J Mol Cell Cardiol 32, 2187–2193 (2000) doi:10.1006/jmcc.2000.1241, available online at http://www.idealibrary.com on
An Immortalized Myocyte Cell Line, HL-1, Expresses a Functional -Opioid Receptor
Claire L. Neilan1, Erin Kenyon1, Melissa A. Kovach1, Kristin Bowden1, William C. Claycomb2, John R. Traynor3 and Steven F. Bolling1 1Department of Cardiac Surgery, University of Michigan, B558 MSRB II, Ann Arbor, MI 48109-0686, USA, 2Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, LA 70112, USA and 3Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109-0632, USA (Received 17 March 2000, accepted in revised form 30 August 2000, published electronically 25 September 2000)
C. L. N,E.K,M.A.K,K.B,W.C.C,J.R.T S. F. B.An Immortalized Myocyte Cell Line, HL-1, Expresses a Functional -Opioid Receptor. Journal of Molecular and Cellular Cardiology (2000) 32, 2187–2193. The present study characterizes opioid receptors in an immortalized myocyte cell line, HL-1. Displacement of [3H]bremazocine by selective ligands for the mu (), delta (), and kappa () receptors revealed that only the -selective ligands could fully displace specific [3H]bremazocine binding, indicating the presence of only the -receptor in these cells. Saturation binding studies with the -antagonist naltrindole 3 afforded a Bmax of 32 fmols/mg protein and a KD value for [ H]naltrindole of 0.46 n. The binding affinities of various ligands for the receptor in HL-1 cell membranes obtained from competition binding assays were similar to those obtained using membranes from a neuroblastoma×glioma cell line, NG108-15. Finally, various - agonists were found to stimulate the binding of [35S]GTPS, confirming coupling of the cardiac -receptor to G- protein. DADLE (D-Ala-D-Leu-enkephalin) was found to be the most efficacious in this assay, stimulating the binding of [35S]GTPS to 27% above basal level. The above results indicate that the HL-1 cell line contains a functionally coupled -opioid receptor and therefore provides an in vitro model by which to study the direct effects of opioids on cardiac opioid receptors. 2000 Academic Press K W: Cardiac myocytes; -opioid receptor; Radioligand binding; [35S]GTPS binding.
Introduction that both delta () and kappa () receptors play an important role.4–6 Opiates and opioid peptides are known to exert The presence of - and -opioid receptor types significant cardiovascular effects including re- in rat cardiomyocyte membrane preparations has gulation of blood pressure and heart rate, via an been demonstrated by a number of groups.7–9 As action on the central nervous system.1 In addition yet no evidence has been found to support the it has been shown that opioids exert a direct effect existence of the mu () opioid receptor in cardiac on the heart, and indeed there are several lines of myocytes.9,10 In addition, the only binding data evidence to suggest that cardiac opioid receptors available has been carried out in either rat or guinea and their ligands play a role in cardioprotection pig cardiac membranes. Several functional studies against prolonged ischemic insult and subsequent have shown that upon activation of cardiac opioid reperfusion.2–4 The exact type(s) of opioid receptors receptors there is an activation of protein kinase involved in mediating cardioprotection is not C,11,12 stimulation of phospholipase C,13,14 increased known, but there is substantial evidence to suggest inositol 1,4,5-triphosphate (IP3), increased in-
Please address all correspondence to: Claire L. Neilan, Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
