No description available.

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at -70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (K(Ca)) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K(Ca) activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V(m)) and 145 ± 13 pS at negative V(m). Glibenclamide (50 μM), Ba2+ (1 mM) and quinine (100 μM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 μM. The identity of this K+ channel with an ATP-sensitive K+ channel (K(ATP)) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 μM). We conclude that plasma membranes of HTB-9 cells contain the K(Ca) and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked K(ATP).

No description available.

No description available.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates migration of various cells and has been linked via Met tyrosine kinase-signaling to transformation and the metastatic phenotype. Migration of transformed MDCK-F cells depends on activation of a charybdotoxin-sensitive, volume-activated membrane K+ current. Thus, we used patchclamp electrophysiology and transwell migration assays to determine whether HGF/SF stimulation of MDCK II cell migration depends on the activation of membrane K+ currents. HGF/SF activated a membrane K+ current that increased over 24 hr, and which could be modulated by increasing intracellular calcium concentration, [Ca2+]i. Charybdotoxin (ChTX, 50 nM), iberiotoxin (IbTX, 100 nM), stichodactyla toxin (Stk, 100 nM) and clotrimazole (CLT, 1 μM) all inhibited this current. HGF/SF (100 scatter units/ml) significantly increased MDCK II cell migration over 8 hr compared to control cells. Addition of ChTX (50 nM), IbTX (100 nm), Stk (100 nM) or CLT (1 μM) inhibited the HGF/SF-stimulated MDCK II cell migration. We conclude that the activation of membrane Ca2+-activated K+ current is necessary for HGF/ SF stimulation of MDCK II cell.

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 μM for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μM (IC50 = 73 μM) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K(ATP) channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10-5 M bromodeoxyuridine. Glibenclamide (100 μM) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K(ATP) channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K(ATP) channel are involved in mechanisms controlling HTB-9 cell growth. However, K(ATP) is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.