Gene polymorphisms in antioxidant enzymes correlate with the efficacy of androgen-deprivation therapy for prostate cancer with implications of oxidative stress.

Background: Oxidative stress mitigated by antioxidant enzymes is thought to be involved in the progression to castration-resistant prostate cancer (CRPC) during androgen-deprivation therapy (ADT). This study investigated the association between genetic variations in antioxidant enzymes and the efficacy of ADT as well as its biological background. Patients and methods: The non-synonymous or promoter-locating polymorphisms of antioxidant enzymes were examined as well as the time to CRPC progression and overall survival in 104 and 92 patients treated with ADT for metastatic and non-metastatic prostate cancer, respectively. In addition, intracellular reactive oxygen species and expression levels of antioxidant enzymes were examined in castration-resistant and enzalutamide-resistant cells. Results: In metastatic prostate cancer, the AG/GG allele in GSTM3 rs7483 and CT/TT allele in CAT rs564250 were associated with a significantly lower risk of progression to CRPC and all-cause death compared with homozygotes of the major AA allele (hazard ratio [HR]; [95% confidence interval (CI)], 0.55 [0.34-0.86], P = 0.0086) and CC allele (HR; [95% CI], 0.48 [0.24-0.88], P = 0.016), respectively. On multivariate analyses, only GSTM3 rs7483 was associated with significant progression risk (AG/GG versus AA; HR; [95% CI], 0.45 [0.25-0.79], P = 0.0047) even after Bonferroni adjustment. In non-metastatic prostate cancer, the AG/GG allele in GSTM3 rs7483 was associated with a significantly lower risk of progression to CRPC (HR; [95% CI], 0.35 [0.10-0.93], P = 0.034) and all-cause death (HR; [95% CI], 0.26 [0.041-0.96], P = 0.043) compared with the AA allele. Intracellular reactive oxygen species levels were increased, accompanied with augmented GSTM3 expression in both castration-resistant and enzalutamide-resistant cells. Conclusions: Differential activity of antioxidant enzymes caused by the polymorphism in GSTM3 may contribute to resistance to hormonal therapy through oxidative stress. The GSTM3 rs7483 polymorphism may be a promising biomarker for prostate cancer patients treated with ADT.

Pinacidil inhibits the ryanodine-sensitive outward current and glibenclamide antagonizes its action in cells from the rabbit portal vein.

Pinacidil, a potassium-channel opener, inhibited the ryanodine-sensitive oscillatory outward potassium current induced by Ca released from an intracellular store. Glibenclamide, a blocker of the ATP-sensitive K-channel, prevented the action of pinacidil, suggesting the presence of an additional site (to K channels) for the vasodilator actions of pinacidil at which glibenclamide can act as an antagonist.

Effects of phosphorylation-related drugs on slow Ca2+ tail current in guinea-pig detrusor cells.

In isolated guinea-pig detrusor cells, large conditioning depolarizations evoke slowly deactivating Ca2+ tail currents, considered to represent the second open state. The possible involvement of channel phosphorylation in this open state was examined. Application of isoprenaline caused a marginal increase in Ca2+ channel current evoked by simple depolarization, while forskolin did not. During application of either drug, slow-tail currents were never observed after simple depolarizations. The conditions necessary to induce slow-tail currents were not changed, even when cyclic AMP, ATP-gamma-S (adenosine 5'-O-(3-thiotriphosphate)), GDP-beta-S (guanosine 5'-O-(2-thiodiphosphate)) (in the pipette) or H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) (to the bathing solution) was applied. The frequent depolarization protocol, known to facilitate Ca2+ current via Ca2+ and cyclic AMP-dependent phosphorylation mechanism(s) in cardiac myocytes, did not induce slow-tail currents. These results suggest that the transition of Ca2+ channels to the second open state during large depolarization is not a result of (voltage-operated) channel phosphorylation itself. Possible underlying mechanisms are discussed.

A target K+ channel for the LP-805-induced hyperpolarization in smooth muscle cells of the rabbit portal vein.

