor agonist, UK 14 304 (0.01-1 micromol L(-1)) inhibited I(Ca2+) and delta[Ca2+]i; maximum inhibition of I(Ca2+) was 40%. UK 14 304 did not affect I(Ca2+) in the presence of SNX-482 or NiCl2 (R-type Ca2+ channel antagonists). UK 14 304 inhibited I(Ca2+) in the presence of nifedipine, omega-agatoxin IVA or omega-conotoxin, inhibitors of L-, P/Q- and N-type Ca2+ channels. UK 14 304 induced inhibition of I(Ca2+) was blocked by pertussis toxin pretreatment (1 microg mL(-1) for 2 h). Alpha2-adrenoceptors couple to inhibition of R-type Ca2+ channels via a pertussis toxin-sensitive pathway in myenteric neurons. R-type channels may be a target for the inhibitory actions of noradrenaline released from sympathetic nerves on to myenteric neurons.

Figure 1

Inhibitory effect of UK 14,304 on I_Ca²⁺. A, Activation of α₂-adrenoceptors with UK 14,304 inhibited I_Ca²⁺. I_Ca²⁺ was activated by depolarizing the membrane potential from −70 mV to −10 mV. B, Concentration-response relationship for UK 14,304 induced inhibition of I_Ca²⁺. Inhibition of I_Ca²⁺ is plotted as amplitude in the presence of each concentration of UK 14,304 normalized to the control I_Ca²⁺. Points represent the mean ± s.e.m. of data from 4−7 cells.

Figure 2

Inhibitory effect of UK 14,304 on I_Ca²⁺ in the presence of the L-type channel antagonist, nifedipine. A, Representative recordings of I_Ca²⁺ showing a reduction by nifedipine and a further inhibition by addition of UK 14,304. B, Effect of UK14,304 (1 μM) to I_Ca²⁺ in the presence of nifedipine (1 μM). Currents recorded in the presence of nifedipine and UK 14,304 were normalized to the control current in the same neuron. The inhibitory effects of UK 14,304 were reversible on washing with nifedipine-containing Krebs’ solution (n = 4). I_Ca²⁺ was activated by depolarizing the membrane potential from −70 mV to −10 mV.

Figure 3

Inhibitory effect of UK 14,304 on I_Ca²⁺ in the presence of ω-agatoxin IVA (ATX), a P/Q-type channel toxin. A, UK 14,304 inhibited I_Ca²⁺ in the presence ATX. I_Ca²⁺ was activated by depolarizing the membrane potential from −70 mV to −10 mV. B, UK14,304 inhibits I_Ca²⁺ in the presence of ATX. Currents recorded in the presence of ATX and UK 14,304 were normalized to the control current in the same neuron. ATX alone reduced I_Ca²⁺ significantly (P < 0.05). In the presence of ATX, UK 14,304 (1 μM) further inhibited I_Ca²⁺ (P < 0.05). The inhibitory effect of UK 14,304 was reversible by washing with ATX-containing Krebs’ solution (n = 4).

Figure 4

Inhibitory effect of UK 14,304 on I_Ca²⁺ in the presence of an N-type Ca²⁺ channel toxin. A, UK 14,304 inhibited I_Ca²⁺ in the presence of CTX. I_Ca²⁺ was activated by depolarizing the membrane potential from −70 mV to −10 mV. B, Inhibitory effect of UK 14,304 on I_Ca²⁺ in the presence of CTX. CTX inhibited I_Ca²⁺ (P < 0.05) and UK 14.304 (1 μM) produced a further reduction in current amplitude (P < 0.05, n = 8). Currents recorded in the presence of CTX with 14,304 were normalized to the current recorded in the presence of CTX in the same neuron.

Figure 5

UK 14,304 does not inhibit I_Ca²⁺ in the presence of R-type Ca²⁺ channel blockers. A and C, Original recordings of I_Ca²⁺ show that UK 14,304 did not alter I_Ca²⁺ in the presence NiCl₂ (A) or SNX-482 (C). B and D, Lack of effect of UK14,304 on I_Ca²⁺ in the presence of R-type Ca²⁺ channel toxins. I_Ca²⁺ was inhibited by NiCl₂ (B, P < 0.05, n = 6) and SNX-482 (D, P < 0.05, n = 5). In the presence of NiCl₂ or SNX-482 (D, n = 5), UK 14,304 did not further reduce I_Ca²⁺ (P > 0.05). In both cases, washing with blocker free Krebs’ solution restored I_Ca²⁺ (n = 3 for NiCl₂ in B, n = 4 for SNX 482 in D). I_Ca²⁺ in the presence of blockers was normalized to the current amplitude recorded under control conditions.

