The major function of the colonic epithelium is to conserve water and electrolytes by maintaining a balance between fluid secretion and absorption. This colonic function is especially important in newborns, where reserves are small. Secretion across most epithelia is governed by C1- transport mechanisms and is regulated by second messengers such as cyclic nucleotides and calcium. Calcium-dependent secretagogues are responsible for the minute-by-minute regulation of ion transport needed in the intestine. However, not much is known about the regulation of C1- secretion in the developing mammal. There is intriguing, albeit sparse, evidence in some tissues that Ca2+ signaling may be postnatally regulated. The few studies on ontogeny of C1- secretion were conducted in the rabbit distal colon and showed that bile acids such as taurodeoxycholate (TDC) stimulate CF transport in the adult but not in the neonate. Ca2+ and cAMP were implicated as mediators of TDC action. To elucidate the cellular basis of the age-related regulation of C1- transport, this laboratory investigated the effects of cAMP- and Ca2+-dependent secretagogues in primary cultures of epithelial cells (colonocytes) isolated from the distal colon of newborn (NBN), weanling (WN) and adult (AD) rabbits. The salient findings were as follows: Cyclic AMP stimulated CF transport at all ages. However Ca2+-dependent secretagogues, such as neurotensin (NT), stimulated C1 transport in AD, but not in WN or NBN colonocytes. In parallel, NT increased [Ca2+], in AD but not in WN colonocytes. Similarly, TDC increased Ca2+, but not cAMP, and stimulated CF transport in AD, but not in WN or NBN colonocytes. In contrast, calcium ionophore stimulated C1- transport at all ages, implying that the distal steps in Ca2+ signaling are functional in the young animal. Examination of the proximal steps, i.e., receptor/G-protein activation of phospholipase C to IP3 to Ca2+ stores, revealed that the weanling animal had sufficient Ca2+ stores to stimulate CF transport. However, NT and TDC increased [IP3]i in AD, but not in WN, colonocytes despite the presence of PLCbeta and gamma proteins at all ages. NT activated Galphaq in both AD and WN colonocytes, but TDC, even in the adult, does not stimulate Galphaq. Therefore, it is hypothesized that multiple pathways in IP3 generation and action are ontogenetically regulated in the postnatal colon to provide protection against excessive loss of fluid in response to its changing milieu. This proposal will test this hypothesis as follows:
Aim I involves identification of the PLC isoforms activated by NT and TDC in AD and determination of the tissue specificity, time course and factors contributing to the age-dependent responsiveness of PLC leading to C1- transport.
Aim II is an examination of whether other steps in Ca2+ signaling, such as IP3 receptors and other inositol phosphates, contribute to the age-dependent appearance of Ca2+-mediated CF transport.
Aim III is a characterization, including age-dependence, of TDC interaction with colonocytes and of the kinase cascade(s) involved in NT- and TDC-stimulated [Ca2+]i and CF transport. By delineating the """"""""protective"""""""" mechanisms that the developing gut has evolved to meet the challenges of its environment, these studies will provide important insights for devising ways to combat the life-threatening chronic diarrheas of infants.
