Insulin inhibits rat hippocampal neurones via activation of ATP-sensitive K+ and large conductance Ca2+-activated K+ channels

In this study, we have used a combination of immunocytochemical and Ca(2+) imaging techniques to determine the functional localisation of insulin receptors as well as the potential role for insulin in modulating hippocampal synaptic activity. Comparison of insulin receptor and MAP2 labelling demonstrated extensive insulin receptor immunoreactivity on the soma and dendrites of cultured hippocampal neurones. Dual labelling with synapsin 1 also showed punctate insulin receptor labelling associated with synapses. In functional studies, insulin inhibited spontaneous Ca(2+) oscillations evoked in cultured hippocampal neurones following Mg(2+) removal. This action of insulin was mimicked by the ATP-sensitive K(+) (K(ATP)) channel opener diazoxide or the large conductance Ca(2+)-activated K(+) (BK) channel activator NS-1619. Furthermore, application of the K(ATP) channel blocker glybenclamide or the BK channel inhibitors iberiotoxin or charybdotoxin attenuated the actions of insulin, whereas prior incubation with a combination of glybenclamide and iberiotoxin completely blocked insulin action. The ability of insulin to modulate the Ca(2+) oscillations was reduced by the inhibitors of MAPK activation PD 98059 and U0126, but not by the PI 3-kinase inhibitors LY 294002 or wortmannin, indicating that a MAPK-driven process underlies insulin action. In conclusion, insulin inhibits spontaneous Ca(2+) oscillations via a process involving MAPK-driven activation of BK and K(ATP) channels. This process may be a useful therapeutic target for the treatment of epilepsy and certain neurodegenerative diseases.     

ATP-sensitive K+ channels in the kidney

ATP-sensitive K⁺ channels (K_ATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K⁺ which, in turn, is a major determinant of cell membrane potential. K_ATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic -cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell.In the renal tubular system, K_ATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K⁺ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the K_ATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed.The pharmacology of K_ATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of K_ATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the K_ATP channels in the pancreatic -cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K⁺ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the K_ATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.     

Substance P induces inward current and regulates pacemaker currents through tachykinin NK1 receptor in cultured interstitial cells of Cajal of murine small intestine

We investigated whether substance P modulates pacemaker currents generated in cultured interstitial cells of Cajal of murine small intestine using whole cell patch-clamp techniques at 30 degrees C. Interstitial cells of Cajal generated spontaneous inward currents (pacemaker currents) at a holding potential of -70 mV. Tetrodotoxin, nifedipine, tetraethylammonium, 4-aminopyridine, or glibenclamide did not change the frequency and amplitude of pacemaker currents. However, divalent cations (Ni2+, Mn2+, Cd2+, and Co2+), nonselective cationic channel blockers (gadolinium and flufenamic acid), and a reduction of external Na+ from normal to 1 mM inhibited pacemaker currents indicating that nonselective cation channels are involved in their generation. Substance P depolarized the membrane potential in current clamp mode and produced tonic inward pacemaker currents with reduced frequency and amplitude in voltage clamp mode. [D-Arg1, D-Trp7,9, Leu11] substance P, a tachykinin NK1 receptor antagonist, blocked these substance P-induced responses. Furthermore, [Sar9, Met(O2)11] substance P, a specific tachykinin NK1 receptor agonist, depolarized the membrane and tonic inward currents mimicked those of substance P. Substance P continued to produce tonic inward currents in external Ca2+-free solution or in the presence of chelerythrine, a protein kinase C inhibitor. However, substance P-induced tonic inward currents were blocked by thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum or by an external 1 mM Na+ solution. Our results demonstrate that substance P may modulate intestinal motility by acting on the interstitial cells of Cajal by activating nonselective cation channels via the release of intracellular Ca2+ induced by tachykinin NK1 receptor stimulation.     

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K⁺ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K⁺ channels and Na⁺/K⁺-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K⁺-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K⁺-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K⁺ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.     

Delta-opioid receptor agonist SNC80 induces peripheral antinociception via activation of ATP-sensitive K+ channels

We investigated the effect of several K+ channel blockers on the antinociception induced by delta-opioid receptor agonist SNC80 using the paw pressure test, in which pain sensitivity is increased by an intraplantar injection (2 microg) of prostaglandin E2 (PGE2). Administration of SNC80 (20, 40 and 80 microg/paw) caused a decrease in the hyperalgesia induced by PGE2, in a dose-dependent manner. The possibility of higher dose of SNC80 (80 microg) causing a central or systemic effect was excluded since administration of the drug into the contralateral paw did not elicit antinociception in the right paw. Specific blockers of ATP-sensitive K+ channels, glibenclamide (20, 40 and 80 microg/paw) and tolbutamide (40, 80 and 160 microg/paw), antagonized the peripheral antinociception induced by SNC80 (80 microg). On the other hand, charybdotoxin (2 microg/paw), a large-conductance blocker of Ca(2+)-activated K+ channels, and dequalinium (50 microg/paw), a small conductance selective blocker of Ca(2+)-activated K+ channels, did not modify the effect of SNC80. This effect also remained unaffected by intraplantar administration of the voltage-dependent K+ channel blockers tetraethylammonium (30 microg/paw) and 4-aminopyridine (10 microg/paw), and of a non-specific K+ channel blocker, cesium (500 microg/paw). This study provides evidence that the peripheral antinociceptive effect of SNC80 result from the activation of ATP-sensitive K+ channels, and the other K+ channels are not involved.     

