The Role of Calreticulin Transacetylase in the Activation of Human ...

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Nov 25, 2008 - ... Suvro CHATTERJEE,b Luciano SASO,c Ashok Kumar PRASAD,d and ..... 17) Desai N. J., Sethna S., J. Org. Chem., 22, 388—390 (1957).
Biol. Pharm. Bull. 32(2) 161—165 (2009)

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The Role of Calreticulin Transacetylase in the Activation of Human Platelet Nitrite Reductase by Polyphenolic Acetates Shvetambri ARORA,a Yogesh Kumar TYAGI,e Ajit KUMAR,a Syamantak MAJUMDER,b Daman SALUJA,f Hanumantharao Guru RAJ,*,a Suvro CHATTERJEE,b Luciano SASO,c Ashok Kumar PRASAD,d and Virinder Singh PARMARd a

Department of Biochemistry, V. P. Chest Institute, University of Delhi; d Department of Chemistry, Bioorganic Laboratory, University of Delhi; f Dr. B. R. Ambedkar Center of Biomedical Research, University of Delhi; Delhi-1100 07, India: b AUKBC Research Center, MIT Campus Anna University; Chennai 600 044, India: c Department of Human Physiology and Pharmacology “Vittorio Erspamer” Sapienza University of Rome; P. le Aldo Moro 5, 00185 Rome, Italy: e School of Basic and Applied Sciences, Guru Gobind Singh Indraprastha University; Kashmere Gate, Delhi-110 403, India. Received June 19, 2008; accepted November 20, 2008; published online November 25, 2008 Our earlier investigations demonstrated the remarkable activation of cytochrome P-450 reductase and nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin, a model polyphenolic acetate by way of acetylation, catalyzed by the Calreticulin. Protein acetyltransferase action of Calreticulin was hence termed Calreticulin transacetylase (CRTAase). Nitric oxide synthase and nitrite reductase are now considered as parts of nitric oxide cycle. The activation of platelets nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin has already been demonstrated by us. Also, there are reports that certain proteins such as cytochrome P-450 reductase and cytochrome P-450 are endowed with the nitrite reductase activity in mammalian cells. Keeping these facts in view, we turned our attention to probe whether 7,8-diacetoxy-4-methylcoumarin could alter the levels of nitric oxide independent of the action of nitric oxide synthase in the human platelets model. The incubation of 7,8-diacetoxy-4-methylcoumarin and nitrite with platelets caused significant elevation of nitric oxide and cyclic guanosine monophosphate levels possibly due to the activation of nitrite reductase. Several polyphenolic acetates were similarly found to activate the nitrite reductase in tune with their affinities as substrate to CRTAase. N-w -Nitro-L-arginine methyl ester, the inhibitor of nitric oxide synthase, failed to reverse such an effect of 7,8-diacetoxy-4-methylcoumarin. Clotrimazole which is known to be an inhibitor of nitrite reductase, effectively abolished the 7,8-diacetoxy-4-methylcoumarin mediated enhancement of nitric oxide levels in platelets as well as the nitric oxide mediated effects; such as cyclic guanosine monophosphate levels as well as adenosine diphospate induced platelets aggregation due to nitrite. Key words

nitrite reductase; cytochrome P-450 reductase; polyphenolic acetate; Calreticulin transacetylase

