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Am J Physiol Heart Circ Physiol 285: H2454-H2462, 2003. First published July 24, 2003; doi:10.1152/ajpheart.00388.2003
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Inhibition of L-type Ca2+ current by C-type natriuretic peptide in bullfrog atrial myocytes: an NPR-C-mediated effect

R. A. Rose,1 A. E. Lomax,1 and W. R. Giles1,2

1Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1; and 2Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0412

Submitted 25 April 2003 ; accepted in final form 23 July 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Single atrial myocytes were isolated from the bullfrog heart and studied under current and voltage clamp conditions to determine the electrophysiological effects of the C-type natriuretic peptide (CNP). CNP (10–8 M) significantly shortened the action potential and reduced its peak amplitude after the application of isoproteronol (10–7 M). In voltage clamp studies, CNP inhibited isoproteronol-stimulated L-type Ca2+ current (ICa) without any significant effect on the inward rectifier K+ current. The effects of cANF (10–8 M), a selective agonist of the natriuretic peptide C receptor (NPR-C), were very similar to those of CNP. Moreover, HS-142-1, an antagonist of the guanylyl cyclase-linked NPR-A and NPR-B receptors did not alter the inhibitory effect of CNP on ICa. Inclusion of cAMP in the recording pipette to stimulate ICa at a point downstream from adenylyl cyclase increased ICa, but this effect was not inhibited by cANF. These results provide the first demonstration that CNP can inhibit ICa after binding to NPR-C, and suggest that this inhibition involves a decrease in adenylyl cyclase activity, which leads to reduced intracellular levels of cAMP.

action potential; C receptor; heart

NATRIURETIC PEPTIDES are a family of molecules that are involved in the regulation of blood pressure and natriuresis. Atrial natriuretic peptide (ANP), which is synthesized and secreted into the circulation from the atria of the heart, was the first member of the family to be discovered (16, 19). This natriuretic peptide family now includes ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). Three separate natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C) have been identified. NPR-A preferentially binds ANP and BNP, whereas NPR-B is more selective for CNP. NPR-A and NPR-B are both membrane bound and include particulate guanylyl cyclase domains. An increase in cGMP occurs when a ligand binds to these receptors. NPR-C has no guanylyl cyclase domain. This receptor subtype has approximately equal affinity for all three natriuretic peptides (6, 12).

NPR-C has traditionally been referred to as a "clearance" receptor. It is thought to function mainly as a buffer system that can modulate concentration of natriuretic peptides in the circulation without activating intracellular second messenger cascades (36, 38). However, in the rat heart and in the gastrointestinal smooth muscle, NPR-C is functionally linked to an adenylyl cyclase enzyme via an inhibitory G (Gi) protein (1, 3, 42, 51, 56). Recent studies in the rat heart have identified a small cytoplasmic domain of NPR-C, which is responsible for the interaction with Gi. Activation of Gi results in the inhibition of adenylyl cyclase (51).

The original experimental work suggested that natriuretic peptides act by binding to NPR-A and NPR-B and then altering intracellular cGMP levels. ANP and CNP can increase cGMP in atrium and ventricle of mammals, including humans (28, 33, 34, 55). CNP is known to have potent negative inotropic effects on the mammalian heart (45) and has been shown to facilitate presynaptic vagal neurotransmission (25). In vascular tissue, CNP is a potent endothelium-independent vasodilator, and it has been shown to have direct effects on canine coronary arteries (12). CNP can also inhibit norepinephrine-induced contractions of both atrial and ventricular strips of heart muscle from the bullfrog (57).

The amino acid sequence and two- and three-dimensional structure (24) of both CNP and its receptors are highly conserved among vertebrate species. For example, there is 96% homology between rat and human forms of CNP (47); and bullfrog CNP has only four amino acid differences from that of the rat (6, 62). CNP was first identified in the brain, but is now known to be present in mammalian heart (61). A variety of techniques have localized this peptide to both the atria and the ventricles (58, 59). NPR-C is also expressed in many tissues (6, 46). In fact, NPR-C makes up ~90% of the total population of NPRs in heart and vascular smooth muscle (5, 37).

