General and Comparative Endocrinology 171 (2011) 301–308

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General and Comparative Endocrinology

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b-Endorphin inhibits phagocytic activity of lizard splenic phagocytes throughl receptor-coupled adenylate cyclase-protein kinase A signaling pathway

Sunil Kumar a, Soma M. Ghorai b, Umesh Rai a,⇑a Department of Zoology, University of Delhi, Delhi 110 007, Indiab Department of Zoology, Hindu College, University of Delhi, Delhi 110 007, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 September 2010Revised 5 February 2011Accepted 17 February 2011Available online 23 February 2011

Keywords:b-EndorphinPhagocytesOpioid receptorAC–cAMP–PKAReptiles

0016-6480/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ygcen.2011.02.008

⇑ Corresponding author.E-mail address: rai_u@rediffmail.com (U. Rai).

The receptor-coupled intracellular signaling mechanism of endogenous opioid peptide b-endorphin (b-end) is explored for the first time in ectothermic vertebrates using wall lizard as a model. b-End inhibitedthe percentage phagocytosis and phagocytic index of lizard splenic phagocytes in a dose-dependent man-ner. The inhibitory effect of b-end on phagocytosis was completely antagonized by non-selective opioidreceptor antagonist naltrexone and also by selective l-receptor antagonist CTAP. However, selectiveantagonists for other opioid receptors like NTI for d-receptor and NorBNI for j-receptor did not alterthe effect of b-end on phagocytosis. This suggests that b-end mediated its inhibitory effect on phagocyticactivity of splenic phagocytes exclusively through l opioid receptors. The l opioid receptor-coupleddownstream signaling cascade was subsequently explored using inhibitors of adenylate cyclase (SQ22536) and protein kinase A (H-89). Both SQ 22536 and H-89 abolished the inhibitory effect of b-endon phagocytosis in a concentration-related manner. Implication of cAMP as second messenger was cor-roborated by cAMP assay where an increase in intracellular cAMP level was observed in response to b-end treatment. It can be concluded that b-end downregulated the phagocytic activity of lizard splenicphagocytes through l opioid receptor-coupled adenylate cyclase-cAMP-protein kinase A pathway.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Beta-endorphin (b-end) encoded by proopiomelanocortin(POMC) gene is expressed primarily in pars distalis and pars inter-media of the pituitary gland. The different forms of b-end are re-ported in jawed vertebrates due to differential post-translationalprocessing of the same POMC gene product depending on animalsand tissue specificity. b-End (1–31) is yielded as an end-product inthe anterior pituitary (pars distalis) while its moderate form (1–26)is also present in the intermediate pituitary (pars intermedia) ofmammals. Further, an extensive N-terminal acetylation of b-endoccurs in the intermediate pituitary cells. In anuran amphibian,N-acetylated small form of b-end (1–8) is shown as a major end-product in the intermediate pituitary cells [17,22,23,37,61,66].The small form of b-end is also demonstrated in other lower ecto-thermic vertebrates [14,19–21,31,34,64]. However, such smallform of b-end is not detected in the pituitary of mammals[24,35], birds [42] and reptiles [16,18]. It is noteworthy that amongthe reptiles, N-acetylated form of b-end is present in turtles [18]but reported absent in lizard Anolis carolinensis [18]. In light of this,non-acetylated form of b-end was used in the present study.

ll rights reserved.

There is growing evidence that b-end secretion in the pituitaryincreases during stress in fishes [62,65], amphibians [40] andmammals [7,8,10,72]. However, studies on physiological signifi-cance of b-end in regulation of immune responses which is highlycompromised during stress are limited to fishes [56,68] and mam-mals [11,43,44,60]. Since reptiles occupy a phylogenic pivotal posi-tion being ancestor to both classes of endotherms, birds andmammals, the present study in wall lizards was aimed to providean insight to develop a comparative understanding on immuno-modulatory role of non-acetylated form of b-endorphin.

