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CARDIOVASCULAR

Functional Adenylyl Cyclase Inhibition in Murine Cardiomyocytes by 2'(3')-O-(N-Methylanthraniloyl)-Guanosine 5'-[-Thio]triphosphate

Department of Pharmacology (D.R., J.M., S.H.) and Center for Molecular Medicine (S.H.), University of Cologne, Köln, Germany; Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey (S.F.V.); and Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany (R.S.)

Received December 11, 2006; accepted February 2, 2007.

	Abstract

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₁-Adrenergic receptor activation stimulates cardiac L-type Ca²⁺ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[-thio-]triphosphate (MANT-GTPS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPS on L-type Ca²⁺ currents (I_Ca,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5^–/–) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPS attenuated I_Ca,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77 ± 13%; 100 nM MANT-GTPS, +43 ± 6%; 1 µM MANT-GTPS, +21 ± 9%; p < 0.05). The leftward shift of current-voltage curves was abolished by 1 µM but not by 100 nM MANT-GTPS. In myocytes from AC5^–/– mice, the residual stimulation of I_Ca,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute -adrenergic stimulation in WT mice. Interestingly, basal I_Ca,L was lowered by 1 µM but not by 100 nM MANT-GTPS. The decrease was less pronounced in myocytes from AC5^–/– mice compared with wild types (–23 ± 1 versus –40 ± 7%), indicating basal I_Ca,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of I_Ca,L by MANT-GTPS, both under basal conditions and following -adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in I_Ca,L activation and that MANT-GTPS predominantly acts via AC5 inhibition.

Although AC5 represents the isoform with the highest catalytic activity in the adult heart (Ishikawa et al., 1992), it remains unclear whether it is the functionally dominant cardiac isoform, in particular regarding acute regulation of L-type Ca²⁺ channel activity and thus modulation of Ca²⁺ influx triggering contraction. Nevertheless, AC5 is an interesting potential drug target in the therapy of heart disease. In this regard, transgenic overexpression of type 6 AC did not induce abnormal histological findings or deleterious changes in the heart (Gao et al., 1999), whereas overexpression of type 5 AC affects basal AC activity and cardiac function (Tepe et al., 1999). These findings indicate that AC5, in contrast to type 6 AC, plays an important role under pathological conditions. Furthermore in AC5-knockout (AC5^–/–) mice, contractile properties and L-type Ca²⁺ currents (I_Ca,L) were impaired in a Ca²⁺-dependent manner (Okumura et al., 2003a), whereas cardiac function was protected against pressure overload induced by aortic banding (Okumura et al., 2003b). Particularly, these findings on AC5 deficiency underline the important role of AC5 in murine hearts and suggest AC inhibitors to be promising pharmacological tools and perhaps even therapeutics. This notion is supported by the finding that excessive activation of the ₁-adrenoreceptor-G_S-AC cascade is detrimental for cardiac function (Engelhardt et al., 1999; Lohse et al., 2003).

Most AC inhibitors known so far are either noncompetitive or nonspecific regarding isoforms (Hanoune and Defer, 2001; Iwatsubo et al., 2004). However, with respect to therapeutic use, both competitiveness and isoform specificity would be helpful properties in minimizing the risk of adverse effects. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl) (MANT) nucleotides as a novel class of potent competitive AC inhibitors (Gille and Seifert, 2003). Intriguingly, MANT-nucleotides inhibit AC5 and AC6 more potently than other cyclases (Gille et al., 2004). Crystallographic, biophysical, and biochemical studies have provided in-depth insight into the molecular interactions of MANT-nucleotides with AC, providing a rational basis for the development of even more potent and selective AC inhibitors than those currently available (Mou et al., 2005, 2006).

