Effect of Stimulators upon Human Hepatic Adenylate Cyclase Activity: A Tool of Nitroimidazole Cytotoxicity Asessment

Nitroimidazole is an antibiotic and radiosensitizer chemical with great potentials in imaging. The hepato-cytoxicity evaluation of nitroimidazole and possible energy status changes in liver cells due to its anti-inflammatory characteristics are crucial in tumor imaging and chemotherapy. Adenylate cyclase is key enzyme to cause energy imbalance leading to cytotoxicity. To evaluate energy status in hepatocytes and Kupffer cells, adenylate cyclase activities in isolated liver cells were compared in presence of stimulators. To evaluate the effect of effectors on adenylate cyclase in cultured hepatocyte cells, adenylate cyclase enzyme was stimulated by different GITP, GTP, progesterone and nitroimidazole effectors. In cultured Kupffer cells, prostaglandin E2 and F2 were used as effectors. The results showed that nitroimidazole decreased adenylate cyclase specific activity in dose-dependent manner after pre-incubation of hepatocytes with the nitroimidazole in medium. Nitroimidazole stimulated adenylate cyclase activities in hepatocytes were mediated by cAMP and determined by cAMP mesurement. The stimulatory effect of nitroimidazole on adenylate cyclase was independent of the GTP presence in the assay system perhaps due to a direct effect on the catalytic subunit of adenylate cyclase enzyme. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the nitroimidazole. In conclusion, nitroimidazole exhibits direct effect on the catalytic subunit of the adenylate cyclase system. The adenylate cyclase was hormone sensitive in liver cells. KEYWORD: progesterone, liver, radiosensitizer, bio-imaging, energy status N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 INTRODUCTION The cyclic AMP (cAMP) status in isolated hepatocytes and Kupffer cells from various animals has been reported to play as secondary messenger (Yang and Li 2003; Zhang, Liu, and Harbrecht 2003; Siu 2004). ATP/cAMP depleted ratio have been shown as initial event of energy insufficiency due to stimulated adenylate cyclase activity in isolated hepatocytes challenged by hormones and drugs (Di Fusco and Anand-Srivastava 2000; Radosavljevic, Todorovic, and Sikic 2004). Moreover, hepatic reconditioning in the presence of oxygen prevents apoptosis to increase the tolerance to hypoxia using adenylate cyclase mediated adenosine release with interaction of adenosine A2A receptors (Carini et al. 2004). In this regard, guanylate cyclase was also reported to play an important role at low concentrations of nitric oxide (NO) to increase the resistance against cell killing and oxidative stress as cause of enhanced hepatic tolerance to hypoxia and reperfusion injury (Joshi, Ponthier, and Lancaster 1999; Nandagopal, Dawson, and Dawson 2001). However, the regulatory role of adenylate cyclase in hormone secretion was widely reported in rat hepatocytes. Progesterone challenge to rat hepatocytes and its action over membrane receptor and binding with adenylate cyclase nucleotide component has been reported as two phase process (Ko, In, and Park-Sarge 1999). Activation and attenuation of adenylate cyclase due to GTP binding protein as messenger in membrane receptor-cyclase binding was reviewed (Van Dyke 2004; Kimura, Nakane, and Nagata 1987). Guanine nucleotides have been shown to be required for adenylate cyclase stimulation by hormones and drugs (Skurat et al. 1985; Sanchez-Yague et al. 1994; Shpakov et al. 2005). The present study extended the information that human hepatocytes have specific receptors for GITP, GTP and Kupffer cells have receptors for prostaglandin E2 and F2. N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 Recently nitroimidazoles have been identified as potential anticancer agents with antibiotic and anti-inflammatory properties (Rossouw et al. 2005; Asikoglu et al. 2000). Till now no information is available on the nitroimidazole action over cyclic AMP status in isolated human hepatocytes. We believe that cAMP may speculate the initial event of nitroimidazole cytotoxicity affecting the cyclic AMP dependent adenylate cyclase and its progesterone hormonal control. The positive correlation between the number of phenylated side-chains and phenolic rings of the imidazole molecule and the efficacy to inhibit adenylate cyclase activity appears due to the possible structure-activity relationship of nitroimidazole compound. In present study, cultured control hepatocytes were compared with hepatocytes exposed with progesterone, GTP, and GITP to characterize their adenylate cyclase