Oxytocin and prostaglandins F<Subscript>2α</Subscript> and E<Subscript>2</Subscript> do not enhance HIV antigen production <Emphasis Type="Italic">in vitro</Emphasis>

Summary.  Oxytocin and prostaglandins (PGs) are hormones involved in labor and are used clinically for its induction. In this study the effect of oxytocin, PGF_2α, and PGE₂ on Humour immunodeficiency virus-1 production in acutely and persistently infected cells was measured. No significant effect on p24 antigen production was found with oxytocin or PGs, except for a transient decrease in persistently infected cells treated with 1 μM PGF_2α. These results showed that oxytocin and PGs could be used clinically for labor induction without any direct enhancement in viral production. Besides, the results with PGF_2α at the highest concentration studied may indicate a pharmacological effect. Received October 10, 2002; accepted October 28, 2002     

Activation of wild-type and ΔF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms

 The cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and its modulation through inhibition of phosphodiesterases (PDE) were studied with the cell-attached patch-clamp technique in Calu-3 cells (expressing endogenous CFTR) and NIH3T3 cells [expressing either wild-type (Wt)-CFTR or ΔF508-CFTR]. In Calu-3 cells, CFTR current was augmented by increasing concentrations of 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (CPT–cAMP) and reached a saturating level at ≥60 μM. Varying the forskolin concentration also modulated CFTR activity; 10 μM was maximally effective since supplemental application of 200 μM CPT–cAMP had no additional effect. Activation of CFTR by increasing the cAMP concentration occurs through an increase of the NP _o (product of the number of functional channels and the open probability) since the single-channel amplitude remains unchanged. In Calu-3 and NIH3T3-Wt cells, PDE inhibitors, milrinone (100 μM), 8-cyclopentyl-1,3-dipropylxanthine (CPX, 25 μM), and 3-isobutyl-1-methylxanthine (IBMX, 200 μM), did not enhance CFTR current initially activated with 10 μM forskolin, but each potentiated CFTR activity elicited with a submaximal forskolin concentration (e.g., 100 nM) and prolonged the deactivation of CFTR channel current upon removal of forskolin. Millimolar IBMX increased the NP _o of both Wt- and ΔF508-CFTR even under maximal cAMP stimulation. Quantitatively, these effects of millimolar IBMX on NP _o approximate those of genistein, which potentiates the cAMP-dependent CFTR activity via a mechanism that does not involve increases in cellular cAMP. Thus, depending on the concentration, PDE inhibitors may affect CFTR through different mechanisms. Received: 27 October 1998 / Accepted: 10 November 1998     

The differential contribution of dopamine D<Subscript>1</Subscript> and D<Subscript>2</Subscript> receptors to μ-opioidergic immunomodulation

Activation of μ-opioid receptors (μ-OR) by the highly selective agonist DAGO (100 μg/kg) significantly increased the immune response in CBA mice. This effect of the μ agonist was prevented by prior blockade of dopamine D₂ receptors with haloperidol (2 mg/kg). In contrast, the selective D₁ receptor antagonist SCH 23390 (1 mg/kg) had no effect on the nature of the immune reaction in response to antigen (sheep erythrocytes, 5·10⁸ cells). However, blockade of both types of dopamine receptor led to the same effect-immunosuppression. These data lead to the suggestion that D₁ and D₂ receptors make different contributions to modulating immunogenesis on activation of μ-OR. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 5, pp. 546–551, May, 2006.     