The resting membrane potential of smooth muscle cells of the rabbit portal vein was -51.2 mV. LP-805 (8-tert-butyl-6,7-dihydropyrrolo[3,2-e] 5-methylpyrazolo [1,5-a] pyrimidine-3-carbonitrile) hyperpolarized the membrane to -62.3 mV (10 microM) and inhibited the burst spike discharges as measured using the microelectrode method. In dispersed smooth muscle cells, LP-805 (10 microM) generated an outward-current with a maximum amplitude of 68 pA at a holding potential of -40 mV in experiments using the voltage-clamp procedure. The reversal potential of the outward current evoked by LP-805 was -82 mV and this value was close to the equilibrium potential for K+ (-80 mV) in the present ionic conditions, suggesting that LP-805 activated the K+ channel. Generation of both the hyperpolarization and the outward current by LP-805 was inhibited by glibenclamide (> or = 1 microM). Using the cell-attached and cell-free patch-clamp (in the presence of GDP) procedures, the maxi-K+ channel current (150 pS) could be recorded in the absence of LP-805; application of LP-805 additionally opened a small conductance K+ channel current (15 pS) without change in the activity of the maxi-K+ channel. The maxi-K+ channel was sensitive to charybdotoxin (0.1 microM) and to intracellular Ca2+ ([Ca2+]i) concentration. The 15 pS channel was insensitive to [Ca2+]i and charybdotoxin, but sensitive to intracellular ATP concentration. Glibenclamide (> 1 microM) inhibited the 15 pS K+ channel activated by LP-805.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of IP3-receptor activation in endothelin-1-induced Ca(2+) influx in rat pulmonary small artery.

We examined the endothelin-1 (ET-1)-induced increase in the intracellular free Ca(2+) concentration ([Ca(2+)]i) in fura-2-loaded rat pulmonary small arteries. ET-1 (30 nM) elicited a long-lasting increase in [Ca(2+)]i in physiological salt solution (PSS). In subsequent experiments, arteries were pretreated with BQ-788, an ETB-specific blocker, to allow us to focus on responses mediated via the ETA receptor, the existence of which was confirmed by immunohistochemistry. In Ca(2+)-free PSS, ET-1 evoked a small transient increase in [Ca(2+)]i, indicating Ca(2+) release from the SR (sarcoplasmic reticulum). After a switch to PSS (containing 2mM CaCl2), ET-1 elicited a long-lasting increase in [Ca(2+)]i that was not inhibited by 1 µM nicardipine, an L-type Ca(2+)-channel inhibitor, suggesting involvement of a Ca(2+)-influx pathway independent of that channel. In arteries preincubated with 30 µM cyclopiazonic acid (CPA) or 2 µM thapsigargin (TG), the ET-1-induced Ca(2+)-release was greatly reduced, and the induced Ca(2+)-influx was attenuated. U-73122, a phospholipase C (PLC) inhibitor, had inhibitory effects similar to those of CPA and TG on the ET-1-induced Ca(2+)-release and Ca(2+)-influx, whereas U-73343, an inactive analogue of U-73122, had no such effects. Two putative membrane-permeable IP3-receptor blockers, 2-aminoethoxydiphenyl borate (2APB, 50 µM) and Xestospongin C (20 µM), (a) almost completely inhibited the ET-1-induced Ca(2+)-release and Ca(2+)-influx, and (b) reduced the ET-1-induced contraction. These results indicate that in rat pulmonary small arteries, ET-1 induces receptor-operated Ca(2+) influx via the ETA receptor, and that this influx interacts with InsP3-receptor activation.

Guanosine diphosphate activates an adenosine 5'-triphosphate-sensitive K+ channel in the rabbit portal vein.