The goal of the present study was to determine whether CNP can modulate electrophysiological responses in isolated bullfrog atrial myocytes. Our findings show that CNP, at physiological concentrations, can strongly inhibit L-type Ca2+ current in atrial myocytes from bullfrog hearts and provide the first evidence that this effect is mediated by the C-type receptor NPR-C.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Single cell preparation. Single myocytes were isolated from bullfrog atrium using methods described previously (31). In brief, adult bullfrogs, Rana catesbeiana, were double pithed, and the heart was excised and placed in normal Ringer solution composed of (in mM) 110 NaCl, 2.5 KCl, 5 MgCl2, 2.5 CaCl2, 10 glucose, and 5 HEPES, and buffered to a pH of 7.4 with NaOH. The atrium was removed, cut into 1-mm2 pieces, and subjected to a series of enzymatic dissociation steps. In the first step, pieces of atrial tissue were placed in 5 ml of nominally Ca2+-free Ringer solution (same composition as normal Ringer solution, except that CaCl2 was 8 x 10–6 M) containing 200 IU/ml of collagenase (type I from clostridium histolyticum, Sigma) combined with 21,000 IU/ml of trypsin (type III from bovine pancreas, Sigma) and gently stirred for 45 min at room temperature. Next, this atrial tissue was transferred into 5 ml of nominally Ca2+-free Ringer solution containing 1 mg/ml bovine serum albumin (Sigma) for 5 min. Finally, the tissue was incubated in a solution of 350 IU/ml of collagenase for an additional 40–60 min. On completion of the final enzyme treatment, the tissue was gently triturated with a large bore (3 mm diameter) pipette to mechanically dissociate it, yielding a population of single atrial myocytes, which were stored in nominally Ca2+-free Ringer solution at 4°C. During the experiment, aliquots of the solution containing the single cells were transferred to the recording chamber, which was superfused with normal Ringer solution at room temperature (22°–23°C). The University of Calgary Animal Resource Center approved all experimental protocols and animal procedures used in this study.

Solutions and drugs. The filling solution for the recording microelectrodes contained the following (in mM): 90 K+ aspartate, 15 KCl, 5 NaCl, 1 MgCl2, 1 EGTA, 3 ATP (dipotassium salt), and 10 HEPES; pH was adjusted to 7.2 with KOH. In some experiments, cAMP was added to the recording pipette at a concentration of 10–5 M. The superfusate consisted of normal frog Ringer solution.

The drugs and chemicals used in this study included tetrodotoxin (TTX), isoproteronol (Iso), and cAMP. All were purchased from Sigma (St. Louis, MO). CNP and NPR-C agonist (cANF) were purchased from Peninsula Laboratories (San Carlos, CA). HS-142-1 was obtained from Kyowa Hakko Kogyo.

Electrophysiological methods. The whole cell configuration of the patch clamp technique (22, 30) was employed to study single cells under both current clamp and voltage clamp conditions. Micropipettes were pulled using a Flaming/Brown pipette puller (model p-87, Sutter Instrument; Novato, CA) from borosilicate glass tubing (1.5 mm outer diameter, World Precision Instruments; Sarasota, FL). The resistance of these pipettes was between 1 and 5 M{Omega} when filled with recording solution.

Microelectrodes were positioned with a hydraulic micro-manipulator (SD Instruments), mounted on the stage of an inverted microscope (Nikon Diaphot). Acceptable seal resistances varied between 2 and 10 G{Omega}. When the membrane under the pipette was ruptured, the series resistance was typically 5–10 M{Omega}. This was compensated (80–85%) using an Axopatch 200 amplifier (Axon Instruments; Foster City, CA). Cell capacitances were 70–100 pF.

Current clamp and voltage clamp signals were digitized using a Digidata 1322A interfaced with pCLAMP 8 software (Axon Instruments). Data were stored on a computer for analysis offline.

Action potentials were recorded from current-clamped atrial myocytes by applying 10-ms depolarizing pulses of ~0.5 nA. These stimuli were applied at a frequency of 0.16 Hz. The peak amplitude as well as the 50% repolarization time (APD50) of these action potentials were measured.

