Blockade of K+ contractures in skeletal muscle by opioid drugs: a nonstereospecific effect

The effect of several opioid drugs was tested on the K+ contractures in frog's skeletal muscle. These contractures are produced by the entrance of extracellular Ca2+ ions via the voltage-dependent, slow Ca2+ channels located in the T tubules. Morphine and other opioid agonists in concentrations ranging from 10(-10) to 10(-5) M inhibited K+ contractures. The stereoisomers, dextrorphan and levorphanol, were found to have identical potency in inhibiting high K+ contractures, suggesting that this was a nonstereospecific blockade of voltage-dependent calcium channels by the opioid drugs despite the low effective drug concentrations. In agreement with this conclusion it was found that the inhibition of K+ contractures by the opioids was not antagonized by naloxone. It also was observed using a sucrose gap apparatus that these opioid drugs in concentrations used to block the high K+ contractures did not reduce the K+-induced membrane depolarization. Raising the bathing solution Ca2+ concentration from 1.08 to 5 mM produced a reversal of the opioid-induced block of K+ contractures. Finally it was shown that while opioids completely blocked K+ contractures, they did not produce any effect on caffeine contractures showing that opioids do not deplete intracellular Ca2+ stores or inhibit the release of Ca2+ from intracellular sarcoplasmic reticulum stores. It was concluded that several opioid drugs in very low concentrations block K+ contractures in frog's skeletal muscle by a nonstereospecific block of voltage-dependent slow calcium channels.     

Complexation and ionophoric properties of taxol and colchicine: complex formation and transport of sodium, potassium, magnesium and calcium ions across a liquid membrane

We report the activities of taxol (an anticancer drug) and colchicine, which are inhibitors of microtubule organization, on the complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane, using a spectrophotometric procedure. Taxol, a diterpenoid compound, that has been demonstrated to possess a potent antitumour activity, is shown to extract Na+, K+, Mg2+ and Ca2+ ions from the aqueous solution to the organic phase with preference for Ca2+ ions. A kinetic study of the transport and complexation of Na+, K+, Mg2+ and Ca2+ ions through a liquid membrane revealed that the K+ ion is more rapidly transported and the Ca2+ ion is more rapidly complexed than other ions. However, colchicine, another alkaloid compound, extracted and transported only the divalent ions tested, Mg2+ and Ca2+. In both complexation and transport, the flux of the ions increases with the concentration of taxol or colchicine. Complexation and ionophoric properties of taxol and colchicine sheds new lights on therapeutic properties of these drugs. The treatment of disease states by the administration of these drugs to alter membrane permeability will prove to be a valuable therapeutic concept.     

Contributions of HERG K+ current to repolarization of the human ventricular action potential

Action potential repolarization in the mammalian heart is governed by interactions of a number of time- and voltage-dependent channel-mediated currents, as well as contributions from the Na+/Ca2+ exchanger and the Na+/K+ pump. Recent work has shown that one of the K+ currents (HERG) which contributes to repolarization in mammalian ventricle is a locus at which a number of point mutations can have significant functional consequences. In addition, the remarkable sensitivity of this K+ channel isoform to inhibition by a variety of pharmacological agents and clinical drugs has resulted in HERG being a major focus for Safety Pharmacology requirements. For these reasons we and others have attempted to define the functional role for HERG-mediated K+ currents in repolarization of the action potential in the human ventricle. Here, we describe and evaluate changes in the formulations for two K+ currents, IK1 and HERG (or IK,r), within the framework of ten Tusscher model of the human ventricular action potential. In this computational study, new mathematical formulations for the two nonlinear K+ conductances, IK1 and HERG, have been developed based upon experimental data obtained from electrophysiological studies of excised human ventricular tissue and/or myocytes. The resulting mathematical model provides much improved simulations of the relative sizes and time courses of the K+ currents which modulate repolarization. Our new formulation represents an important first step in defining the mechanism(s) of repolarization of the membrane action potential in the human ventricle. Our overall goal is to understand the genesis of the T-wave of the human electrocardiogram.     

Essential role of calcineurin in response to endoplasmic reticulum stress

Depletion of calcium ions (Ca2+) from the endoplasmic reticulum (ER) of yeast cells resulted in the activation of the unfolded protein response (UPR) signaling pathway involving Ire1p and Hac1p. The depleted ER also stimulated Ca2+ influx at the plasma membrane through the Cch1p-Mid1p Ca2+ channel and another system. Surprisingly, both Ca2+ influx systems were stimulated upon accumulation of misfolded proteins in the ER even in the presence of Ca2+. The ability of misfolded ER proteins to stimulate Ca2+ influx at the plasma membrane did not require Ire1p or Hac1p, and Ca2+ influx and signaling factors were not required for initial UPR signaling. However, activation of the Ca2+ channel, calmodulin, calcineurin and other factors was necessary for long-term survival of cells undergoing ER stress. A similar calcium cell survival (CCS) pathway operates in the pathogenic fungi and promotes resistance to azole antifungal drugs. These findings reveal an unanticipated new regulatory mechanism that couples ER stress to Ca2+ influx and signaling pathways, which help to prevent cell death and promote resistance to an important class of fungistatic drugs.     

