Roles of Calcium, PKA, and PKG
Address correspondence to Dr. Z. Priel, Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel. Fax: 972-8-6900046; E-mail: alon{at}bgumail.bgu.ac.il
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ABSTRACT |
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Key Words: cilia mucociliary tissue phosphorylation cholinergic cyclic nucleotides
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INTRODUCTION |
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Three ubiquitous second messengers (Ca2+, cGMP, and cAMP) have been shown to participate in the process of ciliary stimulation (Verdugo, 1980; Tamaoki et al., 1989
; Di Benedetto et al., 1991a
,b
; Lansley et al., 1992
; Korngreen and Priel, 1994
; Geary et al., 1995
; Salathe and Bookman, 1995
; Yang et al., 1996
; Runer et al., 1998
). It has become an accepted view that each of these messengers represents an independent pathway, which leads to the CBF enhancement. However, our recent work seriously undermines this conception. Inhibition of the nitric oxide-cGMP-PKG signaling pathway at any of its stages abolished the CBF enhancement in the presence of high intracellular calcium concentration ([Ca2+]i; Uzlaner and Priel, 1999
; Braiman et al., 2000b
). It is well-known that the calciumcalmodulin complex (Ca-CaM) activates nitric oxide (Stuehr, 1999
). Indeed, inhibition of calmodulin nullified ciliary beat frequency enhancement in the presence of high [Ca2+]i (Braiman et al., 2000b
). These findings demonstrate that elevated [Ca2+]i itself, without participation of PKG, cannot enhance CBF.
Recently, a study has been conducted aiming to investigate the effect of acetylcholine (ACh) on the tissue cultures from frog esophagus (Zagoory et al., 2001). It has been found that ACh induces a profound increase in CBF and [Ca2+]i through M1 and M3 muscarinic receptors; that the response in CBF is mediated by PLC and calmodulin (CaM); that the rise in [Ca2+]i is achieved solely through calcium mobilization from the intracellular stores; and that the time course of the CBF enhancement exhibited a clear biphasic pattern. The first phase of the response, which lasted
36 min, was characterized by a well correlated change in both CBF and [Ca2+]i, consisting of a rapid increase followed by a gradual decay. However, in the course of the decay, the correlation between [Ca2+]i and CBF had been gradually lost, indicating transition into the second phase of the response. During this phase, [Ca2+]i declined to its basal level, whereas CBF stabilized at a moderately excited state (between 50 and 100% above the basal level). Finally, inhibition of calmodulin moderately attenuated the response of [Ca2+]i, but abolished completely both the first and the second phase of the CBF enhancement. That was a surprising result due to the fact that the second phase was apparently independent of [Ca2+]i.
These findings strongly suggested that the molecular events underlying the CBF stimulation by ACh combined into a complex mechanism, involving several interacting signaling pathways. The aim of this work is to delineate the chain of events that lead to the biphasic enhancement of CBF induced by ACh.
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MATERIALS AND METHODS |
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Chemicals and Solutions
The simultaneous measurements of [Ca2+]i and CBF were performed in Ringer's solution containing the following (in mM): 120 NaCl, 2.5 KCl, 1.8 CaCl2, 1.8 MgCl2, 5 HEPES, and 0.5 probenecid.
Solutions with low calcium concentration were prepared by adding to Ca2+-free Ringer's solution 0.5 mM EGTA, 1.8 mM Mg2+, and a Ca2+ in concentration calculated, according to known equilibrium constants, to reach the desired free Ca2+ concentration. The external calibration solution for fura-2 was composed of the following (in mM): 115 KCl, 20 NaCl, 5 MgCl2, 5 D-glucose, 5 HEPES, and 10 EGTA, and 1 µM K5Fura-2. All the solutions were adjusted to pH 7.27.4 before use.
Ionomycin and KT8353 were dissolved in ethanol and H-89 was dissolved in the ethanol/water 1:1 mixture as concentrated stock solutions and diluted into Ringer's solution just before use. The final concentration of ethanol in the assay solution did not exceed 0.5%. Thapsigargin was dissolved in DMSO as a concentrated stock solution. The final concentration of DMSO in the assay solution did not exceed 0.05%.
