Effects of thyroxine pretreatment and calcium on the isolated perfused rat heart

Phosphorylase a activity was the same in isolated perfused hearts from euthyroid and thyroxine-pretreated rats. Perfusion with 3.6 mM Ca2+ caused an increase in phosphorylase a in hearts from euthyroid as well as those from thyroxine-pretreated animals, but the Ca2+-induced stimulation of phosphorylase activity was similar in both groups over the time course studied. Greater conversion of phosphorylase b to a occurred with 7.2 mM than with 3.6 mM Ca2+ in both groups, but once again thyroxine pretreatment did not significantly influence the conversion of phosphorylase b to a. Isometric systolic tension increased in response to 3.6 mM and 7.2 mM Ca2+ in hearts from normal and thyrotoxic rats, but thyroxine pretreatment did not appreciably alter the nature of this response. While spontaneous heart rate was higher in hearts from thyroxine-pretreated rats, perfusion with 3.6 mM or 7.2 mM Ca2+ had no significant effect on heart rate in hearts from euthyroid or thyrotoxic rats.     

Mechanisms of the contractile effects of levosimendan in the mammalian heart

In spontaneously beating guinea pig right atria, levosimendan (LS, or R-[[-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)- phenyl]-hydrazono]propanedinitrile) exerted a positive chronotropic effect starting at 0.1 microM. In electrically driven guinea pig left atria, LS (0.1-10 microM) increased force of contraction without changing time parameters of contraction. In electrically driven right papillary muscles, LS (0.1-10 microM) enhanced force of contraction without affecting time parameters of contraction. The maximal effect on force of contraction at 10 microM amounted to 130 +/- 8.6% of predrug value. The positive inotropic effect of LS in papillary muscles was greatly diminished by additionally applied carbachol. In [32P]-labeled guinea pig ventricular cardiomyocytes, LS increased the phosphorylation state of phospholamban, the inhibitory subunit of troponin and C-protein. The maximal effect at 1 microM amounted to 134 +/- 8.6%, 124 +/- 4.2% and 121 +/- 8% of control for phospholamben, the inhibitory subunit of troponin and C-protein, respectively. LS (1 microM) increased cAMP content from 6.3 +/- 0.3 to 8.1 +/- 0.3 pmol/mg protein in guinea pig ventricular cardiomyocytes. Furthermore, whole-cell patch-clamp studies were performed in guinea pig ventricular cardiomyocytes. In this setup, 10 microM LS increased the amplitude of L-type Ca++ current to 402 +/- 86% of predrug value.     

Mechanisms of plumbagin action on guinea pig isolated atria

In electrically driven guinea pig left atria, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; 0.5-10 microM) produced a marked positive inotropic effect that was about 65% that caused by isoprenaline in the same experimental conditions. The effect was mainly not dependent on catecholamine release from adrenergic stores. An EC50 of 3 microM was calculated from the concentration-response curves. The increase in force of contraction was followed by a nonreversible contracture. Plumbagin was reduced by cardiac mitochondrial and soluble reductases with consequent generation of large amounts of superoxide anion. The assay of reduced glutathione/oxidized glutathione content in atria, treated with 10 microM plumbagin and frozen at the appearance of increase in diastolic tension, showed a significant decrease in reduced glutathione (-52% with respect to control atria) and a 5-fold increase in oxidized glutathione levels. Moreover, in the same experimental conditions a significant decrease in adenosine triphosphate (-55% with respect to the controls) and in adenylate energy charge (from 0.92-0.64) was observed. Of the enzymes and transport systems involved in the control of the cardiac contractility, the sarcoplasmic reticulum Ca2+ pump seemed to be a specific target for plumbagin. After 30 min of incubation with cardiac sarcoplasmic reticulum membrane vesicles, plumbagin inhibited Ca2+ uptake by the pump in a concentration-dependent manner (IC50 = 3 microM). On the basis of these results, the increase in diastolic tension caused by plumbagin appears to be related to intracellular Ca2+ accumulation, due both to the low availability of adenosine triphosphate for ionic pumps and direct inhibition of Ca2+ reuptake in sarcoplasmic reticulum.     

