The role of the central thromboxane A 2 in cardiovascular effects of a phospholipase A 2 activator melittin administrated intracerebroventricularly in normotensive conscious rats

The role of the central thromboxane A 2 in cardiovascular effects of a phospholipase A 2 activator melittin administrated intracerebroventricularly in normotensive conscious rats
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  The role of the central thromboxane A 2  in cardiovascular effectsof a phospholipase A 2  activator melittin administratedintracerebroventricularly in normotensive conscious rats Murat Yalcin  a,* , Fusun Ak  a , Melih Erturk  b a Department of Physiology, Faculty of Veterinary Medicine, Uludag University, Gorukle, 16059 Bursa, Turkey b Student, Faculty of Veterinary Medicine, Uludag University, Gorukle, 16059 Bursa, Turkey Received 26 December 2005; accepted 21 January 2006Available online 9 March 2006 Abstract The current study was designed to determine the cardiovascular effect of centrally administrated melittin, a phospholipase A 2 (PLA 2 ) activator, and the mediation of central thromboxane A 2  (TXA 2 ) and its receptors in normotensive conscious rats. Studieswere performed in normotensive male Sprague Dawley rats injected intracerebroventricularly (i.c.v.) with melittin. Melittin (1.5, 3.0,6.0  l g/5.0  l l; i.c.v.) caused dose- and time-dependent increases in mean arterial pressure (MAP) and decrease in heart rate (HR).Maximal effects were observed 5–10 min after 3.0  l g dose of melittin. In order to test the mediation of central TXA 2  and its centralreceptors in the cardiovascular effect of melittin, the rats were pretreated with furegrelate (500.0  l g; i.c.v.), a TXA 2  synthesis inhib-itor, and SQ-29548 (8.0  l g; i.c.v.), a TXA 2  receptor antagonist, 15 min prior to melittin (3.0  l g). Furegrelate or SQ-29548 partiallyinhibited the pressor effect and bradycardia elicited by melittin.In conclusion, our findings show that centrally administered melittin increases MAP and decreases HR in conscious rats. More-over, according to our findings, central TXA 2  and its receptors may in part mediate melittin-induced cardiovascular effects.   2006 Elsevier Ltd. All rights reserved. Keywords:  Brain phospholipase A 2 ; Melittin; Mean arterial pressure; Heart rate; Thromboxane A 2 ; Intracerebroventricular 1. Introduction The phospholipase A 2  (PLA 2 ) form an expandingsuperfamily of enzymes that specifically hydrolyze theacyl ester bond at the  sn -2 position of glycerol in mem-brane phospholipids to produce free fatty acids andlysophospholipids (Akhlaq and Lloyd, 2004). PLA 2 was detected in central nervous system (Molloy et al.,1998) and it was observed that it provides the precursorsfor many of the lipid mediators involved in normal brainfunction and neuroinflammatory pathophysiologicalprocesses (Akhlaq and Lloyd, 2004). Melittin is a poly-peptide component of bee venom that leads to anincrease in arachidonic acid release and subsequentlyin prostaglandin synthesis by activating PLA 2  (Hassidand Levine, 1977). Prostaglandins (PGs) have variousroles on central regulation of cardiovascular systemand these effects of PGs are mostly variable. Thrombox-ane A 2  (TXA 2 ), which is a biologically active PG (Nar-umiya et al., 1999; Wolfe, 1982) synthesized in thecentral nervous system (Kong et al., 1991; Sirko et al.,1989; Wolfe, 1982) has an important role on the regula-tion of cardiovascular and endocrine function (Arm-stead et al., 1988; Brooks et al., 1986; Murakami 0143-4179/$ - see front matter    2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.npep.2006.01.003 * Corresponding author. Tel.: +90 224 4429200x124; fax: +90 2244428025. E-mail address: (M. Yalcin). Neuropeptides 40 (2006) 207–212 Neuropeptides  et al., 1998; Murakami et al., 2002; Tong et al., 1998;Yamaguchi et al., 1998; Yalcin and Savci, 2004).Previously we reported that intracerebroventricularlyinjected TXA 2  causes an increase in blood pressure byactivating central TXA 2  receptors in