To assess the pharmacological effects of MANT nucleotides in murine cardiomyocytes, we took advantage of the pivotal position of AC linking -adrenergic signaling to L-type Ca²⁺ current stimulation. We used the hydrolysis-resistant MANT-nucleotide MANT-GTPS (Fig. 1) as a prototypical competitive AC inhibitor. During whole-cell patch-clamp recordings, the membrane-impermeant compound was delivered by intracellular dialysis. Freshly isolated murine cardiomyocytes were exposed to isoproterenol to study AC5 inhibition under basal conditions and -adrenergic stimulation. We repeated these experiments in AC5^–/– cardiomyocytes to determine the AC5 specificity of the effects of MANT-GTPS.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structural formula of MANT-GTPS. Note the spontaneous isomerization between the 2'- and 3'-MANT-substituted structure.

	Materials and Methods

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Genotyping of Knockout Mice. A tail-clip analysis was performed at 3 to 4 weeks of age. After preparation of genomic DNA, a polymerase chain reaction was run. To genotype AC5^–/–, we used the following primer pairs: wild type (+), forward, 5'-CGC TAC TTC TTC CAC CTG AAC CAG-3'; reverse, 5'-TGA TAA GGA TCA CGC CCA CAG C-3', and knockout (–), forward, 5'-TCG TGC TTT ACG GTA TCG CCG CTC CCG ATT-3'; reverse, 5'-TGA TAA GGA TCA CGC CCA CAG C-3'. Both reactions were run over 40 cycles (saturation). Amplified sequences were 157 base pairs for the WT allele and 443 base pairs for the targeting construct.

Isolation of Cardiac Myocytes. Single ventricular myocytes were isolated by enzymatic dissociation using the method described previously (Foerster et al., 2003). In brief, hearts were perfused with a collagenase solution (Worthington type I and II, 75 U/ml; Cell Systems, St. Katharinen, Germany) in a Langendorff setup, and they were subsequently cut into small chunks. Myocytes were harvested by pouring the suspension through cheesecloth.

Whole-Cell Recording. L-type calcium channel currents were measured at room temperature using the whole-cell configuration of the patch-clamp technique as described previously (Heubach et al., 2001). Whole-cell experiments were performed in an external solution containing 137 mM NaCl, 5.4 mM CsCl, 2 mM CaCl₂, 1.25 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, pH 7.4 with NaOH. Pipettes (1.5 to 3-M borosilicate glass) were filled with 140 mM CsCl, 4 mM MgCl₂, 10 mM HEPES, 10 mM EGTA, and 4 mM Na₂ATP, pH 7.3 with CsOH. Cells were continuously superfused with drug-free bath solution, and then they were switched to a solution containing 100 nM (–)-isoproterenol. Gigaohm seals were formed by gentle suction. The seal resistances were usually between 2 and 5 G. Before series resistance compensation, membrane capacitance was measured by means of fast depolarizing ramp pulses (from –40 to –35 mV; 5-ms duration) at the beginning of each experiment. Compensated access resistance was regularly checked and maintained below 5 M. Series resistance was routinely compensated by 50 to 70%. Membrane currents were low-pass filtered at 2 kHz. Only rod-shaped myocytes with clear striation were used. Whole-cell Ca²⁺ currents were elicited by 200-ms depolarizing voltage steps from a holding potential of –60 mV. The stimulation frequency was 0.2 Hz (EPC-9; HEKA, Lambrecht/Pfalz, Germany). For each cell, a current-voltage relationship was established at the beginning and the end of the experiment. The test potential was +10 mV. For isolation of I_Ca,L from contaminating currents, sodium and T-type calcium currents were inactivated by a 50-ms-long prepulse to –40 mV (holding potential –60 mV), and K⁺ currents were minimized by replacing K⁺ with Cs⁺. To account for variability in cell size, absolute current amplitudes [in picoamperes (pA)] were divided by the respective cell capacitance [in picofarads (pF)], and values are expressed as membrane current I in picoamperes per picofarads. The software PULSE, version 9.12 (HEKA) was used for data acquisition.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effect of 1 µM MANT-GTPS on basal L-type Ca2+ current in wild-type murine cardiomyocytes. A, original traces in the absence and presence of 1 µM MANT-GTPS at the time points 0 and 10 min. B, averaged time course of whole-cell L-type Ca2+ current density in the absence (filled symbols; n = 6) and in the presence of 1 µM MANT-GTPS (open symbols; n = 6). Starting from a holding potential of –60 mV the current was elicited by a test potential of + 10mV preceded by a prepulse to –40 mV.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effect of 1 µM MANT-GTPS on isoproterenol-stimulated L-type Ca2+ current in wild-type murine cardiomyocytes. Cells were superfused by isoproterenol containing bath solution for 12.0 ± 0.1 min (control) and 13.0 ± 0.2 min (MANT-GTPS), respectively. A, original traces before and after 100 nM (–)-isoproterenol stimulation and in the absence and presence of 1 µM MANT-GTPS at the time point 0 min and at the maximal increase of L-type Ca2+ current. B, averaged time course of whole-cell L-type Ca2+ current density from experiments with murine ventricular myocytes before and after 100 nM (–)-isoproterenol stimulation and in the absence (filled symbols; n = 5) and in the presence of 1 µM MANT-GTPS (open symbols; n = 5). Starting from a holding potential of –60 mV, the current was elicited by a test potential of +10 mV preceded by a prepulse to –40 mV. C, control current density-voltage relationship in the absence (filled symbols; n = 5) and in the presence (open symbols; n = 5) of 100 nM (–)-isoproterenol, measured before and 10 min after start of isoproterenol application. D, current density-voltage relationship with 1 µM MANT-GTPS diluted in the pipette solution in the absence (filled symbols; n = 5) and in the presence (open symbols; n = 5) of 100 nM (–)-isoproterenol measured at the beginning and at the end of the experiments. *, p < 0.05 (alternate t test).

