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Cardiovascular Research 2001 51(2):331-343; doi:10.1016/S0008-6363(01)00304-2
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Mechanism for muscarinic inhibition of ICa(L) is determined by the path for elevating cyclic AMP in cardiac myocytes

Yasushi Imai1, Bin Jiang2 and Achilles J Pappano*

Department of Pharmacology, MC-6125, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA

* Corresponding author. Tel.: +1-860-679-2410; fax: +1-860-679-3693 pappano{at}nso1.uchc.edu

Received 15 September 2000; accepted 2 April 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Does carbachol (CCh) require NO/cGMP for inhibition of L-type calcium current (ICa(L)) when either adenylyl cyclase activation or phosphodiesterase suppression is used to raise cAMP? Methods: The effects of the NO donor SIN-1 (3-morpholino-sydnonimine), CCh and atrial natriuretic peptide (ANP) were evaluated when ICa(L) had been stimulated by isoproterenol (ISO) or 3-isobutyl-1-methylxanthine (IBMX) in guinea pig isolated ventricular myocytes (35°C). Results: Carbachol, SIN-1 or ANP did not affect basal ICa(L); each inhibited IBMX-stimulated ICa(L). Dialyzed (30–100 µM) ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one), a soluble guanylyl cyclase (sGC) inactivator, blocked inhibition of IBMX-stimulated ICa(L) by SIN-1 (10 µM) but not by CCh (1–100 µM) or ANP (100 nM). Dialysis with 3 µM LY83583 (6-anilino-5,8-quinolinedione), a particulate (pGC) and sGC inactivator, opposed muscarinic-, ANP- and SIN-1-induced inhibition of IBMX-stimulated ICa(L). Thus CCh can increase cGMP synthesis via pGC. Even with 100 µM [LY83583]pip, CCh inhibited ISO-stimulated ICa(L), an effect referable to suppression of adenylyl cyclase activity. However, 3 µM [LY83583]pip prevented inhibition of ISO-stimulated ICa(L) by ANP. [LY83583]pip did not affect inhibition by 8 bromo-cGMP (100 µM) of ISO- or IBMX-stimulated ICa(L). The observations indicate that: (1) myocytes have ODQ-sensitive sGC activated by NO and LY8353-sensitive pGC activated by ANP, (2) CCh does not inhibit ICa(L) via NO, (3) the mechanism for muscarinic inhibition depends upon the cAMP-elevating agent and (4) LY83583 distinguishes between two pathways for muscarinic inhibition. Conclusion: The nature of the stimulant pathway that increases cAMP determines intracellular transduction of muscarinic inhibition. This hypothesis accords with distinct cyclic nucleotide compartments for the differential expression of muscarinic inhibition of ICa(L).

KEYWORDS Ca-channel; Muscarinic (ant)agonist; Second messengers; Nitric oxide; Signal transduction


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The parasympathetic neurotransmitter acetylcholine (ACh) and muscarinic agonists like carbachol (CCh) inhibit excitation–contraction coupling in vertebrate ventricles (reviewed in Refs. [1–3]). Muscarinic agonists suppress adenylyl cyclase (AC) activity and, by reducing activation of the cAMP/protein kinase A (PKA) cascade, inhibit the L-type Ca2+ current (ICa(L)) and contraction. They also increase the content of cGMP by stimulating guanylyl cyclase (GC) activity in cardiac myocyte-enriched fractions from rat [4] and guinea pig [5].

The role of cGMP in muscarinic inhibition has undergone considerable scrutiny (reviewed in Ref. [6]). While muscarinic agonists increase cardiac cGMP synthesis, the proposed mechanisms differ. Some reported that muscarinic agonist stimulates endothelial NO synthase (eNOS) to produce NO that activates GC to increase cGMP synthesis (reviewed in Ref. [7]). While eNOS is present in mammalian heart tissues [8–10], conclusions differ on the role of NO/cGMP in muscarinic inhibition of the normal myocardium (reviewed in Refs. [3,6,7]). In a transgenic mouse lacking eNOS (eNOS–/–), CCh did not inhibit ICa(L) and contractions stimulated by isoproterenol (ISO) nor did it mimic the NO donor, SIN-1 to increase cGMP production [11]. The increased cGMP synthesis by CCh in eNOS WT mice was prevented by the NOS inhibitor, NG-nitro-L-arginine (L-NNA) and by the inhibitor of soluble GC (sGC), ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one). In contrast, in another study, CCh continued to inhibit ICa(L) and contractions in ventricular myocytes from eNOS–/– mice [12]. Treatment with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) had no effect on muscarinic inhibition of ICa(L) in either WT or eNOS-deficient mice. The eNOS–/– mice used by the two groups have impaired muscarinic regulation of vascular smooth muscle tone. Other experiments gave additional evidence against the NO/cGMP hypothesis. Neither eNOS inhibition by L-NMMA nor sGC inhibition by ODQ changed inhibition of ICa(L) by ACh or of contractions by CCh in feline and human atrial myocytes and in ventricular muscle from normal and failing hearts [10,12,13]. In another study, CCh per se, inhibited ICa(L) in early stage murine embryonic stem cells; NOS inhibitors or the sGC inhibitor ODQ suppressed this effect [14]. In late stage embryonic stem cells, CCh inhibited ICa(L) only when stimulated by ISO; NOS inhibitors did not affect CCh action. Thus, the inhibitory signaling path switched from NOS-dependent in early stage to NOS-independent in late stage embryonic stem cells.

That CCh increases cGMP by an NO-independent path [4] may reflect a species-related phenomenon. NO donors or CCh increased cGMP levels more in rat aorta (fourfold) than in ventricular myocytes (twofold) [15]. The sGC inhibitor ODQ was less effective in preventing increases in cGMP by NO donors or CCh in ventricular myocytes. However, ODQ inhibited cGMP increases by NO donors in cytosolic extracts of rat ventricular myocytes. The high myoglobin content (546±68 mg/100 g) of rat heart cells [16] reportedly binds NO and ODQ [15]. ODQ might inhibit sGC in ventricular myocytes from guinea pig whose heart has a lower myoglobin content (71.6±17.1 mg/100 g).

