Modulation of neuronal evoked responses to the medial septal area of hibernating ground squirrels by some neuropeptides after chronic disconnection between septum and preoptico-hypothalamic structures

Effects of some neuropeptides identified in the brain of hibernators (TSKYR, TSKY, DY) and of monoaminergic neurotransmitters (noradrenaline and serotonin) on responses of the medial septal neurons evoked by intraseptal electrical stimulation were analyzed in slices taken from the ground squirrels with chronic basal undercutting of the septum. Despite the elimination of direct contacts with the preoptic area and afferents ascending in the medial forebrain bundle, the neurons retained almost normal level of reactivity and distribution of the reaction types. The neuropeptides effectively modulated neuronal responses of various types, including oligosynaptic short-latency single-spike responses. The latter were strongly facilitated by the neuropeptides. As a rule, changes in the responses to electrical stimulation were independent of the spontaneous activity shifts (in 78% of the tests). It was suggested that the neuropeptides exert a double influence on the septal neurons: direct nonsynaptic effects on the pacemaker potential responsible for the background activity and modulation of synaptic processes. Our experiments showed that descending influences of the septo-hippocampal system are not crucial for the entrance into the hibernation state and its tonic maintenance. The influences of the thermoregulatory--circadian structures of the preoptico-hypothalamic area determine the paradoxically increased latent excitability of septal neurons that allows the septo-hippocampal system to gate external stimuli and organize arousal of the forebrain during hibernation in case of emergency.     

Regulation of potential-dependent L-type Ca2+ currents by agmatine. Imidazoline receptors in isolated cardiomyocytes

The main goal of the present work was to study the mechanisms of voltage-gated L-type Ca²⁺ currents regulation by agmatine in isolated cardiomyocytes and to determine whether agmatine is involved in mediating the “arginine paradox”. It was shown that agmatine at concentrations from 200 μM to 15 mM inhibited L-type Ca²⁺ currents in isolated cardiomyocytes in a dose-dependent manner. The selective antagonists of α₂-adrenoceptors (α₂-ARs), yohimbine and rauwolscine, did not modulate the effect of agmatine. In contrast, efaroxan and idazoxan known to antagonize both α₂-ARs and type 1 imidazoline receptors (I₁Rs) decreased the efficiency of agmatine almost twofold. The NO synthase inhibitor 7NI insignificantly influenced the suppressive action of agmatine on L-type Ca²⁺ currents, whereas the protein kinase C inhibitor, calphostin C, markedly reduced the effects of agmatine. Arginine did not affect L-type Ca²⁺ currents in the presence of agmatine and vice versa. These data suggest that agmatine is not involved in mediating the “arginine paradox” and that its effects are not due to the activation of endothelial NO synthase (eNOS) followed by cGMP-dependent inhibition of L-type Ca²⁺ current. Most likely, agmatine acts via I₁Rs coupled with the signaling pathway that involves the activation of protein kinase C. Previously nothing was known about possible localization of I₁Rs in isolated cardiomyocytes. Consistently, we have shown that single cardiomyocytes express the nischarin genes homologous to the IRAS gene, which is considered in the modern literature as the major candidate for the gene encoding I₁Rs. To the best our knowledge, this is the first demonstration of I₁Rs expression at the level of individual cells, including cardiomyocytes.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

Modulation of L-type Ca2+ currents and intracellular calcium by agmatine in rat cardiomyocytes

It is shown that agmatine inhibits L-type Ca²⁺ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (I₁Rs) rather than α₂-adrenoceptors (α₂-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca²⁺ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca²⁺ concentration ([Ca²⁺]_in) varied biphasically, depending on the agmatine dose: at 1–500 μM, agmatine decreased [Ca²⁺]_in; at 500 μM-2 mM, [Ca²⁺]_in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca²⁺]_in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of I₁Rs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca²⁺]_in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 μM) or ryanodine (200 μM) were added to the bath to inhibit I₁Rs and ryanodine receptors (RyRs), respectively, [Ca²⁺]_in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 μM), high agmatine doses (10–15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I₁R agonist rilmenidine increased [Ca²⁺]_in in a dose-dependent manner. The effect of rilmenidine was similar to that of agmatine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active cardiomyocytes agmatine at low concentrations decreases [Ca²⁺]_in, does not stimulate I₁Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phospholamban. The effects of high agmatine doses are apparently mediated by I₁Rs and involve RyRs.     

Analysis of ectatomin action on cell membranes.

Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four alpha-helix bundle. At concentrations of 0.05-0.1 microm, ectatomin forms channels in cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 x 10-7 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L-type calcium current was studied. Calcium currents (ICa) in isolated rat cardiac ventricular myocytes were measured using the whole-cell perforated patch-clamp technique. It was shown that ectatomin at concentrations of 0.01-10 nm inhibited ICa after a latency of few seconds. ICa was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of ICa by isoproterenol, an agonist of beta-adrenoreceptors, or forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol- and forskolin-sensitive components of ICa. The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 microm of forskolin, whereas subsequent isoproterenol application did not produce the same effect.     

