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Effect of tetramethylpyrazine on exocrine pancreatic and bile secretion.

AIM
To investigate the effect of tetramethylpyrazine (ligustrazine, TMP) on the secretion of exocrine pancreas (and biliary).


METHODS
In in vivo study, we investigated the effect of TMP on the secretion of pancreatic-bile juice (PBJ) in rats. Using human pancreatic duct cell line, CAPAN-1, combined with the short-circuit current (ISC) technique we further studied the effect of TMP on the pancreatic anion secretion.


RESULTS
Administration of TMP (80 mg/kg, i.p.) significantly increased the secretion of PBJ (P<0.05), but the pH of PBJ and the secretion of pancreatic protein were not significantly affected. Basolateral addition of TMP produced a dose-dependent increase in ISC (EC50=1.56 mmol/L), which contained a fast transient ISC response followed by a slow decay. Apical application of Cl- channel blockers, DPC (1 mmol/L), decreased the response by about 67.1% (P<0.001), whereas amiloride (100 micromol/L), a epithelial sodium channel blockers, had no effect. Removal of extracellular HCO3- abolished TMP-induced increase in ISC by about 74.4% (P<0.001), but the removal of external Cl- did not. Pretreatment with phosphodiesterase inhibitor, IBMX(0.5 mmol/L), decreased the TMP-induced ISC by 91% (P<0.001).


CONCLUSION
TMP could stimulate the secretion of PBJ, especially pancreatic ductal HCO3- secretion via cAMP or cGMP-dependent pathway. It need further study to investigate the roles of cAMP or cGMP in the effect of TMP on the secretion of exocrine pancreas.


INTRODUCTION
Tetramethylpyrazine (TMP, C 8 H 12 N 2 , molecular mass 136.20 u, also known as ligustrazine) is an active alkaloid contained in the rhizome of Chuanxiong [1] . TMP has been widely used for the treatment of patients with cardiovascular and cerebrovascular diseases [1][2][3][4][5] . Its proposed pharmacological actions include antagonizing calcium mobilization [6] , inhibiting platelet aggregation [7] and increasing intracellular cAMP level by inhibiting phosphodiesterase activity [8] . Recently it has been reported that TMP has antioxidant effect, reducing free radical generation [9,10] and decreasing nitric oxide production [11,12] . However, the effect of TMP on the pancreatic exocrine secretion are unknown. Since TMP is known to activate cAMP, it may act similarly to secretin, a physiological regulator of pancreatic secretion by elevating intracellular cAMP to act on pancreatic epithelial CFTR, which is a cAMP/cGMP-regulated Clchannel [13,14] and involved in pancreatic HCO 3 -secretion. In the present study, we investigated the effect of TMP on the secretion of pancreatic-bile juice (PBJ), which includes both pancreatic protein and HCO 3 -. We also undertook the present study using the short-circuit current (I SC ) technique to investigate TMP effect on HCO 3secretion by human pancreatic duct cell line, CAPAN-1, which retains most of the properties of pancreatic duct cells [15,16] .

Methods
Animal preparation Animal experimentation was conducted according to institutional guidelines. Adult male Sprague-Dawley rats, weighing 220-280 g, were housed under controlled temperature (23 ) and fasted for 12 h with free access to water before surgery. Midline abdominal incisions were made under xylazine and ketamine anesthesia (13 and 87 mg /kg body weight, respectively, im), followed by insertion of a polyethylene tube into the proximal duodenum for diversion of PBJ to the duodenum. A pancreatic duct cannula was made by inserting a polyethylene tube at the junction between the pancreatic duct and the duodenal wall for collection of PBJ [17][18][19] . Collection and measurement of exocrine pancreatic-bile secretions The animals were divided into two groups randomly: TMP group (TMP 80 mg/kg, ip. pH 2.3) and control ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol November 15, 2003 Volume 9 Number 11 group (9 g/L NaCl solution, ip. pH 2.3). After a 30-minute stabilization period, pancreatic-bile secretions were collected every 15 minutes for 90 minutes. The volume was measured by a 1mL syringe and the pH value of PBJ was determined by a pH analyzer. 10 µL of PBJ was taken and diluted for pancreatic protein determination using spectra MAX 250 and Brandford reagent. The remaining undiluted PBJ was pumped into the duodenum via the duodenal cannula during the next collection period. Administrations were given after 2-time collections (30 minutes) and the second 15-minute-secretion was the basal PBJ secretion. Cell culture Experiments were performed on the human pancreatic duct cell line, CAPAN-1 at a passage of 50-58. Cells were grown in RPMI 1640 medium with 200 mL/L fetal bovine serum and 10 g/L penicillin-streptomycin (P/S) as described previously [20,21] . Briefly, a volume of 0.2 mL of the cell suspension (1.5×10 9 /L) was plated onto each permeable support, which was made of a millipore filter and a silicon ring with a confined area of 0.45 cm 2 , floating on culture medium and incubated at 37 with 50 mL/L CO 2 and 950 mL/L O 2 for 7-8 days. The cells were used for I SC once the monolayers reached confluence.