Binding of hydrophobic drugs to lipid bilayers and to the (Ca2+ + Mg2+)-ATPase

Microelectrophoretic studies of the binding of a number of commonly used hydrophobic amine drugs to liposomes demonstrated the existence of relatively large surface potentials associated with binding of the protonated forms of the drugs. A theoretical treatment based on Langmuir adsorption isotherms and the Gouy-Chapman theory of the diffuse double layer allows estimation of drug-binding constants from electrophoretic mobility data. Such constants allow calculation of the charge effects arising from drug binding in more complex membrane systems, and it is shown that shifts in the apparent Ca+ affinity of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum in the presence of hydrophobic amine drugs are readily explicable in terms of the electrostatic effects of drug binding.     

Effects of the anti-calmodulin drugs calmidazolium and trifluoperazine on 45Ca transport in plasmalemmal vesicles from gastric smooth muscle

The anti-calmodulin drugs calmidazolium (CMZ) and trifluoperazine (TFP) were shown to have a number of effects on 45Ca transport by plasmalemmal vesicles from gastric smooth muscle. Although these compounds produced the expected dose-dependent inhibition of the plasmalemmal ATP-dependent Ca2+ transport system, they also evoked a Ca2+ release comparable to that observed in the presence of the Ca2+ ionophore, ionomycin. This increased transmembrane Ca2+ flux was so large that it accounted for much of the apparent decrease in 45Ca uptake produced by these agents. Thus, direct effects of CMZ and TFP on ATP-dependent 45Ca uptake could only be reliably assessed for brief (less than or equal to 30 seconds) drug exposures. The explanation for the observed effects of CMZ and TFP on membrane Ca2+ permeability is unclear. The increased transmembrane Ca2+ flux may reflect nonspecific effects on membrane permeability or it may reflect a specific interaction of the anticalmodulin drugs with a Ca2+ release channel or with the Ca2+ transport ATPase. In any case, these results suggest the need for caution in the design and interpretation of studies using both CMZ and TFP as anticalmodulin agents.     

Sequential events in thymocyte differentiation and thymus regeneration revealed by a combination of bromodeoxyuridine DNA labeling and antimitotic drug treatment

The proliferative status of thymocyte cell subsets in vivo was assessed by observing the effect of two antimitotic drugs, hydroxyurea (HU) and demecolcine. Both drugs had the greatest effect on the double-positive (DP) subset followed by the L3T4+ single-positive (SP) subset. However, the decrease in the latter type was delayed by several days, showing that their precursors rather than the cells themselves were killed by HU. Double-negative (DN) cells were less affected, indicating that they contain a resting subset and that they are renewed by emigration rather than by autonomous in situ proliferation. After drug treatment all cycling cells were eliminated from the thymus but, as shown by in vivo and in vitro bromodeoxyuridine incorporation, new cells rapidly reentered in cycle, starting from DN cells and Lyt-2+ SP cells and followed by DP cells. Lyt-2+ SP cycling cells represent an intermediary stage between DN and DP cells, and they are very transient. Injection of HU 24 h after in vivo BrdUrd labeling eliminated most labeled DN cells, but did not prevent the emergence of L3T4+ SP-labeled cells on day 3 as observed in control thymuses. These results suggest that these L3T4+ SP cells are generated from DP cells in the absence of proliferation. Cycling cells in the regenerating thymus were first located at the corticomedullary junction and then in the subcapsular region, suggesting a reverse migration process to that observed after cessation of proliferation. A model is proposed to summarize these sequential events.     

Potentiation of bleomycin cytotoxicity by membrane-interacting drugs and increased calcium ions

The cytotoxic effect of the antitumor antibiotic peplomycin (PEP), a new member of bleomycin group antibiotics, toward HeLa cells and mouse FM3A cells is enhanced by some membrane-interacting drugs such as verapamil, persantin, prenylamine, chlorpromazine and anafranil. The enhancing action of verapamil is selective to this group antibiotics, since it does not potentiate the cytotoxic effects of vincristine, adriamycin, mitomycin C, cis-diamminedichloroplatinum(II) and macromomycin. An enhanced PEP cytotoxicity has been also demonstrated by the treatment of cells in the presence of increased CaCl₂. This enhancing effect of increased CaCl₂ is prevented by the Ca²⁺ transport inhibitor ruthenium red. Since these membrane-directed drugs have been shown to affect Ca²⁺ metabolism, we conclude that potentiation of PEP cytotoxicity by these drugs is mediated by an increase in intracellular Ca²⁺.     