enzyme activities. Nitroimidazole-induced adenylate cyclase activity was analyzed as an indicator of hepatocytotoxicity of nitroimidazole. To establish stimulatory role of nitroimidazole on phagocytosis, the adenylate cyclase activities in Kupffer cells were analyzed in the presence of nitroimidazole, prostaglandins, and amoebic trophozoits. Our focus was to support the evidence of Gs protein-mediated and hormone-sensitive adenylate cyclase enzyme as cytoxicity marker. Other focus was to associate adenylate cyclase activity with nitroimidazole-induced amoebic resistance due to energy imbalance in Kupffer cells. Novelty of this report is that human liver adenylate cyclase showed effector-sensitive and hormone-sensitive nature. MATERIALS AND METHODS Human hepatocytes and Kupffer cell preparation: Human liver biopsies from amoebisis patients advised for liver biopsy (n=12) were collected by Menghini needle N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 inserting in three subsequent directions near the site of right lobe during aspiration of anchovy sauce as per guideline of human research protocol. These biopsy samples were taken out as by products for hepatocytes isolation, in petri dish in phosphate buffered solution containing HEPES buffer and treated with 0.05 % (w/v) Collagenase and Hylouronidase digestion in above buffer containing 5 mM Ca for ten minutes. All perfusion buffers were oxygenated with 100 % oxygen. After perfusion, livers were excised, weighted, minced with rajor blade and placed with five volumes of ice cold perfusion solution and transferred in Potter-Elvehjem homogenizer. Tissue was dispensed by hand with one or two strokes of a hard rubber pestle. The pestle clearance was 2-3 mm. The dispensate was filtered through 100 micron mesh screen to remove aggregated cells. After filtration, dark grey cell suspension was placed in the beaker with bar magnet. Bar magnet was moved for 45 minutes to separate hepatocytes. The cells suspension was centrifuged at 50 x g for five minutes to precipitate hepatocytes. These precipitated hepatocytes were washed three times by successive suspension. For Kupffer cell isolation, supernatant left after hepatocytes was used for pronase in Kreb’s Hansleit buffer (1 mg/ml) digestion for isolating Kupffer cells (Kmiec 2001). Isolated hepatocytes and Kupffer cells were cultured for the experiments on effector stimulation and membrane adenylate cyclase activity measurement in hepatocyte and Kupffer cells. However, whole hepatocytes and Kupffer cells were used instead of membranes due to lesser yield of parenchymal and nonparenchymal cells (Kmiec 2001). Enzyme preparations: Membrane enriched preparations used in adenylate cyclase assay were prepared as follows: hepatocytes were suspended in homogenization buffer and homogenized in Potter-Elvehjem homogenizer (1000 rpm 8 stokes ). Homogenizer buffer N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 contained 0.01 M Tris HCl buffer pH 7.65 containing 1.0 mM dithiotritol. The homogenate was filtered through cheesecloth and centrifuged at 1200 x g for 15 minutes to separate out membranes. The membrane pallet was suspended in buffer and used in adenylate cyclase enzyme assays. Adenylate Cyclase assay: The enzyme reaction incubation mixture contained in total 0.2 ml volume: 22 mM Tris HCl pH 7.65, 10 mM MgCl2 , 0.6 mM thiophyllin, 0.15 mg/ml of bovine serum albumin, 9 mM phosphoenolpyruvate, 35 units/ml of pyruvate kinase, 1.0 mM disodium cyclic AMP, 5.0 mM KCl, 22 mM ammonium sulphate, 0.25 mM dithiothritol, 1.0 mM ATP and P-ATP ( 2 x 10 cpm ). Membrane preparations (50-100 g membrane protein) were added to each sample. After incubation for 15 minutes at 37 C, the reaction was terminated by putting tubes in boiled water for three minutes for each sample. Finally, the levels of P-radioactivity were measured as cyclic P-AMP as described elsewhere (Alvarez et al. 1995). For standard, 2 x 10 cpm P-ATP showed P-radioactivity as 80 cpm. Protein concentrations were measured by the procedure of Lowry et al (Lowry et al. 1951). Isolated hepatocyte cultures and effectors: Hepatocytes in TC 199 cultures were maintained in falcon flasks under carbogen gas 95 % oxygen and 5 % carbon dioxide along with stimulators added as following: Group I: control hepatocyte cultures without any added effectors; Group II: hepatocyte cultures were maintained by incubating progesterone 5.0 mM with hepatocytes in group I cultures. Group III hepatocyte cultures were added with Entamoeba histolytica trophozoits 2 x 10 cells/ml in group I cultures. N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 