Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle

1. Effects on the pinacidil-induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch-clamp method in smooth muscle cells of the rabbit portal vein. 2. A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil-induced current in a concentration-dependent manner with an IC50 of 5.5 microM. Superfusion of Ca2+-free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage-dependent Ba2+ and K+ currents with IC50 values of > 100 microM and 75 microM respectively. Tyrphostin B46 (30 microM), a tyrosine kinase inhibitor, also inhibited the pinacidil-induced current by 70% of the control. 3. Sodium orthovanadate (100 microM), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil-induced and delayed rectifier K+ currents. Daidzein (100 microM), an inactive analogue of genistein, did not inhibit these currents. 4. Neither herbimycin A (1 microM), lavendustin A (30 microM), tyrphostin 23 (10 microM), which are also tyrosine kinase inhibitors, nor wortmannin (10 microM), a phosphatidylinositol 3-kinase inhibitor, had an effect on either the pinacidil-induced or delayed rectifier K+ currents. Epidermal growth factor (EGF; 1 microg ml(-1)) did not induce an outward current or enhance the pinacidil-induced current. 5. Pinacidil alone, in the cell-attached configuration, or pinacidil with GDP, in the inside-out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 microM) reduced the channel's open probability without inducing a change in unitary conductance at any holding potential (-30 to +20 mV). 6. In the inside-out configuration, genistein at 30 microM did not change the mean channel open time, but reduced the burst duration. At 100 microM genistein abolished channel opening. The inhibitory potencies with which 30 and 100 microM genistein acted on the unitary current of the ATP-sensitive K+ channel were similar to those seen in the whole-cell voltage-clamp configuration. 7. Although direct inhibitory actions of genistein on the ATP-sensitive K+ channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP-sensitive K+ channel activity in the rabbit portal vein.     

Assessing interrater agreement from dependent data

The effects of riluzole, a neuroprotective drug, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. At the resting potential, riluzole preferentially blocked TTX-S sodium channels, whereas at more negative potentials, it blocked both types of sodium channels almost equally. The apparent dissociation constants for riluzole to block TTX-S and TTX-R sodium channels in their resting state were 90 and 143 microM, respectively. Riluzole shifted the voltage dependence of activation of TTX-R sodium channels in the depolarizing direction more than that of TTX-S sodium channels. The voltage dependence of the fast inactivation of both types of sodium channels was shifted in the hyperpolarizing direction in a dose-dependent manner, and the apparent dissociation constants for riluzole to block the inactivated channels were estimated to be 2 and 3 microM for the TTX-S and TTX-R sodium channels, respectively, indicating a much higher affinity for the inactivated channels than for the resting channels. Riluzole was equally effective in blocking both types of sodium channels in their slow inactivated state. Since more TTX-S channels are inactivated than TTX-R channels at the resting potential, riluzole blocks TTX-S sodium channels more potently than TTX-R sodium channels. It was concluded that one of the mechanisms by which riluzole exerts its neuroprotective action is to preferentially block the inactivated sodium channel of damaged or depolarized neurons under ischemic conditions, thereby suppressing excess stimulation of the glutamatergic receptors and massive influx of Ca++.     

Selective inhibition of M-type potassium channels in rat sympathetic neurons by uridine nucleotide preferring receptors

