Intracellular zinc protects Kv7 K+ channels from Ca2+/calmodulin-mediated inhibition.

Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7 (KCNQ, M-type) voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca2+/calmodulin (CaM) and zinc-mediated potentiation. We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.

Inhibition of the INa/K and the activation of peak INa contribute to the arrhythmogenic effects of aconitine and mesaconitine in guinea pigs.

Aconitine (ACO), a main active ingredient of Aconitum, is well-known for its cardiotoxicity. However, the mechanisms of toxic action of ACO remain unclear. In the current study, we investigated the cardiac effects of ACO and mesaconitine (MACO), a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs. We showed that intravenous administration of ACO or MACO (25 µg/kg) to guinea pigs caused various types of arrhythmias in electrocardiogram (ECG) recording, including ventricular premature beats (VPB), atrioventricular blockade (AVB), ventricular tachycardia (VT), and ventricular fibrillation (VF). MACO displayed more potent arrhythmogenic effect than ACO. We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes, and observed that treatment with ACO (0.3, 3 µM) or MACO (0.1, 0.3 µM) depolarized the resting membrane potential (RMP) and reduced the action potential amplitude (APA) and durations (APDs) in a concentration-dependent manner. The ACO- and MACO-induced AP remodeling was largely abolished by an INa blocker tetrodotoxin (2 µM) and partly abolished by a specific Na+/K+ pump (NKP) blocker ouabain (0.1 µM). Furthermore, we observed that treatment with ACO or MACO attenuated NKP current (INa/K) and increased peak INa by accelerating the sodium channel activation with the EC50 of 8.36 ± 1.89 and 1.33 ± 0.16 µM, respectively. Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na+ and Ca2+ concentrations. In conclusion, the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak INa via accelerating sodium channel activation and inhibiting the INa/K. These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO, and identify potential novel therapeutic targets for Aconitum poisoning.

Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex.

Kv4 pore-forming subunits co-assemble with ß-subunits including KChIP2 and DPP6 and the resultant complexes conduct cardiac transient outward K+ current (Ito). Compound NS5806 has been shown to potentate Ito in canine cardiomyocytes; however, its effects on Ito in other species yet to be determined. We found that NS5806 inhibited native Ito in a concentration-dependent manner (0.1~30 µM) in both mouse ventricular cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but potentiated Ito in the canine cardiomyocytes. In HEK293 cells co-transfected with cloned Kv4.3 (or Kv4.2) and ß-subunit KChIP2, NS5806 significantly increased the peak current amplitude and slowed the inactivation. In contrast, NS5806 suppressed the current and accelerated inactivation of the channels when cells were co-transfected with Kv4.3 (or Kv4.2), KChIP2 and another ß-subunit, DPP6-L (long isoform). Western blot analysis showed that DPP6-L was dominantly expressed in both mouse ventricular myocardium and hiPSC-CMs, while it was almost undetectable in canine ventricular myocardium. In addition, low level of DPP6-S expression was found in canine heart, whereas levels of KChIP2 expression were comparable among all three species. siRNA knockdown of DPP6 antagonized the Ito inhibition by NS5806 in hiPSC-CMs. Molecular docking simulation suggested that DPP6-L may associate with KChIP2 subunits. Mutations of putative KChIP2-interacting residues of DPP6-L reversed the inhibitory effect of NS5806 into potentiation of the current. We conclude that a pharmacological modulator can elicit opposite regulatory effects on Kv4 channel complex among different species, depending on the presence of distinct ß-subunits. These findings provide novel insight into the molecular design and regulation of cardiac Ito. Since Ito is a potential therapeutic target for treatment of multiple cardiovascular diseases, our data will facilitate the development of new therapeutic Ito modulators.

Local GABAergic signaling within sensory ganglia controls peripheral nociceptive transmission.

