ACh-induced Hyperpolarization Research Articles

Neuron-specific responses to acetylcholine within the spinal dorsal horn circuits of rodent and primate

Excitatory and inhibitory neurotransmission within the spinal dorsal horn is tightly controlled to regulate transmission of nociceptive signals to the brain. One aspect of this control is modulation of neuronal activity through cholinergic signaling. Nociceptive neurons in the dorsal horn express both nicotinic and muscarinic cholinergic receptors and activation of these receptors reduces pain in humans, while inhibition leads to nociceptive hypersensitivity. At a cellular level, acetylcholine (ACh) has diverse effects on excitability which is dependent on the receptor and neuronal subtypes involved. In the present study we sought to characterize the electrophysiological responses of specific subsets of lamina II interneurons from rat and marmoset spinal cord. Neurons were grouped by morphology and by action potential firing properties. Whole-cell voltage-clamp recordings from lamina II dorsal horn neurons of adult rats showed that bath applied acetylcholine increased, decreased or had no effect on spontaneous synaptic current activity in a cell-type specific manner. ACh modulated inhibitory synaptic activity in 80% of neurons, whereas excitatory synaptic activity was affected in less than 50% of neurons. In whole-cell current clamp recordings, brief somatic application of ACh induced cell-type specific responses in 79% of rat lamina II neurons, which included: depolarization and action potential firing, subthreshold membrane depolarization, biphasic responses characterized by transient depolarization followed by hyperpolarization and membrane hyperpolarization alone. Similar responses were seen in marmoset lamina II neurons and the properties of each neuron group were consistent across species. ACh-induced hyperpolarization was blocked by the muscarinic antagonist atropine and all forms of acetylcholine-induced depolarization were blocked by the nicotinic antagonist mecamylamine. The cholinergic system plays an important role in regulating nociception and this study contributes to our understanding of how circuit activity is controlled by ACh at a cellular level in primate and rodent spinal cord.

Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+]i), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca2+ ionophore, ionomycin, and the NO donor, S-nitroso- N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca2+]i induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca2+-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9-/-) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9-/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted efferent mechanism for altering hair cell membrane potential and decreasing membrane resistance that should reduce sensitivity to hair bundle displacements.

Involvement of Hydrogen Sulfide in Endothelium-Derived Relaxing Factor-Mediated Responses in Rat Cerebral Arteries

Background/Aim: H₂S is a novel vasoactivator. To verify the hypothesis that H₂S may act as an endothelium-derived hyperpolarizing factor (EDHF) in the rat cerebrovasculature, the role of H₂S in endothelium-derived relaxing factor (EDRF)-mediated responses was investigated. Methods: Cystathionine-γ-lyase (CSE) was knocked down with an siRNA technique. Artery diameter, hyperpolarization and Ca²⁺-activated K⁺ (K_Ca) current were measured. Results: CSE knockdown was indicated by a decrease in protein and mRNA expression in the rat middle cerebral artery (MCA) and cerebral basilar artery (CBA). Acetylcholine (ACh) induced significant hyperpolarization and vasodilation in endothelium-intact MCA and CBA. Removal of the endothelium abolished these responses. The nitric oxide (NO) synthase inhibitor <smlcap>L</smlcap>-NAME, but not the PGI₂ production inhibitor indomethacin, significantly inhibited ACh-induced hyperpolarization and vasodilation in the CBA. In the presence of <smlcap>L</smlcap>-NAME and indomethacin, ACh-induced hyperpolarization and vasodilation in the MCA and CBA were attenuated. The non-NO/PGI₂-mediated responses were abolished by the K_Ca channel blockers charybdotoxin and apamin. In the cerebral arteries from the CSE knockdown rat, non-NO/PGI₂-mediated responses were significantly attenuated, and the remaining responses were abolished by charybdotoxin and apamin or the CSE inhibitor propargylglycine. CSE knockdown did not affect <smlcap>L</smlcap>-NAME-sensitive responses in the CBA. Sodium hydrosulfide (NaHS) augmented the K_Ca current in CBA vascular smooth muscle cells. Conclusion: EDHF-mediated responses in rat cerebral arteries were due to H₂S activating the K_Ca channel.

