©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subtype-specific Regulation of Muscarinic Receptor Expression and Function by Heterologous Receptor Activation (*)

(Received for publication, March 20, 1995; and in revised form, July 20, 1995)

Darrell A. Jackson Neil M. Nathanson (§)

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Incubation of cultured embryonic chicken heart cells with the beta-adrenergic agonist isoproterenol resulted in a dose-dependent increase in the number of mAChR on the surface of intact cells. The isoproterenol-mediated increase in mAChR number was time dependent and reached a maximum by 48 h. Chick heart cells treated with isoproterenol exhibited a greater than 6-fold increase in the sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity as compared to control. Stimulation of cultured heart cells for 24 h with isoproterenol resulted in a 25-35% increase in cm2 mRNA levels as compared to control cm2 mRNA levels. In contrast, the level of cm4 mRNA was not significantly affected by isoproterenol treatment. cm2 mRNA levels were maximally elevated by 15 h following isoproterenol stimulation and remained elevated for up to 72 h. Incubation of cells with isoproterenol in the presence of Rp-cAMP, an inhibitor of cAMP-dependent protein kinase, blocked the increase in the level of cm2 mRNA. Thus, prolonged activation of beta-adrenergic receptors results in an increase in mAChR number and muscarinic responsiveness in chick heart cells due to a cAMP-dependent protein kinase mediated increase in cm2 mRNA levels.


INTRODUCTION

Muscarinic acetylcholine receptors (mAChRs) (^1)are members of a large family of transmembrane spanning receptors that couple to guanine nucleotide-binding proteins (G-proteins) upon agonist activation. Five different genes encoding the mammalian mAChRs subtypes (m(1)-m(5)) have been identified (Kubo et al., 1986a, 1986b; Peralta et al., 1987a, 1987b; Bonner et al., 1987, 1988; Braun et al., 1987; Shapiro et al., 1988). The chick heart expresses at least three mAChR subtypes (cm2, cm3, and cm4) that share a high degree of homology to the mammalian m(2), m(3), and m(4) mAChR (Tietje et al., 1990; Tietje and Nathanson, 1991; Gadbut and Galper, 1994). Agonist activation of mAChRs have been shown to result in a variety of physiological and biochemical events that include inhibition of adenylyl cyclase (Nathanson et al., 1978), stimulation of guanylyl cyclase (Renaud et al., 1980), stimulation of phospholipase C (Brown and Masters, 1984), and alteration of ion channel conductances (Galper et al., 1982; Hunter and Nathanson, 1986). Activation of phospholipase C results in the generation of the second messengers diacylglycerol and inositol trisphosphate (Orellana and Brown, 1985) and subsequent activation of protein kinase C and increase in intracellular calcium levels (Hirasawa and Nishisuka, 1985). In the continued presence of acetylcholine or other agonists, mAChR in the heart and other tissues undergo sequestration and a subsequent decrease in receptor number (Nathanson, 1987). Long term treatment of embryonic chick heart cells with muscarinic agonists results not only in a down-regulation of muscarinic receptor number, but also in a significant reduction in the levels of mRNA encoding the cm2 and cm4 mAChRs (Habecker and Nathanson, 1992; Habecker et al., 1993). In addition to the agonist-induced down-regulation of mAChRs in the chick heart, activation of adenosine (Ad1) and angiotensin II (AngII) receptors has also been shown to affect chick heart mAChR number and mRNA levels (Habecker and Nathanson, 1992). Chick cardiac cells also express beta-adrenergic receptors which mediate stimulation of adenylyl cyclase activity. Stimulation of mAChRs results in inhibition of beta-adrenergic stimulation of cardiac contractility due at least in part through inhibition of adenylyl cyclase (Hartzell, 1988).

We report here that persistent stimulation with the beta-adrenergic receptor agonist isoproterenol increases mAChR number and muscarinic responsiveness in chick heart cells due to a selective increase in cm2 mRNA levels with no effect on cm4 mRNA levels.