normotensive (Yal-cin et al., 2005a) and hypotensive rats (Yalcin and Savci,2004) and also that central TXA 2  is involved in centralregulation of blood pressure especially in hypotensiveconditions (Yalcin et al., 2005b). Recently, it wasreported that PLA 2  (Yokotani et al., 2000) and TXA 2 (Murakami et al., 2002) activated central sympatho-adrenomedullary outflow and thus increased plasmaadrenalin and noradrenalin. Furegrelate, a TXA 2  syn-thase inhibitor, completely abolished the increase of adrenalin but not that of noradrenalin, induced by mel-ittin (Yokotani et al., 2000).Considering these previous reports, the present studyaimed to determine the cardiovascular effect of melittin,a PLA 2  activator, and mediation of central TXA 2  andits central receptors on the effect of melittin in normo-tensive conscious rats. 2. Methods  2.1. Animals Fifty adult male Sprague Dawley rats (250–300 g)(Experimental Animals Breeding and Research Center,Uludag University, Bursa, Turkey) were used in theexperiments. Rats were housed five per cage, incontrolled conditions of temperature (20–24   C),humidity (60–70%), lighting (12 h light/dark cycle)and provided with food and water ad libitum. Thesurgical and experimental protocols were approvedby the Animal Care and Use Committee of UludagUniversity and were in accordance with the NationalInstitute of Health Guide for the Care and Use of Laboratory Animals (  2.2. Surgical procedures Under ether anesthesia, the left common carotidartery of rats was cannulated with PE 60 tubing filledwith heparinised saline (100 U/ml). For intracerebro-ventricular (i.c.v.) injection of drugs, a burr hole wasdrilled through the skull 1.5 mm lateral to midline and1.0 mm posterior to bregma. A 22-gauge stainless steelhypodermic tubing was directed through the holetowards the lateral ventricle. The cannula was lowered4.2 mm below the surface of the skull and was fixed tothe skull with acrylic cement. After surgery, the ratswere placed in individual cages and allowed to recoverfrom anesthesia for 4–5 h. During this period, the ratsremained calm and without evidence of pain.  2.3. Blood pressure and heart rate recording  After the recovery period, the arterial cannula wasconnected to a volumetric pressure transducer (BPT300, BIOPAC Systems Inc., CA, USA). Blood pres-sure and heart rate of rats were recorded and analyzedusing the MP35 system and AcqKnowledge software(BIOPAC Systems Inc., CA, USA). The blood pres-sure is reported as mean arterial pressure (MAP)(mm Hg) and heart rate (HR) is expressed as beats/min.  2.4. Experimental protocol  In order to determine the cardiovascular effect of mel-ittin, a PLA 2  activator, it was injected i.c.v. at doses of 1.5, 3.0 and 6.0  l g/5  l l in normotensive rats and MAPand HR changes were recorded during 1 h after treat-ment. In order to test the mediation of central TXA 2 and its central receptors in the effect of melittin, TXA 2 synthesis inhibitor furegrelate (500.0  l g; i.c.v.) andTXA 2  receptor antagonist SQ-29548 (8.0  l g; i.c.v.) wereadministrated 15 min before melittin (3.0  l g; i.c.v.)injection, and MAP and HR values were recorded.Furegrelate attenuate the increase in TXA 2  at the hypo-thalamus induced by hemorrhage (Yalcin et al., 2005b).I.c.v. pretreatment with SQ-29548 completely blockMAP and HR responses to U-46619, a TXA 2  analog,in normotensive (Yalcin et al., 2005a) and hypotensiverats (Yalcin and Savci, 2004).  2.5. Drugs Melittin and furegrelate were purchased from Sigma– Aldrich Co. (Deisenhofen, Germany) and weredissolved in 0.9% saline. SQ-29548 was obtained fromCayman Chemical (Ann Arbor, MI, USA) and wasdissolved in saline containing 5% dimethylsulfoxide(DMSO).  