0022–2828/00/122187+07 $35.00/0 2000 Academic Press 2188 C. L. Neilan et al. tracellular calcium and changes in pH.15 In the samples through glass fiber filters (Schleicher and following study the pharmacological charac- Schuell #32, Keene, NH, USA) mounted in a Bran- terization of opioid receptors in an immortalized del 24-well harvester. The filters were subsequently mouse atrial cardiac myocyte cell line is presented. washed three times with ice-cold Tris-HCl, pH 7.4 The study provides evidence for the presence of a and radioactivity determined by scintillation count- functional -opioid receptor and thus provides a ing after addition of 3 ml of Ultima Gold liquid model for the susequent investigation of the direct scintillation fluid. Binding capacities (Bmax) and equi- effects of -opioids on cardiac myocytes. librium dissociation constants (KD) were calculated from non-linear regression using GraphPad Prism, San Diego, CA, USA. Materials and Methods Competition binding assays Cell culture and membrane preparation HL-1 cell membranes (300–500 g protein), or HL-1 cardiac myocytes16 were cultured under a 5% NG108-15 cell membranes (100–150 g protein) CO atmosphere in Ex-Cell 320 medium sup- 2 were incubated at 25°Cin50m Tris-HCl buffer, plemented with 10% fetal bovine serum, 50 g/ pH 7.4 for 1 h with radioligand and varying con- ml endothelial cell growth supplement, 10 g/ml centrations of unlabeled ligand to give a final vol- insulin, 1 retinoic acid, 1×non-essential amino ume of 1 ml (NG108-15 cells) or 500 l (HL-1 acids and 100 norepinephrine to stimulate con- cells). Non-specific binding was defined with 10 tractions. Culture flasks were precoated with 2 g/ naloxone. The reaction was terminated by filtration cm fibronectin/0.02% gelatin solution. NG108-15 as above, and filters were subjected to liquid scin- neuroblastoma cells were grown in Dulbecco’s tillation counting. K values were determined using modified Eagle’s medium (DMEM) supplemented i GraphPad Prism, using K values of 0.20 n for with 10% fetal bovine serum and 2% HAT sup- D [3H]diprenorphine and 0.46 n for [3H]naltrindole plement. Once cells had reached confluency they as determined by saturation assay. For the de- were harvested in HEPES (N-[2-hydroxy- termination of type(s) of opioid receptors present ethyl]piperazine-N′-[2-ethanesulfonic acid], 20 m [3H]bremazocine was used at a concentration of pH 7.4)-buffered saline containing 1 m EDTA 1.0 n. (NG108-15 cells), or trypsin-EDTA (HL-1 cells), then dispersed by agitation and collected by centrifugation at 500×g. The cell pellet was sus- [35S]GTPS binding assays pended in 50 m Tris-HCl buffer pH 7.4, and ho- mogenized with a tissue tearor (Biospec Products, Agonist stimulation of [35S]GTPS binding was Bartlesville, OK, USA). The resultant homogenate measured as described by Traynor and Nahorski.18 was centrifuged for 15 min at approximately Cell membranes (300–500 g protein) were in- 40 000×g at 4°C and the pellet collected, re- cubated for 1 h at 30°CinGTPS binding buffer suspended and recentrifuged. The final pellet was (20 m HEPES, 100 m NaCl, 10 m MgCl , resuspended in 50 m Tris-HCl buffer pH 7.4; sep- 2 pH 7.4). [35S]GTPS (guanosine-5′-O-(3-thio)- arated into 0.5 ml aliquots (0.75–1.0 mg protein) triphosphate) (50 p), and GDP (guanosine 5′-di- and frozen at −80°C. Protein concentration for phosphate) (either 30 or 100 ) were added to cell membrane preparations was determined by the give a final volume of 500 l. The reaction was method of Lowry et al.,17 using a bovine serum terminated by rapid filtration as above except that albumin standard. samples were washed with GTPS binding buffer, pH 7.4, and radioactivity retained on filters ana- lysed by scintillation counting (see above). Basal Saturation binding assays binding was determined in the absence of unlabeled ligand. HL-1 cell membranes (300–500 g protein) were incubated at 25°Cin50m Tris-HCl buffer, pH 7.4 for 1 h with varying concentration of [3H]- Drugs naltrindole in the presence of either water (control) or 10 naloxone to determine total specific bind- [3H]diprenorphine (58 Ci/mmol), [3H]naltrindole ing. The reaction was terminated by filtering the (33 Ci/mmol), [3H]bremazocine (26.5 Ci/mmol) and Characterization of Opioid Receptors in HL-1 Cells 2189
Figure 1 The displacement of specific [3H]bremazocine binding by selective - (DAMGO, 100 n , CTAP, 300 n ), Figure 2 Representative curve of saturation binding of - (DPDPE, DADLE, naltrindole, all 1 ), and - (U69, [3H]naltrindole to receptors in HL-1 cell membranes. ± 593,1 ) opioid ligands. Values represent mean . . . Shown in the inset is the corresponding Scatchard plot. for three experiments performed in duplicate. ∗P<0.05, Student’s t-test.
