Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Background and purpose Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. Experimental approach We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. Key results PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Conclusions and implications Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.


Introduction
According to conventional understanding, G proteins transduce signals from activated G protein-coupled receptors (GPCRs) via second messengers to regulate numerous downstream signalling targets in the cell [1,2]. G proteins are found on the cytoplasmic side of the plasma membrane, composed of a guanine nucleotide binding a subunit (Ga) and a bc dimer (Gbc). Upon GPCR activation, GDP is exchanged for GTP on Ga, and both the GTPliganded Ga and the Gbc dimer regulate downstream targets. Five subfamilies of Ga have been classified, whereof Ga i/o is the only one able to inhibit adenylyl cyclase (AC) and the production of cAMP [3].
The predominant G i -coupled receptor in the heart ventricle is the M 2 muscarinic acetylcholine receptor [4]. Constitutive activity of this receptor exerts a mild, continuous inhibition of AC activity in normal rat ventricular cardiomyocyte membranes [5][6][7]. It is well established that the muscarinic system also antagonizes the inotropic responses mediated by b-adrenoceptors (bARs) [4], known as accentuated antagonism [8,9].
The major stimulatory input in the myocardium comes from the b 1 -and b 2 ARs, whereby activation of these receptors leads to positive inotropic responses. In 1995, Xiao et al. made the intriguing observation that pertussis toxin (PTX), known to cause ADP-ribosylation of the Ga i subunit, uncoupling the GPCRs from G i , enhanced b 2 AR-but not b 1 AR-mediated positive inotropic responses in isolated rat cardiomyocytes [10]. Together with subsequent studies [11][12][13][14], it has been widely accepted that in addition to G s , the b 2 ARs also couple to G i in native systems while the b 1 ARs do not.
However, we recently reported that the isoproterenol-mediated inotropic response in left ventricular muscle strips was potentiated by PTX [15]. When examined separately, both the b 1 -and b 2 ARmediated inotropic response (bAR-IR) and cAMP accumulation in isolated ventricular cardiomyocytes were increased by PTX pretreatment [16]. Additional data from rat [17], guinea-pig [18] and normal [19] as well as transgenic mice overexpressing the b 2 AR [20] indicate that b 1 AR-evoked contractility in the heart may also be regulated by G i activity. Due to conflicting data on the role of G i in b 1 AR signalling, we wanted to further investigate this issue.
It has previously been reported that AC activity evoked by forskolin and agonists at G s -coupled receptors can be increased by prior PTX treatment in various cell types [21][22][23][24], as well as in cardiomyocytes [25] and sarcolemmal membranes from failing human myocardium [26]. However, these studies did not take into account the presence of constitutively active GPCRs which are known to regulate basal AC activity [7,27]. It has been suggested by El-Armouche et al. [28], and shown by Rau et al. [17] through overexpression of Ga i2 as well as by Hussain et al. [15] by the use of phosphodiesterase (PDE) 3 and 4 inhibitors that G i may have receptor-independent effects, whereby it directly inhibits AC activity. We examined the possibility that G i may have receptorindependent effects in the absence of constitutively active receptors by using the unique properties exhibited by stereoisomers of the b 2 AR agonist fenoterol. (R,R)-fenoterol was first characterized by Woo et al. (2009) to selectively activate only the G s pathway of the b 2 AR, whereas the other stereoisomers, including (R,S) used in this study, activate both the G s and G i pathways [29]. On this basis, (R,R)-fenoterol has been used as a tool to differentiate G sand G i -coupling of the b 2 AR [30]. We further studied receptorindependent effects of G i by using the direct AC activator forskolin, as well as the effects of PDE3 and 4 inhibition in the absence and presence of known antagonists or inverse agonists.
Our data indicate that in addition to the traditional role of G i in receptor signalling, this G protein exerts a constant intrinsic inhibition upon AC independent of receptor activation.

Animal care
Experiments and animal care were conducted in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Council of Europe no. 123, Strasbourg 1985) and approved by the Norwegian Animal Research Authority. The animals were housed with a 12/12 h cycle at 21uC, food and water available ad libitum.

