Correspondence to: Jeffrey W. Karpen, Department of Physiology and Biophysics, Box C-240, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. Fax:(303) 315-8110 E-mail:jeffrey.karpen{at}uchsc.edu.
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Abstract |
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Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotidegated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca2+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca2+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the KI of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high Km for cAMP, whereas PDE type I has a low Km for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.
Key Words: adenylyl cyclase, G-protein signaling, calcium influx, GH4 pituitary cells, biosensors
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INTRODUCTION |
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Cyclic nucleotide phosphodiesterases (PDEs),* the crucial terminators of cAMP and cGMP signals, were discovered almost 40 yr ago (
Retinal rod outer segments are the most closely examined second messenger signaling system, in part, because the endogenous CNG channels have been used as real-time detectors of cGMP concentration (
We recently reported that cAMP concentration can be measured accurately with the rat wild-type (WT) olfactory CNG channel, encoded by an adenovirus vector (
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MATERIALS AND METHODS |
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Construction of CNG Channelencoding Adenoviruses
Point mutations were introduced into the subunit of the WT rat olfactory CNG channel (CNG2, CNC
3) using the QuikChange site-directed mutagenesis kit (Stratagene). Overlap extension PCR was used to delete the Ca2+-CaM binding site (amino acids 6190). A replication-defective adenovirus, in which the channel coding sequence containing the E583M mutation replaced the E1 region, was constructed as described previously (
Adenovirus transducing vectors encoding C460W/E583M and 6190/C460W/E583M channels were constructed using the AdEasy system (He, T.-C., et al., 1998). The cDNA encoding the mutant channel was ligated into pShuttle-CMV between the KpnI and XbaI sites. The resultant plasmid was linearized by digestion with PmeI and used to transform Escherichia coli strain BJ5183 that had been transformed with pAdEasy-1. A plasmid containing the adenovirus chromosome encoding the mutated CNG channel was digested with PacI to release the adenovirus chromosome, and this DNA was used to transfect HEK-293 cells. After incubation for 7 d, the virus was released by freezing and thawing, and plaque purified. The purified virus was tested for the presence of the CNG channel cDNA by PCR. A virus containing the channel cDNA was grown in large scale in HEK-293 cells and purified by banding using CsCl step and isopycnic gradients.
Cell Culture and Channel Expression
HEK-293 cells were maintained in culture and infected with adenovirus as described previously (60% confluence in 100-mm culture dishes 24 h before infection with the CNG channelencoding adenovirus constructs (multiplicity of infection = 10 plaque forming units per cell). 2 h after infection, hydroxyurea was added to the cell media at 2 mM final concentration to partially inhibit viral replication. 24 h after infection cells were detached with PBS containing 0.03% EDTA, resuspended in serum-containing medium, and assayed within 12 h.
GH4C1 rat pituitary cells (American Type Culture Collection) were maintained in 13 ml Ham's F-10 medium (Life Technologies Inc.) supplemented with 14.3 mM NaHCO3, 15% donor horse serum (Gemini), and 2.5% FBS, pH 6.8, in 75-cm2 flasks at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were split weekly (1:4) and washed with fresh medium twice weekly. Cells were plated at 60% confluence in 100-mm culture dishes 24 h before infection with the CNG channelencoding adenovirus constructs (multiplicity of infection = 50 plaque forming units per cell). 48 h after infection cells were detached, resuspended in serum-containing medium, and assayed within 12 h.