[35S]GTPS (1250 Ci/mmol) were purchased from DuPont NEN, Boston, MA, USA. The following drugs were generous gifts from the National Institute on Drug Abuse, Rockville, MD, USA: naloxone HCl, SNC80, deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-
Gly-NH2), deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val- 2 5 Gly-NH2), DADLE ([D-Ala , D-Leu ]enkephalin) and DPDPE ([D-Pen2, D-Pen5]enkephalin). CTAP (D-
Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) and nal- trindole were a kind gift from NIH (Bethesda, MD, USA). DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) was Figure 3 The displacement of specific [3H]naltrindole purchased from Tocris Cookson, (Ballwin, MO, binding by various -ligands. [3H]Naltrindole was used USA), DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) from at a concentration of 0.46 n as determined by saturation ± Multiple Peptide Systems (San Diego, CA, USA), and binding. Values represent mean . . . for three ex- periments performed in duplicate. U69,593 from Research Biochemicals International (Natick, MA, USA). CTAP was able to displace only 15% of specific [3H]bremazocine binding. Results Saturation binding of the selective -antagonist [3H]naltrindole to membranes prepared from HL-1 The displacement of the non-selective opioid ligand cells afforded data best fit to a single site with a 3 [ H]bremazocine by various unlabelled opioid lig- binding capacity (Bmax) value of 32 fmols/mg of 3 ands revealed that only -selective ligands, DPDPE, protein, and a dissociation constant (KD) for [ H] DADLE and naltrindole, all at a concentration of 1 naltrindole of 0.46±0.05 n. A representative sat- , were able to fully displace specific [3H]bre- uration binding curve is shown in Figure 2 with mazocine binding from membranes prepared from the Scatchard plot as the inset. HL-1 cells (Fig. 1). Neither the -selective ligand In order to determine if the receptor found in DAMGO nor the -selective ligand U69,593 at HL-1 cells was pharmacologically similar to the concentrations of 100 n and 1 respectively, neuronal -opioid receptor, binding affinities of vari- were able to displace any specifically bound radio- ous -ligands were determined in HL-1 cells and ligand. These concentrations are approximately compared to the values obtained from a 100- and 1000-fold higher than their affinities for neuroblastoma×glioma cell line, NG108-15, the - and -receptor respectively. At a con- which endogenously expresses the mouse -re- centration of 300 n the -selective antagonist ceptor.19 Figure 3 shows the competition binding 2190 C. L. Neilan et al.
3 35 Table 1 Ki values for the inhibition of specific [ H]di- [ S]GTPS binding of 26.6±5.4% above basal level prenorphine binding (NG108-15 cell membranes) and 35 3 3 of [ S]GTP S binding. The non-peptide agonist, [ H]naltrindole binding (HL-1 cell membranes). [ H]Di- prenorphine was used at a concentration of 0.20 n SNC80 (10 ), afforded a stimulation of and [3H]naltrindole at a concentration of 0.46 n,as 14.9±2.2% above basal. The three other -peptides determined by KD values obtained from saturation bind- examined, DPDPE, DSLET, and deltorphin II afforded ing. maximal stimulations of 12.0±2.2, 9.9±6.2, and 12.9±2.7% respectively. Concurrent addition of Ligand NG-108-15 HL-1 the -antagonist naltrindole (1 ) significantly 35 DADLE 3.02±1.30 12.21±3.30 blocked stimulation of [ S]GTPS binding by the DPDPE 1.96±0.76 1.96±0.56 agonists at 1 concentrations, confirming a - Deltorphin 1 2.84±1.07 2.12±0.86 specific agonist effect. Significant differences be- ± ± Naltrindole 0.21 0.03 0.52 0.07 tween individual points were determined by Stu- dent’s t-test. No significant differences were found between stimulation with 10 v 1 agonist, indicating that maximal effect of agonist was achieved at 1 .
