(Received for publication, December 23, 1996, and in revised form, January 28, 1997)
From the Division of Renal Diseases and Hypertension and the Department of Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School, Houston, Texas 77073
Vasodilating agents induce relaxation of mesangial cells, in part through cGMP-mediated activation of large calcium-activated potassium channels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cGMP (Bt2cGMP) is characterized by a biphasic increase and then decrease ("rundown") in open probability. Using the patch-clamp method in conjunction with phosphatase inhibitors, we investigated whether the run-down phase was the result of dephosphorylation by an endogenous protein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentrations, caused a significantly greater and sustained response of BKCa to Bt2cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt2cGMP was greater than the response to these agents added separately. Incubation of mesangial cells with okadaic acid for 20 min at a concentration (5 nM) specific for PP2A increased the basal open probability of BKCa and completely inhibited rundown after activation by Bt2cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BKCa activity. In inside-out patches, Bt2cGMP plus MgATP caused a sustained activation of BKCa that was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common substrate for endogenous cGMP-activated protein kinase, which activates BKCa, and PP2A, which inactivates BKCa, in human mesangial cells.
Mesangial cells, which are excitable and have contractile properties similar to smooth muscle, regulate the glomerular filtration rate by modulating the capillary surface area (1-3). Recent patch-clamp studies of human mesangial cells in culture have shown that the mechanism and ion-selective channels involved in maintaining contractile tone are similar or identical to those of vascular smooth muscle (4, 5).
Large calcium-activated potassium channels (BKCa),1 characterized in mesangial cells (5, 6) and vascular smooth muscle (7), are not involved in setting resting potential but respond in a negative feedback manner to agonist-induced increases in contractile tone. Agonists such as angiotensin II elevate intracellular calcium and depolarize the membrane potential, producing an activation of BKCa. The hyperpolarizing membrane potential inhibits further entry of cell calcium by inactivating voltage-gated calcium channels. The gain in this feedback mechanism is increased by smooth muscle relaxants such as nitric oxide and atrial natriuretic peptide that, via cGMP-activated kinase, lower the voltage and calcium thresholds for activating BKCa (8, 9). However, activation of BKCa "on cell" by vasorelaxants or Bt2cGMP is followed by an inactivation or run-down phase, in which BKCa returns to base line 20 s after peak activity.
Substrate regulation by phosphorylation is a dynamic balance between the forward kinase phosphorylation and the reverse dephosphorylation by a protein phosphatase. Several studies have now shown that vasorelaxants activate BKCa through guanylyl cyclase and cGMP-dependent protein kinase in both smooth muscle and mesangial cells (9-11); however, the role of protein phosphatase has been addressed only very recently (9, 12). Two such studies on tracheal smooth muscle and neurohypophyseal cells supported the notion that cGMP stimulated BKCa by activating protein phosphatase 2A (12, 13), which activated BKCa by dephosphorylation. In contrast, our laboratory previously showed that the mesangial BKCa was activated by cGMP-activated protein kinase in the presence of okadaic acid, an inhibitor of protein phosphatases 1 and 2A (9, 13), suggesting that these phosphatases inactivated, rather than activated, the mesangial BKCa.
The present studies were performed to elucidate the signaling pathways involved in the run-down phase following activation of BKCa by cGMP-dependent protein kinase. Using the cell-attached configuration and established phosphatase inhibitors, we specifically investigated the specific endogenous protein phosphatase involved in regulating BKCa. Using inside-out patches, we then determined the regulation of BKCa by protein phosphatases 1 and 2A.
Human mesangial cells were isolated originally by Abboud and co-workers (14) and cultured using standard techniques. Mesangial cells were plated in Waymouth culture medium, pH 7.4, supplemented with 15 mM HEPES buffer, 2.0 mM glutamine, 0.66 unit/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin (100 units/ml), streptomycin (100 µg/ml), and fetal calf serum (17% v/v). All experiments were performed using subpassages 6-10. During this generation span, mesangial cells maintained a constant phenotype and typical smooth muscle-like spindle shape (2).
