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Cardiovascular Research 2003 60(2):223-225; doi:10.1016/j.cardiores.2003.09.009
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

NO and cholinergic signalling in the heart: divergent routes to regulatory phosphorylation of the cardiac L-type Ca2+ channel

Klaus Groschner*

Department of Pharmacology and Toxicology, SFB-Biomembranes Research Center, Karl-Franzens-University, Universitätsplatz 2, A-8010, Graz, Austria

*Tel.: +43-316-380-5570; fax: +43-316-380-9890. Email address: klaus.groschner{at}uni-graz.at

Received 27 August 2003;
   1. Introduction
Top
 1. Introduction
2. A transgenic animal...
 3. PKG-I may control...
 References
 
Voltage-gated cardiac L-type channels (CaV1.2a) are a pivotal signalling element involved in the control of myocardial function by the autonomic nervous system. Dual control of Ca2+ entry into cardiac myocytes by sympathetic and parasympathetic stimuli is likely to converge on reversible phosphorylation of CaV1.2a channels, a process that is governed by cyclic nucleotide-dependent protein kinases. The molecular basis of this regulation, and in particular the role cGMP-dependent phosphorylation in cholinergic suppression of Ca2+ entry, is still obscure. In this issue of Cardiovascular Research, Schröder et al. [1] report that transgenic over-expression of cGMP-dependent kinase I (PKG I) promotes the inhibitory modulation of cardiac Ca2+ channels by the NO/cGMP pathway but not channel inhibition via muscarinic receptor stimulation. The study confirms the role of cGMP-dependent phosphorylation as an important pathway for downregulation of CaV1.2a channels, but provides also a strong argument against involvement of this mechanism in the control of cardiac Ca2+ entry by the autonomic nervous system.

At the level of intracellular messengers, the opposing modulation of cardiac function by the two branches of the autonomic nervous system has been attributed to opposing effects of the two cyclic nucleotides cAMP and cGMP, according to the "yin-yang" hypotheses proposed in 1975 [2]. This principle was repeatedly demonstrated to govern cardiac (CaV1.2a) Ca2+ channels, and cGMP was proposed as a mediator of cholinergic down-regulation of CaV1.2 channels (for review, see Ref. [3]). Two classical lines of evidence led to the concept of cGMP as a key messenger of cholinergic signalling in the heart. First, cGMP levels were reported to increase in cardiac myocytes during muscarinic receptor stimulation [4], a phenomenon that was later found to result from NO-mediated stimulation of soluble guanylyl cyclase due to cholinergic stimulation of a myocardial NO synthase (NOS III) [5]. Second, exogenous cGMP derivatives and pharmacological tools that promote increases in cellular cGMP mimicked the inhibitory effects of muscarinic receptor stimulation while pharmacological suppression of cGMP signalling was reported to inhibit cholinergic regulation of cardiac Ca2+ entry (see Ref. [3]). Early on, however, the postulated link between cGMP and suppression of cardiac functions was already questioned by studies demonstrating a lack of correlation between cGMP levels and inhibitory effects on contractile as well as electrophysiological parameters [6,7]. Moreover, controversial data on the functional consequences of cGMP-dependent phosphorylation were reported [8,9].


   2. A transgenic animal model challenges the "yin-yang" hypothesis
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 1. Introduction
 2. A transgenic animal...
3. PKG-I may control...
 References
 
When targeted gene knock-out strategies became available, further rigorous tests of the role of the NO/cGMP system in cholinergic signal transduction were conducted, which again led to inconsistent results. Although a NOS III knock-out model initially provided results in favor of an obligatory role of the NO/cGMP system in cholinergic control of cardiac function [10], follow-up investigations on this model failed to confirm the initial conclusions [11,12]. Targeted disruption of the gene encoding type I cGMP-dependent kinase demonstrated its role as a prominent downstream effector system of cGMP in the heart but did not provide any indication for involvement of the kinase in cholinergic inhibition of cardiac functions [13]. In essence, classical pharmacological approaches and gene-knock-out strategies yielded remarkably conflicting results. Considering that pharmacological approaches bear significant uncertainties due to insufficient selectivity, and that interpretation of data obtained from knock-out animal models is complicated by the possible regaining of functions due to compensatory mechanisms, an alternative approach was required. Schröder et al. used a mouse model in which PKG I, used as a sensor for NO/cGMP signals, is over-expressed in the myocardium. Over-expressed PKG I was found to provide a signalling system, which responds to NO/cGMP signals and enables efficient inhibition of CaV1.2a channels by exogenous NO. Interestingly, PKG I over-expression not only promoted the typical "accentuated antagonism", i.e. cGMP-dependent inhibition of Ca2+ channels that are stimulated by cAMP-dependent phosphorylation, but in addition enabled inhibition of basal channel activity by exogenous NO, representing a new type of inhibitory regulation in this experimental model. However, the transgenic NO/cGMP sensor failed to respond to stimulation of muscarinic receptors. Neither was the cholinergic "accentuated antagonism" promoted nor was a basal inhibitory component of cholinergic control of Ca2+ channels uncovered. Based on the assumption that over-expression of the kinase does not generate alterations in subcellular targeting and/or regulation of the enzyme, these results suggest that PKG I does not mediate control of CaV1.2a channels by muscarinic receptor stimulation. Thus, the "yin-yang principle", in terms of reciprocal modulation of CaV1.2a channels by cAMP and cGMP, is not the basis of dual control of cardiac Ca2+ entry by the autonomic nervous system. Cholinergic generation of an endogenous NO signal in mouse myocardium is apparently insufficient to trigger activation of the cGMP/PKG I system to the extent required for CaV1.2 channel regulation, as outlined in the scheme shown in Fig. 1. It is of note that NO (both exogenous and endogenous) may exert inhibitory control of CaV1.2 channels in addition via PKG-independent mechanisms [14,15].