“Arginine paradox” in cardiomyocytes of Sprague Dawley and spontaneously hypertensive rats: α<Subscript>2</Subscript>-adrenoreceptor-mediated regulation of L-type Ca<Superscript>2+</Superscript> currents by <Emphasis Type="Italic">L</Emphasis>-arginine

The “arginine paradox” in cardiomyocytes isolated from the left ventricle of Spraque Dawlay (SD) and spontaneously hypertensive rats (SHR) was studied. With 1 mM L-arginine in the bath, the addition of 5 mM L-arginine to incubation medium increased NO production and inhibited amplitude of L-type Ca²⁺ currents in SD cardiomyocytes. A variety of compounds, including the antagonist of α₂-adrenoceptors yohimbine and inhibitors of PI3 kinase (wortmanine), NO synthase (7NI), and cGMP-dependent protein kinase (KT5823), dramatically weakened the inhibitory effects of 5 mM L-arginine on Ca²⁺ currents. The agonist of α₂-adrenoceptors guanabenz acetate increased NO production and inhibited Ca²⁺ currents, while wortmanine, 7NI, and KT5823 antagonized guanabenz. In SHR cardiomyocytes, the “arginine paradox” was not observed: 5 mM L-arginine affected neither NO production nor Ca²⁺ currents. Consistently, guanabenz acetate did not alter NO production and inhibited Ca²⁺ currents to a much smaller extent in SHR cardiomyocytes as compared to SD cardiomyocytes. Taken together, the data of the inhibitory analysis suggest that millimolar L-arginine serves as an agonist of α₂-adrenoceptors, which are coupled to PI3K-Akt pathway as well as downstream NO-cGMP pathway to control activity of L-type Ca²⁺ channels, thus providing new insights into the “arginine paradox” in cardiomyocytes.     

Development of the myoglobin sensor based on the surface plasmon resonance

Bioaffinic sensor based on the surface plasmon resonance (SPR) with golden layer as a transducer was developed. This sensor was applied for control of antigen-antibodies (Ag-Ab) interactions. For modification of transducer surface the spontaneously organized molecular assemblies of two types of mercaptan were formed. It was shown that the created bioaffinic sensor allowed to detect minimal concentration of antibodies, about 1 ng/ml, to the human heart myoglobin, which preliminary was immobilized on the sensitive surface. The particularities of the dynamics of protein interaction were observed. Duration of incubation with the biospecific component was no more than 5 minutes. After rupturing of specific binding it was possible to obtain initial signal again during 7-9 cycles.     

Effect of chronic disconnection between septal area and hypothalamus on modulation of background activity of septal neurons by biologically-active substances in hibernators

Our previous work demonstrated paradoxically increased excitability of the medial septal (MS) neurons during hibernation of ground squirrels in comparison to waking animals. Recently this was supported by demonstration of higher efficacy of the neuropeptides identified in the brain of hibernators in septal slices of hibernating animals. To decide whether this increased excitability is determined by endogenous properties of the pacemaker septal neurons, or it depends on the influences of thermoregulatory-circadian mechanisms of preoptico-hypothalamic area, testing of the neuropeptides (TSKYR, TSKY, DY) and neurotransmitters participating in control of hibernation (serotonin and noradrenaline) was repeated on septal slices taken from the brain of hibernating animals two weeks after operation disconnecting it from the hypothalamus. Effects of neuropeptides in the deafferented hibernating animals neither quantitatively (low reactivity level), nor qualitatively (distribution of inhibitory and excitatory responses) differed from the data obtained in waking animals. Decrease of reactivity occurred at the expense of the neurons with regular pacemaker-like spontaneous activity. Thus, increased reactivity of the MS neurons to neuropeptides in hibernating animals depends mainly on influence of the hypothalamic centres controlling hibernation behavior upon pacemaker neurons of the MS. Contrary to the neuropeptides, serotonin and noradrenaline were highly effective in deafferented septum. They evoked stronger changes of background activity (shorter latencies and more rapid development of maximal shifts), presumably as a result of development of denervation hypersensitivity after deafferentation.     

The state-dependent action of neuropeptides and monoamines on the background neuronal activity of the medial septal area in hibernating animals

Neuronal activity of the medial septal area (MS-DB) was recorded extracellularly in brain slices from two groups of Yakutian ground squirrels Citellus undulatus--hibernating (winter period) and actively waking (summer period). Effects of three neuropeptides identified in the brain of hibernators (TSKYR, TSKY, and DY) and of two monoamines (serotonin and noradrenaline) on spontaneous activity were analyzed. All neuropeptides reversibly changed the levels of the background activity, but in the hibernating ground squirrels (HGS) the level of reactivity (47-56%) was significantly higher than in the waking ground squirrels (WGS, 25-30%). Serotonin also showed some tendency to higher efficacy in the HGS. Only noradrenaline was equally effective and had absolutely dominating excitatory effect in both states, although the level of excitation in the HGS was higher. All other substances evoked excitatory and inhibitory effects in various proportions. Their distribution was state-dependent, the rate of development, intensity and duration of the effects were greater in the HGS. The experiments confirmed the data on higher excitability and reactivity of the septal neurons in the state of hibernation. It is suggested that the tested neuropeptides may participate in the control of hibernation.     

Analysis of the modal hypothesis of Ca2+-dependent inactivation of L-type Ca2+ channels

A kinetic model of Ca2+-dependent inactivation (CDI) of L-type Ca2+ channels was developed. The model is based on the hypothesis that postulates the existence of four short-lived modes with lifetimes of a few hundreds of milliseconds. Our findings suggest that the transitions between the modes is primarily determined by the binding of Ca2+ to two intracellular allosteric sites located in different motifs of the CI region, which have greatly differing binding rates for Ca2+ (different k(on)). The slow-binding site is controlled by local Ca2+ near a single open channel that is consistent with the "domain" CDI model, and Ca2+ binding to the fast-binding site(s) depends on Ca2+ arising from distant sources that is consistent with the "shell" CDI model. The model helps to explain numerous experimental findings that are poorly understood so far.