Statistics and Data Analysis. Results are given as mean values ± S.E.M. Differences between mean values were tested by alternate t test and considered statistically significant for p < 0.05.

	Results

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Effect of MANT-GTPS on Basal I_Ca,L in Wild-Type Myocytes. We first wanted to assess the effect of MANT-GTPS on basal currents mediated by L-type Ca²⁺ channels in wild-type ventricular myocytes. Under control conditions, I_Ca,L showed a slight decrease that did not exceed 8% of the initial current within 10 min, and it reached stable values for the remaining recording time (from –4.8 ± 0.3 pA/pF to –4.4 ± 0.25 pA/pF after 10 min; N.S., n = 6; Fig. 2B). When 1 µM MANT-GTPS was present in the pipette solution, current density was not instantaneously different compared with control recordings, but it decreased significantly over time (Fig. 2, A and B). After 10 min of recording with MANT-GTPS in the pipette, current density was lowered by 40 ± 7% (Fig. 2, A and B; from –4.0 ± 0.24 pA/pF to –2.5 ± 0.3 pA/pF; p < 0.05, n = 6). This demonstrates that 1 µM MANT-GTPS reduced basal L-type Ca²⁺ current as a function of time, due to intracellular dialysis via the patch pipette. The reduction of basal L-type Ca²⁺ channel activity reached a plateau after 12 to 15 min of recording, suggesting that the reduction of L-type Ca²⁺ current is not due to continuous "run-down."

