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ORIGINAL ARTICLE Inhibitory Effect of Potassium Alum on Smooth Muscle Contraction of Rabbit and Its Mechanism TANG Zhong-yuan (唐中元)△, LIN Yuan (林 原), YANG Xiao-li (杨晓丽), WEI Wei (魏 薇) and TANG Ze-yao (唐泽耀) Objective: To investigate the effects of potassium alum (Alunite ) on smooth muscle contraction ABSTRACT Objective: and phosphorylation of myosin light chain by myosin light chain kinase (MLCK) and to try to find out the clue of Methods:: An isolated rabbit duodenum smooth muscle strip was selected to study the effects its mechanism. Methods of potassium alum on its contractile activity under the condition of Krebs' solution using HW-400S constant temperature smooth muscle trough. The myosin and MLCK were purified from chicken gizzard smooth muscle. Myosin light chain phosphorylation was determined by glycerol-polyacrylamide gel electrophoresis; Results:: Potassium myosin Mg2+-ATPase activity was measured by inorganic phosphate liberation method. Results alum (2.5–20 mmol/L) inhibited the contraction on duodenum in a dose-related and a time-dependent manner; potassium alum could also inhibit the extent of phosphorylation of myosin light chain in a dose-related and a time-dependent manner; and potassium alum inhibited the extent of Mg2+-ATPase activity in a dose-related Conclusions:: Potassium alum inhibited smooth muscle contraction in a way of inhibiting phosphorylation manner. Conclusions of myosin light chain and Mg2+-ATPase activity. This has revealed the molecular mechanism of treatment of gastrointestinal spastic disorders by potassium alum. KEYWORDS potassium alum, smooth muscle contraction, phosphorylation of myosin light chain, myosin Mg2+ATPase, gastrointestinal spastic disorders

As a Chinese traditional medicine, potassium alum (Alunite ) has been used for several hundred years for treatments such as rectal, bladder, esophageal bleeding or as a bactericidal agent when added into water and so on.(1) Recent research indicated that its solution could cure skin disease (aquagenic acrokeratoderma) and decrease serum triglyceride (TG). (2,3) Moreover, potassium alum has been used to treat chronic pelvic inflammatory d i s e a s e . (4) I t c a n a l s o p r e v e n t a u t o i m m u n e diabetes. (5) Furthermore, potassium alum was an adjuvant approved for routine use in humans.(6-9) Its adjuvanticity mechanism is related to the induction of the release of interleukin (IL)-1beta from macrophages and dendritic cells. (10-12) Another mechanism was that potassium alum may promote Th2 immune response.(13) Moreover, its injection could be used as a simple perianal procedure for total rectal prolapse without any severe pain or complication. (14,15) Why potassium alum can treat smooth muscle contractile disorder disease, whether it is related to motor protein (myosin)? This is one of our interests. On the other hand, it was reported that different concentrations of potassium alum solution had different effects on human sperm motility/death,(16) meaning that

it could possibly have a contraceptive action or become an anti-early pregnancy agent in the gynecology department. (17) Accordingly, we are interested in whether its active target is also associated with myosin. It has been known that non-muscle myosin Ⅱ has diverse functions in cell contractility, cytokinesis and locomotion, and myosin ⅡA negatively regulates cell migration.(18) Since human sperm migrates or moves, we hypothesize that myosin would be involved in the movement, and this is the reason why we want to evaluate the effects of potassium alum on the function of myosin. As for its toxicity, it is somewhat disputed. Some report suggested that long-term administration of potassium alum did not exert tumorigenic or any other toxic actions.(19) In a 13-week oral study, its toxicity was not found.(20) However, another report suggested that it could produce gastrointestinal tract response and so

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2014 Supported by the National Natural Science Foundation of China (No. 30772601) Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province (116044), China △ Postgraduate student Correspondence to: Prof. TANG Ze-yao, Tel: 86-411-86110416, E-mail: [email protected] DOI: 10.1007/s11655-014-1330-5

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on, because early symptoms of toxic cases are related to gastrointestinal complications.(21-24) Combining the above-mentioned phenomenon of its effect and side effect aspects, we are interested in its effects on muscle contractile function. Therefore, we propose that potassium alum would be related to smooth muscle contraction in an isolated organ and phosphorylation of myosin light chain by myosin light chain kinase (MLCK) at the same time. Because phosphorylation of myosin light chain by MLCK is generally considered as the primary mechanism for regulating smooth muscle contraction, phosphorylation of myosin light chain can be simply described as an interaction of Ca2+ with calmodulin (CaM) that induces a conformational change of MLCK and activates MLCK. The activated MLCK catalyzes phosphorylation of myosin light chain. The phosphorylation of myosin light chain triggers cycling of myosin cross-bridges along the actin filaments and the development of force. Or there exists Ca2+/CaMindependent of MLC20 by MLCK in our previous work.(25) Now total report is as follows.

METHODS

anesthesia. The duodenum between the pylorus and the Treitz ligament was removed and luminally perused ex vivo as described earlier.(26) Briefly, the perfusion conditions were as follows. The recirculation luminal perfusion with 30 mL of bicarbonate-buffered Tyrode solution (37 ℃, pH 7.2) was equilibrated with 95% O2, 5% CO2, 25 cm hydrostatic pressure, and 50 mL/min flow rate. The segments were kept in a moist chamber. Contractions of the proximal duodenum were directly monitored by both frequency (contractions per minute) and amplitude (increase in pressure). In some cases, a motility index (frequency times mean amplitude) was calculated. Occasional movement artifacts were easily identified as spikes that appeared simultaneously in both recorded channels, and they were eliminated from data analysis. To determine the time at which tolerance developed to the motility effects of potassium alum, we compared the frequency of contractions in control recordings with the frequency of contractions after potassium alum infusion in different groups of animals. Each animal served as its own control, and the recording of intestinal motility was limited to 6 h.

