© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Muscarinic regulation of cardiac L-type Ca2+ current
Pediatric Cardiology, University of California at Los Angeles, School of Medicine, 675 C.E. Young Drive South, MRL Rm. 3754, Los Angeles, CA 90095, USA
* Tel.: +1-310-825-7105; fax: +1-310-825-7458 fchen{at}mednet.ucla.edu
Received 4 September 2000; accepted 7 September 2000
See article by Klein et al. [1] (pages 367–374) in this issue.
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1 Introduction |
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 Top 1 Introduction 2 Role of cGMP...3 Role of nitric...4 ConclusionReferences |
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As discussed by Klein and his colleagues in this issue of Cardiovascular Research [1], cGMP is an important intracellular second messenger of various extracellular stimuli regulating L-type Ca2+ current (ICa-L) in both normal and failing hearts. It has been postulated that muscarinic agonists such as carbachol (CCh) bind to muscarinic receptors to promote formation of an activated inhibitory G protein–guanosine triphosphate (Gi-GTP) complex. Upon GTP binding, the heterotrimeric G protein dissociates into two moieties, Gi
-GTP and Giβ
. Gi inhibits adenylyl cyclase (AC) [2,3] and decreases cyclic AMP (cAMP) concentration and thereby inhibits cAMP-dependent protein kinase (PKA). The final result is the inhibition of the phosphorylation of effectors such as the L-type Ca2+ channel (ICa-L). On the other hand, stimulatory receptors, such as β-adrenergic agonists, catalyze stimulatory G protein (Gs) and stimulate AC. The reaction finally increases cAMP and enhances the Ca2+ current.
However, this archetypal view has been challenged by recent studies, which suggest that muscarinic inhibition of Ca2+ channels requires the presence of the other type of G (Go) protein [4], since there is a specific defect in muscarinic regulation of Ca2+ current in Go knockout (Go–/–) mouse ventricular myocytes. It has also been revealed that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) play important roles in muscarinic regulation of cardiac ICa-L. But the details of muscarinic regulation in cardiac ICa-L is not completely understood. Several important questions, such as how cGMP and cAMP interacts in ICa-L regulation, remain unanswered.
The study by Klein et al. [1] is certainly a welcomed addition to the field. Using the cell-attached configuration of the patch clamp technique, single-channel Ca2+ currents have been recorded to investigate how cGMP antagonizes protein kinase A induced changes of single ICa-L channel gating. They found that isoproterenol (ISO) enhanced single channel peak average current by increasing availability and open probability and by decreasing the time constant of long close times. They also found that the effects of ISO on single ICa-L can be completely reversed by 8-bromo-cGMP (8-Br-cGMP), an activator of PKG, and concluded that PKG may interact either with PKA or directly with the Ca2+ channel protein. These results will help further understanding of muscarinic regulation of ICa-L by defining the cell signaling effectors and interaction between GC/NO-cGMP-PKG and AC-cAMP-PKG pathways.
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2 Role of cGMP and cGMP-dependent protein kinase |
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Top1 Introduction2 Role of cGMP...3 Role of nitric...4 ConclusionReferences |
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It has been found that cGMP and cGMP-dependent protein kinase (PKG) play important roles in muscarinic regulation of ICa-L in heart [5,6]. In cardiomyocytes, it is generally believed that cGMP plays a role antagonistic to that of cAMP. However, the mechanism of cGMP/PKG regulation of the ICa-L is not clear. Sumii et al. [7] found that ICa-L was inhibited by internal perfusion with PKG in young rat ventricular myocytes. The inhibited ICa-L was reversed by ISO, indicating that cAMP and cGMP have opposite interaction in heart, and that the inhibitory effect of PKG exerts a balancing effect between PKA and PKG activation in regulating ICa-L [7].
It has also been found that in guinea pig isolated ventricular myocytes, 3-isobutyl-1-methylxanthine (IBMX, a nonselective PDE inhibitor), increased ICa-L [8]. This effect is similar to the application of cAMP. 8-Br-cGMP, mimicked the effects of muscarinic cholinergic agonist carbachol (CCh), inhibiting ICa-L. Thus, the inhibition of ICa-L by CCh may be caused by cGMP/PKG activation. A recent study has indicated that cGMP causes inhibition of ICa-L by phosphorylation of the 1C subunit of Ca2+ channel at position Ser(533) via the action of PKG [6]. This study provides the evidence that cGMP directly acts on Ca2+ channel protein.
Using G proteins subunit gene knockout mice, our group has recently found the inhibitory effects of CCh may need the inhibitory G protein (Gi) [9]. Perfusion with CCh was found to significantly inhibit the Ca2+ current in WT cells and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca2+ currents in Gi2 gene knockout (Gi2–/–) ventricular myocytes. The results suggest that Gi2–/– cells lack the inhibitory regulation of Ca2+ channels by CCh. The signal pathway distal to Gi protein, e.g. through activation of cGMP/PKG, or decrease of cAMP/PKA, is unknown.
It needs to be pointed out that the results of cGMP on ICa-L from different investigators are variable. In contrast to most reports, one group [10] has found that cGMP facilitates ICa-L via cGMP/PKG in isolated rabbit ventricular myocytes. The exact reasons for this controversy in results of cGMP on ICa-L are not clear. More experiments with reproducible results using the same specie will be beneficial in elucidating the precise action of cGMP/PKG on ICa-L.
