Biomedicine: 2012; 32(3):363 - 368 Effect of nimesulide on cytosolic Phospholipase A, activity in male reproductive organs of mice *Thotakura Balaji, **S, Senthil kumar,***Manickam Ramanathan, *Aruna Subramaniam and *Manickam Subramanian “Thotakura Balaji, Associate Professor, Department of Anatomy, Chettinad Hospital and Research Institute, Chettinad University, Kelambakkam - 60 103, Tamilnadu, India, “*Senthil kumar. 8, Professor, Department of Anatomy, Sif Ramachandra Medical College and Research Institute, Sri Ramachandra University, Poni Chennay 600 0116, Tamilnadu, India “Aruna Subramaniam, Professor, Department of Anatomy, Chettinad Hospital and Research Institute, Chettinad University, Kelambakkam - 603 103, Tamilnadu, India “Manickam Subramanian, Assistant Professor, Department of Anatomy, Cheitinad Hospital and Research Institute, Chettinad University, Kelambakkam - 603 103, Tamilnadu, Indie, “Department of Surgery, Rajah Muthiah Medical College Hospital, Annamalai University, Annamalainagar - 608002, Tamilnadu, India, (Received ™ February, 2012; Revised 12° July, 2012; Accepted 5* August, 2012) Corresponding Author Dr. T.K. Balaji M.Sc., Ph.D. Mobile: 91-9443469106, 9710905221 E-mail: balajitk@yahoo.com Background & Objectives: Ptracthosphotipase AZ (PLA2: 7ir2 Position of membrane phospholipids to release Arachidonic acid (AA). The AA thus re- ig2sed Is converted to prostaglandins (PGs) by the enzyme cyclooxygenase (COX) that ex- ists in two isoforms COX-1 and COX-2. The Plas family consist of cyctosolic phospholipase A2 (cPLA2), secretory phy reaper LA2) both of which are calclum dependent anc Icium independent PLA2 (iPLA2). The PLA2 hes affinity towards AA containing phosph ids. COX-2, which is considered to be upregulated only during inflammation, is constitutively Clean, High eet Of the organ systems. The role of COX? In eae repreductive system Is not fica, High levels of CoX-2 detected in vas deferens of ama are thought to alter the mem- brane properties of sper: t study focuses on the association between COX-2 ane cPLA2 in male reproductive organs of mice. Methods: Adult mice received nimesulide orally. After 3h, 6h, 15 and 45 daye following ni- Resulide administration phospolipid levels and cpla? a vity was measured, Results: There was decrease in phospholipid levete and in vas deferens and epididymis of mice where high levels of COx-2 are detected. Conclusion: Increase in cPLA2 will reduce the Viability of the call as it is thought to Induce weww.Biom Biomedicine - Voi 32; No.3: 201EK Balaji tab Effect of nlmesulide on phospholipase A2 Introduction C yelooxygenase-2 (COX-2) is an isoform of enzyme COX, the other isoform being COX- 1 (1). While Cox-1 is expressed constitutively in most of the tissues, COX-2 in contrast produces prostanoids to physiological stress such as injury and infection (2). The use of COX-2 specific inhibitors led to adverse effects such as acute renal failure (3). The outcome of further experimental studies proved that COX-2 is the constitutive isoform in specific regions of kidney. The constitutively expression of COX-2 in lungs, cardiovascular and nervous system is well documented (4). The available literature on COX-2 and its function in male reproductive ongans is scanty. In humans and primates COX-2 is predominantly expressed in prostate, seminal vesicle and ejaculatory ducts (5) where as in rodents high levels of COX-2 are detected in vas deferens (6). The expression of COX-2 in male reproductive organs of mice in decreasing order is Vas deferens, testis, epididymis and seminal vesicle (7, 8, 9, 10). COX-1 and COX-2 both act on the substrate arachidonic acid*(AA) to produce prostaglandins (PGs). PGs are tissue specific and depend on PG synthetase present in the tissue (11). PGs are called as local hormones and have wide array of effects ‘on the tissue they act. For the production of PGs free AA is required to be released from esterified stores of membrane phospholipids. This is achieved by activation of enzyme phospholipase A, (PLA,). Calcium ion or G protein is required for the activation of PLA,. PLA, cleaves phospholipids fat the sn2 position to release free fatty acids (12). Atleast three types of PLA, are recognized. Cytosolic PLA, (PLA,) and secretory PLA, (GPLA,), both of them are calcium dependent and require calcium at millimolar concentrations to get activated, While the third type is independent PLA, (iPLA,) is not dependent of calcium and in involved in phospholipids remodeling (13). Since cPLA, has affinity for arachidonate containing phospholipids and AA serves as the major substrate for COX, a measure of this particular type of PLA, was considered identical for our study. Targeting the COX-2 molecule is considered effective in www.biomedicineonlineorg the treatment of various types of cancer. Existing data regarding the relationship between COX- 2 and ePLA, is controversial. While the COX-2 expression is high, there is low cPLA, expression in some types of colon carcinoma (14, 