α2C-Adrenergic Receptor Blockade Inhibits Langendorff-Isolated Rat Heart Work.

We studied the effect of selective α2C-adrenergic receptor antagonist JP-1302 in concentrations of 10-9-10-6 M on inotropy, chronotropy, and coronary flow in the Langendorff-isolated rat heart. JP-1302 in all studied concentrations decreased the left-ventricular myocardium force contraction, HR, and coronary flow. The maximum inotropic, chronotropic, and vascular effects were observed when the antagonist was applied to the perfused solution in a concentration of 10-7 M. The least pronounced decrease in the studied parameters was observed at JP-1302 concentrations of 10-8 and 10-9 M. The obtained data indicate the participation of this subtype of α2-adrenergic receptors in the regulation of activity of isolated adult rats heart.

The Effect of α2-Adrenoreceptors Blockade on the Isolated Rat Heart during the Formation of Sympathetic Innervation.

We studied the effects of the α2-adrenergic receptor antagonist yohimbine (10-9-10-6 M) on inotropy, chronotropy, and coronary flow in the Langendorff-isolated heart from 3- and 6-week-old rats. Yohimbine affected all the studied functional parameters of the isolated heart. The force of contraction of the left ventricular myocardium, HR, and coronary flow decreased at all studied concentrations of the antagonist. The maximum chronotropic effect was observed in the heart from 6-week-old rats in the presence of 10-6 M yohimbine in the perfused solution and in the heart from 3-week-old rats in the presence of 10-9 M yohimbine. The minimum chronotropic and inotropic effect was recorded in the hearts from 6-week-old animals after application of 10-9 M α2-adrenergic receptor blocker and in the hearts from 3-week-old animals at yohimbine concentration of 10-7 M. The least pronounced decrease in coronary flow in 3- and 6-week-old rats was observed at the minimum concentration of yohimbine.

Effect of Clonidine Hydrochloride on Isolated Newborn Rat Heart.

The concentration dependenies of the chronotropic response and changes in blood supply to the isolated heart of 7-day-old newborn rats induced by application of α2-adrenergic receptor agonist clonidine hydrochloride in concentrations of 10-9-10-6 M were revealed. The minimum concentration of α2-adrenergic receptor agonist caused tachycardia, while higher concentrations led to bradycardia. The maximum effect manifesting in a decrease in coronary flow was recorded at the minimum concentration of the agonist, while the highest concentration had no effect on the coronary flow. When comparing these results with those obtained in control adult rats, we found that the most pronounced differences in the chronotropic effects were observed after addition of the minimum concentration of the α2-adrenergic receptor agonist: bradycardia in adult rats and tachycardia in newborns. The maximum differences in coronary flow parameters were observed after addition of α2-adrenergic receptor agonist in the maximum concentration that induced a two-phase response in adult rats and had no effect on the blood supply in newborns.

Comparative Analysis of Cardiac Effects of α1A-Adrenoreceptor Stimulation In Vivo and Ex Vivo in Newborn Rats.

The study examined the effects of α1A-adrenoceptor stimulation on chronotropic function of Langendorff-perfused isolated heart ex vivo and on cardiac chronotropy in vivo in 7-day-old rats. α1A-Adrenergic receptor agonist A-61603 reduced heart chronotropy only in the whole organism. No chronotropic effects of selective stimulation of α1A-adrenergic receptors on isolated hearts were observed in ex vivo experiments. These findings suggest that α1A-adrenergic receptors are not implicated in HR regulation in newborn rats. Bradycardia induced by activation of these receptors in vivo is most likely associated with reflex influences on the heart and changes in the vascular tone in the whole organism.

Effect of Stimulation of α2-Adrenergic Receptors on Action Potential of Working Cardiomyocytes in Rat Atrium.

The study examined the effect of clonidine hydrochloride (10-9-10-5 М) on electrical activity of working cardiomyocytes in rat right atrium. Stimulation of α2-adrenergic receptor with clonidine changed electrical activity of the heart. All tested concentrations of the agonist lengthened the action potential and decreased the firing rate of cardiomyocytes in a dose-dependent manner. The maximum effects of clonidine were observed at concentration of 10-5 М.