Cells were subcultured onto individual 22 × 22 mm glass coverslips (Fisher) and maintained for 1-2 days at 37 °C and 5% CO2 in a humidified tissue culture incubator (Queue Scimetrics Inc., Missouri City, TX). The subconfluent cells were placed into an RC-24 perfusion chamber (Warner Instrument Corp., Hamden, CT) and perfused with a physiological salt solution (135 mM NaCl, 5 mM KCl, and 1.0 mM CaCl2, pH 7.4).
Patch-Clamp MethodsMesangial cells were prepared for
analysis of single BKCa channels using standard patch-clamp
techniques previously described (5, 15). Current recordings were made
after obtaining gigaohm seals with the patch electrode on the surface
of the cell (cell attached) or after withdrawing the patch (excised,
inside out). The unitary current (i), defined as zero for
the closed state, was determined as the mean of the best fit Gaussian
distribution of the amplitude histograms. Channels were considered in
an open state when the current was >(n 1/2)i and <(n + 1/2)i,
where n is the maximum number of current levels observed.
The probability of a channel existing in an open state
(Po) is defined as the time spent in the open
state divided by the total time of the recording. In all cases,
Vp implies the holding potential relative to the
pipette.
In all cell-attached
experiments, the pipette solution contained 140 mM KCl plus
10 mM HEPES buffer, pH 7.4, and the bath solution contained
135 mM NaCl, 5 mM KCl, 1 mM
CaCl2, and 10 mM HEPES, pH 7.4. The free
Ca2+ concentration of the bath, initially 1.0 mM, was adjusted to lower concentrations by buffering with
EGTA as described previously (16). In excised patches, the bathing
solution was replaced with 140 mM KCl, 1 µM
CaCl2, 10 mM HEPES, pH 7.4. Okadaic acid, cantharidic acid, and calyculin A, established inhibitors of PP1 and
PP2A, were used to establish the role of endogenous protein phosphatase
in regulation of BKCa. While monitoring BKCa
channels in cell-attached patches, these inhibitors were added to the
bathing solution in concentrations nonspecific for inhibition of PP1
and PP2A. The holding potential (Vp) was either 80 mV or 0 mV, which resulted in outward and inward currents,
respectively. The effects of these inhibitors on the
cGMP-dependent activation of BKCa was
determined by adding inhibitor either in the continued presence of 10 µM dibutyryl cGMP (Bt2cGMP) or at least
30 s before the addition of Bt2cGMP. To determine if
the endogenous protein phosphatase was either 1 or 2A, cells were
incubated in either 5 nM okadaic acid (specific for PP2A)
or 10 nM calyculin A for 10-30 min before obtaining a
seal. Specific inhibition of BKCa by either exogenous PP2A
or PP1 was determined in inside-out patches with 140 mM KCl
in the bath and holding potentials of either 40 mV or
40 mV for
outward or inward currents, respectively. BKCa were
activated either by Bt2cGMP plus MgATP at a holding
potential of 40 mV or by Bt2cGMP plus MgATP plus
cGMP-activated protein kinase at a holding potential of
40 mV. PP2A
(0.5 unit/ml) or PP1 (1 unit/ml) was added in the continued presence of
Bt2cGMP plus MgATP with or without cGMP-activated protein
kinase. Groups were compared for statistical significance using the
paired t test or the ANOVA plus the Student-Newman-Keuls
test as appropriate. Cyclic GMP-activated kinase was purchased from
Promega. All other chemicals used in this study were purchased from
Sigma or Calbiochem.
Experiments were
performed to determine if the run-down phase after adding
Bt2cGMP was the result of either PP1 or PP2A. Fig. 1A shows the effects of cantharidic acid (500 nM), a nonspecific inhibitor of PP1 and PP2A, on the
rundown of Bt2cGMP-activated BKCa in
cell-attached patches in the absence of a phosphatase inhibitor
(upper tracing) and with the simultaneous addition of cantharidic acid (lower tracing). The
Po of BKCa increased from <0.01 to
0.85 after 10 s with the addition of 10 µM
Bt2cGMP and then returned to base line in the next 90 s; after 2 min the Po was <0.01. However, when
Bt2cGMP and cantharidic acid were added together,
BKCa was activated to a Po that was
sustained at 0.78 after 2 min. Note that the channel amplitude
diminished after addition of cantharidic acid. A decrease in the
amplitude of BKCa is the result of a decrease in
electrochemical potential due to the combination of a decrease in the
intracellular potassium concentration and the hyperpolarization of the
membrane potential as BKCa is activated in the cell
membrane (8, 17).