Nitric oxide (NO) is known to play an important role in the regulation of physiological functions. Various cell types are capable of synthesizing NO mainly from L-arginine with the participation of nitric oxide synthase (NOS). The action of NOS on L-arginine is the principal pathway to meet the tissue requirements of NO. The additional demand of NO in certain conditions of stress could be met by inducible nitric oxide synthase (iNOS). Several biological phenomena such as hypoxia is beset with the conditions where NOS is compromised, where the utilization of nitrates for the production of NO is encountered.1) Nitrite reductase (NR) originally described in plants is discernible in microorganisms as well as mammalian cells. Panesar and Chan reported the NR activity in Mouse Leydig Tumor Cells and was attributed to mitochondrial respiratory chain complex III. Also, NO was found to be generated by the utilization of nitrite under basal conditions.2) Nitrate–NR system comprising of protein components such as pyridine nucleotides, flavoproteins and cytochromes are reported to be present in the mitochondria as well as the endoplasmic reticulum for the purpose of NO formation.1) In addition nitrite can function as a signaling molecule independent of the formation of NO.3) The oxidoreductase such as the cytochrome P-450 reductase (CYPR) is known to reduce inorganic nitrate to nitrite and then to NO. The reduction of nitrite to NO is ascribed to various factors including mitochondrial enzymes, polyphenols and protons. Keeping in view of the cardinal role played by NO in the ∗ To whom correspondence should be addressed.

e-mail: [email protected]

physiology of the organisms it has become necessary to study the formation of nitrite and conversion to NO. It is worth mentioning that nitrate, mimics the role of NO in conditions such as normoxia and ischemic-reperfusion.4) NO generated by reduction of nitrite by enzymes of cytochrome P-450 family is also responsible for the activation of cyclic guanosine monophosphate (cGMP) signaling pathway.5,6) The extensive investigations carried out in our laboratory deciphered the identity of TAase with Calreticulin (CRT), a resident protein of the endoplasmic reticulum and consequently the TAase was given a name “Calreticulin transacetylase” (CRTAase).7) Earlier work from our laboratory highlighted the presence of CRTAase in human platelets and activation of platelets NOS by polyphenolic acetates (PA) catalyzed by CRTAase.8) Purified human placental CRTAase mediated acetylation of neuronal nitric oxide synthax (nNOS) by 7,8diacetoxy-4-methylcoumarin (DAMC) was demonstrated.9) Our earlier investigations strongly indicated the CRTAase

Fig. 1.

CRTAase Catalyzed Reaction

Receptor proteins: nitrite reductase has been shown to be the substrate for CRTAase catalyzed reaction by DAMC: A model polyphenolic acetate.

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Fig. 2.

Structure of Polyphenol and Various Polyphenolic Acetate (PA)

Fig. 3.

Nitrite Dependent Elevation of Nitric Oxide in Human Platelets

Different concentrations of nitrite were separately preincubated with platelets for the measurement of NO levels using flowcytometry. Bar-1 indicates fluorescence of platelets. Bar-2 indicates plateletsDCFH-DA. Bar-3 indicates platelets 25 m M nitriteDCFH-DA. Bar-4 indicates platelets50 m M nitriteDCFH-DA. Bar-5 indicates platelets75 m M nitriteDCFH-DA. Bar-6 indicates platelets100 m M nitriteDCFH-DA. Values are mean of four observations with variation 5%.

catalyzed acetylation of CYPR by DAMC culminating in the remarkable hyperbolic activation of the reductase.10) These observations prompted us to examine whether CYPR which is responsible for NR function; could similarly be activated leading to enhancement of intracellular levels of NO upon the addition of sodium nitrite. The present work reports for the first time CRTAase catalyzed activation of cellular NR and inhibition of ADP induced platelet aggregation by nitrite. RESULTS Nitrite Dependent Enhancement of NO Levels in Platelets We examined the effect of DAMC on NO levels in human platelets due to nitrite. Sodium nitrite was preincubated with the platelets followed by incubation with dichloro fluorescein-diacetate (DCFH-DA), the intense fluorescence of DCF was considered proportional to the extent of NO formation.8) NO production in platelets was found to be linear with the concentration of nitrite (Fig. 3). When sodium nitrite alone (devoid of platelets) was dissolved in Phosphate Buffer Saline (PBS) and incubated for 5 min; no NO formation was apparent. Platelets were incubated with sodium nitrite and dimethyl sulfoxide (DMSO) (Fig. 4a) and the concomitant production of NO upon the addition of nitrite and DAMC to platelets was also visualized using the Apollo 4000 NO-Sensor (Fig. 4b). Five fold-enhanced production of NO was observed when DAMC was preincubated with platelets followed by the addition of nitrite (Fig. 4c). The en-

Fig. 4.