EXPERIMENTAL PROCEDURES

Materials

White leghorn chicken eggs were obtained from H & N International (Redmond, WA) and maintained in a humidified 38 °C incubator until the ninth day of incubation. Quinuclidinyl benzilate ([^3H]QNB, 35-44 Ci/mmol) and [^3H]N-methylscopolamine ([^3H]NMS, 85 Ci/mmol) were obtained from Amersham Corp. [^3H]cAMP (36 Ci/mmol) was from ICN, and [alpha-P]UTP (800 Ci/mmol) was obtained from DuPont NEN and [alpha-^3H]UTP (41 Ci/mmol) was from Amersham. The ion-exchange resin AG50W-X4 (200-400 mesh, hydrogen form) used in the cAMP accumulation assay was from Bio-Rad. Cell culture media were purchased from Life Technologies, Inc. and Sigma. All other materials were purchased as described previously (Halvorsen and Nathanson, 1981).

Cell Culture

Heart cells from 9-day embryonic chicken were prepared as described by Subers and Nathanson(1988) in serum-free defined medium containing 98% M-199, 1% penicillin-streptomycin (100 units/ml and 100 µg/ml final concentrations, respectively), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), testosterone (10 nM final concentration), and triiodothyronine (3 nM final concentration). Cardiac cells were plated at a density of 1.0 times 10^5 cells/cm^2 on 100-mm plastic tissue culture dishes and maintained in a 5% CO(2) environment at 37 °C. Isoproterenol, dissolved in 10 µM ascorbic acid, was added once daily, beginning 24 h after culture preparation and until 72 h or otherwise noted. The media was changed on the third day, and assays were performed on the fourth day of culture.

Binding Assay in Membrane Homogenates

The binding of [^3H]QNB to mAChR in crude membrane homogenates was performed according to the method of Halvorsen and Nathanson(1981). Binding of [^3H]NMS to cell surface mAChR on intact chick heart cells in culture was performed as described by Nathanson(1983). In all experiments, nonspecific binding was determined as amount of [^3H]QNB binding remaining in the presence of 1 µM atropine.

cAMP Accumulation Assays

Chick heart cell in cultures were prepared as described above on 60-mm tissue culture plates. Carbachol-mediated inhibition of forskolin-stimulated cAMP accumulation was measured as described by Subers and Nathanson(1988).

RNA Probe Construction

Antisense and sense RNA probes to a BamHI/XbaI fragment encoding the subtype-specific third cytoplasmic loop of the cm2 receptor (Tietje and Nathanson, 1991) and a BalI/SmaI fragment encoding the third cytoplasmic loop of the cm4 receptor (Tietje et al., 1990) were prepared as described by Habecker and Nathanson(1992). Antisense probes were synthesized with [alpha-P]UTP and sense RNA for standards with [alpha-^3H]UTP.

Isolation of RNA from Chick Heart Cells

Total cellular RNA was isolated as described by Peppel and Baglioni(1990) and stored in 2-propanol at -20 °C until use. Samples were resuspended in 0.1% SDS, and the RNA concentration was determined by UV spectrophotometer.

Solution Hybridization

Quantitation of cm2 and cm4 mRNA was measured by hybridization of RNA samples with antisense probes as described by Habecker and Nathanson(1992).


RESULTS

beta-Adrenergic-mediated Increase in mAChR Numbers

Chronic treatment of embryonic heart cells in culture with a high concentration of beta-adrenergic agonist has been reported to result in an increase in mAChR number in membrane homogenates (Reithmann et al., 1992). However, because measurements of mAChRs were performed using the membrane-permeable muscarinic antagonist [^3H]QNB, it was not demonstrated whether chronic beta-adrenergic stimulation resulted in an increase in the number of mAChRs on the cell surface. To determine whether beta-adrenergic stimulation resulted in an increase in mAChRs numbers on the surface of intact cells, we used the membrane-impermeable antagonist [^3H]NMS. Treatment of cultured embryonic chick hearts in defined media with isoproterenol for 72 h resulted in a dose-dependent increase in the number of mAChR on the surface of intact cells as measured with the membrane-impermeable muscarinic antagonist [^3H]NMS (Fig. 1). We also observed similar increases in the number of total cellular mAChR as measured by the binding of the lipophilic muscarinic antagonist [^3H]QNB to membrane homogenates (Fig. 2).


