Rapid effects of corticosteroids on cytosolic protein kinase C and intracellular calcium concentration in human distal colon

Molecular and Cellular Endocrinology 138 (1998) 71 – 79

Rapid effects of corticosteroids on cytosolic protein kinase C and intracellular calcium concentration in human distal colon Christina M. Doolan a,*, Gerald C. O’Sullivan b, Brian J. Harvey a a

Wellcome Trust Cellular Physiology Research Unit, Department of Physiology, Uni6ersity College, Mercy Hospital, Cork, Ireland b Department of Surgery, Mercy Hospital, Cork, Ireland Received 1 December 1997; accepted 28 January 1998

Abstract Recent studies from our laboratory have reported rapid ( B1 min) non-genomic activation of potassium recycling, Na + – H + exchange, protein kinase C (PKC) activity and PKC-sensitive Ca2 + entry by mineralocorticoids in mammalian distal colonic epithelium. Previous studies from other laboratories have described stimulation of the Na + –H + exchanger by PKC activation. Here a rapid non-genomic effect of aldosterone on PKC activity and intracellular free calcium [Ca2 + ]i is demonstrated in human distal colonic epithelium. Rapid activation (after 15 min incubation) of basal PKC activity was observed in cytosolic fractions of human colonic epithelium by aldosterone, fludrocortisone and deoxycorticosterone acetate (DOCA). PKC activation was inhibited by the specific PKC inhibitor bisindolylmaleimide (GF109203X). The glucocorticoid hydrocortisone failed to activate PKC activity. Aldosterone induced a rapid increase in [Ca2 + ]i in isolated human colonic crypts. This stimulatory effect on [Ca2 + ]i was inhibited by the PKC inhibitor chelerythrine chloride. Hydrocortisone and dexamethasone similarly failed to increase [Ca2 + ]i. These results indicate that intracellular signalling for aldosterone involves changes in [Ca2 + ]i via activation of PKC. Since stimulation of PKC activity and increase in [Ca2 + ]i are apparent at normal circulating levels of aldosterone, our findings may have important physiological implications and prompt a reassessment of mineralocorticoid effects on electrolyte homeostasis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Non-genomic steroid action; Mineralocorticoids; Intracellular calcium; Human colonic crypts

1. Introduction Mammalian distal colon is a major target for the mineralocorticoid hormone aldosterone (Edmonds, 1967), and the level of mineralocorticoid receptor gene expression is higher in the distal colon than in other target tissues such as the kidney (Fuller and Verity, 1990). In rat distal colon, aldosterone causes a switch Abbre6iations: Aldo, aldosterone; DOCA, deoxycorticosterone acetate; DTT, dithiothreitol; Fludro, fludrocortisone; Hydro, hydrocortisone; PKC, protein kinase C; PKCI, protein kinase C inhibitor (bisindolylmaleimide GF 109203X, chelerythrine chloride); PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine. * Corresponding author. Tel.: + 353 21 902045; fax: + 353 21 272121; e-mail: [email protected]

from electroneutral NaCl absorption to electrogenic Na + absorption by inducing apical amiloride-sensitive Na + channels and by enhancing basolateral Na + /K + ATPase pump activity (Binder et al., 1989; Turamian and Binder, 1989). In parallel with this mineralocorticoid-dependent change in Na + absorption, net K + absorption in the distal colon of control rats is converted to net K + secretion by aldosterone, which reflects, at least in part, the induction of apical K + channels (Sweiry and Binder, 1989). These effector mechanisms involve binding of aldosterone to intracellular type I mineralocorticoid receptors initiating genomic events. The genomic effects of aldosterone are characterised by a sensitivity to inhibitors of transcription and translation (cyclohexamide, actinomycin D) and a latency5 2–8 h.

