Suppression of cAMP by phosphoinositol/Ca2+ pathway in the cardiac kappa -opioid receptor

Wei-Min Zhang and Tak-Ming Wong

Department of Physiology and Institute of Cardiovascular Science and Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To determine whether the phosphoinositol/Ca2+ pathway interacts with the adenylate cyclase/adenosine 3',5'-cyclic monophosphate (cAMP) pathway in the cardiac kappa -receptor, the effects of U-50488, a specific kappa -receptor agonist, on the intracellular Ca2+ concentration ([Ca2+]i) and forskolin-induced accumulation of cAMP in rat ventricular myocytes were determined after interference of the phosphoinositol/Ca2+ pathway. U-50488 suppressed the forskolin-induced accumulation of cAMP and elevated [Ca2+]i, which were blocked by norbinaltorphimine, a specific kappa -receptor antagonist, and pertussis toxin. The effects of U-50488 were qualitatively similar to those of A-23187, a Ca2+ ionophore, but opposite to those of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), a [Ca2+]i chelator. Abolition of U-50488-induced elevation of [Ca2+]i by BAPTA-AM also abolished the effect of U-50488 on forskolin-induced accumulation of cAMP. Inhibition of the phospholipase C by specific inhibitors, U-73122 and neomycin, abolished the effects of U-50488 on both [Ca2+]i and forskolin-induced accumulation of cAMP. The results showed for the first time that kappa -receptor stimulation may suppress cAMP accumulation via activation of the phosphoinositol/Ca2+ pathway in the rat heart.

adenylate cyclase; intracellular calcium ion; phospholipase C; ventricular myocyte; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IT HAS BEEN SHOWN THAT activation of the phospholipase C (PLC) leading to an increased production of inositol trisphosphate (IP3), which eventually results in mobilization of Ca2+ from its intracellular store, is accompanied by a reduction in adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in a number of receptors, such as bradykinin receptor in NCB-20 cells (4), muscarinic receptor in the pregnant rat myometrium (12), substance K receptor, and P2U-purinergic receptor in C6-2B rat glioma cells (9, 25), alpha 1-adrenergic receptor, and endothelin receptor in the rat cardiac myocyte (5, 13). In these receptors, the production of IP3 is inversely related to the accumulation of cAMP upon receptor stimulation (4, 9, 12). The observations suggest a possible cross talk between the two pathways. It has also been shown that the receptor-mediated inhibition of cAMP accumulation is abolished by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), which deplete Ca2+ of its intracellular store and cytosolic Ca2+ concentration ([Ca2+]i), respectively (4, 9, 11, 12, 25), suggesting that intracellular Ca2+ may mediate the suppressive action of the phosphoinositol/Ca2+ pathway on the adenylate cyclase (AC)/cAMP pathway. In support of a possible mediating role of Ca2+ on the AC/cAMP pathway, [Ca2+]i at a submicromolar concentration range inhibits Ca2+-inhibitable AC in many tissues, including cardiac sarcolemma (6, 27). This is further supported by the successful cloning and expression of Ca2+-inhibitable AC (2, 16, 18, 19, 42).