The innate immune system in vertebrates forms the first line ofhost defense against invading pathogens. In ectothermic verte-brates including reptiles, the innate immune system plays crucialrole in eliminating microbial organisms, as adaptive immune sys-tem is sluggish and takes time to respond against the pathogens[25]. Phagocytes, importantly macrophages and neutrophils, con-stitute the major cellular arms of innate immunity. These cellseliminate the pathogens via phagocytosis and also by releasingcytotoxic substances and proinflammatory cytokines. Thus, thepresent in vitro study in the wall lizard is focused on the role ofb-end in regulation of phagocytosis.

Although opioid receptors are cloned from immune as well asnon-immune tissues of fishes [1,5,6,12,15,46] and mammals[26,32,53,54,70], the functional existence of opioid receptors espe-cially on immune cells is not demonstrated in reptiles. Similarly,

302 S. Kumar et al. / General and Comparative Endocrinology 171 (2011) 301–308

the second messenger system involved in transducing the effect ofopioid peptides intracellularly is yet to be explored in immune aswell as non-immune cells of ectothermic vertebrates. Therefore,this study was undertaken to gain an insight about the existenceof functional opioid receptors on lizard splenic phagocytes, also,an attempt was made to understand the specific opioid receptor-coupled downstream signaling mechanism involved in transducingthe b-end effect on phagocytosis.

2. Materials and methods

2.1. Animals

The Indian wall lizard Hemidactylus flaviviridis weighing 8–10 gwere captured from the vicinity of Delhi, India (Delhi: latitude:28�120–28�530 N, longitude: 76�500–77�230). They were maintainedat room temperature in wooden cages encrusted with wire meshfrom top, sides and front for the proper aeration and light. Priorto experiments, lizards were acclimated to the laboratory condi-tions for a week (12L:12D, lights on at 07:00 h). Food and waterwere provided ad libitum. In the present study, only female lizardswere used owing to their greater immunological responses [39].The guidelines of the Institutional Animal Ethics Committee werefollowed for maintenance and sacrifice of the animals.

2.2. Reagents and culture medium

Molecular biology grade regular use laboratory chemicals werepurchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai, In-dia), Merck Specialties Pvt. Ltd. (Navi Mumbai, India), Central DrugHouse (P) Ltd. (New Delhi, India) and Qualigens Fine Chemicals(Mumbai, India). The cell culture medium RPMI 1640, non-selec-tive opioid receptor blocker, naltrexone (Nalt), adenylate cyclaseinhibitor SQ 22536, protein kinase A inhibitor H-89, phosphodies-terase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), cAMP en-zyme immunoassay kit and MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] were purchased from Sig-ma–Aldrich Co. (St. Louis, MO, USA). Human b-endorphin (b-end)(NeoMPS, Inc., San Diego, CA, USA) and the selective opioid recep-tor antagonists like l-antagonist CTAP (H-D-Phe-Cys-Tyr-Arg-Thr-Pen-Thr-NH2), d1,2-antagonist naltrindole hydrochloride (NTI) andj-antagonist nor-Binaltorphimine dihydrochloride (NorBNI) werethe kind gifts from National Institute on Drug Abuse (NIDA), Na-tional Institute of Health (Bethesda, USA). Since b-end of reptiliansource is commercially unavailable, in this study b-end of humanorigin was used. Moreover, human b-end is reported physiologi-cally active in lizard Podarcis sicula Raf [13].

Cell culture medium RPMI 1640 was supplemented with100 lg/ml streptomycin, 100 IU/ml penicillin, 40 lg/ml gentami-cin, 5.94 mg/ml HEPES buffer {N-(2-hydroxyethyl) piperazine-N0-(2-ehtanesulphonic acid)} and 0.2% sodium bicarbonate. Fetal calfserum (FCS) (Biological Industries, Kibbutz Beit, Haemek, Israel)was heat-inactivated (56 �C for 20 min) and supplemented 2% inculture medium prior to use. Thereafter, culture medium was re-ferred as complete culture medium.