Early stages in the mechanism of action of glucocorticoids on human platelets. Effect of hydrocortisone on platelet aggregation

Hydrocortisone (1–10 μM) has no effect on spontaneous platelet aggregation and induces a 5–10-sec latency after platelet activation with 1 μM ADP. Hydrocortisone inhibits collagen-induced platelet aggregation; this effect is blocked by excess of progesterone. Hydrocortisone potentiates the effect of adenosine on disaggregation: in the absence of the hormone IC₅₀ for adenosine is 2.5 μM, while in the presence of 3 and 10 μM hydrocortisone it drops to 1.7 and 0.4 μM, respectively. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 1, pp. 54–57, January, 1997     

Changes in ATP content in cerebellar granule cells during hyperstimulation of glutamate receptors: Possible role of NO and nitrite ions

In primary 7–8-day culture of cerebellar granule cells, glutamate exposure (100 μM, 10–240 min) induced a 60–30% drop in ATP level; during the postglutamate period ATP level completely recovered after 24 h. Inhibition of NO-synthase with L-NAME during glutamate application resulted in less pronounced decrease in ATP level immediately after its application and had no effect on ATP recovery after 24 h. It was found that hyperstimulation of glutamate receptors elevates concentration of NO products (nitrites and nitrates), while NO₂ ⁻ ions can increase ATP content. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 419–422, April, 2007     

Cell swelling causes the action potential duration to shorten in guinea-pig ventricular myocytes by activating I KATP

 In guinea-pig ventricular myocytes, cell swelling by incubation in hypotonic solution caused a pronounced shortening of the action potential duration (APD₉₀: 15.5±14.6% compared to control; mean ± SD) after a latency of 12 min when the intracellular ATP concentration was 2 mM. This shortening was partially reversible within 10 min after reperfusion with isotonic solution (APD₉₀: 80.5±12.1% compared to control). With 5 mM intracellular ATP in the pipette electrode, the effect of cell swelling on the action potential was significantly reduced. Incubation with 1 μM glibenclamide, a blocker of the ATP-dependent K⁺ current (I _KATP), abolished the swelling-induced shortening of the action potential duration, whereas incubation with 0.5 mM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS), a blocker of the swelling-induced Cl^– current (I _Cl,swell), had no effect on the action potential duration in hypotonic solution. Simultaneous measurements of membrane currents substantiate that I _KATP is the current that underlies this effect. These results suggest that in the ischaemic myocardium I _KATP may be partially activated by cell swelling, resulting in a shortening of the action potential duration before the intracellular ATP concentration has fallen below 2 mM. Received: 30 March 1998 Received after revision: 7 July 1998 Accepted: 25 July 1998     

Antiamnestic effect of α7-nicotinic receptor agonist RJR-2403 in middle-aged ovariectomized rats with Alzheimer type dementia

The effects of chronic combined treatment with α7-nicotinic cholinergic receptor agonist RJR-2403 (1.0 mg/kg intraperitoneally) or α7-nicotinic cholinergic receptor antagonist mecamylamine (1.0 mg/kg intraperitoneally) and 17β-estradiol (0.5 μg per rat intramuscularly) for 10 days on passive avoidance retention were studied in middle-aged (15 months) ovariectomized rats with experimental Alzheimer type dementia. Chronic treatment with RJR-2403 and 17β-estradiol had a pronounced antiamnestic effect under conditions of Alzheimer type dementia in middle-aged ovariectomized rats. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 12, pp. 656–658, December, 2006     

Naloxone-blocked depriming effect of anxiolytic selank on apomorphine-induced behavioral manifestations of hyperfunction of dopamine system

Peptide anxiolytic selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) applied intraperitoneally in doses of 0.01, 0.1, 1.0, and 10.0 mg/kg to mice reduces behavioral manifestations of dopaminergic system induced by apomorphine in the verticalization test. This effect was comparable to that of atypical antipsychotic olanzapine in near-therapeutic doses (0.1 and 1.0 mg/kg, intraperitoneally) and was blocked with nonselective opioid receptor antagonist naloxone (10 mg/kg, intraperitoneally). Radioreceptor assay showed that selank did not displace nonselective D₂-dopamine receptor antagonist ³H-spiperone (EC₅₀>100 μM) and δ-and μ-opioid receptor ligand ³H-DADLE (EC₅₀>40 μM) from specific binding sites on rat brain membranes. It is hypothesized that the revealed behavioral effect of selank is mediated by its modulating effect on the endogenous opioid system and specifically, by its effect on activity of enkephalin-degrading enzymes. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 545–548, November, 2006     