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca 2+ ([Ca 2+] i) and Na + concentrations ([Na +] i) in MPG neurons. The recovery of [Na +] i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na +/K + ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% ( n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca 2+] i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca 2+-activated K + channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na +] i and [Ca 2+] i, which activates Na +/K + ATPase and Ca 2+-activated K + channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.

CILOSTAMIDE PRODUCES HYPERPOLARIZATION ASSOCIATED WITH KATP CHANNEL ACTIVATION, BUT DOES NOT AUGMENT ENDOTHELIUM‐DERIVED HYPERPOLARIZATION IN RAT MESENTERIC ARTERIES

1. Non-nitric oxide/prostaglandin-mediated endothelium-derived hyperpolarization (EDH) is considered to be mediated, in part, by gap junctions and it has been suggested that cAMP increases endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation through the modulation of gap junctions. Cilostamide, which inhibits phosphodiesterase III, has been suggested to augment EDHF-type relaxation by increasing the concentration of cAMP. 2. In the present study, we investigated the effect of cilostamide on EDH per se in mesenteric arteries of Wistar rats using a conventional microelectrode technique. 3. The resting membrane potential of the mesenteric arteries was significantly more negative in the presence of 10(-6) mol/L cilostamide compared with control conditions. Furthermore, EDH in response to 10(-6) mol/L acetylcholine (ACh) in the presence of 10(-5) mol/L indomethacin and 10(-4) mol/L N(G)-nitro-L-arginine was decreased in the presence of 10(-6) mol/L cilostamide by approximately 5 and 3.5 mV in proximal and distal arteries, respectively. 4. Glibenclamide (10(-5) mol/L), an ATP-sensitive potassium channel (K(ATP)) inhibitor, abolished the hyperpolarization to 10(-6) mol/L cilostamide. Furthermore, in the presence of glibenclamide, ACh-induced EDH was unaffected by cilostamide, suggesting that the inhibition of ACh-induced hyperpolarization by cilostamide in the absence of glibenclamide may be due to the smaller driving force for hyperpolarization because of the more negative membrane potential under such conditions. 5. The findings of the present study suggest that cilostamide produces hyperpolarization by activating K(ATP) channels, presumably by increasing cAMP. However, cilostamide alone may not directly affect EDH.

A role for nitroxyl (HNO) as an endothelium‐derived relaxing and hyperpolarizing factor in resistance arteries

Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K(+) channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, L-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with L-NAME, N(omega)-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO(*)) with hydroxocobalamin (HXC) or HNO with L-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and L-cysteine reduced ACh-mediated relaxation, as did L-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with L-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO(*), as HXC and L-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO(*) = HNO > EDHF and EDHF > HNO = NO(*) respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels.

The shy Angeli and his elusive creature: the HNO route to vasodilation

what we now call Angeli's salt (AS) was discovered more than a century ago by the Italian chemist Angelo Angeli (Fig. 1) . He found that “salts of nitroxylaminic acid are readily resolved into the corresponding nitrites and the unsaturated residue nitroxyl” (HNO), proposing their use as suppliers of HNO (3). However, by analogy with the genial but very reclusive personality of the discoverer (17), the physiological properties of HNO didn't draw too much attention until early 1990s. In their seminal work, Fukuto and colleagues (19) reported that HNO elicits vasorelaxation in both the rabbit aorta and bovine intrapulmonary artery by a soluble guanylate cyclase (sGC)- dependent pathway. Later, it was evident that the thiol-donating agent l-cysteine can discriminate the vasodilatative profile of HNO from that of nitric oxide (NO·) or nitrosothiols: AS/HNO action can be blocked by this agent, whereas the NO· effect is potentiated (36). In combination with the suggestion that NO· is the only species able to directly activate sGC (9), these data led to the conclusion that HNO-induced vasorelaxation is due to its intracellular conversion to NO· with subsequent activation of sGC. For several years, this idea has relegated HNO to a mere precursor of the presence and biological activity of NO·, de facto negating to HNO its own right to exist.