Inhibition and active-site modelling of prolidase

Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions.     

Properties of a new calcium ion antagonist on cellular uptake and mitochondrial efflux of calcium ions.

Compound YS 035 [NN-bis-(3,4-dimethoxyphenethyl)-N-methylamine] is a new synthetic compound capable of inhibiting Ca2+ uptake by different cells. The inhibition of Ca2+ uptake by muscle cells isolated from chicken embryo is dose-dependent in the compound YS 035 concentration range 10-30 microM. The new compound also inhibits Ca2+ entry into rat brain synaptosomes and less effectively into baby-hamster kidney cells. Compound YS 035 partially inhibits the slow Ca2+ release induced by Ruthenium Red and the rapid Na+-dependent efflux from heart mitochondria. The inhibition of the Na+/Ca2+ exchange appears to be of a non-competitive type with an apparent Ki of 28 microM. The new Ca2+ antagonist totally inhibits the Ca2+ efflux from liver mitochondria induced by Ruthenium Red, but it does not affect the release induced by uncoupler, respiratory inhibitor or chelator, nor the mitochondrial ATP synthesis and membrane potential. The properties shown by the new compound indicate it to be a Ca2+ antagonist and a useful tool for studies on the mitochondrial Ca2+ transport.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on the rate of calmodulin binding to erythrocyte Ca2+-ATPase

The erythrocyte Ca2+-ATPase shifts reversibly between two states, the calmodulin-deficient A-state and the calmodulin-saturated B-state, dependent on calcium and calmodulin. The effects on this system of the four drugs, trifluoperazine, compound 48/80, TMB-8 and verapamil were studied. All four drugs inhibited the maximum activity of the B -state Ca2+-ATPase and, in addition, trifluoperazine and compound 48/80 in higher doses inhibited the A-state. Furthermore, the four drugs decreased the calmodulin sensitivity of the Ca2+-ATPase in the order of decreasing effect: trifluoperazine greater than compound 48/80 greater than TMB-8 greater than verapamil. In the same order of decreasing effect the drugs increased the time required for full calmodulin activation of the A-state of Ca2+-ATPase, whereas the drugs had only small effects on the rate of deactivation of the B-state, caused by dissociation of calmodulin from the enzyme. It is discussed whether the effects on calmodulin activation were caused by a reduction of free calmodulin due to the formation of drug-calmodulin complexes or whether the drugs, especially trifluoperazine, compound 48/80 and TMB-8, by binding to the Ca2+-ATPase, decreased the rate constants for association of calmodulin and enzyme.     

Ischemia-reperfusion injury changes the dynamics of Ca2+-contraction coupling due to inotropic drugs in isolated hearts.

Positive inotropic drugs may attenuate or exacerbate the deleterious effects of ischemia and reperfusion (IR) injury on excitation-contraction coupling in hearts. We 1) quantified the phase-space relationship between simultaneously measured myoplasmic Ca2+ concentration ([Ca2+]) and isovolumetric left ventricular pressure (LVP) using indexes of loop area, orientation, and position; and 2) quantified cooperativity by linearly modeling the phase-space relationship between [Ca2+] and rate of LVP development in intact hearts during administration of positive inotropic drugs before and after global IR injury. Unpaced, isolated guinea pig hearts were perfused at a constant pressure with Krebs-Ringer solution (37 degrees C, 1.25 mM CaCl2). [Ca2+] was measured ratiometrically by indo 1 fluorescence by using a fiber-optic probe placed at the left ventricular free wall. LVP was measured by using a saline-filled latex balloon and transducer. Drugs were infused for 2 min, 30 min before, and for 2 min, 30 min after 30-min global ischemia. IR injury worsened Ca2+-contraction coupling, as seen from decreased orientation and repositioning of the loop rightward and downward and reduced cooperativity of contraction and relaxation with or without drugs. Dobutamine (4 microM) worsened, whereas dopamine (8 microM) improved Ca2+-contraction coupling before and after IR injury. Dobutamine and dopamine improved cooperativity of contraction and relaxation after IR injury, whereas only dopamine increased cooperativity of relaxation before IR injury. Digoxin (1 microM) improved Ca2+-contraction coupling and cooperativity of contraction after but not before ischemia. Levosimendan (1 microM) did not alter Ca2+-contraction coupling or cooperativity, despite producing concomitant increases in contractility, relaxation, and Ca2+ flux before and after ischemia. Dynamic indexes based on LVP-[Ca2+] diagrams (area, shape, position) can be used to identify and measure alterations in Ca2+-contraction coupling during administration of positive inotropic drugs in isolated hearts before and after IR injury.     

Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential

Many drugs exhibit lipophilic and cationic (basic) characteristics. Previous studies have shown that lipophilic cations can be accumulated into model membrane 'liposomal' (vesicular) systems in response to establishing a membrane potential (inside negative) across the vesicle membrane. We demonstrate here that the anticancer drugs, adriamycin and vinblastine, can be rapidly accumulated into egg phosphatidylcholine large unilamellar vesicles in response to a valinomycin-dependent K+ diffusion potential (delta psi) to achieve high effective interior concentrations. Further, trapping efficiencies approaching 100% can be easily achieved. The influence of lipid composition and the requirement for valinomycin have been examined for adriamycin. Equimolar cholesterol levels inhibit the uptake process at 20 degrees C. However, incubation at higher temperature results in enhanced uptake. Similarly, the presence of egg phosphatidylserine or incubation at elevated temperatures results in significant adriamycin uptake in the absence of valinomycin. It is shown that the adriamycin retention time in the vesicles is enhanced by an order of magnitude or more when actively trapped by the presence of a membrane potential in comparison to passive trapping procedures. It is suggested that such active trapping procedures may be of use for loading liposomal systems for drug delivery applications, and may provide avenues for controlled release of encapsulated material.     

Release kinetics as a tool to describe drug effects on neurotransmitter release

A procedure is developed wherein the time course of neurotransmitter release is used to discern the mechanism of effects of drugs on the release process. It is shown, in agreement with experiments, that the time course of release is insensitive to the temporal distribution of intracellular Ca2+ concentration. The time course is also invariant to the steps in the release process that are Ca2+ dependent. The most influential events in determining the time course of release are shown to be the steps associated with the slowest forward rate constant and all the backward steps. The procedure was examined for its ability to explain the effects on release of the poison curare and those of temperature. It appears that curare probably blocks release by lowering the rate constant associated with the rate limiting step in release. Increasing temperature is predicted to increase both the forward and the backward rate constants, but not to the same extent.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unprecedented Therapeutic Potential with a Combination of A2A/NR2B Receptor Antagonists as Observed in the 6-OHDA Lesioned Rat Model of Parkinson's Disease

In Parkinson''s disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A_2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A_2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients.     

家兔钙调节蛋自-红细胞膜的制备

 本文介绍用等渗咪唑缓冲液溶血,并用低温高速或常速离心机制备出一种带钙调节蛋白(简称CaM)的红细胞膜。它具有与膜稳定结合的CaM,在钙离子存在下可以激活膜上的靶酶——Ca~(2+)+Mg~(2+)-ATP酶活性,为研究CaM功能及有关药物机制提供一种简便而理想的材料。     

Infection with a helminth parasite prevents experimental colitis via a macrophage-mediated mechanism

The propensity of a range of parasitic helminths to stimulate a Th2 or regulatory cell-biased response has been proposed to reduce the severity of experimental inflammatory bowel disease. We examined whether infection with Schistosoma mansoni, a trematode parasite, altered the susceptibility of mice to colitis induced by dextran sodium sulfate (DSS). Mice infected with schistosome worms were refractory to DSS-induced colitis. Egg-laying schistosome infections or injection of eggs did not render mice resistant to colitis induced by DSS. Schistosome worm infections prevent colitis by a novel mechanism dependent on macrophages, and not by simple modulation of Th2 responses, or via induction of regulatory CD4+ or CD25+ cells, IL-10, or TGF-beta. Infected mice had marked infiltration of macrophages (F4/80+CD11b+CD11c(-)) into the colon lamina propria and protection from DSS-induced colitis was shown to be macrophage dependent. Resistance from colitis was not due to alternatively activated macrophages. Transfer of colon lamina propria F4/80+ macrophages isolated from worm-infected mice induced significant protection from colitis in recipient mice treated with DSS. Therefore, we propose a new mechanism whereby a parasitic worm suppresses DSS-induced colitis via a novel colon-infiltrating macrophage population.     

Dihydropyridines and atypical MDR: a novel perspective of designing general reversal agents for both typical and atypical MDR

Multidrug resistance (MDR) is defined as resistance of tumor cells to the cytotoxic action of multiple structurally dissimilar and functionally divergent drugs commonly used in chemotherapy. 1,4-Dihydropyridines (DHP(s)) are one of the major classes of Ca2+ channel blockers, and it has been shown that these agents are a new class of drug resistance reversers in cancer treatment. Analysis of various investigations on MDR reversing effects of DHPs shows that they can be a potential class for designing compounds simultaneously effective on both typical and atypical MDR. Also, it is possible to include some considerations on essential structural features for MDR reversing and further decreasing of Ca2+ channel blocking activity as an adverse effect.