Short-circuit current measurement
The basic principles of the short-circuit current experiments performed in the present study were the same as previously described [20,21]

Statistical analysis
The data were collected and analyzed by SPSS statistical package 10.0. The results were expressed as x±S x . Comparisons between groups of data were carried out using Student's paired or unpaired t-test. Comparisons in one group of data were carried out using One-way ANOVA. A P-value less than 0.05 was considered to be statistically significant. EC 50 values were determined by non-linear regression by GraphPad Prism software. Table 1 shows the volume of pancreatic-bile juice (PBJ) collected at different time points before and after intraperitoneal administration of TMP or 9 mL/L NaCl (control  (Figure 1). However, the same treatment with of 9 mL/L NaCl solution did not produce significant effect (n=12) (Figure1). Protein content in PBJ and pH of PBJ were shown to have no differences between TMP-treated and control groups (data not shown).

DISCUSSION
While TMP has been widely used clinically for treating cardiovascular disorders and cerebrovascular diseases [1][2][3][4][5] , little is known about its effect on pancreatic secretion. In the present study, intraperitoneal application of TMP increased secretion of pancreatic-bile juice (PBJ), but the protein content and pH value were not affected. It is known that PBJ consists of both bile and pancreatic juice, the volume of the basal secretion (or agonist induced) of pancreatic juice is normally very low, only about 9-18 µL in 15 minutes in rat [17,18] , as compared to that of the liver. Although TMP increased the secretion of PBJ by 26-83 µL in 15 minutes, it was difficult to assess the contribution to exocrine pancreatic secretion from our experiments in vivo. Therefore, CAPAN-1 cell line was used to further demonstrate the effect of TMP on the pancreatic secretion. Our data suggested that TMP could directly stimulate HCO 3secretion in human pancreatic duct cells.
According to the HCO 3secretory model [22] , the intracellular HCO 3was accumulated from the tissue fluid via the basolateral membrane [23][24][25] and mainly secreted by a Cl -/HCO 3exchanger [26,27] and/or directly via cAMP/cGMP-dependent Clchannel (CFTR) [13,26,28] . Recently, it was reported that CFTR could also secrete HCO 3as Cl -/HCO 3exchanger [29,30] . The TMP-increased I SC in CAPAN-1 could be blocked by Clchannel blockers, as well as phosphodiesterase inhibitors, which were known to increase cAMP and cGMP, and abolished by removal of extracellular HCO 3 -, suggesting that TMP can stimulate pancreatic HCO 3secretion via CFTR since it has been shown to be activated by both cAMP and cGMP. The TMP-induced increase in the volume of PBJ observed in the rats could be due to an increase in pancreatic HCO 3secretion, as well as bile secretion, leading to enhanced water secretion. However, the inability of TMP to increase the pH of PBJ appeared to contradict to its observed effect on the increase in HCO 3secretion. This could be explained by two possible reasons. One is that the mechanism of HCO 3secretion in human pancreatic duct cells was different from that in rats since the HCO 3concentration of pancreatic juice in rats was only 70 mmol/L while in human was 145 mmol/L [22] . The other is that the HCO 3secreted by the pancreas might be diluted by bile juice and thus having less prominent effect on pH. It is interesting to note that TMP could stimulate HCO 3secretion that could be inhibited by extracellular Clas evidenced by the increase in the TMP-induced I SC observed in the absence of extracellular Cl -. However, the mechanism for the observed inhibition of HCO 3secretion by extracellular Clremains to be elucidated.
In conclusion, the present in vivo and in vitro results suggest that TMP can promote the secretion of PBJ in rats and HCO 3secretion in human pancreatic duct cells, which may be beneficial to improving digestive function.