Spontaneous burst firing is a hallmark attributed to the neuronal network activity. It is known to be accompanied by intracellular calcium [Са2+]i oscillations within the bursting neurons. Studying mechanisms underlying regulation of burst firing is highly relevant, since impairment in neuronal bursting accompanies different neurological disorders. In the present study, the contribution of NMDA and GABA(A) receptors to the shape formation of spontaneous burst -was studied in cultured hippocampal neurons. A combination of inhibitory analysis with simultaneous registration of neuronal bursting by whole-cell patch clamp and calcium imaging was used to assess spontaneous burst firing and [Са2+]i level. Using bicuculline and D-AP5 we showed that GABA(A) and NMDA receptors effectively modulate burst plateau phase and [Са2+]i transient spike which can further affect action potential (AP) amplitudes and firing frequency within a burst. Bicuculline significantly elevated the amplitude and reduced the duration of both burst plateau phase and [Са2+]i spike resulting in an increase of AP firing frequency and shortening of AP amplitudes within a burst. D-AP5 significantly decreases the amplitude of both plateau phase and [Са2+]i spike along with a burst duration that correlated with an increase in AP amplitudes and reduced firing frequency within a burst. The effect of bicuculline was occluded by co-addition of D-AP5 revealing modulatory role of GABA(A) receptors to the NMDA receptor-mediated formation of the burst. Our results provide new evidence on importance of NMDA and GABA(A) receptors in shaping burst firing and Ca2+transient spikes in cultured hippocampal neurons.

Calcium-permeable kainate (CP-KARs) and AMPA (CP-AMPARs) receptors of the brain neurons are active participants of synaptic plasticity and neurotransmitter release trigger. In this paper, CP-KARs and CP-AMPARs were identified in hippocampal neuroglial culture on 14–17 day of cultivation by a characteristic Ca2+ response to a selective agonist of CP-KARs and CP-AMPARs, domoic acid (DA), and to a selective agonist of CP-KARs, ATPA. It was shown that DA at a concentration of 300 nM caused a rapid intracellular Ca2+ concentration increase in two minor subpopulations of neurons. Both subpopulations were found to be GABAergic neurons that were positively stained with antibodies against glutamate decarboxylase 65 and 67 (GAD65/67). The antagonist of CP-AMPARs, NASPM, did not suppress Ca2+ response to DA in the neurons of the first subpopulation. The selective agonist of CP-KARs, ATPA, increased [Ca2+]i to the same extent as DA only in the first subpopulation of GABAergic neurons. An inhibitor of GABA(A) receptors, bicuculline, did not increase the amplitude of Ca2+ response to DA in this subpopulation, indicating the absence of CP-KARs in the postsynaptic membrane, where GABA(A) receptors are located. Thus, these GABAergic neurons can be attributed to neurons containing CP-KARs, which are apparently located in the presynaptic membrane of the GABAergic neurons. The [Ca2+]i increase caused by the DA application in the second subpopulation was completely suppressed by NASPM, an inhibitor of CP-AMPARs. NASPM reduced the Ca2+ oscillations amplitude in the same subset, indicating the involvement of CP-AMPARs in the Ca2+ impulse formation during synchronous calcium activity. For this reason, the neurons of this subpopulation can be attributed to the GABAergic neurons containing CP-AMPARs. Most of the neurons in the hippocampal cell culture (70–85%) were not stained with antibodies against GAD65/67 and responded to the DA by increasing the calcium oscillations frequency with a delay. The amplitude of DA-induced oscillations increased in the presence of NASPM in the subpopulation of inhibitory neurons containing CP-KARs, indicating their innervation by inhibitory neurons containing CP-AMRARs. This increase in the Ca2+ oscillation amplitude in the inhibitory neurons containing CP-KARs correlated with a decrease in the amplitude of synchronous calcium activity in a large subpopulation (42 ± 6% of cells) of glutamatergic neurons, suggesting innervation of the latter by inhibitory neurons containing CP-KARs. Thus, GABAergic neurons containing CP-KARs and CP-AMPARs can work in tandem, controlling the activity of individual subpopulations of neurons.