1. Properties of the pinacidil-sensitive K+ channel in the smooth muscle of the rabbit portal vein were investigated using cell-attached and inside- and outside-out patch clamp techniques. 2. In the cell-attached patch configuration, a K+ channel with a unitary conductance of 150 pS could be recorded when physiological salt solution (PSS) was in the pipette and high-K+ solution was in the bath. Tetraethylammonium (TEA; less than 1 mM) and charybdotoxin (CTX; greater than 50 nM) inhibited the 150 pS K+ channel from the outside of the membrane. This channel was activated by an increase in the concentrations of intracellular Ca2+ but not by pinacidil (less than or equal to 500 microM). 3. In the cell-attached patch configuration, bath application of pinacidil (greater than 3 microM) activated a K+ channel (ATP-sensitive K+ channel) with a unitary conductance of 15 pS and the enhancing action of pinacidil was blocked by glibenclamide. However, in the cell-free patch configuration, pinacidil (100 microM) failed to open the 15 pS K+ channel. With pinacidil in the pipette, the 15 pS K+ channel was completely inactivated within 5 s of the excision of the membrane. Opening of the 15 pS K+ channel also disappeared after saponin treatment (50 micrograms/ml). 4. In the cell-free patch configuration, application of guanosine 5'-diphosphate (GDP; greater than 100 microM) re-activated the inactivated 15 pS K+ channel only when pinacidil was present either in the pipette or bath. GDP increased the mean open time and open probability of the 15 pS K+ channel in a concentration-dependent manner. Simultaneous application of MgCl2 (less than or equal to 1 mM) with GDP did not modify the GDP-induced activation. Neither GDP nor GTP (1 mM) had any effect on the 150 pS K+ channel. 5. Guanosine 5'-triphosphate (GTP; 1 mM) activated the 15 pS K+ channel to a lesser extent that did GDP. Other guanine nucleotides (guanosine 5'-monophosphate, GMP, 1 mM; guanosine 5'-O-(3-thiotriphosphate), GTP gamma S, 100 microM; and guanosine 5'-O-(2-thiodiphosphate), GDP beta S, 1 mM) failed to activate the 15 pS K+ channel. However, GDP beta S, but not GMP or GTP gamma S, inhibited this channel when it was activated by 1 mM-GDP. 6. In the presence of pinacidil, adenosine 5'-triphosphate (ATP; greater than or equal to 10 microM) inhibited the ATP-sensitive K+ channel when it was activated by 1 mM-GDP.(ABSTRACT TRUNCATED AT 400 WORDS)

Nicorandil opens a calcium-dependent potassium channel in smooth muscle cells of the rat portal vein.

The actions of nicorandil on ionic currents recorded from smooth muscle cells of the rat portal vein were investigated using patch-clamp technique. In the whole-cell voltage clamp experiments, nicorandil (greater than 30 microM) produced an outward current in a concentration-dependent manner. The reversal potential was -80 mV. The nicorandil-induced outward current was suppressed in the nominally Ca-free (2.5 mM Mn-containing) solution. Tetraethylammonium (greater than 1 mM) and 4-aminopyridine (greater than 1 mM) inhibited the nicorandil-induced outward current. Recordings of single K-channels showed that there were two types of K currents with different conductance (132 pS and 10 pS) in the smooth muscle cell membrane. Both channels were sensitive to intracellular Ca concentration. The 10-pS K-channel but not the 132-pS K-channel was activated by lowering the ATP concentration inside of the membrane. Low concentrations of tetraethylammonium (less than 1 mM) completely inhibited the 132-pS K-channel, but had no effect on the 10-pS K-channel. On the other hand, 4-aminopyridine (10 mM) inhibited the 10-pS K-channel but not 132-pS K-channel. Nicorandil (500 microM) applied to outside of the membrane increased the mean open-time and number of appearances but reduced slow component of the mean closed time of the 10-pS K-channel current, without affecting their amplitudes. Nicorandil (less than 500 microM) did not modify the 132-pS K-channel. From above results we concluded that nicorandil activates the Ca-dependent and ATP-sensitive K-channel in smooth muscle cells of the rat portal vein.(ABSTRACT TRUNCATED AT 250 WORDS)

Activations of the Ca dependent K channel by Ca released from the sarcoplasmic reticulum of mammalian smooth muscles.

