- Email: [email protected]
Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells
Life Sciences, Vol. 48, pp. 2365-2376 Printed in the U.S.A.
Pergamon Press
MINIREVIEW CYCLIC AMP PHOSPHODIESTERASES AND Ca 2+ C U R R E N T R E G U L A T I O N IN CARDIAC CELLS
Rodolphe Fischmeister" and H. Criss Hartzell t
"Laboratoire de Physiologie Cellulaire Cardiaque, 1NSERM U-241, Universit6 de Paris-Sud, F-91405 Orsay, France tDepartment of Anatomy and Cell Biology, Emory University School of Medicine Atlanta, GA 30322 (Received in final form April 4, 1991)
Summary
At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca 2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca2+-channel current 0c,) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on Ic, amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of Ic, and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.
Ca2+ channels are the main route by which Ca2+ ions enter cardiac cells to regulate some of the fundamental functions of the heart, such as automaticity and contractility. Because both the action potential and contraction are dependent upon Ca2+ influx, studying the characteristics of the transmembrane Ca2+-channel current (Ic,) can provide insights into the participation of Ca2+ ions in each phase of the cardiac action potential and in the initiation and regulation of the amplitude and time course of cardiac contraction. In general, hormones and drugs that modulate Ic, amplitude generally exert similar effects on cardiac contraction. Therefore, when searching for the cellular mechanisms responsible for the cardiac inotropic effect of new substances (e.g. phosphodiesterase inhibitors), one might reasonably first examine the effect of these substances on Ic°. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
2366
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
An important feature of cardiac Ca2+ channels is that their activity is regulated by cAMP-dependent phosphorylation (1,2). Phosphorylation of Ca2+ channels (or a closely associated protein) by cAMP-dependent protein kinase (PK-A) leads to an increase in the mean probability of channel opening 0). Because the steady-state level of phosphorylation of Ca2+ channels is determined by a complex interplay between many enzymatic reactions, any change in the metabolism of cAMP is likely to affect lc,. For example, it is well known that stimulation of adenylyl cyclase activity by fl-adrenergic agonists (4,5) or forskolin (6,7) leads to the stimulation of Ic,. Conversely, one would predict that cAMP phosphodiestera.se~ (PDEs) might also play a substantial role in the physiological regulation of Ic,. However, data supporting this assertion has been mainly indirect until recently. Part of the reason for this is that, unlike adenylyl cyclase, PDEs exist in several isoforms whose identification and distribution in various cardiac tissues remains cloudy. Whether these different isoforms have different functions or how they are regulated differently remains to be determined. Another complication in identifying the function of these different PDEs is that the subcellular distribution of various isoforms of PDEs is very different and many vary between species. Finally, the pharmacological aramentarium available to interfere with PDE activity remains rudimentary. Chemists have only recently developed compounds which bind with a sufficiently high affinity to specific PDE isozymes that allows them to be used in biochemical, pharmacological and electrophysiological studies.
Nomenclature and subcellular localization of cardiac cAMP-PDE isozvmes
Complete information on the purification, identification, characterization, and localization of cardiac PDE isozymes is to be found in articles from Beavo's (8,9) and Weishaar's (10,11) laboratories. The existence of several different classification schemes with different criteria but similar names has impeded communication in this area. The classification scheme we have utilized is taken from Beavo and Reifsnyder (12) and is based on primary protein and eDNA sequences (Table 1). Basically, four different families of PDE participate in the hydrolysis of cAMP in cardiac tissue. Family I. Ca2+-Calmodulin-dependent PDE (Ca/CaM-PDE). It seems likely that there are several forms of Ca/CaM-PDE in heart tissue, because of the differences in substrate specificities and affmities that have been described. Ca/CaM-PDE hydrolyzes cAMP and cGMP with both a low (50 #M, ref. 10) and a high (1 t~M, ref. 10) affinity in guinea pig, but only with a high affinity in human heart (13). In rat heart, a Ca/CaM-PDE appears to specifically hydrolyze cGMP (14). CaJCaM-PDE is activated by CaX+-calmodulin and is inhibited by zaprinast (M&B 22,948) and isobutylmethylxanthine 0BMX) at concentrations < 10 /~M (8,11,15) and by calmodulin antagonists (11). The CaJCaM-PDE is present in the soluble fraction of rat (16), human (13), and guinea pig ventricle (13). Bode et al. (17) have recently found that the Ca/CaM-PDE does not exist in purified cardiac myocytes and thus may be localized in other cell types.
Family II. cGMP-stimulated PDE (cGs-PDE). This isoform was first characterized by Beavo and collaborators (18,19). The cardiac cGs-PDE hydrolyzes both cAMP and cGMP with KIn> 10/~M (8,11,13). In frog heart, 1 /~M cGMP is sufficient to half-maximally activate the enzyme (20). No highly selective inhibitors are known for this family of PDE. However, in guinea pig (10,11) and frog heart (15), the enzyme is inhibited by dipyridamole at concentrations < 10 /~M. High concentrations of IBMX (> 50/zM) also inhibit the cGs-PDE (8). The enzyme seems present both in the soluble (13,16,21) and particulate fractions (20). Family HI. cGMP-inhibited low-Km cAMP-PDE (cGi-PDE). The cardiac form of this enzyme was identified by Harrison et al. (22). The main features of this PDE isozyme are its high
Vol. 48, No. 25, 1991
Phosphodiesterases & Calcium Current
2367
affinity for cAMP as substrate (Km <0.2 /~M, refs. 8,13,22), its high sensitivity to cGMP inhibition (Ki 0.1 /tM, ref. 8), and its high sensitivity to inhibition by a variety of compounds (mostly the bipyridines milrinone, amrinone, indolidan, fenoximone, etc.) which are cardiotonic agents with potential clinical usefulness (8,11,23).
Family IV. cGMP-independent low-Kin cAMP-PDE. Often called cAMP-specific PDE, this isoform of PDE is highly sensitive to inhibition by anti-depressants such as Ro 20-1724 and rolipram (13,23,8). The cAMP-specific PDE hydrolyzes cAMP with Kin<2/~M (13,8). Family V. cGMP-specific PDEs. This class has not been described in heart. Until recently, identification of PDE isoform was based largely on elution profiles from DEAE anion exchange columns and pharmacological data. Because few, if any, of the PDE inhibitors available have absolute specificity for a particular isoform, consideration of the appropriate concentrations of PDE inhibitors used is of paramount importance in evaluating isoform identification based on inhibitor studies alone. For this reason, it is sometimes difficult to establish precisely the family of PDE in question, particularly in older studies. The cellular localization of the two low-Km cAMP-PDEs (PDEs HI and IV) has been extensively studied. Their localization differs significantly from one species to the other and partly explains the species differences found in the activity of PDE inhibitors as inotropic agents. In dog (23-25), rabbit (26-28) and sheep hearts (29), the PDE-III is essentially membrane-bound while most of the PDE-IV is located in the soluble fraction. In guinea pig heart, however, both PDE-HI (13,24) and PDE-IV (13) are in a soluble form. The situation in rat heart is somewhat unclear since only membrane-bound PDE-IV was found in one study (28), while in other studies (23,24) both PDE-III and PDE-IV were found exclusively in the soluble fraction. In the human heart, low-Km cAMP-PDE activity seems located essentially in the soluble fraction, but it is unclear whether this is composed of PDE-III alone (30) or both PDE-III and PDE-IV (13). It also appears that the membrane bound form of low-Kin cAMP-PDEs found in various cardiac preparations is mostly located at the sarcoplasmic reticulum membrane (25-27,29,31) and that localization and sensitivity to inhibitors may vary during development (26). The variety of PDE isozymes and their complex distribution within cardiac cells suggest that cAMP may be compartmentalized inside the cell (24). This would explain the differential effects of various PDE inhibitors on contractile and electrical properties of a given cardiac preparation.
Phvsioloeical reeulation of cardiac cAMP-PDE activitv The idea that phosphodiesterase activity may be physiologically regulated is not new (32,33). There is now abundant evidence that increases in PDE activity (cGMP or cAMP) may be involved in the cellular response to various physiological stimuli such as light in photoreceptors (34), insulin in hepatocytes (35-38) and fat cells (8,39,40), muscarinic agonists in thyroid (41) and astrocytoma cells (42), coneanavalin A in lymphocytes (43), angiotensin H in aortic smooth muscle cells (44). Cyclic AMP PDE activity has also been found to decrease upon application of prostaglandin E1 in fibroblasts (45). Except for the regulation of cGMP-PDE activity in the visual system (34), which involves the direct interaction of a G-protein with the PDE enzyme, the mechanisms responsible for the regulation of PDE activity in other cell types remain unclear and often controversial. Two major
2368
Phosphodiesterases
& Calcium Current
Vol. 48, No. 25, 1991
mechanisms have been proposed: (i) a stimulation of cAMP-PDE activity by cAMP itself (33,38,40,46), possibly via phosphorylation by PK-A (22). This mechanism can be viewed as a form of negative feedback to terminate the cAMP signal generated by activation of adenylyl cyclase. A long term regulation of cAMP-PDE activity by cAMP has also been described that may involve modification in the expression of PDE through changes in mRNA levels (47,48). (ii) cAMP-PDE activity may be regulated via a pertussis-toxin sensitive G-protein (38,49), directly or through activation of phospholipase C, resulting in production of IP3 and subsequent increases in Ca 2+ (42) and diacylglycerol and possible activation of protein kinase C (50,51).
Table 1. cAMP PHOSPHODIESTERASES I N H E A R T *
Type [ I
Description Ca-calmodulin -dependent.
I InhibRors (ICs0)
Effects on
IC.
(ref) t
calmodulin inhibitors, phenothiazines.
unknown
no selective inhibitors.
cGMP inhibits cAMP stimulated Ic,. Effect of cGMP blocked by IBMX (5,56,96).
Not present in myocytes. 1"1
cGMP-stimulated PDE
IBMX (50 pM) cGMP-inhibited PDE
III
Low K~ for cAMP. Specific for cAMP
IV
milrinone (5nM) amrinone (300 nM) indolidan (80 nM)
PDE III and IV inhibitors have no effect on basal Ic,, but stimulate Ic, in presence of low [cAMP] (96).
rolipram (1 tLM) Ro20-1724 (2 I~M)
Glucagon stimulates Ic, (75).