2.6. Intracerebroventricular injection of drugs I.c.v. injections were made with hand-made injectioncannula (28 gauge stainless steel tubing; made by MuratYalcin) connected to a polyethylene tubing, which wasfilled with saline or saline containing the desired doseof the drug of interest in a 10  l l microsyringe. The injec-tion cannula was inserted through the guide cannula.Drugs were then infused slowly within 60 s. The volumeof vehicle or drug-containing vehicle was 5  l l.  2.7. Statistics Data are presented as mean ± SEM. Repeated-mea-sures analysis of variance (RM-ANOVA; two way)was performed for groups. Dunnett’s test was applied 208  M. Yalcin et al. / Neuropeptides 40 (2006) 207–212  as a post hoc test when significant interactions werefound. A  p  value of <0.05 was considered significant. 3. Results 3.1. Cardiovascular effect of melittin in normotensiveconscious rats We firstly investigated whether melittin, a phospholi-pase activator, can influence cardiovascular parametersin normotensive conscious rats. Therefore, melittin(1.5, 3.0 and 6.0  l g) was injected i.c.v. It caused adose-dependent increase in blood pressure (Fig. 1A).Analysis of variance confirmed that melittin produceda significant dose (  f  (3,16) = 7.18;  p  < 0.01), time(  f  (11,176)=8.83;  p  < 0.001) and dose-time (  f  (33,176) =2.48;  p  < 0.01) dependent effect on blood pressure. Themaximum increase was observed 5 min after 3.0  l g mel-ittin as 21 ± 3 mm Hg. Administration of 6.0  l g melittinproduced similar increases to those observed in the3.0  l g melittin administered group (Fig. 1A). The pres-sor effect lasted up to 15 min after 3.0  l g dose of melittin(Fig. 1A). Heart rates of rats receiving melittindecreased dose- and time-dependently {dose (  f  (3,16) =26.7;  p  < 0.001), time (  f  (11,176) = 2.9;  p  < 0.001) anddose-time interaction (  f  (33,176) = 3.7;  p  < 0.01)}(Fig. 1B). The effect lasted for 60 min (Fig. 1B). 3.2. Effect of furegrelate, a TXA  2  synthase inhibitor, pretreatment on the cardiovascular effect of melittin Subsequently, we tested whether blockade of centralTXA 2  synthesis by using TXA 2  synthesis inhibitor fureg-relate could change cardiovascular effect of melittin.Rats were pretreated with furegrelate (500.0  l g; i.c.v.)or with saline as control 15 min before melittin injection(3.0  l g; i.c.v.). Furegrelate pretreatment, itself, did notcauseanychangesintheresting bloodpressureandheartrate of normotensive rats but it partially attenuated thecardiovascular effect of melittin (Fig. 2A and B). 3.3. Effect of SQ-29548, a TXA  2  receptor antagonist, pretreatment on the cardiovascular effect of melittin Lastly, in order to determine the involvement of cen-tral TXA 2  receptors in the cardiovascular effect of mel-ittin, rats were pretreated with TXA 2  receptorantagonist SQ-29548, (8.0  l g; i.c.v.) or its vehicle(5.0  l l saline containing 5% DMSO, i.c.v.) as control15 min before melittin injection (3.0  l g; i.c.v.). SQ-29548 and its vehicle did not change resting blood pres-sure and heart rate of rats. SQ-29548 pretreatmentpartly abolished the pressor and bradycardic effects of mellittin (Fig. 3A and B). 4. Discussion These data show that centrally administered PLA 2 activator melittin increases MAP and decreases HR.Pretreatment with furegrelate, a TXA 2  synthesis inhibi-tor, or SQ-29548, a TXA 2  receptor antagonist, partlyabolished the pressor and bradycardic effect of melittin.I.c.v. injection of melittin produced a rapid and doseand time dependent pressor and bradycardic response innormal rats. These responses began within one minuteand peaked at around 5–10 min. Approximately22 mm Hg increase in MAP and 55 beats/min decreasein HR were observed after injection of 3.0  l g melittin,and the pressor and bradycardic effects were maintainedfor at least 15 and 60 min in normal conscious rats,respectively. It has been reported that i.c.v. melittinleads to increases in MAP and HR, and that brain    M  e  a  n   A  r   t  e  r   i  a   l   P  r  e  s  s  u  r  e   (  m  m    H  g   ) 0.0 µg1.5 µg3.0 µg6.0 µg Melittin (i.c.v.) ***********    H  e  a  r   t   R  a   t  e   (   b  e  a   t  s   /  m   i  n   ) ********************* Time (minute) 0102030405060 Time (minute) 0102030405060240280320360 BA 100110120130140 Fig. 1. Cardiovascular effect of centrally injected melittin. In normalrats, saline (5.0  l l; i.c.v.) or melittin (1.5, 3.0 or 6.0  l g; i.c.v.) wereadministered after baseline blood pressure measurements had beenobtained, then MAP (A) and HR (B) changes were monitored for thenext 60 min. Data are given as means ± SEM of five measurements.Statistical analysis was performed using two-way RM-ANOVA withpost hoc Dunnett’s test.  *  p  < 0.05 significantly different from the valueof the saline group. M. Yalcin et al. / Neuropeptides 40 (2006) 207–212  209  kinins mediated these effects of melittin in anesthetizeddogs (Thomas et al., 1984) and normotensive awake rats(Yang et al., 1989). In anesthetized rats, central melittinadministration gave a biphasic response in MAP inwhich a depressor response was followed by a pressorphase achieved by an increase in HR (Thomas andHiley, 1988). Although the MAP results of these studiesshow compatibility with our results, the HR findings donot. These discrepancies can be due to anesthesia, ani-mal species or the difference in drug administration.Furthermore, it was observed that melittin adminis-trated i.c.v. activated of central sympatho-adrenomedul-lary outflow and increased plasma adrenalin andnoradrenalin, which are hormones regulated cardiovas-cular system, in anaesthetized rats (Murakami et al.,2002). Increasing MAP finding of our and previousstudies are compatible with these hormones increasingevoked melittin.Melittin activates PLA 2  through its interaction withthe membrane bound phospholipid side chains (Nishiya,1991), which results in increased lipid hyrolysis of thePLA 2 -phospholipid complex and thus augments arachi-donic acid release and subsequently raises PG produc-tion. PG is synthesized in the central nervous systemand has important functions on regulation of varioussystems including cardiovascular system. To date, sev-eral studies investigating the mediation of exogenousPGs in central regulation of cardiovascular system have Time (minute) 0102030405060 Time (minute) 0102030405060    M  e  a  n   A  r   t  e  r   i  a   l   P  r  e  s  s  u  r  e   (  m  m    H  g   ) 105110115120125130135140 Saline + salineSaline + melittinFuregrelate + melittin*** + * +    H  e  a  r   t   R  a   t  e   (   b  e  a   t  s   /  m   i  n   ) 260280300320340360 Saline + salineSaline + melittinFuregrelate + melittin***** +++ ** BA ** Fig. 2. Effect of pretreatment with furegrelate, a TXA 2  synthesisinhibitor, on the cardiovascular response to melittin injected i.c.v. Ratswere pre-administered with saline (5.0  l l; i.c.v.) or furegrelate(500.0  l g; i.c.v.). 15 min after pretreatment, melittin (3.0  l g; i.c.v.) orsaline (5.0  l l; i.c.v.) were injected and the MAP (A) and HR (B) of ratswere monitored for a 60 min period. ‘‘0’’ shows time of melittin orsaline injection 15 min after furegrelate or saline pretreatment. Dataare given as means ± SEM of five measurements. Statistical analysiswas performed using two-way RM-ANOVA with post hoc Dunnett’stest.  *  p  < 0.05 significantly different from the value of the saline +saline group and  +  p  < 0.05 significantly different from the value of thefuregrelate + melittin group.    M  e  a  n   A  r   t  e  r   i  a   l   P  r  e  s  s  u  r  e   (  m  m    H  g   ) 105110115120125130135 5%DMSO + saline5%DMSO + melittinSQ-29548 + melittin**** ++    H  e  a  r   t   R  a   t  e   (   b  e  a   t   /  m   i  n   ) 260280300320340360380 5% DMSO + saline5% DMSO + melittinSQ-29548 + melittin** + ** + *** Time (minute) 0102030405060 Time (minute) 0102030405060 BA Fig. 3. Effect of pretreatment with SQ-29548, a TXA 2  receptorantagonist, on the cardiovascular response to melittin injected i.c.v.Rats were administered with vehicle (saline containing 5% DMSO;5.0  l l; i.c.v.) or SQ-29548 (8.0  l g; i.c.v.). 15 min after pretreatments,melittin (3.0  l g; i.c.v.) or saline (5.0  l l; i.c.v.) were injected and theMAP (A) and HR (B) of the rats were monitored for a 60 min period.