Figure 6

PTX blocks the inhibitory effect of UK 14,304 on I_Ca²⁺. A, The inhibitory effect of UK 14,304 (1 μM) on I_Ca²⁺ was blocked by PTX. I_Ca²⁺ activated by depolarizing the membrane potential to − 10 mV from a holding potential of −70 mV. B, PTX pretreatment blocks UK 14,304-induced inhibition of I_Ca²⁺. I_Ca²⁺ was plotted as the mean current normalized to current amplitude before UK 14,304 application. Data are mean ± s.e.m. (n = 5).

Figure 7

Δ[Ca²⁺]_i caused be elevated KCl (60 mM) in cell soma and in varicose nerve fibers. A-C, Images of Fluo-4 fluorescence in two neurons and in a varicose nerve fiber. A shows baseline fluorescence, B shows Δ[Ca²⁺]_i in the presence of KCl (60 mM) and C shows the Δ[Ca²⁺]_i response in the presence of tetrodotoxin (0.3 μM, TTX). Arrows in “B” show the position of varicosities. D, Mean data from 3 experiments similar to that shown in A-C. Data were obtained from 6 neurons and 14 varicosities. TTX significantly reduced Δ[Ca²⁺]_i but did not block this response (P < 0.05, Student's t-test for paired data). These data indicate axonal conduction contributed to the Δ[Ca²⁺]_i response axonal conduction was not an absolute requirement. The Δ[Ca²⁺]_i response was due largely to depolarization-induced activation of voltage-gated Ca²⁺ channels in the cell soma and in varicosities.

Figure 8

Time control study for stability of Δ[Ca²⁺]_i during 3 successive KCl (60 mM) applications. Data are Δ[Ca²⁺]_i caused by KCl depolarization at 10 minute intervals. Data were normalized to the first Δ[Ca²⁺]_i response and are mean + s.e.m. Δ[Ca²⁺]_i did not decline significantly over the time course of this protocol (P > 0.05).

Figure 9

Inhibitory effect of UK 14,304 on Δ[Ca²⁺]_i. A, Recordings Δ[Ca²⁺]_i before and during application of KCl (60 mM) in the absence and presence of UK 14,304 (1 μM) and CdCl₂ (100 μM). UK 14,304 reduced Δ[Ca²⁺]_i in both soma (upper traces) and a varicosity (lower traces). Δ[Ca²⁺]_i evoked by KCl depolarization was blocked CdCl₂. B, Pooled data from experiments similar to that shown in “A”. Control measurements made in individual soma or varicosities were set to a value of “1” and data obtained in the presence of UK 14,304 or CdCl₂ were expressed as a fraction of that control value. Data are mean ± s.e.m. *indicates significantly different from Control (P <0.05). #indicates significantly different from measurements made in the presence of UK 14,304 (P <0.05).

Figure 10

UK 14,304 does not inhibit on Δ[Ca²⁺]_i in the presence of NiCl₂ in soma and varicosities. A, Original recordings of Δ[Ca²⁺]_i evoked by KCl (60 mM). NiCl₂ (50 μM) inhibited Δ[Ca²⁺]_i in the soma (upper tracings) and varicosities (lower traces). Subsequent application of UK 14,304 (1 μM) did not alter Δ[Ca²⁺]_i evoked by KCl depolarization. B, Pooled from experiments illustrated in “A”. NiCl₂ inhibited Δ[Ca²⁺]_i evoked by KCl in the soma and in varicosities while subsequent addition of UK 14,304 did not further reduce Δ[Ca²⁺]_i evoked by KCl depolarization. Control measurements made in individual soma or varicosities were set to a value of “1” and data obtained in the presence of UK 14,304 or NiCl₂ were expressed as a fraction of that control value. Data are mean ± s.e.m. *indicates significantly different from Control (P <0.05).

Figure 11

UK 14,304 does not inhibit on Δ[Ca²⁺]_i in the presence of SNX-482 in soma and varicosities. Original recordings of Δ[Ca²⁺]_i evoked by 60 mM KCl. SNX-482 (0.1 μM) inhibited Δ[Ca²⁺]_i in a soma (upper traces) and a varicosity (lower traces). Further application of UK 14,304 (1 μM) did not alter Δ[Ca²⁺]_i evoked by KCl depolarization.