Measurement of contractility in left ventricular strips
Left ventricular strips (diameter ,1.0 mm) were prepared from male Wistar rats (Taconic, Skensved, Denmark) weighing 300-350 g, mounted in 31uC organ baths containing physiological salt solution with 1.8 mM Ca 2+ , equilibrated and field-stimulated at 1 Hz [31,32]. Contraction-relaxation cycles were recorded and analysed as previously described [33,34]. Maximal development of force (dF/dt) max was measured and inotropic responses were expressed as increase in (dF/dt) max . Concentration-response curves were constructed by estimating centiles (EC 10 to EC 100 ) and calculating the corresponding means, and the horizontal positioning is expressed as -logEC 50 values [33].
In a subset of rats, PTX was administered at a dose of 60 mg/kg i.p. as a single injection three days prior to isolation of the left ventricular muscle strips. Control rats were given a saline injection of equal volume. Data from animals treated with PTX were only included if carbachol inhibition of the bAR-IR was completely abolished (Fig. 1B). To confirm the effectiveness of PTX treatment in vivo (subset of 6 PTX-treated rats and 7 saline-treated rats) and in cardiomyocytes (see below), we measured the level of PTXcatalysed incorporation of [ 32 P]ADP-ribose from [ 32 P]NAD into available G i as previously described [15].

Measurement of cAMP accumulation in left ventricular cardiomyocytes
Adult left ventricular cardiomyocytes were isolated from excised male Wistar rat hearts by retrograde aortic perfusion with a nominally Ca 2+ -free JOKLIK-MEM solution and enzymatic digestion using collagenase (90 U/mL) as previously described [35]. Left ventricular cardiomyocytes were incubated for 20 h in the absence or presence of 1 mg/ml PTX (1.2 ml reaction volume). Experiments were conducted in the presence of the b 1 AR blocker CGP20712 (300 nM), the non-selective PDE inhibitor IBMX (0.5 mM) or PDE3 and 4 inhibitors cilostamide (1 mM) and rolipram (10 mM), as indicated. cAMP accumulation was measured by radioimmunoassay as previously described [36]. Protein was measured with the Coomassie Plus Protein Assay (Pierce, Rockford, IL) according to the manufacturer's protocol and cAMP accumulation was normalized to the amount of protein in each sample.

Measurement of (R,R)-and (R,S)-fenoterol-mediated inotropic responses and ability to activate adenylyl cyclase
To evaluate the b 2 AR-mediated inotropic response, G s -selective (R,R)-fenoterol and G s -and G i -activating (R,S)-fenoterol were assessed by conducting a concentration-response experiment in the presence of a selective b 1 AR antagonist (300 nM CGP20712). Likewise, (R,R)-and (R,S)-mediated activation of AC was assessed by incubating cardiomyocytes for 10 min with increasing concentrations of either stereoisomer (0-100 mM) in the presence of 300 nM CGP20712. All cAMP accumulation experiments in this subset were conducted in the presence of the non-selective PDE inhibitor IBMX (0.5 mM).

Measurement of spontaneous intrinsic G i inhibition upon adenylyl cyclase activity
Receptor-independent G i activity was assessed through forskolin concentration-response curves (50 nM-10 mM) in left ventricular strips and isolated cardiomyocytes in the presence of the nonselective bAR antagonist timolol (1 mM) and a 1 -adrenoceptor antagonist prazosin (0.1 mM, ventricular strips only) with and without prior pre-treatment with PTX (as described above). To determine if spontaneous intrinsic activity of G i regulated basal AC activity, the inotropic response in ventricular strips or cAMP accumulation in cardiomyocytes pre-treated with or without PTX was measured in the presence of both the PDE3 inhibitor cilostamide (1 mM) and PDE4 inhibitor rolipram (10 mM) in the presence of the non-selective bAR antagonist timolol (1 mM) and a 1 -adrenoceptor antagonist prazosin (0.1 mM, ventricular strips only).

Statistics
Data are expressed as mean 6 SEM from n animals. p,0.05 was considered statistically significant (One-way ANOVA and student's t-test). When appropriate, Bonferroni corrections were made to control for multiple comparisons.

