Sahba Fatherazi

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

SummaryATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic β-cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 μm and as high as 500 μm, with maximal activation between 10 and 100 μm ADP in mouse β-cell membrane patches. At concentrations greater than 500 μm, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of β-cell membrane potential, increasing ADP in the 10 and 100 μm range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize β-cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the “ATP/ADP ratio,” and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 μm range as suggested by biochemical studies.

Evidence that TRPC1 contributes to calcium-induced differentiation of human keratinocytes

External calcium ion concentration is a major regulator of epidermal keratinocyte differentiation in vitro and probably also in vivo. Regulation of calcium-induced differentiation changes is proposed to occur via an external calcium-sensing, signaling pathway that utilizes increases in intracellular calcium ion concentration to activate differentiation-related gene expression. Calcium ion release from intracellular stores and calcium ion influx via store-operated calcium-permeable channels are key elements in this proposed signaling pathway; however, the channels involved have not yet been identified. The present report shows that human gingival keratinocytes (HGKs) also undergo calcium-induced differentiation in vitro as indicated by involucrin expression and morphological changes. Moreover, TRPC1, which functions as a store-operated calcium channel in a number of cell types, including epidermal keratinocytes, is expressed in both proliferating and differentiating HGKs. Transfection of HGKs with TRPC1 siRNA disrupted expression of TRPC1 mRNA and protein compared with transfection with scrambled TRPC1 siRNA. Cells with disrupted TRPC1 expression showed decreased calcium-induced differentiation as measured by involucrin expression or morphological changes, as well as decreased thapsigargin-induced calcium ion influx, and a decreased rate of store calcium release. These results indicate that TRPC1 is involved in calcium-induced differentiation of HGKs likely by supporting a store-operated calcium ion influx.

Hypotonically activated chloride current in HSG cells.

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K+ and Cl− containing solutions. An outward current detected at 0 mV, corresponded to a K+ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca2+-activated K+ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl− current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4′-isothiocyanostilbene), and its reversal potentials corresponded to the Cl− equilibrium potentials at high and low external Cl− concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K+ and Cl− currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K+ and Cl− conductances in these cells, and that the activation pathway for the K+ conductance apparently involves [Ca2+], while the activation pathway for the Cl− conductance does not involve [Ca2+] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.

Phosphate Regulates Osteopontin Gene Transcription

Extracellular inorganic phosphate (ePi) is a key regulator of cementoblast behavior, both in vivo and in vitro, and results in a marked increase in osteopontin expression in vitro. To examine the molecular mechanisms involved in ePi induction of osteopontin gene expression, we transfected a series of osteopontin promoter-luciferase constructs into OCCM-30 cementoblasts. Our results demonstrate that ePi can directly induce osteopontin gene transcription. The region responsive to ePi signaling was localized to a 53-bp region of the promoter between −1454 and −1401 that contains a glucocorticoid response element (GRE). Mutation of the GRE abolished the ePi response, suggesting that glucocorticoid receptor (GR) signaling is required for ePi-mediated transcription. In addition, treatment of cells with the GR antagonist RU-486 (Mifepristone) prevented promoter activation by ePi. The results presented support a model demonstrating that inorganic phosphate regulates OPN gene transcription in cementoblasts through a pathway that requires a functional GR.

Evidence that TRPC4 supports the calcium selective ICRAC-like current in human gingival keratinocytes

We previously demonstrated that high external [Ca2+] activated two Ca2+ currents in human gingival keratinocytes (HGKs): an initial small ICRAC-like current and a second large nonspecific cation current (Fatherazi S, Belton CM, Cai S, Zarif S, Goodwin PC, Lamont RJ, Izutsu KT; Pflugers Arch 448:93–104, 2004). It was recently shown that TRPC1, a member of the transient receptor potential protein family, is a component of the store-operated calcium entry mechanism in keratinocytes. To further elucidate the molecular identity of these channels, we investigated the expression of TRPC4 in gingival tissue and in cultured keratinocytes, and the effect of knockdown of TRPC4 expression on the Ca2+ currents and influx. Immunohistochemistry showed TRPC4 was present in gingival epithelium as well as in HGKs cultured in different [Ca2+]s. Results from tissue and cultured HGKs demonstrated TRPC4 expression decreased with differentiation. Knockdown of TRPC4 in proliferating HGKs with antisense oligonucleotides significantly reduced the intracellular [Ca2+] increase obtained upon exposure to high external [Ca2+]. Antisense knockdown of TRPC4 expression was confirmed by reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy of transfected HGKs. Immunofluorescence microscopy and patch clamp measurements in Lucifer-yellow-tagged, antisense-treated HGKs showed attenuation of TRPC4 expression levels as well as attenuation of the ICRAC-like current in the same cell, whereas the large nonspecific cation current was unchanged but significantly delayed. Cells transfected with a scrambled TRPC4 oligonucleotide showed no change in either the ICRAC-like or nonspecific currents. The results indicate that TRPC4 is an important component of the ICRAC-like channel in HGKs.

