Christina M. Doolan

Modulation of Cytosolic Protein Kinase C and Calcium Ion Activity by Steroid Hormones in Rat Distal Colon

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.

Rapid responses to aldosterone in human distal colon

Aldosterone at normal physiological levels induces rapid increases in intracellular calcium and pH in human distal colon. The end target of these rapid signaling responses are basolateral K+ channels. Using spectrofluorescence microscopy and Ussing chamber techniques, we have shown that aldosterone activates basolateral Na/H exchange via a protein kinase C and calcium-dependent signaling pathway. The resultant intracellular alkalinization up-regulates an adenosine triphosphate (ATP)-dependent K+ channel (K(ATP)) and inhibits a Ca2+ -dependent K+ channel (K(Ca)). In Ussing chamber experiments, we have shown that the K(ATP) channel is required to drive sodium absorption, whereas the K(Ca) channel is necessary for both cyclic adenosine monophosphate and calcium-dependent chloride secretion. The rapid effects of aldosterone on intracellular calcium, pH, protein kinase C and K(ATP), K(Ca) channels are insensitive to cycloheximide, actinomycin D, and spironalactone, indicating a nongenomic mechanism of action. We propose that the physiological role for the rapid nongenomic effect of aldosterone is to prime pluripotential epithelia for absorption by simultaneously up-regulating K(ATP) channels to drive absorption through surface cells and down-regulating the secretory capacity by inhibiting K(Ca) channels involved in secretion through crypt cells.

17β-Oestradiol acutely regulates Cl− secretion in rat distal colonic epithelium

1 In this study we used the short circuit current (ISC) technique to measure the non‐genomic effects of the female sex steroid 17β‐oestradiol (E2) on electrogenic transepithelial ion transport in rat distal colonic epithelium. 2 Basal ISC was largely composed of a transepithelial Cl− secretory component with minimal electrogenic Na+ movement. E2 (1‐100 nm) caused a significant decrease in basal ISC after 15 min. In addition, pre‐treating colonic epithelial tissues with E2 (0.1‐100 nm) for 10 min significantly reduced forskolin (20 μm)‐induced Cl− secretion. E2 also down‐regulated Cl− secretion which was pre‐stimulated by forskolin. Cl− secretory responses to the Ca2+‐dependent secretagogue carbachol (10 μm) were also significantly reduced in the presence of E2 (10‐ 100 nm). However, E2 had no effect on amiloride‐sensitive Na+ absorption. 3 The rapid anti‐secretory effect of E2 was abolished in the presence of the intracellular Ca2+ chelator BAPTA (50 μm) or the protein kinase C (PKC) inhibitor chelerythrine chloride (1 μm). However, in the presence of the nuclear oestrogen receptor antagonist tamoxifen (10 μm), E2 still produced an inhibition of Cl− secretion. Testosterone, progesterone and 17α‐oestradiol had no significant effect on colonic Cl− secretion. Also, E2 (100 nm) did not alter Cl− secretion in colonic epithelia isolated from male rats. 4 We conclude that E2 inhibits colonic Cl− secretion via a non‐genomic pathway that involves intracellular Ca2+ and PKC. It is possible that this gender‐specific mechanism contributes to the salt and water retention associated with high E2 states.

Estrogen receptor independent rapid non-genomic effects of environmental estrogens on [Ca2+]i in human breast cancer cells.

The aim of this study was to identify and characterize an alternative pathway through which environmental estrogenic compounds may mediate their intracellular effects. Three human breast cancer cell lines were employed including MCF-7 cells, which express both ERalpha and ERbeta; MDA-MB-231 cells, which express ERbeta but not ERalpha; and SKBR-3 cells, which express neither ERalpha nor ERbeta. The effect of environmental estrogenic compounds on intracellular calcium ion concentration ([Ca(2+)](i)) was measured and compared to that of 17beta-estradiol (E2). A rapid and maintained increase in [Ca(2+)](i) was observed following the application of nanomolar concentrations of environmental estrogens and E2 regardless of the expression of ERalpha and ERbeta. Removal of extracellular Ca(2+) completely abolished the steroid-induced [Ca(2+)](i) increase. Pre-treatment of cells with the estrogen receptor (ER) antagonist ICI 182,780 had no effect on either basal [Ca(2+)](i) or the steroid-triggered [Ca(2+)](i) response. In summary, we have demonstrated ER independent rapid non-genomic effects of environmental estrogenic compounds, at nanomolar concentrations, on [Ca(2+)](i). The results of this study demonstrate an alternative pathway to explain potent intracellular effects of endocrine disrupting chemicals.

Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon

Studies from our laboratory have demonstrated rapid ( < 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC activity and a PKC-dependent Ca(2+) entry through L-type Ca(2+) channels specifically by mineralocorticoids in distal colon. Aldosterone directly stimulates the activity of the PKC alpha isoform (but not PKC delta, PKC epsilon and PKC zeta) in a cell-free assay system containing only purified commercially available enzyme, appropriate substrate peptide, co-factors and lipid vesicles. The primary ion transport target of the non-genomic signal transduction cascade elicited by aldosterone in epithelia is the Na(+)-H(+) exchanger. In isolated colonic crypts, aldosterone produced a PKC alpha sensitive intracellular alkalinisation within 1 min of hormone addition. Intracellular alkalinisation upregulates an ATP-dependent K(+) channel, which is involved in K(+) recycling to maintain the electrical driving force for Na(+) absorption, while inhibiting a Ca(2+) -dependent K(+) channel, which generates the charge balance for Cl(-) secretion. The non-genomic response to aldosterone in distal colon appears to enhance the capacity for absorption while down-regulating the potential for secretion. We have also demonstrated rapid (< 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC alpha activity, and a PKC delta- and PKA-dependent Ca(2+) entry through di-hydropyridine-blockable Ca(2+) channels specifically by 17beta-estradiol in distal colon. These rapid effects are female gender specific and are insensitive to inhibitors of the classical estrogen receptor (ER). 17 beta-Estradiol directly stimulated the activity of both PKC delta and PKC alpha (but not PKC epsilon or PKC zeta) in a cell-free assay system. E2 rapidly inhibited basolateral K(Ca) channel activity which would be expected to result in an acute inhibition of Cl(-) secretion. Physiological concentrations of E2 (0.1-10 nM) reduced both basal and secretagogue-induced Cl(-) secretion. This anti-secretory effect of E2 is sensitive to PKC inhibition, intracellular Ca(2+) chelation, and is female gender specific and insensitive to inhibitors of the classical ER. These observations link rapid non-genomic activation of second messengers with a rapid gender-specific physiological effect in the whole tissue. Aldosterone and E2 differ in their protein kinase signal transduction and both hormones stimulate specific PKC isoforms indicating both common and divergent signalling systems for salt-retaining steroid hormones. The physiological function of non-genomic effects of aldosterone and estradiol is to shift the balance from net secretion to net absorption in a pluripotential epithelium.

A Gαs protein-coupled membrane receptor, distinct from the classical oestrogen receptor, transduces rapid effects of oestradiol on [Ca2+]i in female rat distal colon