Contraction and resting stiffness of isolated cardiac muscle: effects of inotropic agents

The purpose of this study was to test the hypothesis that either hypoxia and its combined effects with extracellular calcium (Ca), digoxin, and ouabain, or these positive inotropic agents acting alone or in combination, influence contraction and resting stiffness of isolated papillary muscle. Stiffness was measured utilizing the sinusoidal forcing function technique. Neither an increase in extracellular calcium concentration (from 2.5 to 4.0 mM) nor digoxin or ouabain in either Ca concentration altered contraction or resting stiffness in the well-oxygenated environment. Resting stiffness for any given resting tension was increased at the end of hypoxia only in the presence of digoxin, and this occurred in both 2.5 mM Ca (P less than 0.02) and in 4.0 mM Ca (P = 0.05). Contraction stiffness for any given tension was increased in 2.5 mM Ca by hypoxia alone (P less than 0.05) and by hypoxia in the presence of digoxin (P less than 0.005) and ouabain (P less than 0.02), but was not increased in any experiments conducted in 4.0 mM Ca. The conclusions from these data are that certain experimental conditions of the study evoked different directional changes in stiffness and contractility. Further, changes in contraction stiffness are not always paralleled by changes in resting stiffness.     

EMD 53998 acts as Ca(2+)-sensitizer and phosphodiesterase III-inhibitor in human myocardium

The effect of EMD 53998 (EMD) (0.1-100 mumol/l), chemically a racemic thiadiazinone derivative, suggested to be a potent Ca(2+)-sensitizer, was studied in human failing and nonfailing left ventricular myocardium. For comparison, the effects of the pyridazinone derivative pimobendan (0.1-300 mumol/l), isoprenaline (Iso) (0.001-3 mumol/l) as well as CaCl2 (1.8-15 mmol/l Ca2+) were investigated. The positive inotropic responses were examined in electrically driven (1 Hz, 37 degrees C) human left ventricular papillary muscle strips from terminally failing hearts (NYHAIV, n = 24) and nonfailing donor hearts (NF, n = 9). The effect of EMD on the Ca(2+)-sensitivity of skinned fiber preparations from the very same human failing hearts were studied as well. EMD and pimobendan increased force of contraction (FOC) in a concentration-dependent manner. As judged from the EC50-values, EMD increased FOC more potently than pimobendan. EMD was significantly more effective than pimobendan to increase FOC in papillary muscle strips from NYHA IV (EMD: +2.5 +/- 0.1 mN; pimobendan: +0.8 +/- 0.2 mN) as well as from nonfailing hearts (EMD: +3.1 +/- 0.5 mN; pimobendan: +1.2 +/- 0.2 mN). Only in terminally failing myocardium, EMD increased FOC as effectively as Iso. After inotropic stimulation with EMD, pimobendan, or Iso, carbachol (1000 mumol/l) reduced FOC in left ventricular papillary muscle strips, indicating a cAMP-dependent mode of action. In skinned fiber experiments, EMD increased Ca(2+)-sensitivity significantly more (p < 0.01) than pimobendan. In conclusion: EMD increases FOC in human myocardium via sensitizing of the contractile proteins towards Ca2+ and by inhibition of phosphodiesterase III-isoenzymes. EMD is a potent calcium sensitizing agent in human myocardium. Thiadiazinone derivatives could be one step in the evolution to more potent and selective calcium-sensitizers.     