Peak inward Ca2+ current (ICa) in frog atrial myocytes was identified and recorded after blocking the sodium current with TTX (5 x 10–8 M) (10, 13). ICa was measured as the difference between the peak inward current and the point at which the current reached steady state (the end of a 200-ms voltage step from a holding level of –80 to +20 mV). It is important to note that the activation kinetics of the delayed rectifier K+ current (IK) are very slow in bullfrog atrial myocytes, so that its contribution to net current changes is negligible during the first 100–200 ms (10). IK is the only repolarizing K+ conductance activated at depolarized potentials in bullfrog atrium, which means it is not necessary to replace K+ when measuring ICa in this preparation. Thus our measurements of ICa during a 200 ms voltage step in the presence of TTX represent "pure" ICa (10). Current voltage (I-V) relationships for peak ICa were generated by applying a series of 10-mV steps between –120 and +80 mV from a holding potential of –80 mV. Isochronal I-V curves for the inward rectifier K+ current (IK1) were plotted from measurements taken at the end of a 200-ms voltage step in the range of –100 to –60 mV (the threshold of activation of ICa).

Statistical analysis. Summary data are presented as means ± SE. The data were analyzed with the use of either ANOVA with Dunnett's multiple-comparison procedure (in most cases) or a paired Student's t-test (Fig. 1) test to identify significant differences. In all instances, a value of P < 0.05 was considered significant.



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  		Fig. 1. Effect of C-type natriuretic peptide (CNP; 10–8 M) on bullfrog atrial myocyte action potentials recorded in the presence of isoproteronol (Iso; 10–7 M). A: representative data showing control action potentials and the effects of Iso (10–7 M) and CNP (10–8 M). Action potentials (elicited at 0.16 Hz, room temperature, 22°C) were recorded as the compounds were added. Iso was added first, and after this CNP was added in combination with Iso. B: average effects of Iso and CNP on 50% repolarization time (APD50) (solid bars) and peak amplitude (open bars). *P < 0.05, values in Iso are significantly greater than control; **P < 0.05, values in Iso + CNP are significantly less than those in Iso alone (n = 7 cells).

 


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of CNP on atrial action potentials. In the initial experiments, CNP was added to the superfusate at physiolological concentrations (21, 59) so that its effects on the action potential of single myocytes could be evaluated. On the basis of the results from the rat heart, which showed that ANP can inhibit adenylyl cyclase (51), isoproteronol was added at the start of each experiment to stimulate adenylyl cyclase and thus maximize the probability of obtaining consistent CNP effects.

Figure 1A shows representative effects of Iso (10–7 M), followed by CNP (10–8 M) on the action potential (stimulated at 0.16 Hz) of an atrial myocyte from bull-frog heart. As expected, Iso lengthened the action potential and increased its amplitude. Both of these effects were markedly reduced by the addition of CNP (10–8 M). These changes, expressed in terms of APD50 and peak amplitude, are shown in Fig. 1B. Iso increased APD50 and peak amplitude by ~100% and 18%, respectively. The subsequent addition of CNP significantly decreased APD50 (~45%) and caused a small reduction in peak amplitude (~5%). Both of these CNP effects were statistically significant.

Effects of CNP on ICa. To explore the ionic mechanism(s) of these CNP effects on the action potential, voltage clamp measurements of ICa were made as described previously (50). The effects of CNP were first measured on basal (unstimulated) ICa. Under these conditions, CNP (10–8 M) caused a small reduction in peak ICa from ~140 to 80 pA (refer to summary IV curve in Fig. 2). The effects of CNP on ICa were also measured after ICa was stimulated with Iso (10–7 M). Under this condition, ICa increased about five- to six-fold (compare control curve in Fig. 2 with Iso-stimulated curve in Figs. 3, 4, 5). Application of CNP (10–8 M) in the presence of Iso produced a significant fivefold reduction in ICa (Fig. 3). All subsequent measurements on ICa were made in the presence of Iso.



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  		Fig. 2. Effect of CNP (10–8 M) on bullfrog atrial myocyte Ca2+ current (ICa) in basal conditions. A: two representative recordings of ICa elicited by a voltage clamp step to +20 mV in control conditions (1) and after the addition of CNP (2). CNP reduced the peak of the current from ~275 to 175 pA. B: summary current-voltage relationship (I-V) curve illustrates the effects of CNP on basal ICa. Note that there was no significant effect on the background inward rectifier K+ current (IK1) measured between –50 and –90 mV (mean ± SE, n = 11 cells). *The membrane voltages at which CNP significantly inhibited ICa.