HEPES, DMSO, EGTA, thapsigargin, and W-7 were obtained from Sigma-Aldrich. Acetylcholine chloride, KCl, NaCl, MnCl2, CaCl2, were obtained from Merck. Ionomycin was obtained from Calbiochem; fura-2/AM was obtained either from Molecular Probes or from Teflabs. K5Fura-2 and pluronic F-127 were purchased from Molecular Probes. All tissue culture media and supplements were supplied by Biological Industries.
Simultaneous Measurement of Intracellular Calcium and Ciliary Beating
Simultaneous measurements of intracellular calcium and ciliary beating were performed as previously described (Korngreen and Priel, 1994). Briefly, the cells were preloaded with fura-2 by incubating the tissue in serum-free growth medium, containing 5 µM fura-2/AM, 0.03% pluronic F-127 and 500 µM probenecid, for 60 min at 32°C in a rotating water bath, followed by washing in Ringer's solution for 30 min. Pluronic F-127 is a nonpolar polymeric detergent that increases solubility of hydrophobic fura-2/AM in aqueous media. Probenecid, an inhibitor of membrane organic anion transporters, is used widely to prevent extrusion of loaded fura-2 from the cells. The dye loaded cells were epi-illuminated with light from a 75-W xenon lamp (Oriel Corp.) filtered through 340- and 380-nm interference filters (Oriel) mounted on a four position rotating filter wheel. The fluorescence, emitted at 510 nm, was detected by a photon counting photomultiplier (model H346053; Hamamatsu). The 340/380 fluorescence ratio, averaged over a period of 1 s, was stored in a computer. CBF was measured by transilluminating the same ciliary area with light at 600 nm (so as not to interfere with fura-2 fluorescence at 510 nm). Scattering of the 600-nm light from the beating cilia created amplitude modulations that were detected by a photomultiplier (model R2014; Hamamatsu).
A calibration curve of the calcium concentration was created by titrating an external calibration solution with a solution of the same composition containing 10 mM CaCl2 (Grynkiewicz et al., 1985; Korngreen and Priel, 1994
). To account for the difference between the fura-2 fluorescence signal in the intracellular medium and in the calibration solution, the maximal and minimal values of the 340/380 fluorescence ratio were measured from the cells. The maximal value was obtained by the addition of 5 µM ionomycin to the cells, which resulted in flooding the cells with Ca2+. The minimal value was obtained by the addition of ionomycin to the cells in a zero-calcium medium. The calibration curve was corrected according to the obtained results. The nonspecific signal was estimated by the addition of ionomycin to the cells in the presence of 1 mM Mn2+, which leads to quenching of fura-2 fluorescence (Grynkiewicz et al., 1985
; Kao, 1994
; Takahashi et al., 1999
). The calcium concentration was calculated directly from the corrected calibration curve by interpolation using a table look-up algorithm.
Before any treatment, Ringer's solution over the tissue culture was changed twice. The tissue was preincubated in a third change of the Ringer's solution for 1530 min before the experiment to prevent any transient effects on ciliary motility.
The basal ciliary beat frequency (Fo) and [Ca2+]i level were measured for 25 min in 900950 µl of the appropriate solution. The results of these measurements were taken as reference values. Then, 50100 µl of solution containing the test substance were added to reach the desired final concentration. In our earlier experiments, an alternative technique also was used: the solution was rapidly changed to the one containing the test substance using a constant flow perfusion system. Our experience indicates that the pattern and the magnitude of the response in either CBF or [Ca2+]i do not depend on the manner of substance addition (Korngreen and Priel, 1994, 1996
; Levin et al., 1997
; Korngreen et al., 1998
; Zagoory et al., 2001
). The frequency (F) and [Ca2+]i were monitored on the same ciliary cell for 10 40 min. All experiments were performed at 23 ± 0.5°C. Beat frequency enhancement was represented as the observed frequency normalized to the reference frequency (Fo), i.e., F/Fo = frequency enhancement. The intracellular calcium elevation was represented by the difference
[Ca2+]i between the observed calcium level and the reference level. The results were presented as an average ± SEM, with n = number of experiments in parentheses. Every experiment was performed using 597 tissue cultures taken from at least two animals. Each tissue culture was used only once. Since the results obtained from tissue cultures grown from either frog esophagus or frog palate were virtually identical, they were combined for the purpose of this presentation.