Electrical Recording
To assess the cyclic nucleotide sensitivity of different CNG channel constructs, excised, inside-out patch recordings were made at room temperature (2021°C) using an Axopatch-200A patch-clamp amplifier (Axon Instruments Inc.). Pipettes were pulled from borosilicate glass and heat polished. Pipettes were lowered onto the cells and gigaohm seals were formed. Patches were excised by shearing cells from the pipette with a jet of liquid. Ionic currents were elicited by 250-ms pulses to membrane potentials of +50 and -50 mV from a holding potential of 0 mV. Current records were sampled at five times the filter setting and stored on an IBM compatible computer. Records were corrected for errors due to series resistance (pipette resistance was 4.1 ± 0.1 M). Both the pipette and bath solutions contained the following (in mM): 130 NaCl, 2 HEPES, 0.02 EDTA, and 1 EGTA, pH 7.6. Cyclic nucleotideinduced currents were obtained from the difference between currents in the presence and absence of cyclic nucleotides. Doseresponse curves for cAMP and cGMP were obtained at +50 and -50 mV in the same patch. The effects of the modifications were assessed using the Hill equation, I/Imax = [cNMP]N/([cNMP]N + K1/2N), where I/Imax is the fraction of maximal current, cNMP represents cyclic nucleotide, K1/2 is the concentration that gives half-maximal current, and N is the Hill coefficient, an index of cooperativity.
Detection of cAMP in Populations of Cells
Homomultimeric CNG channels comprised of the rat olfactory channel subunit are permeable to Ca2+ (
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We used the fluorescent indicator fura-2 to monitor Ca2+ influx in cell populations. Cells were loaded with 4 µM fura-2/AM (the membrane-permeant form) and 0.02% pluronic F-127 at room temperature for 3040 min (HEK-293 cells) or 70 min (GH4C1 cells), in a buffer containing Ham's F-10 medium supplemented with 1 mg/ml BSA fraction V (BSA, Fisher) and 20 mM HEPES, pH 7.4. In some experiments, HEK-293 cells were loaded with a higher concentration of fura-2/AM (16 µM) for comparison (see below). Cells were washed twice, then resuspended in a solution containing the following (in mM): 145 NaCl, 11 D-Glucose, 10 HEPES, 4 KCl, 1 CaCl2, 1 MgCl2, and 1 mg/ml BSA, pH 7.4 (34 x 106 cells /3 ml buffer solution), and assayed using an LS-50B spectrofluorimeter (Perkin Elmer). Additions were made by pipetting stock solutions into a stirred cuvette. The mixing time was estimated to be on the order of 5 s. Fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 510 nm. Under these conditions, Ca2+ influx causes a decrease in fluorescence (F), which was expressed relative to the pre-stimulus fluorescence (F0) to correct for variations in dye concentration, and to allow for comparison of results on different batches of cells. Data were sampled at 0.5 Hz and filtered at 0.1 Hz (see Fig 3A and Fig B) or sampled at 6 Hz and filtered at 1.2 Hz (see Fig 3C and Fig D).
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The measurement of absolute Ca2+ influx rates using fluorescence requires that fura-2 overwhelm endogenous Ca2+ buffers, and that the relation between fluorescence changes and Ca2+ entry be known (
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(1) |
where F represents fura-2, KF the dissociation constant for Ca2+ binding to fura-2, B the endogenous buffer, KB the dissociation constant for Ca2+ binding to the endogenous buffer, and CaT the total Ca2+ concentration that freely exchanges between fura-2 and the endogenous buffer. The equilibrium assumption is reasonable given the time scale of the experiments presented here (tens to hundreds of seconds) and the time scale of binding and unbinding of Ca2+ from fura-2 and endogenous buffers (milliseconds). Equation 1 indicates that with [F] and [B] unchanged by the binding of Ca2+, [CaF] will be a constant fraction of [CaT], and therefore directly proportional to the amount of entering Ca2+. In experiments in which quantitative information was extracted (e.g., the in vivo estimate of KI for PDE inhibitors), we purposely worked at low levels of Ca2+ influx. A typical experiment is shown in Fig 1, in which addition of 0.5 µM forskolin (an adenylyl cyclase activator) and 50 nM rolipram (a PDE inhibitor) caused a rise in cAMP and an increase in Ca2+ influx. There are several lines of evidence that free fura-2 levels did not change significantly at these low influx rates, and that depletion of endogenous buffers did not distort the