In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01–10 μm), in the presence of indomethacin (2.8 μM) and N ω-nitro-l-arginine methyl ester (l-NAME) (100 μM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation ( n = 5). In pressurized arteries, ACh (10 μM), applied intraluminally in the presence of indomethacin (2.8 μM) andl-NAME (100 μM), dilated both PE-stimulated (0.3–0.5 μM; n = 5) and myogenic tone ( n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated ( n = 5) or myogenic tone ( n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation.
] o dilate/relax rat mesenteric arteries, though sensitivities to barium and ouabain dier. K + may be a relaxing factor in this tissue, but its characteristics dier from EDHF. Gap junction inhibitors depress EDHF, implying an important role for myo-endothelial gap junctions.
The effects of chloride channel blockers on pressure‐induced constriction, K+‐induced force, and whole‐cell calcium channel currents were tested in rat cerebral arteries using isobaric and isometric myography, and patch clamp. Under isobaric conditions at 75 mmHg, NPPB), a chloride channel blocker, reversibly depressed the myogenic constriction with an IC50 of 32.8 ± 0.52 μm (mean ± s.e.m., n= 5). Blockers of Ca2+‐activated chloride channels, flufenamic acid (100 μm) and 9‐AC; 1 mm), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel blocker, glibenclamide (100 μm), were without effect in this tissue (n= 3). Under isobaric conditions at 20 mmHg, 37 °C, raising [K+]o to 45 mm induced a constriction which was unaffected by 100 μm NPPB (n= 4). In contrast, at 75 mmHg and 18‐21 °C, 100 μm NPPB completely and reversibly blocked a 45 mm K+‐induced constriction (n= 3). Under isometric conditions, NPPB reversibly depressed a 45 mm K+‐induced force with an IC50 of 10.0 ± 0.76 μm (mean ± s.e.m., n= 5). IAA‐94), another chloride channel blocker, depressed the K+‐induced force with an IC50 of 17.0 ± 1.2 μm (mean ± s.e.m., n= 4). Using whole‐cell patch clamp, 100 μm NPPB or 200 μm IAA‐94 blocked calcium channel currents carried by 10 mm Ba2+ by 79.1 ± 1.7 and 39.8 ± 7.0 %, respectively (mean ± s.e.m., n= 6). In summary, chloride channel blockers depress calcium channel currents in rat cerebral arteries, which could contribute to a reduction in myogenic contraction.
Whole‐cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB). Both high voltage‐activated (HVA) and low voltage‐activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice. The transient LVA calcium current activated rapidly on depolarization (half‐activation, −59 mV) and inactivated during maintained depolarization (half‐inactivation, −89 mV). This T‐type current was observed in somatic recordings but was absent from presynaptic terminals. On the basis of their pharmacological sensitivity, P/Q‐type Ca2+ channels accounted for only 6 % of the somatic HVA, while L‐, N‐ and R‐type Ca2+ channels each accounted for around one‐third of the somatic calcium current. The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current. We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P‐type contribute little to somatic currents but predominate at the synaptic terminal; L‐, N‐ and R‐types dominate at the soma, but contribute negligibly to calcium currents in the terminal.
1 Raised extracellular K + relaxes some arteries, and has been proposed as Endothelium-Derived Hyperpolarizing Factor (EDHF). However, relaxation of rat small mesenteric arteries to K + is highly variable. We have investigated the mechanism of K + -induced dilatation and relaxation of pressurized arteries and arteries mounted for measurement of isometric force. , 10 mM acetycholine still relaxed all arteries. 5 Fifty mM 18a-glycyrrhetinic acid (18a-GA), a gap junction inhibitor, depressed relaxations to both 10 mM acetylcholine and raised [K + ] o , in the presence of 10 mM NPPB. 6 In summary, blockade of a volume-sensitive Cl 7 conductance in small rat mesenteric arteries, using NPPB or hyperosmotic superfusion, reveals a endothelium-dependent, Ba 2+ sensitive dilatation or relaxation of rat mesenteric arteries to raised [K + ] o . We conclude that inwardly rectifying potassium channels on the endothelium underlie relaxations to raised [K + ] o in rat small mesenteric arteries.
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