 


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  		Fig. 3. Effect of CNP (10–8 M) on bullfrog atrial myocyte ICa in the presence of Iso (10–7 M). A: two representative recordings of ICa elicited by a voltage clamp step to +20 mV in Iso (1) and after addition of CNP (2). CNP reduced the peak of the Iso-stimulated current approximately fivefold. The summary I-V curve illustrates the effects of CNP ({blacksquare}) on Iso-stimulated ICa ({circ}). Note that there was no significant effect on the background inward rectifier K+ current (IK1) measured between –50 and –100 mV (means ± SE, n = 7 cells). *Membrane voltages at which CNP significantly inhibited ICa.

 


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  		Fig. 4. Effect of the C receptor-specific agonist (cANF; 10–8 M), on bullfrog atrial myocyte ICa recorded in the presence of Iso (10–7 M). A: superimposed recordings of ICa in Iso elicited by a voltage clamp step to +20 mV (1), followed by the same depolarization in Iso plus cANF (2). Note that the peak of the Iso-stimulated current was decreased approximately threefold. B: summary I-V curve illustrates the effects of cANF on ICa in the presence of Iso. No significant effect on IK1 was observed (means ± SE, n = 12 cells). *Membrane voltages at which cANF significantly reduced ICa.

 


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  		Fig. 5. Effects of CNP (10–8 M) and HS-142-1 (10 µg/ml) on ICa measured in bullfrog atrial myocytes in the presence of Iso (10–7 M). HS-142-1 is a selective inhibitor of the A- and B-type natriuretic peptide receptors (NPR). A: ICa recordings at +20 mV, in the presence of Iso (1), after application of HS-142-1 to block NPR-A and NPR-B (2), and then after addition of CNP (3). Note that although NPR-A and NPR-B (the guanylyl cyclase-linked receptors) were blocked, CNP still reduced peak ICa. B: summary I-V curves illustrate ICa in Iso, HS-142-1 plus Iso, and after the addition of CNP. HS-142-1 had no significant effect on ICa (means ± SE, n = 6 cells). *Membrane potentials at which CNP significantly decreased ICa compared with the HS-142-1 data.

 

In the next series of experiments, an attempt was made to identify the NPR(s) that mediate this electrophysiological effect. cANF was used to test the hypothesis that CNP causes electrophysiological changes by binding to the C receptor. After application of Iso (10–7 M), cANF (10–8 M) was applied and ICa was significantly reduced by about threefold (Fig. 4). Because cANF binds only to NPR-C [and therefore has no capacity to increase cGMP levels (1)], these electrophysiological findings (Figs. 3 and 4) suggest an NPR-C-mediated effect.

As an indication of the selectivity of these effects on ICa, IK1 was also measured in the voltage range of –100 to –50 mV (the threshold of activation of ICa). Neither CNP nor cANF caused any significant changes in this K+ conductance (Figs. 2, 3, 4), confirming that the effects are mainly on Ca2+ influx. This finding is also consistent with the current-clamp experiments in which neither compound affected the resting potential (Fig. 1). It was very difficult to reverse the effects of either CNP or cANF. Even after 20–30 min, during which the myocytes were superfused with normal Ringer solution, ICa remained stable but significantly reduced in peak amplitude.

Effects of blocking NPR-A and NPR-B with HS-142-1. Additional independent evidence for an NPR-C-mediated effect on ICa was obtained by applying the competitive NPR-A and NPR-B receptor antagonist HS-142-1 (10 µg/ml). This compound binds to both of these guanylyl cyclase-linked receptors, thereby blocking the ability of CNP to increase cGMP levels (39). Previous studies (39, 40) have shown that HS-142-1, used at this same concentration, abolished the ability of nanomolar concentrations of CNP to increase cGMP levels.

The raw data and summary I-V curves in Fig. 5 show that HS-142-1 has no significant effect on ICa in the presence of Iso. After 5–10 min of superfusion of HS-142-1, CNP was added, ICa was inhibited by ~50%, and IK1 was unaffected. These findings provide further evidence that CNP inhibits ICa by binding to NPR-C.

CNP effects in presence of elevated intracellular cAMP. Results from biochemical and molecular studies on rat heart strongly suggest that the C receptor is functionally linked to the inhibition of adenylyl cyclase, and that this inhibitory interaction follows the activation of Gi (51). To evaluate this possibility in bullfrog atrium, cAMP (10–5 M) was added to the pipette to increase ICa. Because cAMP production is downstream from the activation of both the G protein and adenylyl cyclase, this maneuver could provide insight into the part of the biochemical pathway that is modulated by CNP. In these experiments, after the cAMP-induced increase in ICa had reached steady state, cANF was added to the superfusate to selectively activate NPR-C. The data in Fig. 6A show that intracellular elevation of cAMP (10–5 M) increased ICa significantly, but that subsequent application of cANF (10–8 M) failed to reduce this increase.



