(Received for publication, August 7, 1995; and in revised form, January 17, 1996)
From the
Studies from our laboratory have demonstrated rapid (<1 min)
non-genomic activation of Na-H
exchange and potassium recycling by mineralocorticoids in human
and rat colonic epithelium. It has previously been demonstrated that
Na
-H
exchange may be stimulated by
protein kinase C (PKC) activation; therefore, we examined the effect of
mineralocorticoids on PKC activity in rat colonic epithelium.
Activation (after 15 min of incubation) of basal PKC activity was
observed in cytosolic fractions of rat colonic epithelium by
aldosterone, fludrocortisone, and deoxycorticosterone acetate. In all
instances, PKC activation was inhibited by the PKC inhibitor
bisindolylmaleimide (GF109203X). Hydrocortisone failed to activate PKC
activity. Stimulation of basal intracellular free calcium
[Ca
]
was observed, in
isolated rat colonic crypts, following aldosterone addition. This
stimulatory effect was inhibited by the PKC inhibitor, chelerythrine
chloride. Hydrocortisone failed to increase
[Ca
]
. These results
indicate that intracellular signaling for aldosterone involves changes
in [Ca
]
via activation
of PKC. Since the stimulation of PKC and increase in
[Ca
]
are apparent at
normal circulating levels of aldosterone, our findings have major
implications for the reassessment of mineralocorticoid effects on
electrolyte homeostasis.
Mineralocorticoid hormones increase sodium reabsorption and promote potassium and hydrogen secretion in high resistance epithelia. This effector mechanism involves binding of aldosterone to intracellular type 1 mineralocorticoid receptors initiating genomic events. The genomic effects of aldosterone are characterized by a latency of 2-8 h and a sensitivity to inhibitors of transcription or translation (cyclohexamide and actinomycin D).
In contrast,
aldosterone has been reported to produce a rapid in vitro effect (acute onset within 1-2 min) on intracellular
electrolyte concentrations and cell volume and activity of the
Na-H
exchanger in human mononuclear
leukocytes(1, 2, 3, 4) . The
inositol 1,4,5-trisphosphate system appears to be involved in rapid
non-genomic effects of aldosterone in human mononuclear
leukocytes(5) . Rapid effects of aldosterone on free
intracellular calcium [Ca
]
in vascular smooth muscle and endothelial cells have
recently been demonstrated(6) . These fast responses to
aldosterone are incompatible with the involvement of the classical
steroid hormone pathway. The unique characteristics of this new pathway
for steroid action include its rapid time course and a 10,000-fold
selectivity for aldosterone over cortisol (hydrocortisone).
Mammalian distal colon has long been considered an
aldosterone-responsive epithelium(7) , and aldosterone has been
shown to induce K conductances in rat distal
colon(8) . Recent studies from our laboratory have demonstrated
rapid (<1 min) non-genomic activation of
Na
-H
exchange and K
recycling by mineralocorticoids in human colon(9, 10) and frog skin(11) . It has also been shown that
Na
-H
exchange may be stimulated by
protein kinase C (PKC) (
)activation(12) .
Many
extracellular signals such as hormones, neurotransmitters, growth
factors and other biologically active substances induce both
Ca mobilization and phosphoinositide turnover in
their target cells(13) . The use of Ca
ionophores has revealed an apparent synergism between
Ca
mobilization and PKC
activation(14, 15) .
The aims of this study were to
investigate the effect of mineralocorticoids on PKC activity in rat
distal colonic epithelium and the effects of aldosterone on
[Ca]
in rat colonic
crypts.
Steroid hormones (except hydrocortisone) were dissolved in methanol and stored in aliquots at -20 °C until required. The final concentration of methanol in all assays was less than or equal to 0.01%, at which concentration methanol was without effect on PKC or intracellular calcium ion activity.
The Ca concentration was calculated
according to ,
where K` is the product of the dissociation constant of
the Ca/Fura-2 complex and a constant related to the
optical characteristics of the particular system, R is the
experimental ratio of F
/F
from which the backround fluorescence has been subtracted, and R
and R
are the values of R in the presence of zero and saturating calcium,
respectively(19) . R
and R
values were obtained using the following
solutions containing: 150 mM KCl, 10 mM EGTA, and 25
µM ionomycin with or without 10 mM CaCl
. A micropipette perfusion system was used to
apply test solutions to isolated colonic crypts plated on a glass
coverslip. Where the effect of the PKC inhibitor was examined, the rat
colonic crypts were preincubated with chelerythrine chloride for 5 min
at room temperature prior to the addition of aldosterone. In all
experiments the test solution was identical to the bathing solution
except for the dose of steroid used.