2.3. Isolation of splenic phagocytes

The splenic phagocyte monolayer was prepared following themethod of Mondal and Rai [39]. In brief, lizards were sacrificed,spleens were excised and pooled in chilled phosphate-buffered sal-ine (PBS, pH 7.2). Single cell suspension of splenocytes was pre-pared by forcing the spleens through nylon mesh (pore size90 lm) in ice-cold complete culture medium. The cell numberwas adjusted to 106 cells/ml. For phagocytic assay, 200 ll of

splenocyte suspension was flooded on each pre-washed slide.Phagocytes were allowed to adhere for 90 min. Non-adhered cellswere washed off with PBS. The viability of adhered cell populationwas above 98% as checked by trypan blue exclusion method. All theexperiments were carried out at 25 �C (±1) in humidified chamber/incubator maintained with 5% CO2.

2.4. Preparation of yeast cell suspension

The yeast cell suspension was prepared by warming commer-cially available Baker’s yeast (Saccharomyces cerevisiae) (1.5 mg/ml PBS) at 80 �C for 20 min. The heat-killed S. cerevisiae cell sus-pension was washed and resuspended in the complete culturemedium.

2.5. Phagocytic assay

Four hundred microlitre yeast cell suspension was flooded oneach slide with adhered phagocyte monolayer. After incubationof phagocytes with yeast cells for 90 min, non-phagocytosed yeastcells were washed off. The monolayer was fixed in methanol,stained with Giemsa and was mounted in DPX. Phagocyte engulf-ing one or more than one yeast cell was considered as positivephagocyte. Without any predetermined sequence or scheme,approximately 200 phagocytes/slide were counted. The experi-menter was blind to technical details of slides while counting.The percentage phagocytosis and phagocytic index were calculatedusing formulae [9]:

(a) Percentage phagocytosis = number of positive phagocytes/100 phagocytes.

(b) Phagocytic index = average number of yeast cells engulfedby each positive phagocytes � percentage phagocytosis.

2.6. Microscopy

The bright field photomicrographs of splenic phagocytesincubated with medium alone (control), and optimally effectiveconcentration of b-end (10�9 M) were captured using ‘NikonEclipse E400 microscope’ attached with ‘Nikon Digital Sight-Fi1 camera’.

2.7. In vitro experiments

2.7.1. Effect of b-endorphinSplenic phagocytes were treated with 10�12, 10�11, 10�10, 10�9

and 10�8 M concentration of b-end for 1 h. After treatment, cellswere washed and processed for phagocytic assay. For control,phagocytes incubated in medium alone for 1 h. The concentrationsof b-end for this experiment were determined based on the plasmaconcentration of b-end reported in different vertebrate groups[38,48,65,73]. Pilot experiment was performed to optimize incuba-tion time for b-end effect on phagocytosis.

2.7.2. Effect of non-selective opioid receptor blockerBased on our observations on concentration-related effect of b-

end, phagocytes were incubated with the most effective concentra-tion of b-end (10�9 M) and a non-selective opioid receptor antago-nist Nalt (10�8 M), simultaneously, for 1 h. Afterward, cells werewashed with PBS, and processed for phagocytic assay. To comparethe results following control groups were made: (a) phagocytesincubated in medium, (b) phagocytes incubated with Nalt(10�8 M), and (c) phagocytes incubated with b-end (10�9 M) for1 h.

Fig. 1. In vitro effect of b-endorphin (b-end) on percentage phagocytosis (bar graph)and phagocytic index (line graph) of splenic phagocytes of the wall lizardHemidactylus flaviviridis. Spleens from 10 lizards were pooled to prepare thephagocyte monolayer for each experiment. The monolayer was treated withdifferent concentrations of b-end for 1 h. Cells incubated in medium alone wereconsidered as control. Each experiment was performed in triplicate and repeatedthree times using different lizards. Data of three independent experiments werepooled, analyzed by one way analysis of variance (ANOVA), and presented asmean ± SEM. Error bars bearing different superscripts(a–d/A–D) differ significantly(Newman–Keuls’ multiple range test, P < 0.05).