In the heart, stimulation of the kappa -receptor by specific kappa -receptor agonist suppresses cAMP (41) and activates PLC, leading to increases in [Ca2+]i (31). It has been shown that, in the heart, Ca2+-inhibitable AC is present (16, 18, 19, 42), while both Ca2+/calmodulin-activated AC and the Ca2+/calmodulin-insensitive AC are not detectable (16). Moreover, cardiac cAMP production is inhibited by Ca2+ (6, 8, 28, 34). The observations suggest the possibility of an inhibitory action of the phosphoinositol/Ca2+ pathway on the AC/cAMP pathway via an increase in [Ca2+]i in the cardiac kappa -opioid receptor. To address this question, we first determined the effects of U-50488 and intracellular Ca2+ on cAMP accumulation induced by forskolin in the rat ventricular myocyte. Second, we studied the effects of the kappa -agonist on cAMP accumulation after interference with the phosphoinositol/Ca2+ pathway. The results indicate that activation of the phosphoinositol/Ca2+ pathway by kappa -receptor stimulation with U-50488 leads to inhibition of cAMP in the heart.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Measurement of [Ca2+]i. Ventricular myocytes were isolated from the heart of male Sprague-Dawley rats (190-210 g) using a collagenase perfusion method described previously (10). Immediately after decapitation, the heart was rapidly removed from the rat and perfused in a retrograde manner at a constant flow rate (10 ml/min) with oxygenated Joklik modified Eagle's medium supplemented with 1.25 mM CaCl2 and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.2, at 37°C for 5 min followed by 5 min in the same medium without Ca2+. Type I collagenase was added to the medium to a concentration of 125 U/ml with 0.1% (wt/vol) bovine serum albumin (BSA). After 35-45 min of perfusion with medium containing collagenase, the atria were discarded, and the ventricular tissue was dissociated by shaking in the same oxygenated collagenase solution for 5 min at 37°C. The ventricular tissue was cut into small pieces with a pair of scissors followed by stirring with a glass rod for 5 min, which separated the ventricular myocytes from each other. The residue was filtered through 250-µm mesh screens, sedimented by centrifugation at 100 g for 1 min, and resuspended in fresh Joklik solution with 2% BSA. More than 80% of the cells were rod shaped and not trypan blue permeable. Ca2+ concentration of the Joklik solution was increased gradually to 1.25 mM in 30 min.

Ventricular myocytes were incubated with fura 2-AM (4 µM) in Joklik solution supplemented with 1.25 mM CaCl2 for 25 min. The unincorporated dye was removed by washing the cells two times in fresh incubation solution. The loaded cells were kept at room temperature (24-26°C) for 30 min before measurement of [Ca2+]i to allow the fura 2-AM in the cytosol to de-esterify.

The ventricular myocytes loaded with fura 2-AM were transferred to the stage of an inverted microscope (Nikon) in a superfusion chamber at room temperature. The inverted microscope was coupled with a dual-wavelength excitation spectrofluorometer (Photo Technical International). Myocytes were perfused with Krebs bicarbonate buffer containing (in mM) 118 NaCl, 5 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose, with 1% dialyzed BSA and a gas phase of 95% O2-5% CO2. Fluorescent signals obtained at 340 nm (F340) and at 380 nm (F380) excitation wavelength were stored in a computer for data processing and analysis. The F340-to-F380 ratio was used to represent [Ca2+]i changes in the myocyte.

For [Ca2+]i, U-50488 at 3 × 10-5 M was used based on previous studies. U-50488 at this concentration significantly increased [Ca2+]i, which was blocked by specific kappa -antagonists (31, 35, 37). The concentrations of A-23187 (26) and U-73122 (17, 32) used in the present study were based on previous studies. Forskolin at 10 µM was chosen based on previous studies that used forskolin at 1-100 µM for similar purposes (14, 29, 36). More importantly, we found that, although U-50488 inhibited the effects of forskolin at 1-10 µM significantly, the inhibitory effect of the kappa -agonist was much more obvious when forskolin at 10 µM was used (Fig. 1A).


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Fig. 1.   Inhibitory effects of U-50488 on forskolin-induced cAMP accumulation in rat myocytes. A: effects of U-50488 at 30 µM on cAMP-accumulation induced by forskolin at 1-10 µM. open circle , Control group; square , forskolin only; triangle , forskolin in the presence of U-50488; n = 8-16. B: dose-related inhibition by U-50488 on cAMP accumulation induced by forskolin at 10 µM; n = 6. Values are means ± SE. # and ## Significantly different from control (no forskolin) at P < 0.05 and P < 0.01, respectively. * and ** Significantly different from the corresponding groups with forskolin only at P < 0.05 and P < 0.01, respectively.