The integration of somatosensory information is generally assumed to be a function of the central nervous system (CNS). Here we describe fully functional GABAergic communication within rodent peripheral sensory ganglia and show that it can modulate transmission of pain-related signals from the peripheral sensory nerves to the CNS. We found that sensory neurons express major proteins necessary for GABA synthesis and release and that sensory neurons released GABA in response to depolarization. In vivo focal infusion of GABA or GABA reuptake inhibitor to sensory ganglia dramatically reduced acute peripherally induced nociception and alleviated neuropathic and inflammatory pain. In addition, focal application of GABA receptor antagonists to sensory ganglia triggered or exacerbated peripherally induced nociception. We also demonstrated that chemogenetic or optogenetic depolarization of GABAergic dorsal root ganglion neurons in vivo reduced acute and chronic peripherally induced nociception. Mechanistically, GABA depolarized the majority of sensory neuron somata, yet produced a net inhibitory effect on the nociceptive transmission due to the filtering effect at nociceptive fiber T-junctions. Our findings indicate that peripheral somatosensory ganglia represent a hitherto underappreciated site of somatosensory signal integration and offer a potential target for therapeutic intervention.

Autoregressive spectral analysis of cortical electroencephalographic signals in a rat model of post-traumatic epilepsy.

OBJECTIVES: Post-traumatic epilepsy (PTE) is a common consequence of traumatic brain injury (TBI) and significant predictor of poor prognosis in TBI patients. To develop clinical interventions for PTE risk reduction, there is a need to elucidate the epileptogenic mechanisms induced by brain injury. METHODS: The iron-induced rat model of epilepsy used here mimics many aspects of human PTE. Intracortical injection of iron results in local neuronal damage and the establishment of an epileptic focus, leading to chronic spontaneous electroencephalographic (EEG) signals and motor seizures, with progressively increasing frequency over many months. Identifying unique aspects of PTE seizure semiology for prognosis and treatment may be aided by novel methods of EEG analysis. Here, autoregressive (AR) methods were compared to the conventional fast Fourier transform (FFT) for processing EEG signals in iron-induced epilepsy. RESULTS: Power spectra obtained using AR showed higher frequency resolution over a given epoch than the spectra obtained using FFT. Moreover, changes in total AR spectral power and frequency distribution over brief successive periods provided convenient indexes for long-term monitoring of seizures. DISCUSSION: Autoregression analysis may prove complementary to FFT for EEG analysis in PTE patients.

Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba(2+) 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K(+), while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K(+) typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba(2+)-sensitive inward rectifier K(+) (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.

Development and validation of HTS assay for screening the calcium-activated chloride channel modulators in TMEM16A stably expressed CHO cells.

Calcium-activated chloride channels (CaCCs), for example TMEM16A, are widely expressed in a variety of tissues and are involved in many important physiological functions. We developed and validated an atomic absorption spectroscopy (AAS)-based detection system for high-throughput screening (HTS) of CaCC modulators. With this assay, Cl(-) flux from CHO cells stably transfected with TMEM16A is assayed indirectly, by measuring excess silver ions (Ag(+)) in the supernatant of AgCl precipitates. The screening process involved four steps: (1) TMEM16A CHO cells were incubated in high-K(+) and high-Cl(-) buffer with test compounds, and with ionomycin as Ca(2+) ionophore, for 12 min; (2) cells were washed with a low-K(+), Cl(-)-free and Ca(2+)-free buffer; (3) CaCC/TMEM16A were activated in high-K(+), Cl(-)-free buffer with ionomycin (10 µmol L(-1)) for 12 min; and (4) excess Ag(+) concentration was measured using an ion channel reader (ICR, an AAS system). The assay can be used to screen CaCC activators and inhibitors at the same time. With this assay, positive control drugs, including NPPB, CaCCinh-A01, flufenamic acid (Flu) and Eact, all had good concentration-dependent effects on CaCC/TMEM16A. NPPB and CaCCinh-A01 inhibited the CaCC/TMEM16A currents completely at 300 µmol L(-1), with IC50 values of 39.35 ± 4.72 µmol L(-1) and 6.35 ± 0.27 µmol L(-1), respectively; and Eact, activated CaCC/TMEM16A, with an EC50 value of 3.92 ± 0.87 µmol L(-1).

Two-electrode voltage clamp.

Two-electrode voltage clamp (TEVC) is a conventional electrophysiological technique used to artificially control the membrane potential (V m) of large cells to study the properties of electrogenic membrane proteins, especially ion channels. It makes use of two intracellular electrodes-a voltage electrode as V m sensor and a current electrode for current injection to adjust the V m, thus setting the membrane potential at desired values and recording the membrane current to analyze ion channel activities. Here we describe the use of TEVC in combination with exogenous mRNA expression in Xenopus oocytes for ion channel recording.

Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells.

We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56 µM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥ 300 µM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62 µM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K(+)-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ∼300 µM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K(+)-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300µM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels.