Dialyzed cGMP or superfused 8-bromo-cGMP inhibits ICa(L) and contraction in amphibian, avian and mammalian ventricles (reviewed in Refs. [3,6,17]). Thus, cGMP mimics muscarinic inhibition in ventricular myocytes stimulated with ISO or the phosphodiesterase (PDE) inhibitor, IBMX. Muscarinic inhibition of the heart may be NO-independent [3]. This does not exclude the possibility that cGMP participates in muscarinic inhibition but rather suggests an alternative path for cGMP synthesis. We tested this possibility with ODQ which inhibits sGC and with LY83583 which inhibits particulate (pGC) and sGC [18]. We asked if atrial natriuretic peptide (ANP), an activator of pGC, mimicked the muscarinic agonist, CCh. Our results indicate that a GC/cGMP limb only partially explains muscarinic inhibition of ICa(L) in guinea pig ventricular myocytes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolation of ventricular myocytes
Single ventricular myocytes were enzymatically isolated from the hearts of guinea pigs (250–450 g) anesthetized with sodium pentobarbital (30 mg/kg, intraperitoneally) and anticoagulated with heparin (1000 IU, i.p.). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Tyrode's solution perfused the heart for 5 min (8–10 ml/min) through an aortic cannula in a Langendorff apparatus. The Tyrode's solution contained (in mM): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1.0, NaH2PO4 0.33, Hepes 10, glucose 20 (pH 7.4 with NaOH). After collagenase and protease disrupted the extracellular matrix, the enzymes were washed out by perfusion with 50 ml of Recovery solution which contained (in mM): K aspartate 130, K2ATP 5, Hepes 5, glucose 20; pH was adjusted to 7.4 with KOH. The ventricles were removed and the cells dispersed in Recovery solution and kept at 4°C for at least an hour. An aliquot of cell suspension was placed in a recording chamber (500 µl volume) on the stage of an inverted microscope. After 10 min, it was superfused with Tyrode's solution (2 ml/min); the glucose concentration was 10 mM. The experimental temperature was 35°C.

2.2 Electrophysiology
An EPC 7 patch clamp amplifier (List Electronics, Germany) applied voltage pulses in patch rupture, whole cell mode. Voltage commands and current data acquisition were controlled by a computer equipped with pClamp software (version 5.5, Axon Instruments, Burlingame, CA) and a Labmaster TL-1 interface (Axon Instruments). The pipette filling solution for glass capillary electrodes (i.d., 1.1 mm; o.d., 1.3 mm) contained (mM): K aspartate 120, KCl 30, Na2ATP 5, MgCl2 1.0, Hepes 5; pH 7.3 (with KOH). The electrode resistance was 1–3 M{Omega}. Series resistance could be compensated up to 70% to values between 1 and 2 M{Omega}.

Membrane voltage was stepped from –80 to –40 mV for 300 ms to inactivate the fast Na+ and T-type Ca2+ currents. A second voltage step to +10 mV for 300 ms elicited ICa(L). The clamp protocol was repeated every 10 s.

2.3 Drugs and application
Drugs were applied to myocytes by superfusion by gravity from a reservoir. The applied solutions were warmed as was the bath (35°C). Atrial natriuretic peptide from rat (rANF, fragment 3-28) and other agonists (CCh, ISO, IBMX, SIN-1) were prepared daily from aqueous stock solutions. When necessary, ODQ and LY83583 were dissolved in the pipette filling solution and present throughout the recording period. Stock solutions of ODQ were prepared in dimethyl sulfoxide; ethanol was the solvent for LY83583. Neither dimethyl sulfoxide nor ethanol, at the highest concentration used of 0.1 vol%, affected ICa(L) when added alone.

2.4 Data analysis
With K+-rich pipette solution, ICa(L) was standardized and reported as peak current at +10 mV sensitive to block by 0.1 mM Cd2+. Measurements are reported as mean±S.E.M. The statistical significance of mean differences was determined by Student's t-test. The difference between concentration–effect relations for CCh in the absence and presence of either ODQ or LY83583 was obtained by ANOVA. P≤0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Experiments with ODQ
In the presence of 100 µM IBMX, ICa(L) increased 2.1-fold (n=6) from an initial value of 0.8±0.17 to 1.7±0.38 nA. (These experiments were done at 30°C.) At peak IBMX effect (~5 min), addition of 10 µM SIN-1 reduced IBMX-stimulated ICa(L) by 51±8.4% (P<0.05). An example is shown in Fig. 1A; SIN-1 inhibited ICa(L) reversibly by 37%. With 30 µM ODQ in pipette solution ([ODQ]pip), basal ICa(L) averaged 0.8±0.11 nA and increased to the same extent (2.4-fold) with IBMX to 1.8±0.20 nA (n=6). However, SIN-1 suppressed the IBMX effect by only 6±2.6%. An example is shown in Fig. 1B where 10 µM SIN-1 reduced IBMX-stimulated ICa(L) by <2% in the presence of 30 µM [ODQ]pip.


Figure 1
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  		Fig. 1 SIN-1 (10 µM) inhibits ICa(L) through an ODQ-sensitive mechanism. Ordinate: ICa(L) in nA; abscissa: time in min. (A) ICa(L) increases with 100 µM IBMX applied in bath solution; addition of 10 µM SIN-1 reversibly inhibits ICa(L). At the end of the experiment, 100 µM Cd2+ is added to block ICa(L). Periods of drug additions are indicated by thin horizontal lines. Current traces on right-hand show Cd2+-sensitive ICa(L) at times indicated by letters (a–e). Thin horizontal line next to trace is zero current level; current and time calibrations shown below. (B) Experiment with [ODQ]pip of 30 µM present from the time of patch rupture; the format is the same as for panel A. While IBMX stimulates ICa(L), addition of 10 µM SIN-1 does not inhibit IBMX effect.

 
Whereas 30 µM [ODQ]pip suppressed inhibition by SIN-1, 100 µM ODQ did not affect CCh-induced reduction of ICa(L). An experiment illustrating the continued inhibition of IBMX-stimulated ICa(L) by CCh is given in Fig. 2A. With 100 µM [ODQ]pip, CCh (1 µM) reversibly suppressed the stimulant effect of IBMX by ~48%. Inhibition by 1 µM CCh in the absence and presence of 30 or 100 µM [ODQ]pip is summarized in Fig. 2B. At 100 µM, ODQ did not significantly interfere with muscarinic inhibition of ICa(L). Thus, ODQ, an inhibitor of sGC, opposed inhibition by the NO donor, SIN-1, but not by CCh.