Discussion
HL-1 cells, derived from a mouse atrial car- diomyocyte tumor, have been shown to retain the phenotypic characteristics of the adult car- diomyocyte, yet are able to proliferate and can be serially passaged.16 The above study provides evidence for the presence of a functional -opioid Figure 4 Stimulation of [35S]GTPS binding by the vari- receptor in these cells. This agrees with previous ous -agonists at 10 and 1 concentrations, and in- findings that opioid receptors exist in the myo- hibition of agonist stimulation (1 ) by naltrindole cardium; low levels of binding of opioids to cardiac (1 ). Values represent mean±... for at least three muscle from guinea pig and rat hearts have been experiments performed in duplicate. Significant dif- reported.20,21 In more recent years several groups ferences were found between stimulation produced by 1 agonist and stimulation produced by 1 agonist have reported binding of - and -opioids to crude 8,22,23 in the presence of naltrindole. ∗P<0.05, ∗∗P<0.01, Stu- membranes prepared from rat heart, from puri- dent’s t-test. fied rat cardiac sarcolemmal preparations7 and from membranes prepared from rat cardiomyocyte cul- tures.9 curves obtained for the displacement of [3H]n- Interestingly, in the HL-1 cell line, the standard altrindole by four -ligands, the peptide agonists -agonist U69,593, did not displace any specific DPDPE, DADLE, and deltorphin 1, and the non- [3H]bremazocine binding. This is contrary to pre- peptide antagonist naltrindole in membranes pre- vious findings that this ligand does bind to cardiac pared from HL-1 cells. All ligands afforded Hill membranes.7,8,22 The difference may be attributable coefficients of close to unity indicating recognition to species differences (rat v mouse) or indeed may of a single binding site. The binding affinities (Ki) be due to the fact that we are using an immortalized values determined for these competition curves are myocyte cell line. In addition, the standard -opioid given in Table 1 and are compared with those peptides DAMGO and CTAP, used at doses selective obtained in NG108-15 cell membranes. for the -receptor, could not displace any specific In order to determine if the receptor present in [3H]bremazocine binding. This is in agreement with HL-1 cells was functionally coupled to G-proteins, previous findings that no appreciable -receptor the ability of various -opioid agonists to stimulate binding can be identified in cardiac membranes.7,8,24 the binding of [35S]GTPS was assessed (Fig. 4). The ability of only the -ligands to fully displace Optimum level of GDP for use in this assay using all [3H]bremazocine binding argues strongly for the the HL-1 cell line was determined as being 30 presence of the -receptor alone in the HL-1 cells. (data not shown). The peptide agonist DADLE, at The level of -opioid receptors is, however, low. a concentration of 10 M, was found to be the most Saturation binding using [3H]naltrindole revealed efficacious in this assay, affording a stimulation of specific, saturable binding to a receptor population Characterization of Opioid Receptors in HL-1 Cells 2191 of 32 fmols/mg protein. A previous study by Ela et acterized, and indeed, there are many reports of 10 al., reported a Bmax of only 12.9 fmols/mg protein direct effects of opioids on cardiomyocyte opioid for the -receptor in rat ventricular cardiomyocytes. receptors. Opioids increase inositol 1,4,5-tri-phos-