Expression of a rapid, low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering.

SummaryDepolarization-activated outward currents ranging in amplitude from 100–1000 pA were studied in cultured, insulinsecreting HIT cells and mouse B-cells using the whole-cell patch clamp. Outward current was identified as a K current since it was blocked by K channel blockers and its tail current reversed nearEK. The K currents of HIT cells dialyzed with internal solutions containing 0.1–10mm EGTA with no added calcium (Ca), or 10mm EGTA with 2mm added Ca, activated rapidly with depolarization. However, the stronger Ca buffer BAPTA (5mm; no added Ca) blocked the rapidly activating current to reveal an underlying more slowly activating K current. With intracellular EGTA, application of the Ca channel blocker cadmium mimicked the effect of intracellular BAPTA. These data suggest that the rapid K current was mediated by low-voltage threshold, Ca-activated K channels while the slower K current was mediated by high threshold delayed rectifier K channels. Mouse B-cells also had both K current components. Dialyzing these cells with either BAPTA (5mm, no added Ca) or high EGTA (10mm with 2mm Ca) blocked the rapid Ca-activated K current observed when cells were filled with 0.1 to 1mm EGTA. It is concluded that the extent of Ca-activated K current activation in either HIT or adult mouse B-cells depends on the degree of intracellular Ca buffering.

Mycoplasma orale infection affects K+ and Cl− currents in the HSG salivary gland cell line

SummaryThe relations between K+ channel and Cl− channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K+ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K+ channels and K+ channel expression as estimated by ionomycin- or hypotonically induced K+ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl− channels, but only the latter decrease was statistically significant. Also, Cl− currents could be elicited more frequently than K+ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K+ channels relatively more than Cl− channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurredin vivo.

Intraseptal morphine potentiates pentobarbital narcosis and hypothermia in the rat

Morphine injected intraseptally in the amounts of 35 and 70 nmol prolonged pentobarbital-induced narcosis in the rat. Pentobarbital-induced hypothermia was also potentiated by intraseptal injection of 70 nmol of morphine. These effects were antagonized when morphine was injected together with naltrexone (29 nmol). Naltrexone injected by itself into the septum did not significantly affect pentobarbital-narcosis and hypothermia. It is concluded that activation of mu opioid receptors in the septal region could affect the actions of pentobarbital.

The oral hypoglycemie agent, U‐56324, inhibits the activity of ATP‐sensitive potassium channels in cell‐free membrane patches from cultured mouse pancreatic B‐cells

U‐56324, a hypoglycemic agent derived from nicotinic acid, inhibited the activity of ATP‐sensitive potassium channels in excised patches from mouse pancreatic B‐cells. The effect of U‐56324 on channel activity was reversible and concentration‐dependent while it had no effect on single channel conductance. The positional isomer, U‐59588, which has relatively little hypoglycemic activity, had no effect on channel properties. U‐56324, at the same concentrations, had no effect on calcium‐activated potassium channels. The basis for the potentially antidiabetic properties of U‐56324 may therefore be due to direct and specific inhibition of ATP‐sensitive potassium channels.

Absence of acetylcholine- and ionomycin-activated Cl- currents in submandibular cells of early postnatal rats.

Abstract Using the whole-cell patch-clamp technique, we investigated developmental changes in the expression of an acetylcholine- (Ach-) activated Cl–conductance in rat submandibular acinar cells. ACh induced an oscillatory inward current in cells isolated from animals older than 5 weeks, but not in animals less than 2–3 weeks of age. The current/voltage (I/V) relationship of the ACh-induced current was that of an outward rectifier, and the current was inhibited by intracellular BAPTA, a Ca2+ buffer, indicating the current was Ca2+ activated. The ACh-induced current was also blocked in the presence of DPC and SITS, two Cl–current inhibitors in other tissues. Ionomycin mimicked the effect of ACh but in a nonoscillatory fashion. The appearance of the ionomycin-induced currents was also age related, as the current was not observed to occur in animals less than 2–3 weeks old. Since both ACh and ionomycin significantly increase cytosolic [Ca2+] in the acinar cells of young animals, the correlation between the age dependence of the ACh-activated Cl–current and the ionomycin-activated Cl–current responses suggests that the lack of responsiveness observed in the young animals is due to the absence of Ca2+-activated Cl–channels, rather than to a deficiency of a cellular mediator.