Prelimilary Study Of Expression Of L-type CaV1.3 Ca²⁺ Channels In Interstitial Cell Of Cajal

BackgroundThe interstitial cell of Cajal (ICC) is found in the gastrointestinal tract and serveed as a pacemaker, in which create the basal electrical rhythm. Abnormality of ICC pacemaker functions lead to gastrointestinal motility disorder, such as chronic intestinal pseudo-obstruction (CIP), diabetic gastroparesis and so on. Ca2+ exists throughout the whole pacemaker time. The channels of calcium sparklet formation are the point at issue in fast-rise phase of calcium transient. In patch clamp recordings of single human intestinal ICC, the largest Ca2+ current is a Ca2+ current carried by L-type Ca2+ channel. Previous studies have suggested pacemaker activity in ICC is associated with robust Ca2+ entry events that are resistant to block by dihydropyridines. And L-type CaV1.3 Ca2+ channel is relatively insensitive to dihydropyridines. We suppose that L-type CaV1.3 Ca2+ channel expressed in ICC, and may be functionally important for the pacemaker activity in ICC, maybe inducing the formation of calcium sparklet.Objective1. Establish a stable method of ICC isolation and cell culture2. Identify the expression of L-type CaV1.3 Ca2+ channel in ICC, and the relation between L-type CaV1.3 Ca2+ channel and pacemaker activity.MethodsPart 1: Cell culture and identificationCell culture: small intestine muscle pieces from BALB/c mice (1-2 d old) were incubated in solution containing 1.0mg/ml collagenase (type II). After passing through the sieve (size: #200), all cell suspension was layered on the surface of a 200 g/L Ficoll density cushion. The cell band located at the interface was plated into dishes with collagen-coated bottoms. The cells resuspended with M199 medium were maintained in 50% CO2 at 37℃.Cell identification: reverse-transcriptase PCR (RT-PCR) was used to detect expression of c-kit mRNA; immunofluorescent staining was used to identify cell location of c-kit; flow cytometry was used to determine purity of cultured ICC; double-immunofluorescent staining was performed to detect ability of proliferation in vitro.Part 2: Expression of L-type CaV1.3 Ca²⁺ channel in ICCExpression of CaV1.3: RT-PCR was used to detect expression of CaV1.3 mRNA; double-immunofluorescent staining was used to detect cell location of c-kit and CaV1.3 in cultured ICC. Small intestine muscle pieces from BALB/c mice (postnatal day 0, 7, 14 and adult) were used in this study. Double-immunofluorescent staining was used to detect cell location of c-kit and CaV1.3. Spontaneous contractile activity was recorded to determine the functional changes of small intestine.ResultPart 1: Cell culture and identificationThe cultured adhesive cells were multangular with large nuclei in the centre and a network was formed by cytoplasmic processes. Cells were alive for more than 10 days, with an increase in the number. C-kit mRNA and protein were detected in cultured ICC by methods of RT-PCR and immunofluorescent staining. The purity was 81.97% using flow cytometry. Result of EdU staining directly showed that there was coexpression of EdU and c-kit protein. Part 2: Expression of L-type CaV1.3 Ca²⁺ channel in ICCCaV1.3 mRNA was detected in cultured cells. C-kit and CaV1.3 proteins coexpressed in ICC cell membrane. Double-immunofluorescent staining of the four growth periods showed that coexpression of the two proteins increased gradually in intensity and the intensity of coexpression from P14 was close to that from adult. The contraction curves became more regular with the processing of growth. Frequency and range of curve from P14 were nearly the same as that from adult.Conclusions1. Using Collagenase - Ficoll method, we established a stable ICC cell culture method, which lays a solid foundation for the basic and clinical research in ICC.2. ICC cultured in vitro had the ability to proliferate.3. L-type CaV1.3 Ca²⁺ channel was abundantly expressed in cell membrane of ICC, and there was a correlation between L-type CaV1.3 Ca²⁺ channel and ICC pacemaker activity.