Measurement of NO Levels in Platelets by NO Sensor

(a) Platelets were incubated with nitrite and DMSO. (b) Platelets were incubated with nitrite and DAMC (100 m M) followed by NO measurement with NO Sensor. (c) The peak value (nA) achieved from the sensor converted to m M concentration of nitric oxide using the standard curve equation. The values are meanS.E.M. ∗∗ p0.001.

hancement of NO levels in platelets by nitrite is solely due to the activation of NR. In this connection no influence of NOS is anticipated, since L-arginine, the substrate of NOS was not included in the reaction mixture. Also, 7-acetoxy-4-methylcoumarin (7-AMC) and 7-acetoxy-3-acyl-2-methylchromone (7-AAMC) on incubation with nitrite could enhance NO production but less than DAMC (Fig. 5a). These observations strongly implicated the role of platelets NR in the NOS independent formation of NO. Incubation of clotrimazole, spe-

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Fig. 5a. PA

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CRTAase Catalyzed Activation of Nitrite Reductase Activity by

Platelets were incubated with PA (100 m M) and nitrite (25 m M), for 30 min and DCF, fluorescence was measured. Bar-1 indicates fluorescence of platelets. Bar-2 indicates plateletsDCFH-DA. Bar-3 indicates platelets25 m M nitriteDCFH-DA. Bar4 indicates platelets25 m M nitrite100 m M DAMCDCFH-DA. Bar-5 indicates platelets25 m M nitrite100 m M 7-AMCDCFH-DA. Bar-6 indicates platelets25 m M nitrite100 m M 7-AAMCDCFH-DA. Values are mean of four observations with variation 5%.

Fig. 6. Inhibition of ADP-Induced Platelets Aggregation by Nitrite: Effect of DAMC and Clotrimazole PRP was incubated with PA (100 m M) and nitrite (25 m M), for 30 min and aggregation was studied. Bar-1 indicates ADP induced platelets aggregation. Bar-2 indicates platelets aggregation inhibited by 5 m M nitrite. Bar-3 indicates platelets aggregation inhibited by 10 m M nitrite. Bar-4 indicates platelets aggregation inhibited by 15 m M nitrite. Bar-5 indicates platelets aggregation inhibited by 5 m M nitrite100 m M DAMC. Bar-6 indicates platelets aggregation by 5 m M nitrite100 m M DAMC10 m M clotrimazole. Bar-7 indicates platelets aggregation by 5 m M nitrite100 m M DAMC500 m M LNAME.

Table 1. The Effect of Several Classes of PA on CRTAase Catalyzed Activation of Platelets Nitrite Reductase Treatment

Fig. 5b. CRTAase Catalyzed Activation of Nitrite Reductase by PA and Its Inhibition by Clotrimazole Platelets were incubated with PA (100 m M) and nitrite (25 m M), for 30 min and DCF, fluorescence was measured. Bar-1 indicates fluorescence of platelets. Bar-2 indicates plateletsDCFH-DA. Bar-3 indicates platelets25 m M nitriteDCFH-DA. Bar-4 indicates platelets25 m M nitrite100 m M DAMCDCFH-DA. Bar-5 indicates platelets25 m M nitrite100 m M DAMC10 m M clotrimazoleDCFH-DA. Bar-6 indicates platelets25 m M nitrite100 m M DAMC500 m M L-NAMEDCFH-DA.