1. UTP and UDP depolarize rat superior cervical ganglion neurons and trigger noradrenaline release from these cells. The present study investigated the mechanisms underlying this excitatory action of uridine nucleotides by measuring whole-cell voltage-dependent K+ and Ca2+ currents. 2. Steady-state outward (holding) currents measured in the amphotericin B perforated-patch configuration at a potential of -30 mV were reduced by 10 microM UTP in a reversible manner, but steady-state inward (holding) currents at -70 mV were not affected. This action of UTP was shared by the muscarinic agonist oxotremorine-M. In current-voltage curves between -20 and -100 mV, UTP diminished primarily the outwardly rectifying current components arising at potentials positive to -60 mV. 3. Slow relaxations of muscarinic K+ currents (IM) evoked by hyperpolarizations from -30 to -55 mV were also reduced by 10 microM UTP (37% inhibition) and oxotremorine-M (81% inhibition). In contrast, transient K+-currents, delayed rectifier currents, fast and slow Ca2+-dependent K+ currents, as well as voltage-dependent Ca2+ currents were not altered by UTP. 4. In conventional (open-tip) whole-cell recordings, replacement of GTP in the pipette by GDPbetaS abolished the UTP-induced inhibition of IM, whereas replacement by GTPgammaS rendered it irreversible. 5. The UTP-induced reduction of IM was half maximal at 1.5 microM with a maximum of 37% inhibition; UDP was equipotent and equieffective, while ADP was less potent (half maximal inhibition at 29 microM). ATP had no effect at < or = 30 microM. 6. The inhibition of IM induced by 10 microM UTP was antagonized by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at > or = 30 microM and by reactive blue 2 at > or = 10 microM, but not by suramin at concentrations up to 30 microM. 7. These results show that rat superior cervical ganglion neurons possess uridine nucleotide preferring P2Y receptors which inhibit KM channels. This effect presumably forms the basis of the excitatory action of uridine nucleotides in rat sympathetic neurons.     

Antioxidative and proapoptotic effects of riluzole on cultured cortical neurons

Riluzole is used clinically in patients with amyotrophic lateral sclerosis. As oxidative stress, in addition to excitotoxicity, may be a major mechanism of motoneuron degeneration in patients with amyotrophic lateral sclerosis, we examined whether riluzole protects against nonexcitotoxic oxidative injury. Probably reflecting its weak antiexcitotoxic effects, riluzole (1-30 microM) attenuated submaximal neuronal death induced by 24-h exposure to 30 microM kainate or NMDA, but not that by 100 microM NMDA, in cortical cultures. Riluzole also attenuated nonexcitotoxic oxidative injury induced by exposure to FeCl3 in the presence of MK-801 and CNQX. Consistent with its antioxidative effects, riluzole reduced Fe3+-induced lipid peroxidation, and inhibited cytosolic phospholipase A2. By contrast, riluzole did not attenuate neuronal apoptosis induced by staurosporine. Rather unexpectedly, 24-48-h exposure to 100-300 microM riluzole induced neuronal death accompanied by nuclear and DNA fragmentations, which was attenuated by caspase inhibitor carbobenzyloxy-Val-Ala-Asp-fluoromethyl ketone but not by protein synthesis inhibitor cycloheximide. The present study demonstrates that riluzole has direct antioxidative actions, perhaps in part by inhibiting phospholipase A2. However, in the same neurons, riluzole paradoxically induces neuronal apoptosis in a caspase-sensitive manner. Considering current clinical use of riluzole, further studies are warranted to investigate its potential cytolethal effects.     

External barium block of Shaker potassium channels: evidence for two binding sites

External barium ions inhibit K+ currents of Xenopus oocytes expressing ShH4 delta 6-46, the non-inactivating deletion of the Shaker K+ channel. At the macroscopic level, Ba2+ block comprises both a fast and a slow component. The fast component is less sensitive to Ba2+ (apparent dissociation constant at 0 mV, K(0), approximately 19.1 mM) than the slow component and is also less voltage dependent (apparent electrical distance, delta, approximately 0.14). The slow component (K(0), approximately 9.4 mM, delta approximately 0.25) is relieved by outward K+ current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast component of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unblocking reaction. In contrast, the slow component appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (tau) produced by 2 mM Ba2+ is approximately 159 s (holding potential, HP = -90 mV). However, the blocking rate of this slow component is not a linear function of external Ba2+ and tends to saturate at higher concentrations. This is inconsistent with a simple bi-molecular blocking reaction. These features of external Ba2+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow component of block.     