Assay of cAMP. Samples containing 3 × 106 to 6 × 106 freshly isolated ventricular myocytes after Ca2+ loading were incubated in an atmosphere of 5% CO2-95% air for 2 h. U-50488 and forskolin were added and incubated for 15 min. U-73122 and U-73433 (10-5 M) were added 5 min before U-50488 and forskolin. At the end of treatment, the cells were centrifuged for 5 s at 100 g. The medium was aspirated, the sediment was resuspended in the ice-cold Krebs solution and then centrifuged again for 5 s at 100 g, and the supernatant was aspirated. Ice-cold ethanol-HCl (1 ml) was added, mixed, and left to stand for 5 min at room temperature. The supernatant was centrifuged at 3,000 g for 5 min and collected with a pipette. The precipitate was washed with 1 ml of ethanol-water (2:1), mixed, and centrifuged at 3,000 g for 5 min. The supernatant was combined to evaporate to dryness at 55°C under a stream of nitrogen. The sediment was stored at -20°C for assay of cAMP. The pellets were neutralized in 0.1 N NaOH for protein determination with the method of Lowry et al. (23), using BSA as a standard.

The determination of cAMP utilized a competitive binding assay with a kit from Amersham. Briefly, 50 µl of 0.5 M tris(hydroxymethyl)aminomethane (4 mM EDTA) were added to 50 µl of each sample on ice, followed by 50 µl of [3H]cAMP and 100 µl of binding protein. The samples were vortexed for 5 s, placed in an ice bath, and allowed to incubate for 2 h. Charcoal suspension (100 µl) was added, and the samples were vortexed for 10 s and centrifuged at 12,000 g for 2 min at 4°C. Samples of 200 µl of supernatant were removed for scintillation counting.

To observe the effect of [Ca2+]i on the action of U-50488, the cell-permeant Ca2+ chelator BAPTA-AM (25 µM) was added for 30 min to chelate intracellular Ca2+ (22), and the Ca2+ ionophore A-23187 was added for 5 min before the administration of the kappa -agonist.

Drugs and chemicals. The substances A-23187, U-50488, fura 2-AM, type I collagenase, neomycin, forskolin, and pertussis toxin (PTX) were purchased from Sigma Chemical. Norbinaltorphimine (Nor-BNI) was from Tocris Cookson. U-73122 and U-73433 were from Research Biochemicals International. The [3H]cAMP assay system was from Amersham International. Fura 2-AM, forskolin, A-23187, U-73122, and U-73433 were dissolved in dimethyl sulfoxide (DMSO), and other chemicals were dissolved in distilled water. The final concentration of DMSO was 0.1%, and at this concentration DMSO had no effect on either [Ca2+]i or cAMP.

Statistical analysis. Data are expressed as means ± SE. For analysis of the differences between means, unpaired Student's t-test was used with the exception of experiments involving the measurement of [Ca2+]i in which the paired Student's t-test was used.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of U-50488 on [Ca2+]i and forskolin-induced accumulation of intracellular cAMP in the rat ventricular myocyte. In agreement with the well-documented observations, forskolin (1-10 µM) dose dependently increased the cAMP content (Fig. 1A). The effects of forskolin were significantly attenuated by U-50488 at 3 × 10-5 M (Fig. 1A). We used forskolin at 10 µM in the following experiments in view of the obvious inhibitory effect of U-50488.

At 10-6 to 5 × 10-5 M, U-50488 dose dependently and significantly inhibited the forskolin-induced accumulation of cAMP (Fig. 1B). At 30 µM, U-50488 also induced an elevation of [Ca2+]i in the quiescent single ventricular myocyte (see Fig. 5, A and B), in agreement with previous observations (31, 35, 37). The effects of U-50488 at 30 µM on cAMP (Fig. 2) and [Ca2+]i (data not shown) were reversed by Nor-BNI (5 µM) and preincubation with 200 ng/ml PTX for 6 h (Fig. 2).


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Fig. 2.   Effects of pretreatment with pertussis toxin (PTX, 200 ng/ml) for 6 h or norbinaltorphimine (Nor-BNI, 5 µM) for 5 min on inhibitory effect of U-50488 (30 µM; filled bars) on forskolin-induced intracellular cAMP accumulation. Open bars, no U-50488. Values are means ± SE; n = 6. ** Significantly different from control at P < 0.01.