G i was essentially inactivated by pertussis toxin
The effectiveness of in vivo PTX treatment to inhibit G i was assessed by measuring PTX-catalysed incorporation of [ 32 P]ADPribose from [ 32 P]NAD into available G i in ventricular tissue. An ,80-90% reduction was seen in the ability of subsequent PTX to ADP-ribosylate G i in vitro in animals pre-treated with PTX compared to animals given saline injection (PTX-treated n = 6, saline-treated control n = 7, see Fig. 1A). It was not possible to distinguish the different Ga i isoforms in the autoradiogram due to the similarity in size (39)(40)(41). Importantly, in the PTX-treated group, data were included only if carbachol-induced inhibition of the bAR-IR was abolished (Fig. 1B). In the (R,R)-and (R,S)fenoterol study, 83% of the PTX-treated rats were included in the study. In the forskolin and PDE inhibitor study 88% of the rats met the criteria and were included.
(R,R)-and (R,S)-fenoterol-stimulated cAMP accumulation was amplified after inactivation of G i (R,R)-and (R,S)-fenoterol-induced AC activation was assessed by measuring cAMP accumulation in isolated left ventricular cardiomyocytes in the presence of the non-selective PDE inhibitor IBMX. Surprisingly, PTX inactivation of G i amplified the concentration-response curve to G s -selective (R,R)-fenoterol (Fig. 1C). (R,R)-fenoterol selectively activated the b 2 AR at the concentration range 10 nM-3 mM, after which it overcame b 1 AR blockade by CGP20712 (not shown). Maximal cAMP accumulation evoked by 3 mM (R,R)-fenoterol was enhanced by 60% from 55 pmol/mg protein in control to 92 pmol/mg protein in the presence of PTX. The EC 50 was not significantly different after PTX treatment (140648 nM in control and 245622 nM after PTX treatment) (Fig. 1C). These EC 50 values were similar to the previously published binding affinity of (R,R)-fenoterol at the b 2 AR (350 6 30 nM [37]).
As expected, (R,S)-fenoterol concentration-response curves were similarly modified by PTX treatment. Maximal cAMP accumulation evoked by the highest selective concentration of (R,S)fenoterol for the b 2 AR (100 mM) was enhanced by 62% from 38 pmol/mg protein in control to 61 pmol/mg protein in the presence of PTX. The EC 50 value was not significantly shifted from 3.560.5 mM in control to 5.060.9 mM after PTX treatment (Fig. 1C). Again, these EC 50 values were similar to the published binding affinity of (R,S)-fenoterol of 3.7 6 0.3 mM [37]. Together, these data demonstrate that PTX amplifies the amount of cAMP produced by both stereoisomers, despite dissimilar G proteincoupling profiles. This suggests that G i exerts inhibitory activity upon AC downstream of receptor coupling to G protein.

Maximal inotropic responses to both (R,R)-and (R,S)-fenoterol were amplified by G i inactivation or PDE inhibition
In untreated (control) left ventricular muscle strips, the highest concentration given of (R,R)-and (R,S)-fenoterol elicited very small inotropic responses of 8.662.7% and 4.462.9% above basal, respectively (b 1 AR antagonist CGP20712 present; Fig. 2A). These values were slightly lower than inotropic responses obtained by b 2 AR stimulation by adrenaline in the presence of CGP20712 (,11% above basal, data not shown).
The inotropic responses evoked by maximal concentrations of both (R,R)-and (R,S)-fenoterol were significantly amplified by either PTX pre-treatment (4466% or 2264% above basal, respectively) or PDE3 and 4 inhibition (96611% or 122619% above basal, respectively) ( Fig. 2A). These data are consistent with the cAMP accumulation data and reinforce the hypothesis that G i may have downstream effects independent of receptor activation in a physiological model using intact, isometrically contracting ventricular muscle. Further, the data reflect that increased cAMP accumulation translates to an increased inotropic response. In the absence of PTX treatment or PDE inhibition it was not possible to determine the EC 50 values for either fenoterol isoform, due to the very small inotropic response. However, in the presence of PTX, the EC 50 of (R,R)-fenoterol was 139618 nM and of (R,S)-fenoterol 4.160.4 mM (Fig. 2B), very similar to that obtained from cAMP accumulation (Fig. 1C) and corresponding to the affinity values reported in the literature [37]. PDE3 and 4 inhibition shifted the concentration-response curves of both stereoisomers to lower concentrations (EC 50 : 58616 nM for (R,R)-and 0.4460.01 mM for (R,S)-fenoterol), indicating that both G i and PDE3 and 4 regulate translation of the cAMP signal to a functional inotropic response.