Figure 1: Concentration-response curve for isoproterenol-mediated increase in [^3H]NMS binding to intact chick heart cell cultures. Embryonic chick hearts cells were cultured in serum-free defined media. Isoproterenol, dissolved in 10 µM ascorbic acid, was added once daily for 72 h. The media was changed on the third day, and assays were performed on the fourth day of culture. The binding of [^3H]NMS to cell surface mAChR on intact chick heart cells in culture was performed as described under ``Experimental Procedures.'' Data are presented as the mean ± S.D. from three separate experiments which each had from six to nine independent determinations. The increase in [^3H]NMS binding is expressed as the percent increase compared to control (10 µM ascorbic acid). The mean value of [^3H]NMS binding to control heart cells was 960 ± 40 fmol/100-mm plate.




Figure 2: Inhibition of isoproterenol stimulation of increase in mAChR by the beta-adrenergic antagonist nadolol and time course of isoproterenol- induced increase of mAChR number. A, antagonism of isoproterenol effect by pretreatment with nadolol. Primary heart cell cultures were pretreated with 2 µM nadolol (final concentration) 10 min prior to addition of isoproterenol. Cells were treated once daily for 3 days with either nadolol, 100 nM isoproterenol, vehicle (10 µM ascorbic acid), or nadolol with isoproterenol. Medium was changed on the third day of culture, and the binding of [^3H]QNB to membrane homogenates was performed on the fourth day. Average [^3H]QNB binding in membrane homogenates of control treated cells was 310 ± 20 fmol/mg protein. The isoproterenol values were significantly different from nadolol values, p < 0.01, and nadolol plus isoproterenol, p = 0.01. Data are presented as the means ± S.D. from three separate experiments which each had from six to eight independent determinations. There were no significant differences between nadolol alone and nadolol plus isoproterenol, p > 0.2. Statistical significance was calculated according to the Student's t test for unpaired observations, with p < 0.05 taken as significant. B, heart cells were treated for 24, 48, or 72 h with 100 nM isoproterenol prior to determination of [^3H]QNB binding.



To ensure that the increase in mAChR numbers by isoproterenol stimulation was due to activation of beta-adrenergic receptors, we pretreated the cells with the beta-adrenergic antagonist nadolol. Pretreatment of embryonic chick cell cultures with nadolol prior to the addition of 100 nM isoproterenol significantly attenuated the increase in mAChR number (Fig. 2A). Treatment of cells with nadolol alone did not produce a significant effect on mAChR number as compared to control (Fig. 2A). In addition, we also observed a time-dependent increase in mAChR numbers in cells stimulated by isoproterenol. Muscarinic receptor number began to increase by 24 h and was maximally elevated by 48 h after isoproterenol stimulation (Fig. 2B).

cAMP Accumulation Assay

In order to determine if the increase in mAChR number following beta-adrenergic agonist stimulation resulted in an increased functional responsiveness to muscarinic agonists, we examined the ability of the mAChR to inhibit adenylyl cyclase activity. The concentration-response curves for carbachol-mediated inhibition of forskolin stimulation of cyclic AMP formation demonstrated that chick heart cells treated with 10 µM isoproterenol exhibited an increased physiological sensitivity to muscarinic agonists (Fig. 3). Forskolin-mediated increase in cAMP formation was maximally inhibited 70% by carbachol in both isoproterenol-stimulated and vehicle-treated heart cells (Fig. 3). However, isoproterenol-treated cells exhibited greater than a 6-fold increase in the sensitivity for carbachol-mediated inhibition of adenylyl cyclase as compared to control (EC values of 3.0 times 10 and 1.9 times 10M, for isoproterenol-treated and control cells, respectively).


Figure 3: Isoproterenol-induced increase in mAChR number results in an increased sensitivity to carbachol-mediated inhibition of forskolin-stimulated cAMP accumulation compared to vehicle-treated cells. Cultures were incubated with either vehicle (closed squares) or 10 µM isoproterenol (open squares) for 72 h. Heart cells were incubated with 100 µM forskolin and the indicated concentrations of carbachol for 5 min and cellular cAMP levels were then determined. Values are presented as percent of control cAMP (± S.D.). The control values of cAMP, measured in the absence of carbachol, were 4330 ± 590 pmol/mg protein and 4370 ± 780 pmol/mg protein for vehicle and 10 µM isoproterenol-treated cells, respectively. Each experiment was performed in triplicate, and the results shown are the average of three separate experiments. At carbachol concentrations of 10 (*) and 10M (**), isoproterenol-treated cell were significantly different compared to control treated cells, p = 0.02 and p < 0.01, respectively.



