Search PubMed⌕ Search

Biomedical subjects

P G Kostyuk

Publications and source records attributed to P G Kostyuk.

At least 19 recordsLinked to original sources
PubMed2005

Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps.

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.

Acetylcholine↗
PubMed2005

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats.

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.

Animals↗
PubMed2004

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells.

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.

Acetylcholine↗
PubMed2003

Effect of hypoxia on calcium channels depends on extracellular calcium in CA1 hippocampal neurons.

Our previous studies have shown that short lasting hypoxia induces an increase of Ca(2+) influx into the cell through high voltage-activated Ca(2+) channels in hippocampal neurons. This effect was abolished by removing of free Ca(2+) from intracellular solution. The aim of this study was to compare hypoxic responses at different extracellular Ca(2+) concentrations ([Ca(2+)](e)) in hippocampal neurons to ascertain whether the hypoxic sensitivity is restricted to Ca(2+) ions. Whole-cell patch-clamp recordings were made from acutely dissociated CA1 hippocampal neurons of rats. Polarographic method for measurements of O(2) partial pressure was used. Here we found that at 2 mM [Ca(2+)](e) the hypoxic effect was significant (up to approximately 94%), whereas [Ca(2+)](e) elevations to 5 and 15 mM resulted in gradual decreasing of the effect. We found, that total Ca(2+) charge carried into the cell under the hypoxia was similar at all [Ca(2+)](e), whereas Ca(2+) charge carried at normoxia was different for different [Ca(2+)](e), being larger at higher [Ca(2+)](e). These data indicated a saturation of the hypoxic effect due to limitation in the channel conductance. Therefore, we suggested that the hypoxic effect can be connected with increase of channel conductance, and the level of channel conductance at normoxia can determine the amplitude of hypoxic effect.

Animals↗
PubMed2003

Intracellular mechanisms of hypoxia-induced calcium increase in rat sensory neurons.

Elevation of cytosolic level of Ca(2+) was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution (pO(2)=10-40 mmHg). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca(2+) to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca(2+) or Na(+) from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca(2+) is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca(2+) and Na(+) channels and Ca(2+) accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca(2+) elevation due to their specific location in these neurons.

Animals↗
PubMed2002

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons.

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.

Animals↗
PubMed2002

Glutamate-receptor-induced modulation of GABAergic synaptic transmission in the hippocampus.

There is a large body of evidence about the short- and long-term changes in GABAergic transmission in the hippocampus produced by the action of different endogenous neuromodulators and in particular neurotransmitters. Both intrinsic hippocampal cells and afferent fibres coming into the hippocampus from various parts of the CNS release substances that are capable of changing inhibitory transmission. This review surveys current understanding of the action of glutamate on the inhibitory transmission mediated in the hippocampus via GABA(A) receptors. Here we pay special attention to the molecular and cellular mechanisms leading to spatio-temporal changes of the glutamate concentration in the extracellular space and to the localization and identity of glutamate receptors involved in this direct modulation of inhibition.

Animals↗
PubMed2002

Selective blockade of N-type calcium channels by levetiracetam.

PURPOSE: We investigated the effect of the new antiepileptic drug (AED) levetiracetam (LEV) on different types of high-voltage-activated (HVA) Ca2+ channels in freshly isolated CA1 hippocampal neurons of rats. METHODS: Patch-clamp recordings of HVA Ca2+ channel activity were obtained from isolated hippocampal CA1 neurons. LEV was applied by gravity flow from a pipette placed near the cell, and solution changes were made by electromicrovalves. Ca2+ channel blockers were used for separation of the channel subtypes. RESULTS: The currents were measured in controls and after application of 1-200 microM LEV. LEV irreversibly inhibited the HVA calcium current by approximately 18% on the average. With a prepulse stimulation protocol, which can eliminate direct inhibition of Ca2+ channels by G proteins, we found that G proteins were not involved in the pathways underlying the LEV inhibitory effect. This suggested that the inhibitory effect arises from a direct action of LEV on the channel molecule. The blocking mechanism of LEV was not related to changes in steady-state activation or inactivation of Ca2+ channels. LEV also did not influence the rundown of the HVA Ca2+ current during experimental protocols lasting approximately 10 min. Finally, LEV at the highest concentration used (200 microM) did not influence the activity of L-, P- or Q-type Ca2+ channels in CA1 neurons, while selectively influencing the activity of N-type calcium channels. The maximal effect on these channels separated from other channel types was approximately 37%. CONCLUSIONS: Our results provide evidence that LEV selectively inhibits N-type Ca2+ channels of CA1 pyramidal hippocampal neurons. These data suggest the existence of a subtype of N-type channels sensitive to LEV, which might be involved in the molecular basis of its antiepileptic action.