Oxidation of pyruvate and palmitoylcarnitine in mitochondria is accompanied by the formation of acetyl-CoA, with its possible participation in the acetylation of various proteins and enzymes that may lead to the inhibition of their functions. This paper studies the effect of the excess of these substrates on respiration and induction of mitochondrial permeability transition pore (MPTP) in mitochondria and liver homogenates of healthy, obese, and type 2 diabetic (T2D) rats and mice. Both substrates produced a reversible inhibition of respiration and induced the opening of MPTP sensitive to cyclosporin A. Induction of MPTP in mitochondria was further activated by calcium ions and inhibited by the NO donor SNAP and NAD–a coenzyme and activator of deacetylation reactions. In obese and T2D animals, the opening of MPTP was stimulated by lower concentrations of L-palmitoylcarnitine than in healthy animals. In these pathologies, an activation effect on the MPTP induction was produced by ammonium ions, in the presence of which the concentration of L-palmitoylcarnitine required for the pore opening was reduced more than twofold. In liver homogenates, an added arginine reduced the probability of the MPTP formation. Analysis of mathematical models has shown that, due to the inhibition of pyruvate dehydrogenase kinase (PDK) by pyruvate, phosphorylation of pyruvate dehydrogenase (PDH) is strongly reduced, and this makes it possible to produce acetyl CoA in a wide range of pyruvate concentrations. The data obtained show that excess substrates that produce acetyl-CoA increase the probability of the MPTP opening, especially in pathologies associated with obesity and T2D. The ability of NO and NAD to inhibit MPTP indicates the participation of phosphorylation and acetylation/deacetylation reactions in this process.

The aim of this work was to study the influence of aging, obesity, metabolic syndrome (MS), hypertension (HT), and type 2 diabetes (T2D) on the endogenous rhythmic activity and the development acetylcholine resistance in aorta rings of male rats. T2D was produced by a free access to fat (lard). It was shown that phenylephrine (PE) or 5-hydroxytryptamine (5-HT) induces two types of rhythmic contractions: with periods T 1 = 3–10 s and T 2 = 50–70 s and amplitudes A 1 = 1–5% and A 2 = 20–40% of the maximal contraction force (F max), respectively. Such periodic modes can be caused by the operation of two known positive feedback loops (PFL) based on the Ca2+-induced activation of IP3 receptor (IP3R) or phospholipase C PFL1 and PFL2, respectively, and are not eliminated by L-NAME. Slow rhythmic activity induced by acetylcholine (Ach) with period T 3 = 7–20 min and amplitude A 3 = 20–30% of F max was observed only in young animals (under 6 months) and can be determined by the operation of PFL3, involving Ca2+, NO, kinase G, cADP-ribose, and the ryanodine receptor (RyR). Fast mode of contractions (T 1, A 1) is maintained regardless of age and the presence of MS and HT (140 mm Hg and higher) and disappears only at later stages of the T2D development. Probability of intermediate mode of contractions (T 2, A 2) decreases to 0.20–0.25 at the age of 14–16 months or during the development of HT and MS. In these circumstances, Ach could cause relaxation of preconstricted rings only to 40 and 60% of F max, respectively. At the stages of the T2D development characterized by high values of arterial pressure (above 150 mm Hg) and of the glucose (10–12 mM), ammonium (120–180 μM), and blood lipid levels, as well as by liver dysfunction (fibrosis/cirrhosis), the rhythmic activity of any type is lost and dysfunction of the initial part of the signaling cascade with the participation of PFL3 is manifested by the absence of responses to Ach or L-NAME. Coenzyme NAD (agonist of the P2Y receptors, К+ channel activator and a precursor of cADP-ribose) can exert a partial relaxation of aorta rings from healthy animals and animals with MS. Nicotinamide (product and an inhibitor of ADP-ribosyl cyclase) and SNP (donor of NO) produce an effective relaxation of aorta rings from healthy animals and animals with T2D. Relaxing effect of nicotinamide may suggest a tandem operation of IP3R and RyR in the control of intracellular Ca2+ stores in vascular cells.