Effects of A-23187, a Ca2+ ionophore, on [Ca2+]i and forskolin-induced accumulation of intracellular cAMP in the rat ventricular myocyte. To determine the effect of an artificial increase in [Ca2+]i on cAMP accumulation, we made use of a Ca2+ ionophore, A-23187, which produced a dose-dependent increase in [Ca2+]i (Fig. 3, A and B). It also decreased the forskolin-induced accumulation of cAMP (Fig. 3B). The effects of A-23187 on both [Ca2+]i and cAMP accumulation were attenuated when the myocytes were incubated in a Ca2+-free medium but not by preincubation with PTX (200 ng/ml) for 6 h (Fig. 4).


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Fig. 3.   Effects of A-23187, Ca2+ ionophore, on intracellular Ca2+ concentration ([Ca2+]i) and intracellular cAMP accumulation in rat ventricular myocytes. A: representative tracings showing effects of A-23187 on [Ca2+]i in single ventricular myocytes. B: relationship between [Ca2+]i in single ventricular myocytes (open circle , n = 6) and intracellular cAMP in ventricular myocytes (square , n = 6) in response to A-23187.


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Fig. 4.   Effects of free extracellular Ca2+ plus ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 1 mM) and pretreatment with PTX (200 ng/ml) for 6 h on inhibitory effect of A-23187 on intracellular cAMP accumulation. Solid and open bars, presence or absence of A-23187, respectively. Values are means ± SE; n = 6. ** Significantly different from control at P < 0.01.

Effects of U-50488 on [Ca2+]i and forskolin-induced accumulation of intracellular cAMP in the rat ventricular myocyte after prior administration of BAPTA-AM, an intracellular Ca2+ chelator. Figure 5 showed that 25 µM BAPTA-AM reduced the basal [Ca2+]i level in the single ventricular myocyte and increased forskolin-induced accumulation of cAMP. When administered 30 min before U-50488, it completely abolished the effects of U-50488 on [Ca2+]i and forskolin-induced accumulation of intracellular cAMP in the rat ventricular myocyte.


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Fig. 5.   Effects of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM, 25 µM), a Ca2+ chelator, for 30 min on U-50488-induced elevation of [Ca2+]i (A: representative tracings; B: effect at 30 min after pretreatment) and attenuation of forskolin-induced cAMP accumulation (C: effect at 30 min after pretreatment) in quiescent ventricular myocytes. Solid and open bars, presence or absence of U-50488, respectively. Values are means ± SE; n = 6. ** Significantly different from corresponding control at P < 0.01. # Significantly different from group with forskolin only at P < 0.05.

Effects of U-50488 on [Ca2+]i and forskolin-induced accumulation of intracellular cAMP in the rat ventricular myocyte after prior administration of U-73122 and neomycin, specific inhibitors of PLC. In agreement with the previous observations (17, 32), U-73122 (but not its structural isomer U-73433) at 10-5 M and neomycin at 1 mM completely blocked the effect of U-50488 on [Ca2+]i (Fig. 6, A and B). These two agents also significantly attenuated the suppressive effect of U-50488 on forskolin-induced accumulation of cAMP in the ventricular myocyte (Fig. 6C).


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Fig. 6.   Effects of U-73122, U-73433, and neomycin on U-50488-induced elevation of [Ca2+]i in quiescent ventricular myocytes (A: representative tracings of the effect of pretreatment with U-73122, U-73433, and neomycin on U-50488-induced elevation of [Ca2+]i in the single quiescent ventricular myocyte; B: change in fluorescence ratio) and effects on U-50488-induced attenuation of forskolin (10 µM)-induced cAMP accumulation (C). Solid and open bars, presence or absence of U-50488, respectively. Values are means ± SE; n = 6. ** Significantly different from control at P < 0.01.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we found that kappa -opioid receptor stimulation with the kappa -receptor agonist U-50488 dose dependently suppressed forskolin-induced accumulation of cAMP in the rat ventricular myocyte, in agreement with the well-documented observations in neural tissues (1, 20) and in the heart (41). The effect was mediated by a PTX-sensitive G protein. We also observed that kappa -receptor stimulation activated the phosphoinositol/Ca2+ pathway, which involves a PTX-sensitive G protein, consistent with previous observations (17, 31, 32, 37).