Figure 2
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  		Fig. 2 Carbachol (1 µM) inhibits IBMX-stimulated ICa(L) in the presence of ODQ. (A) Time course showing IBMX (100 µM) augments ICa(L) and 1 µM CCh inhibits this effect reversibly in a myocyte dialyzed with [ODQ]pip=100 µM. Current traces on right are from times indicated by a–e; ICa(L) is taken as Cd2+-sensitive component. Zero current indicated by thin horizontal line to the left of current traces. (B) Summary of experiments. Ordinate: percent inhibition of IBMX-stimulated ICa(L) by 1 µM CCh; abscissa: results with control pipette solution (CTR) and with [ODQ]pip of 30 and 100 µM. Symbols are mean±S.E.M.; number of cells in parentheses. Addition of 1 µM CCh reduced IBMX-stimulated ICa(L) by 40±8.1% (CTR), by 44±4.3% with 30 µM (P=0.79 compared to CTR) and by 37±6.5% with 100 µM [ODQ]pip (P=0.81 relative to CTR).

 
We tested [ODQ]pip at 30 and 100 µM against the inhibition of ICa(L) by CCh in myocytes exposed to ISO (10 nM). The initial ICa(L) averaged 1.6±0.10 (n=46), 1.7±0.11 (n=20) and 1.9±0.19 nA (n=21) in untreated cells and those subjected to 30 and 100 µM [ODQ]pip, respectively. Addition of 10 nM ISO increased ICa(L) by 246±23, 238±18 and 246±16% under the same conditions, respectively. When CCh was applied at 1, 10 and 100 µM, its inhibition of ISO-stimulated ICa(L) did not change significantly at either 30 or 100 µM [ODQ]pip (Fig. 3). For example, 100 µM CCh suppressed ICa(L) by 77±7.4% in control (n=12) and by 70±4.2% (n=4) with 100 µM [ODQ]pip (P=0.62).


Figure 3
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  		Fig. 3 Summary of experiments testing ODQ on muscarinic inhibition of ISO-stimulated ICa(L). Ordinate: % inhibition of ICa(L); abscissa: CCh concentration. The concentration-dependent effect of CCh in control (CTR) pipette solution (O) averaged 46±8.4, 57±7.0 and 77±7.4% in 1, 10 and 100 µM, respectively. Inhibition by any concentration of CCh did not differ significantly from control in 30 ({blacktriangleup}; 0.82<P>0.52) and 100 ({blacksquare}; 0.67<P>0.24) µM [ODQ]pip. Symbols indicate mean±S.E.M. of data from number of cells shown in parentheses.

 
3.2 Experiments with LY83583
Initially, we tested the effects of external LY83583 on muscarinic inhibition of ISO-stimulated ICa(L) because bath-applied LY83583 increased ICa(L) when applied on top of ISO in frog ventricular myocytes [19]. Neither LY83583 nor ethanol per se had any effect on basal ICa(L) (data not shown). The basal ICa(L) of 1.8±0.15 nA (n=36) increased to 3.5±0.20 (211±11%, n=36) with 10 nM ISO. When LY83583 was added to the superfusion fluid, ICa(L) increased by 18±15, 23±16 and 50±15% at 3 (n=9), 30 (n=13) and 100 µM LY83583 (n=10), respectively. Tests of CCh showed that its inhibition of ICa(L) appeared to diminish significantly only at 100 µM LY83583 (data not shown). The initial current increase in LY83583 might have masked the actual effect of CCh.

Next, we examined the LY83583 effects when added to pipette solution. The data, including results with dialyzed ODQ, are summarized in Fig. 4. Neither [ODQ]pip nor [LY83583]pip significantly affected ICa(L) under any condition. The only exception are the data with 100 µM [LY83583]pip that are statistically significant (P<0.01) under basal condition and with cAMP-elevating agents. Also, 10 nM ISO and 100 µM IBMX increased ICa(L) equally under all conditions.


Figure 4
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  		Fig. 4 Summary of [ODQ]pip and [LY83583]pip experiments on ICa(L) in basal condition and with either 10 nM ISO or 100 µM IBMX. Ordinate: ICa(L) in nA; abscissa: results obtained under basal conditions and with ISO or IBMX. Number of cells at each condition shown in parentheses. ODQ or LY83583 were present from the time of patch rupture in all experiments. Neither ODQ at either concentration nor LY83583 at 3 and 30 µM had a significant effect on basal ICa(L) or on stimulated ICa(L). At 100 µM [LY83583]pip, ICa(L) increased significantly (*P<0.01) in the absence and presence of cAMP-elevating agents.

 
An example of the latter is illustrated in Fig. 5A; 10 nM ISO or 100 µM IBMX increased ICa(L) to the same extent in a single myocyte dialyzed with 30 µM [LY83583]pip. While the effects of cAMP-elevating agents on ICa(L) were the same in LY83583, inhibition by muscarinic agonist depended on the nature of the stimulant. At 1 µM, CCh reversibly inhibited ISO-stimulated ICa(L) by ~45% (see also Fig. 3). After washout of ISO, superfusion with IBMX increased ICa(L) and the addition of the same CCh concentration had no effect on ICa(L) which returned to initial levels upon IBMX washout. This differential effect of [LY83583]pip on ICa(L) inhibition by CCh was independent of the order in which ISO and IBMX were added (half of the trials began with IBMX instead) and reproducible as summarized in Fig. 6. In the absence of LY83583, CCh reduced IBMX-stimulated ICa(L) in a concentration-dependent manner (Fig. 6A). Inclusion of LY83583 in the pipette solution significantly suppressed the CCh effect at each [LY83583]pip as determined by ANOVA (P≤0.03). With 3 µM [LY83583]pip, IBMX increased ICa(L) by 201±12% (n=12) from an initial current of 1.5±0.07 nA (n=27). This is not different from the increase of 231±29% (n=16) of initial current (1.6±0.10 nA, n=46) by IBMX in the absence of LY83583 (see also Fig. 4). Inhibition of IBMX-stimulated ICa(L) by 1 and 10 µM CCh was significantly reduced at 3 µM [LY83583]pip (Fig. 6A). When comparing the results for 100 µM CCh, the effect of LY83583 was marginal at 30 µM (P=0.06) and highly significant at 100 µM (P<0.01).