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  		Fig. 6. Measurement of the effects of cANF (10–8 M) on ICa in the presence of elevated intracellular cAMP (10–5 M). cAMP was included in the recording pipette, and entered the cell by diffusion under conventional whole cell recording conditions. A: time course of the effects of cAMP and cANF on ICa. As indicated by the horizontal bars (top), cAMP was present throughout the experiment, whereas cANF was added 15 min after exposure to cAMP. cANF had no effect on peak ICa in any of these experiments. A: sample recordings of ICa at +20 mV immediately after the rupture of the cell membrane (1), after cAMP had increased ICa at to a stable level (2) and after the addition of cANF (3). Initially, peak ICa measured 342 pA; as cAMP diffused into the cell, peak current increased to 1,809 pA. Approximately 10 min after application of an effective dose of cANF, ICa remained elevated at 1,714 pA. B: summary data illustrating the effects of cAMP, followed by cANF on peak ICa. Control peak ICa (represented by the ICa level immediately on rupture of the cell membrane) is defined as 100%. Note that peak ICa was significantly increased to 413 ± 109% of control in cAMP and remained unchanged at 414 ± 119% of control in cAMP + cANF (means ± SE, n = 10 cells).

 

Figure 6A explores this in detail by illustrating the time course of these effects of cAMP on ICa. Soon after the cell membrane was ruptured (and the cAMP began to diffuse into the myocyte), ICa increased progressively, reaching a steady-state level in ~10 min. Subsequent addition of cANF had no significant inhibitory effect. In our previous experiments (Fig. 4), the inhibitory effects of cANF became apparent within 2 min. In contrast, when intracellular cAMP was increased no reduction of ICa was observed, even after 10 min of superfusion with cANF. On average, cAMP increased ICa approximately fourfold from the baseline level, and the subsequent addition of cANF had no significant inhibitory effects on the cAMP-induced increase in ICa, which remained at 414 ± 119% of control (Fig. 6B).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Natriuretic peptide effects on the heart. Both CNP and the NPR-C receptor have been shown by immunohistochemistry to be present in the heart of mammals, including humans (46, 58), and CNP has a prominent negative inotropic effect on the rat heart (45). CNP can influence heart rate by acting on either presynaptic or postsynaptic sites (25). However, few studies (21, 33, 55) have examined the electrophysiological effects of natriuretic peptides in cardiac tissue, and most of the published data have been obtained using only ANP. A consistent finding is that ANP causes a decrease in ICa. However, all of these results have been previously interpreted in terms of the peptide activating a guanylyl cyclase-linked receptor, with a resulting increase in cGMP.

Our results provide the first electrophysiological evidence that CNP, at physiological concentrations, can inhibit ICa. In addition, we have shown that this effect is mediated via the C receptor (NPR-C) in the heart. Our findings also conclusively demonstrate that NPR-C mediates important physiological functions via the cAMP second messenger system in the heart. Thus, both cANF (a NPR-C-specific agonist), and CNP applied in the presence of HS-142-1 (an NPR-A and NPR-B antagonist) were able to significantly inhibit ICa (Figs. 4 and 5). In combination, these two data sets demonstrate that the inhibition of ICa is mediated by NPR-C. On the basis of these findings, it is no longer appropriate to denote NPR-C as a "clearance receptor" in the heart. Previously, physiological roles for NPR-C have been demonstrated in a variety of cultured cell lines (35, 43, 56). Our conclusion that CNP inhibits ICa by binding to NPR-C relies on the specificity of the agonists and antagonists used in this study. In previous studies (2, 42, 43), cANF has been shown to have no capacity to bind NPR-B or increase cGMP levels and therefore it is used as an NPR-C agonist. Similarly, HS-142-1, at the concentration used in this study, blocks NPR-B and prevents the accumulation of cGMP in the presence of CNP (39, 40). Data obtained with HS-142-1 provide consistent evidence that it is a specific blocker of NPR-B in many tissues, including the heart and vascular smooth muscle (11, 32, 53).