Low I ~ for cAMP. Classification scheme and inhibitor data from ref 12. * Data on Ic, are from frog cardiac myocytes. Mammals may differ. *
Very little is known about the hormonal regulation of cardiac PDEs. Cyclic GMP-stimulation of cAMP hydrolysis has been proposed to participate in the cardiac negative inotropic effect of muscarinic agonists (52-54). Evidence supporting this hypothesis comes from the fact that (i) acetylcholine increases cardiac cGMP levels (2,55,56 for refs.), (ii) cGMP exerts a negative inotropic effect in heart (57-62), (iii) cAMP level is generally reduced when cGMP level increases (52-54), (iv) a cGs-PDE isozyme has been characterized in cardiac tissue (18,19, see above). Such a mechanism could possibly account for the "Yin-Yang" phenomenon (63) where cGMP was found to counterbalance the stimulatory effect of cAMP on various cellular responses to hormones (54,63,64). Conclusive evidence for the participation of a cGs-PDE in the cardiac negative inotropic effect of acetylcholine as well as other hormones known to elevate cGMP level in heart, such as adenosine (65) and atrial natriuretic factor (66,67), has been hindered by the presence of a concomitant direct and strong inhibitory effect of these agonists on cAMP production (66-71). Experiments in which adenylyl cyclase activity could be bypassed were necessary to examine the effects of cGMP. As will be described below, such an approach has permitted the demonstration that stimulation of cAMP-PDE was responsible for the cGMP induced inhibition of Ic, in frog heart (56,72).
Vol. 48, No. 25, 1991
Phosphodiesterases & Calcium Current
2369
A different example of hormonal regulation of cardiac cAMP-PDE activity was provided by Buxton and Brunton (73), who found a stimulation of cAMP hydrolysis in rat heart myoeytes exposed to aradrenergic agonists. This effect appears to be mediated via a G-protein, possibly by activation of phospholipase C and hydrolysis of phosphatidyl inositol 4,5-bisphosphate (74). Consistent with this hypothesis is the recent finding that exposure of rat cardiac microsomes to exogenous phospholipase C stimulates the eAMP-PDE activity in the membrane bound fraction (51). So far, the only example of hormonal inhibition of cardiac PDE activity is the inhibitory effect of glucagon on a particulate low-Km cAMP-PDE in frog ventricle (75). These findings provide a new pathway of regulation of cardiac function and suggest that hormones exerting positive inotropie effects on the heart may act in a similar manner to the synthetic inhibitors. Surprisingly, while glucagon stimulates Ic° both in rat and frog heart (75) and is a positive inotropie agent (76) in rat heart, its action on the latter is not mediated by inhibition of PDE activity but rather by a stimulation of adenylyl eyclase activity (75).
eAMP-PDE activitv and rfgulation of lc. Evidence for the participation of cAMP hydrolysis in the regulation of Ic, was first shown indirectly by the demonstration that massive concentrations (> 500/~M) of IBMX consistently produced a stimulatory effect on cardiac Ico (6,20,77-79) or prolonged action potential duration (80). After various forms of cardiac cAMP-PDEs were characterized biochemically and pharmacologically, a large number of electrophysiological studies were performed to examine the effects of specific or non-specific PDE inhibitors on cardiac electrical properties. Most studies, however, were limited to the effect of these compounds on beat frequency, action potential parameters, or contractility. Evidence for the participation of Ca2+ channels in the positive inotropic and electrophysiological effects of PDE inhibitors was, therefore, indirect. The most studied and highly specific cGi-PDE inhibitors, milrinone and amrinone, produce (i) an increase in beat frequency (81-83), (ii) modifications in action potential amplitude and duration that are sensitive to extracellular Ca:+, dihydropyridines and phenylalkyamines (84,85), (iii) an increase in duration, Vm,x and maximal overshoot of slow calcium-dependent action potentials (86), (iv) an increase in intracellular Ca:+ concentration (87,88), (v) an increase in the positive inotropic effect and in 45Ca uptake that is sensitive to verapamil (89). It is important to mention that various specific PDE inhibitors may exert different effects on contractility and action potentials (90) depending on whether their targets are membrane-bound or soluble (23,91). This could also explain why a given PDE inhibitor (milrinone) modifies cardiac electrical activity in a tissue- and species-dependent manner (92,93). Voltage-clamp studies on the effects of specific PDE inhibitors on cardiac Ica are uncommon. In multicellular preparations, milrinone (86,94) and amrinone (94,95) increased the amplitude of Ic°. However, the concentrations used in these studies were too high to permit an unambiguous conclusion that these effects were due to inhibition of PDE activity rather than to other side effects (see below). Recently, we have examined the effects of several specific inhibitors of the low-Km eAMP-PDEs (milrinone and indolidan for PDE-III and rolipram and Ro 20-1724 for PDE-IV) as well as low concentrations of IBMX on Ic, recorded from isolated cells from frog ventricle (96). We found that each of these compounds increased Ic, when submaximal concentrations of cAMP were perfused internally, but did not increase Ica when exogenous cAMP was not added. These effects were dose-dependent with an apparent K~ for each inhibitor tested (1-3 #M) consistent with the inhibitory effect of the compounds on low Km eAMP-PDE activity (96). These results demonstrated the physiological role of low-Kin eAMP-PDEs in regulating the amplitude of Ica upon a stimulation by a cAMP-dependent mechanism, such as a B-adrenergie
2370
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
stimulation. In other studies, we also demonstrated the participation of another form of cAMP-PDE, the cGs-PDE, in the inhibitory effect of cGMP on Ic, in frog heart (20,56,72). Our finding that cGMP inhibited cardiac Ic, was unexpected, since studies of other investigators had demonstrated the absence of an effect of cGMP on cardiac Ca2+ channels (97-99). Recent evidence, however, has confirmed the inhibition of cardiac Ca2+ channels by cGMP (20,79,100,101), although there are significant species-dependent variations in the mechanisms responsible for this inhibition (72,79,100,101). Four major observations support the role of a cGs-PDE in mediating the effect of cGMP on Ica in frog cardiomyocytes. (i) cGMP was unable to inhibit Ica that had been elevated by the hydrolysis-resistant cAMP analogue, 8-bromo-cAMP. (ii) 8-bromo-cGMP, which does not activate the cGs-PDE in frog heart (20) was unable to inhibit the stimulatory effect of cAMP on Ic~. (iii) Large concentrations of IBMX (> 100/~M) could reverse the inhibitory effect of cGMP on cAMP-elevated Ic°. (iv) A cGs-PDE was identified in this preparation with a similar requirement for cGMP activation as the inhibition of Ic, (20,56,72). Based on our studies, at least three forms of cAMP-PDEs (cGi-PDE, cGs-PDE, and cAMPspecific PDE) participate in the regulation of cardiac Ca2+ channels. However, the capacity to hydrolyze cAMP and/or the localization may vary significantly among the different forms. Indeed, we have found that none of the low-KmcAMP-PDE inhibitors were able to stimulate Ic, when the cGs-PDE was maximally stimulated by cGMP, whereas each of them was effective in the absence of cGMP (96). In addition to the previously mentioned effects of acetylcholine and ANF, several purine and pyrimidine nucleosides and their nucleotide derivatives exert strong pharmacological effects on the heart, as well as coronary vascuiature, that are accompanied by an increase in cGMP levels (e.g. adenosine, ref. 65; ATP and UTP, ref. 102). It is likely that these effects are at least in part mediated by changes in cAMP metabolism.
Additional effects of cAMP-PDE inhibitors One of the major impediments to understanding the function of PDEs in heart and other tissues is the rather poor specificity of PDE inhibitors for PDEs and the poor selectivity of these inhibitors between different forms of PDEs. For example, there are virtually no selective inhibitors of cGs-PDE. Some of the side effects of these drugs could easily confound the interpretation of experiments designed to evaluate the role of PDEs. Several examples follow. The non-specific PDE inhibitor trapidil (a triazulopyrimidine) stimulates Ca2+-dependent slow action potentials (103,104) at "low" concentrations (10-100/zM) as a result of an increase in cAMP. However, at higher concentrations (> 500/~M) trapidil inhibits Ca2+-dependent action potentials (103) even though cAMP levels remain elevated. Similar results have been reported with papaverine (105). In voltage-clamp studies, trapidil and trapidil derivatives, which exert a more potent inhibition of cardiac PDE than wapidil (106), inhibit cardiac Ca current at high concentrations (107,108). The dose-dependency of PDE inhibitors may be complicated by the fact that, as has been demonstrated in the case of the cGs-PDE, inhibitors of the catalytic site of the isozyme also appear to bind with high affinity to the allosteric site on the enzyme and thereby stimulate PDE activity at low inhibitor concentrations (12,109,110). A number of PDE inhibitors, such as caffeine and theophylline (111,112), sulmazole (113,114), and pimobendan (115,116) increase the Ca2+ sensitivity of the troponin C regulatory site in cardiac myofilaments. Surprisingly, these effects were not observed with milrinone
Vol. 48, No. 25, 1991
Phosphodlesterases & Calcium Current
2371
(112,115,117). However, milrinone, and its analogue amrinone, may exert cardiac effects that may be unrelated to PDE inhibition including stimulation of Na/Ca exchange (94,118), Na influx through Na channels (119), membrane Ca-ATPase (120), and the Ca2+-release channel of the sarcoplasmic reticulum (121). It is worth noting that milrinone (but not amrinone) shares structural homologies with thyroid hormone (120), which may partly explain the similarity of action of these substances on cardiac Ca-ATPase (120) and on low-Km cAMP-PDE activity in fat cells and hepatocytes (38 for refs.). Some of the cardiac positive inotropic effects of other PDE inhibitors could also be mediated via other routes than inhibition of PDE activity. For example, methylxanthines, such as IBMX, caffeine, and theophylline, exert strong antagonistic effects on P1 receptors (122,123). These effects result from a structural homology between adenosine and methylxanthines, which share a common ring structure. It has been reported that methylxanthines inhibit nucleoside diphosphate kinase (NDPK, ref. 124). Because NDPK may participate in the control of various effectors such as ionic channels (125-127) and adenylyl cyclase (128, 127 for refs.) by G-proteins, it is conceivable that methylxanthines may interfere with these targets. Consistent with this hypothesis are the findings that IBMX (129) and sulmazole (130,131), which is also a P1 adenosine receptor antagonist (130) and possesses the methylxanthine-like ring structure, block the inhibitory effect of the G-protein, Gi, on adenylyl cyclase (129-131) by affecting the GTP turnover (131). These compounds may, thus, increase cAMP levels by stimulating cAMP production in addition to their inhibitory effect on cAMP degradation.
