Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.
Animals ; Bicuculline ; pharmacology ; Disease Models, Animal ; Glycine ; metabolism ; Hyperalgesia ; drug therapy ; etiology ; metabolism ; Imidazoles ; pharmacology ; Inhibitory Postsynaptic Potentials ; drug effects ; physiology ; Male ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; Neurotransmitter Agents ; pharmacology ; Peripheral Nerve Injuries ; drug therapy ; metabolism ; Phenanthrolines ; pharmacology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology ; Synaptic Transmission ; drug effects ; physiology ; Tissue Culture Techniques ; Touch

Cyproheptadine (CPH), a first-generation antihistamine, enhances the delayed rectifier outward K current (I) in mouse cortical neurons through a sigma-1 receptor-mediated protein kinase A pathway. In this study, we aimed to determine the effects of CPH on neuronal excitability in current-clamped pyramidal neurons in mouse medial prefrontal cortex slices. CPH (10 µmol/L) significantly reduced the current density required to generate action potentials (APs) and increased the instantaneous frequency evoked by a depolarizing current. CPH also depolarized the resting membrane potential (RMP), decreased the delay time to elicit an AP, and reduced the spike threshold potential. This effect of CPH was mimicked by a sigma-1 receptor agonist and eliminated by an antagonist. Application of tetraethylammonium (TEA) to block I channels hyperpolarized the RMP and reduced the instantaneous frequency of APs. TEA eliminated the effects of CPH on AP frequency and delay time, but had no effect on spike threshold or RMP. The current-voltage relationship showed that CPH increased the membrane depolarization in response to positive current pulses and hyperpolarization in response to negative current pulses, suggesting that other types of membrane ion channels might also be affected by CPH. These results suggest that CPH increases the excitability of medial prefrontal cortex neurons by regulating TEA-sensitive I channels as well as other TEA-insensitive K channels, probably I and inward-rectifier Kir channels. This effect of CPH may explain its apparent clinical efficacy as an antidepressant and antipsychotic.
Animals ; Cyproheptadine ; pharmacology ; Female ; Histamine H1 Antagonists ; pharmacology ; Membrane Potentials ; drug effects ; physiology ; Mice, Inbred C57BL ; Patch-Clamp Techniques ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Prefrontal Cortex ; drug effects ; physiology ; Pyramidal Cells ; drug effects ; physiology ; Receptors, sigma ; agonists ; metabolism ; Tetraethylammonium ; pharmacology ; Tissue Culture Techniques

OBJECTIVETo investigate whether the methanol extract of Berberis amurensis Rupr. (BAR) augments penile erection using in vitro and in vivo experiments.

METHODSThe ex vivo study used corpus cavernosum strips prepared from adult male New Zealand White rabbits. In in vivo studies for intracavernous pressure (ICP), blood pressure, mean arterial pressure (MAP), and increase of peak ICP were continuously monitored during electrical stimulation of Sprague-Dawley rats.

RESULTSPreconstricted with phenylephrine (PE) in isolated endotheliumintact rabbit corus cavernosum, BAR relaxed penile smooth muscle in a dose-dependent manner, which was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one, a soluble guanylyl cclase inhibitor. BAR significantly relaxed penile smooth muscles dose-dependently in ex vivo, and this was inhibited by pretreatment with L-NAME H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one. BAR-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, P<0.01), a nonselective K channel blocker, 4-aminopyridine (4-AP, P<0.01), a voltage-dependent K channel blocker, and charybdotoxin (P<0.01), a large and intermediate conductance Ca sensitive-K channel blocker, respectively. BAR induced an increase in peak ICP, ICP/MAP ratio and area under the curve dose dependently.

CONCLUSIONBAR augments penile erection via the nitric oxide/cyclic guanosine monophosphate system and Ca sensitive-K (BK and IK) channels in the corpus cavernosum.

Animals ; Area Under Curve ; Berberis ; chemistry ; Blood Pressure ; drug effects ; Cyclic GMP ; metabolism ; Epoprostenol ; pharmacology ; In Vitro Techniques ; Indomethacin ; pharmacology ; Male ; Models, Biological ; Muscle Relaxation ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Penile Erection ; drug effects ; Phenylephrine ; pharmacology ; Plant Extracts ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Pressure ; Rabbits

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

OBJECTIVETo assess the biological effects of the six-herb mixture Anti-Insomia Formula (AIF) extract using caffeine-induced insomnia Drosophila model and short-sleep mutants.

METHODSCaffeineinduced insomnia wild-type Drosophila and short-sleep mutant flies minisleep (mns) and Hyperkinetic(Y) (Hk(Y)) were used to assess the hypnotic effects of the AIF in vivo. The night time activity, the amount of night time sleep and the number of sleep bouts were determined using Drosophila activity monitoring system. Sleep was defined as any period of uninterrupted behavioral immobility (0 count per minute) lasting > 5 min. Night time sleep was calculated by summing up the sleep time in the dark period. Number of sleep bouts was calculated by counting the number of sleep episodes in the dark period.

RESULTSAIF at the dosage of 50 mg/mL, effectively attenuated caffeine-induced wakefulness (P<0.01) in wild-type Canton-S flies as indicated by the reduction of the sleep bouts, night time activities and increase of the amount of night time sleep. AIF also significantly reduced sleeping time of short-sleep Hk(Y) mutant flies (P<0.01). However, AIF did not produce similar effect in mns mutants.