Effects of calcium antagonist, 6-(N, N-diethylamino) hexyl-3, 4, 5-trimethoxybenzoate, on digitalis-induced arrhythmias and cardiac contractions

6-(N, N-Diethylamino) hexyl-3, 4, 5-trimethylbenzoate (TMB-6) and lidocaine were equipotent (1 mg/kg) in the conversion of ectopic rhythms to normal rhythms in digoxin-toxic dogs. However, TMB-6 had fewer side effects on heart rates and dp/dt than lidocaine. TMB-6 inhibited the contractile force of electrically stimulated dog and guinea-pig atria and ventricles at concentrations ranging from 2.5 X 10(-5) to 1.7 X 10(-4) M. Elevation of extracellular Ca++ concentrations from 2.7 to 5.4 mM produced a significant increase in the ID50 of TMB-6 in atria (from 2.5 X 10(-5) to 5.0 X 10(-5) M in dogs and from 7.2 X 10(-5) to 1.0 X 10(-4) M in guinea pigs). TMB-6 (7.3 X 10(-5) to 2.4 X 10(-4) M) depressed the amplitude of Ca++-dependent action potentials in depolarized dog cardiac Purkinje fibers. These results are discussed with regard to the antagonism of TMB-6 on Ca++ availability in the myocardium which leads to the conversion of cardiac arrhythmias.     

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.     

Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris

The influence of thapsigargin, a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+ ATPase, on the positive inotropic effects of digoxin before and after pretreatment with rimalkalim [(3S,4R)-3-hydroxy-2,2-dimethyl-4-(oxopyrrolidinyl)-6-phenyl-su lfonylchroman hemihydrate (formerly HOE 234)], a known activator of ATP-sensitive K+ channels, was studied in the guinea pig heart. The isolated papillary muscles from the guinea pig heart were used to study these effects. The following parameters were measured: force of contraction (Fc), rate of rise (+dF/dt) and rate of fall (-dF/dt) of Fc, time to peak contraction (ttp) and time to 10% of the total amplitude of force (tt10). After pretreatment with rimalkalim (1 microM), digoxin caused a significant increase in the amplitude of Fc and significant shortening of ttp and tt10 (p < 0.05 compared with the values obtained with digoxin alone). Thapsigargin (1 microM), a selective inhibitor of sarcoplasmic reticulum Ca2+ ATPase, added to rimalkalim, prevented the enhancement of the amplitude of Fc induced by digoxin after pretreatment with rimalkalim but had no significant influence on the effects of digoxin itself. The results demonstrate significant influence of activation of KATP channels on digoxin-induced positive inotropic effects in the guinea pig heart. Attenuation of this effects of rimalkalim by addition of thapsigargin suggests that activation of SR Ca2+ ATPase can be included in this interaction.     

Monensin-induced reversal of positive force-frequency relationship in cardiac muscle: role of intracellular sodium in rest-dependent potentiation of contraction

We have investigated the role of a rest-dependent inotropic factor in determining species-related differences in cardiac force-frequency relationships (FFR). Isolated rat, rabbit or guinea-pig papillary muscles, as well as guinea-pig ventricular myocytes were superfused with 1.8 mM Ca2+ Tyrode. In rat muscles, isometric force amplitude decreased, while in rabbit or guinea-pig muscles force increased with frequency (0.02-1 Hz). Paired-pulse pacing potentiated contraction markedly at all frequencies in rabbit muscles, but not at low frequencies in rat muscles. We tested the hypothesis that high intracellular Na+ levels (Nai) are responsible for negative FFR. The ionophore monensin increased Nai, reversed the FFR of rabbit and guinea-pig muscles from positive to negative, by increasing force mostly at low frequencies, and decreased the paired-pulse potentiation of contraction at low frequencies. Monensin added during rest also reversed rest-induced decay. In isolated myocytes, monensin had qualitatively similar effects on cell shortening as well as on Cai transients. Monensin also decreased the action potential duration (APD) but did not change the pattern of its variation with frequency. Cells intracellularly dialyzed with 20 mM Na+ via a patch pipette also showed rest potentiation of the Cai transients, in contrast to cells dialyzed with 10 mM Na+, which showed rest decay of the transients. APD was also shorter in myocytes dialyzed with 20 mM Na+ than in those dialyzed with lower Na+. The results indicate that in the presence of high Nai, sarcoplasmic reticular Ca2+ load is increased during diastole, possibly via reverse-mode Na+/Ca2+ exchange, and therefore that Nai is an important factor determining the FFR. In addition, the data suggest that short APDs in preparations showing negative FFR may be partly a consequence of increased Nai.     