Concludine remarks
In spite of several limitations in the use of specific PDE inhibitors, these compounds are promising tools to investigate the presence of various cAMP-PDE isozymes in cardiac tissues. Alth0ugh the possibility remains that PDE activity may be different in enzymatically dissociated cells as compared to in situ cells (132), voltage-clamp studies on isolated cardiac myocytes may help to delineate the functional coupling of various cAMP-PDE isozymes to Ca2+ channels as well as to determine their relative potencies to regulate cAMP-dependent phosphorylation of the channels. In addition, the ability to modify the composition of intracellular solution during an experiment, mainly its cyclic nucleotide content, allows one to examine the participation of PDE activity in the regulation of Ca2+ channel activity by hormones and drugs. This experimental approach may provide a celinlar rationale for the physiological effects of these compounds on contractile properties, beat frequency, and action potential, as well as an in vivo counterpart of biochemical studies on regulation of cAMP metabolism and PDE activity.
Acknowled~,ements
Work from our laboratories discussed above was supported by INSERM and NIH grants HL21195 and HL27385 and an exchange program INSERM France/U.S.A.. References °
2.
F. HOFMANN, W. NASTAINCZYK, A. ROHRKASTEN, T. SCHNEIDER, and M. SIEBER, Trends Pharmacol. Sci. 8 393-398 (1987). H. C. HARTZELL, Prog. Biophys. Mol. Biol. 52 165-247 (1988).
2372
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Phosphod±esterases & Calcium Current
Vol. 48, No. 25, 1991
R . W . TSIEN, B. P. BEAN, P. HESS, J. B. LANSMAN, B. NILIUS, and M. NOWYCKY, J. Mol. Cell. Cardiol. 18 691-710 (1986). M. KAMEYAMA, F. HOFMANN, and W. TRAUTWEIN, Pflfigers Arch. 407 285-293 (1985). R. FISCHMEISTER and H. C. HARTZELL, J. Physiol. (Lond.) 376 183-202 (1986). M. KAMEYAMA, J. HESCHELER, F. HOFMANN, and W. TRAUTWEIN, Pfliigers Arch. 407 123-128 (1986). H . C . HARTZELL and R. FISCHMEISTER, Mol. Pharmacol. 32 639-645 (1987). J . A . BEAVO, Adv. See. Mess. Phosphoprot. Res. 22 1-38 (1988). M . D . HOUSLAY and J. A. BEAVO, (eds.) Isozvmes of Cyclic Nucleotid¢ Ph0sphodiesterases. John Wiley & Sons (1990). R . E . WEISHAAR, S. D. BURROWS, D. C. KOBULARZ, M. M. QUADE, and D.B. EVANS, Biochem. Pharmacol. 35 787-800 (1986). R . E . WEISHAAR, M. H. CAIN, and J. A. BRISTOL, J. Med. Chem. 28 537-545 (1985). J.A. BEAVO and D. H. REIFSNYDER, Trends in Pharmacol. Sci. 11, 150-155 (1990). M . L . REEVES, B. K. LEIGH, and P. J. ENGLAND, Bioehem. J. 241 535-541 (1987). W . J . THOMPSON, W. L. TERASAKI, P. M. EPSTEIN, and S. J. STRADA, Adv. Cyclic Nucleot. Res. 10 69-92 (1979). C. LUGNIER, A. Le BEC, C. GAUTHIER, H. SOUSTRE, and J.-C. STOCLET, Arch. Int. Physiol. Biochim. 98 A171 (1990). D . C . BODE, J. R. KANTER, and L. L. BRUNTON, See. Messeng. Phosphoprot. 12 235-240 (1988-89). D.C. BODE, J. KANTER, and L. L. BRUNTON, FASEB J. 3, A1295 (1989) and Circ. Res. (in press). J.A. BEAVO, J. G. HARDMANN and E. W. SUTHERLAND, Biochem. Biophys. Res. Comm. 138 1170-1176 (1971). T . J . MARTINS, M. C. MUMBY, and J. A. BEAVO, J. Biol. Chem. 257 1973-1979 (1982). M . A . SIMMONS and H. C. HARTZELL, Mol. Pharmacol. 33 664-671 (1988). A. LAGRUTrA and H. C. HARTZELL, personal communication. S.A. HARRISON, D. H. REIFSNYDER, B. GALLIS, G. G. CADD, and J. A. BEAVO, Molee. Pharmacol. 29 506-514 (1986). R.E. WEISHAAR, D. C. KOBYLARZ-SINGER, R. P. STEFFEN, and H. R. KAPLAN, Circ. Res. 61 539-547 (1987). R . E . WEISHAAR, D. C. KOBYLARZ-SINGER, and H. KAPLAN, J. Mol. Cell. Cardiol. 19 1025-1036 (1987). R . F . KAUFFMAN, B. G. UTrERBACK, and D. W. ROBERTSON, Circ. Res. 64 1037-1040 (1989). P . A . KITHAS, M. ARTMAN, W. J. THOMPSON, S. J. STRADA, J. Mol. Cell. Cardiol. 21 507-517 (1989). P.A. KITHAS, M. ARTMAN, W. J. THOMPSON, and S. J. STRADA, Circ. Res. 62 782-789 (1988). M. SHAHID, M. WILSON, C. D. NICHOLSON and R. J. MARSHALL, J. Pharmacy Pharmacol. 42 283-284 (1990). M. ARTMAN, D. W. ROBERTSON, L. MAHONY, and W. J. THOMPSON, Mol. Pharmacol. 36 302-311 (1989). H. MASUOKA, M. ITO, T. NAKANO, M. NAKA, and T. TANAKA, J. Cardiov. Pharmacol. 15 302-307 (1990). R . F . KAUFFMAN, V. G. CROWE, B. G. UTI'ERBACK, and D. W. ROBERTSON, Mol. Pharmacol. 30 609-616 (1986).
Vol. 48, No. 25, 1991
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67.
Phosphodlesterases & Calcium Current
2373
E . T . BROWNING, C. O. BROSTROM and V. E. GROPPI, Jr., Mol. Pharmacol. 12 32-40 (1976). J . P . SCHWARTZ and J. V. PASSONEAU, Proc. Natl. Acad. Sci. USA 71 3844-3848 (1974). P . A . McNAUGHTON, Physiol. Rev. 70 847-883 (1990). R.J. MARCHMONT, S. T. AYAD, and M. D. HOUSLAY, Biochem. J. 195 645-652 (1981). M . D . HOUSLAY, Biochem. Soc. Trans. 14 183-193 (1988). C. BENELLI, B. DESBUQUOIS, and B. De GALLE, Eur. J. Biochem. 156 211-220 (1986). J . A . SMOAKE and S. S. SOLOMON, Life Sci. 45 2255-2.268 (1989). H . W . WEBER and M. M. APPLEMAN, J. Biol. Chem. 257 5339-5341 (1982). C.J. SMITH and V. C. MANGANIELLO, Mol. Pharmacol. 35 381-386 (1989). J. Van SANDE, C. ERNEUX, and J. E. DUMONT, J. Cycl. Nucl. Res. 3 335-345 (1977). R.B. MEEKER and T. K. HARDEN, Mol. Pharmacol. 22 310-319 (1982). L. VALE'ITE, A. F. PRIGENT, G. NEMOZ, G. ANKER, O. MACOVSCHI, and M. LAGARDE, Bioch. Bioph. Res. Comm. 169 864-872 (1990). J.B. SMITH and T. M. LINCOLN, Am. J. Physiol. 253 C147-C150 (1987). R. BARBER, K. P. RAY, and R. W. BUTCHER, Biochemistry 19 2560-2567 (1980). E. DEGERMAN, C. J. SMITH, H. TORNQVIST, V. VASTA, P. BELFRAGE, and V. C. MANGANIELLO, Proc. Natl. Acad. Sci. USA 87 533-537 (1990). J . V . SWINNEN, D. R. JOSEPH, and M. CONTI, Proc. Natl. Acad. Sci. USA 86 8197-8201 (1989). B.B. RILEY and S. L. BARCLAY, Proc. Natl. Acad. Sci. USA 87 4746-4750 (1990). C . M . I-IEYWORTI-I, A. M. GREY, S. R. WILSON, E. HANSKI, and M. D. I-IOUSLAY, Biochem. J. 235 145-149 (1986). F. IRV1NE, N. J. PYNE, and M. D. I-IOUSLAY, FEBS Lett. 208 455-459 (1986). M. DUBOIS, G. NEMOZ, M. LAGARDE, and A. F. PRIGENT, Bioch. Biophys. Res. Comm. 170 800-809 (1990). M. ENDOH, Jap. J. Pharmacol. 29 855-864 (1979). F . W . FLITNEY and J. SINGH, J. Mol. Cell. Cardiol. 13 963-979 (1981). U. WALTER, Adv. Cycl. Nucleo. Prot. Phosphoryl. Res. 17 249-258 (1984). W . J . GEORGE, J. B. POLSON, A. G. O'TOOLE, and N. GOLDBERG, Proc. Natl. Acad. Sci. USA 66 398-403 (1970). R. FISCI-IMEISTER and H. C. I-IARTZELL, J. Physiol. 387 453-472 (1987). H. NAWRATH, Nature 267 72-74 (1977). J. DIAMOND, R. E. TEN EICK and A. J. TRAPANI, Biochem. Biophy. Res. Comm. 79 912-917 (1977). J. SINGH and F. W. FLITNEY, Biochem. Pharmacol. 30 1475-1481 (1981). W. T R A I N and G. TRUBE, PflQgers Arch. 366 293-295 (1976). R . D . WILKERSON, R. J. PADDOCK, and W. J. GEORGE, Eur. J. Pharmacol. 36 247-251 (1976). W. TUGANOWSKI and P. KOPEC, Naunyn-Schmied. Arch. Pharmacol. 304 211-213 (1978). N . D . GOLDBERG and M. K. HADDOX, Ann. Rev. Biochem. 46 823-896 (1977). U. WALTER, Rev. Physiol. Biochem. Pharmacol. 113 41-88 (1989). J. SINGI-I and F. W. FLrrNEY, J. Mol. Cell. Cardiol. 12 285-297 (1980). G. CRAMB, R. BANKS, E. L. RUGG, and J. ALTON, Biochem. Biophys. Res. Comm. 148 962-970 (1987). D. McCALL and T. A. FRIED, J. Mol. Cell. Cardiol. 22 201-212 (1990).