The effect of U-50488 on cAMP was similar to that of the Ca2+ ionophore, which increased [Ca2+]i, but opposite to that of BAPTA-AM, a Ca2+ chelator, which reduced [Ca2+]i. We also observed that abolition of the U-50488-induced elevation of [Ca2+]i with BAPTA-AM also abolished the U-50488-induced suppression of cAMP accumulation induced by forskolin. In addition, inhibition of PLC with specific inhibitors U-73122 (not its structural isomer) and neomycin not only abolished the elevation in [Ca2+]i but suppressed cAMP accumulation in response to kappa -receptor stimulation. The observations suggest that activation of the phosphoinositol/Ca2+ pathway may suppress the AC/cAMP pathway. An elevation of [Ca2+]i, which is secondary to the activation of PLC and IP3 upon kappa -receptor stimulation, is the most likely mediator of the suppressive action of the phosphoinositol/Ca2+ pathway on the AC/cAMP pathway. Circumstantial evidence also suggests a similar cross talk between the two signal transduction pathways in bradykinin (4), substance K (9), and alpha 1-adrenergic (5) and muscarinic (12) receptors. The importance of Ca2+ in attenuation of cAMP accumulation has also been observed in kappa -receptor in the spinal cord (1) and delta -receptor in NCB-20 cells (4).

Although the present study did not explore the site(s) of action of Ca2+ leading to suppression of cAMP accumulation, available evidence suggests that Ca2+ inhibition on cAMP may result from a direct action on the AC catalytic subunit in the heart. The evidence is as follows. 1) Two major forms of adenylyl cyclase expressed in the heart (types V and VI adenylyl cyclases) are Ca2+ inhibitable (16, 19, 42). 2) The Ca2+/calmodulin-sensitive adenylyl cyclase and Ca2+/calmodulin-insensitive adenylyl cyclase are not detectable in the heart (16, 30). 3) The Ca2+/calmodulin-stimulated phosphodiesterase (PDE) is not present in the heart (3, 38). 4) Ca2+ inhibits the cardiac cAMP accumulation in the heart (6, 8, 28, 34). 5) Ca2+ directly inhibits the AC catalytic subunit in the heart (7). Further study is needed for verification.

It is of interest to note that a relatively large change in [Ca2+]i induced by the Ca2+ ionophore was needed to cause a significant suppression of cAMP production, whereas a small increase in [Ca2+]i induced by U-50488 was accompanied by an obvious suppression of cAMP accumulation. The discrepancy suggests that [Ca2+]i is probably not the only factor that is responsible for the suppression of cAMP production. It has been shown that activation of protein kinase C (PKC), secondary to the activation of the phosphoinositol pathway, causes a reduction in the receptor-mediated intracellular cAMP increase in rat ventricular myocytes (43) and in NCB-20 cells (15). Further study is needed to determine if PKC is also involved in mediating the suppressive action of the phosphoinositol/Ca2+ pathway on the AC/cAMP pathway upon kappa -receptor stimulation.

That U-50488 at the dose range of 10-6 to 5 × 10-5 M suppressed cAMP production and elevated [Ca2+]i via the phosphoinositol/Ca2+ pathway is in contrast to the obvervation in the cultured embryonic rat heart myoblast that U-50488 at nanomolar concentrations inhibits cAMP without elevating [Ca2+]i, whereas, at micromolar concentrations, it elevates [Ca2+]i and supresses cAMP (21). The signal tranduction processes in the kappa -receptor of different tissues may be different. Further study is needed for verification.

A major concern of the findings is the physiological significance of the effects of kappa -receptor stimulation with high concentrations of U-50488, which is in the order of 10-5 M. Previous studies showed that, in whole animal or isolated perfused heart preparations, myocardial ischemia-reperfusion induces cardiac arrhythmias, which could be reversed by kappa -receptor antagonist, MR-2266 (33, 39), and Nor-BNI (24). The observation suggests that, during myocardial ischemia-reperfusion, there may be an increased release of endogenous kappa -agonists from the heart. The endogenous kappa -opioids may reduce the cardiac contractility, a well-established action of kappa -opioid (40), most likely via suppressing cAMP and/or depleting Ca2+ from its intracellular store. The effect protects the heart as oxygen consumption is reduced. Therefore, in myocardial ischemia-reperfusion, a high concentration of the kappa -opioid is probably secreted for cardiac protection. The occurrence of arrhythmias, an undesirable effect, may be due to excessive release of kappa -agonist during myocardial ischemia-reperfusion.