‘‘0’’ shows time of melittin or saline injection 15 min after SQ-29548 or5% DMSO pretreatments. Data are given as means ± SEM of fivemeasurements. Statistical analysis was performed using two-way RM-ANOVA with post hoc Dunnett’s test.  *  p  < 0.05 significantly differentfrom the value of the vehicle + saline group and  +  p  < 0.05 significantlydifferent from the value of the SQ-29548 + melittin group.210  M. Yalcin et al. / Neuropeptides 40 (2006) 207–212  been reported (Ariumi et al., 2002; Siren, 1982a,b,c;Zhang et al., 2003). It was observed that PGE 2  andPGD 2  increased the MAP and HR of normotensive ratswhen administered i.c.v. but the highest dose of PGE 2 and PGD 2  induced a strong initial hypotensive effect(Ariumi et al., 2002; Siren, 1982a,b). Moreover, pressorand tachycardic effect of arachidonic acid and PGF 2 a administrated i.c.v. in normotensive rats have beenreported as well (Siren, 1982c). These effects of arachi-donic acid and PGF 2 a  were also blockaded with meclo-fenamate i.c.v. pretreatment (Siren, 1982c). TXA 2 ,which is a biologically active PG, has an important rolein the central regulation of cardiovascular system of normotensive and hemorrhaged hypotensive rats aswell. Recently, we reported that i.c.v. injected TXA 2 ,which is a biologically active PG, let an increase inMAP and decrease in HR by activating central TXA 2 receptors in normotensive conscious rats (Yalcin andSavci, 2004; Yalcin et al., 2005a). In the present study,the pressor response induced by central injection of mel-ittin is in agreement with previous studies reporting thatthe arachidonic acid (Siren, 1982c) and PGs includingPGE 2 , PGD 2  (Ariumi et al., 2002; Siren, 1982a,b),PGF 2 a  (Siren, 1982c) and TXA 2  (Yalcin and Savci,2004), which are injected i.c.v., raised MAP. The brady-cardic response we obtained with i.c.v. injection of mel-ittin is in consistent with the response of centrallyinjected U-46619, a TXA 2  analog.Moreover, in the current study, we investigated themediation of central TXA 2  and its central receptor inthe pressor and bradycardic effect of melittin. Furegre-late, a TXA 2  synthesis inhibitor, or SQ-29548, a TXA 2 receptor antagonist, pretreatments were made 15 minbefore melittin injection. Recently, we reported thatfuregrelate attenuated TXA 2  release in the posteriorhypothalamus (Yalcin et al., 2005b) and SQ-29548blocked cardiovascular effect of U-46619, a TXA 2  ana-logue (Yalcin and Savci, 2004; Yalcin et al., 2005a,b).Both furegrelate and SQ-29548 pretreatment partlyattenuated pressor and bradycardic response of mellit-tin. Furthermore, partly diminishing effect of furegrelateon pressor and bradycardic effect evoked by melittin isconsistent with that of SQ-29548. These findings arethe first report revealing that central TXA 2  and its cen-tral receptors may mediate the pressor and bradycardiceffect of melittin in the normotensive conscious rats. Pre-viously, it was reported that PLA 2  (Yokotani et al.,2000) and TXA 2  (Murakami et al., 2002) activated cen-tral sympatho-adrenomedullary outflow and thenincreased plasma adrenalin and noradrenalin. Furegre-late, a TXA 2  synthase inhibitor, completely abolishedthe rise of adrenalin but not that of noradrenalin,induced by melittin (Yokotani et al., 2000).In conclusion, our data show for the first time thati.c.v. administration of melittin, a PLA 2  activator, exertsa clear pressor and bradycardic effect in normal con-scious rats. Central TXA 2  and its central receptors arepartly involved in the pressor and bradycardic effectsof the drug. These findings suggest that melittin admin-istrated i.c.v. may activate central TXA 2  release withother PGs, as well. Acknowledgement This study was supported by grants from TUBITAK(KARIYER 104V116). We thank to Dr. I. Taci Cangulfor his editorial assistance. References Akhlaq, A.F., Lloyd, A.H., 2004. Brain phospholipase A 2 : perspectiveon the history. Prostaglandins Leukot. Essent. Fatty Acids 71, 161– 169.Ariumi, H., Takano, Y., Masumi, A., Takahashi, S., Hirabara, Y.,Honda, K., Saito, R., Kamiya, H., 2002. 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