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells

The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.     

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K+ (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K+ in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45Ca outflow provoked by a rise in the extracellular K+ concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K+ challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca2+ channels.     

Antagonism of the insulinotropic action of first generation imidazolines by openers of K(ATP) channels

The antagonism between K(ATP) channel-blocking insulinotropic imidazolines - phentolamine, alinidine, idazoxan and efaroxan - and K(ATP) channel openers, diazoxide and nucleoside diphosphates, was studied in mouse pancreatic islets and B-cells. In inside-out patches from B-cells, 500muM MgGDP abolished the inhibitory effect of the imidazolines. 300muM diazoxide further increased channel activity. The depolarizing effect of all imidazolines (100muM) on the B-cell membrane potential was practically completely antagonized by 300muM diazoxide. In contrast, diazoxide was unable to decrease the cytosolic Ca(2+) concentration ([Ca(2+)](i)) which was elevated by phentolamine, whereas the [Ca(2+)](i) increases induced by the other imidazolines were promptly antagonized. The effects on [Ca(2+)](i) were reflected by the secretory activity in that the stimulatory effects of alinidine, idazoxan and efaroxan, but not that of phentolamine were antagonized by diazoxide. Metabolic inhibition of intact B-cells by 250muM NaCN, most likely by a decrease of the ATP/ADP ratio, significantly diminished the K(ATP) channel-blocking effect of a low concentration of alinidine (10muM), whereas efaroxan proved to be susceptible even at a highly effective concentration (100muM). This may explain the oscillatory pattern of the [Ca(2+)](i) increase typically produced by efaroxan in pancreatic B-cells. In conclusion, the inhibitory effect of imidazolines on K(ATP) channels, which is exerted at the pore-forming subunit, Kir6.2, is susceptible to the action of endogenous and exogenous K(ATP) channel openers acting at the regulatory subunit SUR, which confers tissue specificity. With intact cells this antagonism can be obscured, possibly by intracellular accumulation of some imidazolines.     

Inhibition of ATP-sensitive K+ channels by taurine through a benzamido-binding site on sulfonylurea receptor 1

ATP-sensitive potassium (K(ATP)) channels in pancreatic beta-cells comprise sulfonylurea receptor (SUR) 1 and inwardly-rectifying potassium channel (Kir) 6.2 subunits. We have evaluated the effect of intracellular taurine on K(ATP) channel activity in rat pancreatic beta-cells using the patch-clamp technique. The mechanism of taurine action was also examined using recombinant K(ATP) channels. The islets and single beta-cells from male Sprague-Dawley rats were collected by collagenase digestion technique. Single K(ATP) channel currents were recorded by the inside-out mode at a membrane potential of -60mV. Cytosolic free-Ca(2+) concentration ([Ca(2+)](c)) and insulin secretory capacity were measured by the dual-excitation fluorimetry and radioimmunoassay, respectively. The native beta-cell K(ATP) channel was directly inhibited by taurine in a dose-dependent manner. Taurine did not influence ATP-mediated inhibition or MgADP-induced activation of the channel activity. The sensitivity of the K(ATP) channel to glybenclamide, but not gliclazide, was enhanced by taurine. Glybenclamide elicited a greater increase in [Ca(2+)](c) and increased insulin secretion in the beta-cells when pretreated with taurine. Taurine did not inhibit Kir6.2DeltaC36 currents, a truncated form of Kir6.2, expressed in Xenopus oocytes without SUR. These results demonstrate that taurine inhibits the K(ATP) channel activity in the beta-cells, interacting with a benzamido-binding site on SUR1, but not Kir6.2.     