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3 Role of nitric oxide synthase |
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Top1 Introduction2 Role of cGMP...3 Role of nitric...4 ConclusionReferences |
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cGMP in the heart seems to be closely related to nitric oxide synthase (NOS) generation. It has previously been shown that NOS is crucial for regulation of cardiac myocyte ICa-L [11]. Using the wild type (WT) mice and mice with targeted disruption of the endothelial NOS (eNOSnull), it is found that muscarinic cholinergic inhibition is absent in eNOSnull cells. In contrast to cells from WT mouse ventricles, CCh produced no measurable suppression on both baseline ICa-L and ICa-L enhanced by ISO. This suggests that eNOS may be coupled with muscarinic receptor and activate cGMP-dependent control of ICa-L in cardiac myocytes.
The effects of NOS donors and NOS inhibitor have also been extensively studied in the different cell preparations. Nitric oxide donors, 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein, increased cGMP generation and suppressed ISO-augmented ICa-L [11]. NOS inhibitors, N(G)-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), have been found to increase ICa-L on guinea-pig ventricular ICa-L [12]. The stimulatory effect of L-NMMA on basal ICa-L was reversed by L-arginine, the natural substrate of NOS. Therefore, inhibitors of NOS are able to modulate the ICa-L in the absence of a receptor-mediated pathway. In human atrial myocytes, however, NO-cGMP pathway may not contribute significantly to the muscarinic regulation of ICa-L [13].
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4 Conclusion |
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Top1 Introduction2 Role of cGMP...3 Role of nitric...4 ConclusionReferences |
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In summary, although the studies have advanced the research in muscarinic regulation of ICa-L in recent decades, we still lack a complete clear view of intracellular signal pathways. As referred by Klein et al. [1], the results concerning the effect of cGMP on ICa-L in mammalian heart are controversial. This could be due to different basal levels of cAMP or PKA activity on specie and age of the animal models used. On the other hand, the technical difficulties in delineating these pathways experimentally must be emphasized. For example, extracellular or intracellular use of cGMP or NOS agonists or antagonists may not function in the same way as de facto physiological conditions, in terms of signal pathway conduction acceleration and augmentation. In addition, to support a second messenger roles of cGMP or cAMP on ICa-L, it is necessary to show that reagents directly activate the cGMP or cAMP pathway through receptor activation. Moreover, extracellular or intracellular use of NOS or cGMP agonists may affect the function of other proteins in intracellular milieu on the signal transduction cascades. Finally, gene knockout animal models, such as G protein subunit and β-adrenergic receptor gene knockout/overexpressed mice, are useful to study the effects of the specific proteins in ICa-L regulation. However, this may affect the other associated protein subunits, which are either structurally or functionally associated with the knockout or overexpressed proteins. More experiments, which mimic closely to actual endogenous signal pathways, will further advance the muscarinic regulation of ICa-L function in heart.
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References |
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Top1 Introduction2 Role of cGMP...3 Role of nitric...4 ConclusionReferences |
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- Klein G., Drexler H., Schröder F. Protein kinase G reverses all isoproterenol induced changes of cardiac single L-type calcium channel gating. Cardiovac Res (2000) 48:367–374.[CrossRef]
- Gilman A.G. G proteins and regulation of adenylyl cyclase. J Am Med Assoc (1989) 262:1819–1825.
[Abstract/Free Full Text] - Hildebrandt J.D., Sekura R.D., Codina J., et al. Stimulation and inhibition of adenylyl cyclases is mediated by distinct proteins. Nature (1983) 302:706–709.[CrossRef][Medline]
- Valenzuela D., Han X., Mende U., et al. Go is necessary for muscarinic regulation of Ca2+ channels in mouse heart. Proc Natl Acad Sci USA (1997) 94:1727–1732.
[Abstract/Free Full Text] - Vila-Petroff M.G., Younes A., Egan J., Lakatta E.G., Sollott S.J. Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ Res (1999) 84:1020–1031.
[Abstract/Free Full Text] - Jiang L.H., Gawler D.J., Hodson N., et al. Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase. J Biol Chem (2000) 275:6135–6143.
[Abstract/Free Full Text] - Sumii K., Imazu M., Yamakido M., Sperelakis N. Cyclic GMP-dependent protein kinase regulates the L-type calcium current in rat ventricular myocytes. Heart Vessels (1997) 12(Suppl):62–65.[Medline]
- Sakai R., Shen J.B., Pappano A.J. Elevated cAMP suppresses muscarinic inhibition of L-type calcium current in guinea pig ventricular myocytes. Cardiovasc Pharmacol (1999) 34:304–315.[CrossRef][Web of Science][Medline]
- Chen F., Spicher K., Birnbaumer L., Wetzel G.T. Lack of muscarinic regulation of Ca2+ channels in Gi2 gene knockout mouse heart. Circulation (1998) 98:I–233.
- Han J., Kim E., Lee S.H., et al. cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes. Pfluger's Arch (1998) 435:388–393.[CrossRef][Web of Science][Medline]
- Han X., Kubota I., Feron O., et al. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proc Natl Acad Sci USA (1998) 95:6510–6515.
[Abstract/Free Full Text] - Gallo M.P., Ghigo D., Bosia A., et al. Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors. J Physiol (Lond) (1998) 506(3):639–651.
[Abstract/Free Full Text] - Vandecasteele G., Eschenhagen T., Fischmeister R. Role of the NO-cGMP pathway in the muscarinic regulation of the L-type Ca2+ current in human atrial myocytes. J Physiol (Lond) (1998) 506(3):653–663.
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