OBJECTIVES: We have shown that Ca2+-dependent regulation of Cl- secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca2+]i, it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl- transport in the developing rabbit distal colon. MATERIALS AND METHODS: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca2+]i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. RESULTS: Serum depletion had no effect on cell number, DNA content, or basal Cl- transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl- transport to a similar extent. EGF maximally stimulated Cl- transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca2+]i. EGF did not alter bethanechol-stimulated Cl- transport or [Ca2+]i. EGF acts via an EGF-receptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl- transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl- transport. CONCLUSIONS: EGF stimulates rabbit colonic Cl- transport via a Ca2+-independent, tyrosine kinase- and MAPK-dependent pathway, and its effects are not age dependent.
OBJECTIVES: We have shown that Ca2+-dependent regulation of Cl- secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca2+]i, it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl- transport in the developing rabbit distal colon. MATERIALS AND METHODS: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca2+]i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. RESULTS: Serum depletion had no effect on cell number, DNA content, or basal Cl- transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl- transport to a similar extent. EGF maximally stimulated Cl- transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca2+]i. EGF did not alter bethanechol-stimulated Cl- transport or [Ca2+]i. EGF acts via an EGF-receptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl- transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl- transport. CONCLUSIONS:EGF stimulates rabbit colonic Cl- transport via a Ca2+-independent, tyrosine kinase- and MAPK-dependent pathway, and its effects are not age dependent.
Authors: Jada C Domingue; Mei Ao; Jayashree Sarathy; Alvin George; Waddah A Alrefai; Deborah J Nelson; Mrinalini C Rao Journal: Physiol Rep Date: 2014-09-28