In conclusion, the present study has provided for the first time several lines of evidence indicating that activation of the phosphoinositol/Ca2+ pathway suppresses the AC/cAMP pathway via [Ca2+]i upon kappa -receptor stimulation with U-50488 in the heart. Further studies are needed to determine 1) whether PKC is also involved and 2) site(s) of action of Ca2+ that result in suppression of cAMP accumulation. The possibility of a feedback from cAMP on the phosphoinositol/Ca2+ pathway also warrants further study.

    ACKNOWLEDGEMENTS

We thank Dr. N. S. Wong for helpful comments and C. P. Mok for technical assistance.

    FOOTNOTES

This study was supported by grants from the Research Grant Council, Hong Kong and The University of Hong Kong.

Address for reprint requests: T. M. Wong. Dept. of Physiology, Faculty of Medicine, The Univ. of Hong Kong, Li Shu Fan Bldg., Sassoon Road, Hong Kong.

Received 18 March 1997; accepted in final form 12 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Attali, B., D. Saya, and Z. Vogel. kappa -Opioid agonists inhibit adenylate cyclase and produce heterologous desensitization in rat spinal cord. J. Neurochem. 52: 360-369, 1989[Medline].

2.   Bakalyar, H. A., and R. R. Reed. Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250: 1403-1406, 1990[Medline].

3.   Bode, D. C., J. R. Kanter, and L. L. Brunton. Cellular distribution of phosphodiesterase isoforms in rat cardiac tissue. Circ. Res. 68: 1070-1079, 1991[Abstract].

4.   Boyajian, C. L., A. Garritsen, and D. M. F. Cooper. Bradykinin stimulates Ca2+ mobilization in NCB-20 cells leading to direct inhibition of adenylylcyclase: a novel mechanism for inhibition of cAMP production. J. Biol. Chem. 266: 4995-5003, 1991[Abstract/Free Full Text].

5.   Buxton, I. L. O., and L. L. Brunton. Action of the cardiac alpha 1-adrenergic receptor: activation of cyclic AMP degradation. J. Biol. Chem. 26: 6733-6737, 1985.

6.   Colvin, R. A., J. A. Oibo, and R. A. Allen. Calcium inhibition of cardiac adenylyl cyclase. Evidence for two distinct sites of inhibition. Cell Calcium 12: 19-27, 1991[Medline].

7.   Cooper, D. M. F. Inhibition of adenylate cyclase by Ca2+---a counterpart to stimulation by Ca2+/calmodulin. Biochem. J. 278: 903-904, 1991[Medline].

8.   Cros, G., A. Molla, and S. Katz. Does calmodulin play a role in the regulation of cardiac sarcolemmal adenylate cyclase activity. Cell Calcium 5: 365-375, 1984[Medline].

9.   DeBernardi, M. A., T. Seki, and G. Brooker. Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA. Proc. Natl. Acad. Sci. USA 88: 9257-9261, 1991[Abstract].

10.   Dong, H., J. Z Sheng, C. M. Lee, and T. M. Wong. Calcium antagonistic and antiarrhythmic actions of CPU-23, a substituted tetrahydroisoquinoline. Br. J. Pharmacol. 109: 113-119, 1993[Medline].

11.   Garritsen, A., Z. Yingxin, A. F. Jordan, D. B. Michael, and D. M. F. Cooper. Inhibition of cyclic AMP accumulation in intact NCB-20 cells as a direct result of elevation of cytosolic Ca2+. J. Neurochem. 59: 1630-1639, 1992[Medline].

12.   Goureau, O., Z. Tanfin, and S. Harson. Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium: interaction between cyclic AMP and Ca2+ signals. Biochem. J. 271: 667-673, 1990[Medline].

