1Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio; and 2Venetian Institute of Molecular Medicine, Padua, Italy
Submitted 10 February 2005 ; accepted in final form 21 March 2005
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ABSTRACT |
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adenovirus; protein kinase A; phosphodiesterase; L-type Ca2+ channel
The ability to measure cAMP accumulation has been an important tool for understanding the signaling mechanisms underlying cAMP-dependent responses. However, earlier studies have relied on techniques that limit both the spatial and temporal resolution of changes in cellular cAMP levels. More recently, a variety of biosensors have been developed that can monitor real-time changes in cAMP activity in living cells. These biological probes are based on modifications of proteins or protein complexes such as cyclic nucleotide-gated (CNG) ion channels or PKA.
The first cAMP biosensor took advantage of the change in fluorescence resonance energy transfer (FRET) that occurs when cAMP levels increase, resulting in the dissociation of catalytic and regulatory subunits of PKA tagged with appropriate fluorescent dyes (1). However, the use of this probe required it to be microinjected into cells. Goaillard et al. (5) demonstrated that it is feasible to dialyze this probe into cardiac myocytes through patch-clamp pipettes. However, difficulties with obtaining sufficient quantities of the probe and problems with diffusion of such a large protein complex remain an impediment to the routine use of this approach.
More recently, a genetically encoded version of this biosensor was created by linking the type II regulatory and catalytic subunits of PKA to cyan (CFP) and yellow fluorescent protein (YFP), respectively. Plasmids encoding each subunit can be expressed in cells using standard transfection techniques (20). This results in targeting of the cAMP biosensor to the same locations where endogenous PKA is found within the cell. This approach has been used to study cAMP responses in neonatal cardiac myocytes (11, 20). However, transfection is not an efficient means of expressing recombinant proteins in some cell types, especially adult cardiac myocytes.
The use of adenoviruses as vectors has proved to be a more reliable way of expressing recombinant proteins in cardiac myocytes (8). In fact, this approach has been used to express modified CNG channels in adult cardiac myocytes (13). These are nonselective cation channels gated by cAMP or cGMP. The mutants created have a higher sensitivity and selectivity for cAMP (12). Increases in cAMP activity can be detected with the use of electrophysiological techniques to monitor changes in the membrane conductance due to activation of these channels. However, because these CNG channels only sense the subsarcolemmal environment, they may be unable to provide information about what is happening to cAMP activity in other locations in the cell, such as where PKA, the primary effector responsible for functional responses, is located.
The present study describes a method utilizing multiple adenoviruses to express the genetically encoded PKA-based cAMP biosensor in adult ventricular myocytes. We then test the hypothesis that the biosensor introduced using this method is sensitive enough to respond to -adrenergic stimulation over a physiologically relevant range of agonist concentrations and that it can detect muscarinic inhibition and as well as muscarinic facilitation of those
-adrenergic responses.
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METHODS |
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Isolation and primary culture of cardiac myocytes.
Cardiac ventricular myocytes were isolated from adult Hartley guinea pigs as previously described (3), using methods in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocols were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University. Briefly, guinea pigs were anesthetized with a pentobarbital injection (150 mg/kg ip). The hearts were quickly excised and the coronary arteries perfused via the aorta with physiological salt solution (PSS) containing (in mM) 140 NaCl, 5.4 KCl, 2.5 MgCl2, 1.5 CaCl2, 11 glucose, and 5.5 HEPES, pH 7.4. After perfusion with nominally Ca2+-free PSS for 5 min, enough collagenase (Type 2; Worthington) was added to achieve a final concentration of 0.5 mg/ml. After 2035 min of digestion at 37°C, the ventricles were removed and placed in a Kraftbrühe solution. The tissue was then minced and single myocytes were obtained by being filtered through nylon mesh.
Microbial contamination of the cell suspension was minimized by gravity sedimentation in a sterile, serum-free, Dulbecco's modified Eagle medium (GIBCO-Invitrogen) containing 1% penicillin-streptomycin (21). The myocytes were then placed in 35-mm plates containing this same culture medium (105 rod-shaped cells/plate) and immediately infected with Ad-RII-CFP, Ad-CAT-YFP, and Ad-TA and incubated at 37°C and 5% CO2 for 1.5 h. After this time, the virus-containing medium was replaced with fresh medium and the cells were further incubated for 4872 h. Relative protein expression was monitored by measurement of CFP and YFP fluorescence.
