R-Type Ca2+ channels couple to inhibitory neurotransmission to the longitudinal muscle in the guinea-pig ileum

Abstract

What is the central question of this study? Subtypes of enteric neurons are coded by the neurotransmitters they synthesize, but it is not known whether enteric neuron subtypes might also be coded by other proteins, including calcium channel subtypes controlling neurotransmitter release. What is the main finding and its importance? Our data indicate that guinea-pig ileum myenteric neuron subtypes may be coded by calcium channel subtypes. We found that R-type calcium channels are expressed by inhibitory but not excitatory longitudinal muscle motoneurons. R-Type calcium channels are also not expressed by circular muscle inhibitory motoneurons. Calcium channel subtype-selective antagonists could be used to target subtypes of neurons to treat gastrointestinal motility disorders. There is evidence that R-type Ca²⁺ channels contribute to synaptic transmission in the myenteric plexus. It is unknown whether R-type Ca²⁺ channels contribute to neuromuscular transmission. We measured the effects of the nitric oxide synthase inhibitor nitro-l-arginine (NLA), Ca²⁺ channel blockers and apamin (SK channel blocker) on neurogenic relaxations and contractions of the guinea-pig ileum longitudinal muscle-myenteric plexus (LMMP) in vitro. We used intracellular recordings to measure inhibitory junction potentials. Immunohistochemical techniques localized R-type Ca²⁺ channel protein in the LMMP and circular muscle. Cadmium chloride (pan-Ca²⁺ channel blocker) blocked and NLA and NiCl₂ (R-type Ca²⁺ channel blocker) reduced neurogenic relaxations in a non-additive manner. Nickel chloride did not alter neurogenic cholinergic contractions, but it potentiated neurogenic non-cholinergic contractions. Relaxations were inhibited by apamin, NiCl₂ and NLA and were blocked by combined application of these drugs. Relaxations were reduced by NiCl₂ or ω-conotoxin (N-type Ca²⁺ channel blocker) and were blocked by combined application of these drugs. Longitudinal muscle inhibitory junction potentials were inhibited by NiCl₂ but not MRS 2179 (P2Y₁ receptor antagonist). Circular muscle inhibitory junction potentials were blocked by apamin, MRS 2179, ω-conotoxin and CdCl₂ but not NiCl₂ . We conclude that neuronal R-type Ca²⁺ channels contribute to inhibitory neurotransmission to longitudinal muscle but less so or not all in the circular muscle of the guinea-pig ileum.

Keywords: calcium channel; nitric oxide; purinergic signaling; synaptic transmission.

Fig. 1

Representative traces showing neurogenic responses after transmural electrical field stimulation of the guinea pig LMMP in vitro. A top, Relaxations were induced in the presence of histamine (1 μM) to increase tone and scopolamine (1 μM) to block muscarinic receptors. Nerve stimulation (ns, 20 Hz, 1 s) caused a relaxation followed by a noncholinergic contraction. A, bottom. The relaxation and contraction were blocked by the non-selective Ca²⁺ channel blocker CdCl₂ (100 μM). B, Inhibition of the relaxation by CdCl₂ was concentration dependent (n=6). The curve was fit to the data points using a 4-parameter (max, min, slope, EC₅₀) non-linear logistic function.

Fig. 2

NiCl₂ inhibits neurogenic relaxations but not neurogenic cholinergic or noncholinergic contractions of the LMMP. A, NiCl₂ and NLA produced a concentration-dependent inhibition of the relaxation evoked by nerve stimulation (20 Hz, 1 s). The control relaxation was approximately 40% of histamine-induced tone. This relaxation was reduced by ~50% by NLA or NiCl₂. Combined application of NiCl₂ and NLA did not produce a greater inhibition then either drug alone (n = 8 for all groups). B, The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CTX, n = 3) and CdCl₂ (n = 6) blocked non-cholinergic contractions of the LMMP (20 Hz 1 s, scopolamine 1 μM present). NiCl₂ produced a concentration dependent increase in the amplitude of the NANC contraction (n = 3). C, Representative recording of contractions of the LMMP evoked by single electrical stimuli. Addition of NiCl₂ did not affect contraction amplitude while subsequent addition of the muscarinic receptor antagonist, scopolamine blocked these contractions completely confirming that they were mediated by nerve released acetylcholine. D, NiCl₂ did not inhibit LMMP contractions caused by single shocks (0.1 Hz, n = 6). These contractions were blocked completely by the muscarinic antagonist scopolamine (not shown). CdCl₂ produced a concentration- dependent inhibition of the neurogenic cholinergic contraction (n = 6).

Fig. 3

Potentiation of LMMP non-cholinergic contractions. A, Non-cholinergic contractions (scopolamine 1 μM present) were evoked by a train of nerve stimulation (ns, 20 Hz, 1 s). NiCl₂ (50 μM) increased the peak amplitude and duration and subsequent addition of NLA (100 μM) did not produce any further increase. However, addition of the SK channel blocker apamin (0.1 μM) further increased contraction amplitude and duration. B, Quantitative data for experiment illustrated in “A”. C, Data from experiment similar to that shown in “A” except the sequence of drug application was altered with NLA application preceding NiCl₂ followed by apamin. NLA increased the contraction while addition of NiCl₂ did not increase the contraction further. Apamin did produce an addition increased in contraction amplitude and duration. D, Sequence of drug application was apamin followed by addition of NLA followed by addition of NiCl₂. Apamin and then NLA produced sequential increased in the contraction while NiCl₂ did not produce a further increase. For all figures * indicates P < 0.05, n=10 for each experiment. All data analyzed by one way ANOVA and Tukey’s post hoc test.