Properties of acetylcholine-induced hyperpolarization in smooth muscle cells of the mouse mesenteric artery

The properties of smooth muscle cell hyperpolarization produced by acetylcholine (ACh) were investigated in mesenteric arteries isolated from mice. The resting membrane potential of the smooth muscle cells was about -60 mV. When ACh (10 microM) was applied for 1 min, the membrane hyperpolarized with a peak amplitude of about 5 mV which was reached in about 1 min, following which the potential slowly reverted to the resting level over about 7 min following withdrawal of ACh from the superfusate (recovery component). Exposure of the artery to 0.5 mM Ba(2+), an inhibitor of inward rectifier K-channels, depolarized the membrane by about 13 mV, increased the amplitude of the ACh-induced hyperpolarization to about 10 mV, and facilitated the visualization of the recovery component. Indomethacine (10 microM), an inhibitor of cyclooxygenase, inhibited the recovery component and as a consequence reduced the duration of the hyperpolarization. The ACh-induced response was not markedly altered by either N(omega)-nitro-L-arginine (10 microM), an inhibitor of nitric oxide (NO) production, or catalase (130 U/ml), a super oxide scavenger. Exogenously applied hydrogen peroxide (H(2)O(2), 300 microM) hyperpolarized the membrane by about 5 mV, which was abolished by catalase. These results suggest that in the mouse mesenteric artery, the ACh-induced hyperpolarization has two components, both an indomethacin-sensitive and an indomethacin-insensitive component. The former component may be produced by prostanoids, while the latter may be produced by factors other than NO or H(2)O(2). The results also suggested that the inward rectifier K-channels may be important for producing the resting membrane potential, but they may not be the main contributor to the ACh-induced hyperpolarization of smooth muscle cell membranes in the mouse mesenteric artery.

Effects of Increased Intracellular Cl− Concentration on Membrane Responses to Acetylcholine in the Isolated Endothelium of Guinea Pig Mesenteric Arteries

ACh-induced membrane responses in vascular endothelial cells that have been reported vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization; the reason for this variation is unknown. Using the perforated whole-cell clamp technique, we investigated ACh-induced membrane currents in freshly isolated endothelial layers having a resting membrane potential of less negative than -10 mV. A group of cells was electrically isolated using a wide-bore micropipette, and their membrane potential was well controlled. ACh activated K(+) and Cl(-) currents simultaneously. The K(+) current was blocked by a combination of charybdotoxin and apamin and appears to result from the opening of IK(Ca) and SK(Ca) channels. The Cl(-) current was partially blocked by tamoxifen, niflumic acid, or DIDS and appears to be produced by Ca(2+)-activated Cl(-) channels. When the pipettes contained 20 mM Cl(-), the ACh-induced K(+) conductance started decreasing during a 1-min application of ACh while the Cl(-) conductance continued, making the ACh-induced hyperpolarization sustained. When the pipettes contained 150 mM Cl(-), both conductances started decreasing during a 1-min application of ACh, making the ACh-induced hyperpolarization small and transient. [Cl(-)](i) is very likely modified by experimental procedures such as the cell isolation and the intracellular dialysis with the pipette solution. Such a variability in [Cl(-)](i) may be one of the reasons for the variations in the ACh-induced membrane response.

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.

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na(+),K(+)-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na(+),K(+)-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na(+),K(+)-ATPase were used. Dose-response curves for ouabain, a specific Na(+),K(+)-ATPase inhibitor, were obtained to ascertain which Na(+),K(+)-ATPase isoform(s) is involved. The ACh dose-response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na(+),K(+)-ATPase was studied in purified lamb kidney Na(+),K(+)-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably alpha2) isoform of Na(+),K(+)-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na(+),K(+)-ATPase. Possible interaction mechanisms are discussed.