Selective modulation of the ATP-sensitive K+ channel by nicorandil in guinea-pig cardiac cell membrane

Summary Effects of a vasodilator, nicorandil (2-nicotinamidoethyl nitrate) on four kinds for cardiac K⁺ channels were investigated in guinea pig ventricular and atrial cells using inside-out patch recording combined with oilgate concentration jump method.Nicorandil of 300 mol/l failed to affect the inward-rectifier K⁺ channel and the Na⁺-activated K⁺ channel. The open probability of the muscarinic K⁺ channel, when activated by the application of GTP, was not changed by the drug. Nicorandil selectively increased the open probability of the ATP-sensitive K⁺ channel that was partly suppressed by intracellular ATP. The median effective concentration (EC50) of nicorandil was 74 mol/l and Hill coefficient was 1.32 in the concentration-open probability relationship. The closing rate of the K⁺ channel by ATP was markedly delayed by the drug, whereas the open rate on removal of ATP was scarcely affected. Nicorandil had only little effect on this channel after run-down. It was concluded that nicorandil selectively activates the ATP-sensitive K⁺ channel mainly by modulating the ATP-dependent gate.Send offprint requests to M. Takano at the above address     

Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K+ current (IKr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K+ channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K+ channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC50) of the antimalarials was: halofantrine (0.04 microM)相似文献   

Pituitary Adenylate Cyclase-activating Polypeptide Inhibits Pacemaker Activity of Colonic Interstitial Cells of Cajal

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K⁺ channel blocker). However, neither N^G-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K⁺ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.     

Effects of U-37883A on intracellular Ca2+ -activated large-conductance K+ channels in pig proximal urethral myocytes

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.     

Role of ATP-sensitive K+ channels in antinociception induced by R-PIA,an adenosine A1 receptor agonist

The influence of several K⁺ channel-acting drugs on antinociception induced by the adenosine A₁ receptor agonist (–)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) was evaluated with a tail flick test in mice. The subcutaneous administration of R-PIA (0.5–8 mg/kg) induced a dose-dependent antinociceptive effect. The ATP-sensitive K⁺ (K_ATP) channel blocker gliquidone (2–8 g/mouse, i.c.v.) produced a dose-dependent displacement to the right of the R-PIA dose-response line, whereas the K_ATP channel opener cromakalim (32 g/mouse, i.c.v.) shifted it to the left. Several K_ATP channel blockers dose-dependently antagonized the antinociceptive effect of R-PIA, the order of potency being gliquidone > glipizide > glibenclamide (i.e., the same order of potency shown by these drugs in blocking K_ATP channels in neurons). In contrast, the K⁺ channel blockers 4-aminopyridine and tetraethylammonium did not antagonize the effect of R-PIA. These data suggest that antinociception produced by adenosine A₁ receptor agonists is mediated by the opening of ATP-sensitive K⁺ channels. The present results, together with those of previous studies, further support a role for K⁺ channel opening in the antinociceptive effect of agonists of receptors coupled to G_i/G_o proteins. Correspondence to: José M. Baeyens at the above address     

Vasopressin-induced activation of human blood platelets: prominent role of Mg2+

Summary Arginine-vasopressin (AVP) caused a marked shape change reaction and rise in [Ca²⁺]_i in human blood platelets only when the extracellular buffer contained Mg²⁺ or Ca²⁺. At physiological concentrations of the cations the potency of AVP was higher in the presence of Mg²⁺ than of Ca²⁺. The amplitude of the shape change reaction was also greater with Mg²⁺ than with Ca²⁺, although the [Ca²⁺]_i-rise was slightly more marked with extracellular Ca²⁺. The concentration of Mg²⁺ at which AVP showed half of its maximal effects was below the physiological plasma level of the cation, whereas the corresponding value for Ca²⁺ was higher. Addition of Ca²⁺ to the Mg²⁺ containing medium did not further enhance the action of AVP on platelet shape. In platelet-rich plasma the potency and efficacy of AVP in causing a shape change were similar in the presence and absence of EGTA, whereas with EDTA in the medium AVP had no effect. In conclusion, Mg²⁺ has an essential physiological role in AVP-induced platelet activation, which is brought about partly by release of intracellular calcium and partly by some other intracellular mechanism.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed.
2. NO (10⁻⁷ M) induced only contraction while 10⁻⁶ M NO induced contraction followed by relaxation. Methylene blue (up to 10⁻⁴ M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10⁻⁶ M NO. Administration of 8-bromo-cyclic GMP (10⁻⁶–10⁻⁴ M) only induced relaxation.
3. Sodium nitroprusside (SNP; 10⁻⁷–10⁻⁵ M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10⁻⁸ M NO and 10⁻⁶ M SNP; it was increased by a factor of 2 during contraction with 10⁻⁷ M NO, and by a factor of 12 during relaxation with 3×10⁻⁶ M NO.
4. The NO-induced contractions were not affected by ryanodine (3×10⁻⁵ M) but were concentration-dependently reduced by nifedipine (10⁻⁸–10⁻⁷ M) and apamin (3×10⁻⁹–3×10⁻⁸ M).
5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca²⁺ influx through L-type Ca²⁺ channels, that might be activated in response to the closure of Ca²⁺-dependent K⁺ channels.

