Small-Conductance Ca2+-Activated K+ Channel 2 in the Dorsal Horn of Spinal Cord Participates in Visceral Hypersensitivity in Rats

Abstract

Visceral hypersensitivity is a highly complex and subjective phenomenon associated with multiple levels of the nervous system and a wide range of neurotransmission. The dorsal horn (DH) in spinal cord relays the peripheral sensory information into the brain. Small conductance Ca²⁺-activated K⁺ (SK) channels regulate neuronal excitability and firing by allowing K⁺ to efflux in response to increase in the intracellular Ca²⁺ level. In this study, we examined the influence of SK2 channels in the spinal DH on the pathogenesis of visceral hypersensitivity induced by colorectal distension (CRD) in rats. Electrophysiological results showed that rats with visceral hypersensitivity presented a decrease in the SK channel-mediated afterhyperpolarization current (I_AHP), and an increase in neuronal firing rates and c-Fos positive staining in the spinal DH. Western blot data revealed a decrease in the SK2 channel protein in the membrane fraction. Moreover, intrathecal administration of the SK2 channel activator 1-EBIO or CyPPA alleviated visceral hypersensitivity, reversed the decrease in I_AHP and the increase in neuronal firing rates in spinal DH in rats that experienced CRD. 1-EBIO or CyPPA effect could be prevented by SK2 channel blocker apamin. CRD induced an increase in c-Fos protein expression in the spinal DH, which was prevented by 1-EBIO. Together, these data suggest that visceral hypersensitivity and pain is associated with a decrease in the number and function of membrane SK2 channels in the spinal DH. Pharmacological manipulation of SK2 channels may open a new avenue for the treatment of visceral hypersensitivity and pain. Highlights: -Neonatal colorectal distension induced visceral hypersensitivity in rats.-Visceral hypersensitivity rats presented a decrease in afterhyperpolarization current (I_AHP) and membrane SK2 channel protein in the spinal dorsal horn.-Visceral hypersensitivity rats presented an increase in neuronal firing rate in the spinal dorsal horn.-Intrathecal administration of SK2 channel activator 1-EBIO or CyPPA prevented visceral hypersensitivity and decrease in I_AHP.

Keywords: colorectal distension; rats; small-conductance Ca2+-activated K+ channel 2; spinal dorsal horn; visceral hypersensitivity.

FIGURE 1

Adult CRD precipitates the visceral hypersensitivity in rats that experienced neonatal CRD. (A) Timeline for CRD and behavioral test in rats. Neonatal CRD were conducted on postnatal days 8, 10, and 12. Adult CRD and behavioral test occurred between weeks 8 and 12. (B) The pain thresholds of rats that experienced neonatal CRD decreased significantly at 1 and 2 h after re-exposed to adult CRD compared with that in sham group (p < 0.01, n = 6 per group). (C) The pain thresholds of rats that experienced neonatal CRD decreased consistently after re-exposed to adult CRD and lasted for 14 days compared with that in sham group (p < 0.01, n = 6 per group). (D) Neonatal CRD rats presented a significant decrease in pain threshold compared with naïve rats or rats that experienced either neonatal or adult CRD (p < 0.01; n = 8 per group). (E) Representative EMG recordings at 20, 40, 60, and 80 mmHg CRD in rats at different condition. Data are expressed as mean ± SEM. ^∗∗p < 0.01.

FIGURE 2

SK2 Channel protein in membrane fraction and I_AHP are reduced in rats that experienced neonatal CRD. (A) Spinal SK2 channel in membrane fraction presented a significant decrease in rats that experienced neonatal and adult CRD compared with that in rats received adult CRD alone (p < 0.05, n = 3 per group). (B) There was no difference in total spinal SK2 channel protein in rats that experienced neonatal or adult CRD as compared to sham rats (p > 0.05, n = 7 per group). (C) The average peak amplitude of I_AHP in neonatal CRD rats significantly reduced compared with that in naïve rats (p < 0.01, n = 16–21 neurons, six rats per group). (D) The spontaneous neuronal firing rate in spinal DH increased significantly in neonatal CRD rats compared with that in naïve rats (p < 0.05, n = 9–10 neurons, five rats per group). (E) 1-EBIO can increase the amplitude of I_AHP in neonatal CRD rats (p < 0.01, n = 10 neurons per group). (F) 1-EBIO can decrease the neuronal firing rate in the spinal DH in rats that experienced neonatal CRD (p < 0.01, n = 10 neurons per group). Data are expressed as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.

FIGURE 3

SK channel activator 1-EBIO prevents the decrease in pain threshold and increase in c-Fos protein expression in rats that experienced neonatal and adult CRD. (A) 1-EBIO prevented the decrease in pain threshold in rats that experienced neonatal and adult CRD (p < 0.01, n = 6 per group). (B) Apamin blocked the effect of 1-EBIO (n = 6). (C) 1-EBIO can prevent neonatal and adult CRD induced c-Fos overexpression (p < 0.01, n = 6 per group). (D) c-Fos immunostaining. Scale bar = 100 μm. Data are expressed as mean ± SEM. ^∗∗p < 0.01.

FIGURE 4

SK2 channel activator CyPPA and blocker apamin affect I_AHP and pain threshold. (A) CyPPA increased the amplitude of I_AHP in rats that experienced neonatal CRD (p < 0.01, n = 10 neurons per group). (B) CyPPA decreased the neuronal firing rate in the spinal DH in rats that experienced neonatal CRD (p < 0.01, n = 14 neurons per group). (C) CyPPA prevented the decrease in pain threshold in rats that experienced neonatal and adult CRD, which could be prevented by apamin (p < 0.01, n = 5 per group). (D) Apamin decreased the amplitude of I_AHP in rats that experienced neonatal CRD (p < 0.01, n = 6 neurons per group). (E) Apamin induced a decrease in membrane SK2 channel protein level (p < 0.01, n = 6 neurons per group). (F) Apamin promoted a decrease in pain threshold in rats that experienced neonatal or adult CRD (p < 0.01, n = 5 per group). Data are expressed as mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01.