Characterization of inhibition by haloperidol and chlorpromazine of a voltage-activated K+ current in rat phaeochromocytoma cells

1. Inhibition by haloperidol and chlorpromazine of a voltage-activated K+ current was characterized in rat phaeochromocytoma PC12 cells by use of whole-cell voltage-clamp techniques. 2. Haloperidol or chlorpromazine (1 and 10 microM) inhibited a K+ current activated by a test potential of +20 mV applied from a holding potential of -60 mV. The K+ current inhibition did not exhibit voltage-dependence when test potentials were changed between -10 and +40 mV or when holding potentials were changed between -120 and -60 mV. 3. Effects of compounds that are related to haloperidol and chlorpromazine in their pharmacological actions were examined. Fluspirilene (1 and 10 microM), an antipsychotic drug, inhibited the K+ current, but pimozide (1 and 10 microM), another antipsychotic drug did not significantly inhibit the K+ current. Sulpiride (1 or 10 microM), an antagonist of dopamine D2 receptors, did not affect the K+ current whereas (+)-SCH-23390 (10 microM), an antagonist of dopamine D1 receptors, reduced the K+ current. As for calmodulin antagonists, W-7 (100 microM), but not calmidazolium (1 microM), reduced the K+ current. 4. The inhibition by haloperidol or chlorpromazine of the K+ current was abolished when GTP in intracellular solution was replaced with GDP beta S. Similarly, the inhibition by pimozide, fluspirilene, (+)-SCH-23390 or W-7 was abolished or attenuated in the presence of intracellular GDP beta S. The inhibition by haloperidol or chlorpromazine was not prevented when cells were pretreated with pertussis toxin or when K-252a, an inhibitor of a variety of protein kinases, was included in the intracellular solution. 5. Haloperidol and chlorpromazine reduced a Ba2+ current permeating through Ca2+ channels. Inhibition by haloperidol or chlorpromazine of the Ba2+ current was not affected by GDP beta S included in the intracellular solution. 6. It is concluded that haloperidol and chlorpromazine inhibit voltage-gated K+ channels in PC12 cells by a mechanism involving GTP-binding proteins. The inhibition may not be related to their activity as antagonists of dopamine D2 receptors or calmodulin antagonists.     

Intracellular divalent cations block smooth muscle K+ channels

The patch-clamp technique was used to examine the sensitivity of delayed rectifier K+ channels to changes in intracellular divalent cations (Mg2+ and Ca2+). During voltage-step and ramp depolarizations, a delayed rectifier K+ current (IK(dr)) was identified in renal, pulmonary, coronary, and colonic smooth muscle cells as a low-noise outward current that activated near -40 mV, was sensitive to 4-aminopyridine (4-AP), and was insensitive to charybdotoxin. During whole-cell voltage-clamp experiments in each of the cell types, the 4-AP-sensitive IK(dr) was significantly less in cells dialyzed with 10 mM Mg2+ as compared with cells in which no Mg2+ was added to the internal dialysis solution (P < or = .05, n > or = 4). In coronary artery cells, 100 microM 2-(2-aminoethyl)pyridine (an H1 receptor agonist) or 10 microM ryanodine, agents that cause an increase in [Ca2+]i, also caused a significant reduction of the 4-AP-sensitive IK(dr) similar to that produced by Mg2+. 4-AP (5 mM) significantly depolarized single renal arterial cells that were dialyzed with Mg(2+)-free solution but not those dialyzed with 10 mM Mg2+ (P < .01, n = 4). In inside-out patches of renal arterial smooth muscle cells, with 200 nM charybdotoxin in the patch pipette to block large conductance Ca(2+)-activated K+ channels, a 59 +/- 10-picosiemen K+ channel that was sensitive to cytoplasmic Mg2+ was identified. In Mg(2+)-free solution, channel open probability was 0.028 +/- 0.012 (n = 8) and 0.095 +/- 0.011 (n = 8) at +40 and +80 mV, respectively. When the bath solution was changed to one containing 5 or 15 mM Mg2+, channel open probability was significantly reduced by 66% and 68% (+40 mV) or 93% and 96% (+80 mV), respectively. This decrease in the open probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. At +40 mV, time constants for the open time distribution were significantly decreased from 5.5 +/- 0.52 to 1.2 +/- 0.14 milliseconds, whereas the closed time constant was significantly increased from 634 +/- 11.1 to 820 +/- 14.4 milliseconds (P < .01, n = 4). It is concluded that a 4-AP-sensitive delayed rectifier K+ channel in both vascular and visceral smooth muscle cells is modulated by changes in intracellular Ca2+ and Mg2+ that may alter membrane potential and the contractile state of smooth muscle.     