     

Relaxation induced by calcium ionophore is impaired in carotid arteries from 2K-1C rats due to failed effect of nitric oxide on the smooth muscle cells

Vascular endothelium generates nitric oxide (NO) in large vessels and induces relaxation of vascular smooth muscle cells (VSMC). The aim of this study was to evaluate the contribution of NO produced in the endothelial cells (EC) to the relaxation induced by the Ca²⁺ ionophore A23187 and whether this relaxation is impaired in renal hypertensive (2K-1C) rat arteries. Concentration-effect curves for A23187 were constructed in intact endothelium isolated carotid rings from 2K-1C and normotensive (2K) in the absence or in the presence of the extracellular NO scavenger haemoglobin or inhibitors of NO-synthase (NOS, L-NOARG), guanylyl-cyclase (GC, ODQ). In carotid rings loaded with Fluo-3AM, both EC and VSMC were simultaneously imaged by a confocal microscope and [Ca²⁺]_c was derived from fluorescence intensities (IF). The maximal relaxation (ME) induced by A23187 was lower in 2K-1C than in 2K arteries. A23187-induced relaxation was abolished by haemoglobin and L-NOARG in both groups. ODQ reduced the ME to A23187 in 2K and abolished its relaxation in 2K-1C. A23187 increased [Ca²⁺]_c in a similar way in 2K and 2K-1C EC, and decreased [Ca²⁺]_c in VSMC, which effect was higher in 2K than in 2K-1C arteries. L-NOARG inhibited the effect of A23187 in VSMC from 2K and abolished it in 2K-1C rats. On the other hand, L-NOARG did not modify the effect of A23187 in EC from 2K and 2K-1C rats.The basal content of cGMP was higher in 2K than in 2K-1C arterial rings that was similarly increased by A23187. In conclusion, the Ca²⁺ ionophore A23187 increases Ca²⁺, activates NOS and NO production in the EC activating GC in VSMC and [Ca²⁺]_c decrease. All these effects are higher in 2K, which contribute to the impaired relaxation to A23187 in 2K-1C rat arteries.     

Induction of vasorelaxation through activation of nitric oxide synthase in endothelial cells by brazilin

The vasorelaxant activity of Caesalpinia sappan L., a traditional Chinese medicine, and its major component brazilin were investigated in isolated rat aorta and human umbilical vein endothelial cells. In isolated rat aorta, C. sappan L. extract and brazilin relaxed phenylephrine-induced vasocontraction and increased cyclic guanosine 3',5'-monophosphate (cGMP) content. Induction of vasorelaxation of brazilin was endothelium-dependent and could be markedly blocked by pretreatment with nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME); N(G)-monomethyl-L-arginine acetate (L-NMMA) and guanylyl cyclase inhibitor, methylene blue; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide (NO) scavenger, hemoglobin. The increasing cGMP content induced by brazilin was also blocked by pretreatment with L-NAME, methylene blue, and the removal of extracellular Ca(2+). In human umbilical vein endothelial cells, brazilin dose-dependently induced an increase in NO formation and NOS activity, which were greatly attenuated by either the removal of extracellular Ca(2+) or the chelating of intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, brazilin dose-dependently induced the influx of extracellular Ca(2+) in human umbilical vein endothelial cells. Collectively, these results suggest that brazilin induces vasorelaxation by the increasing intracellular Ca(2+) concentration in endothelial cells of blood vessels and hence activating Ca(2+)/calmodulin-dependent NO synthesis. The NO is released and then transferred into smooth muscle cells to activate guanylyl cyclase and increase cGMP content, resulting in vasorelaxation.     

Deoxycholic acid inhibits pacemaker currents by activating ATP-dependent K+ channels through prostaglandin E2 in interstitial cells of Cajal from the murine small intestine

1. We investigated the role of deoxycholic acid in pacemaker currents using whole-cell patch-clamp techniques at 30 degrees C in cultured interstitial cells of Cajal (ICC) from murine small intestine. 2. The treatment of ICC with deoxycholic acid resulted in a decrease in the frequency and amplitude of pacemaker currents and increases in resting outward currents. Also, under current clamping, deoxycholic acid produced the hyperpolarization of membrane potential and decreased the amplitude of the pacemaker potentials. 3. These observed effects of deoxycholic acid on pacemaker currents and pacemaker potentials were completely suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. 4. NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, significantly inhibited the deoxycholic acid-induced effects. The treatment with prostaglandin E(2) (PGE(2)) led to a decrease in the amplitude and frequency of pacemaker currents and to an increase in resting outward currents, and these observed effects of PGE(2) were blocked by glibenclamide. 5. We next examined the role of deoxycholic acid in the production of PGE(2) in ICC, and found that deoxycholic acid increased PGE(2) production through the induction of COX-2 enzyme activity and its gene expression. 6. The results suggest that deoxycholic acid inhibits the pacemaker currents of ICC by activating ATP-sensitive K(+) channels through the production of PGE(2).