In mammalian smooth muscles, the outward K current recorded using the whole cell voltage clamp or patch clamp methods can be classified into the Ca-dependent and independent K currents. The former is sub-classified into the extra- and intra-cellular Ca dependent K current. The intra-cellular Ca dependent K current has a close relation to Ca released from the sarcoplasmic reticulum, i.e. Ca released by inositol 1,4,5-trisphosphate (InsP3), ryanodine or Ca ionophores (A23187 or ionomycin) modify the appearance of the K current. The transient (Ca dependent) outward current evoked by depolarization pulses, as measured using the whole cell voltage clamp method, is closely related with after-hyperpolarization of the action potential as recorded using the microelectrode method and is postulated to be due to activations of the Ca-induced Ca release mechanism in the sarcoplasmic reticulum. The oscillatory (Ca dependent) outward K current is closely related with the amount of Ca released from the sarcoplasmic reticulum during the long depolarization induced by electrical stimulation (command pulse) or applications of Ca releasers such as InsP3 or ryanodine. In this review, the Ca dependent K current recorded from smooth muscle cells is compared with the influx and release of Ca.

Ionic currents involved in vasodilating actions of E4080, a newly synthesized bradycardia-inducing agent, in dispersed smooth muscle cells of the rabbit portal vein.

The effects of E4080 [(E)-N-[3-[N'-(2-(3,5-dimethoxyphenyl)ethyl)-N'-methyl)amino)propyl]-4- (4-(1H-imidazol-1-yl)phenyl)-3-butenamide dihydrochloride dihydrate] on ionic currents recorded from the rabbit portal vein were investigated by using the patch-clamp technique. A depolarization of the membrane produced an inward Ca current (ICa), a transient outward current (ITO), a sustained outward current (ISO) and an oscillatory outward current (IOO), whereas a hyperpolarization of the membrane produced a hyperpolarization-activated current (Ih). When ICa was evoked by a depolarizing pulse to 0 mV from the holding potential of -80 mV, 1 microM E4080 increased and higher concentrations (10 microM) inhibited ICa, whereas, at the holding potential of -60 mV, E4080 (greater than 1 microM) consistently inhibited ICa, in a concentration-dependent manner. E4080 inhibited ITO, ISO (greater than or equal to 1 microM) and Ih (greater than or equal to 0.1 microM) concentration dependently. Inasmuch as 1 microM E4080 did not inhibit ICa at the holding potential of -80 mV, the inhibition of ITO induced by 1 microM E4080 was not related to the inhibition of ICa. When a continuous depolarization (0 mV) was applied to the cell, E4080 (1 microM) produced a maintained outward current (14.0 +/- 15.3 pA), which was inhibited by glibenclamide. I infinity was inhibited by E4080 (greater than or equal to 0.1 microM) and application of 1 microM glibenclamide partly restored I infinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of cromakalim on ionic currents recorded from single smooth muscle cells of the rat portal vein.

The effects of cromakalim (BRL34915), a novel K channel opener, on ionic currents in dispersed smooth muscle cells of the rat portal vein were investigated. Application of cromakalim (greater than 3 microM) generated an outward current, the reversal potential of which (-79 mV) was almost the same as the theoretical K equilibrium potential (-80 mV) under the experimental conditions used. When the Ca in the physiological salt solution (PSS) was replaced with Mn, the cromakalim-induced outward current was inhibited markedly. When 4 mM ethylene glycol bis(beta-aminoethyl ether(N,N'-N,N'-tetraacetic acid (EGTA) was added to the pipette solution (bath solution, Ca-free PSS + Mn), cromakalim inhibited the outward current evoked by command pulses. Using a pipette solution containing high Cs with 4 mM EGTA and a bath PSS containing 2.5 mM Ca with 1 microM tetrodotoxin, the Ca inward current was isolated. Cromakalim (greater than 3 microM) inhibited the Ca inward current in a voltage- dependent manner. Cromakalim (30 microM) inhibited the Ca inward current to 0.6 times the control. The decay of the inward Ca current comprised fast and slow components. Cromakalim inhibited the latter component and shifted the voltage-dependent inactivation curve of this current to the left (more hyperpolarized direction) in a parallel manner and delayed recovery from inactivation as estimated using a double pulse protocol. By using a pipette solution containing high Cs with 4 mM EGTA and Ca-free PSS containing 2.5 mM Mn in the bath, Na inward currents, blocked by tetrodotoxin (IC50 = 10 nM), were evoked by depolarizing pulses. Cromakalim (up to 30 microM) had no effect on the Na inward current.(ABSTRACT TRUNCATED AT 250 WORDS)