Animals↗
PubMed2001

Dual action of cytosolic calcium on calcium channel activity during hypoxia in hippocampal neurones.

The role of cytoplasmic calcium level (Ca(i)) in regulation of Ca channel activity during hypoxia was studied in hippocampal neurones from rats. Whole-cell patch clamp recordings in combination with measurements of O(2) partial pressure (pO(2)) were used. Lowering of pO(2) induced a potentiation of HVA Ca channel activity by 25.7% at Ca(i) = 75 nM in comparison with Ca(2+)-free solution. Increase of Ca(i) up to 410 nM slightly increased the effect and significantly slowed the Ca(2+) current run-down. On the other hand, hypoxia increased a steady-state channel inactivation and speeded up the kinetics of Ca(2+) current decay by about 30%. We conclude that moderate hypoxia induces dual action on Ca channels: intracellularly mediated augmentation of Ca influx via Ca channels and their Ca(2+)-dependent inactivation.

Animals↗
PubMed2001

Postsynaptic mechanism may contribute to inhibitory acetylcholine effect on GABAergic synaptic transmission in hippocampal cell cultures.

The effect of acetylcholine (ACh) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. Spontaneous IPSCs and IPSCs evoked by extracellular stimulation of a single presynaptic neuron were recorded. ACh inhibited the evoked IPSCs in most of the connections, although facilitation was also observed. Regardless of inhibitory or facilitatory effects on the evoked IPSCs, an enhanced spontaneous synaptic input to the postsynaptic neurons was usually observed. ACh-induced changes in the evoked IPSCs were usually accompanied by changes in paired pulse depression (PPD), which are thought to reflect presynaptic mechanisms of modulation. However, the time course of PPD changes did not always match that of the IPSC changes, suggesting a contribution of other, possibly postsynaptic, mechanism(s). To analyze this possibility, effects of ACh on responses to direct application of exogenous GABA were studied. In a proportion of the neurons (40%) ACh reversibly decreased GABA responses, indicating that postsynaptic mechanisms may also contribute to the inhibitory ACh effect on GABAergic transmission. We conclude that several different modulatory mechanisms of ACh action participate in the regulation of GABAergic transmission at the level of synaptic connection of a single GABAergic neuron.

Acetylcholine↗
PubMed2001

Subtypes of low voltage-activated Ca2+ channels in laterodorsal thalamic neurons: possible localization and physiological roles.

The macroscopic, low-voltage-activated (LVA or T-type) Ca2+ current in isolated associative (or local-circuit) neurons from the laterodorsal thalamic nucleus of 14-17-day old rats was dissected into two components ("fast" and "slow"), corresponding to the activation of two LVA channel subtypes, based on the difference in the kinetics of inactivation and recovery from inactivation. The steady-state activation and inactivation properties of the channel subtypes endowed slow channels with a substantial window current, whereas fast channels had almost no such current. Fast channels were almost 2 times more sensitive to 30 microM nifedipine (78% inhibition), 10 microM flunarizine (92% inhibition) and 1 microM La3+ (87% inhibition), but about 1.8-fold less sensitive to 100 microM Ni2+ (32% inhibition) than slow channels (40%, 52%, 46% and 56% inhibition respectively). Both channels were almost equally sensitive to 100 microM amiloride (58% and 51% inhibition of fast and slow channels respectively). Comparison of the fast and slow LVA Ca2+ current amplitudes and densities between enzymatically isolated and intact (in brain slices) neurons suggest a predominant localization of the fast channels in soma and the proximal dendrites that remain intact during isolation procedure, whereas the slow channels are more evenly distributed with some preference to the distal areas. These data, together with our previous studies, support the notion of two LVA Ca2+ channel subtypes in associative thalamic neurons and suggest a role for the slow channels in providing the constant Ca2+ influx necessary for the outgrowth of the neurites and for the fast channels in the generation of low-threshold Ca2+ spikes and bursting activity.

Amiloride↗
PubMed2000

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons.

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.