Fluorescence imaging. Infected cells were transferred to a perfusion chamber (234 µl total volume; model 26G, Warner Instruments) mounted on the stage of an inverted microscope (model IX70, Olympus). Cells were bathed in PSS that flowed at a rate of 2 ml/min. Images were obtained using a x40 water-immersion objective (1.3 numerical aperture; Olympus) and a charge-coupled device camera (Orca ER, Hamamatsu). Excitation was achieved using a 175-W xenon arc lamp (Lambda DG-4, Sutter Instruments). CFP/FRET excitation was achieved using a D436/20 band-pass filter with a 455DCLP dichroic mirror. YFP excitation was achieved using an HQ500/20 band-pass filter with a Q515LP dichroic mirror. CFP and YFP emissions were measured using D480/30 and D535/40 band-pass filters, respectively. For ratiometric FRET experiments, CFP and YFP emissions were measured simultaneously using a Dual View Micro-Imager (Optical Insights) equipped with a 505CXR beam splitter. All filters were obtained from Chroma Technology. Exposure times ranged from 0.5 to 2 s. Fluorescence images were acquired using 2 x 2 binning and analyzed with the use of Simple PCI imaging software (Compix).
Changes in cAMP activity were defined as the relative change in the ratio of the background corrected fluorescence intensity at the emission wavelength for CFP and YFP measured for a specified region of interest (ROI). For most experiments, the ROI was the entire cell. However, for some experiments, fluorescence intensity profile measurements were obtained using a rectangular (3.2 µm x 14.1 µm) ROI-oriented parallel to the long axis of the cell. An intensity profile was then obtained by measuring the fluorescence over the entire width of the ROI in 0.32-µm increments along its length.
Electrophysiology.
Whole cell L-type Ca2+ currents were recorded as described before using the conventional patch-clamp technique (2, 3). Microelectrodes had resistances of 24 M when filled with an intracellular solution containing (in mM) CsCl 130, 20 TEA-Cl, 5 EGTA, 5 MgATP, 0.06 TrisGTP, and 5.5 HEPES (pH 7.2). Cells were bathed in PSS. The voltage-clamp protocol employed a holding potential of 80 mV. A 50-ms prepulse to 30 mV was used to inactivate Na+ channels. Changes in the magnitude of the Ca2+ current were monitored by applying a 100-ms test pulse to 0 mV once every 1520 s. The amplitude of the Ca2+ current was determined by measuring the absolute magnitude of the peak inward current during the step depolarization to 0 mV.
Drugs. Isoproterenol bitartarate (Iso; Sigma RBI) and acetylcholine hydrochloride (ACh; Alexis Biochemicals) were prepared as aqueous stock solutions. IBMX (Calbiochem) was prepared as a stock solution in DMSO. To avoid oxidative degradation of Iso, 50 µM ascorbic acid were added to all solutions.
Statistics. All data are expressed as means (SD) of the results obtained from n number of cells. Statistical significance between two groups was defined by paired Student's t-test with P values <0.05.
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RESULTS |
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Responses to phosphodiesterase inhibition.
A consequence of the balance between cAMP synthesis and degradation that exists under basal conditions is that cAMP-dependent responses can be elicited by either stimulating adenylyl cyclase activity or inhibiting phosphodiesterase activity. This is supported by that fact that nonspecific phosphodiesterase inhibitors such as IBMX are capable of producing cAMP-dependent functional responses that are similar in magnitude to those produced by -adrenergic receptor stimulation alone (17). Consistent with this idea, we found that exposure to IBMX (Fig. 2) caused a reversible change in the FRET response. The average magnitude of the response to 100 µM IBMX was a 5.9% (SD 1.75; n = 3) increase in the CFP/YFP fluorescence ratio.
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Responses to muscarinic receptor activation.
In addition to demonstrating that the biosensor is sensitive enough to detect changes in cAMP activity produced by physiologically relevant levels of AR activation, it would also be important to demonstrate whether or not it can be used to detect changes in cAMP activity associated with activation of signaling pathways that modulate
-adrenergic responses. To address this question, we studied the effects of ACh, a muscarinic receptor agonist, on cAMP activity in the absence and presence of various levels of
AR activation. ACh significantly decreased the fluorescence ratio observed in the presence of maximally stimulating concentrations of Iso (Fig. 5). Exposure to 200 nM Iso increased the fluorescence ratio 5.1% (SD 1.66) over baseline, and subsequent addition of 30 µM ACh decreased this value to 2.6% (SD 1.14; n = 3), which represents a 51% (SD 7.0) reduction in measured cAMP activity. This inhibitory effect reversed rapidly on washout of ACh, with the fluorescence ratio returning to the level it was at just before exposure to the muscarinic agonist.