Solution Hybridization Analysis of RNA Isolated from Isoproterenol-treated Cells

We examined if the increase in mAChR number was due to an increased level of mRNA encoding the mAChR. Cultured chick heart cells express two main mAChR subtypes, cm2 and cm4. The levels of cm2 and cm4 mRNA were quantitated by solution hybridization using subtype-specific riboprobes as described by Habecker and Nathanson(1992). Interestingly, isoproterenol stimulation of chick heart cell cultures resulted in a subtype-selective increase in the level of mAChR mRNA. Incubation with 100 nM isoproterenol for 24 h resulted in a 27% increase in cm2 mRNA levels as compared to control cm2 mRNA levels (1.1 ± 0.1 times 10^6 to 1.4 ± 0.1 times 10^6 molecules/µg RNA, p = 0.02). In contrast, the level of cm4 mRNA was not significantly affected by isoproterenol treatment (6.5 ± 0.1 times 10^5 and 6.8 ± 0.1 times 10^5 molecules/µg RNA, p > 0.1). cm2 mRNA levels were maximally elevated by 15 h following isoproterenol stimulation (Fig. 4A) and remained elevated for up to 72 h.


Figure 4: Isoproterenol stimulation of embryonic chick heart cells results in an increase in cm2 mRNA levels. The level of cm2 mRNA was determined by solution hybridization. A, incubation of chick heart cells with 100 nM isoproterenol maximally increased cm2 mRNA levels by 15 h (p = 0.01). B, pretreatment of cell culture with 100 µM Rp-cAMP, 20 min prior to the addition of 100 nM isoproterenol and incubation for 24 h. There was no significant difference between Rp-cAMP-treated cells and Rp-cAMP plus isoproterenol-treated cells, p > 0.1. However, there was a significant difference between isoproterenol-treated cells and cells that were treated with isoproterenol plus Rp-cAMP, p = 0.05. Each point represents the average of three or more independent experiments (± S.D.), each performed in duplicate or quadruplicate.



We used the competitive inhibitor of cAMP-dependent protein kinase, Rp-cAMP (Botelho et al., 1988), to test whether the increase in cm2 mRNA levels by isoproterenol was a result of activation of cAMP-dependent protein kinase. Incubation of cells with isoproterenol in the presence of Rp-cAMP did not lead to an increase in the level of cm2 mRNA (Fig. 4B), demonstrating that the beta-adrenergic receptor-mediated increase inmAChR expression is mediated through cAMP-dependent protein kinase.


DISCUSSION

This work demonstrates that chronic stimulation of cultured chick heart cells beta-adrenergic receptors results in an increased mAChR number and muscarinic responsiveness in chick heart cells due to a selective increase in cm2 mRNA levels with no effect on cm4 mRNA levels. Chick beta-adrenergic receptors appear to be pharmacologically similar to the mammalian beta-1 subtype (Port et al., 1992). Isoproterenol-mediated activation of these beta-adrenergic receptors results in stimulation of adenylyl cyclase activity and a subsequent increase in cAMP accumulation (Port et al., 1992). Pretreatment of cultured heart cells with the cAMP-dependent protein kinase inhibitor, Rp-cAMP, blocked the isoproterenol-mediated increase in cm2 mRNA levels. Thus, the isoproterenol-mediated increase in cm2 mRNA levels involves activation of cAMP-dependent protein kinase. Interestingly, the isoproterenol-mediated increase in mAChRs numbers was due to a differential effect on cm2 and cm4 mRNA levels. Although cm2 mRNA levels were maximally increased by 25-35%, cm4 mRNA did not exhibit any changes following isoproterenol stimulation. Prolonged agonist activation of chick heart mAChRs results in a subsequent decrease in both mAChR number (Galper and Smith, 1980) in mRNA levels encoding both the cm2 and cm4 receptors (Habecker and Nathanson, 1992). Treatment of chick heart cells with muscarinic agonists causes both inhibition of adenylyl cyclase and stimulation of phospholipase C (Brown and Brown, 1984). Coupling of the mAChR to both second messenger pathways is required for agonist-mediated regulation of cm2 and cm4 mRNA. Simultaneous activation of the Ad1 receptor, which results in inhibition of adenylyl cyclase in the chick heart, and AngII receptors, which activates phospholipase C in the chick heart, also resulted in a reduction in mAChR numbers and cm2 and cm4 mRNA levels (Habecker and Nathanson, 1992). These results indicate that there is a complex role of second messenger pathways in the regulation of mAChR gene expression. While the mRNA encoding the cm2 and cm4 receptors are regulated in a similar fashion by activation of mAChR or Ad1 receptors and AngII receptors, the expression of cm2 and cm4 mRNAs are differentially regulated by beta-adrenergic receptor activation.