Group IV hepatocyte cultures were added with nitroimidazole stimulator added 10 g/ml in group I cultures. These cultures were maintained in falcon flasks and after 48 hours incubation, relative adenylate cyclase activity was measured in nano moles per mg protein per 15 minutes in harvested hepatocytes in presence of GTP and GITP stimulators. Isolated Kupffer cell cultures and effectors: Kupffer cells in TC 199 cultures were maintained in falcon flasks under carbogen gas 95 % oxygen and 5 % carbon dioxide along with stimulations added as following: Group I: Control Kupffer cell cultures without any effectors. Group II: Kupffer cell cultures were added Entamoeba histolytica trophozoits 2 x 10 cells/ml in group I cultures. Group III: Kupffer cell cultures were added nitroimidazole stimulator 10 g/ml in group I cultures. These cultures were maintained in falcon flasks and after 48 hours incubation, relative adenylate cyclase activity (in nano moles per mg protein per 15 minutes) and cAMP content (p moles/mg/min) were measured in harvested sticky Kupffer cells. The activities of phosphodiesterase enzyme were measured as indicator of membrane integrity in each group. The activities of prostaglandins E2 and F2 were measured in each group. RESULTS Control group hepatocyte cells showed baseline adenylate cyclase enzyme activities. The hepatocyte yield was (35 + 3) x 10 cells/ml as source livers were normal. N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 In these liver biopsies, the hepatocyte yield and adenylate cyclase activity are shown in Table 1. The addition of stimulators altered the % yield of both liver cells. Activity of adenylate cyclase enzyme is represented in both ways i.e. units per mg protein and units per 10 cells as shown in Figure 1. The protein concentration in hepatocytes is represented as mg/ g wt from liver biopsies (Table 1). Adenylate Cyclase activity in hepatocyte cells was sensitive to additives. The enzyme activity was enhanced in the following order: progesterone < nitroimidazole < trophozoits as shown in Table 2. The enzyme activity is represented as pmoles/mg/15 minutes(units/mg). The stimulators GTP and GITP exhibited additive effect on adenylate cyclase sensitive to both amoebic trophozoits and nitroimidazole chemical agent. The nitroimidazole stimulated the adenylate cyclase enzyme activity and quantitative difference in different group was observed as shown in Figure 1. Progesterone in the presence of GTP in hepatocyte culture medium enhanced the enzyme activity 1.75 fold while progesterone in the presence of GITP also enhanced the enzyme activity but less than GTP addition. It indicated that adenylate cyclase activity was hormone sensitive as well as nitroimidazole sensitive. Other important feature of Entamoeba histolytica trophozoites induced hepatocyte behavior, showed increased the adenylate cyclase enzyme activity in hepatocytes. Thus adenylate cyclase enzyme activity was nitroimidazole-sensitive and showed the enhanced enzyme activity as shown in Table 2 in different cultured hepatocyte groups. Both GTP and GITP stimulators also showed the enhanced adenylate cyclase enzyme activity. However, GITP enhanced adenylate cyclase activity was more than that of GTP induced enzyme activity. The addition of trophozoits enhanced the adenylate N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 cyclase activity in hepatocytes and GITP or GTP stimulatory effects both. The progesterone addition in medium also enhanced the adenylate cyclase enzyme activity. The hepatocyte cultures with progesterone in medium showed 33 % increase in the adenylate cyclase enzyme activity over basal activity. The control group Kupffer cells showed baseline adenylate cyclase enzyme activities and their yield was (17 + 1.0) x 10 cells/ml in normal liver biopsies. Adenylate cyclase activities from Kupffer cells were sensitive to the presence of both amoebic trophozoites and nitroimidazole chemical agent added in the culture media. The phosphodiesterase enzyme activities in Kupffer cells as index of energy turn over showed the significantly enhanced enzyme activity after addition of trophozoits and nitroimidazole at lesser extent. The addition of prostaglandin E2 and prostaglandin F2 in Kupffer cell culture, showed the enhanced kupffer cell adenylate cyclase activities in the following order of additives trophozoites > nitroimidazole > control as shown in Figure 2. DISCUSSION Human liver cells are mainly parenchymal and non-parenchymal cells. The adenylate cyclase enzyme participates in the formation of cAMP from