cific inhibitor of NR with the platelets and nitrite resulted in the inhibition of NR, leading to reduction of NO levels in the cells (Fig. 5b). Reduction of NO levels by the addition of clotrimazole confirms the NOS independent formation of NO. The addition of N-w -nitro-L-arginine methyl ester (LNAME) had no effect on NO production in platelets incubated with nitrite (Fig. 5b). ADP-induced platelets aggregation due to nitrite was also found to be significantly inhibited by DAMC (Fig. 6). Further, clotrimazole was found to abolish the anti-platelet action of nitrite (Fig. 6). Here also LNAME could not inhibit the anti-platelet action of nitrite. These results confirm the activation of nitrite reductase by DAMC. Several PA (Fig. 2) were separately incubated with platelets to examine their relative abilities to activate platelet NR and were found effective in the following order: DAMC7-AMC7-AAMC7, 8-DAF6-AMC5AMC DHMCCPA (Table 1). CPA which is not a substrate for CRTAase was devoid of the ability to NR (Table 1). 7,8-Dihydroxy-4-methylcoumarin (DHMC), deacetylated product of DAMC could not activate NR (Table 1). These results document for the first time the role of CRTAase in the activation of human platelets NR by PA. Nitrite Dependent Enhancement of Cyclic Guanosine Monophosphate (cGMP) Levels in Platelets We studied the stimulatory effects of DAMC on soluble guanylyl cyclases (sGC). sGC are the intracellular receptor for the biological messenger NO.11) On incubation of platelets with DAMC and nitrite intracellular cGMP levels increased due to the stimulation of sGC. 7-AMC could increase the cGMP levels but less than DAMC and CPA which is not a substrate for CRTAase could not activate NR and hence no increase in

Control DAMC 7-AMC 7-AAMC 7,8-DAF 6-AMC 5-AMC DHMC CPA

DCF fluorescence 1021.2 9992.20 7122.1 7002.0 7502.1 5001.3 3002.7 1501.3 1402.98

Platelets were incubated along with DCFH-DA, nitrite (25 m M) and PA (100 m M) followed by the measurement of DCF fluorescence. Changes in DCF fluorescence indicate change in NO levels.

Fig. 7. Nitrite Reductase Catalyzed Modulation of cGMP Levels in Human Platelets: Effect of Clotrimazole Platelets were incubated with 10 m M nitrite, 100 m M PA, 10 m M clotrimazole for 1 h and cell lysate was used for cGMP levels determination. Bar-1 indicates cGMP levels of platelets. Bar-2 indicates cGMP levels of platelets on incubation with 10 m M nitrite. Bar-3 indicates cGMP levels of platelets on incubation with 10 m M nitrite and 100 m M DAMC. Bar-4 indicates cGMP levels of platelets on incubation with 10 m M nitrite and 100 m M 7-AMC. Bar-5 indicates cGMP levels of platelets on incubation with 10 m M nitrite and 100 m M CPA. Bar-6 indicates cGMP levels of platelets on incubation with 10 m M nitrite, 100 m M DAMC and 10 m M clotrimazole. Values are mean of four observations with variation 5%.

cGMP levels was apparent. Also, the incubation of clotrimazole with the platelets resulted in reduction of cGMP levels (Fig. 7). These results document the enhancement of cGMP levels mediated by the activation of NR through augmented NO production. DISCUSSION NO is now well recognized as a crucial signaling molecule playing an important physiological role in neurotransmission, vascular relaxation, platelets aggregation etc. NO mediated