Figure 1
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  		Fig. 1 Cholinergic signaling in the heart is not coupled to the dual (yin-yang) control of cardiac CaV1.2 channels mediated by cyclic nucleotides. Type I cyclic GMP-dependent protein kinase (PKG) mediates inhibition of mouse cardiac Ca2+ channels (CaV1.2) by exogenous NO but not by muscarinic receptor (MR) stimulation, which is expected to be associated with calmodulin (CaM)-dependent stimulation of cardiac nitric oxide synthase (NOS III) and endogenous NO formation. Muscarinic inhibition may occur via inhibition of cAMP signaling (not shown) or alternatively via direct inhibitory effects of NO, but not via PKG I-dependent phosphorylations (AKAP: PKA anchoring protein, PKA: cAMP-dependent protein kinase, PP: protein phosphatase, sGC: soluble guanylyl cyclase, L-Arg: L-arginine).

 

   3. PKG-I may control Cav1.2 channels via multiple target sites
Top
 1. Introduction
 2. A transgenic animal...
 3. PKG-I may control...
References
 
Cyclic AMP/cGMP-dependent dual control of CaV1.2 channels has been suggested to involve multiple regulatory phosphorylations that may reside in the Ca2+ channel {alpha}1 subunit as well as in other regulatory proteins such a protein phosphatases as illustrated in Fig. 1 (see Ref. [16]). Notably, the work of Schröder et al. provides interesting evidence for the existence of multiple PKG I-dependent regulatory phosphorylations that govern the function of CaV1.2a channels. Over-expression of PKG I revealed two qualities of PKG-mediated inhibition. The "accentuated antagonism" observed in cardiomyocytes from wild-type mice was promoted, and cGMP-dependent inhibition of basal Ca2+ currents, which is absent in wild-type cells, was generated. These findings might be interpreted in terms of two distinct regulatory mechanisms based on two phosphorylation sites that are operated at different PKG I activities within the cell. Thus, to understand cyclic nucleotide-mediated dual control of cardiac functions, further studies will be needed to identify the targets of PKG involved in cGMP-induced down-regulation of CaV1.2a channels.


   References
Top
 1. Introduction
 2. A transgenic animal...
 3. PKG-I may control...
 References
 

  1. Schröder F., Klein G., Fiedler B., Bastein M., Schnasse N., Hillmer A., et al. Single L-type Ca2+ channel regulation by cGMP-dependent protein kinase type I (PKG I) in adult cardiomyocytes from PKG I transgenic mice. Cardiovasc. Res. (2003).
  2. Goldberg N.D., Haddox M.K., Nicol S.E., Glass D.B., Sanford C.H., Kuehl F.A. Jr., et al. Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: the Yin Yang hypothesis. Adv. Cycl. Nucleotide Res. (1975) 5:307–330.
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  6. Mirro M.J., Bailey J.C., Watanabe A.M. Dissociation between the electrophysiological properties and total tissue cyclic guanosine monophosphate content of guinea pig atria. Circ. Res. (1979) 45:225–233.[Abstract/Free Full Text]
  7. Groscher K., Holzmann S., Kukovetz W.R. Lack of second messenger function of cyclic GMP in acetylcholine-induced negative inotropism. J. Cardiovasc. Pharmacol. (1986) 8:1154–1157.[Web of Science][Medline]
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  10. Han X., Kubota I., Feron O., Opel D.J., Arstall M.A., Zhao Y.Y., 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. U. S. A. (1998) 95:6510–6515.[Abstract/Free Full Text]
  11. Vandecasteele G., Eschenhagen T., Scholz H., Stein B., Verde I., Fischmeister R. Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase. Nat. Med. (1999) 5:331–334.[CrossRef][Web of Science][Medline]
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  13. Wegener J.W., Nawrath H., Wolfsgruber W., Kuhbandner S., Werner C., Hofmann F., et al. cGMP-dependent protein kinase I mediates the negative inotropic effect of cGMP in the murine myocardium. Circ. Res. (2002) 90:18–20.[Abstract/Free Full Text]
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  15. Poteser M., Romanin C., Schreibmayer W., Mayer B., Groschner K. S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca2+ channels. J. Biol. Chem. (2001) 276:14797–14803.[Abstract/Free Full Text]
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