Bumetanide hyperpolarizes madin-darby canine kidney cells and enhances cellular gentamicin uptake by elevating cytosolic Ca(2+) thus facilitating intermediate conductance Ca(2+)--activated potassium channels.

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La(3+), a non-selective cation channel (NSCC) blocker, and by K(+) channel blockers Ba(2+) and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl(-) channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V r) ~-80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba(2+) or clotrimazole, and absent in elevated [Ca(2+)]i, but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca(2+). Ba(2+) and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current V r near -85 mV, and reduced GTTR uptake by ~20 %. La(3+) alone hyperpolarized the cells by ~-14 mV, reduced the I/V slope with a net current V r near -10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La(3+), bumetanide-caused negligible change in potential or I/V. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca(2+) and thus facilitating activation of the intermediate conductance Ca(2+)-activated K(+) channels.

EGF enhances the migration of cancer cells by up-regulation of TRPM7.

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca(2+) from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200µM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.

Different expression of large-conductance calcium-activated K+ channels in human internal mammary and radial arteries.

AIMS: In this study, we investigated and compared the electrophysiological and molecular properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels between human internal mammary arteries (IMA) and radial arteries (RA). METHODS AND RESULTS: IMA and RA sections were obtained from 79 patients (including 9 females) undergoing coronary artery bypass graft surgery. We examined the effects of K(+) channel blockers tetraethylammonium (TEA), iberiotoxin (IBTX), and 4-aminopyridine (4-AP) on isolated smooth muscle cells (SMCs) using patch clamping. Both TEA (1 mM) and IBTX (0.1 µM) significantly decreased K(+) currents in IMA SMCs and RA SMCs, while 4-AP (1 mM) only had a weak effect. IBTX had a greater K(+)-blocking effect on IMA SMCs than on RA SMCs. Consistently, TEA and IBTX evoked significant constriction of both intact vascular rings. IBTX had a greater constrictor effect on IMA rings (18.5 ± 6.7%, n= 8) than on RA rings (10.6 ± 3.1%, n= 8), P< 0.05. RT-PCR and western blot analysis demonstrated that gene and protein expression of the α-subunit of BK(Ca) channels from IMA was greater than that from RA. CONCLUSION: The density of BK(Ca) channels is greater in human IMA than in RA resulting in greater BK(Ca) currents in SMCs from IMA. This may partly explain the finding of less spasm in IMA grafts than in RA grafts. Our results may be of value in determining the best anti-spasm agent to use peri-operatively.

[Effects of acute hypoxia on the electrophysiological properties of vascular smooth muscle cells of mesenteric artery in guinea pig].

OBJECTIVE: To observe the effects of acute hypoxia on the electrophysiological properties of vascular smooth muscle cells (VSMCs) of mesenteric artery in guinea pig. METHODS: A segment of mesenteric artery (MA) (outer diameter < 100 µm) of guinea pig was digested with collagenase A and its adventitial connective tissue cleaned subsequently with fine tweezers. Whole-cell patch clamp recordings were performed to study the effects of acute hypoxia on the whole-cell membrane current, resting membrane potential (RP), membrane input capacitance (C(input)), and membrane input resistance (R(input) or its reciprocal membrane input conductance G(input)) of VSMC embedded in arteriolar segment. RESULTS: Acute hypoxia induced an outward current with an amplitude of (76 ± 23) pA at holding potential -40 mV and hyperpolarized VSMC from a RP of (-22.5 ± 1.2) mV to (-42.0 ± 2.8) mV (P < 0.01). Acute hypoxia increased the outward current of VSMC in a voltage-dependent manner. And this enhancement was more pronounced at potentials from 0 to +40 mV. The whole-cell membrane current of VSMC induced by step commands (0, +20 and +40 mV) increased from (140 ± 18) pA to (660 ± 124) pA (P < 0.01), (282 ± 23) pA to (1120 ± 186) pA (P < 0.01) and (423 ± 40) pA to (1800 ± 275) pA (P < 0.01) respectively. In the presence of 1 mmol/L tetraethylammonium (TEA, a large conductance Ca(2+)-activated K(+) channel blocker), the enhancement of VSMC membrane current by acute hypoxia was significantly reduced. Acute hypoxia increased the R(input) of VSMC in MA from (446 ± 55) MΩ to (2187 ± 290) MΩ (P < 0.01) and decreased the C(input) from (184.3 ± 75.0) pF to (17.6 ± 2.2) pF (P < 0.01). In the presence of 30 µmol/L 18ß-glycyrrhetinic acid (18ßGA, a gap junction blocker) and 10 mmol/L TEA, the effects of acute hypoxia on the membrane current of VSMCs were almost abolished. CONCLUSION: Acute hypoxia causes vascular hyperpolarization and vasodilation by activating large conductance Ca(2+)-activated K(+) channels of VSMC and inhibits gap junctions between VSMCs so as to improve microcirculation and localize hypoxia-induced damage.

