An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single-channel behavior and conductance properties

Summary An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100mm Cl^–. (2) The channel does not select among monovalent anions but SO ₄ ^2– hardly permeates through the channel. (3) SO ₄ ^2– added to thecis side (the side to which SR vesicles were added) inhibits Cl^– current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO ₄ ^2– is located at about 36% of the way across the channel from thecis entrance.     

Dimensions of the ion channel in neuronal nicotinic acetylcholine receptor as estimated from analysis of conformation-activity relationships of open-channel blocking drugs

Summary Relationship between the size of the molecule in the series of organic ions Et₃N–(CH₂)₅–N⁺R¹R²R³ (R ⁱ -alkyl or cycloalkyl substituents) and their abilities to block nicotinic acetylcholine receptors (AChRs) due to their open-channel blockade in the neurons of autonomic ganglia and in frog end-plate was analyzed.All low-energy equilibrium conformations of the drugs were calculated by the molecular mechanics method. A unique rectangular channel profile 6.1×8.3 Å. for which the best correlation between blocking activity of the drugs and total population of their conformations being able to penetrate into the channel, was deduced from all those tested.     

Electroconvulsive Shock Increases α1b-but Not α1a-Adrenoceptor Binding Sites in Rat Cerebral Cortex

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.     

Beta-adrenergic control of trans-cutaneous evaporative cooling mechanisms in birds

Summary The decreasing effect of -adrenergic blockade on skin resistance to vapor diffusion and the onset of cutaneous water evaporation in the pigeon (Columba livia) was investigated. Oral administration of 1, 2.3 and 5 mg propranolol to pigeons (268±53 g) initiated intensive trans-cutaneous water evaporation (CWE) up to 29.1 mg H₂O·cm^–2·h^–1 in resting birds at 30°C air temperature (Ta), but had only a slight effect on CWE of birds exposed to 50 °C Ta.After 7 h of effective -adrenergic blockade (oral administration of 5 mg propranolol), skin and body temperature stabilized at 39.0±0.5 °C and 41.0±0.7 °C, compared to 40.2±0.8 °C and 41.9±0.6 °C in the control group, respectively. A slight hypothermia was accompanied by feather fluffing.Intradermal injection of 0.001, 0.01 and 0.12 mg propranolol also caused intensive CWE. Local -adrenergic blockade in relatively low blocker doses (0.001 and 0.01 mg propranolol) decreased skin resistance from a high value of 44.5 s·cm^–1 to about 6.0 s·cm^–1, and caused a sharp increase in CWE from a control value of about 4 to a high of 26.4 mg H₂O·cm^–2·h^–1 during the first two hours of exposure to 30°C Ta.The possible role of -adrenergic blockade in regulation of trans-cutaneous water evaporation of latent heat dissipation is discussed.     

Antidepressant Treatments, Including Sibutramine Hydrochloride and Electroconvulsive Shock, Decrease β1 but Not β2-Adrenoceptors in Rat Cortex

The beta 1- and beta 2-adrenoceptor populations in rat cortex were individually quantified by labelling all of the receptors with [3H]dihydroalprenolol and displacing with isoprenaline (200 microM) or CGP 20712A (1-(2-[(3-carbamoyl-4-hydroxy)phenoxy]ethylamino)-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol methanesulphonate; 100 nM) to define total beta-adrenoceptors and beta 1-adrenoceptors, respectively. Binding parameters for beta 2-adrenoceptors were calculated by the difference. Oral administration of the monoamine reuptake inhibitors sibutramine HCl (3 mg/kg), amitriptyline (10 mg/kg), desipramine (10 mg/kg), or zimeldine (10 mg/kg) for 10 days decreased the total number of beta-adrenoceptors present in rat cortex. This effect was entirely due to a reduction in the number of beta 1-adrenoceptors. Similarly, 10 days of treatment with the monoamine oxidase inhibitor tranylcypromine (10 mg/kg p.o.) or five electroconvulsive shocks (ECSs; 200 V, 2 s) spread over this period also down-regulated beta-adrenoceptors by reducing the content of the beta 1-subtype. By contrast, treatment with clenbuterol (5 mg/kg p.o.) for 10 days reduced the number of cortical beta-adrenoceptors by an effect on the beta 2-adrenoceptor population. The effects of short-term treatment with these drugs were also investigated, and, using the doses shown above, the results of 3 days of administration or a single ECS were determined. Sibutramine HCl and desipramine were alone in producing a reduction in number of beta-adrenoceptors after 3 days. Once again, this was exclusively due to a loss of beta 1-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics

In the present study, a simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed to determine TJ0711 hydrochloride, a novel α- and β-receptor blocker. TJ0711 hydrochloride and verapamil hydrochloride (the internal standard) were separated on Knauer Eurospher C₁₈ (250 mm × 4.0 mm i.d., 5 μm) column at 50 °C. The mobile phase was methanol:perchloric acid (12 nM, aq) (56:44, v:v), with a flow rate of 1.0 mL/min. The wavelengths of FD were set at 246 nm for excitation and 300 nm for emission. For plasma samples of rats, the analytes were extracted with acetic ether from alkalinized plasma, and then back-extracted into 10 mM dilute sulfuric acid. The linearity was over a concentration range of 20–10,000 ng/mL. The intra- and inter-day precisions referred by relative standard deviation were less than 2.0% and 4.3%, respectively. The mean analytical recoveries of TJ0711 hydrochloride at different concentrations (50, 1000 and 8000 ng/mL) ranged from 88.3% to 92.9%. The lower limit of quantification (LLOQ) was 20 ng/mL. Finally, this method was successfully applied to the estimation of pharmacokinetic parameters of TJ0711 hydrochloride after intravenous doses of 4, 8 and 16 mg/kg in rats.     

A marine snail neurotoxin shares with scorpion toxins a convergent mechanism of blockade on the pore of voltage-gated K channels.

kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in kappa-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, kappa-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) M-1 s-1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced kappa-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the alpha-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.     

Effects of combined β-adrenergic and cholinergic blockade on the initial ventilatory response to exercise in humans

To elucidate whether combined adrenergic and parasympathetic blockade would affect the ventilatory response to exercise, especially at the initial stage (phase I), six healthy subjects performed a brief and light voluntary bilateral leg extension exercise and passive movements under the conditions of control (before the blockade) and after intravenous administration of combined β-adrenergic (propranolol, 0.2 mg · kg⁻¹) and muscarinic (atropine, 0.04 mg · kg⁻¹) receptor antagonists. The movements were continued only within two breaths after the onset of the motion. Ventilation increased immediately and significantly (P<0.05) within the first breath at the onset of voluntary exercise in all conditions as compared with at rest. However, the magnitude of increase in mean ventilation within two breaths at the start of exercise as against the resting value (delta ventilation) was significantly less (P<0.05) after the combined blockades (2.5 l · min⁻¹) than in the control condition (3.7 l · min⁻¹). Passive movements showed a similar but smaller change as compared with voluntary exercise. The heart rate response to exercise was attenuated by the combined blockade while cardiac output showed a slight change at the onset of exercise. It is concluded that phase I should occur despite the inhibited activity of the β-adrenergic and the cholinergic systems; nevertheless, the response was attenuated by the combined blockade. These results suggest a possible role of the β-adrenergic and/or cholinergic systems in the rapid increase in ventilation that occurs at the start of exercise. Accepted: 2 March 1997     

Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment

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Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.