Formation of giant liposomes promoted by divalent cations: critical role of electrostatic repulsion

Spontaneous formation of giant unilamellar liposomes in a gentle hydration process, as well as the adhesion energy between liposomal membranes, has been found to be dependent on the concentration of divalent alkali cations, Ca2+ or Mg2+, in the medium. With electrically neutral phosphatidylcholine (PC), Ca2+ or Mg2+ at 1-30 mM greatly promoted liposome formation compared to low yields in nonelectrolyte or potassium chloride solutions. When negatively charged phosphatidylglycerol (PG) was mixed at 10%, the yield was high in nonelectrolytes but liposomes did not form at 3-10 mM CaCl2. In the adhesion test with micropipette manipulation, liposomal membranes adhered to each other only in a certain range of CaCl2 concentrations, which agreed with the range where liposome did not form. The adhesion range shifted to higher Ca2+ concentrations as the amount of PG was increased. These results indicate that the divalent cations bind to and add positive charges to the lipids, and that membranes are separated and stabilized in the form of unilamellar liposomes when net charges on the membranes produce large enough electrostatic repulsion. Under the assumption that the maximum of adhesion energy within an adhesive range corresponds to exact charge neutralization by added Ca2+, association constants of PC and PG for Ca2+ were estimated at 7.3 M(-1) and 86 M(-1), respectively, in good agreement with literature values.     

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of 6-dimethylaminopurine

The effects of NS 1619, a newly developed activator of large-conductance Ca2+-activated K+ channels, were investigated on single smooth muscle fibers dissociated enzymatically from rat vas deferens and on contractions of the epididymal half of vas deferens. K+ currents were recorded using whole-cell patch-clamp methods in near-physiological K+ solutions (5.4 mM extracellular K+/145 mM intracellular K+). When cell membrane voltage was stepped to test potentials (-60 to +60 mV) from a holding potential of -10 mV, NS 1619 increased the outwardly rectifying K+ current in a concentration-dependent manner. The increased portion of the K+ current by NS 1619 was totally abolished by charybdotoxin (100 nM) but not by glibenclamide (3 microM). NS 1619 reduced electrically stimulated contractile responses of rat vas deferens in a concentration-dependent manner, and charybdotoxin but not glibenclamide partially inhibited the effect of NS 1619. NS 1619 (50 microM) inhibited the noradrenaline-induced contraction. Charybdotoxin (100 nM) partially reduced the NS 1619-induced inhibition while glibenclamide (3 microM) had no effect. NS 1619 (10-100 microM) reduced the high K+-induced contractions in a noncompetitive manner. The present results indicate that NS 1619 activates charybdotoxin-sensitive Ca2+-activated K+ channels and probably inhibits Ca2+ influx. These two effects might account largely for the observed mechanical inhibition induced by NS 1619 in the epididymal half of isolated rat vas deferens.     

Calcium-sensitive calcium influx in photoreceptor inner segments

The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.     

Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release

Alpha 2-Adrenoceptors are known to inhibit voltage-dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic alpha 2-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10 microM nicotine, or by adding Ca2+ to otherwise Ca(2+)-free medium when cells had been made permeable by the calcium ionophore A23187 or by alpha-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker omega-conotoxin GVIA and application of the alpha 2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with alpha-latrotoxin or A23187. In contrast, the L-type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or alpha-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In omega-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with omega-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented alpha 2-adrenergic effects. Our results indicate that the alpha 2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of omega-conotoxin GVIA-sensitive N-type Ca2+ channels. Mechanisms downstream of these channels and voltage-sensitive Ca2+ channels other than N-type appear not to be important.     