2374
68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81.
82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99.
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
J. SCHRADER, G. BAUMAN, and E. GERLACH, Pflfigers Arch. 372 29-35 (1977). L. BELARDINELLI, S. VOGEL, J. LINDEN, and R. M. BERNE, J. Mol. Cell. Cardiol. 14 291-294 (1982). M.B. ANAND-SRIVASTAVA and M. CANTIN, Biochem. Biophys. Res. Comm. 138 427-436 (1986). R. FISCI-IMEISTER, P.-F. MERY, A. SHRIER, C. PAVOINE, V. BRECHLER, and F. PECKER, Subcellular Basis of Contractile Failure, 39-54, Klfiwer Academic Press, Boston, Dordrecht, London (1990). H . C . HARTZELL and R. FISCI-IMEISTER, Nature 323 273-275 (1986). I . L . O . BUXTON and L. L. BRUNTON, J. Biol. Chem. 260 6733-6737 (1985). I . L . O . BUXTON and L. L. BRUNTON, Am. J. Physiol. 2~1 H307-H313 (1986). P.-F. MERY, V. BRECHLER, C. PAVOINE, F. PECKER, and R. FISCHMEISTER, Nature 34~ 158-161 (1990). A.E. FARAH, Pharmacol. Rev. 35 181-217 (1983). R . W . TSIEN, W. GILES, and P. GREENGARD, Nature 240 181-183 (1972). R . W . TSIEN, Adv. Cyclic Nucleot. Res. 8 363-420 (1977). R . C . LEVI, G. ALLOATH, and R. FISCHMEISTER, Pfliigers Arch. 413 685-687 (1989). D . P . RARDON and A. J. PAPPANO, Am. J. Physiol. 251 H601-H611 (1986). D. BRUNKI-IORST, H. VANDERLEVEN, W. MEYER, R. NIGBUR, C. SCHMIDTSCHUMACHER, and H. SCHOLZ, Nannyn-Schmied. Arch. Pharmacol. 339 575-583 (1989). I. KODAMA, N. KONDO, and S. SHIBATA, Br. J. Pharmacol. 80 511-517 (1983). M. SHAHID and I. W. RODGER, Brit. J. Pharmacol. 98 291-301 (1989). C.J. FRANGAKIS, C. LANNI, K. P. LASHER, R. G. BENTLEY, and A. E. FARAH, J. Cardiovasc. Pharmacol. 13 915-924 (1989). A.E. FARAH and C. J. FRANGAKIS, Basic Res. Cardiol. 84 85-103 (1989). C.O. MALECOT, D. M. BERS, and B. G. KATZUNG, Circ. Res. 59 151-162 (1986). M. ENDOH, T. YANAGISAWA, N. NAIRA, and J. R. BLINKS, Circul. 73 (Suppl. HI) 117-133 (1986). J . P . MORGAN, J. K. GWATHMEY, T. T. DEFOE, and K. G. MORGAN, Circul. 73 (Suppl. HI) 65-77 (1986). E . M . OLSON, D. KIM, T. W. SMITH, and J. D. MARSH, J. Mol. Cell. Cardiol. 19 95-104 (1987). M. GALVAN and C. SCHUDT, Naunyn-Schmied. Arch. Pharmacol. 342 221-227 (1990). R.E. WEISHAAR, D. C. KOBYLARZ-SINGER, M. M. QUADE, R. P. STEFFEN, and H. R. KAPLAN, J. Cyclic Nucleot. Prot. Phosphoryl. Res. 11 513-527 (1987). R . E . WEISHAAR, D. C. KOBYLARZ-SINGER, J. KEISER, S. J. HALEEN, T. C., MAJOR, S. RAPUNDALO, J. T. PETERSON, and R. PANEK, Hypertension 15 528-540 (1990). P.C. CANNIFF, A. E. FARAH, N. SPERELAKIS, and G. M. WAHLER, J. Cardiovasc. Pharmacol. 7 813-821 (1985). J.L. SUTKO, J. L. KENYON, and J. P. REEVES, Circul. 73 (Suppl Ill) 52-58 (1986). C.O. MALECOT, P. ARLOCK, B. G. and KATZUNG, J. Pharmacol. Exp. Ther. 232 10-19 (1985). R. FISCHMEISTER and H. C. HARTZELL, Mol. Pharmacol. 38 426-433 (1990). J. NARGEOT, J. M. NERBONNE, J. ENGELS, and H. A. LESTER, Proc. Natl. Acad. Sci. 80 2395-2399 (1983). W. TRAUTWEIN, J. TANIGUCHI, and A. NOMA, Pflfigers Arch. 392 307-314 (1982). S. RICHARD, J. M. NERBONNE, J. NARGEOT, H. A. LESTF_~, and D. GARNIF_X, Pflfigers Arch. 403 312-317 (1985).
Vol. 48, No. 25, 1991
100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130.
Phosphodiesterases & Calcium Current
2375
G . M . WAHLER, N. J. RUSCH, and N. SPERELAKIS, Can. J. Physiol. 68 531-534 (1990). P.-F. MERY, S. M. LOHMANN, U. WALTER, and R. FISCHMEISTER, Proc. Natl. Acad. Sci. USA 88 1197-1201 (1991). J. SINGH and F. W. FLITNEY, Pflfigers Arch. 392 1-6 (1980). J. AZUMA, H. HARADA, A. SAWAMURA, H. HASEGAWA, S. KISHIMOTO, and N. SPERELAKIS, J. Mol. Cell. Cardiol. 15 43-52 (1983). J. AZUMA, A. SAWAMURA, H. HARADA, T. TANIMOTO, Y. MORITA, N SPERE~LAKIS, and Y. YAMAMURA, Europ. J. Pharmacol 72 199-208 (1981). J . A . SCHNEIDER and N. SPERELAKIS, J. Mol. Cell. Cardiol. 7_867-876 (1975). S. BARTEL, H. TENOR, and E.-G. KRAUSE, Biomed. Biochim. Acta 44 K31-K35 (1985). S. HERIN(}, R. BODEWF_.~, B. SCHUBERT, E.-(}. KRAUSE, and A. WOLLENBERGER, Biomed. Biochim. Acta 44 K37-K41 (1985). F. MARKWARDT and B. NILIUS, Nannyn-Schmied. Arch. Pharmacol. 337 454-458 (1988). C. F_.RNEUX, F. MIOT, P. J. M. Van HAASTERT, and B. JASTORFF, FEBS I_~tt. 142 251-254 (1982). T. YAMAMOTO, S. YAMAMOTO, J. C. OSBOURNE, V. C. MAN(}ANIELLO, and M. VAU(}HAN, J. Biol. Chem. 258 14173-14177 (1983). I . R . WENDT and D. (}. STEPHENSON, PflQgers Arch. 398 210-216 (1983). A.A. ALOUSI, A. M. (}RANT, P. D. ALLEN, and E. D. PAGANI, J. Pharmacol. Exp. Therap. 246 30-37 (1988). R.J. SOLARO and J. C. RUE(}(}, Circ. Res. 51 290-294 (1982). J.W. HERZIG, K. FFJLE, and J. C. RUEGG, Arzneim.-Forsch./Drug Res. 31 188-191 (1981). K. FUJINO, N. SPERELAKIS, and R. J. SOLARO, Circ. Res. 63 911-922 (1988). J . C . RUEGG, G. PFITZER, D. EUBLER, and C. ZEUGNER, Arzneim.-Forsch./Drug Res. 34 1736-1738 (1984). S . T . RAPUNDALO, R. J. SOLARO, and E. G. KRANIAS, Circ. Res. 64 104-111 (1989). A.A. ALOUSI and D. C. JOHNSON, Circul. 73 (Suppl. III) 10-24 (1986). M. GRIMA, M. FREYSS BE(}UIN, E. MILLANVOYE-Van BRUSSEL, N. DECKER, and J. SCHWARTZ, J. Cardiov. Pharmacol. 12 255-263 (1988). K . M . MYLOTrE, V. CODY, P. J. DAVIS, F. B. DAVIS, S. D. BLAS, and M. SCHOENL, Proc. Nail. Acad. Sci. USA 82 7974-7978 (1985). S . R . M . HOLMBERG, P. A. POOLE-WILSON, and A. J. WILLIAMS, Circul. 82 (Suppl. HI) III-519 (1990). B.B. FREDHOLM, Trends Pharmacol. Sci. 1 129-132 (1980). S . H . SNYDER, J. J. KATIMS, Z. ANNAU, R. F. BRAUN, and J. W. DALY, Proc. Nail. Acad. Sci. USA 78 3260-3264 (1981). S.C.-T. LAM, M. A. GUCCIONE, M. A. PACKHAM, and J. F. MUSTARD, Thromb. Haemostas. (Stuttgart) 47 90-95 (1982). A.S. OTERO, G. E. BREITWIESER, and G. SZABO, Science 242 443-445 (1988). H. HEIDBUCHEL, G. CALLEWAERT, J. VEREECKE, and E. CARMELIET, Pflfigers Arch. 416 213-215 (1990). A.S. OTERO, Biochem. Pharmacol. 39 1399-1404 (1990). N. KIMURA and N. SHIMADA, Biochem. Biophys. Res. Comm. 151 248-256 (1988). W.J. PARSONS, V. RAMKUMAR, and G. STILES, Mol. Pharmacol. 34 37-41 (1988). W . J . PARSONS, V. RAMKUNAR, and G. L. STILES, Mol. Pharmacol. 33 441-448 (1988).
2376
131. 132.
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
V. RAMKUMAR and G. L. STILES, Mol. Pharmacol. 34 761-768 (1988). P. ENGFELDT, P. ARNER, and J. OSTMAN, J. Lipid Res. 26 977-981 (1985).
