Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

Abstract

Background: The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia.

Methods: Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry.

Results: Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups.

Conclusions: OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE(2).

Figure 1

Example of short circuit current data from a MUAS-chamber. A: Typical responses from compounds applied apically and/or basolaterally. a: amiloride, b: theophylline, c: indomethacin, e: cAMP (basolateral), g: db-cAMP (basolateral), h: bumetanide, and i: acetazolamide. d: and f: apically applied cAMP and db-cAMP, respectively. B-E: Typical examples of induced SCC by the four model compounds.

Figure 2

Mean short circuit signals. A: Induced SCC by 500 mM of the 4 cNTs presented as mean ± SEM. Control patients: cAMP (N = 7, n = 10), cGMP (N = 5, n = 7), db-cAMP (N = 7, n = 14), db-cGMP (N = 7, n = 12). Neoplasia patients: cAMP (N = 7, n = 12), cGMP (N = 10, n = 20), db-cAMP (N = 10, n = 32), db-cGMP (N = 4, n = 7). B: Responses to application of theophylline and indomethacin. Bars represent Mean ± SEM. Control patients: Theophylline (N = 8, n = 25), indomethacine (N = 8, n = 27). Neoplasia patients: Theophylline (N = 11, n = 37), indomethacine (N = 11, n = 41). C: Responses to application of bumetanide and acetazolamide. Bars represent Mean ± SEM. Control patients: Bumetanide (N = 6, n = 12), acetazolamide (N = 5, n = 11). Neoplasia patients: Bumetanide (N = 9, n = 24), acetazolamide (N = 9, n = 25). N = number of patients and n = number of biopsies. * indicates a statistical significant difference between the two groups.

Figure 3

OATP expression. Expression levels of the OATPs relative to β-actin based on single biopsies from 6 patients in each group. * indicates a statistical significant difference (p < 0.05) between the two groups. Data are presented as means ± SEM.

Figure 4

ABC expression. Expression levels of the ABC-transporters relative to β-actin based on single biopsies from 6 patients in each group. Data are presented as means ± SEM.

Figure 5

OATP-transporter localization. A: OATP1C1, B: OATP2B1, C: OATP4A1, D: OATP4C1. White arrows indicate the location at the bottom of the goblet cells, black arrows staining of endocrine cells and yellow arrows basolateral staining of epithelial cells.

Figure 6

ABC-transporter localization. A: ABCC3, B: ABCC4, and C: ABCC5. Yellow arrows indicate the basolateral staining of ABCC3 in coloumnar epithelial cells and goblet cells. White arrows indicate the localization of ABCC4 in basolateral and perinuclear membranes of goblet cells, and black arrows indicate the basolateral staining of ABCC5 in columnar epithelial cells. D-E: Negative control images of staining with secondary antibody from goat (D) and rabbit (E). No immunohistochemical coloring was obtained for the ABCB1 transporter.

Figure 7

Model of human colon secretory and cAMP turnover systems. A: Schematic presentation of the secretory system of the colon as revealed by this study. The subcellular location of ABCB1 was not determined in this study but has been localized to the apical membrane by others. * Indicates significantly increased expression in CRN patients. B: Schematic presentation of the cAMP-turnover system in a colonic epithelial cell. AC: adenylate cyclase. PDE: phosphodiesterase.