Again the differences observed may be due to species phate (IP3) and elevate intracellular free calcium differences, the use of different heart chambers, by increasing mobilization of calcium from in- or, although more unlikely, the use of a different tracellular stores;15 they cause an activation of radioligand. Interestingly, studies on the relative protein kinase C,11,12 and a stimulation of phos- opioid receptor number in the four chambers of the pholipase C.13,14 Opioids mediate these signal trans- heart have shown that the largest population of duction events via their interaction with G-proteins, opioid receptors is found in the right atrium.8,24 and it has been shown that the effects of opioids To further characterize the receptor found in the on myocardial contraction are blocked by pertussis 29 HL-1 cell line, the binding affinities of the -opioid toxin, indicating that the Gi/Go family of G-proteins peptides DPDPE, DADLE and deltorphin I, and the is involved in this process. Despite all of the reports -non-peptide naltrindole were compared to those on cell signalling mechanisms downstream of G- obtained using membranes prepared from NG108- protein activation, the direct activation of Gi/Go G-
15 cells. DPDPE and deltorphin I had similar Ki proteins in cardiac tissue has not yet been shown. values for the -receptor in both cell lines. DADLE In this study, we chose to use the [35S]GTPS assay appeared to have somewhat lower affinity (12.2 n) in order to determine if -receptor agonists could for the receptor in the HL-1 cells compared to a directly stimulate the binding of [35S]GTPS thus value of 3.0 n for the receptor in NG108-15 cells. demonstrating a functional coupling of the receptor
The antagonist naltrindole also had a slightly lower to Gi/Go. All agonists stimulated the binding of affinity for the receptor in HL-1 cells (0.52 n [35S]GTPS using GDP at a level of 30 .At10 compared to 0.21 n in NG108-15 cells). GDP no stimulation by the agonists was observed, Although no cloning data is available, the ex- and using 100 GDP stimulation dropped sig- istence of -receptor subtypes has been proposed,25,26 nificantly (data not shown). Other studies have based on in vivo antagonism studies using nal- highlighted this dependence on GDP, for example trindole and its analogues, including naltriben stimulation of the binding of [35S]GTPSby- 18 by the (NTB), a putative 2 antagonist, and ben- opioid agonists in SH-SY5Y cells, -receptor 28 zylidenenaltrexone (BNTX), a putative 1 an- in NG108-15 cells, and also by the muscarinic tagonist. DPDPE and DADLE are agonists at the receptor in porcine cardiac membranes.30 Stimu- putative 1 subtype and the peptides DSLET and lation by the agonist was blocked by the concurrent deltorphin II are agonists at the putative 2 subtype. addition of 1 naltrindole, thus confirming a - It has been suggested that the -receptor in NG108- specific response. Naltrindole alone did not produce 27,28 15 cells is of the 2 subtype. Given that the Ki any stimulation (data not shown). The rank order value for DPDPE is similar in both cell lines, and of efficacy between agonists was as follows: that the affinity of DADLE is even lower for the DADLE>SNC80>DPDPE>DSLET>deltorphin II. This receptor in HL-1 cells, it is tempting to propose that corelates with previously reported data using 28 31 32 just the 2 subtype existed in this cell line. In NG108-15 cells, C6 glioma cells and COS cells addition, Hill coefficients were all close to unity both transfected with the rat -receptor. indicating a homogenous receptor population in There has been considerable excitement in recent both cell lines. years following the discovery that opioids (in par- One other possible explanation for the lower ticular - and -ligands) may play an important affinity of DADLE may be the increased presence of role in cardioprotection against prolonged ischemic proteases in this cell line compared to in NG108- insult and