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Recently, studies carried out in extrarenal, non-epithelial cells (human mononuclear leukocytes), indicate rapid in vitro effects of aldosterone (acute onset within 1 – 2 min) on intracellular electrolyte concentrations, cell volume, activity of the Na + – H + exchanger and stimulation of the inositol-1,4,5 trisphosphate system (Wehling et al., 1987, 1989a,b, 1991; Christ et al., 1993). Rapid effects of aldosterone on diacylglycerol production and protein kinase C (PKC), mediated through phospholipase C, and free intracellular calcium [Ca2 + ]i, have also been demonstrated in vascular smooth muscle cells (Wehling et al., 1994; Christ et al., 1995). These rapid aldosterone responses are incompatible with the involvement of the classical steroid hormone pathway. The unique characteristics of this new pathway for steroid hormone action include its rapid time course and a 10000-fold selectivity for aldosterone over hydrocortisone. Recent studies from our laboratory have demonstrated fast (B 1 min) non-genomic activation of K + recycling and Na + – H + exchange by mineralocorticoid hormones in human distal colon (Maguire et al., 1994, 1995). We have also demonstrated rapid stimulation of PKC activity in rat distal colonic epithelium and an increase in intracellular calcium concentration [Ca2 + ]i in isolated rat distal colonic crypts by mineralocorticoids (Doolan and Harvey, 1996a). A rapid non-genomic effect of aldosterone on [Ca2 + ]i in the human colonic epithelial cell line T84 has also been shown (Doolan and Harvey, 1996b). Other investigators have previously demonstrated a direct activation of PKC activity by steroid hormones (Slater et al., 1995) and that the Na + –H + exchanger may be stimulated by PKC activation (Slotki et al., 1990). In this study, the effect of mineralocorticoid hormones on PKC activity in human distal colonic epithelium and the effects of aldosterone on [Ca2 + ]i in single isolated human colonic crypts were investigated.

2. Methods

2.1. Materials [g-32P]ATP (3000 Ci/mmol) and PKC assay kit (RPN 77) were purchased from Amersham (UK). Aldosterone, fludrocortisone, deoxycorticosterone acetate (DOCA) hydrocortisone and dexamethasone were obtained from Sigma (St Louis, MO). Bisindolylmaleimide GF 109203X (Ki, 14 nM) and chelerythrine chloride (Ki, 0.66 mM) were purchased from Calbiochem. Fura-2/AM (acetomethoxy ester) was obtained from Molecular Probes (Eugene, OR). All other chemicals were of the highest purity commercially available.

Steroid hormones (except hydrocortisone) were dissolved in methanol (ethanol for dexamethasone) and stored in aliquots at − 20°C until required. The final concentration of methanol or ethanol in all assays was less than or equal to 0.01% (v/v) at which concentration methanol was without effect on PKC or intracellular calcium ion activity.

2.2. Tissue preparation Normal human colon was obtained from patients undergoing colonic resections. Epithelial sheets containing intact colonic crypts were stripped from the underlying connective tissue by microdissection. The epithelial sheets were homogenised in 3 ml of homogenisation buffer (50 mM Tris–HCl, pH 7.5 containing 5 mM EDTA, 10 mM EGTA, 0.3% (v/v) mercaptoethanol, 10 mM benzamidine, 50 mg/ml PMSF and 50 mg/ml leupeptin) using a Camlab Omni homogeniser at 20000 rpm for 5 × 40 s bursts. The homogenate was spun at 86000× g for 30 min. The supernatant (cytosolic fraction) was retained and the pellet (membrane fraction) resuspended in homogenisation buffer by six passages through a 16 guage needle. Protein content of cytosolic and membrane fractions was determined by the Lowry method (Lowry et al., 1951).