Figure 5
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  		Fig. 5 LY83583 suppresses inhibition by CCh of IBMX- but not ISO-stimulated ICa(L). (A) Time course showing that ISO (10 nM) and IBMX (100 µM) increase ICa(L) to the same extent with [LY83583]pip of 30 µM. Carbachol (1 µM) inhibits effect of ISO as usual but fails to inhibit the effect of IBMX. Drug additions marked by thin horizontal lines; LY83583 present in pipette solution from the time of patch rupture (t=0 min). Individual current traces on right are from experimental periods indicated by letters a–i. (B) At [LY83583]pip of 3 µM, IBMX (100 µM) increases ICa(L) but SIN-1 (10 µM) has no effect. Records taken from a cell different from that shown in A. Individual current traces at experimental points indicated by letters (a–e) shown on right hand of figure. All individual current traces are Cd2+-sensitive ICa(L).

 

Figure 6
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  		Fig. 6 Summary of [LY83583]pip experiments illustrating differential suppression of IBMX- and ISO-stimulated ICa(L) by CCh. (A) Ordinate, CCh concentrations on logarithmic scale; abscissa, percent inhibition of IBMX-stimulated ICa(L). The control concentration–effect relation is shown by ({circ}); CCh reduced IBMX effect by 40±8.1, 57±1.5 and 79±6.3% at 1, 10 and 100 µM, respectively. The concentration-dependent effect of CCh differed significantly from control at [LY83583]pip of 3 (bullet), 30 ({blacktriangleup}) and 100 ({blacksquare}) µM, respectively. Number of cells shown in parentheses. (B) Ordinate, same as in (A); abscissa, percent inhibition of ISO-stimulated ICa(L). Symbols for control and [LY83583]pip are same as in (A). Carbachol inhibited ISO-stimulated ICa(L) by 46±8.4% (1 µM), 57±7.0% (10 µM) and 77±7.4% (100 µM) in the absence of LY83583. There is no difference in CCh effect at any [LY83583]pip.

 
In contrast, inhibition of ISO-stimulated ICa(L) remained unchanged at all [LY83583]pip (Fig. 6B). The concentration-dependence for CCh effect against ISO-stimulated ICa(L) was essentially the same as against IBMX-stimulated ICa(L) in the absence of LY83583. At 3 µM, [LY83583]pip was sufficient to reduce the effect of 1 µM CCh against IBMX by about 50% (Fig. 6A). However, 100 µM [LY83583]pip had no significant effect against 100 µM CCh when ICa(L) was stimulated by ISO (Fig. 6B). There was no difference in the concentration-dependent effect of CCh against ISO-stimulated ICa(L) between control and any [LY83583]pip as determined by ANOVA. This is notable because 100 µM [LY83583]pip increased the current increment but not the percentage increase in ICa(L) by ISO (Fig. 4). At 100 µM [LY83583]pip, ISO increased ICa(L) by 230±14% (n=15) from an initial value of 2.7±0.18 nA (n=29). In the absence of LY83583, ISO increased ICa(L) by 246±23% (n=30) over the control current of 1.6±0.10 nA (n=46). Thus, intrapipette LY83583 opposed the inhibition of IBMX-stimulated, but not of ISO-stimulated ICa(L) by CCh.

We also tested [LY83583]pip at 3 µM on the inhibition by SIN-1 of IBMX-stimulated ICa(L) because LY83583 inactivates sGC and pGC. Under this condition, 10 µM SIN-1 caused a small (~6%) and transient suppression of IBMX-stimulated ICa(L) (Fig. 5B). Addition of 100 µM IBMX increased ICa(L) to 3.1±0.71 nA from an initial value of 1.6±0.21 nA (n=4 cells). At 10 µM, SIN-1 only slightly reduced this current by 7±5.5% (NS). As noted previously, this concentration of SIN-1 inhibited IBMX-stimulated ICa(L) by 51±8.4%. Thus, LY83583 also opposed inhibition of ICa(L) by an NO donor that activates sGC.

3.3 Inhibition of ICa(L) by atrial natriuretic peptide (ANP)
We compared the effects of ANP, a pGC activator, with those of CCh on ICa(L). Atrial natriuretic peptide (10–100 nM), per se, had no effect on basal ICa(L) (data not shown). However, ANP (100 nM) inhibited ICa(L) previously augmented by either IBMX or ISO. An experiment with IBMX is shown in Fig. 7. The increase in ICa(L) by 100 µM IBMX was reduced reversibly by ~40% when 100 nM ANP was added (Fig. 7A). In IBMX experiments, the PDE inhibitor increased ICa(L) from an initial level of 1.5±0.12 to 3.9±0.44 nA (n=5 cells). In control tests, addition of ANP reversibly inhibited the effect of IBMX by 35±4.5%. When this experiment was done with [ODQ]pip of 100 µM, ANP reduced the stimulant effect of IBMX by 30±19.4% (n=4 cells). This was not significantly different from control (P=0.78). In contrast, when LY83583 was present in pipette solution at 3 µM (n=5 cells), ANP reduced IBMX-stimulated ICa(L) by only 6±3.4% (P≤0.01). An example of blockade of ANP effect by LY83583 is shown in Fig. 7B.


Figure 7
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  		Fig. 7 ANP inhibits IBMX-stimulated ICa(L) by an LY83583-sensitive mechanism. (A) ANP (100 nM) reversibly reduced ICa(L) that had been increased by IBMX (100 µM). (B) With [LY83583]pip at 3 µM, ANP has a negligible effect on IBMX-stimulated ICa(L). Individual current traces at experimental points indicated by letters (a–e) shown on right hand of panels (A) and (B). All individual current traces are Cd2+-sensitive ICa(L).