The presence of PKC activity in both cytosolic and membrane fractions isolated from rat colonic epithelium has been identified. Stimulation of PKC activity was observed in the presence of a mixture containing phorbol 12-myristate 13-acetate (6 µg/ml), phosphatidylserine (2 mol%), and calcium acetate (3 mM). The stimulated PKC activity was inhibited by the PKC inhibitor bisindolylmaleimide GF109203X (25 nM), and these results are shown in Fig. 1. Although the intracellular localization of PKC varies with cell type, in most tissues the enzyme is recovered mainly in the soluble fraction and would translocate to the membrane only in the presence of sustained activating signals(20, 21) . In fractionated samples of rat colonic epithelium, approximately 75% of PKC activity is associated with the cytosolic fraction and the remainder localized to the membrane fraction.
Figure 1:
Modulation of PKC activity by phorbol
12-myristate 13-acetate (6 µg/ml), phosphatidylserine (2 mol%), and
protein kinase C inhibitor bisindolylmaleimide GF109203X (25
nM) in cytosol and membrane fractions isolated from rat distal
colonic epithelium. Results are expressed as phosphate transferred
(pmol min mg
protein). Data
represent the mean ± S.E. of six experiments performed in
duplicate. Asterisks indicate significant differences between
values linked by horizontal bars. *, p <
0.0005;**, p < 0.005.
Since it has been
shown that Na-H
exchange may be
stimulated by both mineralocorticoids (4, 22) and PKC
activation (12) , the effect of mineralocorticoids on PKC
activity was investigated in rat colonic epithelium. As we had shown
that PKC activity was recovered mainly in the cytosolic fraction (Fig. 1), this was the fraction chosen to assay for the effect
of steroid hormones on PKC activity. Basal PKC activity was
significantly stimulated, following a 15-min incubation, in the
presence of aldosterone (0.01-100 nM; p <
0.005), fludrocortisone (0.01-100 nM; p <
0.005), and DOCA (0.01-100 nM; p < 0.01) in
cytosolic fractions isolated from rat colonic epithelium. However,
basal PKC activity (29 ± 10, phosphate transferred
min
mg
protein) was not
significantly stimulated by sub-physiological doses of aldosterone (1
pM; 89 ± 35, phosphate transferred min
mg
protein). Hydrocortisone (0.01-100
nM) failed to stimulate basal PKC activity. PKC activation was
only observed when both Ca
(3 mM) and
steroid were present in the assay mixture; no PKC stimulation was
observed when either was present alone (data not shown). These results
indicate that the PKC isozyme present in rat colonic epithelium is
Ca
-dependent. At all doses of steroid examined
(0.01-100 nM), aldosterone and fludrocortisone were
significantly (p < 0.05) more effective as stimulators of
PKC activity than DOCA. The results of these experiments are shown in Table 1. The stimulatory effect of DOCA (0.01-100
nM) on PKC activity was approximately 50% of that observed in
the presence of aldosterone and fludrocortisone (0.01-100
nM).
To examine whether stimulaton of PKC activity could be detected following a shorter incubation time, the effect of aldosterone (0.1 nM) on basal PKC activity was examined following a 5-min aldosterone (0.1 nM) incubation. In these experiments, significant stimulation of basal PKC activity was observed after a 5-min incubation. This stimulatory effect of aldosterone was inhibited by the PKC inhibitor bisindolylmaleimide (25 nM), and these results are shown in Table 2. Aldosterone (0.1 nM) stimulation of basal PKC activity was also observed in membrane fractions isolated from rat distal colonic epithelium. This stimulatory effect of aldosterone was significantly inhibited by the PKC inhibitor bisindolylmaleimide (25 nM), and these data are shown in Table 3. The stimulation of PKC activity in membrane fractions, however, was approximately 4-fold less than that observed in cytosolic fractions. In a further series of experiments, the existence of PKC activity was also established in cytosolic and membrane fractions isolated from rat distal colonic crypts. Significant stimulation of basal PKC activity was observed, following aldosterone (0.1 nM), in both rat crypt cytosolic and membrane fractions, and this stimulation was significantly inhibited by the specific PKC inhibitor bisindolylmaleimide (25 nM). These results are shown in Table 4.
Sex steroid hormones, particularly
-estradiol, have salt-retaining properties, but their possible
non-genomic effects on ion transport or cell signaling have not
previously been examined. Fig. 2shows the stimulation of basal
PKC activity in rat colonic cytosolic fractions in the presence of
-estradiol (0.01-100 nM).