2.3. Protein kinase C acti6ity assay PKC activity was measured in an assay based on the transfer of the terminal phosphate of [g -32P]ATP to a synthetic peptide substrate. Assays were carried out at 25°C in a final volume of 100 ml incubation mixture containing 30 mg protein (either cytosolic or membrane fraction), 225 mg peptide substrate, 7.5 mM DTT, stimulators/inhibitors as appropriate, in 50 mM Tris– HCl, pH 7.5. Following a 2 min preincubation period, the reaction was initiated by addition of 37.5 mM [g -32P]ATP (1–2 × 107 cpm/mmol) containing 11.3 mM Mg acetate. Classical PKC (a, bI, bII and g isozymes) has an absolute requirement for calcium (Asaoka et al., 1992) and magnesium ions, which were present at saturating levels in the assay. Assays were stopped after 15 min and processed by a modification of the method of Witt and Roskoski (1975)—assay mixture was spotted onto P81 phosphocellulose ion exchange chromatography filter papers which were allowed to dry for 30 s and placed in a 75 mM phosphoric acid solution (10 ml/ filter). Filters were then washed (on ice) for 2 ×10 min in phosphoric acid. Incorporated radioactivity was determined by scintillation counting and activity expressed as pmol phosphate transferred/mg protein per min.

C.M. Doolan et al. / Molecular and Cellular Endocrinology 138 (1998) 71–79

2.4. Isolation of human colonic crypts Human distal colonic epithelium was placed in crypt isolation buffer pH 7.4 (NaCl (96 mM), KCl (1.5 mM), HEPES–Tris (10 mM), EDTA (27 mM), sorbitol (55 mM), sucrose (44 mM), DTT (1 mM)) at room temperature for 15 min. The crypts were detached after the incubation by vigorous shaking followed by centrifugation at 1000× g for 10 min. The pellet of crypts obtained was resuspended in Krebs solution (NaCl (140 mM), KCl (5 mM), MgCl2 (1 mM), CaCl2 (2 mM), HEPES (10 mM), Tris – HCl (10 mM), glucose (10 mM)).

2.5. Ca 2 + spectrofluorescence Colonic crypts were loaded with 5 mM FURA-2/AM for 30 min at 22°C. The crypts were washed 3× in Krebs solution (1000 rpm for 5 min) and transferred to glass cover slips treated with a 1:10 dilution of poly-Llysine. The coverslips were mounted on an inverted epi-fluorescence microscope (Diaphot 200, Nikon). The light from a Xenon lamp (Nikon) was filtered through alternating 340 and 380 nm interference filters (10 nm bandwidth, Nikon). The resultant fluorescence was passed through a 400 nm dichroic mirror, filtered at 510 nm and then collected using an intensified CCD camera system (Darkstar, Photonic Science). Images were digi-

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tised and analysed using the Starwise Fluo system (Imstar, Paris, France) as described in detail (Donnadieu et al., 1992). The Ca2 + concentration was calculated from: [Ca2 + ]= K%(R− Rmin/Rmax − R), where K% is the product of the dissociation constant of the Ca2 + /Fura-2 complex and a constant related to the optical characteristics of the particular system, R is the experimental ratio of F340/F380 from which the backround fluorescence has been subtracted and Rmin and Rmax are the values of R in the presence of zero and saturating calcium respectively (Donnadieu et al., 1992). Rmin and Rmax values were obtained using the following solutions: (1) Ca2 + free, 150 mM KCl, 10 mM EGTA and 25 mM ionomycim; (2) high Ca2 + , 150 mM KCl, 25 mM ionomycim and 10 mM CaCl2. A micropipette system was used to apply test solutions to isolated colonic crypts plated on a glass cover slip. Where the effect of the PKC inhibitor was examined, the human colonic crypts were pre-incubated with chelerythrine chloride (1 mM) 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 concentration of steroid used. All experiments were carried out at room temperature (20–22°C) to minimise dye leakage and colonic crypt disintegration.

2.6. Statistical analysis Measurements of PKC and Ca2 + activities are presented as mean values9SE mean of n experiments. Statistically significant differences were determined by an unpaired Student’s t-test and differences were deemed significant if P5 0.05.

3. Results

Fig. 1. Modulation of unstimulated PKC activity by PMA (6 mg/ml), PS (2 mol%) and PKCI bisindolylmaleimide GF109203X (25 nM) in cytosol and membrane fractions isolated from human distal colonic epithelium. Results are expressed as pmol phosphate transferred/mg protein per min. Data represent the mean 9 SE mean of four experiments carried out in duplicate. Asterisks indicate significant differences between values linked by horizontal bars: *** PB 0.005, ** P B 0.01, * P B 0.025.