 
Isoproterenol (10 nM) increased ICa(L) from 1.5±0.24 to 4±0.75 nA (n=5 cells). In control conditions, ANP (100 nM) reduced the stimulant effect of ISO by 28±5.9%. Again, [ODQ]pip at 100 µM had no effect on the response to ANP inasmuch as this peptide reduced the ISO-stimulated ICa(L) by 21±4.7% (P=0.34 compared to control). With [LY83583]pip of 3 µM, ANP was able to decrease ISO-stimulated ICa(L) by only 5±3.9% (P=0.01; n=5 cells).

Thus, ANP, like CCh, inhibited either IBMX- or ISO-stimulated ICa(L) by an ODQ-resistant mechanism. Further, inhibition of IBMX-stimulated ICa(L) by ANP was blocked by LY83583 as was the effect of CCh. Atrial natriuretic peptide, unlike CCh, lost its ability to inhibit ISO-stimulated ICa(L) when LY83583 was present in pipette solution.

3.4 Experiments with 8 Bromo-cGMP (8 Br-cGMP)
In order to ascertain the selectivity of LY83583 action, we tested its effects against 8 Br-cGMP-induced inhibition of ICa(L). At 100 µM, this stable cGMP analogue had no effect on basal ICa(L) (data not shown). In the presence of 10 nM ISO, 8 Br-cGMP suppressed ICa(L) by 32±7.1% (n=7). This is shown in Fig. 8A where 8 Br-cGMP reversibly suppressed ICa(L) by ~31%. In another cell with [LY83583]pip of 3 µM, 8 Br-cGMP inhibited ISO-stimulated ICa(L) by 32% (Fig. 7B). The average inhibition by 8 Br-cGMP amounted to 24±7.0% (n=7) and this did not differ from that seen in the absence of LY83583 (P=0.46).


Figure 8
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  		Fig. 8 Inhibition of ISO-stimulated ICa(L) by 8 Br-cGMP is resistant to LY83583. (A) 8 Br-cGMP (100 µM) reversibly inhibits ICa(L) stimulated by 10 nM ISO. (B) Inhibition by 8 Br cGMP persists with [LY83583]pip of 30 µM. Individual current traces shown on right correspond to letters above time course data on left. All currents are Cd2+-sensitive ICa(L).

 
The stimulant effect of 100 µM IBMX decreased by 34±3.6% in the presence of 8 Br-cGMP (n=6). In the presence of 3 µM [LY83583]pip (n=4), 8 Br-cGMP suppressed IBMX-stimulated ICa(L) by 39±7.8% (P=0.54). Therefore, LY83583 had no effect against 8 Br-cGMP, an activator of cGMP-dependent protein kinase (PKG).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We find that: (1) ODQ, an inhibitor of sGC, prevents block of IBMX-stimulated ICa(L) by SIN-1 but not CCh or ANP, (2) LY83583, an inhibitor of sGC and pGC, opposes block of IBMX-stimulated ICa(L) by SIN-1, CCh or ANP and (3) neither ODQ nor LY83583 opposes CCh block of ISO-stimulated ICa(L). However, LY83583 prevents ANP block of ISO-stimulated ICa(L). Can these results conform to the cGMP hypothesis for muscarinic inhibition of L-type Ca2+ channels?

4.1 ODQ discriminates between NO- and CCh-induced inhibition of ICa(L)
Our SIN-1 results on IBMX-stimulated ICa(L) replicate those of others [20]. SIN-1 generates NO and ODQ blockade of SIN-1 effect accords with the hypothesis that NO activates sGC [21], increases cGMP and, via PKG, inhibits ICa(L) [6,20,22]. ODQ blocks inhibition of ISO-stimulated ICa(L) by the NO donor, SNAP [19]. The negative inotropic effects of SIN-1 and SNAP are additive [23] as are the negative dromotropic effects of SIN-1 and CCh [24].

SIN-1 effect via NO depends on the cGMP target. NO from SIN-1 activates ODQ-sensitive GC; the cGMP increases the hyperpolarization-activated current, If, to cause a positive chronotropic effect in rabbit sinoatrial node [25]. Superoxide dismutase increased pacemaker stimulation by SIN-1 by protecting NO against oxidation and allowing greater cGMP synthesis [25]. Superoxide dismutase, alone or with catalase, augmented SIN-1 inhibition of ICa(L) and contractions in guinea pig ventricular myocytes [20]. The cGMP target, PKG, caused ICa(L) to decrease [20]. Formation of NO metabolites (peroxynitrite, nitrosothiols) can increase ICa(L) or contractions by an ODQ-resistant process that complicates SIN-1 action [19,23,26]. We find SIN-1 only inhibited IBMX-stimulated ICa(L) by an ODQ-sensitive mechanism and conclude that SIN-1 acted through an NO/sGC/cGMP path.

ODQ did not oppose inhibition by CCh. In frog ventricular and human atrial myocytes, ODQ did not affect muscarinic inhibition of ISO-stimulated ICa(L) [19,27]. Muscarinic agonists increase cGMP in isolated cardiac myocytes (reviewed in Ref. [6]) without activating NOS in guinea pig [5,9] and rat [15] ventricular myocytes. The high myoglobin content of adult rat ventricle acts as a scavenger for ODQ and NO [15]. In newborn rat ventricles, with one-third the adult myoglobin content, ODQ prevented inhibition of contractions by SNAP [28]. (Neonatal [29] and adult rat [30] ventricular myocytes in culture displayed NOS activity whose suppression by methylene blue or hemoglobin prevented muscarinic inhibition of ISO-stimulated ICa(L). This discrepancy might be resolved if myoglobin content falls within 24 h of placing cells in culture.) The sevenfold lower myoglobin content of guinea pig ventricle [16] could explain SIN-1 suppression of ICa(L) and ODQ's effectiveness against SIN-1. Activation of ODQ-inaccessible NOS by CCh seems unlikely; inhibition of ICa(L) by ACh in cat atrial myocytes is NO-independent yet rebound stimulation of ICa(L) by ACh occurs via NO activation of ODQ-sensitive sGC [10]. We propose that muscarinic agonists activate pGC to increase cGMP. Results with ANP, which activates pGC, sustain this hypothesis because ODQ did not oppose ANP-induced inhibition.

