Differential p16/INK4A cyclin-dependent kinase inhibitor expression correlates with chemotherapy efficacy in a cohort of 88 malignant pleural mesothelioma patients.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and essentially incurable malignancy most often linked with occupational exposure to asbestos fibres. In common with other malignancies, the development and progression of MPM is associated with extensive dysregulation of cell cycle checkpoint proteins that modulate cell proliferation, apoptosis, DNA repair and senescence. METHODS: The expression of cyclin-dependent kinase inhibitor p16/INK4A was evaluated by immunohistochemistry using tumour biopsy specimens from 88 MPM cases and a semi-quantitative score for p16/INK4A expression was obtained. Post-diagnosis survival and the survival benefit of chemotherapeutic intervention was correlated with p16/INK4A expression. RESULTS: A low, intermediate and high score for p16/INK4A expression was observed for 45 (51.1%), 28 (31.8%) and 15 (17.1%) of the MPM cases, respectively. Those cases with intermediate or high p16/INK4A tumour expression had a significantly better post-diagnosis survival than those cases whose tumours lost p16 expression (log-rank P<0.001). Those patients with sustained p16/INK4A expression who received chemotherapy also had a better survival than those treated patients whose tumours had lost p16/INK4A expression (log-rank P<0.001). CONCLUSIONS: Sustained p16/INK4A expression predicts better post-diagnosis survival in MPM and also better survival following chemotherapeutic intervention.

Parameter estimates for invasive breast cancer progression in the Canadian National Breast Screening Study.

BACKGROUND: The aim of screening is to detect a cancer in the preclinical state. However, a false-positive or a false-negative test result is a real possibility. METHODS: We describe invasive breast cancer progression in the Canadian National Breast Screening Study and construct progression models with and without covariates. The effect of risk factors on transition intensities and false-negative probability is investigated. We estimate the transition rates, the sojourn time and sensitivity of diagnostic tests for women aged 40-49 and 50-59. RESULTS: Although younger women have a slower transition rate from healthy state to preclinical, their screen-detected tumour becomes evident sooner. Women aged 50-59 have a higher mortality rate compared with younger women. The mean sojourn times for women aged 40-49 and 50-59 are 2.5 years (95% CI: 1.7, 3.8) and 3.0 years (95% CI: 2.1, 4.3), respectively. Sensitivity of diagnostic procedures for older women is estimated to be 0.75 (95% CI: 0.55, 0.88), while women aged 40-49 have a lower sensitivity (0.61, 95% CI: 0.42, 0.77). Age is the only factor that affects the false-negative probability. For women aged 40-49, 'age at entry', 'history of breast disease' and 'families with breast cancer' are found to be significant for some of the transition rates. For the age-group 50-59, 'age at entry', 'history of breast disease', 'menstruation length' and 'number of live births' are found to affect the transition rates. CONCLUSION: Modelling and estimating the parameters of cancer progression are essential steps towards evaluating the effectiveness of screening policies. The parameters include the transition rates, the preclinical sojourn time, the sensitivity, and the effect of different risk factors on cancer progression.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis: in vitro evidence in cultured epithelial cells.

BACKGROUND: Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. OBJECTIVES: The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. METHODS: Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. RESULTS: Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway. CONCLUSIONS: This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours.

BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)α activity in clinical breast cancer was established by relating the expression of cPLA(2)α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.

Rapid anti-secretory effects of glucocorticoids in human airway epithelium.