We have identified the presence of PKC activity in cytosolic and membrane fractions isolated from human distal colonic epithelium. Basal (unstimulated) PKC activity was significantly increased (cytosol PB0.01, membrane PB 0.005) in the presence of a cocktail containing PS (2 mol%), PMA (6 mg/ml) and calcium acetate (3 mM) and this stimulated PKC activity was inhibited in the presence of the specific PKC inhibitor bisindolylmaleimide GF109203X (25 nM). These results are shown in Fig. 1. The intracellular localisation of PKC varies between cell types but in most tissues the enzyme is recovered mainly in the soluble (cytosolic) fraction and would translocate to the membrane only in the presence of sustained activating signals (Anderson et al., 1985; Kikkawa et al., 1982). In this study, using fractionated samples of human distal colonic epithelium, we have determined that :75% of PKC activity is associated with the cytosolic fraction with the remainder being localised to the membrane fraction.

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Table 1 Modulation of PKC activity, within 15 min, by corticosteroids in cytosolic fractions isolated from human distal colonic epithelium Steroid (nM)

Aldosterone

Fludrocorti- DOCA sone

Hydrocortisone

100 10 1 0.1

3.2 90.7 3.0 90.7 3.2 90.9 3.6 91.1

3.5 9 0.9 3.59 1 3.49 0.9 3.6 9 1.2

1.3 9 0.15 1.2 9 0.03 1.29 9 0.2 1.17 9 0.02

3.4 9 0.8 3.2 9 0.7 4.29 1 3.5 9 1.2

Results are expressed as fold stimulation over basal activity. Data represent the mean 9SE mean of three experiments carried out in duplicate.

Since previous studies have demonstrated that the Na + –H + exchanger may be stimulated by PKC activation (Slotki et al., 1990) and by mineralocorticoid hormones (Maguire et al., 1994, 1995), we examined the effect of mineralocorticoid hormones on PKC activity in human colonic epithelium. From the results described in Fig. 1 we found that PKC activity was recovered mainly in the cytosolic fraction and this fraction was therefore chosen to assay for the effect of steroid hormones on human colonic PKC activity. Basal PKC activity (44 93.8 pmol phosphate transferred/mg protein per min) was significantly stimulated, following a 15 min incubation, in the presence of the mineralocorticoid hormones aldosterone (0.1 – 100 nM, P B 0.05), fludrocortisone (0.1 – 100 nM, P B0.05) and DOCA (0.1–100 nM, P B0.05) in cytosolic fractions isolated from human distal colonic epithelium. However, the glucocorticoid hormone, hydrocortisone (0.1– 100 nM), failed to stimulate basal cytosolic PKC activity. These results are summarised in Table 1. A comparison of the effect of a physiological concentration of mineralocorticoid hormones (0.1 nM) versus a 100-fold higher concentration of hydrocortisone is shown in Fig. 2. From these experiments, it appears that PKC activity, in cytosolic fractions isolated from human colonic epithelium, is specifically activated by mineralocorticoid hormones and not by glucocorticoid hormones. Activation of human colonic cytosolic PKC activity was observed only when both the steroid hormone and calcium (3 mM) were present in the assay mixture. No activation of unstimulated PKC activity was observed when either was present alone (data not shown), indicating the presence of a Ca2 + -dependent PKC isozyme in cytosolic fractions of human distal colonic epithelium. In a further set of experiments we examined whether cytosolic PKC activity could be activated by hormone following a shorter incubation time (5 min). Aldosterone (0.1 nM) significantly (P B0.025) stimulated basal PKC activity, following a 5 min incubation, in cytosolic fractions isolated from human colonic epithelium. The aldosterone-induced stimulation of PKC ac-

Fig. 2. Modulation of PKC activity, within 15 min, by aldosterone (0.1 nM), fludrocortisone (0.1 nM), DOCA (0.1 nM) and hydrocortisone (100 nM) in cytosolic fractions isolated from human distal colonic epithelium. Results are expressed as fold stimulation over basal activity. Data represent the mean 9 SE mean of three experiments carried out in duplicate. Asterisks indicate significant differences between the mineralocorticoid hormones compared with hydrocortisone: * P B0.05.