Inward rectifier K + ‐channel and electrical coupling form the conduction mechanism of the EDHF‐induced arterial hyperpolarization

Focal hyperpolarization and dilation induced by EDHF-releasing agent (e.g., ACh) can conduct along the vessel but the cellular mechanism for such conduction remains unclear. Using intracellular or whole-cell recording combining with cell labeling techniques on guinea pig in vitro spiral modiolar artery, we found: 1) The initial resting potentials (RP) in sampled cells showed a bimodal distribution peaked near −75 (high RP) and −40 mV (low RP). A cell initially having a low RP may swiftly shift to high RP state and vice versa. 2) Ba2+ (1–100 μM) always caused a shift from high RP to low RP by its wash-in and a reversed shift by its wash-out. An overshot was often seen in the end of fast shift in both directions. Whole-cell current showed an inward rectification that was sensitive to Ba2+. 3) ACh-induced a robust hyperpolarization or outward current at ~−40 mV, that was blocked by a gap junction blocker 18β-glycyrrhetinic acid plus Ba2+in smooth muscle (SMC), but not in endothelial cells (EC). 4) Data of dual cell recording, GRA effects on K+-induced hyperpolarization and cell labeling indicated a heterogeneous electrocoupling among cells and a macroscopic myoendothelial coupling efficiency of 0.49. We conclude that the vascular SMCs express abundant inward rectifier K+-channels (Kir); the ACh-induced hyperpolarization originates in the EC, electrotonically spreads to the muscular layer and in turn activates the Kir in the SMCs; the intercellular electrocoupling and the positive feed back by the loop of hyperpolarization→Kir disinhibition→further hyperpolarization in muscle cells form the cellular basis for the conductive inhibition. Supported by grant of NIH NIDCD DC004716.

Reduced hyperpolarization in endothelial cells of rabbit aortic valve following chronic nitroglycerine administration

This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the hyperpolarization induced by acetylcholine (ACh) in aortic valve endothelial cells (AVECs) of the rabbit and, if so, whether antioxidant agents can normalize this downregulated hyperpolarization. ACh (0.03-3 microM) induced a hyperpolarization through activations of both apamin- and charybdotoxin-sensitive Ca2+-activated K+ channels (K(Ca)) in rabbit AVECs. The intermediate-conductance K(Ca) channel (IK(Ca)) activator 1-ethyl-2-benzimidazolinone (1-EBIO, 0.3 mM) induced a hyperpolarization of the same magnitude as ACh (3 microM). The ACh-induced hyperpolarization was significantly weaker, although the ACh-induced [Ca2+]i increase was unchanged, in NTG-treated rabbits (versus NTG-untreated control rabbits). The hyperpolarization induced by 1-EBIO was also weaker in NTG-treated rabbits. The reduced ACh-induced hyperpolarization seen in NTG-treated rabbits was not modified by in vitro application of the superoxide scavengers Mn-TBAP, tiron or ascorbate, but it was normalized when ascorbate was coadministered with NTG in vivo. Superoxide production within the endothelial cell (estimated by ethidium fluorescence) was increased in NTG-treated rabbits and this increased production was normalized by in vivo coadministration of ascorbate with the NTG. It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced hyperpolarization in rabbit AVECs, possibly through chronic actions mediated by superoxide.

Myoendothelial Coupling Is Not Prominent in Arterioles Within the Mouse Cremaster Microcirculation In Vivo