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2.7.3. Effect of selective opioid receptor blockersTo decipher the involvement of selective-opioid receptor in

mediation of b-end effect, splenic phagocytes were treated with10�9 M b-end and 10�8 M selective opioid receptor antagonist(l-antagonist CTAP/d-antagonist NTI/j-antagonist NorBNI), simul-taneously, for 1 h. Phagocyte monolayer was washed and pro-cessed for phagocytic assay. In parallel, phagocytes were alsoincubated in medium/b-end/selective antagonist for l-/d-/j-opioidreceptor for 1 h to compare the results. The following controlgroups were run in parallel to compare the results: (a) phagocytesincubated in medium (b) incubated with b-end, and (c) selectiveantagonist for l-/d-/j-opioid receptor for 1 h.

2.7.4. Effect of inhibitors for adenylate cyclase and protein kinase AThe specific opioid receptor-coupled downstream signaling

mechanism involved in mediation of b-end action in phagocytosiswas investigated using inhibitors of adenylate cyclase and proteinkinase A. Splenic phagocyte monolayer was pre-incubated withvarying concentrations of adenylate cyclase inhibitor, SQ 22536(2.5, 5.0, 7.5, and 10.0 nM) or protein kinase A inhibitor, H-89(25, 50, 75, and 100 nM) for 30 min. Then, cells were treated with10�9 M b-end + SQ 22536/H-89 for 1 h. Monolayer was washed andprocessed for phagocytic assay. In order to compare the results, fol-lowing control groups were run in parallel: (a) phagocytes incu-bated in medium alone for 90 min (b) pre-incubated withmedium alone for 30 min and then with 10�9 M b-end for 1 h (c)incubated with 10 nM SQ 22536/100 nM H-89 for 90 min. The con-centration range of the SQ 22536 and H-89 was determined basedon the literature available [50,51] and pilot experiments performedusing lizard splenic phagocytes.

2.8. cAMP assay

The intracellular cAMP level was measured in b-end-treatedsplenic phagocytes following the manufacturer’s protocol (Sig-ma–Aldrich, St. Louis, MO, USA). In brief, 100 ll splenocyte suspen-sion was added to each well of 96 well tissue culture plate. Cellswere incubated for 90 min for the preparation of monolayer.Non-adhered cells were washed off using PBS (pH 7.2). Instead ofcomplete culture medium, PBS was used for further incubations.The adhered cells were incubated with phosphodiesterase inhibi-tor, IBMX (10�4 M) for 30 min to inhibit the degradation of cAMP.Thereafter, phagocytes were incubated with PBS alone (control) or10�9 M b-end for 1 h. After removing supernatant, cells werewashed and lyzed with 0.1 M HCl. To remove the cells debris, celllysate was centrifuged at 600g for 10 min at room temperature.The intracellular cAMP content was estimated in cell lysate usingcAMP enzyme immunoassay kit.

2.9. MTT assay

One hundred microlitre splenic cell suspension was added to 96well tissue culture plate for the preparation of phagocyte mono-layer. After 90 min incubation, non-adhered cells were washedwith PBS. Adhered phagocytes were incubated with b-end for vary-ing concentrations. Phagocytes incubated in medium alone for 1 hwere considered as control. The viability of b-end-treated phago-cytes was assessed following MTT assay [41]. This assay is basedon the ability of mitochondrial enzyme succinate dehydrogenasein live cells to reduce MTT into formazan, the membrane imperme-able and insoluble purple crystals. The amount of formazan pro-duced is directly related to the number of live cells. Aftertreatment with different concentrations of b-end for 1 h, splenicphagocytes were washed and incubated in 100 ll of medium con-taining 0.5 mg/ml MTT for 2 h. Cells were washed with PBS, andpermeabilized by adding 20 ll of 0.1% Triton-X 100. The reduced

product formazan was solubilized by adding 150 ll of DMSO. Fol-lowing incubation at room temperature for 15 min, the absorbancewas measured at 570 nm using ELISA plate reader (MS 5608A, ECIL,India).