Pergamon Press
MINIREVIEW CYCLIC AMP PHOSPHODIESTERASES AND Ca 2+ C U R R E N T R E G U L A T I O N IN CARDIAC CELLS
Rodolphe Fischmeister" and H. Criss Hartzell t
"Laboratoire de Physiologie Cellulaire Cardiaque, 1NSERM U-241, Universit6 de Paris-Sud, F-91405 Orsay, France tDepartment of Anatomy and Cell Biology, Emory University School of Medicine Atlanta, GA 30322 (Received in final form April 4, 1991)
Summary
At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca 2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca2+-channel current 0c,) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on Ic, amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of Ic, and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.
Ca2+ channels are the main route by which Ca2+ ions enter cardiac cells to regulate some of the fundamental functions of the heart, such as automaticity and contractility. Because both the action potential and contraction are dependent upon Ca2+ influx, studying the characteristics of the transmembrane Ca2+-channel current (Ic,) can provide insights into the participation of Ca2+ ions in each phase of the cardiac action potential and in the initiation and regulation of the amplitude and time course of cardiac contraction. In general, hormones and drugs that modulate Ic, amplitude generally exert similar effects on cardiac contraction. Therefore, when searching for the cellular mechanisms responsible for the cardiac inotropic effect of new substances (e.g. phosphodiesterase inhibitors), one might reasonably first examine the effect of these substances on Ic°. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
2366
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
An important feature of cardiac Ca2+ channels is that their activity is regulated by cAMP-dependent phosphorylation (1,2). Phosphorylation of Ca2+ channels (or a closely associated protein) by cAMP-dependent protein kinase (PK-A) leads to an increase in the mean probability of channel opening 0). Because the steady-state level of phosphorylation of Ca2+ channels is determined by a complex interplay between many enzymatic reactions, any change in the metabolism of cAMP is likely to affect lc,. For example, it is well known that stimulation of adenylyl cyclase activity by fl-adrenergic agonists (4,5) or forskolin (6,7) leads to the stimulation of Ic,. Conversely, one would predict that cAMP phosphodiestera.se~ (PDEs) might also play a substantial role in the physiological regulation of Ic,. However, data supporting this assertion has been mainly indirect until recently. Part of the reason for this is that, unlike adenylyl cyclase, PDEs exist in several isoforms whose identification and distribution in various cardiac tissues remains cloudy. Whether these different isoforms have different functions or how they are regulated differently remains to be determined. Another complication in identifying the function of these different PDEs is that the subcellular distribution of various isoforms of PDEs is very different and many vary between species. Finally, the pharmacological aramentarium available to interfere with PDE activity remains rudimentary. Chemists have only recently developed compounds which bind with a sufficiently high affinity to specific PDE isozymes that allows them to be used in biochemical, pharmacological and electrophysiological studies.
Nomenclature and subcellular localization of cardiac cAMP-PDE isozvmes
Complete information on the purification, identification, characterization, and localization of cardiac PDE isozymes is to be found in articles from Beavo's (8,9) and Weishaar's (10,11) laboratories. The existence of several different classification schemes with different criteria but similar names has impeded communication in this area. The classification scheme we have utilized is taken from Beavo and Reifsnyder (12) and is based on primary protein and eDNA sequences (Table 1). Basically, four different families of PDE participate in the hydrolysis of cAMP in cardiac tissue. Family I. Ca2+-Calmodulin-dependent PDE (Ca/CaM-PDE). It seems likely that there are several forms of Ca/CaM-PDE in heart tissue, because of the differences in substrate specificities and affmities that have been described. Ca/CaM-PDE hydrolyzes cAMP and cGMP with both a low (50 #M, ref. 10) and a high (1 t~M, ref. 10) affinity in guinea pig, but only with a high affinity in human heart (13). In rat heart, a Ca/CaM-PDE appears to specifically hydrolyze cGMP (14). CaJCaM-PDE is activated by CaX+-calmodulin and is inhibited by zaprinast (M&B 22,948) and isobutylmethylxanthine 0BMX) at concentrations < 10 /~M (8,11,15) and by calmodulin antagonists (11). The CaJCaM-PDE is present in the soluble fraction of rat (16), human (13), and guinea pig ventricle (13). Bode et al. (17) have recently found that the Ca/CaM-PDE does not exist in purified cardiac myocytes and thus may be localized in other cell types.
Family II. cGMP-stimulated PDE (cGs-PDE). This isoform was first characterized by Beavo and collaborators (18,19). The cardiac cGs-PDE hydrolyzes both cAMP and cGMP with KIn> 10/~M (8,11,13). In frog heart, 1 /~M cGMP is sufficient to half-maximally activate the enzyme (20). No highly selective inhibitors are known for this family of PDE. However, in guinea pig (10,11) and frog heart (15), the enzyme is inhibited by dipyridamole at concentrations < 10 /~M. High concentrations of IBMX (> 50/zM) also inhibit the cGs-PDE (8). The enzyme seems present both in the soluble (13,16,21) and particulate fractions (20). Family HI. cGMP-inhibited low-Km cAMP-PDE (cGi-PDE). The cardiac form of this enzyme was identified by Harrison et al. (22). The main features of this PDE isozyme are its high
Vol. 48, No. 25, 1991
Phosphodiesterases & Calcium Current
2367
affinity for cAMP as substrate (Km <0.2 /~M, refs. 8,13,22), its high sensitivity to cGMP inhibition (Ki 0.1 /tM, ref. 8), and its high sensitivity to inhibition by a variety of compounds (mostly the bipyridines milrinone, amrinone, indolidan, fenoximone, etc.) which are cardiotonic agents with potential clinical usefulness (8,11,23).
Family IV. cGMP-independent low-Kin cAMP-PDE. Often called cAMP-specific PDE, this isoform of PDE is highly sensitive to inhibition by anti-depressants such as Ro 20-1724 and rolipram (13,23,8). The cAMP-specific PDE hydrolyzes cAMP with Kin<2/~M (13,8). Family V. cGMP-specific PDEs. This class has not been described in heart. Until recently, identification of PDE isoform was based largely on elution profiles from DEAE anion exchange columns and pharmacological data. Because few, if any, of the PDE inhibitors available have absolute specificity for a particular isoform, consideration of the appropriate concentrations of PDE inhibitors used is of paramount importance in evaluating isoform identification based on inhibitor studies alone. For this reason, it is sometimes difficult to establish precisely the family of PDE in question, particularly in older studies. The cellular localization of the two low-Km cAMP-PDEs (PDEs HI and IV) has been extensively studied. Their localization differs significantly from one species to the other and partly explains the species differences found in the activity of PDE inhibitors as inotropic agents. In dog (23-25), rabbit (26-28) and sheep hearts (29), the PDE-III is essentially membrane-bound while most of the PDE-IV is located in the soluble fraction. In guinea pig heart, however, both PDE-HI (13,24) and PDE-IV (13) are in a soluble form. The situation in rat heart is somewhat unclear since only membrane-bound PDE-IV was found in one study (28), while in other studies (23,24) both PDE-III and PDE-IV were found exclusively in the soluble fraction. In the human heart, low-Km cAMP-PDE activity seems located essentially in the soluble fraction, but it is unclear whether this is composed of PDE-III alone (30) or both PDE-III and PDE-IV (13). It also appears that the membrane bound form of low-Kin cAMP-PDEs found in various cardiac preparations is mostly located at the sarcoplasmic reticulum membrane (25-27,29,31) and that localization and sensitivity to inhibitors may vary during development (26). The variety of PDE isozymes and their complex distribution within cardiac cells suggest that cAMP may be compartmentalized inside the cell (24). This would explain the differential effects of various PDE inhibitors on contractile and electrical properties of a given cardiac preparation.
Phvsioloeical reeulation of cardiac cAMP-PDE activitv The idea that phosphodiesterase activity may be physiologically regulated is not new (32,33). There is now abundant evidence that increases in PDE activity (cGMP or cAMP) may be involved in the cellular response to various physiological stimuli such as light in photoreceptors (34), insulin in hepatocytes (35-38) and fat cells (8,39,40), muscarinic agonists in thyroid (41) and astrocytoma cells (42), coneanavalin A in lymphocytes (43), angiotensin H in aortic smooth muscle cells (44). Cyclic AMP PDE activity has also been found to decrease upon application of prostaglandin E1 in fibroblasts (45). Except for the regulation of cGMP-PDE activity in the visual system (34), which involves the direct interaction of a G-protein with the PDE enzyme, the mechanisms responsible for the regulation of PDE activity in other cell types remain unclear and often controversial. Two major
2368
Phosphodiesterases
& Calcium Current
Vol. 48, No. 25, 1991
mechanisms have been proposed: (i) a stimulation of cAMP-PDE activity by cAMP itself (33,38,40,46), possibly via phosphorylation by PK-A (22). This mechanism can be viewed as a form of negative feedback to terminate the cAMP signal generated by activation of adenylyl cyclase. A long term regulation of cAMP-PDE activity by cAMP has also been described that may involve modification in the expression of PDE through changes in mRNA levels (47,48). (ii) cAMP-PDE activity may be regulated via a pertussis-toxin sensitive G-protein (38,49), directly or through activation of phospholipase C, resulting in production of IP3 and subsequent increases in Ca 2+ (42) and diacylglycerol and possible activation of protein kinase C (50,51).
Table 1. cAMP PHOSPHODIESTERASES I N H E A R T *
Type [ I
Description Ca-calmodulin -dependent.
I InhibRors (ICs0)
Effects on
IC.
(ref) t
calmodulin inhibitors, phenothiazines.
unknown
no selective inhibitors.
cGMP inhibits cAMP stimulated Ic,. Effect of cGMP blocked by IBMX (5,56,96).
Not present in myocytes. 1"1
cGMP-stimulated PDE
IBMX (50 pM) cGMP-inhibited PDE
III
Low K~ for cAMP. Specific for cAMP
IV
milrinone (5nM) amrinone (300 nM) indolidan (80 nM)
PDE III and IV inhibitors have no effect on basal Ic,, but stimulate Ic, in presence of low [cAMP] (96).
rolipram (1 tLM) Ro20-1724 (2 I~M)
Glucagon stimulates Ic, (75).