ATP catalyzed by phosphodiesterase enzyme. Phophodiesterase enzyme plays role as membrane integrity marker. The cAMP plays a regulatory roles as a secondary messenger in the synthesis of active proteins by enzyme protein kinase C (PKC) in the presence of effectors as represented in Figure 3. However, human hepatocytes lack Gs protein indicating the active role of Gs-GTP coupled -adrenergic receptors playing role in basal cAMP generation. The resulting excessive rise in intracellular cAMP leads to lots of fluid accumulation. The N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 role of intracellular cGMP in cultured hepatocytes is well understood to regulate cyclic GMP-dependent kinase as shown in Figure 4. However, the role of increased cAMP-dependent adenylate cyclase and phosphodiesterase are not well known. The role of prostaglandins and steroid hormones suggest the activation of adenylate cyclase as a biochemical mechanism of defense (Berstein, Pravosudov, and Kryukova 1995; Melien et al. 1988). Although liver cell separation is widely reported, human hepatocytes and Kupffer cells isolation and culture methods are still poorly reported. Most of the reports are available on rat liver cells. Isolated hepatocyte cultures have been known very promising tool for drug monitoring studies and enzyme regulatory studies (Elaut et al. 2006). In present study, isolation and culture of hepatocytes by collagenase digestion, yielded sufficient number of heaptocytes comparable with the yield by other studies. However, the adenylate cyclase enzyme activities of human hepatocytes were lower than the earlier reported enzyme activity (Zippin et al. 2004). The present study shows the high yield of the viable hepatocytes and adenylate cyclase enzyme estimations. We have attempted in present study to isolate hepatocytes by collagenase digestion, and Kupffer cells by pronase digestion to achieve high enzyme yield. The cultured hepatocytes were viable and sufficient for enzyme estimations. The number of hepatocytes isolated from liver biopsies was in the range of 10 cells/ml. The yield of Kupffer cells was in the range of 10 cells/ml. In recent years, human liver transplantation and liver cell reconditioning and replacement therapeutic techniques have become a promising tool. Adenylate cyclase regulatory properties: Adenylate cyclase enzyme in isolated hepatocytes has been reported as tool of regulatory studies of hormones, enzymes in the N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 presence of Gs bound receptors (Small et al. 2000). Membrane adenylate cyclase activity is defined due to its multi-pass transmembrane protein made of two cytosolic segments as shown in Figure 4. These segments play active roles in association of Gs-GTP. The GTP induced Gs binding with adenylate cyclase can be a key mechanism of adenylate cyclase stimulation or inhibition during progesterone bound -adrenergic receptor (Sunahara et al. 1997). The possible mechanism of adenylate cyclase activation or inhibition by progesterone is shown in Figure 4. The membrane adenylate cyclase activity can be an indicator of membrane viability. However, the enzyme activity depends on the type and nature of effecter. The behavior of liver cells in culture in the presence of effectors may indicate the regulatory effect of stimulators in vivo. However, in cultures, hepatocytes and Kupffer cells may likely experience different physiological environment different from in vivo. In controlled liver cell culture conditions of medium, the liver cells showed adenylate cyclase stimulation in a specific manner by effectors such as progesterone, nitroimidazole, GTP, GITP, and prostaglandins (Aoshiba, Rennard, and Spurzem 1997). The receptors for prostaglandins interact with Gs subunits. Gs-GTP complex binds with -adrenalgic receptor made of seven subunits as shown in Figure 5. Binding of receptor with Gs-GTP protein inhibits adenylate cyclase activity and cAMP formation. The present study highlights the behavior of liver cells and the characteristic features of their cyclic AMP dependent adenylate cyclase regulatory system in culture. Progesterone modulation of adenylate cyclase activity: Progesterone modulates the activity of adenylate cyclase and adenylate cyclase response is hormone sensitive (Gilman 1984). The progesterone stimulates the receptor activating the adenylate cyclase by N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 stimulatory Gs-GTP complex resulting with formation of cAMP. Hepatocytes with progesterone added in medium exhibited the enhanced enzyme activity. The addition of