Naringenin inhibits the growth of Dictyostelium and MDCK-derived cysts in a TRPP2 (polycystin-2)-dependent manner.

BACKGROUND AND PURPOSE: Identifying and characterizing potential new therapeutic agents to target cell proliferation may provide improved treatments for neoplastic disorders such as cancer and polycystic diseases. EXPERIMENTAL APPROACH: We used the simple, tractable biomedical model Dictyostelium to investigate the molecular mechanism of naringenin, a dietary flavonoid with antiproliferative and chemopreventive actions in vitro and in animal models of carcinogenesis. We then translated these results to a mammalian kidney model, Madin-Darby canine kidney (MDCK) tubule cells, grown in culture and as cysts in a collagen matrix. KEY RESULTS: Naringenin inhibited Dictyostelium growth, but not development. Screening of a library of random gene knockout mutants identified a mutant lacking TRPP2 (polycystin-2) that was resistant to the effect of naringenin on growth and random cell movement. TRPP2 is a divalent transient receptor potential cation channel, where mutations in the protein give rise to type 2 autosomal dominant polycystic kidney disease (ADPKD). Naringenin inhibited MDCK cell growth and inhibited cyst growth. Knockdown of TRPP2 levels by siRNA in this model conferred partial resistance to naringenin such that cysts treated with 3 and 10 µM naringenin were larger following TRPP2 knockdown compared with controls. Naringenin did not affect chloride secretion. CONCLUSIONS AND IMPLICATIONS: The action of naringenin on cell growth in the phylogenetically diverse systems of Dictyostelium and mammalian kidney cells, suggests a conserved effect mediated by TRPP2 (polycystin-2). Further studies will investigate naringenin as a potential new therapeutic agent in ADPKD.

myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line.

Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) inhibit apical Ca(2+)-activated Cl(-) conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P(4) levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P(4) levels were therefore specifically elevated, independently of receptor activation, by incubating cells with a cell-permeant bioactivable analogue, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester (Bt(2)Ins (3,4,5,6)P(4)/AM). The latter inhibited Ca(2+)-activated Cl(-) secretion by 60%. We next used nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from electrochemically dominating ion movements through the apical membrane. Thus, we studied autonomous regulation of apical Cl(-) channels in situ. The properties of Cl(-) flux across the apical membrane were those expected of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold greater permeability of I(-) over Cl(-). Following nystatin-treatment, we elevated intracellular levels of Ins(3,4,5,6)P(4) with either purinergic agonists or with Bt(2)Ins(3,4,5,6)P(4)/AM. Both protocols inhibited Ca(2+)-activated Cl(-) secretion (up to 70%). These studies provide the first demonstration that, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P(4)-inhibited CaCC.

Carbachol-stimulated chloride secretion in mouse colon: evidence of a role for autocrine prostaglandin E2 release.

We used the short-circuit current technique to investigate the possible facilitatory role of epithelium-derived prostaglandin E2 (PGE2) release on Cl- secretion in the mouse colon. Carbachol- (CCh)-stimulated Cl- secretion was reduced by pretreatment with either indomethacin (10 microM), or TTX (1 microM), and when added together, these inhibitors revealed net CCh-stimulated K+ secretion. CCh-stimulated Cl- secretion was partially restored to TTX/indomethacin-treated colons by addition of a subsecretory concentration of PGE2 (1 nM). In acutely isolated, unstimulated crypt cells, we measured PGE2 release at a similar level. We conclude that autocrine release of PGs from epithelial cells is sufficient to support the CCh-induced Cl- secretory response and is a likely co-factor in this response.

Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate.

Ca2+-activated Cl- channels are inhibited by inositol 3,4,5, 6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 1-kinase activity that was either (i) in lysates of AR4-2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [3H]inositol-labeled AR4-2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3, 4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P3 2.7-fold, while [3H]Ins(3, 4,5,6)P4 levels increased 2-fold; there were no changes to levels of other 3H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1, 3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 microM). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3, 4)P3-mediated inhibition of the 1-kinase. We also found that Ins(1,3, 6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 microM, respectively). The non-physiological trisphosphates, D/L-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 microM), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P3 acts in an important signaling cascade.

Dissociated adult rat subfornical organ neurons maintain membrane properties and angiotensin responsiveness for up to 6 days.

We have utilised standard dissociation techniques to obtain a preparation of subfornical organ (SFO) cells that have been maintained in tissue culture for up to 1 week. Stable (> 15 min) whole cell recordings were obtained from 80 cells displaying rapid (<2 ms) voltage-dependent sodium currents (blocked by tetrodotoxin in 10 of 10 cells tested), and current evoked action potentials, which were thus classified as SFO neurons. These neurons had a resting membrane potential of-63.8 +/- 1.3 mV (mean +/- SEM), spike amplitude of 86.8 +/- 2.5 mV, and input resistance of 1.2 +/- 0.1 G omega, characteristics which did not change significantly in recordings obtained for up to 6 days after dissociation. Current clamp recording showed that of 65 cells tested with bath application of angiotensin (ANG; 1,000-10nM), 41 responded to this peptide with decreases in input resistance (control 1.4 +/- 0.16 G omega, after ANG 0.78 +/- 0.1 G omega, p < 0.0001), and depolarisations (mean 18.3 +/- 2.0 mV, p < 0.0001). Similar recordings were obtained from viable cells up to 6 days after initial cell dissociation. These studies provide the first description of the basic membrane properties of dissociated SFO neurons. The responsiveness of these cells to ANG supports the conclusion that their properties are similar to those in vivo. These data suggest that use of this technique will permit systematic analysis of the membrane events underlying the actions of multiple ligands on this uniquely specialised group of CNS neurons.