Fig. 4

Nitrergic and SK channel mediated components of the neurogenic relaxation. A, Representative experiment showing the effect of NLA/NiCl₂ (100 μM/50 μM) and apamin (0.1 μM) and on the neurogenic relaxation. B, Quantification of the data for experiment illustrated in “A”. Control relaxation was approximately 60% of histamine-induced tone. Addition of NLA/NiCl₂ caused approximately 30% reduction of the peak relaxation amplitude and cumulative addition of apamin abolished the remaining response (n = 7; *P <0.05). C, Similar experiment as shown in A and B but apamin was applied first. Apamin produced 21% reduction of peak relaxation amplitude and this effect was significant when compare to control (n=12; *P<0.05). Cumulative application of NLA/NiCl₂ abolished the apamin-resistant relaxation (*P<0.05). All data analyzed by one way ANOVA and Tukey’s post hoc test.

Fig. 5

Contributions of P2Y₁ receptors, nitric oxide, N-type and R-type Ca²⁺ channels to neurogenic relaxation of the longitudinal muscle. A, The P2Y₁ receptor antagonist, MRS2179 (10 μM) did not affect the neurogenic relaxation while NLA (100 μM) inhibited the relaxation (*P < 0.05 vs Control and MRS 2179). Apamin (0.1 μM) produced a further reduction in relaxation amplitude although this was not different from NLA alone (*P < 0.05 vs Control and MRS 2179, n=7). B, NLA alone inhibited the neurogenic relaxation while cumulative application of NiCl₂ and ω-CTX (0.1 μM) did not produce any further inhibition of the relaxation (*P<0.05 vs. Control). C, Cumulative addition of MRS 2179 followed by NiCl₂ did not alter the neurogenic relaxation while subsequent addition of ω-CTX reduced the relaxation by ~52% (*P<0.05 vs. NiCl₂, n=7). D, MRS 2179 did not inhibit the neurogenic relaxation while subsequent cumulative application of ω-CTX reduced the relaxation by 56% while cumulative addition of NiCl₂ reduced the relaxation by an additional 40% (*P< 0.05, n=7). All data analyzed by one way ANOVA and Tukey’s post hoc test. In these experiments, the Y axis represents % of the control (maximal) relaxation (in the absence of antagonist drugs) as the % relaxation normalized to histamine- induced tone was greater than 100% in some tissues.

Fig. 6

NiCl₂ inhibits inhibitory (IJP) but not excitatory (EJP) junction potentials recorded from longitudinal smooth muscle cells. A, Representative recordings of an IJP and EJP from a longitudinal smooth muscle cell. NiCl₂ (50 μM) inhibits the IJP but not the EJP. B, Summary data showing NiCl₂ inhibits the IJP (n = 4). C, NiCl₂ does not inhibit the EJP (n=5). D, The P2Y₁ receptor antagonist, MRS 2179, did not alter the IJP but reduced the EJP by ~50% (n=6). All data in this figure were analyzed using a paired t-test (*P < 0.05 vs. control).

Fig. 7

CdCl₂, ω-CTX but not NiCl₂ inhibit IJPs recorded from circular smooth muscle cells. A, IJPs were evoked by a 10 Hz, 1 s stimulus train. These IJPs were blocked by tetrodotoxin (TTX, 0.3 μM) the P2Y₁ receptor antagonist, MRS2179 (10 μM) and by the SK channel blocker, apamin (0.1 μM). The stimulus artifacts have been truncated for clarity. B, Concentration inhibition curves for the N-type Ca²⁺ blocker ω-CTX, and CdCl₂ and NiCl₂. NiCl₂ (up to 50 μM) did not inhibit the IJP (n=4 for each drug). C. Circular muscle IJPs evoked by 5 Hz stimulation are not affected by NiCl₂ (50 μM) or NLA (100 μM). D, NiCl₂ or NiCl₂ plus NLA did not change the amplitude of the single stimuli IJP (n=9). Subsequent addition of apamin (0.1 μM) inhibited the IJP (n=4, *P<0.05 vs. Control).

Fig. 8

Immunohistochemical localization of Ca_V2.3 and neuronal NOS. A, Image shows distribution of Ca_V2.3-immunoreactive varicose nerve fibers in myenteric ganglia and in the tertiary plexus (tp) that supplies the longitudinal muscle. B,C,D, High magnification confocal images of a single nerve fiber in the tertiary plexus that co-expresses the α1E subunit of Ca_V2.3 and nNOS-immunoreactivity. E,F,G, Localization of NOS-immunoreactivity and α1E subunit of Ca_V2.3 immunoreactivity in the deep muscular plexus of the circular muscle. NOS and Ca_v2.3 α1E immunoreactivity are localized to different nerve fibers in the deep muscular plexus. H, Ca_V2.3 subunit in the myenteric plexus of the mouse colon. There are brightly fluorescent varicose nerve fibers that appear to encircle nerve cell bodies not labelled by the Ca_V2.3 antibody. I, Ca_V2.3 positive nerve fibers are not detected in myenteric ganglia in tissues from Ca_V2.3 KO mice. Insets show higher magnification images of α1E positive varicose nerve fibers in the ganglia from the WT but not Ca_V2.3 KO mouse.