Conformational Locking Induced Enantioselective Diarylcarbene Insertion into B-H and O-H Bonds Using a Cationic Rh(I)/Diene Catalyst.

Transition-metal-catalyzed enantioselective transformations of aryl/aryl carbene are inherently challenging due to the difficulty in distinguishing between two arene rings in the reaction process thus remain largely less explored. The few successful examples reported so far, without exception, have all been catalyzed by Rh(II)-complexes. Herein, we describe our successful development of a novel cationic Rh(I)/chiral diene catalytic system capable of efficient enantioselective B-H and O-H insertions with diaryl diazomethanes, allowing the access to a broad range of gem-diarylmethine boranes and gem-diarylmethine ethers in good yields with high enantioselectivities. Notably, previously unattainable asymmetric diarylcarbene insertion into the O-H bond was achieved for the first time. A remarkable feature of this newly designed Rh(I)/diene catalyst bearing two ortho-amidophenyl substitutents is that it can distinguish between two arene rings of the diaryl carbene through a stereochemically selective control of π-π stacking interactions. DFT calculations indicate that the rotation-restricted conformation of Rh(I)/diene complex played an important role in the highly enantioselective carbene transformations. This work provides an interesting and unprecedented stereocontrol mode in diaryl metal carbene transformations.

Visible-light induced C(sp3)-H arylation of glycine derivatives by cerium catalysis.

A Ce(III)-catalyzed, visible-light induced aerobic oxidative dehydrogenative coupling reaction between glycine derivatives and electron-rich arenes is disclosed. The protocol proceeds efficiently under mild conditions, providing an efficient method for the rapid synthesis of α-arylglycine derivatives without the need for an external photosensitizer and additional oxidant. Moreover, this protocol could be performed on a 5 mmol scale, without obvious reduction of the efficiency.

Optogenetic Control of Mouse Outer Hair Cells.

Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects.

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.

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.

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.

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.

Electrical coupling and release of K+ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery.

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N(omega)-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba(2)(+) (100 microm) and ouabain (1 microm) each attenuated ACh-hyperpolarization by approximately 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba(2)(+) and 18beta-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K(+) (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49 : 1. Ba(2)(+) blocked the K(+)-induced hyperpolarization by approximately 85% in both cell types, whereas ouabain inhibited K(+)-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18beta-Glycyrrhetinic acid blocked the high K(+)-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca(2)(+)-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b) activation of inward rectifier K(+) channels (K(ir)) and Na(+)-K(+) pump current by elevated interstitial K(+) released from the endothelial cells, these being responsible for about 60% and 40% of the hyperpolarization, respectively. The role ratio of K(ir) and pump current activation is at 8 : 1 or less.

Basal nitric oxide production contributes to membrane potential and vasotone regulation of guinea pig in vitro spiral modiolar artery.

Nitric oxide (NO) is a potent vasodilating agent implicated in cochlear blood flow regulation. We recently demonstrated that exogenously applied NO donor DPTA-NONOate hyperpolarizes both endothelial and smooth muscle cells of in vitro spiral modiolar artery (SMA) via activation of ATP-sensitive K+ channels (K(ATP)). Also, NO was detected in the SMA cells by NO indicator dye in the in vitro basal condition. Using intracellular recording techniques, electrochemical NO-sensing measurement, and a vaso-diameter video tracking method, we investigated the basal release of NO from the in vitro SMA and its role in the vascular function. We found that (1) 300 microM L-NAME, a NO synthase inhibitor, and 3 microM glipizide caused a depolarization of approximately 4.5 and approximately 3.2 mV, respectively, in cells with a resting potential less negative than -60 mV; (2) NO sensor in the close vicinity of the SMA detected a NO concentration of approximately 50 nM that was suppressed by L-NAME and enhanced by L-arginine (1-1000 microM); (3) NO donor DPTA-NONOate (0.1-30 microM) applications produced about 8-245 nM of NO in the recording bath. These data indicate a NO concentration-hyperpolarization relation, with an EC50 of 22 nM. (4) Finally, L-NAME but not glipizide produced a 4.8% reduction in SMA diameter (approximately 50 microm) in the majority of SMAs, whereas NONOate (10 microM) always caused a dilation. Both the induced constriction and dilation were not significantly affected by 3 microM glipizide. We conclude that a significant amount of NO (> 50 nM) is tonically released from the in vitro SMA, which is above the EC50 for activation of K(ATP), and thus contributes to the membrane polarization. The basal release of NO also contributes to vasotone relaxation, but the K(ATP) activation appears to play little role in the relaxation of the in vitro SMA.

Nitric oxide induces hyperpolarization by opening ATP-sensitive K(+) channels in guinea pig spiral modiolar artery.

Nitric oxide (NO) hyperpolarizes vascular smooth muscle cells and dilates blood vessels of various beds, but little is known on cochlear vasculatures. Using in vitro preparations of the spiral modiolar artery (SMA), intracellular electrical recording and labeling techniques, we found that the NO donor DPTA-NONOate (10 microM) caused a hyperpolarization of approximately 9 mV in all the cells that had a low resting potential (RP) level near -40 mV. The hyperpolarization amplitude was concentration-dependent, with a 50% effect concentration (EC(50)) of 1 microM. The responses occur in both smooth muscle and endothelial cells, neither of which was blocked by 18beta-glycyrrhetinic acid. The induced hyperpolarization was completely blocked by glipizide, but not by charybdotoxin, apamin, barium, 4-aminopyridine or tetraethylammonium. The hyperpolarizing responses were imitated by pinacidil (EC(50)=30 microM). The pinacidil-induced response was also blocked by glipizide but not by the other K(+) channel blockers mentioned above. Both DPTA-NONOate and pinacidil had little membrane potential effect on cells that had a high RP level near -75 mV. However, when the high RP cells were depolarized to a level beyond -45 mV by barium, both DPTA-NONOate and pinacidil hyperpolarized these cells not differently from those that initially had a low RP. It is concluded that NO hyperpolarizes the SMA primarily by activating K(ATP) channels in both muscle and endothelial cells.