Sean M. Wilson

ClC-3 is a fundamental molecular component of volume-sensitive outwardly rectifying Cl- channels and volume regulation in HeLa cells and Xenopus laevis oocytes.

Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells andX. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes.

SCD1 Inhibition Causes Cancer Cell Death by Depleting Mono-Unsaturated Fatty Acids

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells

Capacitative Ca2+ entry (CCE) has been speculated to contribute to Ca2+ influx during hypoxic pulmonary vasoconstriction (HPV). The aim of the present study was to directly test if acute hypoxia causes intracellular Ca2+ concentration ([Ca2+]i) rises through CCE in canine pulmonary artery smooth muscle cells (PASMCs). In PASMCs loaded with fura‐2, hypoxia produced a transient rise in [Ca2+]i in Ca2+‐free solution, indicating Ca2+ release from the intracellular Ca2+ stores. Subsequent addition of 2 mm Ca2+ in hypoxia elicited a sustained rise in [Ca2+]i, which was partially inhibited by 10 μm nisoldipine. The dihydropyridine‐insensitive rise in [Ca2+]i was due to increased Ca2+ influx, because it was abolished in Ca2+‐free solution and hypoxia was shown to significantly enhance the rate of Mn2+ quench of fura‐2 fluorescence. The dihyropyridine‐insensitive rise in [Ca2+]i and the increased rate of Mn2+ quench of fura‐2 fluorescence were inhibited by 50 μm SKF 96365 and 500 μm Ni2+, but not by 100 μm La3+ or 100 μm Gd3+, exhibiting pharmacological properties characteristic of CCE. In addition, predepletion of the intracellular Ca2+ stores inhibited the rise in [Ca2+]i induced by hypoxia. These results provide the first direct evidence that acute hypoxia, by causing Ca2+ release from the intracellular stores, activates CCE in isolated canine PASMCs, which may contribute to HPV.

Pregnancy Upregulates Large-Conductance Ca 2+ -Activated K + Channel Activity and Attenuates Myogenic Tone in Uterine Arteries

Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca2+-activated K+ (BKCa) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K+ current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K+ currents to the same extent in uterine arterial myocytes. The BKCa channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BKCa channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17&bgr;-estradiol and progesterone caused a significant increase in the BKCa channel current density. Western blot analyses demonstrated a significant increase of the &bgr;1, but not &agr;, subunit of BKCa channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the &bgr;1, but not &agr;, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the &bgr;1 subunit and BKCa channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.

Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells

Experiments were performed to determine whether capacitative Ca2+ entry (CCE) can be activated in canine pulmonary and renal arterial smooth muscle cells (ASMCs) and whether activation of CCE parallels the different functional structure of the sarcoplasmic reticulum (SR) in these two cell types. The cytosolic [Ca2+] was measured by imaging fura‐2‐loaded individual cells. Increases in the cytosolic [Ca2+] due to store depletion in pulmonary ASMCs required simultaneous depletion of both the inositol 1,4,5‐trisphosphate (InsP3)‐ and ryanodine (RY)‐sensitive SR Ca2+ stores. In contrast, the cytosolic [Ca2+] rises in renal ASMCs occurred when the SR stores were depleted through either the InsP3 or RY pathways. The increase in the cytosolic [Ca2+] due to store depletion in both pulmonary and renal ASMCs was present in cells that were voltage clamped and was abolished when cells were perfused with a Ca2+‐free bathing solution. Rapid quenching of the fura‐2 signal by 100 μM Mn2+ following SR store depletion indicated that extracellular Ca2+ entry increased in both cell types and also verified that activation of CCE in pulmonary ASMCs required the simultaneous depletion of the InsP3‐ and RY‐sensitive SR Ca2+ stores, while CCE could be activated in renal ASMCs by the depletion of either of the InsP3‐ or RY‐sensitive SR stores. Store depletion Ca2+ entry in both pulmonary and renal ASMCs was strongly inhibited by Ni2+ (0.1–10 mM), slightly inhibited by Cd2+ (200–500 μM), but was not significantly affected by the voltage‐gated Ca2+ channel (VGCC) blocker nisoldipine (10 μM). The non‐selective cation channel blocker Gd3+ (100 μM) inhibited a portion of the Ca2+ entry in 6 of 18 renal but not pulmonary ASMCs. These results provide evidence that SR Ca2+ store depletion activates CCE in parallel with the organization of intracellular Ca2+ stores in canine pulmonary and renal ASMCs.