The mechanisms of hyperexcitability of neuronal networks by ammonium ions and inhibition of this activity by coenzyme NAD were investigated on mixed neuro-glial cultures of rat hippocampus. Ammonium ions cause activation of silent or spontaneously active neuronal networks inducing a bursting electrical activity of neurons and high-frequency synchronous calcium oscillations. In control conditions NAD completely inhibits spontaneous activity of the neuronal network. NAD added after NH4Cl disrupts synchronous oscillation in neurons and splits the network into five populations of neurons. In 4% of cells NAD decreased the amplitude of Ca2+ oscillations, preserving initial mode of oscillations. In 32% of cells, a transient suppression of the neuronal oscillations was observed: inhibition was followed by restoration of the synchronous periodic activity. In 10% of cells, NAD produced a gradual decrease of Ca2+ oscillations down to a complete termination of the initial periodic activity induced by ammonium. Fast and total inhibition of Ca2+ oscillations by NAD was observed in two small groups of neurons. First group (A) participated in the initial spontaneous network activity (5% of cells) with a period of 66–100 s. Second group (B), on the contrary, did not participate in the spontaneous activity. This group of neurons began to pulse with a high frequency (with a period of 6–8 s) synchronously with other neurons in the network after the addition of NH4Cl. Based on the comparison of calcium responses of different cell groups to the depolarization caused by KCl and NH4Cl and to the application of domoic acid, as well as on the results obtained in experiments with fluorescent antibodies against GAD 65/67, parvalbumin, calretinin, and calbindin, we propose that neurons of populations (A) and (B) may belong to GABAergic neurons containing calbindin and parvalbumin, respectively. Further analysis of specificity of the NAD effect on these neuronal groups may allow identification of the main targets of the ammonium toxic action in the brain. Thus, we have shown that NAD selectively inhibits neuronal activity and high-frequency synchronous Ca2+ oscillations in GABAergic neurons containing calcium-binding proteins. The inhibition is accompanied by desynchronization of oscillations and dissociation of neuronal network into several populations.

Цель. В сравнительных экспериментах исследовать влияние фенилэфрина (РЕ) и ацетилхолина (Ach) на сократительные свойства аорты крыс в условиях гипертензии (ГТ). Материалы и методы. Использованы здоровые крысы линии WKY (8-10 недель и 10 месяцев) и спонтанно-гипертензивные крысы SHR (возраст 10 месяцев, артериальное давление AD > 200 mmHg) с недостаточностью сердца; стандартные методы регистрации AD и методы регистрации силы сокращений образцов аорты и папиллярных мышц миокарда. Результаты. Показано, что у крыс SHR, с недостаточностью сердца, регистрируемой как отсутствие «эффекта паузы» и изменения зависимости силы сокращений(F) от частоты стимуляции (f), отсутствует расслабляющий эффект Achна сокращение сосудов, вызываемое РЕ. Введение донора NO-SNP или ингибитора ADP-рибозилциклазы (или CD38) – никотинамида (NAM) обеспечивает полное сокращение сосудов в этих условиях. Прием многокомпонентного состава «Хелпер-1» улучшает характеристики «эффекта паузы» в папиллярных мышцах без улучшения сократительных свойств образцов аорты(F(f)). Выводы. Дисрегуляция автокаталитического Са2+/NOS/NO/sGS/cGMP/PKG/CD38/cADPr/RyR/Ca2 + -сигнального пути в эндотелии и миоцитах сосудов может быть одним из основных факторов дисфункции эндотелия и гипертензии сосудов.