Expression cloning and biochemical characterizations of recombinant cyclophilin proteins from Schistosoma mansoni

Cannabinoid receptor agonists inhibit electrically evoked isometric contractions of the myenteric plexus--longitudinal muscle preparation of the guinea-pig small intestine (MPLM), probably by reducing release of acetylcholine (ACh) through the activation of prejunctional CB1 receptors. As CB1 receptors are thought to be negatively coupled through Gi/o proteins to both N-type Ca2+ channels and adenylate cyclase, we have now further investigated the involvement of CB1 receptors by monitoring the effects of forskolin, 8-bromo-cAMP, 3-isobutyl-1-methylxanthine (IBMX), and extracellular Ca2+ on the ability of the cannabinoid agonist, (+)-WIN 55212 to inhibit electrically evoked contractions of the MPLM (0.1 Hz, 0.5 ms, and 110% maximal voltage). Some experiments were performed with normorphine instead of (+)-WIN 55212. At 10(-7) M, forskolin, 8-bromo-cAMP, and IBMX were found to reduce significantly the maximum inhibitory response to (+)-WIN 55212 by 49.4, 48.4, and 40.2%, respectively, without affecting control contractions or responses to exogenous ACh. Low external Ca2+ (0.64 mM) significantly increased the maximum response to (+)-WIN 55212 and shifted the curve slightly leftwards, whereas high external Ca2+ (5.08 mM) reduced the maximum response by 27.2%. The concentration-response curve to normorphine, which also reduces evoked contractions of this preparation as a result of a presynaptic inhibition of ACh release via opioid mu receptors, was affected similarly. These results support the hypothesis that cannabinoid-induced inhibition in the MPLM is mediated by CB1 receptors.     

Aconitum sp. alkaloids: the modulation of voltage-dependent Na+ channels, toxicity and antinociceptive properties

Alkaloids from Aconitum sp., used as analgesics in traditional Chinese medicine, were investigated to elucidate their antinociceptive and toxic properties considering: (1) binding to Na+ channel epitope site 2, (2) alterations in synaptosomal Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), (3) arrhythmogenic action of isolated atria, (4) antinociceptive and (5) acute toxic action in mice. The study revealed a high affinity group (Ki 1 microM) and a low affinity group (Ki 10 microM) of alkaloids binding to site 2. The compounds of the high affinity group induce an increase in synaptosomal [Na+]i and [Ca2+]i (EC50 3 microM), are antinociceptive (ED50, 25 microg/kg), provoke tachyarrhythmia and are highly toxic (LD50 70 microg/kg), whereas low affinity alkaloids reduce [Ca2+]i, induce bradycardia and are less antinociceptive (ED50 20 mg/kg) and less toxic (LD50 30 mg/kg). These results suggest that the alkaloids can be grouped in Na+ channel activating and blocking compounds, but none of the alkaloids seem to be suitable as analgesics because of the low LD50/ED50 values.     

The inhibitory effect of gallamine on muscarinic receptors

1 The inhibitory effect of gallamine (1.1 muM-1.1 mM) on negative inotropic responses to acetylcholine (ACh) or carbachol (CCh) was investigated in isolated electrically stimulated atria of the guinea-pig. Gallamine caused parallel rightward shifts of the dose-response curves to the agonists, with no depression of the maximal response. 2 Gallamine (0.11 - 1.1 mM) produced a greater degree of antagnism towards CCh than towards ACh. With either agonist, the degree of antagonism produced by gallamine in high concentrations was less than that expected for a competitive antagonist.. 3 Similar findings were made when either negative inotropic or chronotropic responses were recorded in spontaneously beating guinea-pig atria. The inhibitory effect of gallamine against the negative inotropic response to cholinomimetics in electrically stimulated atria was not altered either in the presence of propranol (17 muM) or in atria obtained from guinea-pigs pretreated with diisopropylphosphorofluoridate (DEP) 12.5 mumol/kg, in divided doses over 3 days). 4 When ACh was used as the agonist, combination of gallamine with atropine (0.05-0.4 muM) produced dose-ratios which were less than expected for combination of two competitive antagonists. The same phenomenon was observed in atria obtained from guinea-pigs pretreated with DFP. 5 It is suggested that the antagonism produced by gallamine is a type of non-competitive inhibition, which has been termed "metaffinoid antagonism". An antagonist of this type allosterically alters the affinity of the agonist for its binding site, rather than changing the effectiveness of the agonist-receptor interaction.     