Fig. 1B shows current tracings of BKCa in a
cell-attached patch, illustrating the BKCa activity at a
Vp of 80 and 0 mV with cantharidic acid and
cantharidic acid plus Bt2cGMP. In the control,
BKCa were quiescent at 0 mV, and the
Po was <0.001 at 80 mV. Approximately 2 min
after the addition of 500 nM cantharidic acid, the
Po increased to 0.084 at 80 mV and 0.005 at 0 mV. Note the difference in open probability between 80 and 0 mV,
demonstrating the voltage dependence of BKCa. In the
continued presence of cantharidic acid, Bt2cGMP further
increased BKCa after 2 min to a sustained Po of 0.40 and 0.035 at 80 and 0 mV,
respectively. Thus, cantharidic acid eliminated the rundown in the
response of BKCa to Bt2cGMP. Moreover, the
effects of Bt2cGMP and phosphatase inhibitor were potentiating and independent of the holding potential.
A summary of the separate and combined effects on BKCa of cantharidic acid and Bt2cGMP are shown in Fig. 1C. Two minutes after the separate additions of Bt2cGMP and 500 nM cantharidic acid, the open probability of BKCa was 0.011 ± 0.006 and 0.16 ± 0.05, respectively. Two minutes after the addition of Bt2cGMP plus cantharidic acid, the Po increased to a significantly higher value of 0.48 ± 0.10. This value was more than twice the value of the sum of the separate additions, suggesting a potentiated effect of Bt2cGMP and cantharidic acid.
Fig. 2 shows the effects of 100 nM okadaic
acid and 100 nM calyculin A on cGMP-activated
BKCa in cell-attached patches. At these concentrations, the
phosphatase inhibitors are nonspecific for PP2A and PP1 and are
effective within 120 s. As shown in Fig. 2A
(Vp = 0 mV), BKCa activated within
5 s from <0.001 to 0.10 in response to 10 µM
Bt2cGMP and then returned to base line after approximately
5 more seconds. In the continued presence of Bt2cGMP, the
addition of okadaic acid reactivated BKCa to a greater and
sustained value of 0.83 after 120 s. Similar results were obtained
with 100 nM calyculin A (Fig. 2B,
Vp = 80 mV). The Po of
BKCa increased from <0.001 to 0.42 within 5 s after
the addition of Bt2cGMP and then returned to base line within the next 20 s. However, after the addition of
Bt2cGMP plus 100 nM calyculin A, the
Po increased and remained at 0.65. The increase
in channel amplitude at 0 mV and the reduction in amplitude at 80 mV
shows that the membrane potential is hyperpolarizing, as
BKCa is activated by either okadaic acid or calyculin
A.
Pharmacological Differentiation between Endogenous PP1 and PP2A
Low concentrations of okadaic acid (5 nM) and calyculin A (10 nM) were used to determine if the endogenous phosphatase was PP1 or PP2A. Although IC50 values for phosphatase inhibition for okadaic acid and calyculin A are dependent on the substrate and purity of inhibitor, in general okadaic acid is at least 30-fold more potent for PP2A than PP1 (18, 19), and calyculin A is 30-fold more potent than okadaic acid for inhibiting PP1 (19, 20). Therefore, if PP1 were the endogenous phosphatase, it would be expected that BKCa would be activated by calyculin A more than okadaic acid. If PP2A were the endogenous phosphatase, it would be expected that BKCa would be activated more by okadaic acid than calyculin A.
In the experiments of Fig. 3, okadaic acid (5 nM) and calyculin A (10 nM) were added to the
bathing medium 20-50 min before obtaining a cell-attached patch. As
shown by the upper current tracing of Fig. 3A, in
the presence of okadaic acid, BKCa was active
(Po = 0.063) after obtaining the seal at 80 mV.
The addition of Bt2cGMP increased the
Po further to a sustained value of 0.32. As
shown in Fig. 3B, BKCa was relatively quiescent
(Po < 0.005) after the addition of calyculin A
and was activated transiently to 0.227 on the addition of
Bt2cGMP. This rundown to base line approximately 20 s
after peak activation was similar to control experiments (see Fig.
