Expression of the Ca2+-channels ECaC1 and ECaC2 in small intestines of suckling and weaned piglets
Functional data from postnatal development of the intestinal Ca -absorption in pigs were collected and the Ca -channels which are responsible for the Ca -uptake across the brush border membrane of enterocytes were investigated qualitatively and age-dependent quantitatively.
2+
2+ 2+
To collect the functional data brush border membrane vesicles were prepared from enterocytes of the proximal small intestine of suckling and weaned piglets as well as porkers by combined Mg2+-precipitation method and differential centrifugation and the Ca2+-uptakes were investigated by the rapid filtration technique using the radiotracer 45Ca.
Epithelial Ca2+-channels (ECaC) were identified with RT-PCR using primers deduced from well known sequences of other species.
For the relative quantification of ECaC on mRNA levels investigations were carried out with preparations of suckling and weaned piglets by northern blots and real-time-PCRs.
Following results are to be emphasised:
(1) The kinetic of the Ca2+-uptake into the duodenal brush border membrane vesicles could be fitted best with a “one-site-binding” model. The results did not support directly the assumption of the presence of different ECaCs in the small intestine of pigs.
(2) Suckling piglets had a significantly higher maximal Ca2+-uptake (Vmax) compared with weaned piglets (p < 0.01) or porkers (p < 0.05). Vmax values were 3.64 ± 0.45 nmol.mg-1 protein.30s-1 in suckling piglets, 1.89 ± 0.20 nmol.mg-1 protein.30s-1 in weaned piglets and 2.13 ± 0.20 nmol.mg-1 protein.30s-1 in porkers (x ± SEM, n = 5).
(3) Suckling piglets had a significantly higher apparent Km compared with weaned piglets or porkers (p < 0.05). Km values were 1.44 ± 0.06 mmol.l-1 in suckling piglets, 1.14 ± 0.05 mmol.l-1 in weaned piglets and 1.13 ± 0.09 mmol.l-1 in porkers (x ± SEM, n = 5).
(4) Partial sequences from the Ca2+-channels were detected which show to some extent sequence overlay. That can be analysed as an evidence for the presence of two different Ca2+-channels in porcine duodenum. One of the partial sequences had a higher homology to the ECaC1 of rat (95 %) compared with ECaC2 of human (33 %). The other partial sequence exhibited a higher homology to the hECaC2 (88 %) compared with the rECaC1 (81 %).
(5) With the real-time-PCR method differentiation of ECaC1 and ECaC2 was enabled. The mRNA for ECaC1 was present in suckling and weaned piglets in rather small extent but without a difference. The mRNA level for ECaC2 was lower by 66 % in suckling piglets compared with weaned piglets.
In summary, the results show that two different Ca2+-channels are involved in Ca2+-absorption from the small intestines, similarly to those shown recently in other mammalian species. The mRNA levels of these channels can be quantified using the real-time-PCR method and the results for the ECaC2 point to same kind of postnatal adaptation, which should be further elucidated in following investigations.
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