| Ofloxacin (OFLX) is one of the candidates of fluoroquinolone-antibiotic with a broad antimicrobial spectrum that may have a potential role in the treatment of bacterial diarrhea. The mammalian colon is the final station of the gastrointestinal tract, where water and electrolyte transport still can be regulated and, the water and salt contents of the feces will have the final chance to be modified, therefore colon plays an important role in the maintenances of electrolyte balance. However little is known about the effects of Ofloxacin on the ion transport of the colonic mucosa and the cellular signaling mechanism underlying its' action.Aim: To investigate the effects of Ofloxacin on the ion transport in rat distal colonand its underlying mechanisms.Methods: The effect of Ofloxacin on the ion transport was investigated in rat distalcolon mounted in Ussing chambers. Adult female SD rats, weighing 180-230g, were used. Segments of distal colon about 5cm long were removed, the serosa, submucosa, and muscular layer were stripped away with fine forceps to obtain a mucosa preparation, consisting of epithelium and connective tissue. Sheets of tissue were mounted on a modified Ussing chamber, bathed with a volume of 5ml buffer solution on each side of the mucosa, and the solution was gassed with 5% CO2 in 95% Oi and kept at 37? and continuously short-circuited by an automatic voltage-clamp device(VCC MC6,San Diego, USA) with correction for solution resistance. At 142msintervals, amplitude -0.1 mV was applied to the tissue and the changes in short -circuit current(/sc) was measured. The changes in /sc was define as changes of total charges ( C/cm2). Data are presented as means SE. Statistical significance of comparisons between two experimental groups under various conditions were made with the Student's t-test. A "P" vale of less than 0.05 was considered statistically significant.Results: Basolateral addition of Ofloxacin(0.0875 -5.6mmol/L, n=6 for allconcentrations) produced a concentration-dependent increase in /sc(EC5o=2.02mmol/L), which contained a fast transient peak followed by a slower but more sustained plateau. The prominent response was reached at 2.8mmol/L. In all the following experiments, the Ofloxacin concentration of 2.8mmol/L was chosen to achieve a distinct effect of the Ofloxacin.Apical application of Na channel blocker, amiloride(10 mol/L) did not significantly affect the Ofloxacin-induced changes in /sc response(n=6, P>0.05). The Cl'-channels blocker DPC(lmmol/L, apical) completely inhibited the Ofloxacin-induced changes in /sc(n=6, P<0.001). Removal of external C1- decreased the response by about 45%(n=4, PO.05). Removal of HCO3- from buffer solution did not affect the Ofloxacin-induced changes in isc significantly(n=6, P>0.05). Pretreatment of the colonic mucosa with the carbonic anhydrase inhibitor, acetazolamide(lmmol/L,both sides) did not alter the Ofloxacin-induced changes in /sc (n=5, P>0.05). The CFTR blocker glibenclamide (Immol/L, apical) decreased the response by about 60%(n=6, PO.01). Apical application of Ca-dependent Cl'channels blockers, DIDS( 1 00 u mol/L, n=5), or a Ca chelator, BAPTA-AM(100 u mol/L, n=6), didn't affect the Ofloxacin-induced changes in /sc (P>0.05). Pretreatment with bumetanide(100 u mol/L, basolateral), a blocker of Na+-K+-2Cl"-cotransport, significantly reduced the Ofloxacin-induced changes in /sc by 60% (n=5, PO.05). The Ofloxacin-induced change in /sc was almost completely inhibited by basolateral exposure to Ba(lmmol/L, a nonspecific K channel blocker) (n=5, PO.001).Conclusions:1. Ofloxacin concentration-dependently stimulates C1- secretion in rat distal colon.2. Ofloxacin-induced /sc response is mediated by apical Cl" channels, probably CFTR, as well as basolateral NKCC and K channels.3. Introcellular Ca seems not involved in the Ofloxacin-induced Cl" secretion. The involved secondary messenger might be the cAMP.4. Ofloxacin-produced colonic Cl" secretion might be related to that the diarrhea, one of its side effects, induce... |
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