Effect of β-hydroxy-β-methylglutaryl coenzyme a reductase inhibitor atorvastatin on contractility of the isolated rat heart under normal conditions and during oxidative stress

Long-term administration of β-hydroxy-β-methylglutaryl coenzyme A reductase inhibitor atorvastatin to rats was accompanied by an increase in the relative weight of the heart and decrease in the rate of pressure development in the isovolumic heart. During oxidative stress induced by addition of 100 μM H₂O₂ to the perfusate, the decrease in contractile function was more pronounced that in the control. Our results indicate that administration of atorvastatin is accompanied by a decrease in myocardial contractility, which becomes more pronounced under conditions of oxidative stress. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 383–385, April, 2007     

Role of cannabinoid receptor agonists in mechanisms of suppression of central pain syndrome

We studied the effect of cannabinoid receptor agonists anandamide and WIN 55,212-2 on the central pain syndrome induced by intraspinal injection of penicillin sodium salt in rats. Cannabinoids suppressed allodynia and spontaneous attacks in rats with the central pain syndrome. The analgesic effect was most pronounced after intrathecal injection of cannabinoid receptor agonist in a dose of 100 μg in 10 μl. After systemic treatment the analgesic effect was produced by only WIN 55,212-2 in a dose of 1 mg/kg. WIN 55,212-2 was superior to anandamide by the duration and intensity of the effect on allodynia and spontaneous attacks. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 47–50, July, 2006     

Use of electrochemical impedance measurements to monitor β-adrenergic stimulation of bovine aortic endothelial cells

 Due to the high permeability of endothelial cell layers derived from macrovascular vessels, precise determination of their barrier function towards ion movement requires refined experimental techniques. We thus cultured bovine aortic endothelial cells (BAEC) directly on thin gold-film electrodes and measured the electrochemical impedance to study their passive electrical properties in general and during β-adrenergic stimulation. Impedance spectra (10–2·10⁶ Hz) of confluent cell monolayers revealed that the electrical characteristics of the cells can be modelled by a simple resistor-capacitor parallel network. Under control conditions the overall resistance of confluent BAEC monolayers was 3.6±0.6 Ω·cm² (n=30) and the capacitance was 0.6±0.1 μF/cm². Both quantities are discussed with respect to morphological characteristics of these cells. Stimulation of BAECs with the synthetic β-adrenoceptor agonist isoproterenol leads to a concentration-dependent, highly specific increase of the cell layer resistance characterized by a concentration for half-maximal response (EC₅₀) of 0.3±0.1 μM. The cell layer capacitance, however, remained unaffected. Using impedance measurements at a single frequency, we analysed the response of BAECs to treatment with isoproterenol in comparison with several chemically unrelated compounds known to stimulate the adenosine 3’,5’-cyclic monophosphate (cAMP)-dependent signal transduction cascade. These studies confirmed that the enhancement of the cell layer resistance after β-adrenergic stimulation is mediated by an increase in intracellular cAMP. Received: 23 September 1998 / Received after revision: 23 November 1998 / Accepted: 28 January 1999     

Effect of PAR1 agonist on acetylcholine secretion in a newly formed neuromuscular synapse in mice

Peptide agonist of PAR1 in a concentration of 10 μM significantly facilitated neuromuscular transmission in newly formed synapses in mice. The absence of changes in the amplitude of miniature end-plate potentials attests to presynaptic mechanism of the effect of PAR1 agonist. The effect of the peptide was blocked by protein kinase A inhibitor H89 (1 μM). Blockade of inositol-1,4,5-trisphosphate receptors with 2-aminoethoxydiphenylborate (30 μM) did not prevent the effects of PAR1 agonist. Inhibition of protein kinase C with bisindolylmaleimide (1 μM) facilitated neuromuscular transmission in newly formed synapses. Protein kinase C inhibition was associated with reversal of the object of PAR1 agonist: transmission inhibition instead of facilitation. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 490–494, November, 2007     