Forskolin responses are enhanced by inactivation of G i
Forskolin, a direct activator of AC, was used to study receptorindependent effects of G i on AC activity. To eliminate the influence of constitutive bAR activation of G s (known to enhance forskolin responses), all experiments were conducted in the presence of the bAR inverse agonist timolol. cAMP accumulation in response to the maximum concentration of forskolin tested (10 mM; this concentration was sufficient to reach an asymptotic response) was enhanced by 85% by PTX treatment from 269641 pmol/mg protein in control to 498669 pmol/mg protein in the presence of PTX (Fig. 3A).
In control left ventricular strips, forskolin (10 mM) evoked an inotropic response 120615% above basal with an EC 50 of 2.2 mM. After pre-treatment with PTX, the response to forskolin was significantly shifted to 10-fold lower concentrations, yielding an EC 50 of 0.22 mM, with no change in the maximum inotropic response (103620% above basal) at maximal tested concentrations (asymptote reached in range from 1-10 mM, Fig. 3B). Together, these data demonstrate that forskolin-stimulated AC activity and functional response in the absence of G protein-coupled receptor activation is modified by PTX treatment.

Simultaneous inactivation of PDE3 and PDE4 produced a robust cAMP-dependent inotropic response in myocardium only after prior inactivation of G i
The following experiments were conducted in the presence of bAR blockade (timolol, 1 mM), to eliminate a possible effect of residual endogenous noradrenaline or constitutive activation of G s through b 1 -or b 2 ARs. As previously reported [15], and replicated in this study, concomitant PDE3 and PDE4 inhibition (1 mM cilostamide and 10 mM rolipram) was sufficient to elicit a large inotropic response in ventricular strips (77614% above basal) after PTX treatment (Fig. 4A,C right trace). However, simultaneous inhibition of PDE3 and 4 did not cause an inotropic effect in control hearts (0.4260.19% above basal) (Fig. 4A,C left trace), indicating the absence of constitutively active G s -coupled receptors. To test whether the effect of PTX occurred as a result of removing constitutive receptor activation of G i , we evaluated the effect of two established G i -coupled receptor systems in the heart. Neither the muscarinic inverse agonist atropine (1 mM) nor the non-selective adenosine inverse agonist CGS 15943 (1 mM), shown to be an inverse agonist at the G i -coupled A 1 adenosine receptor [38], alone or in combination evoked a change in contractile force above basal (Fig. S1).
To investigate the relationship between cAMP and the inotropic response evoked by PDE3 and 4 inhibition after PTX treatment, cAMP accumulation was measured in isolated ventricular cardiomyocytes with or without PTX pre-treatment (Fig. 4B). Simultaneous inhibition of PDE3 and 4 increased cAMP levels in control cardiomyocytes (5.560.8 pmol/mg protein above basal).
The level of cAMP accumulation evoked by simultaneous inhibition of PDE3 and 4 was significantly increased (,2-fold) in cardiomyocytes pre-treated with PTX (to 12.761.3 pmol/mg protein above basal, Fig. 4B). The effect of PTX was not due to removal of constitutively active G i -coupled receptors, since neither atropine (1 mM) nor CGS 15943 (1 mM) alone or in combination in the presence of simultaneous inhibition of PDE3 and 4 mimicked the effect of PTX treatment (Fig. 4B).

Discussion and Conclusions
Data from this study indicate that G i tonically inhibits basal cAMP production, limiting functional responses. This inhibition appears to be independent of constitutive receptor activation of G s and G i upon AC. In support, (1) PTX treatment increased responses evoked by both the G s -selective (R,R)-and the dually coupled (R,S)-fenoterol isoforms (Fig. 1C & 2A); this should not occur if the PTX effect was dependent upon receptor activity, since b 2 ARs stimulated by (R,R)-fenoterol have been shown to only activate G s [29,39]. (2) PTX treatment amplified forskolinevoked cAMP accumulation and increased the potency of forskolin to evoke an inotropic response (Fig. 3A,B). Responses to forskolin, being a direct activator of AC [40], should not have been enhanced by inactivation of G i ; and (3) PTX treatment revealed intrinsic AC activity upon basal responses only after inhibition of PDE3 and 4, whereas inverse agonists at muscarinic receptors and adenosine receptors were without effect after PDE3 and 4 inhibition (Fig. 4A,B). This indicates that PDE3 and 4 inhibition, even in the presence of the bAR inverse agonist timolol, increased cAMP levels in PTX-treated tissue sufficiently to produce an inotropic response (Fig 4A,B,C). If constitutively active G s -coupled receptors were mediating this effect, an inotropic response should have occurred after PDE3 and 4 inhibition alone in the absence of PTX treatment. Further, PDE3 and, at least in the rat ventricle, PDE4 activity (Fig. 4A,B; [41]) normally degrades low basal levels of cAMP, maintaining basal contractile force. These data highlight the necessity to remove the tonic inhibition of G i upon AC prior to PDE inhibition to allow for the enhanced cAMP signal to be transduced into a functional response. We propose that PTX removes an intrinsic inhibition of G i upon AC that is independent of G i -coupled receptors. That neither the inverse agonist atropine (muscarinic M 2 ) nor the non-selective adenosine receptor antagonist CGS 15943, alone or in combination, elicits effects similar to PTX (Fig. 4B, Fig. S1) indicates that PTX does not simply remove the effect of constitutively active G i -coupled receptors.