Effect of Neuropeptide Y on Action Potential of Working Right Atrial Cardiomyocytes in Early Postnatal Ontogeny.

The effects of neuropeptide Y (10-9-10-6 M) on electrical activity of right atrial cardiomyocytes of rats aging 7, 21, and 100 days were examined in vitro. Neuropeptide Y affected the amplitude-temporal parameters of the action potential in these cells. It decreased the duration of repolarization phase in 7-day-old rats in concentrations of 10-8 and 10-7 M, in 21-day-old rats at 10-8 and 10-6 M, and in 100-day-old at 10-6 M. The data indicate elevation of total membrane potassium current under the action of neuropeptide Y.

Effect of If Current Blockade on Newborn Rat Heart Isolated According to Langendorff.

The study examined the effects of hyperpolarization-activated funny current (If) on HR and coronary flow in Langendorff-isolated hearts from newborn rats. Blockade of If current with ZD7288 changed the examined cardiac parameters. The blocker in a concentration of 10-9 M decreased HR by 26.8% (p≤0.05). In concentrations 10-8, 10-7, 10-6, and 10-5 M ZD7288 produced minor differently directed effects. In a concentration of 10-5 M, ZD7288 reduced coronary flow in the isolated heart (p≤0.01). In other concentrations, the blocker produced no significant effects on coronary flow.

Abnormal Membrane Localization of α2 Isoform of Na,K-ATPase in m. soleus of Dysferlin-Deficient Mice.

Dysferlin protein plays a key role in the multimolecular complex responsible for the maintenance of sarcolemma integrity and skeletal muscle cell functioning. We studied the membrane distribution of nicotinic acetylcholine receptors and α2 isoform of Na,K-ATPase in motor endplates of m. soleus in dysferlin-deficient Bla/J mice (a dysferlinopathy model). Endplates of Bla/J mice were characterized by increased area (without changes in fragmentation degree) and reduced density of the membrane distribution of nicotinic acetylcholine receptors in comparison with the corresponding parameters in control С57Bl/6 mice. The density of the membrane distribution of α2 isoform of Na,K-ATPase was also reduced, but the level of the corresponding mRNA remained unchanged. It can be hypothesized that abnormal membrane localization of α2 isoform of Na,K-ATPase results from adaptive skeletal muscle remodeling under conditions of chronic motor dysfunction.

Correction to: Abnormal Membrane Localization of α2 Isoform of Na,K-ATPase in m. soleus of Dysferlin-Deficient Mice.

The third author's name should read: V. V. Matchkov.

Effect of Neuropeptide Y on Action Potential Generation in Working Cardiomyocytes of the Right Atrium in Rat Heart.

We studied the effect of neuropeptide Y in concentrations of 10-8-10-6 M on electrical activity of adult rat right atrial cardiomyocytes with preserved spontaneous activity. Neuropeptide Y was found to modulate the amplitude-time parameters of action potential: in concentrations of 10-7 and 10-6 M it reduced the membrane potential, increased the amplitude of action potential and duration of the repolarization phase, and reduced the frequency of action potential generation. In concentration of 10-6 M, neuropeptide Y produced stronger effect on the analyzed parameters, while in concentration of 10-8 M it produced no significant changes.

Effect of α2-Adrenoceptor Stimulation on Functional Parameters of Langendorff-Isolated Rat Heart.

We studied the effect of α2-adrenoreceptor agonist clonidine hydrochloride in concentrations of 10-9-10-6 M on inotropy, chronotropy, and coronary flow in Langendorff-isolated heart of adult rats. It was found that α2-adrenoreceptor agonist changed all studied parameters. Left ventricular myocardium contraction force decreased after application of all tested concentrations, the maximum effect was observed at a concentration of 10-6 M. Stimulation of α2-adrenergic receptors in concentrations of 10-8, 10-7, and 10-6 M produced a two-phase effect (initial increase and a subsequent decrease) on the coronary flow. Clonidine hydrochloride in the maximum concentration (10-6 M) caused a decrease in HR in one group and an increase in the other.