13.   Hilal-Dandan, R., K. Urasawa, and L. L. Brunton. Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes. J. Biol. Chem. 267: 10620-10624, 1992[Abstract/Free Full Text].

14.   Hohl, C. M., W. Stephan, R. H. Fertel, D. K. Wimsatt, G. P. Brierley, and R. A. Altschuld. Hyperthyroid adult rat cardiomyocytes. I. nucleotide content, beta - and alpha -adrenoreceptors, and cAMP production. Am. J. Physiol. 257 (Cell Physiol. 26): C948-C956, 1989[Abstract/Free Full Text].

15.   Hollingsworth, E. B, and J. W. Daly. Inhibition of receptor-mediated stimulation of cyclic AMP accumulation in neuroblastoma-hybrid NCB-20 cells by a phorbol ester. Biochim. Biophys. Acta 930: 272-278, 1987[Medline].

16.   Ishikawa, Y., S. Katsushika, L. Chen, N. J. Halnon, J. Kawabe, and C. J. Homcy. Isolation and characterization of a novel cardiac adenylylcyclase cDNA. J. Biol. Chem. 267: 13553-13557, 1992[Abstract/Free Full Text].

17.   Jin, W., N. M. Lee, H. H. Loh, and S. A Thayer. Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells. J. Neurosci. 14: 1920-1929, 1994[Abstract].

18.   Katsushika, S., L. Chen, J. Kawabe, R. Nilakantan, N. J. Halnon, C. J. Homcy, and Y. Ishikawa. Cloning and characterization of a sixth adenylyl cyclase isoform: types and constitute a subgroup within the mammalian adenylyl cyclase family. Proc. Natl. Acad. Sci. USA 89: 8774-8778, 1992[Abstract].

19.   Krupinski, J., T. C. Lahman, C. D. Frankenfield, J. C. Zwaagstra, and P. Watson. Molecular diversity in the adenylyl cyclase family: evidence for eight forms of the enzyme and cloning of type VI. J. Biol. Chem. 267: 24858-24862, 1992[Abstract/Free Full Text].

20.   Kurose, H., T. Katada, T. Amano, and M. Ui. Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha -adrenergic, cholinergic and opiate receptors in neuroblastoma x glioma hybrid cells. J. Biol. Chem. 258: 4870-4875, 1983[Abstract/Free Full Text].

21.  Lau, S. Y., T. M. Wong, and N. S. Wong. Stimulation of kappa -receptor in cultured cardiomyoblast produced different combinations of intracellular signals at low and high concentrations of specific kappa -agonists (Abstract). 27th Meeting of the International Narcotics Research Conference, Long Beach, CA. 1996, p. 89.

22.   Lew, V. L., R. Y. Tsien, C. Miner, and R. M. Bookchin. Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator. Nature 298: 478-481, 1982[Medline].

23.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

24.   McIntosh, M., K. Kane, and J. Parratt. Effects of selective opioid receptor agonists and antagonists during myocardial ischaemia. Eur. J. Pharmacol. 210: 37-44, 1992[Medline].

25.   Munshi, R., M. A. Debernardi, and G. Brooker. P2U-Purinergic receptors on C6-2B rat glioma cells: modulation of cytosolic Ca2+ and cAMP levels by protein kinase C. Mol. Pharmacol. 44: 1185-1191, 1993[Abstract].

26.   Murray, J. J., P. W. Reed, and J. G. Dobson, Jr. Biochemical changes accompanying enhanced cardiac contractility by ionophore A-23187. Am. J. Physiol. 249 (Heart Circ. Physiol. 18): H1204-H1210, 1985[Medline].

27.   Neil, S. M., T. Lakey, and S. Tomlinson. Calmodulin regulation of adenylate cyclase activity. Cell Calcium 6: 213-226, 1985[Medline].

28.   Potter, J. D., M. T. Piascik, P. L. Wisler, S. P. Robertson, and C. L. Johnson. Calcium dependent regulation of brain and cardiac muscle adenylate cyclase. Ann. NY Acad. Sci. 356: 220-231, 1980[Abstract].