Low I ~ for cAMP. Classification scheme and inhibitor data from ref 12. * Data on Ic, are from frog cardiac myocytes. Mammals may differ. *
Very little is known about the hormonal regulation of cardiac PDEs. Cyclic GMP-stimulation of cAMP hydrolysis has been proposed to participate in the cardiac negative inotropic effect of muscarinic agonists (52-54). Evidence supporting this hypothesis comes from the fact that (i) acetylcholine increases cardiac cGMP levels (2,55,56 for refs.), (ii) cGMP exerts a negative inotropic effect in heart (57-62), (iii) cAMP level is generally reduced when cGMP level increases (52-54), (iv) a cGs-PDE isozyme has been characterized in cardiac tissue (18,19, see above). Such a mechanism could possibly account for the "Yin-Yang" phenomenon (63) where cGMP was found to counterbalance the stimulatory effect of cAMP on various cellular responses to hormones (54,63,64). Conclusive evidence for the participation of a cGs-PDE in the cardiac negative inotropic effect of acetylcholine as well as other hormones known to elevate cGMP level in heart, such as adenosine (65) and atrial natriuretic factor (66,67), has been hindered by the presence of a concomitant direct and strong inhibitory effect of these agonists on cAMP production (66-71). Experiments in which adenylyl cyclase activity could be bypassed were necessary to examine the effects of cGMP. As will be described below, such an approach has permitted the demonstration that stimulation of cAMP-PDE was responsible for the cGMP induced inhibition of Ic, in frog heart (56,72).
Vol. 48, No. 25, 1991
Phosphodiesterases & Calcium Current
2369
A different example of hormonal regulation of cardiac cAMP-PDE activity was provided by Buxton and Brunton (73), who found a stimulation of cAMP hydrolysis in rat heart myoeytes exposed to aradrenergic agonists. This effect appears to be mediated via a G-protein, possibly by activation of phospholipase C and hydrolysis of phosphatidyl inositol 4,5-bisphosphate (74). Consistent with this hypothesis is the recent finding that exposure of rat cardiac microsomes to exogenous phospholipase C stimulates the eAMP-PDE activity in the membrane bound fraction (51). So far, the only example of hormonal inhibition of cardiac PDE activity is the inhibitory effect of glucagon on a particulate low-Km cAMP-PDE in frog ventricle (75). These findings provide a new pathway of regulation of cardiac function and suggest that hormones exerting positive inotropie effects on the heart may act in a similar manner to the synthetic inhibitors. Surprisingly, while glucagon stimulates Ic° both in rat and frog heart (75) and is a positive inotropie agent (76) in rat heart, its action on the latter is not mediated by inhibition of PDE activity but rather by a stimulation of adenylyl eyclase activity (75).
eAMP-PDE activitv and rfgulation of lc. Evidence for the participation of cAMP hydrolysis in the regulation of Ic, was first shown indirectly by the demonstration that massive concentrations (> 500/~M) of IBMX consistently produced a stimulatory effect on cardiac Ico (6,20,77-79) or prolonged action potential duration (80). After various forms of cardiac cAMP-PDEs were characterized biochemically and pharmacologically, a large number of electrophysiological studies were performed to examine the effects of specific or non-specific PDE inhibitors on cardiac electrical properties. Most studies, however, were limited to the effect of these compounds on beat frequency, action potential parameters, or contractility. Evidence for the participation of Ca2+ channels in the positive inotropic and electrophysiological effects of PDE inhibitors was, therefore, indirect. The most studied and highly specific cGi-PDE inhibitors, milrinone and amrinone, produce (i) an increase in beat frequency (81-83), (ii) modifications in action potential amplitude and duration that are sensitive to extracellular Ca:+, dihydropyridines and phenylalkyamines (84,85), (iii) an increase in duration, Vm,x and maximal overshoot of slow calcium-dependent action potentials (86), (iv) an increase in intracellular Ca:+ concentration (87,88), (v) an increase in the positive inotropic effect and in 45Ca uptake that is sensitive to verapamil (89). It is important to mention that various specific PDE inhibitors may exert different effects on contractility and action potentials (90) depending on whether their targets are membrane-bound or soluble (23,91). This could also explain why a given PDE inhibitor (milrinone) modifies cardiac electrical activity in a tissue- and species-dependent manner (92,93). Voltage-clamp studies on the effects of specific PDE inhibitors on cardiac Ica are uncommon. In multicellular preparations, milrinone (86,94) and amrinone (94,95) increased the amplitude of Ic°. However, the concentrations used in these studies were too high to permit an unambiguous conclusion that these effects were due to inhibition of PDE activity rather than to other side effects (see below). Recently, we have examined the effects of several specific inhibitors of the low-Km eAMP-PDEs (milrinone and indolidan for PDE-III and rolipram and Ro 20-1724 for PDE-IV) as well as low concentrations of IBMX on Ic, recorded from isolated cells from frog ventricle (96). We found that each of these compounds increased Ic, when submaximal concentrations of cAMP were perfused internally, but did not increase Ica when exogenous cAMP was not added. These effects were dose-dependent with an apparent K~ for each inhibitor tested (1-3 #M) consistent with the inhibitory effect of the compounds on low Km eAMP-PDE activity (96). These results demonstrated the physiological role of low-Kin eAMP-PDEs in regulating the amplitude of Ica upon a stimulation by a cAMP-dependent mechanism, such as a B-adrenergie
2370
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
stimulation. In other studies, we also demonstrated the participation of another form of cAMP-PDE, the cGs-PDE, in the inhibitory effect of cGMP on Ic, in frog heart (20,56,72). Our finding that cGMP inhibited cardiac Ic, was unexpected, since studies of other investigators had demonstrated the absence of an effect of cGMP on cardiac Ca2+ channels (97-99). Recent evidence, however, has confirmed the inhibition of cardiac Ca2+ channels by cGMP (20,79,100,101), although there are significant species-dependent variations in the mechanisms responsible for this inhibition (72,79,100,101). Four major observations support the role of a cGs-PDE in mediating the effect of cGMP on Ica in frog cardiomyocytes. (i) cGMP was unable to inhibit Ica that had been elevated by the hydrolysis-resistant cAMP analogue, 8-bromo-cAMP. (ii) 8-bromo-cGMP, which does not activate the cGs-PDE in frog heart (20) was unable to inhibit the stimulatory effect of cAMP on Ic~. (iii) Large concentrations of IBMX (> 100/~M) could reverse the inhibitory effect of cGMP on cAMP-elevated Ic°. (iv) A cGs-PDE was identified in this preparation with a similar requirement for cGMP activation as the inhibition of Ic, (20,56,72). Based on our studies, at least three forms of cAMP-PDEs (cGi-PDE, cGs-PDE, and cAMPspecific PDE) participate in the regulation of cardiac Ca2+ channels. However, the capacity to hydrolyze cAMP and/or the localization may vary significantly among the different forms. Indeed, we have found that none of the low-KmcAMP-PDE inhibitors were able to stimulate Ic, when the cGs-PDE was maximally stimulated by cGMP, whereas each of them was effective in the absence of cGMP (96). In addition to the previously mentioned effects of acetylcholine and ANF, several purine and pyrimidine nucleosides and their nucleotide derivatives exert strong pharmacological effects on the heart, as well as coronary vascuiature, that are accompanied by an increase in cGMP levels (e.g. adenosine, ref. 65; ATP and UTP, ref. 102). It is likely that these effects are at least in part mediated by changes in cAMP metabolism.
Additional effects of cAMP-PDE inhibitors One of the major impediments to understanding the function of PDEs in heart and other tissues is the rather poor specificity of PDE inhibitors for PDEs and the poor selectivity of these inhibitors between different forms of PDEs. For example, there are virtually no selective inhibitors of cGs-PDE. Some of the side effects of these drugs could easily confound the interpretation of experiments designed to evaluate the role of PDEs. Several examples follow. The non-specific PDE inhibitor trapidil (a triazulopyrimidine) stimulates Ca2+-dependent slow action potentials (103,104) at "low" concentrations (10-100/zM) as a result of an increase in cAMP. However, at higher concentrations (> 500/~M) trapidil inhibits Ca2+-dependent action potentials (103) even though cAMP levels remain elevated. Similar results have been reported with papaverine (105). In voltage-clamp studies, trapidil and trapidil derivatives, which exert a more potent inhibition of cardiac PDE than wapidil (106), inhibit cardiac Ca current at high concentrations (107,108). The dose-dependency of PDE inhibitors may be complicated by the fact that, as has been demonstrated in the case of the cGs-PDE, inhibitors of the catalytic site of the isozyme also appear to bind with high affinity to the allosteric site on the enzyme and thereby stimulate PDE activity at low inhibitor concentrations (12,109,110). A number of PDE inhibitors, such as caffeine and theophylline (111,112), sulmazole (113,114), and pimobendan (115,116) increase the Ca2+ sensitivity of the troponin C regulatory site in cardiac myofilaments. Surprisingly, these effects were not observed with milrinone
Vol. 48, No. 25, 1991
Phosphodlesterases & Calcium Current
2371
(112,115,117). However, milrinone, and its analogue amrinone, may exert cardiac effects that may be unrelated to PDE inhibition including stimulation of Na/Ca exchange (94,118), Na influx through Na channels (119), membrane Ca-ATPase (120), and the Ca2+-release channel of the sarcoplasmic reticulum (121). It is worth noting that milrinone (but not amrinone) shares structural homologies with thyroid hormone (120), which may partly explain the similarity of action of these substances on cardiac Ca-ATPase (120) and on low-Km cAMP-PDE activity in fat cells and hepatocytes (38 for refs.). Some of the cardiac positive inotropic effects of other PDE inhibitors could also be mediated via other routes than inhibition of PDE activity. For example, methylxanthines, such as IBMX, caffeine, and theophylline, exert strong antagonistic effects on P1 receptors (122,123). These effects result from a structural homology between adenosine and methylxanthines, which share a common ring structure. It has been reported that methylxanthines inhibit nucleoside diphosphate kinase (NDPK, ref. 124). Because NDPK may participate in the control of various effectors such as ionic channels (125-127) and adenylyl cyclase (128, 127 for refs.) by G-proteins, it is conceivable that methylxanthines may interfere with these targets. Consistent with this hypothesis are the findings that IBMX (129) and sulmazole (130,131), which is also a P1 adenosine receptor antagonist (130) and possesses the methylxanthine-like ring structure, block the inhibitory effect of the G-protein, Gi, on adenylyl cyclase (129-131) by affecting the GTP turnover (131). These compounds may, thus, increase cAMP levels by stimulating cAMP production in addition to their inhibitory effect on cAMP degradation.
