Hyperpolarization-activated cyclic nucleotide-gated cation channel subtypes differentially modulate the excitability of murine small intestinal afferents

AIM:To assess the role of hyperpolarization-activated cyclic nucleotide-gated cation(HCN) channels in regulating the excitability of vagal and spinal gut afferents.METHODS:The mechanosensory response of mesenteric afferent activity was measured in an ex vivo murine jejunum preparation.HCN channel activity was recorded through voltage and current clamp in acutely dissociated dorsal root ganglia(DRG) and nodose ganglia(NG) neurons retrogradely labeled from the small intestine through injection of a fluorescent marker(DiI).The isoforms of HCN channels expressed in DRG and NG neurons were examined by immunohistochemistry.RESULTS:Ramp distension of the small intestine evoked biphasic increases in the afferent nerve activity,reflecting the activation of low-and high-threshold fibers.HCN blocker CsCl(5 mmol/L) preferentially inhibited the responses of low-threshold fibers to distension and showed no significant effects on the high-threshold responses.The effect of CsCl was mimicked by the more selective HCN blocker ZD7288(10 μmol/L).In 71.4% of DiI labeled DRG neurons(n = 20) and 90.9% of DiI labeled NG neurons(n = 10),an inward current(I h current) was evoked by hyperpolarization pulses which was fully eliminated by extracellular CsCl.In neurons expressing I h current,a typical "sag" was observed upon injection of hyperpolarizing current pulses in current-clamp recordings.CsCl abolished the sag entirely.In some DiI labeled DRG neurons,the I h current was potentiated by 8-Br-cAMP,which had no effect on the I h current of DiI labeled NG neurons.Immunohistochemistry revealed differential expression of HCN isoforms in vagal and spinal afferents,and HCN 2 and HCN 3 seemed to be the dominant isoform in DRG and NG,respectively.CONCLUSION:HCNs differentially regulate the excitability of vagal and spinal afferent of murine small intestine.

Deletion of Gpr128 results in weight loss and increased intestinal contraction frequency

AIM:To generate a Gpr128 gene knockout mouse model and to investigate its phenotypes and the biological function of the Gpr128 gene.METHODS:Bacterial artificial chromosome-retrieval methods were used for constructing the targeting vector.Using homologous recombination and microinjection technology,a Gpr128 knockout mouse model on a mixed 129/BL6 background was generated.The mice were genotyped by polymerase chain reaction(PCR)analysis of tail DNA and fed a standard laboratory chow diet.Animals of both sexes were used,and the phenotypes were assessed by histological,biochemical,molecular and physiological analyses.Semi-quantitative reverse transcription-PCR and Northern blotting were used to determine the tissue distribution of Gpr128mRNA.Beginning at the age of 4 wk,body weights were recorded every 4 wk.Food,feces,blood and organ samples were collected to analyze food consumption,fecal quantity,organ weight and constituents of the blood and plasma.A Trendelenburg preparation was utilized to examine intestinal motility in wild-type(WT)and Gpr128-/-mice at the age of 8 and 32 wk.RESULTS:Gpr128 mRNA was highly and exclusively detected in the intestinal tissues.Targeted deletion of Gpr128 in adult mice resulted in reduced body weight gain,and mutant mice exhibited an increased frequency of peristaltic contraction and slow wave potential of the small intestine.The Gpr128+/+mice gained more weight on average than the Gpr128-/-mice since 24 wk,being 30.81±2.84 g and 25.74±4.50 g,respectively(n=10,P<0.01).The frequency of small intestinal peristaltic contraction was increased in Gpr128-/-mice.At the age of 8 wk,the frequency of peristalsis with an intraluminal pressure of 3 cmH2O was 6.6±2.3 peristalsis/15 min in Gpr128-/-intestine(n=5)vs 2.6±1.7peristalsis/15 min in WT intestine(n=5,P<0.05).At the age of 32 wk,the frequency of peristaltic contraction with an intraluminal pressure of 2 and 3 cmH2O was 4.6±2.3 and 3.1±0.8 peristalsis/15 min in WT mice(n=8),whereas in Gpr128-/-mice(n=8)the frequency of contraction was 8.3±3.0 and 7.4±3.1peristalsis/15 min,respectively(2 cmH2O:P<0.05 vs WT;3 cmH2O:P<0.01 vs WT).The frequency of slow wave potential in Gpr128-/-intestine(35.8±4.3,36.4±4.2 and 37.1±4.8/min with an intraluminal pressure of 1,2 and 3 cmH2O,n=8)was also higher than in WT intestine(30.6±4.2,31.4±3.9 and 31.9±4.5/min,n=8,P<0.05).CONCLUSION:We have generated a mouse model with a targeted deletion of Gpr128 and found reduced body weight and increased intestinal contraction frequency in this animal model.