Glucocorticoids are anti-inflammatory molecules classically described as acting through a genomic pathway. Similar to many steroid hormones, glucocorticoids also induce rapid non-genomic responses. The present paper provides a general overview of the rapid non-genomic effects of glucocorticoids in airway and will be mainly focused on a retrospective of the authors work on rapid effects of glucocorticoids in airway epithelial cell transport. Using fluorescence microscopy, short circuit current measurements in human bronchial epithelial (16HBE14o(-)) cells, we reported rapid non-genomic effects of dexamethasone on cell signalling and ion transport. Dexamethasone (1 nM) rapidly stimulated Na(+)/H(+) exchanger activity and pH(i) regulation in 16HBE14o(-) cells. Dexamethasone also produced a rapid decrease of intracellular [Ca(2+)](i) to a new steady state concentration and inhibited the large and transient [Ca(2+)](i) increase induced by apical adenosine tri-phosphate (ATP). Dexamethasone also reduced by 1/3 the Ca(2+)-dependent Cl(-) secretion induced by apical ATP. The rapid effects of dexamethasone on intracellular pH and Ca(2+) were not affected by inhibitors of transcription, cycloheximide or by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The rapid responses to glucocorticoid were reduced by the inhibitors of adenylated cyclase, cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (ERK1/2). Our results demonstrate, that dexamethasone at low concentrations, rapidly regulates intracellular pH, Ca(2+) and PKA activity and inhibits Cl(-) secretion in human bronchial epithelial cells via a non-genomic mechanism which neither involve the classical glucocorticoid nor mineralocorticoid receptor.

Cl- permeability of the basolateral membrane of the Rana esculenta epithelium: activation of Cl-/HCO3- exchange by alkaline intracellular pH.

We have investigated Cl- transport mechanism(s) located in the basolateral membranes of the frog skin epithelium and in particular activation of Cl-/HCO3- exchange following an alkaline load. We found that 87% of the total 36Cl uptake by the epithelial cells occurs across the basolateral membranes (JbCl-) and submitting the epithelium to an alkaline load (HCO3(-)-Ringer solution, pH 8.1) increased JbCl-. Intracellular Cl- activity (aiCl-), measured with ion-sensitive microelectrodes, increased when the Ringer solution bathing the basolateral membranes was changed from a Ringer solution equilibrated in air (pH 7.4) to one containing CO2/HCO3- (pH 7.4). pHi recovery following an alkaline load was dependent on Cl- since it did not occur in serosal Cl(-)-free media, indicating the presence of a Cl(-)-dependent regulatory mechanism. Acid loading of the epithelial cells (5% CO2, HCO3(-)-free Ringer) produced no change in JbCl- but stimulated an amiloride-sensitive 22Na uptake across the basolateral membranes of the epithelium, compatible with an activation of a Na+/H+ exchanger, previously described in this tissue. JbCl- was partially blocked by SITS (5 x 10(-4) mmol/I), niflumic acid (5 x 10(-5) mmol/I), furosemide or bumetanide. Simultaneous addition of furosemide and niflumic acid produced an inhibition of JbCl- which was not different with furosemide alone. Substitution of Na+ by choline had no effect on JbCl- and furosemide did not block the 22Na+ uptake, suggesting that JbCl- is not a Na(+)-dependent process (cotransport). We conclude that a significant Cl- permeability at the basolateral membranes of the epithelial cells is due to the presence of a Cl-/HCO3- exchanger which is essential for the recovery of pHi following an alkaline load.

Effects of intracellular signals on Na+/K(+)-ATPase pump activity in the frog skin epithelium.

The effects of intracellular signals (pHi, Na+i, Ca2+i, and the electrical membrane potential), on Na+ transport mediated by the Na+/K+ pump were investigated in the isolated Rana esculenta frog skin. In particular we focussed on pHi sensitivity since protons act as an intrinsic regulator of transepithelial Na+ transport (JNa) by a simultaneous control of the apical membrane Na+ conductance (gNa) and the basolateral membrane K+ conductance (gK). pHi changes which modify JNa, gNa and gK, do not affect the Na+ transport mediated by the pump as shown by kinetic and electrophysiological studies. In addition, no changes were observed in the number of 3H-ouabain binding sites in acid-loaded epithelia. Our attempts to modify cellular Ca2+ (by using Ca(2+)-free/EGTA Ringer solution or A23187 addition) also failed to produce any significant effects in the Na+ pump turnover rate or the number of 3H-ouabain binding sites. The Na+ pump current was found to be sensitive to the basolateral membrane potential, saturating for very positive (cell) potentials and a reversal potential of -160 mV was calculated from I-V relationships of the pump. Changes in Na+i considerably affected the Na+ pump rate. A saturating relationship was found between pump rate and Nai+ with maximal activation at Nai+ greater than 40 mmol/l; a high dependence of the pump rate and of the number of 3H-ouabain binding sites was observed in the physiological range of Nai+. We conclude that protons (in the physiological pH range) which act directly and simultaneously on the passive transport pathways (gNa and gK), have no direct effect on the Na+/K+ pump rate. After an acid load, the inhibition of JNa is primarily due to the reduction of gNa. This results in a reduction of Nai and the pump turnover rate then becomes dependent on other pathways of Na+ entry such as the basolateral membrane Na+/H+ exchanger.

