| Tetramethylpyrazine (TMP) is the major active component extracted from the Chinese herbal medicine ligustium wollichii franchat which has been used for centuries. Traditionally, TMP is commonly used to treat cardiovascular and cerebral disorders because of its multiple biological effects including activating blood circulation, increasing coronary flow, decreasing oxygen consumption, lowering blood pressure, improving microcirculation. During the past few years exploration about the cellular signaling mechanism underlying its action showed TMP could influence the intracellular Ca2+ and cAMP, motivate synthesis and release of PGI2. Observation about distribution of 3H-TMP in animal body revealed the sensitive target of TMP is digestive system. Despite the substantial amount of data available on the various effects of TMP, little is known about its influence on the mucosa of the mammalin colon and the cellular signaling mechanism underlying its action.The mammalian colon is the final station of the gastrointestinal tract, where the organism has the last possibility to modify the electrolyte contents of the feces and thereby to regulate water and salt transport, thus plays an important role in the maintenances of electrolyte balance. It has been reported that Bak Foong Pills ethanol extract exerted a stimulatory effect on the human colonic epithelia cell line Tg4 Cl- secretion. TMP is the major active component in Bak Foong Pills. We thereforeinvestigated the influence of TMP on water and electrolyte transport properties of native rat colon and the cellular signaling mechanism underlying its action using Isc technique, radioimmunoassay analysis in conjunction with pharmacological agents and specific inhibitors, in order to determine is there any potential target for pharmacological intervention in colonic disorders. The present study is the first to provide scientific evidence for the pharmacological action of TMP on the colon, which may explore a new insight in treatment of gastrointestinal disorders.Adult male SD rats were used. Segments of distal colon about 5cm were removed, the serosa, submucosa, and muscular layer were stripped away with fine forceps to obtain a mucosa preparation, consisting of epithelium and underlying connective tissue. Flat sheets of distal colon with intact mucosa were mounted between two halves of modified Ussing chambers to measure the Isc in vitro. The influences of different activators or inhibitors on the Isc can be obtained. Cytosolic cAMP concentrations were measured by radioimmunoassay. The data were expressed as means 盨E. Comparisons between groups of data were made via Student's Mest or Wilcoxon test when appropriate. A "P" value of less than 0.05 was considered statistically significant. ResultsThe basal PD and Isc in rat distal colon in vitro were -5.510.8 (mV) (n=8) and 60.3 ?.7( μ A/cm2) (n=16), respectively. Rt that could be calculated from these valuesaccording to Ohm's law was 94.9?.9 ( cm2) (n=16). Addition of TMP (0.01-5mM) tothe basolateral membrane produced a concentration-dependent increase in Isc with an apparent EC50 of about 0.5mM (n=16). The TMP-elicited Isc depended on Cl- and HCO3, and was inhibited by apical application of Diphenylamine-2,2'- dicarboxylic acid (DPC, 2mM, a nonspecific Cl" channel blocker) and glibenclamide (ImM, CFTR blocker), but resistant to 4-4'-Didsothiocyanostilbene- 2,2'-disulfonic acid (DIDS, l00μM, CaCC blocker). The Isc response could be elicited in normal mice (genotype: +/+), but was abolished in CF mice expressing F508 mutation (genotype: -/-). Removal of Na+ from basolateral or bilateral solution almost completely abolished the Isc response to TMP, but it was insensitive to apical Na+ replacement or apical Na+ channel blocker, amiloride(10μM). Pretreating the basolateral membrane with bumetanide (lOOuM), NKCC inhibitor or DIDS (l00μM), as basolateral NBC and C17HCO3 exchange inhibitor, markedly attenuated the Isc response to TMP by 89.3% (n=6, P<0.001) and 68.9% (n=4, P<0.
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