Effects of MANT-GTPSonI_Ca,L Stimulation by Isoproterenol in Wild-Type Myocytes. Isoproterenol is known to stimulate currents mediated by L-type Ca²⁺ channels due to increased protein kinase A activity following enhanced cAMP production catalyzed by AC. We therefore obtained I_Ca,L stimulated by isoproterenol in the absence and the presence of the AC inhibitor MANT-GTPSat1 µM. Under control conditions, 100 nM isoproterenol increased maximal I_Ca,L by 77%. This increase was attenuated to only +21% in the presence of MANT-GTPS(+77 ± 13 versus +21 ± 9%; p < 0.01, n = 10; Figs. 3, A and B, and 5D). When MANT-GTPS was applied, the leftward-shift of I/V curves typically caused by isoproterenol was absent (Fig. 3, C and D). It is noteworthy that after 15 min of isoproterenol perfusion, currents still were significantly increased above predrug values in the absence of MANT-GTPS. In contrast, in the presence of the AC inhibitor, current density declined below values obtained before drug application (Fig. 3B). This could be due to an ongoing reduction of basal current, of isoproterenol-stimulated current, or both. To discriminate between these possibilities, washout experiments were performed. Isoproterenol was applied early in the course of the experiment and washed out 10 min later (Fig. 4, A and B). Compared with controls (Fig. 4B), calcium current decrease by isoproterenol washout was less pronounced in the presence of MANT-GTPS, indicating smaller remaining agonist effect at that time. These data match our findings showing a decrease in current density by MANT-GTPS even in the absence of -adrenergic stimulation (see above). In summary, MANT-GTPS significantly and almost completely diminished the increase of I_Ca,L due to isoproterenol.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Effect of 100 nM MANT-GTPS on isoproterenol-stimulated L-type Ca2+ current in wild-type murine cardiomyocytes. Cells were superfused by isoproterenol containing bath solution for 8.4 ± 0.1 min. A, original traces before and after 100 nM (–)-isoproterenol stimulation and in the presence of 100 nM MANT-GTPS at the time point 0 min and at the maximal increase of L-type Ca2+ current. B, averaged time course of whole-cell L-type Ca2+ current density from experiments with murine ventricular myocytes before and after 100 nM (–)-isoproterenol stimulation in presence of 100 nM MANT-GTPS (filled symbols; n = 5). Starting from a holding potential of –60 mV, the current was elicited by a test potential of +10 mV preceded by a prepulse to –40 mV. C, current density-voltage relationship with 100 nM MANT-GTPS diluted in the pipette solution in the absence (filled symbols; n = 5) and in the presence (open symbols; n = 5) of 100 nM (–)-isoproterenol measured at the beginning and at the end of the experiments, respectively. *, p < 0.05 (alternate t test). D, maximal increase of L-type Ca2+ current density by isoproterenol (percentage of predrug values) compared with basal current in the absence (black bar; n = 10) and in the presence of 100 nM (gray bar; n = 5) or 1 µM (white bar; n = 11) MANT-GTPS, respectively. *, p < 0.05 versus control (alternate t test).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Reversibility of the effect of isoproterenol on L-type Ca2+ current in wild-type murine cardiomyocytes. A, averaged time course of whole-cell L-type Ca2+ current density from experiments with myocytes before and after 100 nM (–)-isoproterenol stimulation, followed by isoproterenol washout in the presence of 1 µM MANT-GTPS (filled symbols; n = 5). Open symbols show basal L-type Ca2+ current time course (cf. Fig. 2B) to illustrate reversibility of isoproterenol stimulation in the presence of MANT-GTPS. Starting from a holding potential of –60 mV the current was elicited by a test potential of +10 mV preceded by a prepulse to –40 mV. B, filled symbols represent the averaged (n = 5) time course of similar washout experiments, performed in the absence of MANT-GTPS.