GTP stimulator did not change the adenylate cyclase activity. In contrast, GITP stimulated cells exhibited an apparent adenylate cyclase activation. Unlike the adenylate cyclase enzyme activity in the presence of different effectors in stimulated hepatocytes, the rates of cyclic AMP production was delayed in control hepatocytes. However, progesterone addition to hepatocytes did not exhibit significant delay in GITP stimulated adenylate cyclase activity. Thus reason can be speculated that progesterone binds with stimulatory receptors on hepatocytes which lead to increase in intracellular cyclic AMP accumulation through adenylate cyclase activation via guinine nucleotide regulated mechanism (Cohen-Tannoudji et al. 1991). The Kupffer cells also showed similar guanine regulatory enzyme properties. The progesterone response of adenylate cyclase activity is a two phase process (Ko, In, and Park-Sarge 1999; Martin, Farndale, and Wong 1987). First, progesterone receptor and catalytic subunit (C complex) of adenylate cyclase occupy the place on membrane followed by Gs protein interaction with enzyme to make G protein C complex (Ko, In, and Park-Sarge 1999). However, the addition of GITP stimulated the adenylate cyclase to a greater extent in hepatocytes more than pre-exposed hepatocytes to progesterone. Recently, G protein-enzyme complex formation by GTP/GITP stimulation has been reviewed (Niu et al. 2003). To our present knowledge, present study represents first human hepatocyte adenylate cyclase regulatory properties and enzyme stimulation by nitroimidazole, progesterone, and GTP/GITP stimulators. It suggests the possible Gs protein-GTP N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 complex formation as progesterone stimulation over adenylate cyclase stimulation. In the presence of progesterone, stimulation of adenylate cyclase is associated with phosphodiesterase activation indicating adenylate cyclase as hormone-sensitive. It corroborates with similar view originally reported immunosuppression in cultured hepatocytes due to high deoxycortisone from pregnanolone and hydroxylation (Houslay et al. 1992). However, Progesterone bound with adenylate cyclase is regulated by Gs-GTP complex of enzyme and possibly progesterone receptor from cell surface leads to dose dependent progesterone stimulation of hepatocyte adenylate cyclase in the presence of Mg (Bockaert and Sebben-Perez 1983). The adenylate cyclase stimulatory effects were effector specific and additive except nitroimidazole that did not alter GITP/GTP stimulations. Nitroimidazole stimulation of adenylate cyclase activity: Nitroimidazole is recently emerged as anti-tumor and radiosensitizer compound. It is used in tumor imaging, therapy and anti-amoebic applications. Nitroimidazole has phenolic rings and phenolic side chains as shown in Figure 6. So, it is obvious that the side chains interfere with the dephosphorylation of ATP by phosphodiesterase enzyme. It appears to result with inhibition of hepatocyte adenylate cyclase catalytic subunit function in the presence of nitroimidazole. The cytotoxicity of nitroimidazole is poorly studied and reported. However, its anti-amoebic effect has been proven to decrease hepatic and intestinal infection rate. The nitroimidazole is widely reported as single dose in treatment of amoebic liver abscess. Moreover, nitroimidazole induced cytotoxic effect on human hepatocytes are still not known. DNA strand breakage, energy regulatory enzymes, phagocytosis, and N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 immune response have been reported as nitroimidazole induced cytotoxic effects in animals (Zheng and Olive 1997; Whitaker and McMillan 1992; Singh, Nair, and Pradhan 1991). The role of nitroimidzole is understood to restore energy regulatory process involving adenylate cyclase, phosphodiesterase, cAMP formation in hepatocytes and reported as hormone and GTP specific in action. The nitroimidazole derivatives inhibited anterior pituitary cell function apparently by their direct effect on the catalytic subunit of the adenylate cyclase holoenzyme (Stalla et al. 1989). The nonparenchymal Kuffer cells play a significant role of phagocytosis sensitive to prostaglandins. Recently, nitroimidazole derivatives have been identified as anticancer potential agents. Still the role of imidazoles as anticancer agent is not established (Anderson et al. 2006). The present study indicates the association of imidazole ring structure with the cyclic AMP dependent adenylate cyclase catalytic subunit regulatory mechanism in liver cells. The antimaoebic effect has been