Histamine-induced cytosolic calcium increase in porcine articular chondrocytes.

Chondrocytes have been shown to possess two types of histamine receptors, H1 and H2. The application of histamine to isolated porcine chondrocytes was found to significantly increase intracellular calcium and this increase was partially dependent upon the presence of extracellular calcium. This, therefore, implies that there is some role for a plasma membrane calcium transport system in the increase of cytosolic calcium in response to histamine. The increase in intracellular calcium in response to the application of histamine was found to be reduced by both H1 and H2 receptor antagonists.

Role of Na+/H+ exchange in the control of intracellular pH and cell membrane conductances in frog skin epithelium.

Ion-sensitive microelectrodes and current-voltage analysis were used to study intracellular pH (pHi) regulation and its effects on ionic conductances in the isolated epithelium of frog skin. We show that pHi recovery after an acid load is dependent on the operation of an amiloride-sensitive Na+/H+ exchanger localized at the basolateral cell membranes. The antiporter is not quiescent at physiological pHi (7.1-7.4) and, thus, contributes to the maintenance of steady state pHi. Moreover, intracellular sodium ion activity is also controlled in part by Na+ uptake via the exchanger. Intracellular acidification decreased transepithelial Na+ transport rate, apical Na+ permeability (PNa) and Na+ and K+ conductances. The recovery of these transport parameters after the removal of the acid load was found to be dependent on pHi regulation via Na+/H+ exchange. Conversely, variations in Na+ transport were accompanied by changes in pHi. Inhibition of Na+/K+ ATPase by ouabain produced covariant decreases in pHi and PNa, whereas increases in Na+ transport, occurring spontaneously or after aldosterone treatment, were highly correlated with intracellular alkalinization. We conclude that cytoplasmic H+ activity is regulated by a basolateral Na+/H+ exchanger and that transcellular coupling of ion flows at opposing cell membranes can be modulated by the pHi-regulating mechanism.

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.

UNLABELLED: Inward-rectifier K channel: using macroscopic voltage clamp and single-channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current-voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell-attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. CONCLUSIONS: under physiological ionic gradients, a 15-pS inward-rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.

Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium.

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.

Cost-effectiveness of breast cancer screening policies using simulation.

In this paper, we study breast cancer screening policies using computer simulation. We developed a multi-state Markov model for breast cancer progression, considering both the screening and treatment stages of breast cancer. The parameters of our model were estimated through data from the Canadian National Breast Cancer Screening Study as well as data in the relevant literature. Using computer simulation, we evaluated various screening policies to study the impact of mammography screening for age-based subpopulations in Canada. We also performed sensitivity analysis to examine the impact of certain parameters on number of deaths and total costs. The analysis comparing screening policies reveals that a policy in which women belonging to the 40-49 age group are not screened, whereas those belonging to the 50-59 and 60-69 age groups are screened once every 5 years, outperforms others with respect to cost per life saved. Our analysis also indicates that increasing the screening frequencies for the 50-59 and 60-69 age groups decrease mortality, and that the average number of deaths generally decreases with an increase in screening frequency. We found that screening annually for all age groups is associated with the highest costs per life saved. Our analysis thus reveals that cost per life saved increases with an increase in screening frequency.

Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca(2+)](i) by 17beta-oestradiol in female rat distal colon.