Effect of MANT-GTPS on Basal I_Ca,L in AC5^–/– Myocytes. To determine AC5-specific effects of MANT-nucleotides and to characterize the functional role of AC5, experiments obtained with WT cardiomyocytes were repeated in ventricular myocytes from mice lacking AC5 (AC5^–/–). In ventricular myocytes from AC5^–/– mice, 1 µM MANT-GTPS decreased I_Ca,L (after 10 min, –23 ± 1 versus 8 ± 2% without MANT-GTPS; p < 0.01, n = 6; Fig. 6, A and B). This reduction of basal I_Ca,L in AC5^–/– myocytes was significantly less pronounced than in WT myocytes (after 10 min, –41 ± 7%; p < 0.05, n = 6), indicative for an AC5-specific effect of MANT-GTPS.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effect of 1 µM MANT-GTPS on basal L-type Ca2+ current in AC5–/– murine cardiomyocytes. A, original traces in the absence and presence of 1 µM MANT-GTPS at the time points 0 and 10 min. B, averaged time course of whole-cell L-type Ca2+ current density from experiments with murine ventricular myocytes lacking type 5 AC (AC5–/–) in the absence (filled symbols; n = 6) and in the presence of 1 µM MANT-GTPS (open symbols; n = 6). Starting from a holding potential of –60 mV the current was elicited by a test potential of +10 mV preceded by a prepulse to –40 mV.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Effect of 1 µM MANT-GTPS on isoproterenol-stimulated L-type Ca2+ current in AC5–/– murine cardiomyocytes. Cells were superfused by isoproterenol containing bath solution for 8.2 ± 0.1 min (control) and 8.2 ± 0.1 min (MANT-GTPS), respectively. A, original traces before and after 100 nM (–)-isoproterenol stimulation and in the absence and presence of 1 µM MANT-GTPS at the time point 0 min and at the maximal increase of L-type Ca2+ current. B, averaged time course of whole-cell L-type Ca2+ current density from experiments with murine ventricular myocytes lacking type 5 AC (AC5–/–) before and after 100 nM (–)-isoproterenol stimulation and in the absence (filled symbols; n = 5) and in the presence of 1 µM MANT-GTPS (open symbols; n = 5). Starting from a holding potential of –60 mV, the current was elicited by a test potential of +10 mV preceded by a prepulse to –40 mV. C, maximal increase of L-type Ca2+ current density (percentage) compared with basal current (=0) in the absence (black bar; n = 5) and in the presence (white bar; n = 5) of 1 µM MANT-GTPS. D, control current density-voltage relationship from AC5–/– myocytes in the absence (filled symbols; n = 5) and in the presence (open symbols; n = 5) of 100 nM (–)-isoproterenol measured at the beginning and at the end of the experiments. E, current density-voltage relationship from AC5–/– myocytes with 1 µM MANT-GTPS diluted in the pipette solution in the absence (filled symbols; n = 5) and in the presence (open symbols; n = 5) of 100 nM (–)-isoproterenol measured at the beginning and at the end of the experiments, respectively.

	Discussion

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In our present study, we characterized the effect of the novel competitive AC inhibitor MANT-GTPS on murine ventricular I_Ca,L. MANT-GTPS is well suited for electrophysiological studies using intracellular dialysis because it is hydrolysis-resistant. Our findings indicate AC5 to be the major functional isoform mediating acute ₁-adrenergic stimulation of I_Ca,L. In the presence of MANT-GTPS, we find an attenuation of I_Ca,L increase following -adrenergic stimulation via isoproterenol, confirming previous findings obtained with AC5-deficient mice (Okumura et al., 2003a). Most importantly, we found that in AC5-deficient mice, 1 µM MANT-GTPS did not further attenuate maximal I_Ca,L response to isoproterenol. Considering the similar affinities of MANT-GTPS to the cardiac isoforms AC5 and AC6 (Gille et al., 2004), we conclude that AC5 plays a major role in mediating immediate -adrenergic stimulation of L-type Ca²⁺ channels in ventricular cardiomyocytes.

Using the lower concentration of MANT-GTPS, 100 nM, a moderate inhibition of isoproterenol effects was obtained without concomitant inhibition of basal currents. Furthermore, I_Ca,L increase following isoproterenol was maintained here, whereas in the presence of 1 µM nucleotide, I_Ca,L decreased continuously during the course of experiments. This indicates that the isoproterenol-stimulated current is more sensitive toward the AC inhibitor than the basal current. The only minimal effect of MANT-GTPS on basal current in AC5-deficient mice reveals that part of the inhibition of basal current in WT mice is due to AC5 inhibition. Additional mechanisms, such as involvement of a less sensitive functional AC isoform in basal current regulation, or other non-specific effects of the nucleotide cannot be further elucidated by our experiments. Thus, our data reveal AC5 to activate tonically basal Ca²⁺ current activity. This interpretation might at first glance contrast to the unchanged basal current values reported for AC5^–/– mice by group comparison (Okumura et al., 2003a), but long-term compensatory mechanisms are to be expected in such genetic models. Considering the lower isoproterenol effect on I_Ca,L in AC5^–/– myocytes, one might also speculate on decreased abundance of potential phosphorylation sites, e.g., by lower expression of the poreforming, protein kinase A-sensitive 1C subunit coded by exon 1a (van der Heyden et al., 2005) or altered composition of the heteromeric channel complex by pore-forming 1C and auxiliary -subunits (Bünemann et al., 1999).