Chronic Hypoxia Suppresses Pregnancy-Induced Upregulation of Large-Conductance Ca2+-Activated K+ Channel Activity in Uterine Arteries

Our previous study demonstrated that increased Ca2+-activated K+ (BKCa) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BKCa channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BKCa channel activity and suppressed BKCa channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BKCa channel &bgr;1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BKCa channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the &bgr;1 subunit and BKCa channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BKCa channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BKCa channel &bgr;1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Role of insp3 and ryanodine receptors in the activation of capacitative ca2+ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Experiments were performed to determine if capacitative Ca2+ entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP3 receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP3‐ and ryanodine‐sensitive sarcoplasmic reticulum (SR) Ca2+ stores in these cells.

CaV3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

Rationale: T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results: Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Role of basal extracellular Ca2+ entry during 5-HT-induced vasoconstriction of canine pulmonary arteries.

1 Measurements of artery contraction, cytosolic [Ca2+], and Ca2+ permeability were made to examine contractile and cytosolic [Ca2+] responses of canine pulmonary arteries and isolated cells to 5‐hydroxytryptamine (5‐HT), and to determine the roles of intracellular Ca2+ release and extracellular Ca2+ entry in 5‐HT responses. 2 The EC50 for 5‐HT‐mediated contractions and cytosolic [Ca2+] increases was ∼10−7 M and responses were inhibited by ketanserin, a 5‐HT2A‐receptor antagonist. 3 5‐HT induced cytosolic [Ca2+] increases were blocked by 20 μM Xestospongin‐C and by 2‐APB (IC50=32 μM), inhibitors of InsP3 receptor activation. 4 5‐HT‐mediated contractions were reliant on release of InsP3 but not ryanodine‐sensitive Ca2+ stores. 5 5‐HT‐mediated contractions and cytosolic [Ca2+] increases were partially inhibited by 10 μM nisoldipine, a voltage‐dependent Ca2+ channel blocker. 6 Extracellular Ca2+ removal reduced 5‐HT‐mediated contractions further than nisoldipine and ablated cytosolic [Ca2+] increases and [Ca2+] oscillations. Similar to Ca2+ removal, Ni2+ reduced cytosolic [Ca2+] and [Ca2+] oscillations. 7 Mn2+ quench of fura‐2 and voltage‐clamp experiments showed that 5‐HT failed to activate any significant voltage‐independent Ca2+ entry pathways, including store‐operated and receptor‐activated nonselective cation channels. Ni2+ but not nisoldipine or Gd3+ blocked basal Mn2+ entry. 8 Voltage‐clamp experiments showed that simultaneous depletion of both InsP3 and ryanodine‐sensitive intracellular Ca2+ stores activates a current with linear voltage dependence and a reversal potential consistent with it being a nonselective cation channel. 5‐HT did not activate this current. 9 Basal Ca2+ entry, rather than CCE, is important to maintain 5‐HT‐induced cytosolic [Ca2+] responses and contraction in canine pulmonary artery.

Purine nucleotides modulate proliferation of brown fat preadipocytes

Abstract. The hypothesis that purine nucleotides and nucleosides affect brown fat preadipocyte proliferation was tested using isolated rat interscapular brown fat preadipocytes in culture. Daily addition of 100 μM adenosine triphosphate (ATP) (n= 4) to cultures enhanced the relative DNA content by 1.5‐fold compared to control cultures (P < 0.05) measured using CyQUANT‐GR fluorescence. Higher concentrations of ATP inhibited growth and 500 (n= 2) or 1000 μM ATP (n= 3) almost completely inhibited growth. ATP (100 μM) did not affect while 250–1000 μM ATP decreased protein content relative to control cultures. Adenosine (100 μM; n= 3) did not affect DNA or protein content, but 500 μM and 1000 μM adenosine suppressed brown adipocyte proliferation and inhibited protein synthesis. Cultured brown adipocytes quickly removed or degraded ATP in the culture media as determined by luciferin–luciferase bioluminescence, suggesting that the inhibitory effects of high ATP concentrations may result from its breakdown to adenosine. The results support the conclusion that ATP promotes and adenosine inhibits brown adipocyte proliferation.