Dysfunction of Neuromuscular Synapses in the Genetic Model of Alzheimer's Disease.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction.

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Peculiar Aspects in Influence of α1-Adrenoceptor Stimulation on Isolated Rat Heart.

The study examined the effect of α1-adrenoceptor stimulation with methoxamine on chronotropic function of isolated heart perfused ex vivo according to Langendorff and cardiac chronotropy in vivo. Stimulation of α1-adrenoceptors in isolated heart induced gradually developing bradycardia, which progressed during several minutes. Similar stimulation in vivo produced a short-term bradycardia probably terminated by the compensatory influences in the whole organism. Comparison of the data obtained in both experimental paradigms during α1-adrenoceptor stimulation revealed unidirectional changes in cardiac chronotropy characterized with time-related peculiarities.

Selective Blockade of α2-Adrenoceptor Subtypes Modulates Contractility of Rat Myocardium.

The study examined the dose-dependent effects of selective antagonists of α2A/D-, α2B-, and α2C- adrenoceptors applied in concentrations of 10-9-10-5 M on atrial and ventricular contractility of rat myocardium in vitro. Selective blockade of each α2-adrenoceptor subtype affected the contractile force of the atrial and ventricular strips. Various concentrations of α2A/D- and α2C-adrenoceptor antagonists produced positive inotropic effect on ventricular strips and negative effect on atrial strips. α2B-Adrenoceptor blocker in the majority of the tested concentrations produced a positive inotropic effect in both atria and ventricles.

Ketamine-Midazolam Anesthesia Induces Total Inhibition of Cortical Activity in the Brain of Newborn Rats.

The effects of general anesthetics ketamine and midazolam, the drugs that cause neuroapoptosis at the early stages of CNS development, on electrical activity of the somatosensory cortex in newborn rats were studied using extracellular recording of local field potentials and action potentials of cortical neurons. Combined administration of ketamine (40 mg/kg) and midazolam (9 mg/kg) induced surgical coma and almost completely suppressed early oscillatory patterns and neuronal firing. These effects persisted over 3 h after injection of the anesthetics. We concluded that general anesthesia induced by combined administration of ketamine and midazolam profoundly suppressed cortical activity in newborn rats, which can trigger neuroapoptosis in the developing brain.

Effect of ICa,L Blockade on Adrenergic Stimulation in Developing Heart.

The effect of verapamil-induced blockade of L-type calcium ionic currents (ICa,L) on the action of non-selective adrenergic cardiac stimulation by norepinephrine was examined during different periods of early postnatal ontogeny. In 1-week-old rats, intravenous norepinephrine induced a short-term tachycardia both with and without preliminary injected verapamil. In 3-week-old rats, norepinephrine alone produced no chronotropic effect; in contrast, it induced a biphasic tachycardia in verapamil-treated rats. In 6- and 20-month-old rats, norepinephrine induced a short-term tachycardia, which could be prevented by verapamil. The age-related peculiarities of chronotropic action of non-selective adrenergic stimulation are indicative of the role of L-type calcium ionic channels in the development of sympathetic control over the heart.

Myosin accelerates synaptic vesicle recycling in the motor nerve endings.

Neurotransmitter release and exocytosis of synaptic vesicles in the motor nerve endings of the frog cutaneous-pectoris muscle were studied using electrophysiological and optical methods under the conditions of inhibition of the myosin light-chain kinase and non-muscle myosin by the specific inhibitors ML-7 (12 µM) and (-)-blebbistatin (100 µM). At high-frequency stimulation (20 pulses/s), these inhibitors strengthened suppression of transmitter release during the first 20-25 s and slowed down the release of the fluorescent dye FM 1-43. The obtained results indicate that myosin accelerates rapid synaptic vesicle recycling upon high-frequency stimulation.