1. In this study, the effect of 17beta-oestradiol on adenosine 3' : 5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PKA) activity was investigated. 2. Rapid (within 15 min) activation of basal PKA activity was observed in cytosolic fractions by 17beta-oestradiol but not by 17alpha-oestradiol, progesterone or testosterone. This stimulation was abolished by the specific PKA inhibitor PKI but not by the classical oestrogen receptor antagonist tamoxifen. 3. 17beta-Oestradiol did not stimulate basal PKA activity in membrane fractions or in cytosolic fractions from male rats. 4. The increase in cytosolic PKA activity was indirect as (i) it was inhibited by the adenylyl cyclase inhibitor SQ22536, (ii) it was mimicked by forskolin and (iii) 17beta-oestradiol did not cause a stimulation of basal PKA activity in either type I or type II commercially available PKA holoenzymes. 5. Protein kinase Cdelta (PKCdelta) was directly activated by 17beta-oestradiol. The specific PKC inhibitor, bisindolylmaleimide I (GF 109203X), abolished the 6. 17beta-oestradiol-induced PKA activation. 17beta-Oestradiol stimulate an increase in free intracellular calcium ion concentration ([Ca(2+)](i)) in isolated female but not male rat colonic crypts. This was inhibited by verapamil, nifedipine and zero extracellular [Ca(2+)] but unaffected by tamoxifen. 17alpha-Oestradiol, testosterone and progesterone failed to increase [Ca(2+)](i). 7. PKC and PKA inhibitors abolished the 17beta-oestradiol-induced increase in [Ca(2+)](i). 8. These results demonstrate the existence of a novel 17beta-oestradiol-specific PKA and Ca(2+) signalling pathway, which is both sex steroid- and gender-specific, in rat distal colonic epithelium.

Rapid activation of basolateral potassium transport in human colon by oestradiol.

1. We investigated the effect of oestradiol on basolateral potassium channels in human colonic epithelium. 2. Ion transport was quantified using short circuit current (I:(sc)) measurements of samples mounted in Ussing chambers. Serosal K transport was studied using nystatin permeabilization of the apical membrane. Intracellular pH changes were quantified using spectroflouresence techniques. 3. Experiments were performed with either 10 nM or 1 microM Ca(2+) in the apical bathing solution. With 10 nM Ca(2+) in the apical bathing solution addition of oestradiol (1 nM) to the basolateral bath produced a rapid increase in current (delta I(K)=11.2+/-1.2 microA.cm(-2), n=6). This response was prevented by treatment of the serosal membrane with tolbutamide (1 microM). With 1 microM Ca(2+) in the apical bathing solution addition of oestradiol produced a rapid fall in current (delta I(K)=-12.8+/-1.4 microA.cm(-2)), this response was prevented by treatment of the basolateral membrane with tetra-pentyl-ammonium (TPeA). These responses were rapid and occurred independently of protein synthesis. 4. Inhibition of basolateral Na(+)/H(+) exchange with either amiloride or a low sodium bathing solution prevented this response. These responses were prevented by inhibition of protein kinase C (PKC) with bis-indolyl-maleimide. 5. Oestradiol (1 nM) produced a rapid intracellular alkanization (mean increase=0.11 pH units; n=6; P<0.01). 6. These results suggest that oestradiol rapidly modulates serosal K transport in human colon. These effects depend upon intact Na(+)/H(+) exchange and protein kinase C. We propose a non-classical, possibly membrane linked, mechanism for oestradiol action in human colonic epithelium.

17beta-oestradiol stimulates capacitative Ca2+ entry in human endometrial cells.

Oestrogen plays an essential role in regulating growth and differentiation in the human endometrium which undergoes dynamic morphological and functional changes during the menstrual cycle in preparation for implantation. In this tissue, it has been suggested that intracellular calcium could be a key signal in transducing early responses to steroid hormones. Here, we have investigated the rapid effects of 17beta-oestradiol on [Ca2+]i in a human endometrial cell line (RL95-2). Using confocal imaging microscopy, we show that physiological concentrations of 17beta-oestradiol trigger rapid and transient increases in [Ca2+]i. Our results demonstrate that 17beta-oestradiol-induced [Ca2+]i variations are critically dependent on calcium influx via lanthanum-sensitive calcium channels. Moreover, the 17beta-oestradiol-induced Ca2+ influx is significantly increased by the depletion of intracellular stores by thapsigargin and decreased by chelerythrine chloride, an inhibitor of protein kinase C. These data indicate a non-genomic action of 17beta-oestradiol to stimulate capacitative Ca2+ entry through store-operated calcium channels via a PKC-sensitive pathway.