measurements. First, after a brief delay (during which cAMP rose to steady level), the Ca2+ influx rate was constant for a substantial period (linear fit in Fig 1). Second, the changes in fluorescence over which Ca2+ influx rates were measured were always a very small fraction of the total change in fluorescence measured when saturating Ca2+ was admitted into the cells (by adding 30 µl of 10% Triton to the cuvette). The fluorescence changes used in the linear fits were generally <10% of the saturated fura-2 response, indicating that unbound fura-2 was predominant. At high levels of Ca2+ influx, nonlinearities in the traces are likely due to depletion of fura-2 and Ca2+ pumping mechanisms. Third, increasing the external fura-2/AM concentration from 4 to 16 µM significantly altered the intracellular fura-2 concentration but had no effect on the measurement of relative Ca2+ influx rates or estimates of PDE inhibitor KI. The intracellular concentrations of fura-2 were not determined, but a previous study of neuroblastoma cells and isolated pulmonary artery endothelial cells found that 60 min loading with 10 µM fura-2/AM yielded intracellular fura-2 concentrations of 130 µM (
510 µM). Thus, the ratio [B]/KB in Equation 1 was estimated to be 75. KF for fura-2 was estimated to be 150 nM. Thus, the [F]/KF values under our two loading conditions are expected to be 233 and 933. At the higher loading condition, fura-2 may be overwhelming the low affinity cellular buffers. However, as pointed out above, this is not necessary as long as the buffers are not being depleted. It is very likely that low affinity buffers are present at a high enough concentration that they are not being depleted by the low amounts of entering Ca2+. Two of the lines of evidence cited above indicate that buffers (including any high affinity buffers that may be present) are not affecting the proportion of Ca2+ detected by fura-2: the linearity of the influx traces (Fig 1) and the observation that increasing fura-2 by a factor of four does not alter the measurement of relative Ca2+ influx rates. These results strongly suggest that any high affinity buffers are being overwhelmed by the fura-2 concentrations used.
Unless otherwise stated, data are representative of four experiments, and quantified results are given as mean ± standard error. Forskolin, VIP, nimodipine, and PDE inhibitors were obtained from Calbiochem. Fura-2/AM and pluronic F-127 were purchased from Molecular Probes. Unless otherwise noted, all other chemicals were obtained from Sigma-Aldrich.
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RESULTS |
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Improving the cAMP-sensing Capabilities of CNG Channels
We have previously shown that adenovirus-expressed rat olfactory CNG channels have several properties that make them excellent cAMP sensors (
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We first introduced the mutation E583M into the rat olfactory CNG channel subunit, guided by the work of
subunit of the bovine retinal rod CNG channel (D604M) increased the sensitivity to cAMP and decreased the sensitivity to cGMP. The olfactory channel has a higher overall sensitivity to cyclic nucleotides than the rod channel, and, in excised patches, the E583M channel displayed increased sensitivity for cAMP and decreased sensitivity for cGMP (Table II; compare Fig 2, panel A with B). Furthermore, cGMP was only a partial agonist of the E583M channel. The current elicited by saturating cGMP was
40% of the current elicited by saturating cAMP (Table 2 and Fig 2 B).
To further increase the sensitivity to cAMP, we constructed a second mutant, C460W/E583M, guided by studies showing that modifications to C460 (or C481 in the retinal channel) increased the sensitivity of CNG channels to cyclic nucleotides (10-fold lower K1/2, Table 2 and Fig 2 C).
To remove regulation of the channel by Ca2+-CaM binding, we deleted residues 6190, described previously by 6190/C460W/E583M, is almost as sensitive to cAMP as the E583M channel (Table 2 and Fig 2 D), yet it is virtually insensitive to cGMP. In fact, the current elicited by saturating cGMP was <20% of the current elicited by saturating cAMP (Table 2 and Fig 2 D). Thus, we have generated two very useful channel constructs for the measurement of cAMP: the
6190/C460W/E583M channel that is sensitive to cAMP at the upper end of the physiological range (
150 µM), and the C460W/E583M channel that is sensitive to cAMP at the lower end of the physiological range (
0.15 µM).