Quantitative Determination of Cyclic Nucleotides
The esophagus tissue was cut into four to six pieces (30 mg each). These pieces were placed in stimulation medium (Ringer's solution supplemented with the tested materials). Before any treatment, Ringer's solution over the tissue culture was changed twice. The tissue was preincubated in a third change of the Ringer's solution for 1530 min before the experiment to prevent any transient effects. The stimulation was stopped by freezing in liquid nitrogen. To prevent build-up of an icy layer over the ciliary tissue, the thin layer of liquid was absorbed off the tissue by lint-free paper before freezing. The ciliary side of the frozen tissue was scrubbed three times with a scalpel and cells were collected into 0.8 ml of 0.1 N HCl. Cells were homogenized by grinding at 300 rpm for 40 s and the homogenate was centrifuged. Two samples of 100 µl from the supernatant were taken for the quantitative determination of cyclic nucleotide concentration.
Determination of the cyclic nucleotide concentration was done by using a commercial kit: Correlate-EIA direct cyclic AMP enzyme immunoassay kit, or Correlate-EIA direct cyclic GMP enzyme immunoassay kit. Briefly, the method is based on ELISA, a competitive immunoassay for the quantitative determination of the relevant nucleotide in samples treated with 0.1 N HCl. According to the protocol supplied with the kit, the samples, as well as the standards, underwent acetylation. Since the antibody better recognizes the acetylated nucleotides, this procedure increased the sensitivity of the analysis. The measurements were done in duplicate. At the final step, the optical density of samples and standards were measured. The amount of the nucleotide in each sample was calculated based on a standard curve. The protein concentration of the supernatant was determined by Bio-Rad assay. It is important to note that the amount of the cyclic nucleotides may vary with sex, age, and the seasons of the year. For instance, during the winter time, the levels of cAMP were very high (five times higher than in the spring). Due to those variations, the concentrations of the cyclic nucleotides for each experiment were presented as relative values normalized to the levels obtained from the tissues subjected to the same treatment, but without application of the stimulant.
The cross reactivities for cAMP or cGMP was determined by Assay Designs, Inc. The cross reactivities of cGMP and cAMP were <0.05%, as determined by Correlate-EIA direct cAMP enzyme immunoassay kit and Correlate-EIA direct cGMP enzyme immunoassay kit, respectively. The endogenous levels of cGMP were near the low end of the kit sensitivity. Therefore, in the cGMP detection experiments, the tissues were stimulated in the presence of a phosphodiesterase inhibitor, IBMX (1 mM).
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RESULTS |
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We examined the ability of ACh to alter the cytosolic levels of the cAMP and cGMP in the ciliary tissue from frog esophagus. As can be seen (Fig. 1), 10 µM ACh elevated the cytosolic levels of cAMP or cGMP in a time dependent manner. The time course of the elevation in cGMP was characterized by a bell-shaped pattern. An initial slight decrease in the cGMP levels was followed by a steady elevation and the maximal response of a 4.50 ± 1.3-fold increase was achieved in 45 min after stimulation. Subsequently, the cytosolic level of cGMP gradually declined, attaining its basal value at 5 min after reaching the maximum.
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Ca-CaM Mediates Activation of Guanylate Cyclase and Adenylate Cyclase
To assess the role of Ca-CaM in the elevation of the cGMP and cAMP levels induced by ACh, several sets of experiments were performed. First, the cells were treated with a calmodulin inhibitor W-7 (150 µM; Hidaka et al., 1981) for 1013 min before application of ACh. In the cells pretreated with the inhibitor, the elevation in both cGMP and cAMP levels induced by ACh was strongly attenuated, being 1.52 ± 0.09-fold and 1.22 ± 0.16-fold, respectively (Fig. 2).