Autoantibodies associated with Type I diabetes mellitus persist after diagnosis in children

We have examined the effects of riluzole, a neuroprotective drug which stabilizes voltage-dependent sodium channels in their inactivated state and inhibits the release of glutamate in-vivo and in-vitro, on the release of newly taken up [3H]dopamine induced by ouabain, a potent and selective inhibitor of Na+/K+-ATPase in mouse striatal slices in-vitro. Riluzole potently (IC50 (concentration resulting in 50% inhibition) = 0.9+/-0.3 microM) and dose-dependently antagonized ouabain-stimulated [3H]dopamine release, the effect being observed at low concentrations. Tetrodotoxin (1 microM) and nomifensine (10 microM) also abolished ouabain-induced [3H]dopamine release. Blockade of glutamate receptors with dizocilpine (1 microM) and 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione (YM-90K; 10 microM), alone or in combination, was without effect. Incubation of striatal slices with 50 microM La3+, which blocks voltage-dependent calcium channels, did not inhibit [3H]dopamine release induced by ouabain. The potent effects of riluzole observed in this model are probably related to its ability to block voltage-dependent sodium channels. The consequences of this activity are critically discussed in relation to the protective action of riluzole previously reported in various models of Parkinson's disease and other neurodegenerative disorders.     

Voltage- and ligand-gated ion channels in floor plate neuroepithelia of the rat

Whole-cell patch-clamp recordings were used to characterize the membrane properties and ion channel complement of floor plate neuroepithelia in embryonic and neonatal rats. The average resting potential was close to -60 mV, the capacitance was approximately 7 pS and the membrane time constant averaged 31 ms, in both neonates and embryos. Two types of K+ current were identified (i) a slowly activating, slowly inactivating current that was present in all cells, and (ii) a rapidly inactivating current that was present in 39% of cells from neonates and 64% of cells from embryos. K+ currents were significantly larger in neonates than embryos. Na+ currents were absent from all neuroepithelial cells examined. In contrast, the majority of floor plate cells exhibited a significant Ca2+ current. Biophysically this current activated at potentials positive to 60 mV and exhibited fast, voltage-dependent, inactivation. The Ca2+ current was equipermeant to Ca2+ and Ba2+, sensitive to 40-120 microM Ni2+ and only slightly inhibited by 100 microM Cd2+. These and other observations indicated this current is mediated by low-voltage-activated (i.e. T-type) Ca2+ channels. The majority of floor plate cells tested also exhibited responses to the neurotransmitter GABA which produced robust inward currents at negative membrane potentials, in chloride-loaded cells. Both the pharmacology and voltage-dependence of the GABA-activated currents indicated they arose from activation of GABA(A) receptors.     

Gating of I(sK) channels expressed in Xenopus oocytes

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+ current, I(Ks), in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopus oocytes results in I(Ks)-like currents, I(sK), due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of I(sK) suggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40 mV from holding potentials between -60 and -120 mV showed an unusual "cross-over," with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100 ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac I(Ks) may suppress premature action potentials.     

