ß-Citronellol, an alcoholic monoterpene with inhibitory properties on the contractility of rat trachea

β-Citronellol is an alcoholic monoterpene found in essential oils such Cymbopogon citratus (a plant with antihypertensive properties). β-Citronellol can act against pathogenic microorganisms that affect airways and, in virtue of the popular use of β-citronellol-enriched essential oils in aromatherapy, we assessed its pharmacologic effects on the contractility of rat trachea. Contractions of isolated tracheal rings were recorded isometrically through a force transducer connected to a data-acquisition device. β-Citronellol relaxed sustained contractions induced by acetylcholine or high extracellular potassium, but half-maximal inhibitory concentrations (IC50) for K+-elicited stimuli were smaller than those for cholinergic contractions. It also inhibited contractions induced by electrical field stimulation or sodium orthovanadate with pharmacologic potency equivalent to that seen against acetylcholine-induced contractions. When contractions were evoked by selective recruitment of Ca2+ from the extracellular medium, β-citronellol preferentially inhibited contractions that involved voltage-operated (but not receptor-operated) pathways. β-Citronellol (but not verapamil) inhibited contractions induced by restoration of external Ca2+ levels after depleting internal Ca2+ stores with the concomitant presence of thapsigargin and recurrent challenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacin or tetraethylammonium did not change the relaxing effects of β-citronellol. Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) receptors with selective antagonists caused no change in the effects of β-citronellol. In conclusion, β-citronellol exerted inhibitory effects on rat tracheal rings, with predominant effects on contractions that recruit Ca2+ inflow towards the cytosol by voltage-gated pathways, whereas it appears less active against contractions elicited by receptor-operated Ca2+ channels.

The methyl ester of rosuvastatin elicited an endothelium-independent and 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent relaxant effect in rat aorta

The relaxant effect of the methyl ester of rosuvastatin was evaluated on aortic rings from male Wistar rats (250-300 g, 6 rats for each experimental group) with and without endothelium precontracted with 1.0 µM phenylephrine. The methyl ester presented a slightly greater potency than rosuvastatin in relaxing aortic rings, with log IC50 values of -6.88 and -6.07 M, respectively. Unlike rosuvastatin, the effect of its methyl ester was endothelium-independent. Pretreatment with 10 µM indomethacin did not inhibit, and pretreatment with 1 mM mevalonate only modestly inhibited the relaxant effect of the methyl ester. Nω-nitro-L-arginine methyl ester (L-NAME, 10 µM), the selective nitric oxide-2 (NO-2) inhibitor 1400 W (10 µM), tetraethylammonium (TEA, 10 mM), and cycloheximide (10 µM) partially inhibited the relaxant effect of the methyl ester on endothelium-denuded aortic rings. However, the combination of TEA plus either L-NAME or cycloheximide completely inhibited the relaxant effect. Inducible NO synthase (NOS-2) was only present in endothelium-denuded aortic rings, as demonstrated by immunoblot with methyl ester-treated rings. In conclusion, whereas rosuvastatin was associated with a relaxant effect dependent on endothelium and hydroxymethylglutaryl coenzyme A reductase in rat aorta, the methyl ester of rosuvastatin exhibited an endothelium-independent and only slightly hydroxymethylglutaryl coenzyme A reductase-dependent relaxant effect. Both NO produced by NOS-2 and K+ channels are involved in the relaxant effect of the methyl ester of rosuvastatin.

Ca2+-activated K+ Current in Freshly Isolated c-Kit Positive Cells in Guinea-pig Stomach