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DISCUSSION |
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The subcellular expression pattern of the probe introduced using adenoviruses (see Fig. 1) is consistent with the expression pattern observed in neonatal cardiac myocytes, where RII-CFP and C-YFP were found near the Z line of the sarcomere (20). This is also consistent with the expression pattern of endogenous PKA, which is determined by the interaction of the RII subunit with A kinase anchoring proteins (19). A kinase anchoring proteins, in turn, interact with other myocyte proteins, such as L-type Ca2+ channels and ryanodine receptors, which are concentrated in or near the T tubule network (7). Although targeting the biosensor to specific subcellular locations limits its ability to detect changes in cAMP activity throughout the intracellular compartment, expression in the same location as endogenous PKA is likely to improve the correlation between cAMP responses detected by this probe and functional responses mediated by PKA.
The sensitivity of this biosensor to changes in cAMP is also a function of its binding affinity for cAMP. The actual affinity of the PKA RII subunit for cAMP is 100300 nM (11). Again, because the probe is based on PKA, the primary effector for cAMP-dependent responses in ventricular myocytes, one would expect it to respond to levels of
-adrenergic stimulation that produce functional responses. Consistent with this idea, we found that the apparent sensitivity of the probe to
AR stimulation closely correlated with the sensitivity of L-type Ca2+ channels in cells expressing the same probe. It is also important to note that expression of the probe did not appear to adversely affect PKA-dependent functional responses. In previous studies (2, 3), we have determined that
AR activation stimulates the L-type Ca2+ current in adult guinea pig ventricular myocytes with an EC50 of
1 nM and that maximally stimulating concentrations of agonist increases the magnitude of the current by 200250% over baseline. This is consistent with the sensitivity and the magnitude of the L-type Ca2+ channel response observed in cells expressing the exogenous PKA-based biosensor described in the present study.
CNG ion channels have also been used to detect cAMP changes in adult rat ventricular myocytes. However, they appear to exhibit a distinctly different sensitivity to agonist stimulation. Rochais et al. (13) demonstrated that the CNG channel expressed in cardiac myocytes responds only to concentrations of Iso >10 nM. This is 100-fold less sensitive than the PKA-based probe used in the present study (see Fig. 4). Furthermore, the CNG channel was unable to detect changes in cAMP activity in myocytes exposed to concentrations of IBMX (100 µM) expected to maximally inhibit PDE activity (13). Again, this is distinctly different from the response we observed, where the same concentration of IBMX produced a robust change in the fluorescence ratio due to cAMP activation of the PKA-based probe (see Fig. 2). The most likely explanation for the difference in the apparent sensitivity of the two biosensors has to do with the location within the cell where they are expressed and/or their affinity for binding cAMP. Other than being in the plasma membrane, the exact location that CNG channels are found when expressed in cardiac myocytes is not known. However, their low sensitivity to agonist induced responses suggests that they may be located in a domain that is separated from the source of cAMP generation. Furthermore, these modified CNG channels have a significantly lower affinity (110 µM) for cAMP (12). Together, these factors are likely to affect the ability of CNG channels to detect small changes in cytosolic cAMP activity in cardiac ventricular myocytes.
The fact that the cAMP biosensor used in the present study produced reversible responses indicates that reassociation of the recombinant PKA subunits takes place when cAMP levels return to baseline and that mixing of endogenous and recombinant subunits does not occur to any significant degree. More recent studies (18) suggest that the consistent reversibility of these responses might be because cAMP binding does not necessarily result in complete dissociation of regulatory and catalytic subunits, especially with type II PKA. The reversibility of the biosensor's response to cAMP made it feasible to use this probe to detect the more complex temporal responses to muscarinic receptor activation.
In the present study, we found that exposure to ACh produced a decrease in cAMP activity but only after cAMP levels had first been elevated by AR activation (see Fig. 5). Furthermore, washout of ACh produced a transient increase in cAMP activity but only in the presence of a submaximally stimulating concentration of Iso (see Fig. 6B). There was no rebound increase in cAMP on washout of ACh in the absence of Iso (Fig. 6A) or in the presence of a maximally stimulating concentration of Iso (see Fig. 5). These results are consistent with the idea that the effect that muscarinic receptor activation has on cAMP-dependent functional responses in cardiac myocytes is actually much more complex than simply inhibition of
AR-induced cAMP production. The net response is a balance between inhibitory and stimulatory mechanisms (6). During muscarinic receptor activation, the dominant effect is inhibition of cAMP responses due to inhibition of adenylyl cyclase type 5 and/or 6 by the
-subunit of Gi. However, muscarinic receptor activation can produce a stimulatory response that is consistent with Gi
-subunits activating adenylyl cyclase type 4 and/or 7 (3). This stimulatory effect can be observed as a transient rebound response on washout of ACh. The fact that this type of rebound response in ventricular myocytes can only be observed in the presence of a submaximally stimulating concentration of
AR agonist supports the idea that such effects involve the stimulation of cAMP production. However, because of the transient nature of the response, it has not been possible to prove this hypothesis by directly measuring changes in cAMP activity until now.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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