1A). These results are summarized in Fig. 3B. In
control cells, the Po of BKCa was
increased by Bt2cGMP to a value of 0.19 ± 0.04 after
5 s and ran down to base line after 120 s (n = 6). After incubation with okadaic acid, the basal Po was 0.18 ± 0.07 and increased to
0.29 ± 0.05 and 0.25 ± 0.05 after 5 and 120 s,
respectively (n = 4). After incubation with calyculin A
(n = 4), the Po increased from a
basal of 0.002 ± 0.001 to 0.20 ± 0.05 after 5 s and
returned near the base line value (0.008 ± 0.006) after 120 s. Using the ANOVA plus the Student-Newman-Keuls test, the effects of
okadaic acid at basal and Bt2cGMP (120 s) were
significantly greater than the respective Po
values for control and calyculin A. These results indicate that
BKCa are maintained quiescent in cell-attached patches by
endogenous PP2A. Moreover, specific inhibition of PP2A (by 5 nM okadaic acid) prevents the rundown after activation of
BKCa by Bt2cGMP in cell-attached patches.
Effects of Exogenous PP2A and PP1
The inside-out patch
configuration was used to determine the effects of exogenous PP2A and
PP1 on BKCa. Fig. 4 shows the inactivation of cGMP-activated BKCa by PP2A. As shown in the continuous
tracing of Fig. 4A, dibutyryl cyclic GMP plus MgATP
activated BKCa from 0.029 to 0.332, and the subsequent
addition of PP2A inactivated BKCa to 0.098. However, there
was no effect of PP1 on cGMP-activated BKCa (not shown).
These data are summarized in the bar graph of Fig.
4B. In each experiment, after activation of BKCa
by Bt2cGMP plus MgATP in either the absence or presence of
cGMP-activated protein kinase, PP2A (n = 5) decreased
the Po from 0.57 ± 0.11 to 0.40 ± 0.14 (p < 0.025, paired t test). However,
the Po was 0.66 ± 0.11 and 0.70 ± 0.13 (n = 3; not significant) before and after the
addition of PP1.
This study further defined the signal transduction pathways for regulating BKCa channels in a contractile cell. Three phosphatase inhibitors, in concentrations that inhibit both PP1 and PP2A, caused a larger and more sustained increase in open probability of BKCa in response to Bt2cGMP, the second messenger mediator for relaxation by nitric oxide and atrial natriuretic peptide. Basal open probability of BKCa was increased by okadaic acid in concentrations specific for PP2A but not by calyculin A, a more potent inhibitor of PP1, indicating that endogenous PP2A but not PP1 was maintaining BKCa in a dephosphorylated quiescent state. It was shown that exogenous PP2A, but not PP1, applied to the cytosolic side of BKCa in inside-out patches can specifically inhibit the activation of BKCa by cGMP-dependent protein kinase.
Biphasic Response of BKCa to cGMPIn both smooth muscle and mesangial cells, BKCa are activated by cGMP-dependent protein kinase (7, 9). However, after activation by Bt2cGMP in cell-attached patches, the open probability of the mesangial BKCa rapidly runs down to base-line levels. The present study shows that the run-down phase is due to the presence of PP2A, which would dephosphorylate BKCa. Phosphatase-induced channel rundown has been more commonly described for channels in excised patches. Kubokawa et al. (21) found that renal Kd(ATP) channels run down in excised patches due primarily to the presence of PP2A. However, phosphatase-induced rundown is not only found in excised patches; it was also shown that okadaic acid prevents rundown of Ba2+ current (whole cell) in dissociated helix neurons (22).
