title: [Expression and function of T-type α1H Ca(2)(+) channels in the rat model of Hirschsprung disease].
abstract: OBJECTIVE
  To investigate the role of T-type α1H Ca(2+) channels(Cav 3.2) in the pathogenesis of Hirschsprung disease(HD).
  
  
  METHODS
  Eighty neonatal SD rats 6 to 8 days of age were randomly divided into 2 groups, 40 in each. Microinjector catheters were carefully placed into the bowl of one group, and 0.2% benzalkonium chloride (BAC) solution was injected to establish HD rat model. Control group was treated with saline instead of benzalkonium chloride. At postoperative 4, 6 and 8 weeks, ten rats were sacrificed randomly and examined through general observation, histopathological observation and immunofluorescent staining of PGP 9.5 to identify the animal models. Meanwhile, the distribution of Cav 3.2 in abnormal colon of HD rat model was studied by immunohistochemical staining, Western blot, and the co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Besides, function of Cav 3.2 was surveyed with the model rats tensile force and frequency of colonic muscle in vitro.
  
  
  RESULTS
  After 8 weeks of BAC treatment, the rat model was successfully established according to the results of histopathological and immunofluorescent staining, which showed decrease or lack of ganglion cells within the area of BAC treatment. The distribution of Cav 3.2 was detected by immunohistochemical staining. In the normal colon, Cav 3.2 were mainly distributed between circular muscle and longitudinal muscle, and showed continuous distribution. However, in the narrow segment of HD rat model, the distribution of Cav 3.2 was decreased significantly, and its continuity was destroyed . The results of Western blot were quite consistent with immunohistochemistry staining, in the narrow segment of HD rat model, the expression of Cav 3.2 was decreased gradually after the BAC treatment, especially at postoperative 8 weeks. The relative expression of Cav 3.2 of the control group was 0.63±0.06, 0.62±0.09, 0.63±0.06 at postoperative 4, 6 and 8 weeks respectively. While that of HD group was 0.63±0.06, 0.38±0.06, 0.26±0.07 respectively, which was significantly lower as compared with that of control group at postoperative 6 and 8 weeks respectively (t-value 5.27 and 8.63 respectively, both P<0.05). The co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Similarly, compared with control groups, the co-localization of Cav 3.2 and c-kit were reduced obviously or vanished in the narrow segment of HD rat model. In motility studies, the control rats show cyclic depolarization regularly. However, the cyclic depolarization largely disappeared in model rats. Besides, on the premise of cyclic depolarization in control muscle strips, when we added ZnCl2, known to inhibit Cav 3.2 selectively among three T-channel isoforms, into tissue chambers, the pattern of muscle contractions appear similar to HD model rats.
  
  
  CONCLUSIONS
  The abnormal alteration of Cav 3.2 probably mediate the functional change of interstitial cells of cajal in the HD, and finally induce the intestinal dysfunction of HD.
authors.affiliation: Department of Pediatrics, Tangdu Hospital, the Fourth Military Medical University, Xi'an 710038, China.
authors.forename: N N
authors.initials: NN
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Tian
authors.forename: Y
authors.initials: Y
authors.lastname: Lin
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: C K
authors.initials: CK
authors.lastname: Hu
authors.forename: L C
authors.initials: LC
authors.lastname: Zeng
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: B X
authors.initials: BX
authors.lastname: Wang
authors.forename: Z
authors.initials: Z
authors.lastname: Shu
authors.forename: X
authors.initials: X
authors.lastname: Jiang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D003106:Colon / Q000378:metabolism; D006627:Hirschsprung Disease / Q000139:chemically induced / Q000378:metabolism*; D007150:Immunohistochemistry; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: C509958:Cacna1h protein, rat; D020747:Calcium Channels, T-Type