Animals↗
PubMed2000

Long-term depolarization changes morphological parameters of PC12 cells.

It is well known that neuronal differentiation is strongly dependent on the intracellular level of free calcium ions ([Ca2+]i). In the present study the morphological and intracellular free calcium concentration changes were compared on PC12 pheochromocytoma cells cultured in control conditions and in a medium with high KCl level. Culturing PC12 cells in a medium with 20-30 mM KCl deprived of nerve growth factor supported cell proliferation and rapid growth of small neurite-like processes. However, their lengths did not increase with prolongation of the time of culturing. During culturing with 40 mM KCl the growth of these processes became blocked; the cells stopped proliferating and showed signs of degeneration. Measurements of [Ca2+]i level during the first days of PC12 cells culturing in a hyperpotassium medium indicate that such changes in this level could be an important factor in the induction of the observed morphological alterations; however, other effects induced by membrane depolarization may also be responsible for them.

Animals↗
PubMed2000

Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line.

Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl(-) was the primary charge carrier (termed I(Cl,swell)). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br(-) approximately I(-) > Cl(-) > F(-) > methanesulfonate >> glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC conductance was determined at 9.6 +/- 1.8 pS. Conventional Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) and DIDS (100 microM) inhibited whole cell I(Cl,swell) in a voltage-dependent manner, with the block decreasing from 39.6 +/- 9.7% and 71.0 +/- 11. 0% at +50 mV to 26.2 +/- 7.2% and 14.5 +/- 6.6% at -100 mV, respectively. Verapamil (50 microM), a standard Ca(2+) antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I(Cl,swell) (genistein with an IC(50) of 2.6 microM and lavendustin A by 60 +/- 14% at 1 microM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 microM) stimulated I(Cl,swell) by 54 +/- 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

High-threshold calcium channel activity in rat hippocampal neurones during hypoxia.

Whole-cell patch clamp recordings in combination with direct control and measurements of O2 tension (pO2) in bath solution were used to determine the sensitivity of Ca2+ channels of cultured hippocampal neurones to hypoxia in glucose free solution. In all tested neurones, a lowering of pO2 to 4/50 mmHg did not induce changes either in magnitude, kinetics or voltage-current relations of total Ca2+ currents, which composed mainly from two types, L-type (64%) and N-type (31%) components. Hypoxia only induced a delay of Ca2+ current run-down about 27.5% and 39% at 50 and 4 mmHg pO2 respectively that presumably depended on changes in cytoplasmic channel-modulatory metabolites. The obtained results demonstrate that Ca2+ channel molecules in cultured hippocampal neurones are themselves insensitive to short-lasting (10-20 min) oxygen and glucose deprivation, and that they are not a principal target for hypoxic influences on hippocampal function.

Animals↗
PubMed1999

Divalent cation selectivity of the subtypes of low voltage-activated Ca2+ channels in thalamic neurons.

LVA Ca2+ current in isolated associative neurons from the laterodorsal thalamic nucleus of 14- to 17-day-old rats was dissected into two, 'fast' and 'slow', components based on the difference in the kinetics of inactivation. The selectivity of the channel responsible for the fast LVA current for Ca2+, Sr2+ and Ba2+ (I(Ca):I(Sr):I(Ba) = 1.0:1.23:0.94) as well as the shifts of the I-V produced by these ions were found to be almost identical to those observed for LVA channels in other preparations. The channel responsible for the slow LVA current showed selectivity more characteristic of HVA Ca2+ channels (I(Ca):I(Sr):I(Ba) = 1.0:2.5:3.4), although the ability of Ca2+, Sr2+ and Ba2+ to shift its voltage dependence remained the same as for the fast channel.

Animals↗
PubMed1999

Low-voltage activated calcium channels: achievements and problems.

Low-voltage activated Ca2+ channels, which possess unique properties quite different from those of common (high-voltage activated) channels, were discovered 15 years ago but the first alpha1 subunit has only recently been identified which might provide their structural basis. However, simultaneously, extensive data are being accumulated on the functional diversity of low-voltage activated Ca2+ currents with regard to their pharmacological sensitivity, ionic selectivity, activation and inactivation kinetics. Such diversity corresponds to equally prominent heterogeneity in the location and function of the channels. This commentary summarizes the data available in an attempt to predict a possibly wider structural subdivision of low-voltage activated Ca2+ channels into subtypes.

Animals↗