It is noteworthy that CNP caused a small, but significant, reduction in basal Ca2+ current (Fig. 2). This is consistent with the supposition that there is some turnover of the enzyme adenylyl cyclase under basal conditions (26). Application of CNP can, therefore, activate Gi and reduce basal adenylyl cyclase activity that is normally present in cardiac myocytes (20).

Gisbert and Fischmeister (21) first reported that atrial natriuretic factor (as ANP is sometimes referred to) can strongly inhibit ICa in frog ventricular myocytes; however, they did not specifically examine which NPR subtype was involved. Their papers also showed that including cAMP in the recording pipette can prevent the inhibitory effect of atrial natriuretic factor. For these reasons, they concluded that atrial natriuretic factor acted by binding to NPR-B, increasing cGMP levels, and activating a cGMP-dependent phosphodiesterase, which would be expected to decrease cAMP levels (21, 29). The reductions in ICa we have illustrated in Figs. 4 and 5 (which were obtained by blocking any possible contribution from the NPR-B/cGMP pathway) were slightly smaller than the effect illustrated in Fig. 3 (where both NPR-B and NPR-C could have been stimulated). It is therefore possible that activation of NPR-B could make a small contribution to the reduction in ICa. Nevertheless, our results show that NPR-C is the main receptor responsible for decreasing ICa. Available cell signaling and molecular data suggest that this occurs after activation of Gi and the inhibition of adenylyl cyclase as opposed to the well-known NPR-B-mediated increase in intracellular cGMP (51).

A recent study by Doyle et al. (17) on NPR expression in the rat heart has shown that NPR-B (the main guanylyl cyclase-linked CNP receptor) is primarily expressed in a nonmyocyte population of cells. In this study, anti-receptor antibody staining was detected in the smooth muscle of the vasculature and interstitial cells, but not the myocytes themselves. The same antibody detected NPR-B in immunoblots of protein extracts from nonmyocytes and recognized an equivalent protein in cardiac fibroblasts. Thus it appears that expression of NPR-B in the heart is localized mainly to fibroblasts (17).

Intracellular signaling pathway associated with NPR-C. An elegant series of papers examining the effects of natriuretic peptides on isolated myocardial preparations from rat heart (3, 4, 51) allow our findings to be put into the context of well-defined biochemical cascades in mammalian heart. These biochemical studies demonstrated that ANP significantly inhibited adenylyl cyclase in a dose-dependent fashion in cultured atrial and ventricular myoctyes from neonatal rats (1). The inhibitory effect of ANP on adenylyl cyclase was attenuated by pertussis toxin (4), suggesting that a Gi protein was responsible for the ANP-mediated inhibition of adenylyl cyclase.

More recent molecular investigations (3, 51) have demonstrated that adenylyl cyclase and NPR-C can interact in mammalian heart. Anand-Srivastava and co-workers (3) raised polyclonal rabbit antisera against the 37 amino acid sequence corresponding to the cytoplasmic domain of bovine NPR-C and showed that application of this antibody markedly suppressed the ANP-mediated inhibition of adenylyl cyclase. This result implicates the cytoplasmic domain of NPR-C in the inhibition of adenylyl cyclase. As a more direct test, a synthetic peptide corresponding to the 37-amino acid sequence of NPR-C was used. This peptide alone significantly reduced adenylyl cyclase activity, an effect that was completely abolished by pertussis toxin. Pagano and Anand-Srivastava (51) subsequently demonstrated that the cytoplasmic domain of NPR-C contains specific Gi activator sequences that can directly inhibit adenylyl cyclase, as first described by Okamoto and Nishimoto (48). Most recently, the precise 17-amino acid sequence of the intracellular domain of NPR-C that is responsible for the direct activation of Gi has been identified with the use of site-directed mutagenesis techniques (63). Thus the cytoplasmic domain of NPR-C can directly activate the Gi protein and inhibit adenylyl cyclase.

In our experiments, when Iso (which increases cAMP via a stimulatory G protein) was used to increase ICa, both CNP and cANF inhibited the Ca2+ current by at least 50%. However, including cAMP in the pipette (which bypasses adenylyl cyclase and provides the cell with a continuous supply of cAMP) resulted in an increase in ICa that was sustained during subsequent application of cANF. These results suggest that CNP acts via the Gi protein and adenylyl cyclase, rather than through cGMP-dependent phosphodiesterase.