29.   Rocha-Singh, K. J., D. K. Hines, N. Y. Honbo, and J. S. Karliner. Concanavalin A amplifies both beta -adrenergic and muscarinic cholinergic receptor-adenylate cyclase-linked pathways in cardiac myocytes. J. Clin. Invest. 88: 760-766, 1991[Medline].

30.   Rosenberg, G. B., and D. R. Storm. Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclase. J. Biol. Chem. 262: 7623-7628, 1987[Abstract/Free Full Text].

31.   Sheng, J. Z., and T. M. Wong. Chronic U50,488H abolishes inositol 1,4,5-triphosphate and intracellular Ca2+ elevations evoked by kappa -opioid receptor in rat myocytes. Eur. J. Pharmacol. 307: 323-329, 1996[Medline].

32.   Sheng, J. Z., N. S. Wong, H. X. Wang, and T. M. Wong. Pertussis toxin, but not tyrosine kinase inhibitors abolishes effects of U-50,488H on cytosolic Ca2+ in myocytes. Am. J. Physiol. 272 (Cell Physiol. 41): C560-C564, 1997[Abstract/Free Full Text].

33.   Sitsapesan, R., and J. R. Parratt. The effects of drugs interacting with opioid receptors on the early ventricular arrhythmias arising from myocardial ischaemia. Br. J. Pharmacol. 97: 795-800, 1989[Abstract].

34.   Tada, M., M. A. Kirchberger, J. M. Iorio, and A. M. Katz. Control of cardiac sarcolemmal adenylate cyclase and sodium, potassium-activated adenosine triphosphatase activities. Circ. Res. 36: 8-17, 1975[Abstract].

35.   Tai, K. K., C. F. Bian, and T. M. Wong. kappa -Opioid receptor stimulation increase intracellular free calcium in isolated rat ventricular myocytes. Life Sci. 51: 909-913, 1992[Medline].

36.   Taouis, M., R. S. Sheldon, R. J. Hill, and H. J. Duff. Cyclic AMP-dependent regulation of the number of [3H]batrachotoxinin benzoate binding sites on rat cardiac myocytes. J. Biol. Chem. 266: 10300-10304, 1991[Abstract/Free Full Text].

37.   Ventura, C., H. Spurgeon, E. G. Lakatta, C. Guarnieri, and M. C. Capogrossi. kappa and delta  Opioid receptor stimulation affects cardiac myocyte function and Ca2+ release from an intracellular poll in myocytes and neurons. Circ. Res. 70: 66-81, 1992[Abstract].

38.   Weishaar, R. E., D. C. Kobylarz-Singer, and H. R. Kaplan. Subclasses of cyclic AMP phosphodiesterase in cardiac muscle. J. Mol. Cell. Cardiol. 19: 1025-1036, 1987[Medline].

39.   Wong, T. M., A. Y. S. Lee, and K. K. Tai. Effects of drugs interacting with opioid receptors during normal perfusion or ischaemia and reperfusion in the isolated rat heart---an attempt to identify cardiac opioid receptor subtype(s) involved in arrhythmogenesis. J. Mol. Cell. Cardiol. 22: 1167-1175, 1990[Medline].

40.   Xia, Q., J. Z. Sheng, K. K. Tai, and T. M. Wong. Effects of chronic U50,488H treatment on binding and mechanical responses of the rat hearts. J. Pharmacol. Exp. Ther. 268: 930-934, 1994[Abstract].

41.   Yamada, K., S. Yoshida, and Y. Shimada. Atrial natriuretic polypeptide secretion via selective activation of kappa -opioid receptor: role of dynorphin. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E293-E297, 1991[Abstract/Free Full Text].

42.   Yoshimura, M., and D. M. F. Cooper. Cloning and expression of a Ca2+-inhibitable adenylyl cyclase from NCB-20 cells. Proc. Natl. Acad. Sci. USA 89: 6716-6720, 1992[Abstract].

43.   Zheng, J.-S., A. Christie, M. N. Levy, and A. Scarpa. Ca2+ mobilization by extracellular ATP in rat cardiac myocytes: regulation by protein kinase C and A. Am. J. Physiol. 263 (Cell Physiol. 32): C933-C940, 1992[Abstract/Free Full Text].


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