subsequent reperfusion. It has been 15 cells. No displacement of [3H]naltrindole from shown that isolated rabbit hearts pre-treated with receptor in HL-1 cells was obtained by the peptide DADLE exhibit improved functional recovery fol- [Leu5]-enkephalin unless a protease inhibitor cock- lowing cardioplegic ischemia and reperfusion.4 Sim- tail was present in the binding buffer, and the ilar results were observed by Kevelaitis et al.,33 who incubation temperature decreased to 4°C (data not reported improved functional preservation of cold- shown). It may be that DADLE is similarly sensitive stored rat hearts pre-treated with DADLE. Opioids to proteases in HL-1 cells and therefore is partially also appear to mimic the effects of ischemic pre- degraded upon incubation with the membrane frac- conditioning.2,3 The exact mechanism by which tion. opioids afford cardioprotection is not known, al- The cell signalling events that occur upon agonist though it is believed to be a result of an action on 5,33,34 activation of opioid receptors have been well char- the KATP channel. All of the above studies 2192 C. L. Neilan et al. have been carried out using either whole hearts or and the involvement of protein kinase C. Br J Pharm primary myocyte cultures. The results of the present 1998; 124: 600–606. 12. V C, P G. Opioid peptide gene expression study clearly show that the HL-1 cell line contains in the primary hereditary cardiomyopathy of the a functionally coupled -opioid receptor and thus Syrian hamster. I. Regulation of prodynorphin gene provides a valid model with which to study the expression by nuclear protein kinase C. J Biol Chem direct effects of opioids on cardiac -receptors whilst 1997; 272: 6685–6692. eliminating lengthy tissue preparation times and 13. S J-Z, W NS, W H-X, W TM. Pertussis toxin, but not tyrosine kinase inhibitors, abolishes complications of mixed tissues. + effects of U50,488H on [Ca2 ] in myocytes. Am J Physiol 1997; 272: C560–C564. 14. Z WM, W TM. Suppression of cAMP by Acknowledgements phosphoinositol/Ca2+ pathway in the cardiac opioid receptor. Am J Physiol 1998; 274: C82–C87. This study was funded by NIH grant 0000HL 15. V C, S H, L EG, G C, C MC. and opioid receptor stimulation 58781. The authors would like to thank Marsha affects cardiac myocyte function and Ca2+ release Gallagher for her additional input. from an intracellular pool in myocytes and neurons. Circ Res 1992; 70: 66–81. 16. C WC, L NA, S BS, E- DB, D JB, B A, I NI. HL-1 References cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult car- 1. H JW. Cardiovascular effects of endogenous diomyocyte. Proc Natl Acad Sci USA 1998; 95: 2979– opiate systems. Ann Rev Pharmacol Toxicol 1983; 23: 2984. 541–594. 17. L OH, R NG, F AL, R RJ. 2. S JJ, R E, Y Z, G GJ. Evidence for Protein measurement with the Folin phenol reagent. involvement of opioid receptors in ischemic pre- J Biol Chem 1951; 139: 265–275. conditioning in rat hearts. Am J Physiol 1995; 268: 18. T JR, N SR. Modulation by mu-opioid H2157–H2161. ′ 35 agonists of guanosine-5 -O-(3-[ S]thio)triphosphate 3. S JJ, H AK, G GJ. Ischemic pre- binding to membranes from human neuroblastoma conditioning and morphine-induced cardio- SH-SY5Y cells. Mol Pharm 1995; 47: 848–854. protection involve the delta ()-opioid receptor in 19. E CJ, K DE, M H, M K, the intact rat heart. J Mol Cell Cardiol 1997; 29: E RH. Cloning of a delta opioid receptor by 2187–2195. functional expression. Science 1992; 258: 1952– 4. B SF, S T-P, C KF, N X-H, H N, 1955. K K, O PR. The use of hibernation 20. S R, C SR, S SH. (3H) Opiate induction triggers for cardiac transplant pre- servation. Transplantation 1997; 63: 326–329. binding: anomalous properties in kidney and liver membranes. Mol Pharm 1978; 14: 69–76. 