Reithmann et al.(1992) previously reported that chick cardiac cells which were treated with beta-adrenergic agonists did not exhibit an increased magnitude of inhibition of adenylyl cyclase activity in response to a single high concentration of carbachol. However, because of the presence of spare receptors, an increase in mAChR number should lead not to an increase in extent of inhibition but to a decrease in the concentration of carbachol required for inhibition of adenylyl cyclase activity (Halvorsen and Nathanson, 1981; Hunter and Nathanson, 1986). Indeed, we show here that the isoproterenol-mediated increase in mAChR numbers leads to a greater responsiveness to carbachol-mediated inhibition of forskolin stimulation of adenylyl cyclase (Fig. 3).

We have shown here that chronic stimulation of beta-adrenergic receptors by isoproterenol causes an increase in mAChR numbers in heart cells. In addition, these cells exhibit an increased sensitivity for mAChR-mediated inhibition of forskolin stimulation of adenylyl cyclase. Furthermore, we have shown that beta-adrenergic activation results in a selective increase in mRNA levels encoding the cm2 mAChR subtype. This increase in cm2 mRNA levels is dependent upon activation of cAMP-dependent protein kinase.

Long term treatment with beta-adrenergic antagonists has been shown to induce an increase in beta-adrenergic receptor number and a concomitant decrease in muscarinic m2 receptor number and functional responsiveness in mammalian heart (Motomura et al., 1990; Marquetant et al., 1992). These changes in neurotransmitter receptor levels are likely to contribute to the adverse clinical effects of abrupt withdrawal of chronic beta-adrenergic antagonist administration (Prichard et al., 1983; Fishman, 1987). The regulation of mAChR expression by beta-adrenergic receptors thus is not only an interesting example of the regulation of neurotransmitter receptors by heterologous receptor activation but also has important clinical implications.


FOOTNOTES

*
This study was supported by Grant HL 30639 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230.

(^1)
The abbreviations used are: mAChR, muscarinic acetylcholine receptors; QNB, quinuclidinyl benzilate; NMS, N-methylscopolamine; G-protein, GTP-binding protein; AngII, angiotensin II; Ad1, adenosine 1.


ACKNOWLEDGEMENTS

We thank Lise McKinnon for technical advice.