3. Statistical analysis

Each treatment was performed in triplicate. All the experimentswere repeated three times to verify the reproducibility of results.The data of three independent experiments were pooled, analyzedby one-way analysis of variance (ANOVA) followed by Newman–Keuls’ multiple range test, and represented as mean ± SEM. Stu-dent’s t-test was used to compare the means of two groups in caseof cAMP estimation.

4. Results

4.1. Effect of b-endorphin

Splenic phagocytes incubated with different concentrations ofb-end ranging from 10�12 to 10�8 M showed concentration-re-lated significant variation in phagocytic activity when analyzedby one way analysis of variance (ANOVA, P < 0.001). b-End inhib-ited the percentage phagocytosis and phagocytic index in dose-dependent manner (Fig. 1). b-End-induced significant decreasein phagocytosis was observed at 10�12 M concentration as com-pared to control (P < 0.05). The inhibitory effect of b-end becamemore pronounced with an increase of concentration. The maxi-mum inhibitory effect of b-end on phagocytic activity was noticedat 10�9 M concentration. It is noteworthy that b-end at any of itsconcentration did not affect the viability of phagocytes as as-sessed by MTT assay (Fig. 2). Furthermore, the bright field imageof splenic phagocytes incubated with medium alone (control cul-ture), and optimally effective concentration of b-end (10�9 M)was captured. As compared to control culture (Fig. 3A), phago-cytes incubated with b-end (10�9 M) (Fig. 3B) showed decreasedphagocytic activity.

Fig. 2. Effect of b-end on cell viability of splenic phagocytes following MTT assay.Data represents mean of the three independent experiments. Error bars bearingsame superscript do not differ significantly.

Fig. 4. Antagonistic effect of non-selective opioid receptor antagonist naltrexone(Nalt) on b-end-induced-inhibition of percentage phagocytosis (bar graph) andphagocytic index (line graph) of splenic phagocytes. Spleens of 10 lizards werepooled to make spleen cell suspension for the preparation of phagocyte monolayer.Phagocytes were treated with 10�8 M Nalt and 10�9 M b-end, simultaneously for1 h. Each treatment was carried out in triplicate. The experiment was repeatedthree times using different spleen samples (10 lizards/spleen sample). Data of threeindependent experiments were pooled, analyzed (ANOVA), and presented asmean ± SEM. Error bar bearing different superscripts(a–b/A–B) differ significantly(Newman–Keuls’ multiple range test, P < 0.01).

304 S. Kumar et al. / General and Comparative Endocrinology 171 (2011) 301–308

4.2. Effect of non-selective opioid receptor blocker

Although non-selective opioid receptor blocker, naltrexone,alone did not affect the phagocytic activity of splenic phagocytes(Nalt vs control, not significant, Fig. 4), it completely antagonizedthe inhibitory effect of b-end on percentage phagocytosis andphagocytic index (b-end vs Nalt + b-end, P < 0.01, Fig. 4). The phag-ocytic activity of splenic phagocytes treated with b-end + Nalt wascomparable to control.

4.3. Effect of selective opioid receptor blocker

The selective l-receptor antagonist, CTAP, completely blockedthe inhibitory effect of b-end on percentage phagocytosis andphagocytic index (Fig. 5). The phagocytic activity of b-end + -CTAP-treated phagocytes was similar to that of control. Unlike tothat of CTAP, the antagonist for d-receptor (NTI) or j-receptor(NorBNI) did not antagonize the inhibitory effect of b-end onphagocytosis. However, none of these antagonists alone had anyeffect on phagocytic activity of splenic phagocytes.