2-Aminoethoxydiphenyl borate blocks electrical coupling and inhibits voltage-gated K+ channels in guinea pig arteriole cells.

2-Aminoethoxydiphenyl borate (2-APB) analogs are potentially better vascular gap junction blockers than others widely used, but they remain to be characterized. Using whole cell and intracellular recording techniques, we studied the actions of 2-APB and its potent analog diphenylborinic anhydride (DPBA) on vascular smooth muscle cells (VSMCs) and endothelial cells in situ of or dissociated from arteriolar segments of the cochlear spiral modiolar artery, brain artery, and mesenteric artery. We found that both 2-APB and DPBA reversibly suppressed the input conductance (G(input)) of in situ VSMCs (IC(50) ≈ 4-8 µM). Complete electrical isolation of the recorded VSMC was achieved at 100 µM. A similar gap junction blockade was observed in endothelial cell tubules of the spiral modiolar artery. Similar to the action of 18ß-glycyrrhetinic acid (18ß-GA), 2-APB and DPBA depolarized VSMCs. In dissociated VSMCs, 2-APB and DPBA inhibited the delayed rectifier K(+) current (I(K)) with an IC(50) of ∼120 µM in the three vessels but with no significant effect on G(input) or the current-voltage relation between -140 and -40 mV. 2-APB inhibition of I(K) was more pronounced at potentials of ≤20 mV than at +40 mV and more marked on the fast component than on the slow component, which was mimicked by 4-aminopyridine but not by tetraethylammonium, nitrendipine, or charybdotoxin. In contrast, 18ß-GA caused a linear inhibition of I(K) between 0 to +40 mV, which was similar to the action of tetraethylammonium or charybdotoxin. Finally, the 2-APB-induced inhibition of electrical coupling and I(K) was not affected by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C. We conclude that 2-APB analogs are a class of potent and reversible vascular gap junction blockers with a weak side effect of voltage-gated K(+) channel inhibition. They could be gap junction blockers superior to 18ß-GA only when Ca(2+)-actived K(+) channel inhibition by the latter is a concern but inositol 1,4,5-trisphosphate receptor and voltage-gated K(+) channel inhibitions are not.

Effects of Ginkgo biloba extracts on NMDA-activated currents in acutely isolated hippocampal neurons of the rat.

Ginkgo biloba extracts (GBE) have long been used as a traditional herbal medicine for treating central nervous system diseases and peripheral vascular diseases, but the underlying mechanisms have yet to be elucidated. Furthermore, traditional GBE is in the form of microsomes and only dissolves in organic solvents; its clinical applications have been greatly limited. Therefore, in the present study, nanometer GBE (nGBE) was prepared utilizing supercritical anti-solvent (SAS) upon CO(2) -supercritical fluid extraction (CO(2) -SPF). Using whole-cell patch clamp techniques, the effects of different preparations of GBE on N-methyl-D-aspartate (NMDA)-activated currents (I(NMDA) ) from acutely isolated rat hippocampal neurons were investigated and the difference in protective potency between nGBE and mGBE evaluated. The results showed that the inward current activated by NMDA could be depressed by mGBE and nGBE. The inhibitory rates were 40% ± 17% and 64% ± 15%, and the half-inhibition concentrations (IC(50) ) were 0.0210 ± 0.0055 and 0.0262 ± 0.0038 mg/mL, respectively. In comparison, the modulatory effect of nGBE (dissolved in extracellular solution) on NMDA-activated current was significantly greater than that of mGBE (dissolved in DMSO) (p < 0.05). This indicated that the modulatory effects of GBE on NMDA-activated current may contribute to the neuroprotective effects of GBE and the modulatory effect of nGBE on NMDA-activated current was greater than that of mGBE.