Assessment of the Relative cAMP Sensitivity of Channel Constructs In Vivo
We next wished to assess the ability of each channel construct to detect increases in local cAMP concentration. Changes in cAMP concentration were detected using fura-2 to monitor Ca2+ influx through CNG channels (see MATERIALS AND METHODS for details). We measured Ca2+ influx induced by different concentrations of forskolin, an AC activator, in the presence of 100 µM IBMX, a nonselective PDE inhibitor. This approach allowed us to compare the cAMP sensitivities of the different channels to changes in cAMP, regardless of variations in expression levels between experiments. In HEK-293 cells expressing the WT channel, addition of forskolin was followed by a brief delay and an increase in Ca2+ influx (Fig 3 A). The delay decreased and the rate of Ca2+ influx increased in a dose-dependent manner. Neither effect was saturated at 50 µM forskolin. In cells expressing the E583M channel, addition of forskolin was also followed by a brief delay and an increase in Ca2+ influx (Fig 3 B). As with the WT channel, the delay and the initial rate of Ca2+ influx were dose-dependent. However, both effects saturated at 20 µM forskolin (the 20- and 50-µM forskolin traces overlap; Fig 3 B). In cells expressing the C460W/E583M channel, both the delay and the rate of Ca2+ influx were saturated at 5 µM forskolin (Fig 3 C). In cells expressing the 6190/C460W/E583M channel, addition of forskolin caused similar increases in Ca2+ influx as the E583M channel, including saturation of the delay and rate of influx at 20 µM (Fig 3, compare B and D). The increased response of the modified channels to forskolin treatment is consistent with the apparent cAMP affinities measured in excised patches in the absence of Ca2+-CaM (C460W/E583M > E583M
6190/C460W/E583M > WT channels). There are several factors that may have contributed to the apparent lack of a Ca2+-CaM effect on the channels. As pointed out in MATERIALS AND METHODS, the concentrations of fura-2 being used are likely overwhelming high affinity cellular Ca2+ buffers, in terms of the fraction of incoming Ca2+ that is bound and, therefore, fura-2 is probably significantly reducing the effects of Ca2+-CaM. It is also possible that HEK-293 cells do not produce enough CaM to regulate heterologously expressed CNG channels, or that the CaM concentration within microdomains is too low to significantly regulate the channels. Very small forskolin-induced changes in Ca2+ influx were observed in HEK-293 cells not expressing CNG channel constructs (Fig 4).
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PDE Activity in HEK-293 Cells
As described earlier, CNG channels monitor cAMP produced in subcellular compartments near the plasma membrane. To assess the extent to which PDE activity affects cAMP levels, we measured forskolin-induced Ca2+ influx in the presence and absence of PDE inhibitors. Initially we examined the effects of the nonselective PDE inhibitor, IBMX, using each CNG channel construct as a cAMP sensor. In cells expressing the WT channel, there was little or no change in Ca2+ influx in response to an intermediate forskolin concentration (10 µM; Fig 5 A). After a 3-min pretreatment with 100 µM IBMX only a modest forskolin-induced increase in Ca2+ influx was observed (Fig 5 A), arising from Ca2+ entry through WT channels. In cells expressing the E583M channel, a moderate forskolin-induced increase in Ca2+ influx was observed in the absence of PDE inhibitors (Fig 5 B). After pretreatment with IBMX, forskolin caused a robust increase in Ca2+ influx. In cells expressing the C460W/E583M channel, forskolin-induced increases in Ca2+ influx were readily observable, even in the absence of PDE inhibitors (Fig 5 C). Thus, this channel is capable of detecting cAMP in cells with low AC activity. Using the 6190/C460W/E583M channel as a sensor gave similar forskolin-induced changes in Ca2+ influx as the E583M channel, both in the absence and presence of IBMX (Fig 5, compare B and D). These results demonstrate that, in HEK-293 cells, basal PDE activity limits the accumulation of cAMP after a moderate stimulus. However, even in the absence of PDE inhibitors, after stimulation of AC with high concentrations of forskolin (50100 µM), cAMP reaches levels high enough to activate WT CNG channels (not shown).