Functional expression of sperm angiotensin II type I receptor in Xenopus oocyte: modulation of a sperm Ca2+-activated K+ channel

gamma-Hydroxybutyric acid (GHB) is an abused substance that occurs naturally in the basal ganglia. Electrophysiological recordings of membrane voltage and current were made to characterize the effects of GHB on dopamine neurons in the ventral tegmental area of the rat midbrain slice. Perfusate containing GHB caused a concentration-dependent membrane hyperpolarization (EC50 = 0.88 +/- 0.21 mM) and a reduction in input resistance (EC50 = 0.74 +/- 0.21 mM). The highest concentration of GHB studied (10 mM) hyperpolarized neurons by 20 +/- 3 mV and reduced input resistance by 58% +/- 9%. Changes in membrane potential and input resistance were blocked by the gamma-aminobutyric acid antagonist CGP-35348 (300 microM), but neither bicuculline (30 microM) nor strychnine (10 microM) was an effective antagonist. Voltage-clamp recordings demonstrated that GHB (1 mM) evoked 80 +/- 6 pA of outward current (at -60 mV) that reversed at -110 mV (in 2.5 mM K+). Increasing concentrations of extracellular K+ progressively shifted the reversal to more depolarized potentials. In tetrodotoxin (0.3 microM) and tetraethylammonium (10 mM), depolarizing voltage steps (to -30 mV) evoked calcium-dependent current spikes that were completely blocked by GHB (1 mM). These data suggest that GHB is an agonist at gamma-aminobutyric acid receptors and would be expected to inhibit DA release by causing K+-dependent membrane hyperpolarization.     

Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones. 2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1-100 microM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na+ currents (TTXR). From a holding potential (Vh) of -90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC50 value of approximately 103 microM. 3. The inhibitory effect of 4030W92 on TTX(R) was both voltage- and use-dependent. Currents evoked from a Vh of -60 mV were inhibited by 4030W92 with a mean IC50 value of 22 microM, which was 5 fold less than the value obtained at -90 mV. Repeated activation of TTX(R) by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 microM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX(R) and markedly slowed the recovery from channel inactivation. 4. Fast TTX-sensitive Na+ currents (TTXs) were also inhibited by 4030W92 in a voltage-dependent manner. The IC50 values obtained from Vhs of -90 mV and -70 mV were 37 microM and 5 microM, respectively. 4030W92 (30 microM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTXs. 5. High threshold voltage-gated Ca2+ currents were only weakly inhibited by 4030W92. The reduction in peak Ca2+ current amplitude produced by 100 microM 4030W92 was 20+/-6% (n=6). Low threshold T-type Ca2+ currents were inhibited by 17+/-8% and 43+/-3% by concentrations of 4030W92 of 30 microM and 100 microM, respectively (n=6). 6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10-30 microM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7+/-1.5 to 4.2+/-1.0 and 2.6+/-1.1 in the presence of 10 microM and 30 microM 4030W92, respectively (n=5). 7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na+ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na+ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.     

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BK(Ca)) in smooth muscle cells isolated from the rat mesenteric artery

1. To assess the action of nitric oxide (NO) and NO-donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2. Inside-out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212+/-5 pS (n = 12) in symmetrical (140 mM) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK(Ca). 3. In five out of six inside-out patches, NO (45 or 67 microM) produced an increase in BK(Ca) activity. In inside-out patches, BK(Ca) activity was also enhanced in some patches with 100 or 200 microM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 microM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4. In inside-out patches, 100 microM SIN-1 failed to increase BK(Ca) activity (in all 4 patches tested), while at a higher (500 microM) concentration SIN-1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside-out patches increased (P < 0.05) BK(Ca) activity in two patches. 5. In the majority of cells (6 out of 7), application of NO (45 or 67 microM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 microM). With SIN-1, the increase in current was blocked with the BK(Ca) channel blocker, iberiotoxin (50 nM). 6. With conventional whole-cell voltage clamp, the increase in the outward K+ current evoked with SIN-1 (50-300 microM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+/-10.2% was recorded at 40 mV. 7. In cell-attached patches, large conductance voltage-dependent K+ channels were stimulated by SIN-1 (100 microM) applied to the cell (n = 5 patches). 8. These data indicate that NO and its donors can directly stimulate BK(Ca) activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK(Ca) channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.     

HERG-like K+ channels in microglia

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.     