proven as effective for amoebic liver abscess as nitroimidazole single dose sufficiently decreasing the hepatic infection rate (Blessmann et al. 2003). In tinidazole treated animals, the phagocytosis, DNA-strand damage, and immune response studies are well known in animals in vivo (Katsitadze et al. 1990). Still it is not known if cytokines in immunopathology are affected by nitroimidazole. In this direction, the present study indicated the role of nitroimidazole to obstruct GTP or GITP (in medium) binding with adenylate cyclase enzyme catalytic unit and GTP/GITP receptors on enzyme active site corroborating with earlier study. The Gs protein can activate both adenylate cyclase and guanylate cyclase enzymes (Johnson and Corbin 1991). Recently, the role of guanulate cyclase activation was reported as a two-step mechanism: a conformational change by ATP in the presence of high Ca concentrations (Yamazaki et N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 al. 2003). It indicates the role of high Ca concentrations on adenylate cyclase. GTP and GITP stimulation: Effect of GTP and GITP stimulators upon adenylate cyclase enzyme activity in hepatocyte membranes was characteristic. The control hepatocytes with progesterone in medium exhibited the enhanced adenylate cyclase enzyme activity and delayed cAMP production. However, no effect was seen on GTP stimulated states. In contrast, GITP stimulated state exhibited an apparent activation. However, progesterone addition to hepatocyte cultures did not cause any significant delay in GITP stimulated enzyme activity. Thus reason can be speculated that progesterone may bind with specific GTP and GITP receptors on hepatocyte membrane which lead to increased intracellular cyclic AMP accumulation through adenylate cyclase activation via guanine nucleotide regulated mechanism. Perhaps the adenylate cyclase catalytic site is sensitive to different effectors and appears occupied in different specific ways by different additives in hepatocyte cultures. The progesterone response of adenylate cyclase activity may be explained as two-phase process of G protein interaction. First, progesterone receptor and catalytic subunit of adenylate cyclase occupy the place on hepatocyte membrane followed by second phase of G proteins interaction with C complex to make G protein-C complex. However GITP stimulates adenylate cyclase to a much greater extent in membranes pre-exposed to progesterone. The role of G protein-C complex formation by GTP/GITP stimulation has been reviewed earlier (Johnson and Corbin 1991). Kupffer cell enzyme regulatory properties: In present study, the amoebic trophozoits were N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9 added in cultured non-parenchymal liver cells to observe the enzyme phosphodiesterase changes in vivo in presence of protaglandins as regulatory effectors. However, the in vivo trophozoit infection induced adenylate cyclase enzyme changes were appreciably higher than earlier animal liver cell reports (Yamazaki et al. 2003). These animals suggested high rate of immunopathological changes and membrane damage in liver. Entamoeba histolytica infection seems to disrupt hepatocyte membrane as reported widely (Soid-Raggi, Torres-Marquez, and Meza 1998). Membrane adenylate cyclase activity serves as indicator of membrane integrity as drug-sensitive or effector sensitive. The present study shows 5’ nitroimidazole specific cytotoxicity due to adenylate cyclase stimulation. as hormone sensitive. The adenylate cyclase stimulation was also hormone sensitive and possibly dependent on Gs protein interaction with subsequent stimulatory effect of GTP/GITP stimulators. Progesterone bound with adenylate cyclase appears to be regulated by guanine nucleotide component of enzyme. Possibly, the progesterone effects the -adrenergic receptors from hepatocyte cell surface leading to dose dependent progesterone stimulation of adenylate cyclase in hepatocytes. The stimulatory effects were effector-specific and additive while nitroimidazole altered GITP/GTP stimulation as minimal. In conclusion, the hepatocyte adenylate cyclase provides cAMP as a second messenger. The adenylate cyclase activity in membrane is progesterone hormone-sensitive through receptor in the presence of Gs-GTP complex. The interaction of Gs-GTP with adenylate cyclase catalytic subunit regulates the available cAMP. The nitroimidazole enhances adenylate cyclase stimulation as a result of its cytotoxicity and resistance to amoebic trophozoits. N at ur e P re ce di ng s : h dl :1 01 01 /n pr e. 20 09 .2 85 0. 1 : P os te d 4 F eb 2 00 9