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We next sought to identify pharmacologically the PDE type(s) that regulates cAMP levels near CNG channels. The C460W/E583M channels were used to monitor forskolin-stimulated cAMP accumulation in the presence and absence of a series of PDE inhibitors. The reported IC50 values of inhibitors used in this study for each PDE type are given in Table 1. The PDE inhibitor concentrations used were typically at least fivefold higher than the most potent IC50. Either vehicle or PDE inhibitors were added at 0 s and 1 µM forskolin was added at 180 s (Fig 6). In the absence of PDE inhibitors, little or no forskolin-induced Ca2+ influx was observed; whereas, in the presence of IBMX (10 or 100 µM), or the PDE type-IVspecific inhibitor RO-20-1724 (10 µM) significant forskolin-induced Ca2+ influx was observed (Fig 6A, Fig B, and Fig G). Two other PDE type-IV inhibitors significantly increased the forskolin-induced Ca2+ influx, rolipram (see Fig 7 C) and etazolate (not shown). Inhibitors specific to other PDE families did not affect forskolin-induced Ca2+ influx. Similar results were obtained in HEK-293 cells expressing either the WT or 6190/C460W/E583M channels by stimulating cAMP production with higher forskolin concentrations (10 µM, not shown), or when prostaglandin E1 (1 µM) was used to stimulate AC activity (not shown). In these experiments, we used inhibitor concentrations at which specific PDE types should be inhibited; however, many PDE inhibitors are not completely specific (Table 1). Also, PDE types VIII and IX are insensitive to IBMX as well as most PDE inhibitors (see references in Table 1). Unfortunately, dipyridamole, the inhibitor to which they are most sensitive, fluoresces and, as such, cannot be used in this assay.
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In Vivo Estimates of PDE Inhibitor KI
To further establish that PDE type IV is responsible for the observed IBMX-sensitive PDE activity, we estimated the KI's (inhibition constants) of IBMX and two PDE type-IV inhibitors (RO-20-1724 and rolipram) in vivo. This required developing a quantitative framework to assess the relationship between cAMP synthesis, hydrolysis, and redistribution throughout the cell. Thus, we adopted the following formalism:
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(2) |
where C is the steady-state rate of cAMP synthesis by AC, Vmax is the maximal rate of cAMP hydrolysis, Km is the Michaelis constant for PDE, and kf is the rate constant of cAMP flux out of the microdomain. To estimate KI for the PDE inhibitors, we made two assumptions. First, we assumed that at low levels of AC stimulation and PDE inhibition, the concentration of local cAMP is low and diffusion of cAMP out of the microdomain is negligible. Second, we assumed that cAMP levels reach steady-state shortly after AC stimulation (i.e., equal rates of synthesis and hydrolysis). With these assumptions, Equation 2 can be simplified to:
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(3) |
Interestingly, this equation reveals that when the inhibitor concentration is equal to KI the cAMP concentration is twice that in the absence of inhibitor. At low cAMP concentrations (<K1/2 for the channel), the cAMP concentration is proportional to the square root of the Ca2+ influx rate (the Hill coefficient for channel activation is 2). Thus, KI can be estimated using a linear fit to the square root of the slopes of the fluorescence traces as a function of inhibitor concentration (Fig 7).
The high cAMP affinity C460W/E583M channels were used to detect changes in cAMP concentration after pretreatment with PDE inhibitors (added at 0 s) and modest forskolin stimulation (0.5 µM added at 180 s). IBMX, RO-20-1724, and rolipram were completely equilibrated across the plasma membrane of HEK-293 cells in <180 s (data not shown). The assumption that cAMP levels reach steady state is supported by the long-lasting linear rise in Ca2+ concentration after forskolin stimulation (a steady Ca2+ influx rate reflects a constant cAMP level; Fig 1). Doseresponse relations for the three PDE inhibitors are shown in Fig 7. Based upon fits to this data with Equation 3 (Fig 7, AC, insets), the KI values were estimated to be 11 ± 2 µM (IBMX), 0.13 ± 0.02 µM (RO-20-1724), and 0.07 ± 0.02 µM (rolipram), n = 4, which are consistent with published IC50 values from in vitro experiments (Table 1). To ensure that the results were independent of channel construct, we also estimated the KI for RO-20-1724 using the lower cAMP affinity 6190/C460W/E583M channels. The KI (0.15 ± 0.02 µM, n = 3) was indistinguishable from that estimated using C460W/E583M channels. These data strongly suggest that PDE type IV is primarily responsible for the IBMX-sensitive component of PDE activity monitored using CNG channels and, thus, for regulating localized cAMP concentration in HEK-293 cells.