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.

Berberine inhibits ion transport in human colonic epithelia.

The effects of berberine on ion transport in both human colonic mucosal epithelia and an intestinal epithelial cell line (T84) were examined. Berberine (concentration range 0-500 microM) reduced both basal and stimulated ion transport responses in human colonic mucosae in a manner which was non-specific for Ca2+ -or cAMP-mediated signals. Similarly, in cultured intestinal epithelial monolayers, berberine inhibited Ca2+ -and cAMP-mediated responses indicating an inhibitory activity directly at the level of the epithelium rather than an indirect effect through other mucosal element(s). Berberine did not alter the rate of generation of cAMP by adenylyl cyclase or the activity of protein kinase A, the effector enzyme of the cAMP pathway. Berberine inhibited carbachol-stimulated 86Rb+ efflux from T84 monolayers. Berberine also inhibited K+ conductance in apically-permeabilised re-sected mucosae. These results indicate i) that berberine exerts an anti-secretory action directly upon epithelial cells and ii) the mechanism of action may be at the level of blockade of K+ channels.

Renin-angiotensin system expression and secretory function in cultured human ciliary body non-pigmented epithelium.

BACKGROUND: Renin-angiotensin system (RAS) components have been identified in human ciliary body and aqueous humour, pointing to a role for the RAS in the regulation of aqueous humour dynamics. Here, the authors examine the functional expression of a RAS and the effects of angiotensin II (AII) on a signal transduction pathway and ion secretion mechanism in cultured human ciliary body non-pigmented epithelium (HNPE). METHODS: RAS expression was examined in cultured HNPE cells using polymerase chain reaction (PCR) analysis. Secretory function was determined using spectrofluorescence imaging microscopy to measure cell calcium (Ca(2+)(I)) and volume responses. Single channel patch clamp techniques were employed to investigate ion channel activity. RESULTS: PCR analysis demonstrated the expression of angiotensinogen and the AT(1b) receptor in HNPE cells. A large conductance potassium (BK) channel (mean 190 (SEM 5.6) pS, n = 22 cells), was observed in plasma membrane patches. This channel was calcium sensitive with channel open probability (Po) increasing with increasing Ca(2+)(I) (K(0.5) 10.79 (0.44) microM Ca(2+), Hill coefficient of 1.04 (0.04)). AII (100 nM) increased the number (N) of active BK channels in HNPE cells and also the probability of channel opening (Po). N.P(o) increased from 0.008 (0.002) to 1.38 (0.4) following the addition of AII (p=0.0064). AII also induced a rapid rise in Ca(2+)(I) from resting values of 112 (14) nM to a peak of 992 (106) nM (p<10(-4)). A simultaneous cell volume reduction of 24.70% (3.34%) (p<10(-4)) occurred during this calcium signal. Losartan (1 microM) significantly blocked the AII induced BK channel activation (p=0.0131), the Ca(2+)(I) response (p<10(-4)), and the AII induced volume effect (p=0.0046). CONCLUSION: It was demonstrated that AII activates a Ca(2+)(I) signalling system which subsequently increases potassium ion channel activity. These effects are accompanied simultaneously by cell volume loss, indicating that AII acts as receptor operated secretagogue in HNPE cells. The ability of an AT(1) receptor antagonist to inhibit these processes may thus offer a new family of pharmaceutical agents to the current armamentarium in the treatment of glaucoma.

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.

Chloride dependent intracellular pH effects of external ATP in cultured human non-pigmented ciliary body epithelium.

PURPOSE: To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. RESULTS: In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient. CONCLUSION: We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.