Our observations and previous studies provide a basis for identifying some of the intracellular effector proteins involved in this pathway (refer to Fig. 7). The production of cAMP is dependent on adenylyl cyclase activity. The adenylyl cyclase V and VI isoforms are expressed at high levels in the heart (23, 54). Inhibition of adenylyl cyclase occurs via the Gi proteins (Gi{alpha}1, Gi{alpha}2, and Gi{alpha}3), and Gi{alpha}2 and Gi{alpha}3 are expressed in the heart (3). Thus our working hypothesis is that in cardiac myocytes CNP binds to NPR-C and then activates either Gi{alpha}2 or Gi{alpha}3, thus inhibiting adenylyl cyclase V or VI. Inhibition of adenylyl cyclase results in a decrease in the level of phosphorylation of the ICa by protein kinase A, which decreases Ca2+ influx (27).



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  		Fig. 7. Schematic illustration of a working hypothesis for the effects of CNP on cardiac myocytes. CNP binds to NPR-C, which contains inhibitory G protein (Gi) activator sequences in its cytoplasmic domain. Activation of Gi{alpha} subunits (Gi{alpha}2 and Gi{alpha}3 are present in the heart) inhibits adenylyl cyclase (AC; type V and VI are expressed in the heart and are subject to regulation by Gi{alpha} subunits) and thus reduces cAMP. The decrease in cAMP reduces the phosphorylation of L-type calcium channels mediated by protein kinase A (PKA) and thus inhibits ICa.

 

Significance of our electrophysiological findings. It is intriguing that the effects of CNP observed in this study are similar to the effects of acetylcholine (18, 20, 44). Acetylcholine causes well-characterized negative inotropic effects that are mediated via a sarcolemmal muscarinic (M2) receptor, which is distinct from that used by CNP. In a variety of cardiac cells, including murine atrial cells, it has been clearly demonstrated that acetylcholine decreases cAMP levels that have been previously increased with catecholamines (8, 9). Many previous studies have established that acetylcholine, when bound to the M2 muscarinic receptor, stimulates Gi protein and inhibits adenylyl cyclase, which reduces cAMP levels (27). The similarity of CNP and acetylcholine effects illustrates a level of redundancy in signaling pathways within the heart. Multiple hormones and neurotransmitters activate very similar biochemical cascades and mediate the same electrophysiological effects. Furthermore, recent findings (7) clearly demonstrate that cAMP signaling occurs in distinct intracellular compartments. This suggests that different G protein-coupled receptor-signaling pathways may utilize different pools or sources of intracellular second messengers such as cAMP (7). The NPR-C effects described in this study may involve this phenomenon of intracellular compartmentalization.

Acetylcholine is also known to bind to the M2 muscarinic receptor and activate an inwardly rectifying K+ conductance via the release of the {beta}{gamma}-subunits of the heterotrimeric G protein (60). CNP is unlikely to mimic this acetylcholine effect because NPR-C is not a traditional heterotrimeric G protein and is not known to elicit any G protein-mediated {beta}{gamma}-subunit effects. Rather, NPR-C contains specific Gi activator domains, which directly modulate Gi and the enzyme adenylyl cyclase (51, 63).

In humans, pathological conditions such as congestive heart failure can result in significant increases in the release of all natriuretic peptides (59). Elevated BNP levels, which are documented after acute myocardial infarction (41), are considered to be a strong risk factor for mortality (14, 49). Accordingly, plasma BNP levels are now used as a diagnostic tool for patients with myocardial infarctions and congestive heart failure (15, 52). NPR-C binds to all of the natriuretic peptides with similar affinity, therefore it is anticipated that ANP and BNP would also decrease ICa in the same way we have demonstrated for CNP.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by Canadian Institute of Health Research Grant 69-6100 (to W. R. Giles) and the Heart and Stroke Foundation of Alberta Research Chair held by W. R. Giles.

A. E. Lomax is the recipient of postdoctoral fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research. R. A. Rose is the recipient of Research Studentship Awards from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research.


   ACKNOWLEDGMENTS
 
The HS-142-1 compound was generously provided by Kyowa Hakko Kogyo (Shizuoka, Japan).


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. R. Giles, Dept. of Physiology and Biophysics, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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