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Forskolin Elevates the Endogenous Levels of cAMP and cGMP
It is well-known that forskolin is a powerful activator of AC. Indeed, application of 25 µM forskolin enhanced the endogenous level of cAMP by ninefold (Fig. 3). Surprisingly, forskolin also induced a strong increase in cGMP concentration, by 3.54-fold (Fig. 3). Since forskolin, via activated PKA, elevates [Ca2+]i in the discussed system (Braiman et al., 1998), it is possible that the rise in the cGMP level induced by forskolin is secondary to this [Ca2+]i elevation. To examine this possibility, the cells were exposed to 1 µM thapsigargin inducing strong elevation of [Ca2+]i (Braiman and Priel, 2001
). It is important to emphasize that no apparent loss of viability was detected in the ciliary cells and the cilia continued beating vigorously for at least 2 h after application of thapsigargin. As expected, high [Ca2+]i induced a rise in endogenous cGMP levels (Fig. 3). However, this rise was considerably lower than the elevation in cGMP induced by either forskolin or ACh (Fig. 3).
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PKG Activity Is Essential for CBF Enhancement Induced by ACh
To evaluate the role of the cGMP-PKG pathway in the process of CBF enhancement induced by ACh, two inhibitors were used: a GC inhibitor LY83583 (IC50 = 2 µM; Fleisch et al., 1984) and a PKG inhibitor KT5823 (IC50 = 0.234 µM; Grider, 1993
). The responses in [Ca2+]i and CBF to 10 µM ACh were monitored simultaneously from the same cell, which was either untreated, or pretreated with 50 µM LY83583, or pretreated with 0.5 µM KT5823. Addition of 10 µM ACh to tissue cultures pretreated with either the inhibitor of GC or the inhibitor of PKG resulted in a partial attenuation of the [Ca2+]i rise (by
25 and 50%, respectively) and in a complete inhibition of the CBF enhancement (Figs. 4 and 5). The decrease in the [Ca2+]i rise produced by the inhibitors is incomparable to their deleterious effect on the CBF enhancement. Moreover, the results shown below (see Figs. 6 and 7) and published elsewhere (Korngreen and Priel, 1996
; Levin et al., 1997
) indicate that a [Ca2+]i rise of a similar magnitude can evoke a strong CBF enhancement. The mechanism of the cGMP/PKG involvement in the dynamic regulation of [Ca2+]i is not yet known, and is beyond the scope of this work. Nevertheless, it is possible to conclude that PKG activity is essential for CBF enhancement induced by ACh, even in the presence of high [Ca2+]i.
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The responses in [Ca2+]i and CBF to 10 µM ACh were monitored simultaneously from the same cell, which was either untreated, or pretreated with 5 µM H-89, or pretreated with 100 µM Rp-8-Br-MB-cAMPS. Both inhibitors produced essentially identical effects. The response to ACh of the cells pretreated with the inhibitors was attenuated by 4050% and significantly shortened (Figs. 6 and 7). The rise in [Ca2+]i was diminished by 4050% and followed by a relatively fast decay (within 13 min after reaching the maximum) to its basal level. The CBF enhancement was also attenuated, and the degree of the attenuation was similar to the degree of inhibition in the [Ca2+]i elevation. In addition, the second phase of the CBF enhancement was completely abrogated, and CBF declined to its basal level in a striking accordance with [Ca2+]i (Figs. 6 and 7).
According to the IC50 values of H-89 in mammalian tissues (0.05 µM for PKA and 0.5 µM for PKG), this blocker, used at 5 µM, is expected to inhibit the PKG activity as well. However, since the effect of H-89 (Figs. 6 and 7) was strikingly different from the effect of the PKG and GC inhibitors (Figs. 4 and 5), and, at the same time, it was virtually identical to the effect produced by the highly specific PKA inhibitor Rp-8-Br-MB-cAMPS (Fig. 7), it is safe to assume that H-89 did not inhibit PKG in our preparation. The discrepancy may be a result of the differences in the enzyme sensitivity or in the membrane permeability to H-89 between the mammalian and the frog tissue. These results suggest that PKA activity contributes to the duration and the magnitude of the first phase and is obligatory for the existence of the second phase of the response to ACh.