This study was designed to isolate Ca2+-activated K+ current (IKCa) and elucidate its physiological significance in freshly isolated interstitial cells of Cajal (ICCs) of guinea-pig stomach. Single ICC was freshly isolated by enzymatically dissociating from myenteric border of gastric antrum free of circular muscles, and conventional whole-cell voltage clamp technique including immunohistochemical techniques were employed to characterize the cells: In myenteric border of gastric antrum, ICC-MY (ICCs from myenteric border) were detected by immunohistochemical reactivity, and single ICC-MY which has many branches was immunohistochemically c-Kit positive. Under K+-rich and 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid pipette solution, ICC produced spontaneous inward current (-256+/-92.2 pA). When step-depolarizing pulse from -80 to +80 mV was applied at holding potential (Vh) of -80 mV, voltage-dependent outward currents were recorded with superimposed spontaneous transient outward currents (STOCs). Both STOCs and outward currents were reversibly affected by tetraethylammonium chloride (TEA) and iberiotoxin (IbTX); 2 mM TEA and 200 nM IbTX completely abolished STOCs and significantly inhibited outward K+ current over the whole potential range tested for current/voltage (I/V) relationship. In addition, TEA delayed repolarization phase of spontaneous inward current. The present results indicate the presence of IKCa in a single ICC, and it might be involved in regulation of repolarizing phase of spontaneous inward current in guinea-pig stomach.

Ptychodiscus brevis toxin decreases the spontaneous activity of rat right atria involving muscarinic receptors and potassium channels.

Marine dinoflagellate Ptychodiscus brevis toxin (PbTx), is known to produce toxic effects on cardiovascular system. The present experiments were conducted to evaluate the effect of synthetic phosphorus containing Ptychodiscus brevis toxin on spontaneously beating right atrium in vitro. The PbTx (0.84-84 microM) decreased the rate and force of right atrial contractions in a concentration-dependent manner. Ethanol, a vehicle present in highest concentration of PbTx, had no effect on atrial rate or force of contraction. Pretreatment with atropine blocked the PbTx-induced decrease in atrial rate and force of contraction. The tetraethylammonium, a potassium channel blocker, blocked the PbTx-induced decrease in atrial rate and force, where as, L-type of calcium channel blocker, nifedipine blocked the PbTx-induced force of contraction but not the rate changes. The results indicate that the PbTx decreased the atrial rate and force of contraction via cholinergic receptors involving K+ channel.

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic â-cells in situ

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic â-cells within intact mouse islets of Langerhans at 37C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I Cav), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of â-cell electrical activity. The temperature dependence of I Cav was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on â-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I Cav contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping â-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.

blocking activity of different toxins against potassium channels Kv3.4 in RLE cells

K[+] channel toxins are essential tools for the first purifications, analysis of subunit structures and brain localization of voltage-gated K[+] [Kv] channels. The effects of a lot of toxins on Kv are not fully known. Using whole-cell patch clamping technique the action of a series of toxins on Kv3.4 current in rat liver cells with expressed Kv3.4 channels [RLE] cloned cells was investigated. The cells were grown in Williams E medium and after 6-8 days, they were suitable for patch clamping. A family of currents was recorded during voltage-clamp steps to various potentials applied from a holding potential of-60 mV to 60-80 mV in 10 mV increments. Upon depolarization, all channels were opened with a sigmoidal time course, reached to the peak within a few 10[th] of milliseconds and then slowly inactivated. Bath application of tetraethyl ammonium [TEA] or 3, 4-diaminopyridine [DAP] reduced the current dose dependently and inhibited it completely at 3 mM and 25 micro M respectively. The Bunodosoma granulifera [BgK] and Heteractis magniflca [HmK] toxins at concentrations up to 30 and 10 micro M respectively could not completely inhibit the current. On the hand, toxins such as beta-bungarotoxin, corotoxin, novel toxin and dendrotoxins I [DIP] and K [DPK] even in high concentrations [up to 100 mM] had not any significant effect on Kv3.4 current. Comparison of chemical structures of these effective agents with other reported effective toxins such as blood depressing substances [BDS 1 and II] show no homology between them, but specially the potency of 3, 4-DAP is comparable with these toxins. These results showed that, the Kv3.4 is more sensitive than other K[+] channels to 3, 4-DAP. The sensitivity of this channel to the TEA is low [at mM concentration]. More investigation is necessary to find more selective and potent inhibitor of Kv3.4 channels

Evaluation of vasopressin mediated effects on hemostatic mechanisms: relationship with aquaporins and caveolin proteins.