At higher PDE inhibitor concentrations, the square root of Ca2+ influx rate, which is proportional to cAMP concentration, deviates from linearity (Fig 7). It is likely that at higher inhibitor concentrations cAMP may reach levels K1/2 of the channel. At these concentrations, the relationship between Ca2+ influx rate and cAMP concentration deviates from a simple square law. Importantly, this does not explain why at high levels of PDE inhibition the Ca2+ influx rate reaches a plateau. When PDE activity is completely inhibited, forskolin-stimulated cAMP accumulation would continually increase in a confined region of free diffusion. This, in turn, would lead to higher Ca2+ influx rates. To ensure that the plateau was not primarily due to channel saturation or Ca2+ homeostatic mechanisms, we measured Ca2+ influx rates induced by 50 µM forskolin and maximal PDE inhibition in the same experiment. Under these conditions, the Ca2+ influx rate greatly exceeded the influx rates induced by 0.5 µM forskolin with PDE inhibition. At increased cAMP concentrations, cAMP efflux from the microdomain is expected to increase; this increased efflux could create the observed plateau. This would be consistent with the diffusionally restricted microdomain model (
PDE Activity in GH4C1 Cells
To further test the utility of this approach, we have examined PDE activity in excitable GH4C1 pituitary cells. In all the experiments shown (Fig 8 Fig 9 and Table 3), 1 µM nimodipine was added at time zero to block Ca2+ influx though voltage-gated Ca2+ channels (triggered by membrane depolarization due to Ca2+ and Na+ influx through CNG channels). This concentration of nimodipine was sufficient to block Ca2+ influx through voltage-gated Ca2+ channels activated by membrane depolarization in 24 mM external KCl, and did not alter forskolin or pCPT-cGMP induced Ca2+ influx through CNG channels expressed in HEK-293 cells (data not shown). Fig 8 Fig 9 each depict experiments done on a single batch of cells. As before, similar results were obtained on three other batches.
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Fig 8 A shows the forskolin-induced responses of cells expressing WT channels. Either vehicle or 100 µM IBMX was added at 0 s and 50 µM forskolin was added at 180 s. After the addition of forskolin, there was a short delay followed by an increased Ca2+ influx. The slope of the Ca2+ influx was greater in the presence of IBMX, indicating higher cAMP levels. In control cells (cells not expressing CNG channel constructs), no forskolin-induced Ca2+ influx was observed in either the presence or absence of IBMX (Fig 8 B). This was true of controls done for all experimental protocols.
We next examined forskolin-induced responses and the effects of IBMX in cells expressing the high cAMP affinity construct, C460W/E583M. In these experiments, either vehicle or 100 µM IBMX was added at 60 s (Fig 8C and Fig D). Interestingly, the addition of IBMX triggered Ca2+ influx that was not observed in either control cells or cells expressing the WT channel. Subsequent addition of 10 µM forskolin (Fig 8 C) or 100 nM vasoactive intestinal peptide (VIP; Fig 8D) caused additional cAMP accumulation and Ca2+ influx. A comparison of responses measured with the WT and C460W/E583M channels indicates that the IBMX-induced response was not due to an increase in local cGMP concentration. Moreover, 100 µM IBMX did not alter CNG channel activity monitored in excised patches (data not shown). Thus, these data indicate that the IBMX-induced Ca2+ influx was due primarily to an increase in cAMP arising from basal AC activity.