Figure 2:
Modulation of PKC activity, within 15 min
by -estradiol (100-0.01 nM) in cytosolic fractions
isolated from rat distal colonic epithelium. Results are expressed as
phosphate transferred (pmol min
mg
protein). Data represent the mean ± S.E. of three
experiments performed in duplicate. Asterisks indicate
significant differences between values linked by horizontal bars. *, p < 0.05;**, p <
0.025.
The stimulatory effect
of all of the above steroid hormones on PKC activity from rat distal
colonic cytosolic fractions was inhibited by the PKC inhibitor
bisindolylmaleimide (25 nM). The effects of aldosterone
(0.01-100 nM), on PKC activity, in the absence and
presence of the PKC inhibitor bisindolylmaleimide (25 nM) are
shown in Table 5. Bisindolylmaleimide (25 nM) also
inhibited the PKC stimulatory effect obtained in the presence of
fludrocortisone, DOCA, and -estradiol (0.01-100 nM)
(data not shown).
As rapid effects of aldosterone on free
[Ca]
have recently been
demonstrated in endothelial cells(6) , we determined the effect
of both aldosterone and hydrocortisone on free
[Ca
]
in single isolated crypts
from rat colonic epithelium. We also examined whether the effect of
aldosterone to increase [Ca
]
was due to protein kinase C activation, by use of the PKC
inhibitor chelerythrine chloride. Fig. 3shows a typical rat
colonic crypt with surface cells attached. The regions of analysis
corresponding to Fig. 4and Table 6and Table 7are
indicated. (2 mM Ca
was present in the
external bathing solution in all cases unless otherwise stated.)
Figure 3:
Rat
colonic crypt with surface cells attached (magnification, 40).
Regions of interest corresponding to Ca
measurements
in Table 6, Table 7, and Fig. 4are indicated as
follows: B, base of crypt; M1-M3, mid-regions
of crypt; SC, surface cells.
Figure 4:
Modulation of
[Ca]
, within 16 min,
by aldosterone (1 µM) and hydrocortisone (1
µM) in rat colonic crypts, in the presence of 2 mM Ca
externally. Basal conditions indicate the
basal values prior to the addition of steroid to individual crypt
preparations. Results are expressed as
[Ca
]
(nM).
Data represent the mean ± S.E. of six experiments. Asterisks indicate significant differences between values linked by horizontal bars. *, p < 0.05;**, p <
0.025.
Basal free [Ca]
was
significantly increased in all regions of the rat colonic crypt, 16 min
following aldosterone (1 µM) addition. In contrast, no
increase in basal free [Ca
]
was
observed in any region of the isolated rat colonic crypt following
hydrocortisone (1 µM) addition. These results are shown in Fig. 4.
No increase in free
[Ca]
was observed following
aldosterone (1 µM) addition when the crypts were present
in 0 mM Ca
in the external bathing solution
(data not shown).
Stimulation of basal free
[Ca]
was observed throughout
the length of the rat colonic crypt following the addition of low
concentrations of aldosterone (10 nM, 1 nM). Table 6(parts a and b, respectively) summarizes these results.
Basal free [Ca]
was
significantly increased in all regions of the rat colonic crypt, 16 min
following aldosterone (0.1 nM) addition. When the rat colonic
crypts were preincubated (5 min) with the PKC inhibitor chelerythrine
chloride (1 µM), this stimulatory effect of aldosterone on
[Ca
]
was abolished. These
results are shown in Table 7.
In the present study, the involvement of PKC and
Ca as possible second messengers in rapid
aldosterone effects was investigated.
This study demonstrates for
the first time, rapid stimulation (after 5 and 15 min of incubation) of
PKC activity and an increase in [Ca]
by mineralocorticoid hormones in rat distal colonic epithelium.
Both of these steroid hormone effects were inhibited in the presence of
a PKC inhibitor. These results indicate that intracellular signaling
for aldosterone involves changes in
[Ca
]
via activation of PKC
activity.
In cytosolic fractions isolated from rat distal colonic
epithelium, basal PKC activity was significantly stimulated by
aldosterone (0.01-100 nM). A dose-response relationship
was not observed, as all doses of steroid stimulated PKC activity to
the same extent. These results imply that under these conditions PKC
activity is maximally stimulated at physiological concentrations of
aldosterone. Sub-physiological (pM) concentrations of
aldosterone failed to stimulate PKC activity in a significant manner.