4.2 Dialyzed LY83583 differentially suppresses muscarinic inhibition
LY83583, which inhibits pGC and sGC [31], increased ISO- or IBMX-stimulated ICa(L) when applied externally at 100 µM [8,19,20] and opposed muscarinic inhibition of ISO-stimulated ICa(L). LY83583 does not inhibit PDE [20] and does not block either mAChR or the inhibitory guanine nucleotide binding protein, Gi [19]. External LY83583 reduced ACh inhibition of ISO-stimulated ICa(L), an effect attributed to superoxide anion [19]. Also, LY83583 inhibits GC via oxygen radical formation [18] and could negate SIN-1 effect on GC by inactivating NO [32].

Clearer distinctions emerged when LY83583 was dialyzed. At 3 µM, [LY83583]pip did not affect ‘basal’ ICa(L) yet opposed muscarinic inhibition of IBMX-stimulated ICa(L). At 100 µM [LY83583]pip, CCh inhibited ISO-stimulated ICa(L) equally well as in its absence. This illustrates the importance of applying LY83583 internally [20,33]. Assuming that dialyzed LY83583 suppresses GC activity, activation of a GC/cGMP path by CCh inhibits ICa(L) when cAMP is elevated by PDE inhibition but not by AC stimulation (Figs. 5 and 6Go). Alternatively, LY83583 might selectively block CCh effect against IBMX-stimulated ICa(L) by superoxide anion action independent of GC. This mechanism does not explain how [LY83583]pip opposes inhibition of ISO- and IBMX-stimulated ICa(L) by ANP but not by 8 Br-cGMP.

Different subcellular cGMP compartments appear to mediate inhibition [14,34]. Thus, NO from SIN-1 activates ODQ-sensitive sGC while CCh and ANP activate LY83583-sensitive pGC. First, neither inhibition of NOS nor of sGC opposed cGMP formation by CCh [5,35]. When L-NMMA or L-NNA suppressed NO synthesis, basal ICa(L) increased and was inhibited by CCh in the absence of receptor ligands [9]. In the presence of L-NMMA, muscarinic agonist inhibited contractions and pacemaker activity in rat and guinea pig atrial and sinoatrial preparations, respectively [36]. Second, ODQ did not affect cGMP accumulation by CCh in rat ventricular myocytes [15]. Lastly, LY83583 prevented ICa(L) inhibition by ANP in either ISO or IBMX (Fig. 7). LY83583, but not ODQ, inhibits cGMP synthesis by pGC activators [37]. In platelets and vascular smooth muscle, ODQ prevented cGMP synthesis by SNAP but not by ANP [38], a pGC activator [39]. In rabbit ventricular myocytes, LY83583 prevents cGMP synthesis and cell volume regulation by ANP but not by 8 Br-cGMP [40].

4.3 Dual pathway hypothesis for muscarinic inhibition of ICa(L)
We propose that the reaction causing ICa(L) stimulation determines the muscarinic inhibition path. With ISO, CCh acts proximally to inhibit AC activity. With IBMX, CCh acts distally via the pGC/cGMP/PKG cascade [20,33,41]. That LY83583 selectively blocks inhibition by CCh of IBMX-stimulated ICa(L) supports this and excludes many proteins (β-adrenoceptor, mAChR, AC, PKA, PKG and L-type Ca2+ channel) as targets of LY83583 and its oxidation products.

Distinct cAMP/PKA compartments occur in vertebrate ventricular myocytes exposed to different cAMP-elevating agents [42,43]. In dog myocytes, 10 nM ISO or 100 µM IBMX similarly increased particulate cAMP and intracellular Ca2+ transients and decreased the t1/2 for transients' decline [44]. We find 10 nM ISO or 100 µM IBMX caused ICa(L) to double. Either 10 nM ISO or 100 µM IBMX increased contractions and relaxation rate about twofold and also increased phospholamban phosphorylation in guinea pig myocytes [45]. However, only ISO promoted phosphorylation of troponin I and C protein. cAMP compartments cannot be excluded for differential muscarinic inhibition of ICa(L).

How can the putative cGMP accumulation by CCh fail to inhibit ISO-stimulated ICa(L) when ANP and 8 Br-cGMP are effective? LY83583 opposed ANP but not CCh-induced inhibition of ICa(L) in ISO. Perhaps ANP stimulates pGC inaccessible to CCh. Alternatively, CCh may stimulate pGC only weakly such that it would inhibit IBMX-stimulated ICa(L) because IBMX also prevents cGMP hydrolysis. Also, mimickry need not imply that CCh and 8 Br-cGMP use the same signal pathway. Cardiac cells display compartments of membrane delimited and intracellular signaling molecules for G protein-coupled receptors [46]. Equieffective concentrations of β1- and β2-adrenoceptor agonist similarly increased cAMP, the contraction and relaxation rate of rat myocyte contractions [47]. Carbachol, by signals distal to Gi, inhibited all β1-adrenoceptor agonist effects but only the increased rate of cell shortening caused by β2-adrenoceptor agonist. Thus, CCh suppressed AC stimulation by β1- but not by β2-adrenoceptor agonist.

4.4 Limitations
SIN-1 is a source of superoxide anion [8,23]. That SIN-1 acted like SNAP [23,25], which does not produce superoxide anion, reduces concern about this in our experiments. The defining role of LY83583 in the dual pathway hypothesis depends upon its inhibiting pGC as well as sGC while ODQ action is restricted to inhibiting sGC. If LY83583 selectively inhibits sGC as some report [48,49], one would have to posit two sGC isoforms, one sensitive to ODQ and activated by NO and another sensitive to LY83583 and activated by CCh. Our experiments cannot distinguish these possibilities. The use of ANP can help in this regard but the form of ANP we used reportedly inhibited ISO-stimulated ICa(L) by reducing AC activity [50]. We favor the view that ANP activates pGC. The validity of the dual pathway hypothesis requires additional scrutiny.

4.5 Summary
Intracellular transduction of muscarinic inhibition depends on the stimulus that increases cAMP such that ISO-, but not IBMX-stimulated ICa(L) is more likely regulated by changes in AC activity. When a GC/cGMP limb for muscarinic inhibition is evident, pGC appears responsible and this extends the model previously reported [20,33]. Calveolae-directed signaling could target mAChR to distinct pathways for ICa(L) inhibition even without participation of eNOS [51].