CONCLUSIONAIF might be able to rescue the abnormal condition caused by mutated modulatory subunit of the tetrameric potassium channel, but not rescuing the abnormal nerve firing caused by Shaker gene mutation. This study provides the scientific evidence to support the use of AIF in Chinese medicine for promoting sleep quality in insomnia.

Animals ; Caffeine ; Chromatography, High Pressure Liquid ; Disease Models, Animal ; Drosophila melanogaster ; drug effects ; physiology ; Hypnotics and Sedatives ; pharmacology ; therapeutic use ; Mutation ; genetics ; Potassium Channels ; genetics ; Sleep ; drug effects ; Sleep Initiation and Maintenance Disorders ; drug therapy ; Wakefulness ; drug effects

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.
Animals ; Basilar Artery ; drug effects ; Blood Pressure ; Cerebral Arteries ; drug effects ; Chalcones ; pharmacology ; Cyclic GMP ; physiology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; physiology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Systole

BACKGROUNDCyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.

METHODSIn an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.

RESULTSDose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP.

CONCLUSIONScAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Chloride Channels ; antagonists & inhibitors ; Ciliary Body ; drug effects ; physiology ; Colforsin ; pharmacology ; Cyclic AMP ; physiology ; Humans ; In Vitro Techniques ; Potassium Channel Blockers ; pharmacology ; Sodium Channel Blockers ; pharmacology

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, alpha-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.
4-Aminopyridine/pharmacology ; Action Potentials ; Animals ; Calcium Channel Blockers/pharmacology ; Calcium Channels/metabolism ; Cells, Cultured ; Ketanserin/pharmacology ; Male ; Mesenteric Arteries/drug effects/*metabolism/physiology ; Muscle Contraction ; Muscle, Smooth, Vascular/cytology/drug effects/metabolism/physiology ; Myocytes, Smooth Muscle/drug effects/metabolism/physiology ; Nifedipine/pharmacology ; Potassium Channel Blockers/pharmacology ; Potassium Channels, Voltage-Gated/antagonists & inhibitors/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptor, Serotonin, 5-HT2A/*metabolism ; Serotonin/*pharmacology ; Serotonin 5-HT2 Receptor Antagonists/pharmacology ; Spiperone/pharmacology ; *Vasoconstriction ; src-Family Kinases/antagonists & inhibitors/*metabolism

PURPOSE: Despite the fact that desflurane prolongs the QTC interval in humans, little is known about the mechanisms that underlie these actions. We investigated the effects of desflurane on action potential (AP) duration and underlying electrophysiological mechanisms in rat ventricular myocytes. MATERIALS AND METHODS: Rat ventricular myocytes were enzymatically isolated and studied at room temperature. AP was measured using a current clamp technique. The effects of 6% (0.78 mM) and 12% (1.23 mM) desflurane on transient outward K+ current (I(to)), sustained outward current (I(sus)), inward rectifier K+ current (I(KI)), and L-type Ca2+ current were determined using a whole cell voltage clamp. RESULTS: Desflurane prolonged AP duration, while the amplitude and resting membrane potential remained unchanged. Desflurane at 0.78 mM and 1.23 mM significantly reduced the peak I(to) by 20+/-8% and 32+/-7%, respectively, at +60 mV. Desflurane (1.23 mM) shifted the steady-state inactivation curve in a hyperpolarizing direction and accelerated inactivation of the current. While desflurane (1.23 mM) had no effects on I(sus) and I(KI), it reduced the L-type Ca2+ current by 40+/-6% (p<0.05). CONCLUSION: Clinically relevant concentrations of desflurane appear to prolong AP duration by suppressing Ito in rat ventricular myocytes.
Action Potentials/*drug effects ; Anesthetics, Inhalation/*pharmacology ; Animals ; Calcium Channels, L-Type/physiology ; Heart Conduction System/drug effects/physiology ; Heart Ventricles/drug effects ; Isoflurane/*analogs & derivatives/pharmacology ; Myocardial Contraction/*drug effects/physiology ; Myocytes, Cardiac/*drug effects/physiology ; Patch-Clamp Techniques ; Potassium Channels/physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo establish a perforated patch-clamp technology with amphotericin B and beta-escin and to research the regulation of small conductance calcium-activated potassium channel SK2 currents by calcium ions.

METHODSSingle human atrial myocytes were enzymatically isolated from the right atrial appendage. Amphotericin B and / or beta-escin were used by perforated electrode liquid. The regulation of SK2 current by calcium ions in human atrial myocytes was performed with the perforated patch-clamp technique. The intracellular calcium changes were measured by the intracellular calcium test system.

RESULTSMixed perforated electrode liquid compared with 150 microg/ml amphotericin B or 6.88 microg/ml beta-escin alone, it was easy to seal cells and activate SK2 current by the former method. Moreover, the ration of F340/380 was consistent with the change of intracellular free calcium ion concentration increase after the formation of perforation. The ration of F340/380 was measured by intracellular calcium test system.

CONCLUSIONThe appropriate concentration of amphotericin B mixed with beta-escin can form a stable whole-cell patch recording technology that is appropriate for the research of SK2 current regulation by intracellular calcium.

Amphotericin B ; pharmacology ; Calcium ; metabolism ; Electric Conductivity ; Escin ; pharmacology ; Humans ; Myocytes, Cardiac ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; drug effects