tivity was significantly inhibited (PB 0.005) in the presence of the specific PKC inhibitor bisindolylmaleimide (25 nM). These results are summarised in Table 2. The stimulatory effect on PKC activity of all the mineralocorticoid hormones examined was inhibited in the presence of the PKC inhibitor bisindolylmaleimide (25 nM, data not shown). Previous studies in extrarenal, non-epithelial cells (Wehling et al., 1994), in rat distal colonic crypts (Doolan and Harvey, 1996a) and in the human colonic epithelial cell line T84 (Doolan and Harvey, 1996b) have demonstrated a rapid effect of aldosterone to increase free [Ca2 + ]i. Here, we compared the effects of the mineralocorticoid hormone aldosterone with those of the natural glucocorticoid hormone hydrocortisone and the synthetic glucocorticoid hormone dexamTable 2 Modulation of PKC activity, within 5 min, by aldosterone (0.1 nM) in cytosolic fractions isolated from human distal colonic epithelium Unstimulated PKC activity

61 9 8.3

Aldosterone (0.1 nM) Aldosterone (0.1 nM)+PKCI (25 nM)

102 98.7 (PB0.025) (vs basal) 10 95.5 (PB0.005) (vs aldosterone)

Results are expressed as pmol phosphate transferred/mg protein.min. Data represent the mean 9SE mean of three experiments carried out in duplicate.

C.M. Doolan et al. / Molecular and Cellular Endocrinology 138 (1998) 71–79

Fig. 3. Human colonic crypt with surface cells attached (magnification, × 40). Regions of interest corresponding to Ca2 + measurements in Figs. 4 – 6 and Table 3 are indicated as follows: (B) base of crypt; M1 – M3, mid-regions of crypt; SC, surface cells.

ethasone, on free [Ca2 + ]i in single isolated crypts from human distal colonic epithelium (hydrocortisone may be oxidised to its inactive 11-keto form by the microsomal enzyme 11b-hydroxysteroid dehydrogenase (11bHSD) which exists in the colon, however, dexamethasone is not a substrate for this microsomal oxoreductase enzyme). In a further set of experiments we tested, using the membrane permeable PKC inhibitor chelerythrine chloride (1 mM), whether the effect of aldosterone to increase [Ca2 + ]i in isolated colonic crypts was due to the activation of PKC. A typical isolated human colonic crypt with surface cells attached is shown in Fig. 3. The regions of interest corresponding to Table 3 and Figs. 4 – 6 are indicated. In all experiments where the effect of steroid hormones on [Ca2 + ]i was examined, calcium (2 mM) was present in the external bathing solution unless otherwise stated. A significant increase in [Ca2 + ]i was observed in all regions of the human distal colonic crypt following aldosterone addition. Intracellular calcium concentration was significantly increased at physiological concentrations of aldosterone (0.1 nM). This stimulatory effect of aldosterone on [Ca2 + ]i was abolished when the colonic crypts were pre-incubated (5 min) in the presence of the PKC inhibitor chelerythrine chloride (1 mM). The results of these experiments are summarised in Table 3 (a) and (b) respectively. Modulation of free [Ca2 + ]i in the lower middle (M1) region of the human distal colonic crypt by aldosterone (0.1 nM) in the absence and presence of the membrane permeable PKC inhibitor chelerythrine chloride (1 mM) is shown in Fig. 4.