Concludine remarks
In spite of several limitations in the use of specific PDE inhibitors, these compounds are promising tools to investigate the presence of various cAMP-PDE isozymes in cardiac tissues. Alth0ugh the possibility remains that PDE activity may be different in enzymatically dissociated cells as compared to in situ cells (132), voltage-clamp studies on isolated cardiac myocytes may help to delineate the functional coupling of various cAMP-PDE isozymes to Ca2+ channels as well as to determine their relative potencies to regulate cAMP-dependent phosphorylation of the channels. In addition, the ability to modify the composition of intracellular solution during an experiment, mainly its cyclic nucleotide content, allows one to examine the participation of PDE activity in the regulation of Ca2+ channel activity by hormones and drugs. This experimental approach may provide a celinlar rationale for the physiological effects of these compounds on contractile properties, beat frequency, and action potential, as well as an in vivo counterpart of biochemical studies on regulation of cAMP metabolism and PDE activity.
Acknowled~,ements
Work from our laboratories discussed above was supported by INSERM and NIH grants HL21195 and HL27385 and an exchange program INSERM France/U.S.A.. References °
2.
F. HOFMANN, W. NASTAINCZYK, A. ROHRKASTEN, T. SCHNEIDER, and M. SIEBER, Trends Pharmacol. Sci. 8 393-398 (1987). H. C. HARTZELL, Prog. Biophys. Mol. Biol. 52 165-247 (1988).
2372
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Phosphod±esterases & Calcium Current
Vol. 48, No. 25, 1991
R . W . TSIEN, B. P. BEAN, P. HESS, J. B. LANSMAN, B. NILIUS, and M. NOWYCKY, J. Mol. Cell. Cardiol. 18 691-710 (1986). M. KAMEYAMA, F. HOFMANN, and W. TRAUTWEIN, Pflfigers Arch. 407 285-293 (1985). R. FISCHMEISTER and H. C. HARTZELL, J. Physiol. (Lond.) 376 183-202 (1986). M. KAMEYAMA, J. HESCHELER, F. HOFMANN, and W. TRAUTWEIN, Pfliigers Arch. 407 123-128 (1986). H . C . HARTZELL and R. FISCHMEISTER, Mol. Pharmacol. 32 639-645 (1987). J . A . BEAVO, Adv. See. Mess. Phosphoprot. Res. 22 1-38 (1988). M . D . HOUSLAY and J. A. BEAVO, (eds.) Isozvmes of Cyclic Nucleotid¢ Ph0sphodiesterases. John Wiley & Sons (1990). R . E . WEISHAAR, S. D. BURROWS, D. C. KOBULARZ, M. M. QUADE, and D.B. EVANS, Biochem. Pharmacol. 35 787-800 (1986). R . E . WEISHAAR, M. H. CAIN, and J. A. BRISTOL, J. Med. Chem. 28 537-545 (1985). J.A. BEAVO and D. H. REIFSNYDER, Trends in Pharmacol. Sci. 11, 150-155 (1990). M . L . REEVES, B. K. LEIGH, and P. J. ENGLAND, Bioehem. J. 241 535-541 (1987). W . J . THOMPSON, W. L. TERASAKI, P. M. EPSTEIN, and S. J. STRADA, Adv. Cyclic Nucleot. Res. 10 69-92 (1979). C. LUGNIER, A. Le BEC, C. GAUTHIER, H. SOUSTRE, and J.-C. STOCLET, Arch. Int. Physiol. Biochim. 98 A171 (1990). D . C . BODE, J. R. KANTER, and L. L. BRUNTON, See. Messeng. Phosphoprot. 12 235-240 (1988-89). D.C. BODE, J. KANTER, and L. L. BRUNTON, FASEB J. 3, A1295 (1989) and Circ. Res. (in press). J.A. BEAVO, J. G. HARDMANN and E. W. SUTHERLAND, Biochem. Biophys. Res. Comm. 138 1170-1176 (1971). T . J . MARTINS, M. C. MUMBY, and J. A. BEAVO, J. Biol. Chem. 257 1973-1979 (1982). M . A . SIMMONS and H. C. HARTZELL, Mol. Pharmacol. 33 664-671 (1988). A. LAGRUTrA and H. C. HARTZELL, personal communication. S.A. HARRISON, D. H. REIFSNYDER, B. GALLIS, G. G. CADD, and J. A. BEAVO, Molee. Pharmacol. 29 506-514 (1986). R.E. WEISHAAR, D. C. KOBYLARZ-SINGER, R. P. STEFFEN, and H. R. KAPLAN, Circ. Res. 61 539-547 (1987). R . E . WEISHAAR, D. C. KOBYLARZ-SINGER, and H. KAPLAN, J. Mol. Cell. Cardiol. 19 1025-1036 (1987). R . F . KAUFFMAN, B. G. UTrERBACK, and D. W. ROBERTSON, Circ. Res. 64 1037-1040 (1989). P . A . KITHAS, M. ARTMAN, W. J. THOMPSON, S. J. STRADA, J. Mol. Cell. Cardiol. 21 507-517 (1989). P.A. KITHAS, M. ARTMAN, W. J. THOMPSON, and S. J. STRADA, Circ. Res. 62 782-789 (1988). M. SHAHID, M. WILSON, C. D. NICHOLSON and R. J. MARSHALL, J. Pharmacy Pharmacol. 42 283-284 (1990). M. ARTMAN, D. W. ROBERTSON, L. MAHONY, and W. J. THOMPSON, Mol. Pharmacol. 36 302-311 (1989). H. MASUOKA, M. ITO, T. NAKANO, M. NAKA, and T. TANAKA, J. Cardiov. Pharmacol. 15 302-307 (1990). R . F . KAUFFMAN, V. G. CROWE, B. G. UTI'ERBACK, and D. W. ROBERTSON, Mol. Pharmacol. 30 609-616 (1986).
Vol. 48, No. 25, 1991
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67.
Phosphodlesterases & Calcium Current
2373
E . T . BROWNING, C. O. BROSTROM and V. E. GROPPI, Jr., Mol. Pharmacol. 12 32-40 (1976). J . P . SCHWARTZ and J. V. PASSONEAU, Proc. Natl. Acad. Sci. USA 71 3844-3848 (1974). P . A . McNAUGHTON, Physiol. Rev. 70 847-883 (1990). R.J. MARCHMONT, S. T. AYAD, and M. D. HOUSLAY, Biochem. J. 195 645-652 (1981). M . D . HOUSLAY, Biochem. Soc. Trans. 14 183-193 (1988). C. BENELLI, B. DESBUQUOIS, and B. De GALLE, Eur. J. Biochem. 156 211-220 (1986). J . A . SMOAKE and S. S. SOLOMON, Life Sci. 45 2255-2.268 (1989). H . W . WEBER and M. M. APPLEMAN, J. Biol. Chem. 257 5339-5341 (1982). C.J. SMITH and V. C. MANGANIELLO, Mol. Pharmacol. 35 381-386 (1989). J. Van SANDE, C. ERNEUX, and J. E. DUMONT, J. Cycl. Nucl. Res. 3 335-345 (1977). R.B. MEEKER and T. K. HARDEN, Mol. Pharmacol. 22 310-319 (1982). L. VALE'ITE, A. F. PRIGENT, G. NEMOZ, G. ANKER, O. MACOVSCHI, and M. LAGARDE, Bioch. Bioph. Res. Comm. 169 864-872 (1990). J.B. SMITH and T. M. LINCOLN, Am. J. Physiol. 253 C147-C150 (1987). R. BARBER, K. P. RAY, and R. W. BUTCHER, Biochemistry 19 2560-2567 (1980). E. DEGERMAN, C. J. SMITH, H. TORNQVIST, V. VASTA, P. BELFRAGE, and V. C. MANGANIELLO, Proc. Natl. Acad. Sci. USA 87 533-537 (1990). J . V . SWINNEN, D. R. JOSEPH, and M. CONTI, Proc. Natl. Acad. Sci. USA 86 8197-8201 (1989). B.B. RILEY and S. L. BARCLAY, Proc. Natl. Acad. Sci. USA 87 4746-4750 (1990). C . M . I-IEYWORTI-I, A. M. GREY, S. R. WILSON, E. HANSKI, and M. D. I-IOUSLAY, Biochem. J. 235 145-149 (1986). F. IRV1NE, N. J. PYNE, and M. D. I-IOUSLAY, FEBS Lett. 208 455-459 (1986). M. DUBOIS, G. NEMOZ, M. LAGARDE, and A. F. PRIGENT, Bioch. Biophys. Res. Comm. 170 800-809 (1990). M. ENDOH, Jap. J. Pharmacol. 29 855-864 (1979). F . W . FLITNEY and J. SINGH, J. Mol. Cell. Cardiol. 13 963-979 (1981). U. WALTER, Adv. Cycl. Nucleo. Prot. Phosphoryl. Res. 17 249-258 (1984). W . J . GEORGE, J. B. POLSON, A. G. O'TOOLE, and N. GOLDBERG, Proc. Natl. Acad. Sci. USA 66 398-403 (1970). R. FISCI-IMEISTER and H. C. I-IARTZELL, J. Physiol. 387 453-472 (1987). H. NAWRATH, Nature 267 72-74 (1977). J. DIAMOND, R. E. TEN EICK and A. J. TRAPANI, Biochem. Biophy. Res. Comm. 79 912-917 (1977). J. SINGH and F. W. FLITNEY, Biochem. Pharmacol. 30 1475-1481 (1981). W. T R A I N and G. TRUBE, PflQgers Arch. 366 293-295 (1976). R . D . WILKERSON, R. J. PADDOCK, and W. J. GEORGE, Eur. J. Pharmacol. 36 247-251 (1976). W. TUGANOWSKI and P. KOPEC, Naunyn-Schmied. Arch. Pharmacol. 304 211-213 (1978). N . D . GOLDBERG and M. K. HADDOX, Ann. Rev. Biochem. 46 823-896 (1977). U. WALTER, Rev. Physiol. Biochem. Pharmacol. 113 41-88 (1989). J. SINGI-I and F. W. FLrrNEY, J. Mol. Cell. Cardiol. 12 285-297 (1980). G. CRAMB, R. BANKS, E. L. RUGG, and J. ALTON, Biochem. Biophys. Res. Comm. 148 962-970 (1987). D. McCALL and T. A. FRIED, J. Mol. Cell. Cardiol. 22 201-212 (1990).