ACh-induced depolarization in inner ear artery is generated by activation of a TRP-like non-selective cation conductance and inactivation of a potassium conductance.

Adequate cochlear blood supply by the spiral modiolar artery (SMA) is critical for normal hearing. ACh may play a role in neuroregulation of the SMA but several key issues including its membrane action mechanisms remain poorly understood. Besides its well-known endothelium-dependent hyperpolarizing action, ACh can induce a depolarization in vascular cells. Using intracellular and whole-cell recording techniques on cells in guinea pig in vitro SMA, we studied the ionic mechanism underlying the ACh-depolarization and found that: (1) ACh induced a DAMP-sensitive depolarization when intermediate conductance KCa channels were blocked by charybdotoxin or nitrendipine. The ACh-depolarization was associated with a decrease in input resistance (R(input)) in high membrane potential (V(m)) ( approximately -40 mV) cells but with no change or an increase in R input in low Vm ( approximately -75 mV) cells. ACh-depolarization was attenuated by background membrane depolarization from approximately -70 mV in the majority of cells; (2) ACh-induced inward current in smooth muscle cells embedded in a SMA segment often showed a U-shaped I/V curve, the reversal potential of its two arms being near EK and 0 mV, respectively; (3) ACh-depolarization was reduced by low Na+, zero K+ or 20mM K+ bath solutions; (4) ACh-depolarization was inhibited by La3+ in all cells tested, by 4-AP and flufenamic acid in low Vm cells, but was not sensitive to Cd2+, Ni2+, nifedipine, niflumic acid, DIDS, IAA94, linopirdine or amiloride. We conclude that ACh-induced vascular depolarization was generated mainly by activation of a TRP-like non-selective cation channel and by inactivation of an inward rectifier K+ channel.

Dihydropyridines inhibit acetylcholine-induced hyperpolarization in cochlear artery via blockade of intermediate-conductance calcium-activated potassium channels.

Acetylcholine (ACh) induces hyperpolarization and dilation in a variety of blood vessels, including the cochlear spiral modiolar artery (SMA) via the endothelium-derived hyperpolarization factor (EDHF). We demonstrated previously that the ACh-induced hyperpolarization in the SMA originated in the endothelial cells (ECs) by activating a Ca(2+)-activated K(+) channel (K(Ca)); the hyperpolarization in smooth muscle cells was mainly an electrotonic spread via gap junction coupling. In the present study, using intracellular recording, immunohistology, and vascular diameter tracking techniques on in vitro SMA preparations, we found that 1) ACh-induced hyperpolarization was suppressed by intermediate-conductance K(Ca) (IK) blockers clotrimazole (IC(50) = 116 nM) and nitrendipine and by the calmodulin antagonist trifluoperazine, but it was not suppressed by the big-conductance K(Ca) blocker iberiotoxin. The immunoreactivity to anti-SK4/IK1 antibody was localized mainly in ECs. 2) The three dihydropyridines--nifedipine, nitrendipine, and nimodipine--all concentration-dependently inhibited the ACh-induced hyperpolarization, with an IC(50) value of 455, 34, and 3.2 nM, respectively. 3) Among other L-type Ca(2+) channel (I(L)) blockers, 10 microM verapamil exerted a 20% inhibition on ACh-induced hyperpolarization, whereas diltiazem and the metal ion Ca(2+) channel blockers Cd(2+) and Ni(2+) had no effect. 4) Nitrendipine and charybdotoxin abolished ACh-induced dilation in the SMA. We conclude that ACh-induced hyperpolarization in the SMA is generated mainly by activation of the IK in the ECs, and dihydropyridines suppress the EDHF-mediated hyperpolarization by blocking the IK channel, not the I(L) channel. The clinical relevance of this dihydropyridine action is discussed.

Characteristics of P2X purinoceptors in the membrane of rat trigeminal ganglion neurons.