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by USPHS Grant HL-13339.


   Notes
 
1 Present address: Department of Internal Medicine (Cardiology), Teikyo University Ichihara Hospital, Ichihara City, Chiba 299-0111, Japan. Back

2 Present address: Department of Cardiology, The First Affiliated Hospital, Suzhou Medical College, Suzhou, Jiangsu 215006, PR China. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Löffelholz K., Pappano A.J. The parasympathetic neuroeffector junction of the heart. Pharmacol. Rev. (1985) 37:1–24.[Abstract]
  2. Hartzell H.C. Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems. Prog. Biophys. Mol. Biol. (1988) 52:165–247.[CrossRef][Web of Science][Medline]
  3. Brodde O.E., Michel M.C. Adrenergic and muscarinic receptors in the human heart. Pharmacol. Rev. (1999) 51:651–690.[Abstract/Free Full Text]
  4. MacDonell K.L., Tibbits G.F., Diamond J. cGMP elevation does not mediate muscarinic agonist-induced negative inotropy in rat ventricular myocytes. Am. J. Physiol. (1995) 269:H1905–H1912.[Web of Science][Medline]
  5. Stein B., Drogemüller A., Mülsch A., Schmitz W., Scholz H. Ca2+-dependent constitutive nitric oxide synthase is not involved in the cyclic GMP-increasing effects of carbachol in ventricular cardiomyocytes. J. Pharmacol. Exp. Ther. (1993) 266:919–925.[Abstract/Free Full Text]
  6. Méry P.-F., Abi-Gerges N., Vandecasteele G., et al. Muscarinic regulation of the L-type calcium current in isolated cardiac myocytes. Life Sci. (1997) 60:1115–1120.
  7. Kelly R.A., Balligand J.-L., Smith T.W. Nitric oxide and cardiac function. Circ. Res. (1996) 79:363–380.[Free Full Text]
  8. Zakharov S.I., Pieramici S., Kumar G.K., Prabhakar N.R., Harvey R.D. Nitric oxide synthase activity in guinea pig ventricular myocytes is not involved in muscarinic inhibition of cAMP-regulated chloride channels. Circ. Res. (1996) 78:925–935.[Abstract/Free Full Text]
  9. 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. (1998) 506:639–651.[Abstract/Free Full Text]
  10. Wang Y.G., Rechenmacher C.E., Lipsius S.L. Nitric oxide signaling mediates stimulation of L-type Ca2+ current elicited by withdrawal of acetylcholine in cat atrial myocytes. J. Gen. Physiol. (1998) 111:113–125.[Abstract/Free Full Text]
  11. Han X., Zhao Y.Y., Kubota I., 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]
  12. Vandecasteele G., Eschenhagen T., Scholz H., et al. Muscarinic and β-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]
  13. Kilter H., Schwinger R.H., Lenz O., et al. Evidence against a role of nitric oxide in the indirect negative inotropic-effect of M-cholinoceptor stimulation in human ventricular myocardium. Naunyn Schmiedebergs Arch. Pharmacol. (1995) 352:308–312.[Web of Science][Medline]
  14. Ji G.J., Fleischmann B.K., Bloch W., et al. Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition. FASEB J. (1999) 13:313–324.[Abstract/Free Full Text]
  15. Wegener J.W., Closs E.I., Förstermann U., Nawrath H. Failure of 1H-[1,2,4] oxadiazolo [4,3-1] quinoxalin-1-one (ODQ) to inhibit soluble guanylyl cyclase in rat ventricular cardiomyocytes. Br. J. Pharmacol. (1999) 127:693–700.[CrossRef][Web of Science][Medline]
  16. O'Brien P.J., Shen H., McCutcheon L.J., et al. Rapid, simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin: use in various animals indicates functional role of myohemoproteins. Mol. Cell. Biochem. (1992) 112:45–52.[Web of Science][Medline]
  17. Pappano A.J. Cardiac electrophysiology: from cell to bedside. Zipes D.P., Jalife J., eds. (1995) 2nd ed. Philadelphia PA: WB Saunders. 411–422.
  18. Lüönd R.M., McKie J.H., Douglas K.T. A direct link between LY83583, a selective repressor of cyclic GMP formation, and glutathione metabolism. Biochem. Pharmacol. (1993) 45:2547–2549.[CrossRef][Web of Science][Medline]
  19. Abi-Gerges N., Hove-Madsen L., Fischmeister R., Méry P.-F. A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K+ currents in frog cardiac myocytes. Br. J. Pharmacol. (1997) 121:1369–1377.[CrossRef][Web of Science][Medline]
  20. Wahler G.M., Dollinger S.J. Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. Am. J. Physiol. (1995) 268:C45–C54.[Web of Science][Medline]
  21. Garthwaite J., Southam E., Boulton C.L., et al. Potent and selective inhibition of nitric oxide-sensitive guanylyl cylase by 1H-[1,2,4] oxadiazolo [4,3-1] quinoxalin-1-one. Mol. Pharmacol. (1995) 48:184–188.[Abstract]
  22. Méry P.-F., Pavoine C., Belhassen L., Pecker F., Fischmeister R. Nitric oxide regulates cardiac Ca2+ current. J. Biol. Chem. (1993) 268:26286–26295.[Abstract/Free Full Text]
  23. Chesnais J.M., Fischmeister R., Méry P.-F. Positive and negative inotropic effects of NO donors in atrial and ventricular fibres of the frog heart. J. Physiol. (1999) 518:449–461.[Abstract/Free Full Text]
  24. Han X., Kobzik L., Zhao Y.Y., et al. Nitric oxide regulation of atrioventricular node excitability. Can. J. Cardiol. (1997) 13:1192–1201.
  25. Musialek P., Lei M., Brown H.F., Paterson D.J., Casadei B. Nitric oxide can increase heart rate by stimulating the hyperpolarization-activated inward current If. Circ. Res. (1997) 81:60–68.[Abstract/Free Full Text]
  26. Campbell D.J., Stamler J.S., Strauss H.C. Redox modulation of L-type calcium channels in ferret ventricular myocytes. J. Gen. Physiol. (1996) 108:277–293.[Abstract/Free Full Text]