With a view to evaluate the role of AQP-1 and caveolin proteins in the hemostatic actions of vasopressin, hemostasis was evaluated by bleeding and clotting time respectively.Groups of mice and guinea pigs were treated with arginine vasopressin (AVP) and 1-deamino-8D-AVP (DDAVP) to evaluate their effects on the hemostasis. DDAVP and AVP were able to appreciably reduce the bleeding and clotting time after sodium thiopentone, but not effectively after TEA treatment. Animal groups were pretreated with aquaporin-1 (AQP-1) blockers or water deprived to enhance the expression of AQP-1 water channels. Another group of animals were treated with caveolin protein modulators, cholera toxin (CTX) and the effect of vasopressin analogues evaluated. The results suggest that AQP-1 water channels and caveolin proteins contribute to modulate the hemostatic mechanisms of vasopressin.

Inhibitory Effects of Potassium Channel Blockers on Carbachol-induced Contraction in Rat Detrusor Muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 micrometer to 5 micrometer. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 micrometer thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potasssium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.

Mechanisms of relaxation of coronary artery by hypoxia

This study was designed to clarify the dependency of hypoxic coronary vasodilation (HCD) on the endothelium and the role of the K+ channels on HCD in the rabbit coronary artery. HCD was investigated in an isolated left circumflex coronary artery precontracted with prostaglandin F2 alpha. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit (KH) solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and was maintained for 15 approximately 25 min. Hypoxia elicited a vasodilation in the precontracted coronary artery with and without endothelium. There was no difference between the amplitude of the HCD induced by two consecutive hypoxic challenges and the effects of 20% O2 + 5% CO2 + 75% N2 and 95% O2 + 5% CO2 control K-H solution of subsequent responses to hypoxia. Inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HCD. Blockades of the tetraethylammonium chloride-sensitive K+ channel abolished HCD. Apamin, a blocker of the small conductance Ca(2+)-activated K+ (KCa) channel, and iberiotoxin, a blocker of the large conductance KCa channel had no effect on HCD, respectively. Glibenclamide, a blocker of the ATP-sensitive K+ (K+ATP) channel, reduced HCD. Cromakalim, an opener of the K+ATP channel, relaxed the coronary artery precontracted with prostaglandin F2 alpha. The degree of relaxation by cromakalim was similar to that by hypoxia while glibenclamide reduced both hypoxia- and cromakalim-induced vasodilatations. In conclusion, these results suggest that HCD is independent on endothelium and HCD is considered to be induced by activation of K+ATP channel.

Effects of hypoxia on pulmonary vascular contractility

Although hypoxic pulmonary vasoconstriction (HPV) has been recognized by many researchers, the precise mechanism remains unknown. As isolated pulmonary arteries will constrict in vitro in the response to hypoxia, the oxygen sensor/transduction mechanism must reside in the pulmonary arterial smooth muscle or in the endothelium, or in both. Unfortunately, much of the current evidence is conflicting, especially as to the dependency of HPV on the endothelium and the role of a K+ channel. Therefore, this experiment was attempted to clarify the dependency of HPV on the endothelium and the role of a K+ channel on HPV in rat pulmonary artery. The effects of hypoxia were investigated in isolated main pulmonary arteries precontracted with norepinephrine. Vascular rings were suspended for isometric tension recording in an organ chamber filled with a Krebs-Henseleit solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and this was maintained for 20 min. Hypoxia elicited a vasoconstriction in arteries with endothelium. Mechanical disruption of the endothelium abolished HPV. There was no difference between the amplitude of the HPV induced by two consecutive hypoxic challenges and the effect of normoxic and hyperoxic control Krebs-Henseleit solution on a subsequent response to hypoxia. Inhibition of NO synthesis by treatment with N(omega)-nitro-L-arginine reduced HPV, but inhibition of a cyclooxygenase pathway by treatment with indomethacin had no effect on HPV. Blockades of a tetraetylammonium chloride-sensitive K+ channel abolished HPV. Verapamil, a Ca2+ entry blocker reduced HPV. In conclusion, these results suggest that HPV was dependent on the endothelium and that HPV can be considered to be induced by inhibition of the mechanisms of NO-dependent vasodilation such as the opening of a K+ channels.