To identify the PDE family or families that regulate the local cAMP concentration in GH4C1 cells, cAMP accumulation was monitored in the presence and absence of PDE inhibitors (Table 3). Again, the high cAMP affinity C460W/E583M channels were used. PDE inhibitors were added at 60 s. Although the addition of IBMX alone triggered Ca2+ influx, we added 10 µM forskolin at 240 s as a positive control for CNG channel activity. Only the nonspecific PDE inhibitor, IBMX, and the PDE type-Ispecific inhibitor, 8-methoxymethyl-IBMX, induced Ca2+ influx through CNG channels (Table 3). This PDE is likely to have a low Km for cAMP because it is capable of regulating cAMP at concentrations too low for the WT or 6190/C460W/E583M channels to detect. Thus, it is likely that a Ca2+-CaMstimulated PDE (type I) is primarily responsible for controlling basal cAMP signals in GH4C1 cells.
We also monitored PDE activity using 6190/C460W/E583M channels to detect changes in cAMP concentration (Fig 9). PDE inhibitors and forskolin were added as described above. With this construct, we were unable to observe changes in cAMP concentration due to inhibition of PDE activity alone. However, PDE inhibitors caused an increase in forskolin-induced Ca2+ influx. As observed using the high cAMP affinity C460W/E583M channels, both IBMX and the PDE type-Ispecific inhibitor 8-methoxymethyl-IBMX significantly inhibited PDE activity (Fig 9A and Fig B). Interestingly, the PDE type-IVspecific inhibitors, RO-20-1724 and rolipram, also inhibited PDE activity (Fig 9C and Fig D). Inhibitors specific to other PDE types had no effect on the forskolin-induced Ca2+ influx (data not shown). No RO-20-1724- or rolipram-sensitive PDE was observed using the high cAMP affinity channel (Table 3). It is likely that Ca2+ influx through the C460W/E583M channels was saturated at cAMP concentrations below the Km for this PDE. Thus, there are likely two different PDE types that regulate local cAMP signals in GH4C1 cells: a low Km, 8-methoxymethyl-IBMXsensitive PDE (type I) and a high Km, RO-20-1724-rolipramsensitive PDE (type IV).
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DISCUSSION |
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We have made several modifications to the WT rat olfactory CNG channel that significantly enhance its utility as a cAMP sensor. Three novel constructs were tested. The E583M channel is more sensitive to cAMP and less sensitive to cGMP. Two additional mutations produced channels that make even better cAMP sensors. The C460W/E583M channel has a high affinity for cAMP, allowing it to detect cAMP at levels which activate PKA. The 6190/C460W/E583M channel is almost as sensitive to cAMP as E583M. However, the
6190/C460W/E583M channel has two important advantages: it is not inhibited by Ca2+-CaM, and it is barely activated by cGMP.
Using both WT and modified CNG channels, we have developed a convenient assay for detecting changes in local cAMP concentration, and used this assay to examine PDE activity in two cell types. There are several advantages of measuring Ca2+ influx through CNG channels as a way to monitor changes in cAMP. First, this assay is very simple to implement in either cell populations or single cells. Second, the measurement has a high signal-to-noise ratio. Third, it is sensitive to small changes in AC activity. This is due, in part, to the fact that CNG channels detect local rather than total cellular cAMP. With traditional assays, high agonist and PDE inhibitor concentrations are often required to detect cAMP accumulation. In contrast, the channels described here readily detect changes in cAMP caused by subsaturating agonist concentration in the absence of PDE inhibitors. Overall, the assay presented here can be used as an initial screen for changes in cAMP in response to physiological stimuli or pharmacological agents.