15). Various studies have stated high expression of PLA, during tumerogenesis. In contrast High PLA, expression attenuates the viability of the cell. The present study measures the activity of cPLA, following suppression of COX-2 using nimesulide a selective COX-2 inhibitor. Whether cell death following COX-2 suppression is ePLA, mediated? The study focuses on the relation between COX-2 and cPLA, which helps us to determine whether to target COX- 2 in the effective treatment of cancer. Materials and Methods ‘Adult male albino mice of Swiss strain with body weight 25 + 2 gm and approximately 90 days old bred in central animal house ( Annamalai University, Tamilnadu, India) were used in this study. All the animals were fed on standard pellet diet (Agro Corporation Private Limited, Bangalore, India), Water was available ad libitum. The animals were housed in plastic cages under controlled conditions of 12 hours light/12 hours dark eycle, 50% relative humidity and at temperature of 30 + 2 °C, They were maintained in accordance with the guidelines of National Institute of Nutrition (Indian Council of Medical Research, Hyderabad, India) and the study was approved by Animal Ethical Committee, Annamalai University. Nimesulide was purchased from Sigma chemicals (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) which served as a vehicle, The final concentration of DMSO in water was 0.1%. All the drugs were administered orally; Group! served as control received only 0.1% DMSO. Group 2 and 3 received a single oral dose of nimesulide 12 mg/kg body weight and maintained for 3 and 6 hours. Group 4 and 5 received nimesulide 12 mg/kg twice a day for 15 and 45 days respectively. Control group was maintained and sacrificed along with group5 ‘After the treatment period animals were sacrifice¢ by cervical dislocation and processed for following Biomedicine - Vol 32; No.3: 201:parameters. Total phospholipids in the tissue were estimated by the method of Zilversmit and Davis (1950). Cytosoliephospholipase A,(cPLA,) activity assay cPLA, activity was measured using an assay kit (Cayman Kit Cat. No. 765021). The kit works on the following principle. Arachidonoyl Thio-PC 2 synthetic substrate which is supplied with the kit is hydrolysed by the PLA, present in the sample. This releases free thiol which is detected by DTNB (5,5¢-dithiobis-(2-nitrobenzoic acid)). Prior to assay the tissues were processed in the following way. Animals were perfused with PBS (phosphate buffer saline) pH 7.4 containing 0.16 mg/mL heparin to remove any red blood ceils and clots. The tissues were dissected and homogenized in 5 mL of cold 50. mM Hepes bufier pH 7.4 containing 1 mM EDTA per gram tissue, The homogenate was centrifuged at 10,000’ for 15 min at 4 °C. The resultant supernatant was collected and stored at -80 °C until assayed. The assay was performed according to manufacturer instructions. Any iPLA, (calcium independent) activity was removed using bromoenol lactone supplied with the kit, in the same way sPLA, activity was removed prior to assaying using membrane filter with molecular weight cut-off of 30,000. The plate was developed according to kit instruction and read at 414 nm using a plate reader. The cPLA, activity was calculated using the following formula EK Balaji et al: Efect of nimesulide on phospholipas Statistical analysis Statistical analysis was performed using one way analysis of variance: (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) by using statistical package for social science (SPSS) version 10.0 for windows. The values are mean + SD for six samples in each group. P values <0.05 were considered as ievel of significance. Results fable 1 shows the total phospholipid ieveis of testis, epididymis, vas deferens and seminal vesicle in control and nimesulide treated groups. Total phospholipid levels decreased significantly in testis, cauda epididymis and vas deferens by 6 hrs of nimesulide treatment. The phospholipid levels were almost reduced to half in 45 days nimesulide treated group with vas deferens showing least phospholipid levels. The phospholipid levels of seminal vesicle were not affected in any of the nimesulide treated groups. Discussion The Constitutive expression of COX-2 in epididymis and vas deferens of mice is thought to be involved in lipid remodelling of sperm. The secretory epithelium of male reproductive tract is involved in the secretion of PGs through COX-2 Aaa (sample)-A 4, (blank) Adl4/min = 6 min A . PLA? activity = Friel BE Daa sample dilution 10.66 hi“? 