Genistein inhibits slow component delayed-rectifier K currents via a tyrosine kinase-independent pathway

In single guinea pig ventricular cells, genistein, a potent inhibitor of protein tyrosine kinase (PTK), was found to suppress the delayed-rectifier K (IK) current. The present study was carried out to examine the underlying mechanism. Ventricular myocytes were voltage-clamped in the conventional whole-cell mode (36 degrees C). The amplitudes of tail and steady-state (2-s pulse) currents were measured as IK. Genistein (10-100 microM) reversibly inhibited both basal and intrapipette cAMP (1 mM)-enhanced IK currents in a concentration-dependent manner with a half-maximum inhibitory concentration (IC50) at approximately 30 microM. In contrast, lavendustin A (10 microM; n = 5) and tyrphostin 51 (100 microM; n = 5) had no effect on the currents. The inhibitory action of genistein was also seen after IK currents were activated by forskolin (500 nM) plus intrapipette orthovanadate (500 microM). The intrapipette cAMP-enhanced IK was also reduced to a lesser degree by daidzein, an inactive analogue of genistein. Envelope tail and short pulse protocols revealed that genistein inhibits the slow component of IK (IKs). Thus, the inhibitory action of genistein is not mediated via an inhibition of PTK but may be due to the block of IKs channels.     

Selective block by glyceryl nonivamide of inwardly rectifying K+ current in rat anterior pituitary GH3 cells

The effects of glyceryl nonivamide (GLNVA) on ionic currents were compared and examined in rat pituitary GH3 cells. Hyperpolarization-activated K+ currents in GH3 cells bathed in high-K+ Ca2+-free external solution were studied to assess effects of GLNVA on the an inwardly rectifying K+ current (I(K(IR))). GLNVA is very potent in blocking I(K(IR)) in a concentration-dependent manner, with a half maximal concentrations of 0.1 microM. The complete block of I(K(IR)) achieved with concentrations > or = 1 microM revealed the presence of a non-inactivating current. We also found that GLNVA at a concentration above 30 microM inhibited L-type voltage-dependent Ca2+ current and two components of K+ outward currents, while GLNVA (< or = 3 microM) did not have any effect on them. This study shows that GLNVA, in addition to retaining the capability of eliciting peptidergic neurons, is a selective block of I(K(IR)) in GH3 cells and will provide a useful tool for characterizing I(K(IR)) and understanding its physiological function. In addition, the carefulness should be taken about the interpretation of GLNVA-mediated responses in vivo or in vitro.     

Effect of age on the vasorelaxation elicited by cromakalim. Role of K+ channels and cyclic GMP

The objective of this study was to investigate whether age induces changes on vasodilator response induced by cromakalim, an ATP-sensitive K+ (K(ATP)) channel opener, as well as the underlying mechanism involved in this possible alteration. For this purpose, aortic segments from young (3-5 months) and old (3 years) rabbits were used, which were precontracted with noradrenaline (NA, 1 microM). The vasodilator response induced by cromakalim (0.01-100 microM) was reduced in intact segments from old rabbits, and endothelium removal reduced and did not modify this effect in young and old animals, respectively. In both groups of animals, glibenclamide (10 microM), a blocker of K(ATP) channels, significantly reduced the response elicited by cromakalim, which was not modified by the large conductance Ca2+-activated K+ channel blocker charybdotoxin (ChTX, 0.4 microM). Acetylcholine (ACh, 10 microM) and sodium nitroprusside (SNP, 100 microM) induced a lesser vasodilator effect in aortic segments from old compared with young rabbits. In segments precontracted with NA, 10 microM ACh or 100 microM SNP similarly increased cGMP levels in both groups of animals. However, basal cGMP level was reduced in segments from old rabbits. Incubation with 8-bromo-cGMP (100 microM) increased the response induced by cromakalim in both groups of animals, reaching similar maximum values in young and old rabbits. The response induced by cromakalim plus 8-bromo-cGMP was markedly decreased by glibenclamide and unmodified by ChTx in both types of animals. These results suggest that aging decreases the vasodilator response to cromakalim, mechanism in which appears to be involved the maintained low cGMP levels observed in old rabbits, and that this messenger modulates the degree of K(ATP) channel activation.