Fludrocortisone (0.01-100 nM) produced similar
significant stimulation of PKC activity, whereas the stimulatory effect
of DOCA (0.01-100 nM) was 50% of that observed in the
presence of aldosterone or fludrocortisone. In membrane fractions,
isolated from rat distal colonic epithelium, PKC could also be
stimulated by aldosterone (0.1 nM). The stimulation observed
was approximately 25% of that seen in cytosolic fractions. These
results were expected, inasmuch as, in most tissues, PKC resides mainly
in the cytosolic fraction and is translocated to the membrane
post-activation. From these results it appears, therefore, in the rat
colonic epithelium, that aldosterone stimulates the enzyme in both
fractions. Protein kinase C activity in isolated rat colonic crypts was
also significantly stimulated by aldosterone (0.1 nM). The sex
steroid -estradiol (0.01-100 nM) also produced a
significant stimulation of PKC activity, but only to 20% of that seen
with either aldosterone or fludrocortisone. In contrast to the
stimulatory effects observed in the presence of the above steroids, no
stimulation of PKC activity, in cytosolic fractions isolated from rat
colonic epithelium, was observed in the presence of the glucocorticoid
hydrocortisone (0.01-100 nM).
Previous studies have
shown that Na-H
exchange may be
stimulated by PKC activation (12) and by
mineralocorticoids(4, 22) . The results obtained in
the first part of this study demonstrate a significant stimulation of
basal PKC activity by aldosterone over a concentration range similar to
that found to be effective to stimulate
Na
-H
exchange and inositol
1,4,5-trisphosphate generation in vascular smooth muscle cells and
human mononuclear leukocytes(5, 22) . Recent studies
from our laboratory provide evidence for rapid non-genomic activation
of ATP-regulated K
(K
) channels by
aldosterone in human distal colon and frog
skin(9, 10, 11) . Rapid activation of
basolateral K
channels by the hormone occurs within 5 min
with an EC
of
0.8 nM. This effect is
insensitive to spironolactone, cyclohexamide, or actinomycin D, but can
be prevented by pretreatment of the epithelium with PKC inhibitors or
by inhibition of Na
-H
exchange
(amiloride or Na
-free medium). The estrogen
-estradiol (100 nM) also produced an immediate (<5
min) activation of K
channels in human colonic epithelium (23) . This rapid effect of
-estradiol on
potassium-dependent SCC was abolished by inhibition of basolateral
Na
-H
exchange (100 µM amiloride or Na
-free medium). It is possible
therefore that stimulation of PKC activity, by steroid hormones, may in
turn lead to activation of Na
-H
exchange.
In the second part of this study the effect of
aldosterone on free [Ca]
was
investigated in isolated rat colonic crypts. Rapid stimulation of basal
free [Ca
]
was observed in all
regions of the rat colonic crypt following aldosterone addition (1
µM, 10 nM, 1 nM, and 0.1 nM),
and this increase in [Ca
]
appeared to plateau within 12-16 min after aldosterone
addition. This stimulatory effect of aldosterone (0.1 nM) on
[Ca
]
was abolished in the
presence of the PKC inhibitor chelerythrine chloride (1
µM) or in a Ca
-free medium. No
stimulation of [Ca
]
was
observed following hydrocortisone (1 µM). Thus,
aldosterone appears to stimulate the influx of extracellular
Ca
via a PKC-sensitive pathway.
Clearly these
rapid effects of aldosterone to: 1) stimulate
Na-H
exchange in leukocytes and
vascular smooth muscle, 2) increase
[Ca
]
in vascular smooth muscle,
3) stimulate inositol 1,4,5-trisphosphate generation, and 4) in this
study, stimulate PKC activity and increase
[Ca
]
in rat colonic epithelium,
are incompatible with classical genomic mechanisms of steroid action
but indicate a non-genomic pathway with high affinity for
mineralocorticoids and very low affinity for hydrocortisone. More
importantly, the results obtained in our study demonstrate that the
non-genomic signal transduction mechanism is operative in a classical
steroid hormone target epithelium. These results therefore give
additional support to the hypothesis of a novel rapid pathway for
aldosterone action.
The physiological significance of non-genomic
aldosterone action is supported by the low apparent K for the rapid in vitro effects of aldosterone, which are
consistent with the physiological concentration of free circulating
aldosterone (
0.1 nM). The selectivity for aldosterone is
important, since it may explain the different effects of
mineralocorticoids and glucocorticoids on sodium homeostasis.