4.4. Effect of inhibitors of adenylate cyclase and protein kinase A

Both, SQ 22536 (an adenylate cyclase inhibitor, Fig. 6A) and H-89 (a protein kinase A inhibitor, Fig. 6B), decreased the inhibitoryeffect of b-end on phagocytosis in a dose-dependent manner.Although these inhibitors did not affect the phagocytosis at thelowest concentrations used in this study (2.5 nM SQ22536/25 nM

Fig. 3. Bright field image of splenic phagocytes incubated with (A) medium alone, and (Bkilled yeast cells are indicated by solid arrow ( ) and arrow head ( ), respectively. Phaginvolved in phagocytosis are demonstrated with dotted arrow ( ) (Original magnificati

H-89), a marked decrease in b-end-induced inhibition of phagocy-tosis was observed when phagocytes were incubated with 5 nm SQ22536 or 50 nm H-89 (b-end vs b-end + 5 nM SQ 22536/50 nM H-89, P < 0.05). The inhibitory effect of b-end on phagocytosis wascompletely abrogated at the higher concentrations of SQ 22536or H-89 (b-end vs b-end + 7.5 nM/10 nM SQ22536, P < 0.05; b-endvs b-end + 100 nM H-89, P < 0.05). The phagocytic activity of sple-nic phagocytes treated with 10�9 M b-end + 7.5 or 10 nM of SQ22536/100 nM of H-89 was comparable to that of control (mediumalone).

4.5. Intracellular cAMP

The intracellular cAMP level in b-end-treated splenic phago-cytes was significantly (P < 0.05) higher than that of control (Fig. 7).

5. Discussion

The non-acetylated and N-acetylated forms of b-end secretedfrom corticotrophs of pars distalis and melanotrophs of pars inter-media, respectively, have been implicated in the regulation ofstress responses. Interestingly, stress-associated corticotrophinreleasing hormone (CRH) is reported to increase the production

) optimally effective concentration of b-end (10�9 M). Splenic phagocytes and heat-ocytes showing phagocytic activity are shown by solid triangle (N) while those not

on 400�).

Fig. 5. Effect of selective l-/d-/j-receptor antagonists, CTAP/NTI/NorBNI, respectively, on b-end-induced inhibition of phagocytosis. Phagocytes were incubated with 10�9 Mb-end + 10�8 M concentration of CTAP/NTI/NorBNI, simultaneously for 1 h. Each treatment was performed in triplicate, and repeated three times using different spleensamples. Data of three independent experiments were pooled for analysis (ANOVA), and presented in the form of mean ± SEM. Error bars bearing same superscript do notdiffer significantly (Newman–Keuls’ multiple range test, P < 0.01).

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of non-acetylated b-end from corticotrophs in fish [64] and mam-mal [45,71]. In reptiles, the effect of stress or stress-associatedhypothalamic hormone on the production of b-end is not explored.Further, N-acetylated form of b-end is shown to be present in tur-tles [18] but absent in lizard A. carolinensis [18]. With regard to therole of b-end in physiological homeostasis including immune re-sponses, the literature is confined only to fishes [56,68] and mam-mals [11,43,44,60]. The present in vitro study in the wall lizarddemonstrates the immunomodulatory role of b-end for the firsttime in reptiles. The non-acetylated form of b-end used in the pres-ent study inhibited the phagocytic activity of splenic phagocytes ina concentration-dependent manner. Our results are in contrast tothat reported in fishes rainbow trout Onchorhynchus mykiss [68],common carp Cyprinus carpio [68] and spotted murrel Channapunctatus [56]. b-End is shown to stimulate the phagocytic activityof phagocytes isolated from head kidney of rainbow trout O. my-kiss, common carp C. carpio and spleen of spotted murrel C. punct-atus. Although reptiles are ectotherms as fishes, the response ofphagocytes to b-end with regard to phagocytic activity in the pres-ent study in wall lizards is alike to that observed with b-end or itsreceptor agonists (l-receptor agonists) in most of the studies inmammals [3,4,11,44,47,63,69]. b-End is reported to inhibit thephagocytic activity of human blood monocytes [44]. Likewise, l-receptor agonist morphine is shown to downregulate the phago-cytic activity of murine peritoneal macrophages [11,47,63] and hu-man blood neutrophils [69]. The similar response is described inrat microglia cells [4] and human monocytic cell line THP-1 [3]using other l-receptor agonists endomorphin 1 and 2. Surpris-ingly, Ortega and group [43] have shown the stimulatory effectof b-end on phagocytic activity of peritoneal exudate cells fromBALB/c mice. This discrepancy may be strain-related as observedby Stanojevic and group [60] that b-end inhibited the phagocyticactivity in Albino Oxford strain, while it showed concentration-re-lated differential effects, stimulatory at lower concentration andinhibitory at higher concentration, in Dark Agouti.