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In contrast to these results, a 10000-fold higher dose of the glucocorticoid hormones, hydrocortisone (1 mM) or dexamethasone (1 mM), failed to increase [Ca2 + ]i in any region of the human colonic crypt over a time course of 8 min. Modulation of [Ca2 + ]i in the lower middle (M1) region of the colonic crypt by aldosterone (0.1 nM), hydrocortisone (1 mM) and dexamethasone (1 mM) are shown in Fig. 5. In the lower middle (M1) region: (i) basal [Ca2 + ]i = 2529 57 nM, and 8 min post-hydrocortisone addition [Ca2 + ]i = 2599 62 nM; (ii) basal [Ca2 + ]i = 1009 17 nM, and 8 min post-dexamethasone addition [Ca2 + ]i = 1029 20 nM. It could be that the lack of effect of hydrocortisone on [Ca2 + ]i in these experiments may be due to its metabolism by the enzyme 11b-HSD which is present in the colon (Whorwood et al., 1992). However, since dexamethasone (which is not a substrate for metabolism by 11b-HSD) also failed to produce an increase in [Ca2 + ]i in human distal colonic crypts it would appear that the lack of effect observed in the presence of hydrocortisone was not due to its metabolism. It can be concluded therefore that glucocorticoid hormones do not exhibit rapid non-genomic effects in human distal colonic crypts. Also, at the high concentrations of glucocorticoid hormones tested, hydrocortisone and dexamethasone would have been expected to bind to the non-specific type I mineralocorticoid receptor. The inability of the glucocorticoid hormones to elevate [Ca2 + ]i reinforces our conclusion that the rapid effects of low concentrations of mineralocorticoid hormones are not mediated by the classical intracellular mineralocorticoid receptor. Extracellular calcium was required for the aldosterone response since no increase in [Ca2 + ]i was observed following aldosterone (10 nM) addition when isolated colonic crypts were incubated in an external bathing solution containing 0 mM Ca2 + and 5 mM EGTA. In these experiments, crypts were preincubated in Ca2 + -free solutions for 1 min followed by aldosterone addition. Intracellular Ca2 + was recorded over an 8 min time period. Fig. 6 shows that aldosterone (10 nM) had no significant effect on [Ca2 + ]i in the lower middle M1 region of the human colonic crypt in the absence of extracellular calcium. A similar result was obtained in all other regions of the crypt (data not shown).

4. Discussion In this study we investigated the involvement of PKC and Ca2 + as possible second messengers of rapid non-genomic effects of aldosterone in human colon and have demonstrated, for the first time, a rapid stimulation (B 5 min) of PKC activity and an increase in [Ca2 + ]i in human distal colonic epithelium by mineralocorticoid hormones.

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Table 3 Modulation of free [Ca2+]i, within 16 min, by aldosterone (0.1 nM) in (a) the absence and (b) presence of the protein kinase C inhibitor chelerythrine chloride (1 mM), in various regions of the human colonic crypt corresponding to Fig. 3 Crypt region

Base

M1

M2

M3

Surface cells

Basal (a) Aldosterone (0.1 nM)

140948 245927 (PB0.01)

128 9 32 2379 28 (PB0.05)

133 9 29 248 9 41 (PB0.05)

120 926 227 916 (PB0.025)

47 916 118 929 (PB0.05)

Basal (b) Aldosterone (0.1 nM)+PKCI

105916 93937

121 933 1299 40

116 929 111 9 48

83 927 83 948

38 928 61 945

Data represent the mean 9 SE mean of four independent experiments each for (a) and (b). Results are expressed as [Ca2+]i (nM).

In cytosolic fractions isolated from human distal colonic epithelium, basal PKC activity was significantly stimulated by aldosterone (0.1 – 100 nM). A similar significant stimulation of PKC activity was also observed in the presence of the mineralocorticoid hormones fludrocortisone (0.1 – 100 nM) and DOCA (0.1 –100 nM). No clear dose-response relationship was observed and all doses of steroid examined stimulated PKC activity to a similar extent. These results are similar to those previously observed for the effect of mineralocorticoid hormones and 17 b-oestradiol on PKC activity in cytosolic fractions isolated from rat distal colonic epithelium (Doolan and Harvey, 1996a). The results of these studies imply that PKC activity is maximally stimulated at physiological steroid concentrations, under the conditions of these assays. In contrast to the stimulatory effects on PKC activity observed in the presence of mineralocorticoid hor-