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signal transduction occurs through guanylyl cyclase-coupled receptors, resulting in the increased levels of intracellular cGMP which can engage multiple downstream targets, including kinases, ion channels, and phosphodiesterases, to produce various biological effects. NO is principally produced by the action of NOS on L-arginine. The chemical agents that can enhance intracellular NO levels obviously assume importance. Several studies have highlighted that naturally occurring chemicals such as polyphenols are endowed with the ability to produce NO from nitrite. Such conclusion was apparent from the beneficial action of food products like wine and fruits.4) Previous work carried out in our laboratory projected polyphenolic acetates (PA) as the enhancers of NO in cells such as human platelets and rat tracheal smooth muscle cells.8,21) In this connection polyphenolic acetate such as DAMC was found to produce NO from nitrite to a greater extent compare to parent polyphenol, DHMC. Our persistent investigations demonstrated for the first time the remarkable activation of CYPR by DAMC (Fig. 1).10) Later we elaborated the role of CRTAase in mediating the acetylation of NOS by DAMC.9) The acetylation of NOS resulted in the enhanced intracellular levels of NO. We have also reported the CRTAase mediated activation of CYPR by DAMC and other PA (Fig. 2.) by way of acetylation.12—14) We then turned our attention towards the activation of NR by DAMC and alteration of the cellular levels of NO independent of the action of NOS in platelets (Fig. 3). NO formed due to the activation of NR by DAMC catalyzed by CRTAase was also detected using NO-sensor (Figs. 4b, c). Also, the nitrite dependent NO formation in platelets was further activated by DAMC. These observations strongly implicated the role of platelets NR in the NOS independent formation of NO by nitrite (Fig. 5a). The activation of platelets NR by PA catalyzed by CRTAase by way of acetylation is obvious from the results presented in this report. There are several reports on the existence of nitrite reductase in mammalian cells.5,6) CYPR, an important flavoprotein of the endoplasmic reticulum mediates the reduction of biomolecules such as CYP and also Fe3. Some reports have ascribed the role of NR to CYPR.6) Our earlier work has unequivocally established the CRTAase catalyzed remarkable activation of CYPR due to PA.10,14) We have also demonstrated the purified placental CRTAase catalyzed acetylation of CYPR using antiacetyl lysine antibody.9) Clotrimazole shown to be an effective inhibitor of NR caused the cessation of NO levels (Fig. 5b). The role of NOS in nitrite mediated enhancement of NO levels (Fig. 5b) is ruled out since no L-Arginine the substrate of NOS was included in the incubation mixture. Cotrimazole being the effective inhibitor of CYPR is endowed with the ability to abolish the effect of nitrite. Hence clotrimazole alone can be expected to abolish the aforementioned DAMC mediated effects such as inhibition of ADP induced platelets aggregation (Fig. 6) and stimulation of cGMP levels (Fig. 7). Also, L-NAME could not abolish the NR mediated effects on platelet aggregation (Fig. 6). These results further substantiate the activation of NR by DAMC. The role of CRTAase in mediating the activation of NR by PA is further strengthened by the structure–activity relationship studies (Table 1). The results reported here document for the first time enhancement of nitrite dependent platelet NO levels by PA.

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CONCLUSION Nitrite reductase activity is known in mammalian cells. But for the first time we have reported in ex-vivo CRTAase mediated activation of platelets nitrite reductase by polyphenolic acetates. MATERIALS AND METHODS Synthesis and Characterization of Various Polyphenolic Acetates Synthesis of 7,8-dihydroxy-4-methylcoumarin (DHMC); 7,8-diacetoxy-4-methylcoumarin (DAMC) and 7AMC as reported in our earlier paper15); 7-AAMC was synthesized according to literature procedures16); 6-acetoxy-4methylcoumarin (6-AMC) and 5-acetoxy-4-methylcoumarin (5-AMC) were synthesized and characterized according to the method of Dixit and Padukone.17) The compound 7,8-diacetoxyisoflavone (DAF) was obtained from the collection of the (Late) Professor T. R. Seshadri, FRS, former Head, Department of Chemistry, University of Delhi, India. Catechin commercially obtained from Sigma-Aldrich, U.S.A. and acetylated with acetic anhydride/pyridine method as reported in our earlier publication.18) Chemicals Reagents DCFH-DA, L-NAME, L-arginine, clotrimazole were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Sodium nitrite was purchased from Thomas Baker Chemicals Ltd. (Mumbai, India). All other chemicals used were of high purity and were obtained from local suppliers. Subjects The human subject experiments were performed at V. P. Chest Institute (Delhi, India). Ethical permission No. 00170 was provided by the Animal Research Ethical Committee of V. P. Chest Institute, University of Delhi (India). Isolation of Platelets Rich Plasma (PRP) The citrated blood was used for the preparation of PRP by the method of Vickers and Thompson.19) Nine milliliters of venous blood was collected from one healthy human volunteer (Blood from 20 healthy human volunteers has been collected from this study) and was mixed with 1.0 ml of 3.8% trisodiumcitrate (anticoagulant). The citrated blood was centrifuged at 180g for 10 min at room temperature. The upper two-third fraction of plasma (PRP) was transferred to another centrifuge tube leaving behind lower one-third layer to avoid contamination with WBC’s and RBC’s. PRP was then further centrifuged at 4000g for 5 min to produce a platelets button. The platelets button was then homogenized using a hand homogenizer. Assay of NOS by Flow Cytometry The method outlined by Imrich and Kobzik was followed for the assay of NOS by flow cytometry.20,21) Measurements were made with a 488 nm laser based flow cytometer (FACS calibur, Becton and Dickinson, U.S.A.) and data (light scatter and green fluorescence) was acquired using the Cell Quest software (Becton and Dickinson, U.S.A.). Analysis was performed by applying appropriate gates with reference to the auto fluorescence measured under similar conditions. Platelets isolated as described earlier were suspended in PBS and platelets count was adjusted to 106/ml using electronic particle counter (SYSMEX, Model no. FA 20). DCFH-DA (20 m M) different concentrations of sodium nitrite were then added to