Identification of ClC-2-like chloride currents in pig pancreatic acinar cells.

We used the whole-cell patch-clamp technique to identify a hyperpolarization-activated Cl- current (approximately 50 pA/pF at -60 mV) in acutely isolated, voltage-clamped, single, pig pancreatic acinar cells. This current had characteristic properties of inward rectification, a Cl- = Br->I- selectivity sequence and activation by extracellular hypotonicity. These properties are similar to those reported for the ClC-2 Cl- channel recently cloned from rat and expressed in oocytes. An antiserum raised against the C-terminus of ClC-2 localized the channel to secretory granules containing amylase that were situated exclusively at the apical pole of the pig pancreatic acinar cells, but the channel was not localized in the basolateral membrane. Our study combines a functional assessment and immunohistochemical localization of ClC-2-like channels in a native mammalian cell. The data suggest that the ClC-2-like Cl- channel may function as a Cl- efflux pathway in pancreatic acinar cells.

Control of Ca2+ entry into rat lactotrophs by thyrotrophin-releasing hormone.

1. Lactotrophs are adenohypophysial cells that synthesize and secrete prolactin (PRL), a hormone principally involved in mammalian milk production. An increase in the intracellular Ca2+ concentration ([Ca2+]i) is an important signal for PRL secretion. Thyrotrophin-releasing hormone (TRH) generates Ca2+ signals derived from both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+, the latter being particularly important for PRL secretion. The identity of this TRH-sensitive Ca2+ entry pathway is unknown and therefore the subject of the present study. 2. [Ca2+]i was measured by video imaging of fura-2 loaded into single rat anterior pituitary cells. Ca2+ influx was detected by quenching of fura-2 fluorescence by external Mn2+. All data are from lactotrophs isolated from lactating female rats. Individual lactotrophs were identified by postexperimental immunofluorescent detection of PRL in fixed cells. 3. TRH induced the release of Ca2+ from intracellular stores and also stimulated Mn(2+)-permeable Ca2+ influx. U73122 (1 microM), a phospholipase C inhibitor, prevented the Ca(2+)-mobilizing actions of TRH. The chemically similar but inactive analogue, U73343 (1 microM), had no effect on TRH responses. U73122 did not act as a global G protein inhibitor because the reduction of basal [Ca2+]i by dopamine (1 microM, a G protein-mediated event) was not affected. 4. TRH-stimulated Mn2+ influx occurred either immediately after addition of TRH (early entry) or after a delay of about 130 s (late entry). There were no statistically significant differences in the magnitude or temporal characteristics of the Ca2+ signals evoked from cells showing early or late Mn2+ entry. 5. The identity of Ca2+ channels permeable to Mn2+ was investigated. Cell depolarization with 50 mM KCl stimulated Ca2+/Mn2+ influx and was prevented by nifedipine (1 microM). Bay K 8644 (1 microM) also stimulated Mn2+ influx. Thus, the presence of Mn(2+)-permeable L-type voltage-operated Ca2+ channels is likely. A second Mn(2+)-permeable pathway was present in lactotrophs. Depletion of Ca2+ stores by thapsigargin (1 microM) stimulated a Ca2+ signal and Mn2+ influx. This 'capacitative entry pathway' was insensitive to nifedipine (1 microM), indicating that putative L-type Ca2+ channels were not activated. 6. TRH-stimulated Mn2+ influx was not prevented by nifedipine (1 microM). TRH added during KCl-induced Mn2+ influx reduced the quench rate within the time frame of the TRH-induced Ca2+ spike. TRH may therefore inhibit putative L-type Ca2+ channels. 7. Addition of thapsigargin in Ca(2+)-free medium transiently increased [Ca2+]i and prevented subsequent Ca2+ responses to TRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular ATP activates calcium entry and mobilization via P2U-purinoceptors in rat lactotrophs.

Extracellular ATP has been previously shown to activate calcium signalling in pituitary cell populations [1] but the particular cell types involved have not been identified. We used video imaging of Fura-2 loaded into single rat pituitary cells and identified as lactotrophs to study the effects of extracellular ATP on [Ca2+]i. ATP does not permeabilize the cells as shown by exclusion of propidium iodide. ATP causes two types of calcium transients in lactotrophs. The most common response is a rapid increase in [Ca2+]i that decays slowly and is terminated by washout of ATP. This type of response is also seen in calcium-free medium, demonstrating mobilization of calcium stores dependent upon the presence of the agonist. ATP also stimulates calcium entry as detected by Mn(2+)-quenching of Fura-2. ATP in Mg(2+)-free medium and ATP gamma S are effective agonists suggesting ATP4- is the active form. The presence of P2-purinoceptors is apparent because ATP, ADP and AMP increase [Ca2+]i in decreasing order of potency and adenosine has no effect. ATP-induced calcium transients are reduced by the P2-purinoceptor antagonists suramin and quinidine. UTP is equipotent with ATP and defines the receptor subtype as P2U. We conclude that ATP4- acts on rat lactotrophs via P2U-purinoceptors to elevate [Ca2+]i from intracellular and extracellular sources.

The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells.

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.