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Biomedical subjects

P A Doroshenko

Publications and source records attributed to P A Doroshenko.

At least 19 recordsLinked to original sources
PubMed1997

Routes of Cl- transport across the trophectoderm of the mouse blastocyst.

The blastocyst stage of embryo development is characterized by a fluid-filled cavity called the blastocoel. Blastocoel formation requires vectorial Na+ and Cl- transport and the accompanying osmotic accumulation of fluid. We found under conditions of low external Cl- that inhibitors of Cl- transport mechanisms inhibited blastocoel expansion, indicating a possible transcellular route for Cl- uptake across the outer epithelial layer (the trophectoderm). Using the Cl--sensitive fluorophore, N-(6-methoxyquinolyl)acetoethyl ester, we found that Cl- efflux from the blastocoel can occur via pathways with properties that resemble both HCO-3/Cl- exchange and Cl- channels, as well as by another yet uncharacterized pathway. In contrast, Cl- re-uptake into Cl--depleted blastocoels (the physiologically relevant direction for Cl- transport during blastocoel expansion) occurred only via the channel-like mechanism. Patch-clamp recordings detected a component of current carried by apical Cl- channels. Intracellular pH measurements during external Cl- removal detected HCO-3/Cl- exchange activity in collapsed blastocysts but little in intact blastocysts, suggesting predominantly basolateral HCO-3/Cl- exchange activity. This was corroborated by the immunolocalization of the AE2 isoform of HCO-3/Cl- exchanger to the basolateral surface of the trophectoderm. Thus, it appears that Cl- transport into the blastocoel may occur via apical Cl- channels, while efflux also involves a basolateral HCO-3/Cl- exchanger.

Animals↗
PubMed1997

Pharmacologically distinct presynaptic calcium channels in cerebellar excitatory and inhibitory synapses.

We have used whole-cell patch clamp recordings and pharmacological blockers of Ca channels to compare the pharmacology of Ca channels that mediate synaptic transmission at the three types of synapses innervating Purkinje cells in rat cerebellar slices. Both parallel fiber and climbing fiber excitatory synapses were sensitive to the P-type Ca channel blocker, omega-AgaIVA and the P/Q/N-type channel blocker, omega-conotoxin MVIIC. Transmission at inhibitory interneuronal synapses was not suppressed by these toxins, or by the N-type (omega-conotoxins GVIA and MVIIA) or L-type (nimodipine) channel blockers. Inhibitory transmission could be inhibited by Ni2+ and amiloride, but only at concentrations (IC50 approximately 300 microM) that affect other types of Ca channels. These results indicate that excitatory and inhibitory presynaptic terminals of the cerebellar cortex possess different types of voltage-gated Ca channels. The excitatory terminals contain P-type, Q-type and N-type Ca channels, with P-type channels playing the most prominent role. The inhibitory terminals possess quite different type(s) of Ca channel. The heterogeneous distribution of Ca channel types should impart unique properties to transmitter release from the excitatory and inhibitory terminals.

Animals↗
PubMed1993

A functional role for GTP-binding proteins in synaptic vesicle cycling.

The squid giant synapse was used to test the hypothesis that guanosine-5'-triphosphate (GTP)-binding proteins regulate the local distribution of synaptic vesicles within nerve terminals. Presynaptic injection of the nonhydrolyzable GTP analog GTP gamma S irreversibly inhibited neurotransmitter release without changing either the size of the calcium signals produced by presynaptic action potentials or the number of synaptic vesicles docked at presynaptic active zones. Neurotransmitter release was also inhibited by injection of the nonhydrolyzable guanosine diphosphate (GDP) analog GDP beta S but not by injection of AIF4-. These results suggest that a small molecular weight GTP-binding protein directs the docking of synaptic vesicles that occurs before calcium-dependent neurotransmitter release. Depletion of undocked synaptic vesicles by GTP gamma S indicates that additional GTP-binding proteins function in the terminal at other steps responsible for synaptic vesicle replenishment.

Aluminum↗
PubMed1992

Effects of serotonin and cAMP on calcium currents in different neurones of Helix pomatia.