We examined the hypothesis whether rapid non-genomic effects of oestradiol (E2) on [Ca2+]i are mediated via a membrane-located oestrogen receptor (ER) and further elucidated the signalling pathways involved in rapid non-genomic effects of E2 on [Ca2+]i in distal colonic crypts. Basal [Ca2+]i was significantly increased, within minutes, in response to physiological concentrations of E2. Oestradiol linked to bovine serum albumin (E2–BSA), which renders the E2 membrane impermeable, rapidly increased [Ca2+]i suggesting mediation by a membrane surface receptor. A classical ER is not involved however, as no inhibition of either the E2 or E2–BSA [Ca2+]i response was seen in the presence of the classical ER antagonist ICI 182,780. Treatment with the Gαs inhibitor cholera toxin abolished both E2 and E2–BSA induced Ca2+ increases. In contrast, treatment with pertussis toxin, an inhibitor of Gαi and Gαo, had no inhibitory effect. Following subsequent additions of E2 and E2–BSA, no further increases in [Ca2+]i were observed, indicating receptor desensitisation. The E2-induced increase in [Ca2+]i was completely abolished by the PKCδ-specific inhibitor rottlerin, whereas Go6976, an inhibitor of Ca2+-sensitive PKC isoforms, was without inhibitory effect. The phospholipase A2 antagonist, quinacrine, and the COX1 inhibitor, indomethacin, abolished the E2-induced increase in [Ca2+]i. MAP kinase activation is not involved in rapid stimulatory effects of E2 on [Ca2+]i as the specific inhibitor PD98059 did not inhibit the E2 response. These results demonstrate that rapid E2-induced stimulation of [Ca2+]i, in femal rat distal colonic crypts, occurs via a CTx-sensitive Gαs-coupled membrane receptor distinct from the classical ER. PKCδ and fatty acids are involved in the E2 signalling pathway. In contrast, PKCα and MAP kinase are not required.

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

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.

Rapid responses to steroid hormones: from frog skin to human colon. A homage to Hans Ussing

Fifty years ago, Hans Ussing described the mechanism by which ions are actively transported across frog skin. Since then, an enormous amount of effort has been invested in determining the cellular and molecular specifics of the transport mechanisms and their regulatory pathways. Ion transport in high-resistance epithelia is regulated by a variety of hormonal and non-hormonal factors. In vertebrates, steroid hormones such as mineralocorticoids, glucocorticoids and estrogens are major regulators of ion and water transport and hence are central to the control of extracellular fluid volume and blood pressure. Steroid hormones act through nuclear receptors to control the transcriptional activity of specific target genes, such as ion channels, ion transporters and ion pumps. These effects are observed after a latency of several hours and can last for days leading to cellular differentiation that allows a higher transport activity. This pathway is the so-called genomic phase. However, in the past 10 years, it has become apparent that steroid hormones can regulate electrolyte and water transport in tight epithelia independently of the transcription of these ion channels and transporters by regulating ion transporter activity in a non-genomic fashion via modulation of various signal transduction pathways. The molecular mechanisms underlying the steroid hormone-induced activation of signal transduction pathways such as protein kinase C (PKC), protein kinase A (PKA), intracellular calcium, intracellular pH and mitogen-activated protein kinases (MAPKs) and how non-genomic activation of these pathways influences epithelial ion transport will be discussed in this review.

Protein Kinase C Isoforms as Non-Genomic Receptors

It has been suggested that estrogen receptors (ERs) at the plasma membrane may be involved in rapid non-genomic effects of 17β-estradiol (E2) in several cell types (9,27,21,25,34). Studies from several laboratories have demonstrated rapid non-genomic effects of steroids via membrane receptors which are distinct from ERα and ERβ (1,29,31,22) and evidence against the existence of a membrane localised receptor for rapid effects of E2 has also been published (37,10,32,5,11,12). It is clear therefore, that although the general second messenger signalling pathways utilised by steroids are often similar, specific actions and the receptors involved in signal transduction may be specific to the steroid, cell, tissue, species and gender.

Ion Channels and Cell Signaling in Cell Cultures

Two major diseases, namely cancer and cystic fibrosis (CF), have driven the development of immortalized human airway epithelial cell lines. Primary cultures of airway epithelial cells have also played a central role in the study of other diseases such as airway inflammation and viral infection. However, primary lung culture is limited by the number of viable cells that can be generated from the tissue and by the availability of fresh tissue. Immortalized cell lines of airway epithelium were developed from lung carcinomas or from primary cultures transformed in vitro using viruses (SV40). The development of cell lines has greatly enhanced our understanding of the biochemical and proliferation properties of lung epithelium because these characteristics are not affected by viral transformation. However, many transformed cells lose certain differentiated functions, such as tight junction formation or physiological ionic transport properties. For this reason, most of the knowledge of ionic transport mechanisms and regulation come from studies on primary cell culture.