Animals, Drugs and Instrument The experiments were performed according to the rules of animal care and were approved by the Animal Care Committee of Dalian Medical University with the certification No. SCXK (Liao) 2008-0002. Rabbits (New Zealand, 1.5–2.0 kg) were used in all experiments. Animals were housed five per cage in a temperaturecontrolled room with a 12-h light-dark cycle (lights on at 7:00 A.M.). Food and water were free to access. Drug of aluminum potassium sulfate was purchased from Sigma-aldrich (Substance Name CAS # SARA 313, USA). The ingredient of the Krebs's solution (mmol/L) was as following: sodium chloride 114.0, potassium chloride 4.7, magnesium chloride 1.2, calcium chloride 2.5, sodium dehydrogenate phosphate 1.8, glucose 11.5, sodium bicarbonate 18.0, pH 7.4±0.5. Type HW400S constant temperature smooth muscle trough and type BL-420F biology function experiment system were products of Chengdu Taimeng Science and Technology Ltd., Co. (China).

Preparation, Perfusion of Isolated Duodenal Segments and Measurement of Gastrointestinal Motility The abdomen was opened under urethane

Drug Administration Potassium alum was administered by injecting into the experimental organ chamber with mini syringes according to the articles.(16,23,24) The dosage for continuous administration of potassium alum was chosen to establish a steady-state concentration in the isolated intestinal organ approximately equal to the 50% effective dose (ED50) of potassium alum that results in 50% increasing of transit. After initiation of potassium alum infusion, we compared the frequency and amplitude of contractions in control recordings with the one at various times. Potassium alum infusion resulted in a significant decrease in contractility of the duodenum within 1 h, and its effects persisted for 12 to 15 h. Tolerance to continuous administration of potassium alum in the contractile activity of the intestine was assessed by two means, (1) by measuring the time required for the duodenum to return to normal frequency of contractions and; (2) by recording the loss of effectiveness of administration of potassium alum (2.5, 5, 10, 20 mmol/L), which suggested that the contractile activity of the intestine was tolerant to the effects of continuous potassium alum administration

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(2.5, 5, 10, 20 mmol/L).

Protein Purification The myosin and MLCK used in our study were purified from the chicken gizzard smooth muscle to homogeneity as described previously.(25) The purity analysis of protein samples was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Phosphorylation of Myosin Light Chain Phosphorylation of myosin light chain was carried out in a 20 mmol/L Tris-HCl (pH 7.4) buffer containing 1 mmol/L dithiothreitol (DTT), 5 mmol/L MgCl2, 60 mmol/L KCl, 2 mmol/L ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA), 4 μmol/L myosin and 2 mmol/L ATP. Various concentrations of MLCK in different incubation time and incubation temperature for of myosin light chain phosphorylation were described in detail in the corresponding figure legends.

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was used to measure phosphorus in the range of 0.2–15 nmol/mL of the ATPase reaction mixture. An ATPase reaction mixture was quenched with an equal volume of 0.6 mol/L PCA; the centrifuged supernatant was mixed with an equal volume of the malachite green/molybdate reagent containing 2 g of sodium molybdate, 0.3 g of malachite green and 0.5 g of Triton X-100 or Sterox SE in 1 L of 0.7 mol/L HCl, and the absorbance at 650 nm was then measured after a 35–40 min incubation at 25 ℃. Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately by using relatively low concentrations of native myosin. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8–0.9 mol per site in 0.1 mmol/L KCl, 10 mmol/L MgCl2 (pH 8.0) at 25 ℃ for fresh enzyme preparations.

Other Procedures Determination of Myosin Light Chain Phosphorylation Glycerol-polyacrylamide gel electrophoresis (Glycerol-PAGE) was used to measure the extent of the of myosin light chain phosphorylation. Glycerol PAGE was made using the method: the separating gel containing 13.96% acrylamide, 0.372% bis-acrylamide, 40% (v/v) glycerol, and 0.375 mol/L Tris (pH 8.7); and the stacking gel containing 5.72% acrylamide, 0.152% bisacrylamide, 10% (v/v) glycerol, and 0.125 mol/L Tris-HCl (pH 6.7). Myosin samples which contained 7.5 mol/L urea were added to the sample buffer containing 6 mol/L urea, 20% gycerol, 0.05 mol/L Tris (pH 6.7), 14 mmol/L β-mercaptoethanol, and a moderate amount of 0.01% bromophenol blue (BPB). The reaction mixture was loaded onto the gel. The densitometry extent of myosin light chain phosphorylation was measured with the Scion Image software from Scion Co., Ltd. The extent of di-phosphorylation of myosin light chain (DIP) of MLC20 was selected as the control (calculated as 100%); the extent of mono-phosphorylation of myosin light chain (MIP) of MLC20 was a relative value calculated from MIP/DIP.

Measurement of Myosin Mg2+-ATPase Activity ATPase activities of the fractionated dynein were measured by a malachite green method as described. (26,27) The malachite green method for determination of inorganic phosphate (PI) liberation

Protein concentrations were determined by the method of Bradford.(28) The graphs of phosphorylation of myosin light chain and myosin Mg 2+ -ATPase activities were obtained with Microsoft Excel 2005.

Statistical Analysis The results were expressed as means±standard deviation (x–±s ). Statistical analysis was performed with unpaired t test in the organ chamber experiment, and with one-way ANOVA in other experiments. P