  27. 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. (1998) 506:653–663.[Abstract/Free Full Text]
  28. Vulcu S.D., Wegener J.W., Nawrath H.G. Functional differences in the NO-sGC/cGMP pathway in cardiomyocytes from newborn and adult rats. Biophys. J. (2000) 78:222A. Abstract.
  29. Balligand J.-L., Kelly R.A., Marsden P.A., Smith T.W., Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc. Natl. Acad. Sci. USA (1993) 90:347–351.[Abstract/Free Full Text]
  30. Balligand J.-L., Kobzik L., Han X., et al. Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes. J. Biol. Chem. (1995) 270:14582–14586.[Abstract/Free Full Text]
  31. O'Donnell M.E., Owen N.E. Role of cyclic GMP in atrial natriuretic factor stimulation of Na+, K+, Cl– cotransport in vascular smooth muscle cells. J. Biol. Chem. (1986) 261:15461–15466.[Abstract/Free Full Text]
  32. Barbier A.J.M., Lefebvre R.A. Effect of LY 83583 on relaxation induced by nonadrenergic nocholinergic nerve stimulation and exogenous nitric oxide in the rat gastric fundus. Eur. J. Pharmacol. (1992) 219:331–334.[CrossRef][Web of Science][Medline]
  33. Mubagwa K., Shirayama T., Moreau M., Pappano A.J. Effects of PDE inhibitors and carbachol on the L-type Ca current in guinea pig ventricular myocytes. Am. J. Physiol. (1993) 265:H1353–H1363.[Web of Science][Medline]
  34. Kumar R., Joyner R.W., Komalavilas T., Lincoln T.M. Analysis of expression of cGMP-dependent protein kinase in rabbit heart cells. J. Pharmacol. Exp. Ther. (1999) 291:967–975.[Abstract/Free Full Text]
  35. Levi R.C., Alloatti G., Penna C., Gallo M.P. Guanylate-cyclase-mediated inhibition of cardiac ICa by carbachol and sodium nitroprusside. Pflügers Arch. (1994) 426:419–426.[CrossRef][Web of Science][Medline]
  36. Nawrath H., Baumner D., Rupp J., Oelert H. The ineffectiveness of the NO-cyclic GMP signaling pathway in the atrial myocardium. Br. J. Pharmacol. (1995) 116:3061–3067.[Web of Science][Medline]
  37. Olson L.J., Knych E.T. Jr., Herzig T.C., Drewett J.G. Selective guanylyl cyclase inhibitor reverses nitric oxide-induced vasorelaxation. Hypertension (1997) 29:254–261.[Abstract/Free Full Text]
  38. Moro M.A., Russell R.J., Cellek S., et al. cGMP mediates the vascular and platelet actions of nitric oxide: Confirmation using an inhibitor of the soluble guanylyl cyclase. Proc. Natl. Acad. Sci. USA (1996) 93:1480–1485.[Abstract/Free Full Text]
  39. Waldman S.A., Rapoport R.M., Murad F. Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J. Biol. Chem. (1984) 259:14332–14334.[Abstract/Free Full Text]
  40. Clemo H.F., Feher J.J., Baumgarten C.M. Modulation of rabbit ventricular cell volume and Na+/K+/2Cl– cotransport by cGMP and atrial natriuretic factor. J. Gen. Physiol. (1992) 100:89–114.[Abstract/Free Full Text]
  41. Sakai R., Shen J.-B., Pappano A.J. Elevated cAMP suppresses muscarinic inhibition of L-type calcium current in guinea pig ventricular myocytes. J. Cardiovasc. Pharmacol. (1999) 34:304–315.[CrossRef][Web of Science][Medline]
  42. Hayes J.S., Brunton L.L., Brown J.H., Reese J.B., Mayer S.E. Hormonally specific expression of cardiac protein kinase activity. Proc. Natl. Acad. Sci. USA (1979) 76:1570–1574.[Abstract/Free Full Text]
  43. Jurevicius J., Fischmeister R. Acetylcholine inhibits Ca2+ current by acting exclusively at a site proximal to adenylyl cyclase in frog cardiac myocytes. J. Physiol. (1996) 491:669–675.[Abstract/Free Full Text]
  44. Hohl C.M., Li Q. Compartmentation of cAMP in adult canine ventricular myocytes. Circ. Res. (1991) 69:1369–1379.[Abstract/Free Full Text]
  45. Rapundalo S.T., Solaro R.J., Kranias E.G. Inotropic responses to isoproterenol and phosphodiesterase inhibitors in intact guinea pig hearts: comparison of cyclic AMP levels and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins. Circ. Res. (1989) 64:104–111.[Abstract/Free Full Text]
  46. Ostrom R.S., Post S.R., Insel P.A. Stoichiometry and compartmentation in G protein-coupled receptor signaling: Implications for therapeutic interventions involving Gs. J. Pharmacol. Exp. Ther. (2000) 294:407–412.[Abstract/Free Full Text]
  47. Aprigliano O., Robinson R.B., Rybin V.O., Steinberg S.F., Pak E. β1- and β2-adrenergic receptors exhibit differing susceptibility to muscarinic accentuated antagonism. Am. J. Physiol. (1997) 272:H2726–H2735.[Web of Science][Medline]
  48. Eskinder H., Hillard C.J., Flynn N., et al. Role of guanylate cyclase-cGMP systems in halothane-induced vasodilation in canine cerebral arteries. Anesthesiology (1992) 77:482–487.[CrossRef][Web of Science][Medline]
  49. Ali N., Yousufzai Y.K., Abdel-Latif A.A. Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. Cell. Signal. (2000) 12:491–498.[CrossRef][Web of Science][Medline]
  50. Gisbert M.-P., Fischmeister R. Atrial natriuretic factor regulates the calcium current in frog isolated cardiac cells. Circ. Res. (1988) 62:660–667.[Abstract/Free Full Text]
  51. Okamoto T., Schegel A., Scherer P.E., Lisanti M.P. Caveolins, a family of scaffolding proteins for organizing ‘preassembled signaling complexes’ at the plasma membrane. J. Biol. Chem. (1998) 273:5419–5422.[Free Full Text]

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