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DISCUSSION |
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We previously have shown that CBF enhancement induced by ACh crucially relies on rise in [Ca2+]i (Zagoory et al., 2001). ACh achieves this effect by mobilization of Ca2+ from the intracellular stores. In this work, we demonstrate that ACh also elevates the cytosolic levels of cGMP and cAMP. It was shown that inhibition of calmodulin abolishes the CBF enhancement induced by ACh in the presence of high [Ca2+]i (Zagoory et al., 2001
), conforming to the findings that calmodulin is essential for the CBF enhancement normally induced by ATP or by a calcium ionophore in the tissue cultures from frog esophagus and from rabbit trachea (Braiman et al., 2000b
). These results indicate that calcium ions themselves cannot enhance CBF. On the other hand, it is well-known that Ca-CaM triggers a multitude of cellular events, including activation of GC and AC (Hinrichsen, 1993
; Antoni, 1997
; Stuehr, 1999
; Chin and Means, 2000
; Groves and Wang, 2000
). Activated GC and AC increase the intracellular levels of the correspondent cyclic nucleotides cGMP and cAMP. Indeed, activation of muscarinic receptors by ACh induced a strong, time-dependent rise in the cytosolic levels of cGMP and cAMP (Fig. 1). Moreover, the rise in the concentrations of both nucleotides was strongly attenuated by pretreatment of the cells with a calmodulin inhibitor or by impeding the [Ca2+]i rise (Fig. 2). In addition, the time course of the cGMP elevation closely resembles the response in [Ca2+]i induced by ACh (Figs. 1 and 47). Taken together, these results indicate that the rise in the cGMP level is mainly mediated by Ca-CaM.
The interplay between cAMP and Ca-CaM appears to be more complex. Inhibition of CaM impedes the rise in the cAMP level (Fig. 2), suggesting that the rise in the cAMP level is also mediated by Ca-CaM. On the other hand, the time course of the cAMP rise exhibits a different behavior as compared with [Ca2+]i (Figs. 1 and 47). The level of cAMP reaches its maximum in 10 min after stimulation and is maintained elevated for a relatively long time after [Ca2+]i decays to its basal level. These results suggest that Ca-CaM is essential for initiation of the rise in cAMP, probably via activation of CaM-dependent AC. However, the ACh receptors utilize an additional, Ca2+-independent pathway to maintain high levels of cAMP. It is tempting to suggest that activation of the muscarinic receptors leads to inhibition of cAMP-specific phosphodiesterases, since preincubation of the tissue with the phosphodiesterase inhibitor IBMX strongly inhibited the ability of ACh to increase further the cAMP concentration (not shown).
Comparison between the time courses of the response in cytosolic Ca2+, cGMP, and cAMP levels and CBF (Figs. 1 and 47) demonstrates that during the initial strong CBF enhancement followed by a partial decay, the concentrations of all three second messengers are elevated. However, during the first phase, the rise in the concentrations of cytosolic Ca2+ and cGMP are more pronounced and highly correlated with the CBF enhancement. On the other hand, the second phase of the response (a sustained moderate enhancement of CBF) seems to be an outcome of the elevation in the cAMP concentration, probably via PKA activation.
The Role of PKG and PKA in the ACh-induced Ciliary Stimulation
Obviously, the rise in cGMP and cAMP concentrations leads to activation of the correspondent kinasesPKG and PKA. Indeed, inhibition of either PKG or GC abolished completely the CBF enhancement induced by ACh (Figs. 4 and 5). It is important to emphasize that inhibition of either PKG or GC also attenuated the [Ca2+]i elevation induced by ACh. Therefore, one might speculate that the abolishment of CBF enhancement resulted from the attenuation in [Ca2+] rise. However, whereas the maximal rise in [Ca2+]i induced by ACh in the cells treated with either the PKA inhibitors or the GC/PKG inhibitors were statistically indifferent, a significant enhancement in CBF was observed in the cells treated with the PKA inhibitors. Thus, the partial attenuation in [Ca2+] rise is unlikely to account for the complete abolishment of CBF enhancement obtained following GC/PKG inhibition. In addition, results published elsewhere (Korngreen and Priel, 1996; Levin et al., 1997
) also indicate that the [Ca2+]i rise of a similar magnitude or less can evoke a strong CBF enhancement. Therefore, abolishment of the CBF enhancement induced by the PKG or GC inhibitors is not a result of a decrease in the [Ca2+]i response.