Effects of application of serotonin (5-HT) and intracellular administration of cyclic adenosine monophosphate (cAMP) on voltage-gated calcium current (ICa) were studied in isolated, intracellularly perfused Helix pomatia neurones. Two types of the effects of 5-HT (1-10 microM) were observed in different neurones: reversible inhibition (by about 20%) or reversible potentiation (up to 50%) of the current amplitude. Some cells did not respond to 5-HT application. In cells with the potentiating effect of 5-HT, ICa could also be increased by intracellular introduction of cAMP (100 microM), but not the guanosine analogue, cGMP (50-100 microM). These effects were not additive and could be potentiated by theophylline (5 mM) and 3-isobutyl-1-methylxanthine (IBMX, 100-500 microM); they could be mimicked by forskolin (10-50 microM) and abolished by tolbutamide (1-5 mM) or protein kinase inhibitor (500 micrograms/ml), indicating that cAMP-dependent phosphorylation mediates the potentiating action of 5-HT on ICa. In neurones showing inhibitory effect of 5-HT, neither cAMP nor forskolin increased ICa. Methiothepin (10-50 microM), a 5-HT1,2 receptor antagonist, irreversibly inhibited the potentiating effect of 5-HT, while antagonists of 5-HT2 receptors cyproheptadine (10-50 microM) or ketanserine (10-60 microM) and of 5-HT3 receptors ISC 205-930 (10-50 microM) or cocaine (5-25 microM) had no effect on ICa and its enhancement by 5-HT. It is suggested that in certain snail neurones the possibility of cAMP-dependent up-regulation of ICa correlates with the presence of 5-HT1-like receptors in the neuronal membrane.

Animals↗
PubMed1990

[The effect of cAMP on the calcium currents of mollusk neurons possessing different sensitivities of their calcium conductance to serotonin action].

The action of cAMP (100 microM) and serotonin (5-HT, 1-10 microM) on the calcium current (Ica) in intracellularly perfused Helix pomatia neurons was studied with voltage clamp method. Three types of 5-HT-induced changes in the calcium current were observed in different cells: reversible blockade, increase and no changes in the current amplitude. Intracellular introduction of exogenous cAMP (100 microM) affected Ica only in cells with the stimulatory effect of 5-HT; cAMP-induced increase in the current amplitude was not additive to that elicited by 5-HT while both of these effects were similarly potentiated by cyclic nucleotide-phosphodiesterase inhibitor. The data presented show that the stimulatory action of 5-HT on the potential-activated calcium current is mediated by an increase in intracellular cAMP. Existence of two types of calcium channels differing in their dependence on cAMP metabolism is suggested in the snail neurons. The presence of the cAMP-dependent calcium channels seems to correlate with the existence of the definite type of 5-HT receptors in the cell membrane. A new approach to the investigation of isolated neurons is suggested: their functional identification.

Animals↗
PubMed1989

gamma-Aminobutyric acid elevates cytosolic Ca in bovine chromaffin cells.

Measurements of the cytosolic Ca concentration ([Ca]i) with the Ca-sensitive dye, fura-2, showed that in intact, but not in voltage-clamped, bovine chromaffin cells gamma-aminobutyric acid (GABA, 10 microM) elicited a transient increase in [Ca]i. The Ca transient of intact cells was inhibited by bicuculline (20 microM), by removal of extracellular Ca or by treatment with the Ca channel blocker cobalt (2.5 mM), and enhanced by lowering the extracellular Cl. We conclude, that GABA elevates [Ca]i by inducing a GABAA-receptor-linked Cl current which depolarizes the cell membrane sufficiently to activate potential-operated Ca channels and cause Ca entry into the cell.

Adrenal Medulla↗
PubMed1988

Modulation of calcium current by calmodulin antagonists.

The short-term effects of bath applied calmodulin antagonists--chlorpromazine, trifluoperazine and calmidazolium (R24571)--on potential-dependent calcium channels in the membrane of intracellularly perfused snail neurons were studied in voltage clamp conditions. All the drugs affected the calcium inward current peak value, the effects being reversible and dependent on the concentration used. Submicromolar concentrations (0.1-1 microM) increased the current amplitude (the maximal effect was on the average 20% at 0.5 microM), whereas higher concentrations inhibited the current. Analysis of the dose-effect curve for the blockade suggests positive cooperativity in the interaction of the drugs with the channel; experimental data on chlorpromazine action (10-100 microM) are well approximated by a binding curve for two molecules with the effective Kd = 70 microM. The efficiency of the blockade depended neither on the current-carrying cations (calcium or barium) nor on the intracellular introduction of 10 mM EGTA. The presence of calmodulin antagonists influenced the blockade of the calcium current by inorganic blockers: 50 microM chlorpromazine decreased the Kd value from 90 to 50 microM for the current blockade by Cd ions. It is suggested that calmodulin antagonists interact with two sites in the calcium channel, with high and low binding affinity (responsible for enhancement and inhibition of the current, respectively). The interaction induces changes in binding of penetrating cations in the channel, thereby producing modulation of the calcium current amplitude.

