Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABA(B) receptors, but not α2 adrenergic receptors

Abstract

GABA(B) , μ-opioid and adrenergic α(2) receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABA(B) receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high-frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABA(B) agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low-frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α(2) adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low-frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABA(B) receptors, but not by α(2) receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels.

Fig. 1. Confocal images of NK1R neurons in lamina I after noxious stimulation of the hind paw in vivo

Rats received a noxious stimulus (clamp for 30 sec) in the hind paw to induce substance P release and were fixed 10 min later for NK1R immunohistochemistry. Images were taken from sections of the L4–L5 spinal segments. A, B: Rats received an intrathecal injection of saline 10 min before the stimulus; A and B correspond to the contralateral and ipsilateral dorsal horns, respectively, of the same histological section. B: Rats received an intrathecal injection of DAMGO (2 nmol) 10 min before the stimulus; ipsilateral dorsal horn. C: Rats received intrathecal injections of DAMGO (2 nmol) and PP1 (10 nmol) 10 min before the stimulus and of PP1 (10 nmol) 70 min before the stimulus; ipsilateral dorsal horn. Main panels: images taken with a 10x objective, with a voxel size of 830 × 830 × 5983 nm and 2 confocal planes. Insets: images of lamina I neurons taken with a 63x objective, with a voxel size of 132 × 132 × 383 nm and 3 confocal planes. Neurons with NK1R internalization are indicated with “*” and neurons without internalization by “o”. Scale bars (in panel D) are 100 μm for the main panels and 10 μm for the insets.

Fig. 2. NK1R internalization induced by 100 Hz dorsal root stimulation of spinal cord slices: SFK inhibitors reversed the inhibition by DAMGO and baclofen

Spinal cord slices (n=3–4) were stimulated at the dorsal root at 100 Hz to induce substance P release. A. Slices were superfused with aCSF (control), 10 μM PP1, 1 μM DAMGO or DAMGO + PP1. B. Slices were superfused with aCSF, 30 μM baclofen, baclofen + 10 μM PP1 or baclofen + 10 μM PP2. Treatments with PP1 and PP2 were preceded by 1 hr preincubation with these drugs at 10 μM. Two-way ANOVA: p<0.0001 for stimulus and drugs. Bonferroni’s post-hoc test: *, p<0.05, ***, p<0.001 compared to control; †††, p<0.001 as indicated.

Fig. 3. NK1R internalization induced by 1 Hz dorsal root stimulation of spinal cord slices: a SFK inhibitor did not reverse the inhibition by DAMGO

Spinal cord slices were stimulated at the dorsal root at 1 Hz to induce substance P release while being superfused with the peptidase inhibitors captopril (10 μM) and thiorphan (10 μM) alone (control), with 1 μM DAMGO or with 1 μM DAMGO + 10 μM PP1. Treatment with PP1 was preceded by 1 hr preincubation with 10 μM PP1. Two-way ANOVA: p<0.0001 for stimulus, drugs and interaction. Bonferroni’s post-hoc test: ***, p<0.001 compared to control. Number of slices (n) for each data set is given inside the columns.

Fig. 4. SFK inhibitors reverse the inhibition by DAMGO and baclofen of NK1R internalization induced by noxious stimulation in vivo

Rats (n=3) received an i.t. injection of 3 nmol PP1 (PP1*: 10 nmol), 3 nmol PP3, or no injection. One hour later they received an i.t. injection of DAMGO (2 nmol) or baclofen (50 nmol) with or without PP1 or PP3, as indicated. Noxious stimulation (hindpaw clamp for 30 sec) was delivered 10 min after the second injection, and rats were fixed 10 min later. NK1R internalization was measured in the L4–L5 spinal segments. Two-way ANOVA: p<0.0001 for ‘drugs’ and ‘stimulus’. Bonferroni’s post-hoc test: ***, p<0.001, **, p<0.01 compared to control; †††, p<0.001 as indicated.

Fig. 5. Inhibition of NK1R internalization by α₂ adrenergic agonists in spinal cord slices

Spinal cord slices were stimulated at the dorsal root to evoke substance P release. The α₂ agonists medetomidine and guanfacine were superfused to the slices at the concentrations indicated. PP1 (10 μM) was applied in a 1 hr preincubation and then added to guanfacine in the superfusate. Number of slices (n) for each data set is indicated by numbers inside the columns. A. 100 Hz stimulation; two-way ANOVA: p<0.0001 for ‘stimulus’, p=0.25 for ‘drugs’, p=0.66 for their interaction. B. 1 Hz stimulation in the presence of peptidase inhibitors (captopril and thiorphan, 10 μM); two-way ANOVA: p<0.0001 for ‘stimulus’, p=0.0002 for ‘drugs’, p=0.0002 for their interaction. Bonferroni’s post-hoc tests: **, p<0.01, ***, p<0.001 compared to control.

Fig. 6. Mechanisms of inhibition of substance P release from primary afferent terminals

Substance P (SP) and glutamate (Glu) are released when Ca²⁺ enters the terminal through Ca(V) channels. MORs, GABA_B receptors (GABA_BRs) and α_2A receptors inactivate (-) Ca(V)2.2 through Gβ γ. MORs and GABA_BRs also inactivate Ca(V)2.2 by Tyr (Y)-phosphorylation by SFKs. NE: norepinephrine; enk: enkephalin.