2374
68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81.
82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99.
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
J. SCHRADER, G. BAUMAN, and E. GERLACH, Pflfigers Arch. 372 29-35 (1977). L. BELARDINELLI, S. VOGEL, J. LINDEN, and R. M. BERNE, J. Mol. Cell. Cardiol. 14 291-294 (1982). M.B. ANAND-SRIVASTAVA and M. CANTIN, Biochem. Biophys. Res. Comm. 138 427-436 (1986). R. FISCI-IMEISTER, P.-F. MERY, A. SHRIER, C. PAVOINE, V. BRECHLER, and F. PECKER, Subcellular Basis of Contractile Failure, 39-54, Klfiwer Academic Press, Boston, Dordrecht, London (1990). H . C . HARTZELL and R. FISCI-IMEISTER, Nature 323 273-275 (1986). I . L . O . BUXTON and L. L. BRUNTON, J. Biol. Chem. 260 6733-6737 (1985). I . L . O . BUXTON and L. L. BRUNTON, Am. J. Physiol. 2~1 H307-H313 (1986). P.-F. MERY, V. BRECHLER, C. PAVOINE, F. PECKER, and R. FISCHMEISTER, Nature 34~ 158-161 (1990). A.E. FARAH, Pharmacol. Rev. 35 181-217 (1983). R . W . TSIEN, W. GILES, and P. GREENGARD, Nature 240 181-183 (1972). R . W . TSIEN, Adv. Cyclic Nucleot. Res. 8 363-420 (1977). R . C . LEVI, G. ALLOATH, and R. FISCHMEISTER, Pfliigers Arch. 413 685-687 (1989). D . P . RARDON and A. J. PAPPANO, Am. J. Physiol. 251 H601-H611 (1986). D. BRUNKI-IORST, H. VANDERLEVEN, W. MEYER, R. NIGBUR, C. SCHMIDTSCHUMACHER, and H. SCHOLZ, Nannyn-Schmied. Arch. Pharmacol. 339 575-583 (1989). I. KODAMA, N. KONDO, and S. SHIBATA, Br. J. Pharmacol. 80 511-517 (1983). M. SHAHID and I. W. RODGER, Brit. J. Pharmacol. 98 291-301 (1989). C.J. FRANGAKIS, C. LANNI, K. P. LASHER, R. G. BENTLEY, and A. E. FARAH, J. Cardiovasc. Pharmacol. 13 915-924 (1989). A.E. FARAH and C. J. FRANGAKIS, Basic Res. Cardiol. 84 85-103 (1989). C.O. MALECOT, D. M. BERS, and B. G. KATZUNG, Circ. Res. 59 151-162 (1986). M. ENDOH, T. YANAGISAWA, N. NAIRA, and J. R. BLINKS, Circul. 73 (Suppl. HI) 117-133 (1986). J . P . MORGAN, J. K. GWATHMEY, T. T. DEFOE, and K. G. MORGAN, Circul. 73 (Suppl. HI) 65-77 (1986). E . M . OLSON, D. KIM, T. W. SMITH, and J. D. MARSH, J. Mol. Cell. Cardiol. 19 95-104 (1987). M. GALVAN and C. SCHUDT, Naunyn-Schmied. Arch. Pharmacol. 342 221-227 (1990). R.E. WEISHAAR, D. C. KOBYLARZ-SINGER, M. M. QUADE, R. P. STEFFEN, and H. R. KAPLAN, J. Cyclic Nucleot. Prot. Phosphoryl. Res. 11 513-527 (1987). R . E . WEISHAAR, D. C. KOBYLARZ-SINGER, J. KEISER, S. J. HALEEN, T. C., MAJOR, S. RAPUNDALO, J. T. PETERSON, and R. PANEK, Hypertension 15 528-540 (1990). P.C. CANNIFF, A. E. FARAH, N. SPERELAKIS, and G. M. WAHLER, J. Cardiovasc. Pharmacol. 7 813-821 (1985). J.L. SUTKO, J. L. KENYON, and J. P. REEVES, Circul. 73 (Suppl Ill) 52-58 (1986). C.O. MALECOT, P. ARLOCK, B. G. and KATZUNG, J. Pharmacol. Exp. Ther. 232 10-19 (1985). R. FISCHMEISTER and H. C. HARTZELL, Mol. Pharmacol. 38 426-433 (1990). J. NARGEOT, J. M. NERBONNE, J. ENGELS, and H. A. LESTER, Proc. Natl. Acad. Sci. 80 2395-2399 (1983). W. TRAUTWEIN, J. TANIGUCHI, and A. NOMA, Pflfigers Arch. 392 307-314 (1982). S. RICHARD, J. M. NERBONNE, J. NARGEOT, H. A. LESTF_~, and D. GARNIF_X, Pflfigers Arch. 403 312-317 (1985).
Vol. 48, No. 25, 1991
100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130.
Phosphodiesterases & Calcium Current
2375
G . M . WAHLER, N. J. RUSCH, and N. SPERELAKIS, Can. J. Physiol. 68 531-534 (1990). P.-F. MERY, S. M. LOHMANN, U. WALTER, and R. FISCHMEISTER, Proc. Natl. Acad. Sci. USA 88 1197-1201 (1991). J. SINGH and F. W. FLITNEY, Pflfigers Arch. 392 1-6 (1980). J. AZUMA, H. HARADA, A. SAWAMURA, H. HASEGAWA, S. KISHIMOTO, and N. SPERELAKIS, J. Mol. Cell. Cardiol. 15 43-52 (1983). J. AZUMA, A. SAWAMURA, H. HARADA, T. TANIMOTO, Y. MORITA, N SPERE~LAKIS, and Y. YAMAMURA, Europ. J. Pharmacol 72 199-208 (1981). J . A . SCHNEIDER and N. SPERELAKIS, J. Mol. Cell. Cardiol. 7_867-876 (1975). S. BARTEL, H. TENOR, and E.-G. KRAUSE, Biomed. Biochim. Acta 44 K31-K35 (1985). S. HERIN(}, R. BODEWF_.~, B. SCHUBERT, E.-(}. KRAUSE, and A. WOLLENBERGER, Biomed. Biochim. Acta 44 K37-K41 (1985). F. MARKWARDT and B. NILIUS, Nannyn-Schmied. Arch. Pharmacol. 337 454-458 (1988). C. F_.RNEUX, F. MIOT, P. J. M. Van HAASTERT, and B. JASTORFF, FEBS I_~tt. 142 251-254 (1982). T. YAMAMOTO, S. YAMAMOTO, J. C. OSBOURNE, V. C. MAN(}ANIELLO, and M. VAU(}HAN, J. Biol. Chem. 258 14173-14177 (1983). I . R . WENDT and D. (}. STEPHENSON, PflQgers Arch. 398 210-216 (1983). A.A. ALOUSI, A. M. (}RANT, P. D. ALLEN, and E. D. PAGANI, J. Pharmacol. Exp. Therap. 246 30-37 (1988). R.J. SOLARO and J. C. RUE(}(}, Circ. Res. 51 290-294 (1982). J.W. HERZIG, K. FFJLE, and J. C. RUEGG, Arzneim.-Forsch./Drug Res. 31 188-191 (1981). K. FUJINO, N. SPERELAKIS, and R. J. SOLARO, Circ. Res. 63 911-922 (1988). J . C . RUEGG, G. PFITZER, D. EUBLER, and C. ZEUGNER, Arzneim.-Forsch./Drug Res. 34 1736-1738 (1984). S . T . RAPUNDALO, R. J. SOLARO, and E. G. KRANIAS, Circ. Res. 64 104-111 (1989). A.A. ALOUSI and D. C. JOHNSON, Circul. 73 (Suppl. III) 10-24 (1986). M. GRIMA, M. FREYSS BE(}UIN, E. MILLANVOYE-Van BRUSSEL, N. DECKER, and J. SCHWARTZ, J. Cardiov. Pharmacol. 12 255-263 (1988). K . M . MYLOTrE, V. CODY, P. J. DAVIS, F. B. DAVIS, S. D. BLAS, and M. SCHOENL, Proc. Nail. Acad. Sci. USA 82 7974-7978 (1985). S . R . M . HOLMBERG, P. A. POOLE-WILSON, and A. J. WILLIAMS, Circul. 82 (Suppl. HI) III-519 (1990). B.B. FREDHOLM, Trends Pharmacol. Sci. 1 129-132 (1980). S . H . SNYDER, J. J. KATIMS, Z. ANNAU, R. F. BRAUN, and J. W. DALY, Proc. Nail. Acad. Sci. USA 78 3260-3264 (1981). S.C.-T. LAM, M. A. GUCCIONE, M. A. PACKHAM, and J. F. MUSTARD, Thromb. Haemostas. (Stuttgart) 47 90-95 (1982). A.S. OTERO, G. E. BREITWIESER, and G. SZABO, Science 242 443-445 (1988). H. HEIDBUCHEL, G. CALLEWAERT, J. VEREECKE, and E. CARMELIET, Pflfigers Arch. 416 213-215 (1990). A.S. OTERO, Biochem. Pharmacol. 39 1399-1404 (1990). N. KIMURA and N. SHIMADA, Biochem. Biophys. Res. Comm. 151 248-256 (1988). W.J. PARSONS, V. RAMKUMAR, and G. STILES, Mol. Pharmacol. 34 37-41 (1988). W . J . PARSONS, V. RAMKUNAR, and G. L. STILES, Mol. Pharmacol. 33 441-448 (1988).
2376
131. 132.
Phosphodiesterases & Calcium Current
Vol. 48, No. 25, 1991
V. RAMKUMAR and G. L. STILES, Mol. Pharmacol. 34 761-768 (1988). P. ENGFELDT, P. ARNER, and J. OSTMAN, J. Lipid Res. 26 977-981 (1985).