Animals↗
PubMed1988

[Action of forskolin on the calcium current in the membrane of nerve cells in mollusks].

The action of forskolin, adenylate cyclase activator, on the calcium inward current was studied on intracellularly perfused nonidentified snail neurons. Extracellular application of 5-20 mumol/l of forskolin was shown to cause about 20% increase of the calcium current amplitude and this confirms the previous suggestion that the stimulation of the cellular synthesis of cAMP leads to enhancement of the calcium current through the cell membrane.

Animals↗
PubMed1986

Transmembrane outward hydrogen current in intracellularly perfused neurones of the snail Helix pomatia.

The ionic nature and pharmacological properties of the outward current activated by membrane depolarization were studied on isolated neurones of the snail Helix pomatia, placed in Na+- and Ca2+-free extracellular solutions and intracellularly perfused with K+-free solution ("nonspecific outward current"). It was shown that the amplitude and reversal potential of this current (estimated from instantaneous current-voltage characteristics) are determined mainly by the transmembrane gradient for H+ ions. Lowering of pHi induced an increase in the current amplitude and a shift of the reversal potential to more negative values; the shift magnitude was comparable with that predicted for the hydrogen electrode. Raising pHi, as well as lowering pHo, induced a decrease in the current amplitude and a displacement of the current activation curve to more positive potentials. Addition of EGTA (8 mmol/l) to the intracellular perfusate did not affect the current amplitude. Extracellular 4-aminopyridine (10 mmol/l), verapamil (0.25 mmol/l) or Cd2+ (0.5 mmol/l) blocked the current. It is concluded that the current studied is carried mainly by H+ ions. In the same neurones the nature of the fast decay of the calcium inward current was also studied (in the presence of extracellular Ca2+ ions). This decay considerably slowed when pHi was raised or pHo was lowered, and it became less pronounced upon extracellular application of 4-aminopyridine or upon intracellular introduction of phenobarbital (4 mmol/l) and tolbutamide (3 mmol/l). It is suggested that the fast decay of the calcium inward current is due to activation of a Ca-sensitive component of the hydrogen current which depends on accumulation of Ca2+ ions. The possible physiological role of the transmembrane hydrogen currents is discussed.

4-Aminopyridine↗
PubMed1985

Fast decrease of the peak current carried by barium ions through calcium channels in the somatic membrane of mollusc neurons.

In experiments on nonidentified intracellularly perfused snail neurons the effects of replacing external divalent cations on the function of potential-dependent Ca channels have been studied. Ba substitution for Ca in the external medium caused a rapid decline (with half-times of about 2-3 min) in peak inward current amplitude when the current was activated from holding potential levels close to the resting potential. The decline could be reversed by membrane hyperpolarization. Barium current declined to a steady-state level which resembled in both relative amplitude (10-30% of the initial current amplitude) and insensitivity to intracellular introduction of exogenous cAMP the steady-state Ca current reached during the "wash out" process. It is suggested that two populations of Ca channels exist in snail neuronal membrane, one of which is dependent on cAMP metabolism and is reversibly switched off by the passage of Ba ions.

Animals↗
PubMed1984

Intracellular protein kinase and calcium inward currents in perfused neurones of the snail Helix pomatia.