The characteristics of purinoceptors in the membrane of rat trigeminal ganglion (TG) neurons were studied by using whole- cell patch clamp technique. The results showed that most of neurons examined (78.9%, 142/180) were responsive to ATP in a concentration-dependent manner; the others (21.1%, 38/180) were ATP insensitive. Of the ATP-sensitive cells, the majority (95.1%, 135/142) responded to ATP with an inward current, a few (2.1%, 3/142) with an outward current, and the rest (2.8%, 4/142) with biphasic current. Small sized cells (<30 mum) responded to ATP with a rapid desensitizing inward current and were highly sensitive to vanilloid; the medium sized cells (30~40 mum) responded to ATP with slow desensitizing inward current and were not sensitive to vanilloid; while the majority of large sized cells (>40 mum) did not respond to ATP and vanilloid. The waveform of ATP-activated inward currents was related to the cell diameter. The I-V curves for both small and medium sized cells manifested obvious inward rectification. Furthermore, we studied the kinetic features of ATP-activated currents and the effects of P2 purinoceptor agonists and antagonists on I(ATP). The findings suggest that ATP receptor-ion channels are expressed differently among different types of rat TG neurons.

Effects of dopamine, SKF-38393 and R(-)-NPA on ATP-activated currents in rat DRG neurons.

This study aimed to investigate the effect of the activation of dopamine (DA) receptors on ATP-activated currents (IATP) in freshly isolated dorsal root ganglion (DRG) neurons of rats using whole-cell patch clamp technique in combination with intracellular dialysis. Extracellular application of DA inhibited IATP in half of the neurons tested (39/77, 50.6%), enhanced IATP in a small subset of the neurons (22/77, 28.6%), and had no effect on IATP in the rest (16/77, 20.8%). To investigate the DA receptor subtypes that mediate these modulations, the effects of R(-)-NPA, a D2 receptor agonist, and SKF-38393, a D1 receptor agonist, were examined. Preapplication of R(-)-NPA inhibited IATP in most of the cells tested (53/57, 93.0%) and had no effect in the rest (4/57, 7.0%); no potentiating effect was observed. Preapplication of SKF-38393 inhibited IATP in a majority of the cells tested (57/77, 74.0%), potentiated IATP in some cells (12/77, 15.6%), and had no effect in the remainder (8/77, 10.4%). Further study of the inhibitory effect of R(-)-NPA and SKF-38393 revealed that both of them acted in a noncompetitive manner, shifting the concentration-response curve for IATP downwards with the maximal response markedly reduced and EC50 basically unchanged; and the inhibition was independent of the holding potential. Intracellular dialysis of GDP-beta-S and H-7 abolished R(-)-NPA inhibition of IATP completely, and SKF-38393 inhibition of IATP was removed by intracellular application of H-7 but not by H-9. These results suggest that the activation of DA receptors dominantly inhibits IATP in dorsal root ganglion cells, and this inhibition may be involved in the modulation of afferent information by the diencephalon-derived DA in the primary sensory neurons.

5-HT2 receptor mediated the potentiation of GABA-activated current in the membrane of the dorsal root ganglion neurons of rat.

AIM: To explore the modulation of 5-HT on GABA-activated current (I(GABA)) in the membrane of rat dorsal root ganglion (DRG) neurons and its mechanism. METHODS: Rat DRG neurons were isolated mechanically and enzymatically, on which whole-cell patch clamp recording and repatch technique for intracellular dialysis were performed. RESULTS: In the majority of neurons examined (92.0%, 69/75) GABA induced a concentration-dependent inward current. In neurons sensitive to GABA preapplication of 5-HT produced potentiation effect (82.6% , 57/69) on I(GABA). Preapplication of 5-HT at concentrations of 1 x 10(-6), 1 x 10(-5), 1 x 10(-4) and 1 x 10(-3) mol x L(-1) potentiated I(GABA) by (35 +/- 8)% (n=8), (47 +/- 11)% (n=10), (65 +/- 17)% (n=9) and (75 +/- 18)% (n=11), respectively. This effect was mimicked by alpha-methyl-5-HT (1 x 10(-6) mol x L(-1)), a specific 5-HT2 receptor agonist, and reversed by cyproheptadine, a selective 5-HT2 receptor antagonist. The potentiation of I(GABA) by 5-HT was irrespective to whether the I(5-HT) presents or not in a subset of neurons. The concentration-response curves for GABA before and after pretreatment with 5-HT manifested the same threshold value and similar EC50 (2.0 x 10(-5) and 1.9 x 10(-5) mol x L(-1), respectively) , while the maximal value of I(GABA) for the latter was 33.6% higher than that for the former. Intracellular dialysis with GDP-beta-S or H-7 abolished the potentiation of I(GABA) by 5-HT, while H-9 did not. CONCLUSION: 5-HT can potentiate GABA-activated current via PKC-dependent phosphorylation of GABA(A) receptor following the activation of 5-HT2 receptor.