The different members of the opioid receptor family, l, d and j,have been cloned from the immune cells of mammals[26,53,54,70]. In case of ectothermic vertebrates, functional exis-tence of opioid receptors on immune cells is pharmacologicallydemonstrated in fishes [29,56–59,68] using selective opioid recep-tor agonists and antagonists or radioligand binding technique. Re-cently, cDNA for opioid receptors is cloned and characterized fromimmune cells of common carp C. carpio [12] and are shown to sharea high degree of homology with d, l and j opioid receptors ofmammals. However, studies on localization of opioid receptorson immune cells are totally lacking in amphibians and reptiles.

To our surprise, no effort has been made till date to clone the cDNAfor opioid receptors either from immune or even non-immune cellsof reptiles. The present in vitro study pharmacologically demon-strated the existence of opioid receptor on lizard splenic phago-cytes as the non-selective opioid receptor antagonist naltrexonecompletely antagonized the inhibitory effect of b-end on phagocy-tosis. Our results are in concurrence to various studies in mammals[3,4,11,44,47,63,69] except a study in mouse in which opioidreceptor-independent mechanism is implicated for b-end effecton phagocytosis of latex particles by peritoneal macrophages[28]. Interestingly, the diversity in specificity of target cells e.g.Candida albicans/Escherichia coli/latex particles, to be phagocytosedhas been contemplated for this discrepancy. The opioid receptor-mediated effect of b-end on phagocytic activity is demonstratedalso in fishes [56,68]. Although b-end is considered to be l receptoragonist, it is shown to involve other type of opioid receptor also inmodulating the respiratory burst activity of fish splenic phagocytes[56]. Nonetheless, exclusively l receptor is implicated in transduc-ing the effect of b-end on phagocytosis in teleost C. punctatus. Thisis in agreement to that reported in mice where only l receptor ago-nist, and not the d- and j-opioid receptor agonist, influenced thephagocytic activity of murine peritoneal macrophages [63]. Theinvolvement of l opioid receptor in regulation of phagocytosisseems to be conserved since selective l-receptor antagonist CTAPcompletely nullified the effect of b-end on phagocytosis whileselective d-/j-receptor antagonist failed to do so in lizard splenicphagocytes.

The cloning and sequence analysis of opioid receptors revealedthat they belong to the G-protein coupled receptor (GPCR) super-family [32]. Although intracellular Ca2+ level and K+ ion channelhave been implicated in opioid receptor downstream signalingmechanism, AC–cAMP–PKA pathway is shown to be involved lar-gely in transducing the effect of opioids [32]. Opioid receptorsupon activation have long been known to downregulate the aden-ylate cyclase activity and consequently, cAMP production in num-ber of neuronal cells [32]. However, contradictory results arereported in the immune cells. Activation of opioid receptors isshown to inhibit the cAMP production in mouse R1.1 thymoma cellline [33] and human T-cell line [55], while it led to increase ofcAMP production in human lymphocyte cell lines [27], murinesplenic T-cells [67] and human monocytes [2]. Further, concentra-tion- and time-related differential effects of opioid peptide, stimu-latory at low concentration (10�12 M) for 15 min while inhibitoryat high concentration (10�9 M) for 2 h, are demonstrated on cAMPproduction by human peripheral lymphocytes [36]. Similarly,depending on basal level of cAMP, differential effect of b-end is