Fig. 4. Modulation of free [Ca2 + ]i within 16 min, in the lower middle (M1) region of the human distal colonic crypt by aldosterone (0.1 nM) in the “ absence and  presence of the membrane permeable PKC inhibitor chelerythrine chloride (1 mM). Results are expressed as [Ca2 + ]i (nM). This is a representative experiment which was carried out 4× with similar results.

mones, no stimulation of PKC activity was observed in the presence of the glucocorticoid hormone hydrocortisone (0.1–100 nM). It appears therefore that PKC activity is specifically activated by mineralocorticoid hormones. Aldosterone has also been reported to activate and translocate PKC-a from the cytosolic to the membrane fraction in rat vascular smooth muscle cells (Christ et al., 1995). In contrast Sato et al. (1997) have demonstrated a significant inhibition of basal and PMA-stimulated PKC activity by aldosterone (1 nM) in neonatal rat cardiomyocytes. However, in rat cardiac fibroblasts aldosterone did not alter basal or PMA-stimulated PKC activity. In a previous study by Sato and Funder (1996, 1996) mineralocorticoid receptor mediated hypertrophy of rat neonatal cardiomyocytes by aldosterone was abolished by the mineralocorticoid receptor antagonist spironolactone and the specific PKC inhibitor bisindolylmaleimide GF109203X. It appears

Fig. 5. Modulation of free [Ca2 + ]i within 8 min, in the lower middle (M1) region of the human distal colonic crypt by aldosterone (0.1 nM),  hydrocortisone (1 mM) and “ dexamethasone (1 mM). Results are expressed as [Ca2 + ]i (nM). This is a representative experiment which was carried out 4 × with similar results.

C.M. Doolan et al. / Molecular and Cellular Endocrinology 138 (1998) 71–79

Fig. 6. Modulation of free [Ca2 + ]i within 8 min, in the lower middle (M1) region of the human colonic crypt by aldosterone (10 nM) in the “ presence of extracellular Ca2 + (2 mM) or  in the absence of extracellular calcium ( + 5 mM EGTA). Results are expressed as [Ca2 + ]i (nM). This is a representative experiment which was repeated 4× with similar results.

therefore, that both genomic and rapid non-genomic mineralocorticoid hormone action may be regulated by PKC activity. The varying PKC responses observed in the different cell types may be explained by tissue and transport pathway specificity. Previous studies from other laboratories have demonstrated a direct activation of PKC activity by steroid hormones (Slater et al., 1995). In these experiments, using a cell free assay system with purified PKC, the metabolite of vitamin D3, 1 a, 25-dihydroxyvitamin D3 (1,25-D3), at physiological concentrations, directly and potently activated PKC activity in a manner similar to the stimulation observed in the presence of the natural activator of PKC, diacylglycerol. The study described here demonstrates a significant increase in basal PKC activity by aldosterone at a similar concentration range to that found to be effective to stimulate Na + – H + exchange and inositol 1,4,5trisphosphate generation in human mononuclear leukocytes and vascular smooth muscle cells (Wehling et al., 1989a, 1991; Christ et al., 1993) and to stimulate PKC activity in rat distal colonic epithelium (Doolan and Harvey, 1996a). Rapid (within 5 min) non-genomic activation of ATP-regulated K + (KATP) channels has been observed in our laboratory in human distal colon and frog skin (Maguire et al., 1994, 1995; Urbach et al., 1996) by aldosterone, with an EC50 of  0.8 nM. This activation is insensitive to inhibitors of transcription and translation such as cyclohexamide and actinomycin D or to the classical type I mineralocorticoid receptor antagonist, spironolactone. However, the rapid effect of aldosterone on KATP channel activity may be prevented by inhibition of PKC activity or inhibition of the basolateral membrane Na + – H + exchanger either in