It is not understood why the effects of cGMP-dependent kinase are transient and ultimately overcome by a phosphatase that presumably dephosphorylates and inactivates BKCa despite the continued presence of Bt2cGMP. However, several mechanisms could be involved in the temporary inhibition and then activation of a protein phosphatase to initiate the run-down phase. A similar type of biphasic activation was demonstrated for Ca2+/calmodulin-dependent protein kinase II, also a substrate for PP2A (23). An increase in intracellular Ca2+ in the rat brain is accompanied by a sequential autophosphorylated increase and then decrease in phosphorylation level of Ca2+/calmodulin-dependent protein kinase II. The decrease in phosphorylation was blocked by 1 nM okadaic acid. It was suggested by these authors that an increase in intracellular calcium autophosphorylated a serine/threonine protein kinase (described by Guo et al. (24)) that would temporarily phosphorylate and inhibit PP2A. In time, PP2A would autodephosphorylate and inactivate Ca2+/calmodulin-dependent protein kinase II. A similar mechanism may be involved whereby cGMP temporarily activates an inhibitor of PP2A. Although a recent study described inhibition of PP1 by cGMP-dependent protein kinase (25), a cGMP-activated inhibitor of PP2A has not been described.
Regulation of BKCa by PhosphatasesAlthough several studies have described regulation of ion-selective channels by cAMP- and cGMP-dependent protein kinases, the reversal of channel phosphorylation by phosphoprotein phosphatases has been investigated only recently (26-31). The present study is one of a few that have now implicated PP2A as a physiological regulator of BKCa channels (12, 13, 32). However, for cGMP-activated protein kinase-activated BKCa, at least two previous studies using three different cell types (12, 13) have shown either that PP2A activates BKCa or cGMP-activated protein kinase does not activate BKCa in the presence of inhibitors of PP2A. These results contrast with our study which showed that BKCa was inactivated by PP2A and activated by either cGMP-activated protein kinase or inhibitors of PP2A.
Our disparate results may be explained by another study by Reinhart et al. (32) who have shown that BKCa from the brain expresses two types of channels in planar bilayers with respect to PP2A regulation. Type 1 channels are activated by cAMP-activated protein kinase and inactivated by PP2A. Type 2 BKCa channels are inactivated by cAMP-activated protein kinase and activated by PP2A. Although this was a protein kinase A and not a cGMP-activated protein kinase-activated mechanism, it is possible that there are two types of BKCa with respect to regulation of phosphorylation and dephosphorylation by cGMP-activated kinase. However, these type 2 BKCa channels have not been observed in mesangial cells.2 When the properties of the PP2A-activated (type 2) channels in tracheal smooth muscle (12) are compared with the mesangial BKCa of this study, we find that the reported single channel conductance (in symmetrical 140 mM KCl) of the tracheal BKCa (257 ± 13 picosiemens) is somewhat larger than the mesangial BKCa (206 ± 18 picosiemens, see Ref. 5). However, voltage-gated activation response in 1 µM Ca2+ is similar (see Refs. 5 and 12). It remains to be determined if these are distinct isoforms of BKCa.
The dominant type of BKCa in smooth muscle could be determined by the contractile response to low concentrations of okadaic acid. Since PP1 dephosphorylates myosin light chain kinase (33), higher concentrations of okadaic acid would be expected to induce contractions. If type 1 channels predominate, it would be expected that okadaic acid in concentrations of 1-10 nM would inhibit smooth muscle contraction. That range of concentrations of okadaic acid would induce smooth muscle contraction if type 2 channels predominate. In support of type 1 channels, at least two studies have shown that low concentrations of okadaic acid relax vascular smooth muscle (34, 35). Although the kinase specific for activation of the mesangial BKCa appears identical to smooth muscle, the effects of okadaic acid on mesangial contraction have not been determined.
It would not be surprising if BKCa is regulated by different mechanisms in different cell types. Tseng-Crank et al. (36) found that HSLO, the human gene encoding the calcium-activated potassium channel, contains multiple splice variants in the brain. It was recently found that the mesangial BKCa contains at least two of these variants of HSLO (37). It is therefore feasible that the differential expression of these vari-ants would confer different sites for regulation by kinases and phosphatases.
In summary, we have demonstrated that the mesangial BKCa is not regulated by an all-or-none phosphorylation or dephosphorylation mechanism but rather by a dynamic enzymatic balance between cGMP-activated protein kinase and protein phosphatase 2A, which specifically activate and inactivate BKCa, respectively. It remains to be established, however, if there are multiple molecular variants of BKCa that are differentially modulated by kinase/phosphatase signal transduction mechanisms.
The human mesangial cells were a generous gift from Hanna Abboud of the University of Texas, San Antonio. We thank Dr. Shirish Shenolikar for the advice regarding the use and specificity of protein phosphatase inhibitors.