С использованием методов флуоресцентной микроскопии, иммунохимии и ПЦР-анализа проведено сравнительное исследование особенностей динамического поведения и экспрессии генов IP3 и NO/cGMP зависимых Са2+-сигнальных и метаболических путей адипоцитов здоровых мышей и животных с ожирением и диабетом 2-го типа (D2T). Показано, что культивируемые адипоциты (9DIV), выделенные из эпидидимальных депо белой жировой ткани здоровых мышей при аппликации 1–10 мкМ ацетилхолина (Ach) или норадреналина (NE) способны генерировать разнообразные Са2+-колебания, триггерные переключения и Са2+-спайки. Периоды колебаний растут при увеличении размеров клеток до 150–200 мкм и накоплении в них липидов. Культивируемые адипоциты животных с ожирением способны расти и накапливать липиды только в присутствии жирных кислот в среде. В таких клетках Ach и NE (15–30 мкМ) приводят только к генерации Са2+-спайков с выходом на плато. У животных с Д2Т зрелые гипертрофированные адипоциты имеют малый объем свободной, не занятой липидами цитоплазмы. В таких клетках Ach и NE также могут вызвать только Са2+-импульсы малой амплитуды или монотонный рост [Са2+]i. Утрата клетками способности генерировать разнообразные типы Са2+-ответов при ожирении и Д2Т может быть связана с наблюдаемыми сдвигами и подавлением экспрессии ряда генов, контролирующих Са2+-сигнальные пути (в первую очередь с участием NO/cGMP/cADPr) и метаболические пути синтеза и распада липидов клеток жировой ткани.

Precipitation of Ehrlich ascites tumor cells (EATC) by centrifugation causes ATP secretion. ATP secretion is accompanied by an increase of calcium concentration in the cytosol and persists for a long time (minutes) after centrifugation during the storage of cells at a low temperature. During prolonged storage (for more than 1.5 h), the concentration of extracellular ATP decreases to the level of ∼100 nM due to termination of secretion and ATP hydrolysis by surface ATPases. The rate of ATP hydrolysis exponentially falls with the temperature decrease from 36 to 8°C. ARL67156, a selective inhibitor of E-NTPDase-1, effectively suppresses the extracellular ATP hydrolysis. The intensity of ATP secretion does not correlate with the calcium ions concentration in the cytosol and Ca2+ mobilization from endoplasmic reticulum but correlates with the intensity of Ca2+ influx into the cells. The temperature dependences of ATP secretion intensity and Ca2+ entry rate coincide.

341 Compensated influx and efflux of Ca 2+ ions provide the maintenance of constant Ca 2+ ion concentration in cytoplasm of quiescent cells in variable external conditions. Pumping of calcium from cells is carried out mainly by plasma membrane Ca 2+ -ATPase (PMCA). As far as at physiological levels of cytosolic calcium PMCA is constitutively active, there is also constantly active Ca 2+ channel. It is known that activity of PMCA as an enzyme, unlike other Ca 2+ -transporting systems, has characteristic strong dependence on temperature [1]. It is known also that concentration of cytosolic Ca 2+

Compensated influx and efflux of calcium ions maintain the constancy of Ca2+ concentration in cytoplasm of quiescent cells under variable external conditions. In cell plasma membrane there exist several types of Ca2+ channels with different properties, regulation mechanisms, and pharmacology. Using fluorescent Ca2+-sensitive probes, we have shown here that in T-lymphocytes under resting conditions, Ca2+ influx occurs through special constitutively active Ca2+ channels, permeable to Ni2+ and Mn2+. These channels differ from the receptor-activated SOC channels, from Ca2+ channels activated by arachidonic acid, and from calmidazolium-activated channels. Ca2+ influx rate in quiescent cells increases with a rise in temperature (Q10 =1.9). The strong dependence of the constitutively active channel activity on temperature coincided with the plasma membrane Ca2+-ATPase dependence, indicating that intracellular enzymes regulate the channel activity. To identify the constitutively active channel, we analyzed the effects of L-type Ca2+ channels, SOC channels, Ca2+-independent phospholipase A2, and calmodulin inhibitors. Of all inhibitors listed only dihydropyridine blocker of L-type voltage-dependent Ca2+ channels, isradipin, at a concentration of 1.5 μM completely suppressed calcium influx. However, the channels did not exhibit sensitivity to changes in membrane potential. Our observations testify to the existence of a new nonselective Ca2+ channel in T-lymphocyte plasma membrane and characterize the new channels pharmacologically. The results obtained are important for understanding the regulation mechanisms of Ca2+ channels in plasma membrane of non-excitable cells.