G i has a propensity to be in a spontaneously active conformation
There is a good basis indicating that G i has receptorindependent activity. Lutz et al. [26] reported that in sarcolemmal membranes from failing human myocardium, G i rapidly released GDP in a time-and temperature-dependent manner. When GDPbS was used in place of GDP to hold G i in the inactive state, AC activity increased, suggesting relief of tonic inhibition. This effect was replicated by Mn 2+ or high Mg 2+ , which prevent AC inhibition by G i . The authors suggest that AC was inhibited by an empty but apparently active G i (even in the absence of activating GTP), further highlighting the inhibitory potential of presumably inactivated G i . [26].
Further, Piacentini et al. [42] reported that the synthetic GTP analogue Gpp(NH)p had no effect on basal AC activity. With the G s :G i ratio ranging from ,1:10 to 1:40 [43,44], G i would be the predominant G protein activated under these conditions. Since AC may already be maximally inhibited by spontaneously active G i , it is expected that no additional effect of Gpp(NH)p is seen. However, when the stable GDP analogue GDPbS was used, basal AC activity increased, presumably through relief of tonic G i inhibition. Under this condition, subsequent addition of Gpp (NH)p caused inhibition of AC, presumably through competition with GDPbS [42]. Thus, AC inhibition could be relieved by introduction of synthetic GDP-analogues and re-introduced by synthetic GTP-analogues, providing further support for receptorindependent G i intrinsic activity.
Regulators of G protein signalling (RGS) are GTPase-accelerating proteins that promote rapid GTP hydrolysis and consequently inhibit signal transduction by inactivating G proteins and returning them to the GDP-bound heterotrimeric state. The effect of mutated, RGS-insensitive Ga i2 has been studied in cardiomy- , spontaneously active G i dominates, inhibiting basal AC activity and maintaining low cAMP levels which are incapable of eliciting an inotropic response even after inhibition of PDE3 and 4 (inhibition marked by X). After PTX treatment (bottom panel), G i is not only inactivated (through ADP-ribosylation, marked by X) removing its spontaneous intrinsic inhibition upon AC, but also sequesters a large proportion of the shared Gbc pool. This indirectly increases the proportion of receptor-independent spontaneously active G s , leading to increased basal AC activity and cAMP that is normally readily degraded by PDE3 and 4. However, inhibition of PDE3 and 4 (marked by X) allows for translation of this cAMP increase into an inotropic response (see Fig. 4A). The net result is a shift from predominantly spontaneous G i activity towards G s activity, increasing basal AC activity and promoting activation of AC and positive inotropic effects of all inotropic agents working through increased cAMP signalling. (B) NDPK-hypothesis: In the absence of PTX (top panel), NDPK-activation of G i dominates due to excess of G i protein levels over G s , resulting in low cAMP production readily degraded by PDE3 and 4. In the presence of PTX (bottom panel), G i is inactivated by permanent ADP-ribosylation (marked by X). Thus, NDPK B could predominantly activate G s , leading to both increased basal contractile force and cAMP accumulation that becomes revealed after PDE3 and 4 inhibition (marked by X). doi:10.1371/journal.pone.0106608.g005 ocytes derived from embryonic stem cells [45] and mice homozygous for the mutated, RGS-insensitive Ga i2 [46], providing a model for studying active G i . Interestingly, basal inotropic and lusitropic responses were reduced by ,10% in these mice compared to wildtype [46]. Also, G s -dependent stimulation of beating rate by the b 2 -AR agonist procaterol was nearly abolished in foetal RGS-insensitive Ga i2 -cardiomyoctes via a PTX-sensitive mechanism, further indicating that G i exerts tonic inhibition on AC [45].
Chen-Goodspeed et al. [47] reported that human AC5 has high basal activity when expressed in Sf9 cell membranes, which was concentration-dependently inhibited by GTPcS-activated Ga i . AC6 basal activity was low, but could be inhibited by Ga i if previously activated with GTPcS-Ga s [47]. Thus, the two most common AC isoforms in the heart have the tendency to be spontaneously active, providing a substrate for opposition by intrinsic G i activity.
In addition, data suggest that AGS proteins (activators of G protein signalling) may promote G protein activation by either directly acting as a guanine nucleotide exchange factor or by interfering with subunit association/dissociation/trafficking (both dependent and independent of nucleotide exchange) preventing heterotrimeric Gabc formation [48]. Together, these effects of AGS could potentially provide another plausible mechanism for mediation of receptor-independent G i activity. Consistent with this hypothesis, Graham et al. [49] have shown that AGS1, which is selective for Ga i2 and Ga i3 , inhibited cAMP accumulation evoked by constitutively active G s or forskolin in 293T cells [49].