MANT-nucleotides represent novel experimental tools in the search for potential therapeutic compounds because they are competitive AC inhibitors (Gille and Seifert, 2003). Recent high-resolution crystallographic data underline their eminent role as lead compounds for the development of potent and isoform-specific AC inhibitors (Mou et al., 2005, 2006). Our present data show that isoproterenol effects are reduced by MANT-GTPS via concentration-dependent inhibition of AC5. Considering the sympathetic overdrive in heart failure, this indicates MANT-nucleotides as promising starting points regarding innovative therapeutic strategies. MANT-GTPS as a prototypical competitive AC inhibitor shows AC5-mediated effects, as proven by its lack of effect on isoproterenol-stimulation of I_Ca,L in AC5^–/– cardiomyocytes. Because AC5 deficiency seems to be protective against heart failure due to pressure overload (Okumura et al., 2003b), this demonstrates that MANT-nucleotides could serve as a novel approach in the therapy of cardiac diseases. This is supported by the known benefits of -adrenoceptor antagonists in the treatment of heart failure. -Adrenoceptor antagonists have become standard treatment of heart failure, although it is unclear what the exact underlying mechanism is. Some data suggest that signaling via ₂-adrenoceptors would be beneficial (Dorn et al., 1999; Du et al., 2000), whereas ₁-adrenoceptor signaling obviously is detrimental (Engelhardt et al., 1999; Lohse et al., 2003). This divergence is supported by findings on the differential signaling cascades and L-type Ca²⁺ channel regulation by ₁- and ₂-adrenoceptors (Foerster et al., 2003, 2004; Xiao et al., 2006). A major difference is dual signaling via G_i and G_s proteins by ₂-adrenoceptors, and only G_s in case of ₁-adrenoceptors. Hence, adenylyl cyclase inhibitors might be an attractive alternative to -adrenoceptor antagonists due to their selective prevention of an (excessive) increase of cAMP.

In future studies, we will have to design potent cell-permeable AC5 inhibitors because the currently available AC inhibitors are not cell membrane-permeable. A feasible approach is the design of pronucleotides that can be deprotected and phosphorylated within cells (Laux et al., 2004). Moreover, we have already shown that MANT-nucleoside 5'-diphosphates are phosphorylated to the corresponding triphosphates by cellular kinases (Gille et al., 2004). It should be emphasized that the concept of AC5 inhibition for the treatment of cardiovascular disease is quite different from the current concept of ₁-adrenoceptor blockade because ACs integrate the input from multiple receptor systems (Hanoune and Defer, 2001). Our present study shows that the combination of pharmacological studies with gene knockout studies is very powerful at delineating signal transduction pathways and establishing novel therapeutic concepts.

	Acknowledgements

 
We thank Dr. Andreas Gille (University of Heidelberg, Heidelberg, Germany) for helpful discussions and Sigrid Kirchmann and Jens Reifenrath for skillful technical support.

	Footnotes

 
This work was supported by Deutsche Forschungsgemeinschaft Grant Se 529/5-1 (to R.S.), National Institutes of Health Grants HL069020, AG023137, AG028854, AG014121, HL033107, HL59139, and HL069752 (to S.F.V.), Köln-Fortune KF 186/2004 (to S.H.), and Zentrum für Molekulare Medizin der Universität zu Köln A5 (to S.H.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.118422.

ABBREVIATIONS: AC, adenylyl cyclase; I_Ca,L, L-type calcium current; MANT, 2'(3')-O-(N-methylanthraniloyl); GTPS, guanosine 5'-[-thio]-triphosphate; WT, wild-type; I/V, current-voltage.

Address correspondence to: Dr. Stefan Herzig, Department of Pharmacology, University of Cologne, Gleueler Str. 24, D-50931 Köln, Germany. E-mail: stefan.herzig{at}uni-koeln.de

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