A smooth muscle hyperpolarization is essential for endothelium-dependent hyperpolarizing factor-mediated dilations. It is debated whether the hyperpolarization is induced by a factor (endothelium-derived hyperpolarizing factor) and/or is attributable to direct current transfer from the endothelium via myoendothelial gap junctions. Here, we measured membrane potential in endothelial cells (EC) and smooth muscle cells (SMC) in vivo at rest and during acetylcholine (ACh) application in the cremaster microcirculation of mice using sharp microelectrodes before and after application of specific blockers of Ca2+-dependent K+ channels (K(Ca)). Moreover, diameter changes in response to ACh were studied. Membrane potential at rest was lower in EC than SMC (-46.6+/-1.0 versus -36.5+/-1.0mV, P<0.05). Bolus application of ACh induced robust hyperpolarizations in EC and SMC, but the amplitude (11.1+/-0.9 versus 5.1+/-0.9mV, P<0.05) and duration of the response (10.7+/-0.8 versus 7.5+/-1.0s, P<0.05) were larger in EC. Blockers of large conductance K(Ca) (charybdotoxin or iberiotoxin) abrogated ACh-induced hyperpolarizations in SMC but did not alter endothelial hyperpolarizations. In contrast, apamin, a blocker of small conductance K(Ca) abolished ACh-induced hyperpolarizations in EC and had only small effects on SMC. ACh-induced dilations were strongly attenuated by iberiotoxin but only slightly by apamin. We conclude that myoendothelial coupling in arterioles in vivo in the murine cremaster is weak, as EC and SMC behaved electrically different. Small conductance K(Ca) mediate endothelial hyperpolarization in response to ACh, whereas large conductance K(Ca) are important in SMC. Because tight myoendothelial coupling was found in vitro in previous studies, we suggest that it is differentially regulated between vascular beds and/or by mechanisms acting in vivo.

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.

Dependency of endothelial cell function on vascular smooth muscle cells in guinea-pig mesenteric arteries and arterioles

Using guinea-pig mesenteric arteries and arterioles, we investigated the membrane potential of endothelial cells at rest and during application of acetylcholine (ACh) with and without the smooth muscle layers attached. When smooth muscle and endothelial layers were in close apposition, the resting membrane potentials of the two types of cells were closely related and were slightly more negative in the smooth muscle cells than in the endothelial cells. Once the endothelial layer was separated from the smooth muscle layer, the endothelial cells depolarized (the average, -4.2 mV). In the isolated endothelial layer, ACh did not induce a membrane hyperpolarization as expected, but did induce a quick depolarization soon after conventional whole-cell recording was started. However, as the pipette solution (high K+) gradually diffused into the endothelial layer, the membrane response to ACh gradually changed toward hyperpolarization. ACh-induced hyperpolarization was also observed after incubating preparations in a high-potassium bath solution. Our results indicate that vascular smooth muscle cells and endothelial cells are influencing each other as a functional unit and that the endothelial cells rely on the smooth muscle cells for their intracellular ionic composition and resting membrane potential.

Effects of insulin on the acetylcholine-induced hyperpolarization in the guinea pig mesenteric arterioles

Background: Insulin induces endothelium-dependent vasodilatation, which may be casually related to the insulin resistance and hypertension. Endothelium-derived nitric oxide (NO) is the most important mechanism of insulin-induced vasodilatation, and a possible contribution of endothelium-derived hyperpolarizing factor (EDHF) is also considered. Attempts were made to observe the effects of insulin on acetylcholine (ACh)-induced hyperpolarization in the submucosal arteriole of the guinea pig ileum, the objective being to investigate possible involvement of EDHF in the actions of insulin. Methods: Conventional microelectrode techniques were applied to measure the membrane potential of smooth muscle cells in the submucosal arteriole. EDHF-induced hyperpolarization was elicited by ACh in the presence of both N ω-nitro- l-arginine ( l-NNA) (100 μM) and diclofenac (1 μM). Results: The resting membrane potential was −70.9 mV, and Ba 2+ (0.5 mM) depolarized the membrane to −33.0 mV. Insulin (10 μU/ml to 100 mU/ml) did not change the membrane potential in the absence or presence of Ba 2+. In the presence of Ba 2+, ACh (3 μM) hyperpolarized the membrane with two components, an initial large hyperpolarization followed by a slow and small one. Low concentration of insulin (100 μU/ml) did not alter the ACh-induced hyperpolarization. High concentration of insulin (100 mU/ml) shortened the time required to reach the peak amplitude and tended to increase the peak amplitude of the ACh-induced hyperpolarization. Conclusions: The data show that insulin enhances the ACh-induced hyperpolarization in the submucosal arterioles of the guinea pig ileum. The results suggested that EDHF also accounts for one of the endothelial factors involved in the insulin-induced vasodilatation.

Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels

The present study was designed to elucidate whether the conduction of vasomotor responses mediated by endothelium-derived hyperpolarizing factor (EDHF) in rat mesenteric arteries is altered during hypertension. Iontophoresed acetylcholine (ACh; 500 ms) caused EDHF-mediated hyperpolarization and vasodilatation at the local site and these responses spread through the endothelium to remote sites in 12-week-old Wistar-Kyoto rats (WKY). Conducted responses were significantly attenuated in age-matched spontaneously hypertensive rats (SHR) although the rate of decay with distance did not change. Inhibition of inwardly rectifying potassium (Kir) channels (30 microM barium) eliminated the difference between WKY and SHR by attenuating conducted responses in WKY but not SHR. At the local site, barium (30 microM) significantly reduced the duration but not the amplitude of ACh-induced hyperpolarization in WKY only. Barium had no effect when the iontophoretic stimulus was reduced to 350 ms. After blockade of EDHF in SHR, ACh elicited a depolarization which our indirect data suggest spreads along the vessel in the endothelium. Messenger RNA expression of Kir2.0 genes did not differ between the strains nor did the amplitude of K(+)-induced hyperpolarization, which was abolished by disruption of the endothelium. Immunohistochemistry revealed a decrease in connexin (Cx)37 but not Cx40 or Cx43 protein in endothelial cells of SHR compared to WKY. Results suggest that conduction of EDHF-mediated responses in WKY, but not in SHR, is facilitated by activation of Kir channels at the site of ACh application and not by differences in endothelial connexin expression. Lack of Kir channel involvement in hypertension may result from reduction in the duration of the hyperpolarization due to the development of ACh-mediated depolarization, rather than to any difference in Kir subunit expression or function.

Sodium–calcium exchanger contributes to membrane hyperpolarization of intact endothelial cells from rat aorta during acetylcholine stimulation

1. The role of sodium-calcium exchanger in acetylcholine (Ach)-induced hyperpolarization of intact endothelial cells was studied in excised rat aorta. The membrane potential was recorded using perforated patch-clamp technique. 2. The mean resting potential of endothelial cells was -44.1+/-1.4 mV. A selective inhibitor of sodium-calcium exchanger benzamil (100 microm) had no significant effect on resting membrane potential, but reversibly decreased the amplitude of sustained Ach-induced endothelial hyperpolarization from 20.9+/-1.4 to 5.7+/-1.1 mV when applied during the plateau phase. 3. The blocker of reversed mode of the exchanger KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate, 20 microm) reversibly decreased the amplitude of sustained Ach-induced hyperpolarization from 20.5+/-2.9 to 7.5+/-1.8 mV. 4. Introduction of tetraethylammonium (10 mm) in the continuous presence of Ach decreased the sustained phase of hyperpolarization from 17.9+/-1.5 by 12.9+/-0.9 mV. Subsequent addition of 20 microm KB-R7943 further depolarized endothelial cells by 4.8+/-1.1 mV. 5. Substituting external sodium with N-methyl d-glucamine during the plateau phase of Ach-evoked hyperpolarization reversibly decreased the hyperpolarization from -61.8+/-2.7 to -54.2+/-1.9 mV. In the majority of preparations, the initial response to removal of external sodium was a transient further rise in the membrane potential of several mV. Sodium ionophore monensin hyperpolarized endothelium by 10.3+/-0.7 mV. 6. The inhibitory effect of benzamil on Ach-induced endothelial sustained hyperpolarization was observed in endothelium mechanically isolated from smooth muscle. 7. These results suggest that the sodium-calcium exchanger of intact endothelial cells is able to operate in reverse following stimulation by Ach, contributing to sustained hyperpolarization. Myoendothelial electrical communications do not mediate the effect of blockers of sodium-calcium exchanger.