Biochemical and electrophysiological studies on the mechanism of action of PNU-151774E, a novel antiepileptic compound.
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PNU-151774E [(S)-(+)-2-(4-(3-fluorobenzyloxy)benzylamino)propanamide methanesulfonate], a new anticonvulsant that displays a wide therapeutic window, has a potency comparable or superior to that of most classic anticonvulsants. PNU-151774E is chemically unrelated to current antiepileptics. In animal seizure models it possesses a broad spectrum of action. In the present study, the action mechanism of PNU-151774E has been investigated using electrophysiological and biochemical assays. Binding studies performed with rat brain membranes show that PNU-151774E has high affinity for binding site 2 of the sodium channel receptor, which is greater than that of phenytoin or lamotrigine (IC50, 8 microM versus 47 and 185 microM, respectively). PNU-151774E reduces sustained repetitive firing in a use-dependent manner without modifying the first action potential in hippocampal cultured neurons. In the same preparation PNU-151774E inhibits tetrodotoxin-sensitive fast sodium currents and high voltage-activated calcium currents under voltage-clamp conditions. These electrophysiological activities of PNU-151774E correlate with its ability to inhibit veratrine and KCl-induced glutamate release in rat hippocampal slices (IC50, 56.4 and 185.5 microM, respectively) and calcium inward currents in mouse cortical neurons. On the other hand, PNU-151774E does not affect whole-cell gamma-aminobutryic acid- and glutamate-induced currents in cultured mouse cortical neurons. These results suggest that PNU-151774E exerts its anticonvulsant activity, at least in part, through inhibition of sodium and calcium channels, stabilizing neuronal membrane excitability and inhibiting transmitter release. The possible relevance of these pharmacological properties to its antiepileptic potential is discussed. (+info)
Reduced coronary NO production in conscious dogs after the development of alloxan-induced diabetes.
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The role of nitric oxide (NO) in the control of coronary blood flow (CBF) during the development of diabetes is unknown. To study this, mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and CBF. With heart rate controlled (150 beats/min), veratrine (1-10 micrograms/kg) caused dose-dependent increases in CBF; e.g., 5 mirograms/kg of veratrine increased CBF by 57 +/- 7% from 41 +/- 1.3 ml/min (P < 0.05). The dogs developed diabetes 4-5 wk after injection of alloxan (40-60 mg/kg iv, blood glucose levels were 384 +/- 18 mg/dl). After diabetes the same doses of veratrine caused smaller increases in CBF; i.e., 5 micrograms/kg of veratrine increased CBF by 32 +/- 2% (P < 0.05 compared with control) from 28 +/- 4 ml/min. ACh- and adenosine-induced coronary vasodilation were reduced after diabetes as well. In anesthetized dogs after diabetes, vagal stimulation caused smaller increases in CBF. ACh and bradykinin caused smaller increases in NO(-)(2) production in coronary microvessels from diabetic dogs. Furthermore, despite the fact that mRNA for endothelial cell NO synthase from the aorta was increased twofold with the use of Northern blotting, the protein for aortic endothelial constitutive NO synthase was reduced by 66% after diabetes, as determined by Western blotting. Our results indicate that the NO-dependent coronary vasodilation by the Bezold-Jarisch reflex is impaired in conscious dogs after diabetes. The mechanism responsible for the impaired endothelium-dependent coronary vasodilation is most likely the decreased release of NO from the endothelium. (+info)
The emetic reflex in a reptile (Crocodylus porosus).
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The emetic (vomiting) reflex in a crocodilian, Crocodylus porosus, was characterised for the first time using the plant alkaloid veratrine (5 mg kg(-)(1) i.v. or i.p.) as an emetic stimulus. The latency to the onset of vomiting was 8.0+/-0.9 min (mean +/- s.e.m., N=5 animals). Vomiting was preceded by a clearly defined set of prodromal behaviours including, in temporal sequence, rhythmic contraction of the pharynx, sneezing and jaw snapping. Expulsion of vomitus was not particularly forceful and was accompanied by lateral shaking of the head. Physiological studies revealed that vomiting was accompanied by oscillatory (9.1+/-0.7 oscillations over 29.7+/-3. 6 s, N=9 episodes in three animals) increases in intraperitoneal pressure (7.0+/-0.9 kPa, cf. 0.7+/-0.1 kPa during respiration). The significance of these results is discussed in the context of the role(s) of vomiting as a protective reflex and as a mechanism for removal of indigestible food residues (e.g. fur, claws) from the gut. (+info)
Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs.
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Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg. kg(-1). day(-1)) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 +/- 1.2 to 7. 79 +/- 1.3 microM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MVO(2)) decreased from 6.8 +/- 0.6 to 5.9 +/- 0.4 ml/min (P < 0.05); NO-dependent regulation of MVO(2) in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MVO(2). (+info)
Role of cardiac nerves in the cardiovascular response to cocaine in conscious dogs.
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BACKGROUND: Although the cardiovascular toxicity of cocaine is well recognized, considerable controversy remains as to the relative contribution of local norepinephrine reuptake inhibition versus central stimulatory effects of cocaine in eliciting its cardiovascular actions. The purpose of the present study was to determine the role of cardiac nerves in mediating the left ventricular (LV) and coronary hemodynamic responses to cocaine. METHODS AND RESULTS: We studied the cardiovascular response to acute cocaine administration (1 mg/kg) in 10 intact, conscious dogs and 6 dogs with ventricular denervation (VD). There were no significant differences in baseline hemodynamic parameters or plasma catecholamines between the 2 groups. In response to acute cocaine, LV and coronary hemodynamic responses were enhanced in the VD dogs. The enhanced systemic pressor and heart rate responses in VD dogs suggest that cardiac nerves mitigate the response to cocaine through ventricular mechanoreceptors rather than mediating the responses. CONCLUSIONS: These data suggest that peripheral blockade of norepinephrine reuptake is not the principal mechanism of the acute cardiac effects of cocaine. Rather, cardiac nerves modulate the effects of cocaine through baroreflex mechanisms. Thus, individual differences in baroreflex sensitivity may explain the hemodynamic variability observed in response to cocaine. (+info)
LC-EI-MS determination of veratridine and cevadine in two fatal cases of Veratrum album poisoning.
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We report two fatal poisonings due to the ingestion of plant material. The two deceased were discovered in the water of a mountain lake about one month after the postmortem immersion of the corpses. Macroscopic examination of the stomachs revealed the presence of a very large number of small blackish granules, which were later identified as seeds of a Veratrum species. Veratridine and cevadine were identified and quantitated by high-performance liquid chromatography-electrospray ionization-mass spectrometry. Measured blood concentrations were 0.17 and 0.40 ng/mL for veratridine and 0.32 and 0.48 ng/mL for cevadine. The absence of other toxic substance led to the assumption that this massive ingestion was the cause of death, although the circumstances surrounding intake remained unknown. (+info)
Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.
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The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent cations by the ionophore. (+info)
Immunohistochemical demonstration of c-fos protein in neurons of the medulla oblongata of the musk shrew (Suncus murinus) after veratrine administration.
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We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting. (+info)