Mrinalini C. Rao - US grants

The long-term goal is to elucidate the molecular mechanisms by which the second messengers cAMP, cGMP and Ca regulate electrolyte and water transport in epithelia. Sodium- or Na/K-dependent mechanisms of Cl permeation into cells are important both in the vectorial transport of salt and water across epithelia and in volume regulation in a variety of tissues. Malfunctioning of ion transport processes, including cation-coupled Cl entry mechanisms, has been implicated in a number of diseases, including glaucoma, cystic fibrosis, diarrhea and essential hypertension. In epithelia, net fluid transport represents a balance between active absorption and active secretion and Na/Cl or Na/K/Cl cotransport mechanisms are necessary components of both processes. An increase in the intracellular content of either Ca, cAMP or, in some cases, cGMP, regulates Na/Cl transport in most epithelia, leading to a decrease in fluid absorption in absorptive epithelia and to an increase in fluid secretion in secretory epithelia. These second messengers probably act via specific protein kinases. This proposal aims to correlate Ca-, cAMP- and cGM-mediated changes in Na/K/Cl cotransport with phosphorylation of specific proteins in intact tissues and to characterize the transport-related phosphoproteins. The model system is the intestine of the winter flounder, where Na/K/Cl cotransport is important in osmoregulation. This simple epi thelium is relatively homogeneous, and possesses a well-characterized Na/K/Cl cotransport mechanism that can be regulated by all three second messengers. Changes in phosphorylation and cotransport wil be correlated on the basis of time, dose dependency and reversibility of effects. Specific phosphoprotein substrates will be identified by one- and two-dimensional gel electrophoresis followed by autoradiography, and transport will be assessed by radioisotope fluxes. Specific substrates and kinases will be characterized by various techniques, including subcellular fractionation, photoaffinity labelling, peptide mapping, and phosphoaminoacid analysis. The overall objective is to identify the molecular nature of ion transport components; second-messenger-specific phosphoproteins could be the transporter per se or modulaters thereof. These studies will provide an excellent model for examining similar transport mechanisms in more complex epithelia such as mammalian ileum and the thick ascending limb of Henle's loop in mammalian kidney.

This proposal will characterize the unique regulation of C1 transport in normal, human colonocytes in primary cultures and study its differences along the cephalocaudal axis. Models ranging from animal species to human colon carcinoma cell lines have been used to understand human colonic C1 transport. Animal models are not always applicable to humans since there are species differences in colonic ion transport along the cephalocaudal axis. Human colon carcinoma cell lines cannot be used to study segmental differences. In addition, being "transformed" cells, they often demonstrate small intestinal better than colonic function. Recognizing these limitations, the complexities of cellular C1 transport regulation discerned from these models will have to be relate to events in primary cultures of human colonocytes and ultimately to those in the intact, normal, colon. This laboratory has developed primary, 24 hr cultures of human colonocytes which exhibit cAMP, Ca2+ and cGMP-sensitive C1 transport, features that resemble the intact colon more closely than other colonic models. In contrast tot he colonic cell line, T-84, C1 transport in human colonocytes is activated by phorbol esters and human, but not rabbit, colonocytes have a cGMP-activated C1 transport. Therefore, the hypothesis to be tested is that the cellular regulation of C1 transport in primary cultures of human colonocytes resembles that of the intact human colon, shows segmental differences and is distinct from that seen in other model systems. The first aim is to define the unique differences in C1 permeabilities in cells from different colonic segments. To conserve tissues, fluorimetric techniques will be used to study ion specificities, sensitivity to inhibitors and kinetics. The influence of extracellular matrices (ECM_) on C1 transport will be studied. The second aim is to determine if the regulation of Ci permeabilities by cAMP, Ca2+ and cGMP, is influenced by segmental differences and/or by the ECM. Specific aspects of the cGMP and protein kinase C (PKC) cascades will be analyzed as their presence/role varies amongst the different colonocyte models. It will be determined if cGMP is acting via a cAMP-PK or a cGMP-PK and if there is cross-talk between the cAMP?PKC cascades. Sequential steps in these cascades will be examined including changes in intracellular mediators, the effects of inhibitors of protein kinases and phosphatases and changes in phosphorylation of transporters such as CFTR and the Na+-K+-2C1 cotransporter. The third aim is to relate the C1 transport characteristics of colonocytes in culture with those of the intact epithelium using human colonic epithelial sheets, obtained at the time of surgery. Specifically, segmental differences and cross-talk between second messenger-activated systems in regulating transepithelial C1 transport will be studied. These studies will provide invaluable information about normal colonic cell physiology which can be used to understand the molecular pathophysiology of toxin-mediated diarrheas, cystic fibrosis an inflammatory disorders.

DESCRIPTION (Applicant's Abstract): 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.

A long-term goal at the University of Illinois at Chicago (UIC) is to increase the number, participation and leadership status of women in academic science and engineering through institutional transformation. Fundamental to this goal is the commitment and active participation of UIC administration at the highest level, leaders working closely with faculty and staff to create and actualize individual departmental programs in the eleven disciplinary areas of science, technology, engineering and mathematics (STEM). The practices to be developed for these disciplines will have broader use and implications across campus. Under this award, UIC will focus its energies on instituting practices in the STEM disciplines to promote gender equity and leadership with a special focus on increasing the recruitment, promotion and tenure, and retention rates of STEM women faculty and advancing them into leadership positions. This will be done through the training and efforts of dedicated faculty facilitators in each of the disciplines. In addition, current efforts to improve networking, mentoring, and evaluating support services for women in STEM disciplines will be augmented.

This award is supported by the NSF ADVANCE Program. The goal of the ADVANCE program is to increase the participation of women in the scientific and engineering workforce through the increased representation and advancement of women in academic science and engineering careers. To meet this goal, the ADVANCE program provides award opportunities for both individuals and organizations and seeks to support new approaches to improving the climate for women in U.S. academic institutions and to facilitate women's advancement to the highest ranks of academic leadership.

The goal of the Women in Science and Engineering System Transformation (WISEST)
project, at the University of Illinois at Chicago (UIC), is to increase the number, participation,
and leadership status of women, majority and minority, in eleven science and engineering
(STEM) departments through institutional transformation. WISEST will use an innovative
approach of a network of faculty facilitators from all STEM departments working with
department heads and an executive committee of key administrators and a social scientist.
This network will carry out five integrated and mutually reinforcing strategies: warm the
climate and decrease the isolation of women STEM faculty; recruit minority women faculty
through an unique mentored postdoctoral program; transform STEM departments to foster
diversity and womens leadership; promote womens scholarship and teaching; and improve
the ability to track and report on gender equity. Proposed outcomes for STEM women
faculty include: increased numbers of majority and minority faculty; improved retention
rate; salary equity with men of similar accomplishments and productivity; increased
percentage of leadership positions; improved job satisfaction; and increased national visibility
for our scholars. The intellectual merit of WISEST is that it will assess the impact of
systemic change to erase gender stereotyping rather than individual remediation and it will
specifically extend the focus of action to include the postdoctoral level to recruit faculty. Its
broader impact will be the creation of a life-friendly work climate for all UIC faculty.
WISEST will share its experiences nationally, and serve as an exemplary model for fostering
gender equity and diversity in academe.