5. F RM, H AK, E JT, N H, G GJ. Opioid-induced second window of cardioprotection. 21. B J. Naloxone in shock. Lancet 1981; 1: 942. 22. X Q, S JZ, T KK, W TM. Effects of chronic Potential role of mitochondrial KATP channels. Circ Res 1999; 84: 846–851. U50,488H treatment on binding and mechanical 6. W S, L HY, W TM. Cardioprotection of pre- responses of the rat hearts. J Pharmacol Exp Ther conditioning by metabolic inhibition in the rat vent- 1994; 268: 930–934. 23. J W-Q, T KK, C TKY, W TM. Further ricular myocyte. Involvement of the -opioid 3 receptor. Circ Res 1999; 84: 1388–1395. characterization of [ H]U69593 binding sites in the 7. V C, B L, B P, C CM, rat heart. J Mol Cell Cardiol 1995; 27: 1507–1511. G C. Opioid receptors in rat cardiac sar- 24. K SA, F AI, F G. Opiate binding colemma: effect of phenylephrine and isoproterenol. in rat hearts: modulation of binding after Biochem Biophys Acta 1989; 987: 69–74. hemorrhagic shock. Biochem Biophys Res Comm 8. T KK, J W-Q, C TKY, W TM. Charac- 1985; 127: 120–128. terization of [3H]U69593 binding sites in the rat 25. S M, P PS, T AE. Dif- heart by receptor binding assays. J Mol Cell Cardiol ferential antagonism of delta opioid agonists by nal- 1991; 23: 1297–1302. trindole and its benzofuran analog (NTB) in mice: 9. Z R, G D, E H, M Z, R evidence for delta opioid receptor subtypes. J Phar- B, G S, E C, E Y, V Z, B J. Expression macol Exp Ther 1991; 257: 676–680. of opioid receptors during heart ontogeny in nor- 26. J Q, T AE, S M, P PS, motensive and hypertensive rats. Circulation 1996; B WD, M HI, P F. Differential 93: 1020–1025. antagonism of opioid delta-antinociception by [D- 10. E C, B J, V Z, H Y, E Y. Distinct ala2, leu5, cys6]enkephalin and naltrindole 5′-iso- components of morphine effects on cardiac myocytes thiocyanate: evidence for delta-receptor subtypes. J are mediated by the and opioid receptors. JMol Pharmacol Exp Ther 1991; 257: 1069–1075. Cell Cardiol 1997; 29: 711–720. 27. L PY, MG TM, W MJ, E LJ, E 11. B JS, W HX, Z WM, W TM. Effects CJ, L HH. Analysis of delta opioid receptor activities of kappa-opioid receptor stimulation in the heart stably expressed in CHO cell clones: function of Characterization of Opioid Receptors in HL-1 Cells 2193
receptor density? J Pharmacol Exp Ther 1994; 271: 31. C MJ, E PJ, M A, A H, W 1686–1694. JH, P PS, R AE, M F. 28. S PG, T JR. Delta opioid modulation Opioid efficacy in a C6 glioma cell line stably ex- of the binding of guanosine-5′-O-(3-[35S]thi- pressing the delta opioid receptor. J Pharm Exp Ther o)triphosphate to NG108-15 cell membranes: 1997; 283: 501–510. characterization of agonist and inverse agonist ef- 32. B K, T L, K BL. [35S]GTPS bind- fects. J Pharmacol Exp Ther 1997; 283: 1276–1284. ing: a tool to evaluate functional activity of a cloned 29. W H, S B, T H. Diminution opioid receptor transiently expressed in COS cells. of contractile response by -opioid receptor agonists Neurochem Res 1996; 21: 1301–1307. in isolated rat ventricular cardiomyocytes is me- 33. K E, P J, M C, L J-M, M- diated via a pertussis toxin-sensitive G-protein. Nuan P. Opening of potassium channels. The com- Schmied Arch Pharmacol 1998; 358: 360–366. mon cardioprotective link between preconditioning 30. H G, G P, J KH. Muscarinic acetyl- and natural hibernation? Circulation 1999; 99: choline receptor-stimulated binding of guanosine5′- 3079–3085. O-(3-thiotriphosphate) to guanosine-nucleotide- 34. L BT, G GJ. Direct preconditioning of cardiac
binding proteins in cardiac membranes. Eur J Bio- myocytes via opioid receptors and KATP channels. chem 1989; 186: 725–731. Circ Res 1999; 84: 1396–1400.