There are a few shortcomings to this approach that should be mentioned. The temporal resolution is somewhat limited. It is also difficult to extract the precise cAMP concentration from this assay because the responses cannot be calibrated (with known intracellular cAMP), and because Ca2+ handling and depletion of free fura-2 become issues at high Ca2+ influx rates. When more precise measurements of local cAMP are required, cAMP concentration can be determined using electrophysiological methods (
We have previously demonstrated in HEK-293 and C62B glioma cells that CNG channels measure cAMP produced in subcellular compartments or microdomains near the plasma membrane, and that diffusion of cAMP between the microdomains and the bulk cytosol is severely hindered (
Much has been learned about the 2-D localization of proteins involved in cellular signaling. For example, certain isoforms of PDE are differentially distributed between particulate (membrane-localized) and supernatant (cytosolic) cellular fractions; PDE kinetics may vary with cellular localization (
However, 2-D localization within the plasma membrane does not account for the 3-D compartmentalization of cAMP signals. We have shown that proximity to AC does not provide sufficient cAMP concentrations for activation of effector proteins because, in essence, each cAMP molecule diffuses away more rapidly than the next molecule is synthesized (
In the current study, we have used Ca2+ influx through CNG channels to detect changes in cAMP concentration in both nonexcitable HEK-293 cells and excitable pituitary GH4C1 cells. The specific goal was to assess the PDE subtypes responsible for shaping cAMP signals near the plasma membrane. To study the effects of PDEs, it is important to be able to first detect cAMP changes in the absence of PDE inhibitors. We had no difficulty with this in either cell type, despite the modest levels of cAMP accumulation in response to forskolin or VIP stimulation reported previously (6190/C460W/E583M channels, inhibition of either PDE type I or type IV increased forskolin-induced cAMP accumulation. These observations point to the presence of two different PDE types: a low Km, PDE type I and a higher Km, PDE type IV. Based on the apparent cAMP affinities of the two channel constructs, we estimate the two Km values to be <1 µM and >5 µM.
Interestingly, the data in GH4C1 cells indicate the presence of a "futile cycle" of cAMP synthesis and hydrolysis. Obviously, restricting this phenomenon to subcellular compartments would be advantageous energetically, but why expend the additional energy at all? One possibility is that constant AC and PDE activity allow the system to respond rapidly to a stimulus. In essence, the enzymes are poised to respond to changes in active G-protein or changes in internal Ca2+, allowing for rapid increases or decreases in cAMP levels. A similar situation exists in light-adapted photoreceptor cells; for a detailed discussion see
The existence of PDE type I in GH4C1 cells is consistent with previous work showing that CaM antagonists inhibit the hydrolysis of cAMP (
If the two PDE types coexist in the same subcellular compartment then the low Km PDE would be likely to regulate cAMP levels under basal conditions. Upon further activation of AC, this PDE would be overwhelmed, and large concentrations of cAMP would accumulate locally. The high Km PDE would be able to efficiently hydrolyze cAMP at these elevated concentrations. Furthermore, these PDE types could be differentially regulated, allowing different feedback mechanisms to control cAMP levels within this compartment. If the activity of the two PDE types is compartmentalized, then distinct cyclic nucleotide signals could occur simultaneously in different regions of the cell. Although this has not yet been demonstrated, it seems likely because cAMP can be excluded from areas of the cell by PDE activity (
In the future, it should be possible with the improved sensors to measure local cAMP signals in response to a variety of extracellular stimuli. The ultimate goal is to understand how cAMP can differentially regulate >200 cellular targets. This requires a quantitative knowledge of how cAMP signals are initiated and terminated. Different PDE subtypes are likely to play crucial roles in shaping the signals in different cellular compartments. Within these compartments, cAMP levels are likely to vary dynamically (
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Footnotes |
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* Abbreviations used in this paper: AC, adenylyl cyclase; AKAP, A-kinase anchoring protein; CaM, calmodulin; HEK-293, human embryonic kidney cell line; IBMX, 3-isobutyl-1-methylxanthine; NO, nitric oxide; pCPT-cGMP, 8-p-chlorophenylthio-cGMP; PDE, phosphodiesterase; RO-20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone; VIP, vasoactive intestinal peptide; WT, wild-type.
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Acknowledgements |
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We would like to thank Dr. R.R. Reed for providing the cDNA encoding the olfactory CNG channel, Dr. K.-W. Yau for helpful advice concerning the Ca2+-CaM deletion, Dr. M.M. Tamkun for providing HEK-293 cells, Dr. Y. He and X. Wang for technical assistance, and Dr. D.M.F. Cooper for helpful discussions and for use of spectrofluorimeter and tissue culture facilities.
This work was supported by the National Institutes of Health grants EY09275, NS28389, HL58344, and DC00385, and American Heart Association grant DM0020554Z.
Submitted: 22 December 2000
Revised: 18 May 2001
Accepted: 21 May 2001
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References |
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