Changes in the amplitude of the calcium inward current caused by intracellular administration of tolbutamide (an inhibitor of the cyclic AMP-dependent protein kinase activity) or catalytic subunits of cAMP-dependent protein kinases from rabbit myocardium were studied on internally perfused nerve cells of the snail, Helix pomatia. Intracellular administration of 7 mM tolbutamide caused a rapid decline of the amplitude of the calcium current that had been stabilized by theophylline; the effect was practically completely reversible. In contrast, addition to the perfusing solution of exogenous catalytic subunits of cyclic AMP-dependent protein kinases (about 0.7 microM of protein) together with 2 mM adenosine 5'-triphosphate and 3 mM MgCl2, led to stabilization of the calcium conductance of the cell membrane or restored it if it had declined during the perfusion with basic solution. The effect depended largely on the presence of adenosine 5'-triphosphate. Its time course was very slow (dozens of minutes) due probably to slow diffusion of the protein inside the cell. Heat-inactivated catalytic subunits did not produce such a stabilizing or restoring action on the calcium conductance. The results substantiate the suggestion that the normal functioning of calcium channels depends on phosphorylation catalyzed by cyclic AMP-dependent protein kinases.

Adenosine Triphosphate↗
PubMed1984

Inactivation of calcium current in the somatic membrane of snail neurons.

The decline of calcium inward currents evoked by a long-lasting membrane depolarization was studied on isolated snail neurons internally perfused with a K+-free solution. Two exponential components superimposed on a steady inward current could be distinguished, a slow decline with a time constant of several hundreds of milliseconds, observed at all the testing potentials used, and a fast one with a time constant of several dozens of milliseconds, which appeared at depolarizations to about -10 mV and above. When the calcium current was blocked by extracellular Cd2+ or verapamil, an outward current could be recorded at the same depolarizations. Subtraction of the latter current from the total current, recorded prior to the blockage, largely reduced the fast component of the decline of the total current. An increase in pHi from 7.3 to 8.1 led to the elimination of both the outward current and the fast component of the calcium current decline. The slow component remained practically unchanged, with its rate depending upon the current amplitude. It was slowed following intracellular administration of EDTA, and after equimolar substitution of Ba2+ for Ca2+. It is concluded that the fast component of the calcium inward current decline is mainly due to the superposition of the outward current produced by low selective channels. Only the slow component represents an actual decline of the inward current through calcium channels; it is due to ion accumulation at the inner surface of the cell membrane.

Animals↗
PubMed1984

[Role of calcium ions in the generation of abdominal sense organ response in the scallop].

The effect of a Ca2+-free solution, Cd2+ ions and theophylline on compound response recorded from the nerve which originates from the abdominal sensory organ of the scallop (Patinopecten yessoensis) was studied during mechanical stimulation of this organ. It was shown that the elimination of Ca2+ ions from the extracellular medium or addition of 10 mmol/l Cd2+ caused complete reversible block of the response. The addition of 5 mmol/l theophylline to the medium resulted in a significant increase in the amplitude of the response. These data show participation of Ca2+ ions in the process of the mechanical stimulus transformation into electrical signal as well as a possible regulation of this process by intracellular cAMP-dependent reactions.

Animals↗
PubMed1982

[Blocking of the Ca-dependent inward current in the somatic membrane of mollusk neurons by an elevated intracellular pH].

The action of elevated intracellular pHi (pHi) on the transmembrane ionic currents in the somatic membrane was studied in intracellularly perfused nerve cells from Helix pomatia. Following a change in pHi from 7.3 to 9.0 the amplitude of potassium outward current recorded simultaneously with the calcium inward current was significantly reduced. This was accompanied by a shift of its I-V curve to more positive membrane potential values. In case of the calcium inward current blocking by external Cd2+ ions no reduction of the outward current was observed. Only a shift of its I-V curve along the potential axis remained. The calcium inward current was practically the same. It is suggested that the elevated pHi selectively blocks the Ca-dependent component of the potassium outward current.

Animals↗
PubMed1982

[Inactivation of calcium channels in the somatic membrane of mollusk neurons].

The time-dependent decrease of calcium inward currents (inactivation) at long (about 1s) depolarizing membrane potential shifts was studied on isolated dialysed neurons of the mollusc Helix pomatia. It is shown that the time course of this decrease can be approximated by two exponents with corresponding time constants 20-70 and 250-350 ms. The fast component of the decay disappeared completely after pH increase of the intracellular solution to 8.5. The kinetics of the slow component did not change. A conclusion is made that the fast decay of the recorded current does not represent variations in the calcium current but is due to parallel activation of a nonspecific outward current whereas the slow decay represents true inactivation of the calcium current. It is shown that the inactivation velocity of this current is related to the maximal value of the latter but not to the level of membrane depolarization, and depends on conditions of accumulation of calcium ions at the inner surface of the cell membrane.

Animals↗