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

Abstract

Recent studies from our laboratory have reported rapid (<1 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.

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 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 latency≤2–8 h.

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, Wehling et al., 1989a, Wehling et al., 1989b, Wehling et al., 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 10 000-fold selectivity for aldosterone over hydrocortisone.

Recent studies from our laboratory have demonstrated fast (<1 min) non-genomic activation of K⁺ recycling and Na⁺–H⁺ exchange by mineralocorticoid hormones in human distal colon (Maguire et al., 1994, Maguire et al., 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.