Similar results were obtained using extracellular ATP as an agonist in tissue cultures from frog esophagus or in tissue cultures from rabbit trachea (Uzlaner and Priel, 1999; Braiman et al., 2001
), suggesting the generality of these findings. Furthermore, we previously have shown that the AC-stimulating agent forskolin activates CBF in a Ca2+-dependent and a Ca2+-independent manner (Braiman et al., 1998
). Both effects are mediated through PKA. Yet, the PKG inhibitor KT5823 abolished both modes of the forskolin-induced CBF enhancement (unpublished data). These results indicate that PKG is a universal key player in the process of ciliary stimulation. The PKG activity is necessary for the CBF enhancement, whether it is mediated through Ca2+ or PKA. Elevation in the cGMP concentration, observed after application of forskolin (Fig. 3), further supports this idea.
Inhibition of PKA produced a profound change in the time course of the CBF enhancement induced by ACh (Figs. 6 and 7). The rise in CBF was transient, rapidly decaying to its basal level within 23 min after application of ACh. The rise in [Ca2+]i was also considerably shortened and attenuated. Similar transient response, accompanied by a high correlation between CBF and [Ca2+]i, was observed as a normal response to ACh in tissue cultures from ovine trachea (Salathe et al., 1997; Salathe and Bookman, 1999
). It is tempting to suggest that such a discrepancy may be due to either an under activation of cAMP/PKA pathway in the tissue culture used by Salathe et al. (1997)
or over activation of this pathway in our tissue culture.
It was shown that, in tissue cultures from frog esophagus, PKA induced a release of Ca2+ from the intracellular stores (Braiman et al., 1998), most probably by phosphorylation of the IP3 receptor (Burgess et al., 1991
). Therefore, it is not surprising that inhibition of PKA shortened and attenuated the rise in [Ca2+]i induced by ACh, which consequently led to the reduction of a comparable magnitude in the CBF enhancement during the first phase of the response. Apparently, PKA facilitates Ca2+ mobilization from the intracellular stores and, thereby, prolongs and augments the first, Ca2+-dependent phase of the CBF enhancement.
We have shown that the rise in [Ca2+]i is essential for initiation, but not maintenance, of the second phase of the CBF enhancement induced by ACh (Zagoory et al., 2001). However, despite a significant initial rise in [Ca2+]i, the second phase of the response did not develop in the cells pretreated by the PKA blockers (Figs. 6 and 7). These results further support the conclusion that PKA controls the second phase of the CBF enhancement induced by ACh, namely the sustained moderately excited state of CBF. To support this idea even further, it is worth mentioning that application of forskolin to tissue cultures from frog esophagus also produces a biphasic response in CBF with a second phase being virtually identical to the one produced by ACh (Braiman et al., 1998
). In addition, we have shown here that application of either ACh, or forskolin or the calcium mobilizing agent thapsigargin lead to an increase in the cGMP levels (Fig. 3). However, the increase in cGMP induced by either ACh or forskolin, which also profoundly increases the cAMP concentration, is significantly stronger than the increase in cGMP induced by the calcium elevation alone. Apparently, in addition to the ability of PKA to elevate the cGMP concentration through Ca2+ mobilization, another mode exists for the cAMP pathway to stimulate the cGMP pathway. The nature of this mode is yet to be found.
Molecular Events Induced by ACh
Combining the results of this work with the results published previously (Zagoory et al., 2001), we suggest the following cascade of molecular events that underlies the biphasic CBF enhancement induced by ACh (Fig. 8). Stimulation of muscarinic receptors by ACh leads to activation of phospholipase C (PLC) and mobilization of Ca2+ from the intracellular stores. Elevation in [Ca2+]i results in formation of Ca-CaM, which activates GC and AC. The increased levels of cGMP and cAMP activate the corresponding kinases, PKG and PKA. The latter augments and prolongs the response by facilitation of Ca2+ release from the intracellular stores. In addition, PKA or cAMP enhances the elevation in cGMP by an unknown mechanism. The concerted action of PKG, PKA, and elevated [Ca2+]i results in a strong CBF enhancement (the first phase). At the same time, muscarinic receptors utilize an additional pathway to maintain a high level of cAMP, possibly through inhibition of cAMP degradation. As a result, whereas the [Ca2+]i gradually decreases, the cAMP concentration and, consequently, the PKA activity remain high. The active PKA maintains a stable moderately excited state of CBF in a Ca2+-independent manner (the second phase). The unimpaired functioning of PKG is required for this step to develop.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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Submitted: 23 October 2001
Revised: 15 February 2002
Accepted: 19 February 2002
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REFERENCES |
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