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Na + -free medium or in the presence of amiloride. Previous work from other laboratories has demonstrated the activation of KATP channels in rabbit and human ventricular myocytes (Hu et al., 1996; Liu et al., 1996) and the inhibition of Ca2 + -sensitive K + channels (KCa) in T84 colonic epithelial cells and vascular smooth muscle cells, by PKC activation (Reenestra, 1993; Slater et al., 1995; Ren et al., 1996; Satoh, 1996). It may be possible therefore that aldosterone enhances net salt reabsorption in the mammalian distal colon, by simultaneously stimulating the pathway for Na + reabsorption via activation of the KATP channel while down regulating the Cl − secretory pathway via inhibition of the KCa channel. In the second part of this study the effects of the mineralocorticoid hormone aldosterone and the glucocorticoid hormones hydrocortisone and dexamethasone on [Ca2 + ]i were examined in single isolated human distal colonic crypts. PKC involvement in the stimulatory response to aldosterone was investigated using the membrane permeable PKC inhibitor chelerythrine chloride. A rapid increase in basal free [Ca2 + ]i was observed at all regions of isolated single human distal colonic crypts following aldosterone addition. The increase in [Ca2 + ]i was immediate upon aldosterone addition, which is clearly incompatible with a genomic response. Inhibition of PKC activity, by chelerythrine chloride, abolished the stimulatory effect of aldosterone (0.1 nM) on [Ca2 + ]i. Similarly, no increase in [Ca2 + ]i was observed in a Ca2 + free bathing medium. High concentrations of the glucocorticoid hormones hydrocortisone (1 mM) or dexamethasone (1 mM) did not induce an increase in [Ca2 + ]i in any region of the human distal colonic crypt. From these results, aldosterone appears to stimulate the influx of extracellular Ca2 + via a PKC sensitive pathway. These results are similar to our observations where aldosterone stimulated PKC-dependent Ca2 + influx in isolated rat distal colonic crypts (Doolan and Harvey, 1996a) and a PKCdependent, verapamil-sensitive Ca2 + channel in the human colonic epithelial cell line T84 (Doolan and Harvey 1997). Studies from other laboratories have established that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) as well as acting as a classical steroid hormone via a receptor-mediated nuclear mechanism of activation (Norman et al., 1982; Minghetti and Norman, 1988) also exerts rapid actions in classical, as well as non-classical, target tissues independent of gene activation (Nemere et al., 1984; de Boland and Boland, 1987; Liebherr, 1987; Nemere and Norman, 1987; Baran and Kelly, 1988). Evidence has been obtained that these non-genomic effects of 1,25 (OH)2D3 may be mediated by the activation of cell membrane voltage-dependent Ca2 + channels (de Boland and Boland, 1987; de Boland et al., 1989; Tornquist and Tashjian, 1989.) via PKC activa-

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tion (Yamaguchi et al., 1987; Wali et al., 1990). The increase in [Ca2 + ]i elicited by the 1,25 (OH)2D3 in isolated duodenal cells (de Boland and Norman, 1990) and the osteosarcoma cell line UMR-106 (Yamaguchi et al., 1987) was due to Ca2 + influx from the extracellular medium, since it was blocked in the presence of the Ca2 + chelator EGTA (5 mM) in the extracellular medium and by the Ca2 + channel blocker nifedipine. It may be possible therefore, that a common Ca2 + signalling pathway exists for non-genomic steroid hormone action. The reported rapid in vitro effects of aldosterone (Wehling et al., 1987, 1989a,b, 1991; Christ et al., 1993; Wehling et al., 1994; Maguire et al., 1994; Christ et al., 1995; Maguire et al., 1995; Doolan and Harvey, 1996a,b) are incompatible with classical genomic mechanisms of steroid hormone action but indicate a nongenomic pathway with high affinity for mineralocorticoid hormones and very low affinity for hydrocortisone. Importantly, the results obtained in our study demonstrate the existence of non-genomic steroid hormone action in a human classical steroid hormone target epithelium. The rapid in vitro effects of aldosterone are evident within the physiological concentration range of free circulating hormone ( 0.1 nM) and this would lend support to the physiological significance of these results. The selectivity for aldosterone is important, since it may explain the different effects of mineralocorticoids and glucocorticoids on sodium homeostasis.

Acknowledgements This work was funded by Wellcome Trust Programme Grant (040067/Z/93) and a Health Research Board (IRL) Inflammatory Bowel Disease Unit Grant.

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