Does PTX treatment shift the balance of intrinsic G i and G s activity upon AC towards G s ?
Although the current data support the hypothesis that G i exerts intrinsic receptor independent inhibition upon spontaneous AC activity, the mechanism is still unknown. Further, we cannot completely rule out the possibility that PTX treatment removes an unknown constitutively active G i -coupled receptor. Despite these limitations, it is reasonable to hypothesize that the ratio of G s :G i has important implications for the regulation of basal AC activity. As reported here, a minimum of 80-90% ADP-ribosylation of G i was necessary to reveal functional effects of PTX treatment in our models. Under this scenario, the normal reported ratio of G s :G i of ,1:10 to ,1:40 would be altered to ,1:1 to ,1:4, giving G s more favourable terms to compete for binding to AC. This is consistent with Lutz et al. [26] who suggested that the AC system may be designed to operate from a predominantly off position (high intrinsic G i activity), as both basal AC activity and G s -coupled receptor activation would be enhanced by PTX inactivation of G i . The mechanism that mediates these effects of PTX treatment remains to be determined.
It has recently been reported that G s and G i compete for an apparently limited pool of bc [50]. Based on this finding, it is possible that as inactive GDP-bound G i becomes ADP-ribosylated by PTX, G i sequesters bc from the total shared pool. Under this scenario, over time, there will be less bc available for G s , consequently increasing the probability of G s to be in its active state (GTP-bound) in addition to the corresponding removal of intrinsically active G i . A plausible hypothesis would be that G i acts as a bc sink. In support, overexpression of bARK-ct, a bc scavenger, resulted in increased AC stimulation [51]. The net effect of PTX treatment would be to shift the balance from largely intrinsic inhibition to a greater intrinsic stimulation upon AC, thereby sensitising AC and enhancing/amplifying those systems known to activate AC (Fig. 5A).
Alternatively, based on the findings of Hippe et al. [52], PTX treatment may shift the ability of endogenous nucleoside diphosphate kinase B (NDPK B) to activate the G i and G s proteins. NDPK B is a G protein histidine kinase that regulates cAMP synthesis and cardiomyocyte contractility independent of GPCRs through the formation of an NDPK B-G bc complex that activates G protein through a phosphotransfer from NDPK B to His-266 in G b [52]. The phosphate is then transferred onto GDP, and the resultant GTP leads to receptor-independent G protein activation [52][53][54]. In the normal state, due to the excess of G i over G s , NDPK B would predominantly activate G i and the stimulatory effects of G s upon basal cAMP generation may be neutralized [52]. After PTX treatment, G i is inactivated by permanent ADP-ribosylation. Thus, NDPK B would predominantly activate (potentiate) G s activity leading to both increased basal contractile force and cAMP accumulation that becomes revealed after PDE3 and 4 inhibition (Fig. 5B). That we also observe enhancement of other cAMP signalling inotropes and forskolin is consistent with this hypothesis. To this end, experiments are currently underway to test both hypotheses, and should provide greater clarity of the mechanism behind intrinsic receptorindependent inhibitory activity of G i upon AC. Figure S1 Effect of PTX, bAR inverse agonist timolol (1 mM), non-selective muscarinic inverse agonist atropine (1 mM) or non-selective adenosine receptor inverse agonist CGS-15943 (1 mM) upon PDE3 (cilostamide, 1 mM) and PDE4 (rolipram, 10 mM)-evoked inotropic response. Data are mean 6 SEM. * P,0.05 vs. PTX, One-way ANOVA with Bonferroni post test adjustment for multiple comparisons. (TIF)