REFERENCES

  1. Bonner, T. I., Buckley, N. J., Young, A. C., and Braun, M. R. (1987) Science 237,527-532 [Medline] [Order article via Infotrieve]
  2. Bonner, T. I., Young, A. C., Braun, M. R., and Buckley, N. J. (1988) Neuron 1,403-410 [Medline] [Order article via Infotrieve]
  3. Botelho L. H., Rothermel, J. D., Coombs, R. V., and Jastorff, B. (1988) Methods Enzymol. 159,159-172 [Medline] [Order article via Infotrieve]
  4. Braun, T., Schofield, P. R., Shivers, B. D., Pritchett, D. B., and Seeburg, P. H. (1987) Biochem. Biophys. Res. Commun. 149,125-132 [Medline] [Order article via Infotrieve]
  5. Brown, J. H., and Brown, S. L. (1984) J. Biol. Chem. 259,3777-3781 [Abstract/Free Full Text]
  6. Brown, J. H., and Masters, S. B. (1984) Fed. Proc. 43,2613-2617 [Medline] [Order article via Infotrieve]
  7. Fishman, W. H. (1987) Am. J. Cardiol. 59,26F-32F [Medline] [Order article via Infotrieve]
  8. Gadbut, A. P., and Galper, J. B. (1994) J. Biol. Chem. 41,25823-25829
  9. Galper, J. B., and Smith, T. W. (1980) J. Biol. Chem. 255,9571-9579 [Free Full Text]
  10. Galper, J. B., Dziekan, L. C., Miura, D. S., and Smith, T. W. (1982) J. Gen. Physiol. 80,231-256 [Abstract]
  11. Habecker, B. A., and Nathanson, N. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,5035-5038 [Abstract]
  12. Habecker, B. A., Wang, H., and Nathanson, N. M. (1993) Biochemistry 32,4986-4990 [Medline] [Order article via Infotrieve]
  13. Hirasawa, K., and Nishizuka, Y. (1985) Annu. Rev. Pharmacol. 25,147-170 [CrossRef][Medline] [Order article via Infotrieve]
  14. Halvorsen, S. W., and Nathanson, N. M. (1981) J. Biol. Chem. 256,7941-7948 [Abstract/Free Full Text]
  15. Hartzell, H. C. (1988) Prog. Biophys. Mol. Biol. 52,165-247 [CrossRef][Medline] [Order article via Infotrieve]
  16. Hunter, D. D., and Nathanson, N. M. (1986) J. Neurosci. 6,3739-3748 [Abstract]
  17. Kubo, T., Fukuda, K., Mikami, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1986a) Nature 323,411-416 [Medline] [Order article via Infotrieve]
  18. Kubo, T., Maeda, K., Sugimoto, K., Akiba, I., Mikami, A., Takahashi, H., Haga, K., Ichiyama, A., Kangawa, K., Matsuo, H., Hirose, T., and Numa, S. (1986b) FEBS Lett. 209,367-372 [CrossRef][Medline] [Order article via Infotrieve]
  19. Marguetant, R., Brehm, B., and Strasser, R. H. (1992) J. Mol. Cell Cardiol. 24,535-548 [Medline] [Order article via Infotrieve]
  20. Motomura, S., Deighton, N. M., Zerkowski, H.-R., Doetsch, N., Michel, M. C., and Brodde, O.-E. (1990) Br. J. Clin. Pharmacol. 30(Suppl.),112S-114S _ [Medline] [Order article via Infotrieve]
  21. Nathanson, N. M. (1983) J. Neurochem. 41,1545-1549 [Medline] [Order article via Infotrieve]
  22. Nathanson, N. M. (1987) Annu. Rev. Neurosci. 10,195-236 [CrossRef][Medline] [Order article via Infotrieve]
  23. Nathanson, N. M., Klein, W. L., and Nirenberg, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75,1788-1791 [Abstract]
  24. Orellana, S. A., and Brown, J. H. (1985) Biochem. Pharmacol. 34,1321-1324 [Medline] [Order article via Infotrieve]
  25. Peppel, K., and Baglioni, C. (1990) BioTechniques 9,711-713 [Medline] [Order article via Infotrieve]
  26. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandra, J., and Capon, D. J. (1987a) EMBO J. 6,3923-3929 [Abstract]
  27. Peralta, E. G., Winslow, J. W., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandra, J., Schimerlik, M. I., and Capon, D. J. (1987b) Science 236,600-605 [Medline] [Order article via Infotrieve]
  28. Port, J. D., Debellis, C. C., Klein, J., Peeters, G. A., Barry, W. H., and Bristow, M. R. (1992) J. Pharmacol. Exp. Ther. 262,217-224 [Abstract]
  29. Prichard, B. N. C., Tomlinson, B., Walden, R. J., and Bhatta-Charjee, P. (1983) J. Cardiovasc. Pharmacol. 5,S56-S62
  30. Reithmann, C., Panzer, B., & Werdan, K. (1992) Naun.-Schm. Arch. Pharmacol. 345,530-540
  31. Renaud, J. F., Barhanin, J., Cavey, D., Fosset, M., and Lazdunski, M. (1980) Dev. Biol. 78,184-200 [Medline] [Order article via Infotrieve]
  32. Shapiro, R. A., Scherer, N. M., Habecker, B. A., Subers, E. M., and Nathanson, N. M. (1988) J. Biol. Chem. 263,18397-18403 [Abstract/Free Full Text]
  33. Subers, E. M., and Nathanson, N. M. (1988) J. Mol. Cell Cardiol. 20,131-140 [Medline] [Order article via Infotrieve]
  34. Tietje, K. M., and Nathanson, N. M. (1991) J. Biol. Chem. 266,17382-17387 [Abstract/Free Full Text]
  35. Tietje, K. M., Goldman, P. G., and Nathanson, N. M. (1990) J. Biol. Chem. 265,2828-2834 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.