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the cell suspension and separately preincubated at 37 °C for 30 min while rotating (10 rpm) to prevent adherence during assay. To stop the assay, samples were placed on ice for 10 min in dark. Relative green DCF fluorescence was measured. Similarly, several classes of PA were separately preincubated with nitrite and their relative fluorescence measured thereafter. NO Measurements Using NO Sensitive Electrode Direct NO measurements were carried out at 37 °C, pH 7.4 using Apollo 4000, an optically isolated multi-channel free radical analyzer with a NO selective membrane. Electrodes were allowed to equilibrate for at least 30 min in PBS before use. Platelets were suspended in PBS and incubated with PA for 30 min. Next, the electrode was placed in the well containing the platelets in suspension and incubated with nitrite (5—25 m M), followed by a real-time acquisition of NO production through a single-board computer that displays the experimental data. Platelets Aggregation Studies PRP was prepared by the above-mentioned method. Platelets count was adjusted to 250000/m l with homologous platelet-poor plasma (PPP). PPP was prepared by centrifugation of the remainder of blood at 2500 g for 10 min. Platelets counts (PC) were adjusted according to the following formula: PC (PRP)ml PRP 250000ml (PRP 250000). Nitrite (25 m M), DAMC (100 m M) and clotrimazole (10 m M, dissolved in methanol) were preincubated for 30 min with PRP to make the final volume of 0.18 ml. Platelets aggregation was induced by the addition of 5 m M ADP. Platelets aggregation studies were performed using BIODATA Corporation, Platelet Aggregation Profiler, Model No. PAP-4D. Platelets aggregation was expressed as the maximum percentage of light transmittance change (%max) from the baseline at the end of the recording time, using PPP as a reference. Platelets aggregation curves were recorded for 8 min and analyzed according to internationally established standards.22) cGMP Level Measurement cGMP levels were measured using cGMP EIA kit of Cayman Chemical Company, Ann Arbor U.S.A., Cat. No. 581021. Statistical Analysis Student t-test was used to analyze the data. The level of significance applied to the data was p0.05. Acknowledgement This work was supported by Department of Biotechnology, Government of India and by Italian Ministry of University and Research, General Management of Strategies and Development of Internationalization of Scientific and Technological Research. Shvetambri Arora is thankful to Council of Scientific and Industrial Research (CSIR), Govt. of India, New Delhi for financial support. Ajit Kumar is thankful to Department of Science and Technology, Govt. of India, New Delhi for financial support.

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