0.01 in cPLA? activity was expressed as nmoles/min/mL. www biomedicineonline.org icine - Vol 32; No.3: 2012EK Balaji eal: Effect of nimesulide om phospholipase 42 366 Table 1. Total phospholipid comtent (mg/g tissue) of male reproductive organs in control and nimesulide treated groups Controt 3 hrs 6 hrs 15 days 45 days Testis 9.2 £0.70" 892067 6.02045 SS£041% 5.24039" Cauda epididymis 6.5 = 0.49" 622047 422032 392029 3,520.26 Vas deferens 78 £0.59 722058 632047 412031¢ 4.620.348 Seminal vesicte 85 40.68 8320.63" 7920.60 8.240624 8.340634 \alues are given as mean + S.D. of six experiments in each group ‘Values not sharing & common supersript differ significant Assay of cPLA, activity COX-2 suppression led to an increase in cPLA2 activity. Moderate increase in cPLA2 activity testis and cauda epididymis, whereas in vas deferens the cPLA2 activity increased significantly following 6 hrs of nimesulide administration. No furrther increase or decrease was observed in 15 and 45 days nimesulide and appeared to be the same as that of 6 hrs nimesulide treated group. No ch was observed in any of the nimesulide treated groups (Fig. 1) at p $0.05 (DMRT), was observed in treated groups ange in cPLA? activity of seminal vesicle Table 2, cPLA2 activity (nmoles/min/mL, of testis, cauda epididymis, vas deferens and seminal vesicle in controt and nimesulide treated groups Testis Epididymis Vas deferens Seminal Vesicle Control 4.9 40.37" 10.9 £0.82" 17.5 #133 11.20.85" 3 Hrs 5.14038" 11.00.83 17.9 41.36" 11.4 40.86 6 Hrs 63 40.47 20,341.54 31.3.42.38 11.0 40.83 15 Days 6.6 20.50" 20.241.54¢ 30.2. 42.30" 11.6 40.88" 45 Days 654045 20.4 #1.55¢ 15 40.885 31.4 42.398 ‘Values ate given as mean + S.D. of six experiments in each group. ‘Values not sharing pathway (16). Through our previous studies it was evident that suppression of COX-2 lead to increase of AA as well as PG levels (7, 8, 10). In the present study total phospholipids levels lowered following COX-2 suppression. Decrease in phospholipids level was much higher in vas deferens when compared to testis and epididymis. Obviously no changes were observed in seminal vesicle since the expression of »wm biomedicineontine.org ‘comnon superseript differ significantly at p£ 0.05 (DMR), ‘COX.2 is minimal, Decreased levels of phospholipids indicate phospholipids are rapidiy degraded due to increased PLA, activity. Our study showed increased activity of cPLA, in testis, epididymis and vas deferens following COX-2 suppression which might be due to the following reasons. The high molecular weight cPLA, activity is receptor mediated. An increase in cPLA, activity might Biomedicine - Vol 32; No.3: 2012be due to increased PG production (17) which was well observed in our study. The PGs thus produced might couple with G-protein and induce influx of calcium. Intracellular rise in calcium is a prerequisite for cPLA, activation, The other possibility for increased cPLA, activity is that, an inverse relation is observed between cPLA, and COX-2. This feature is best observed in cancer cells which express high levels of COX-2 but low cPLA, activity (14), The reason for low cPLA, activity in cancer ceils is thought to be responsible for maintaining viability of the cell. COX-2 is thought regulate apoptosis in testis, epdidymis and vas deferens of mice (18). High cPLA, sotivity teads to creased AA which is cytotoxic. An initial decline COX-2 levels and increased cPLA, is observed after kinate induced seizures in hippocampus (19). Increase in cPLA, activity causing ceil death is also being reported (20). it is thought that COX-1 is usually coupled to cPLA, in immediate production of PGs. Where as sPLA, is coupled to COX-2 but not COX-1 and is responsible for time delayed proxiuction of PGs (21). The reason for sPLA, affinity towards COX-2 might be due to following reason. COX-1 is away from the vicinity of sPLA, and sPLA, is much associated with cell membrane rather than cPLA,, which is associated to nuclear membrane and ER (12). But an initiai induction of cPLA, is necessary for SPLA, activation and there also exists a cross talk between the two (22). In mai of mice various subtyy D, HE and HIF are jocali is and vas deferens (23). The iocalization , coincides with mPGES-} and so sPLA might be responsible for the production of PGE (24). Upregulation of sPLA, in cancer ceils is also observed by many researchers (25) which states COX-2 and sPLA, may be directly proportional to each other. Further clarification is required whether there is an increase or decrease in sPLA, following COX-2 suppression. reproductive sPLA, c sach lly Conclusion Itis clearly evident that suppression of COX-2 leads to increased cPLA? activity in male reproductive iomedicineonline.org EK.Balafi etal: Effect of nimesulide on phospholipase A2 organs of mice. This fact can be applied for the treatment of various types of cancer. Specifically targeting the COX-2 molecule in tumor ceils using COX-2 inhibitors might hold good for in vitro studies but extensive research is required prior to targeting the COX-2 in tumor ceils in vivo. References Habemicht AJ, Goerig M, Grudich J, Rothe D, Gronwoid R, Loth U, et al. Human platelet derived growth factor timulates prostaglandin synthesis by activation and by rapid denovo synthesis of eyelooxygenase. J Clin inv 785; 75: 1381-1387. mith WL, Dewitt DL and Garavito RM. Cycloox Structural, celiular and molecular biology. Anna Biochem 2000; 69: 145-182. 3. 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