Protective effect of l-cis-diltiazem on hypercontracture of rat myocytes induced by veratridine

The protective effect of l-cis-diltiazem, the stereoisomer of d-cis-diltiazem, was studied against the veratridine-induced hypercontracture of rat myocytes. Veratridine increased both [Na+]i and [Ca2+]i, but did not cause hypercontracture in the absence of extracellular Ca2+. Both l-cis-diltiazem (0.1-10 microM) and d-cis-diltiazem (10-30 microM) inhibited the hypercontracture and the increase in [Ca2+]i in a concentration-dependent manner. However, l-cis-diltiazem did not exert a negative inotropic effect in K+ (20 mM)-depolarized rat papillary muscles even at a dose of 10 microM. As seen in the case of tetrodotoxin, l-cis-diltiazem and d-cis-diltiazem also suppressed the increase in [Na+]i. The results show that l-cis-diltiazem prevents the veratridine-induced hypercontracture of myocytes by suppression of the [Ca2+]i increase. The attenuation of the [Ca2+]i increase by l-cis-diltiazem was not dependent on inhibition of Ca2+ channels, but was partly due to inhibition of excessive Na+ entry via veratridine-modified Na+ channels.     

Hemolytic properties of Ca(2+)-treated human erythrocytes under hydrostatic pressure

The effect of intracellular Ca2+ on high pressure-induced hemolysis of human erythrocytes was examined. Red cells were incubated with Ca2+ (0.01-1 mM) in the presence of ionophore A23187. The Ca(2+)-loaded cells were subjected to a pressure of 200 mPa. Treatment with 0.1 mM Ca2+ had the greatest suppressive effect on the hemolysis. On removal of intracellular Ca2+, red cells showed a morphological change from echinocytes to normal discocytes but the hemolysis remained unaltered. Measurement of intracellular K+ and viscosity demonstrated that the suppressive effect of Ca2+ on the hemolysis is irreversible and is largely associated with the increase of intracellular viscosity induced by K+ efflux.     

Expression of endothelin 3 by mesenchymal cells of embryonic mouse caecum

The mechanism of radiolabeled levofloxacin ([3H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentration ratio of about 4 to 6. Levofloxacin accumulated in PMNs parallel to the extracellular concentration, without saturation, over the range of 2.5 to 200 mg/liter (linear regression analysis: r = 0.92; P < 0.001). The activation energy was low (36 +/- 7.2 kJ/mol). Levofloxacin uptake was increased in Ca(2+)-depleted, EGTA-containing medium by approximately 33% (P = 0.022), while Ni2+, a Ca2+ channel inhibitor, inhibited it in a concentration-dependent manner, with the concentration that inhibited 50% of control uptake being approximately 2.65 mM. Verapamil (an L-type Ca2+ channel inhibitor) and other pharmacologic agents which modify Ca2+ homeostasis did not modify levofloxacin uptake. Interestingly, Ca2+ and Mg2+ inhibited levofloxacin uptake in a concentration-dependent manner. EGTA, Ni2+, and verapamil did not modify levofloxacin efflux; thapsigargin, a Ca2+ pool-releasing agent, modestly increased the intracellular retention of levofloxacin. In addition, contrary to other fluoroquinolones, probenecid at 1 to 10 mM did not modify either levofloxacin uptake or efflux. These data are consistent with a mechanism of passive accumulation of levofloxacin in PMNs. Extracellular Ca2+ and Mg2+ may influence the structural conformation of levofloxacin or the lipophilicity of PMN membranes, thus explaining their effect on levofloxacin uptake.