A

B

Fig. 6. The concentration-related effect of inhibitors of adenylate cyclase SQ 22536 (A)/protein kinase A H-89 (B) on b-end-induced inhibition of phagocytic activity.Phagocytes were first treated with different concentrations of SQ 22536/H-89 for 30 min, and thereafter incubated with b-end + varying concentrations of SQ 22536/H-89 for1 h, simultaneously. Each treatment was carried out in triplicate and the experiment was repeated three times. Data of three independent experiments were pooled andanalyzed by one way analysis of variance (ANOVA), and presented as mean ± SEM. Error bars bearing same superscript(a–d/A–D) do not differ significantly (Newman–Keuls’multiple range test, P < 0.05).

306 S. Kumar et al. / General and Comparative Endocrinology 171 (2011) 301–308

shown on cAMP production by human peripheral blood mononu-clear cells [30]. However, all these studies are carried out in mam-mals and no report is available in the non-mammalian vertebrates.Nevertheless, an increase in intracellular level of cAMP is shown tohave inhibitory effect on phagocytosis in wall lizards H. flaviviridis[49]. In the present study, an adenylate cyclase inhibitor SQ 22536abrogated the inhibitory effect of b-end on phagocytosis. Similarresults were achieved using PKA inhibitor H-89. Taken together,AC–cAMP–PKA pathway seems to be involved in transducing theinhibitory effect of b-end on phagocytosis through l-opioid recep-tor. The role of AC–cAMP–PKA pathway in regulation of phagocyto-sis is also suggested in fish C. punctatus where cortisol [50] andmelatonin [51] are reported to increase the intracellular cAMP le-vel and decrease the phagocytic activity of splenic phagocytes. In

light of these reports in fish [50,51] as well as from the findingsof present study, we can speculate the possible reason regardingthe contradictory effects of b-end on phagocytosis in fish as com-pared to that in reptiles and mammals. b-End would have down-regulated the AC–cAMP–PKA pathway and consequentlystimulated the phagocytosis in spotted murrel fish C. punctatus.Nonetheless, an increase in intracellular cAMP content in responseto b-end in the present study affirmed the involvement of cAMP inregulating phagocytosis.

6. Conclusion

In conclusion, b-end appears to directly regulate the phagocytefunctions across the vertebrates, though its effect on phagocytosis

Fig. 7. Effect of b-end on intracellular cAMP production in splenic phagocytes ofwall lizards. Cells were treated with b-end for 1 h. The intracellular cAMP contentwas estimated using enzyme immunoassay. Data was analyzed by Student’s t-test(⁄P < 0.05) and represented as (mean ± SEM).

S. Kumar et al. / General and Comparative Endocrinology 171 (2011) 301–308 307

in lower vertebrates is opposite as compared to that in higher ver-tebrates. In fish, C. punctatus, b-end differentially regulates thephagocytic and cytotoxic activities of splenic phagocytes. It stimu-lated the phagocytosis and respiratory burst activity, while inhib-ited the nitric oxide production, and thus, helps in maintainingimmune homeostasis [54]. However, in most of the studies inmammals, b-end is shown to downregulate the phagocytic[44,63] and proinflammatory activities [52]. Since effect of b-endon phagocytic activity of lizard splenic phagocytes is similar to thatreported in mammals, we speculate that b-end helps the animal tosurvive from deleterious septic shock during acute infection bydecreasing phagocytosis and consequently proinflammatory re-sponse. Regarding signaling mechanism, l receptor-coupled AC–cAMP–PKA pathway is solely implicated in transducing the effectof b-end since l receptor antagonist or an inhibitor of AC/PKA com-pletely eliminated the b-end-induced inhibition of phagocyticactivity of lizard splenic phagocytes.

Acknowledgments

This research work is financially supported by University GrantsCommission, New Delhi, India and Department of Science andTechnology, New Delhi, India. Authors are indebted to NationalInstitute on Drug Abuse (NIDA), National Institute of Health(NIH) Bethesda, USA, for the kind gift of b-endorphin and its recep-tor antagonists.

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