On the mechanism of the enhancement of delayed rectifier K+ current by extracellular ATP in guinea-pig ventricular myocytes

 The effects of extracellular adenosine 5′-triphosphate (ATP) on the delayed rectifier K⁺ current (I _K) were studied in guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique. ATP increased I _K concentration dependently with a concentration eliciting a half-maximal response of 1.86 μM and a maximal increase of about 1.8-fold. The enhancement of I _K developed slowly, the effect reaching a maximum in about 1.6 min after application of ATP. The rank order of agonist potency in enhancing I _K was 2-methylthio-ATP≥ ATP>>α,β-methylene-ATP. The ATP response was attenuated in guanosine 5′-O-(2-thiodiphosphate) (GDPβS)- loaded cells, but was not affected by pertussis toxin (PTX)-pre-treatment, indicating that a PTX-insensitive G protein is involved in the response. These features are consistent with operation of P_2Y-type purinoceptors. ATP produced a further increase in I _K stimulated maximally either by isoprenaline (1 μM) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 μM) did not affect the ATP response, suggesting that PKA and PKC do not mediate the response. ATP irreversibly enhanced I _K in cells loaded with adenosine 5′-O-(3-thiotriphosphate) (ATPγS, 5 mM) or okadaic acid (10 μM), a phosphatase inhibitor, suggesting that a phosphorylation step is present after the receptor stimulation. Genistein, an inhibitor of tyrosine phosphorylation, suppressed the ATP response significantly, while daidzein, an inactive analogue of genistein, had little effect on it, although both genistein or daidzein alone decreased I _K. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of cardiac I _K by P_2Y-purinergic stimulation. Received: 10 August 1998 / Received after revision: 14 November 1998 / Accepted: 4 December 1998     

On the mechanism of genistein-induced activation of protein kinase A-dependent Cl– conductance in cardiac myocytes

Genistein, an inhibitor of protein tyrosine kinase (PTK), enhanced the activation of the cardiac isoform of the protein kinase A (PKA)-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– conductance in guinea-pig ventricular cells. We examined the mechanism(s) underlying this excitatory action of genistein by using patch-clamp techniques. The CFTR Cl^– conductance, activated by isoproterenol (ISO, 10 nM; [Cl^–] 153 mM extracellular, 21 mM intracellular; 36 °C), was enhanced by 20 μM genistein. Daidzein, a structural analogue of genistein with little inhibitory action on PTK, also enhanced CFTR Cl^– currents. After maximal activation of the Cl^– conductance by a cocktail of adenosine 3’,5’-cyclic monophosphate, 3-isobutyl-1-methylxanthine and okadaic acid or vanadate plus forskolin in the pipette, genistein was no longer stimulatory but was rather slightly inhibitory at 100 μM. Direct exposure of myocytes to higher concentrations of genistein (50–100 μM) elicited outwardly rectifying currents with a reversal potential of –47 mV in the absence of ISO. In the presence of 50 μM H-89, a PKA inhibitor, genistein had no effect. Vanadate in the pipette at a concentration (100 μM) inhibiting phosphotyrosine phosphatases alone did not prevent the action of genistein. In contrast, no conductance was activated by tyrphostins B42 or 51 or lavendustin A, other PTK inhibitors. Genistein’s stimulation of cardiac CFTR Cl^– conductance appears to be independent of the PTK pathway and to be due to its direct interaction with CFTR Cl^– channels. Received: 22 January 1999 / Received after revision: 9 April 1999 / Accepted: 22 April 1999     

Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a messenger molecule with multiple clinical implications. Understanding the activation of sGC is an important step for establishing new therapeutic principles. We have now overexpressed sGC in a baculovirus/Sf9 system optimized for high protein yields to facilitate spectral and kinetic studies of the activation mechanisms of this enzyme. It was expressed in a batch fermenter using a defined mixture of viruses encoding the α₁ and β₁ subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosolic fraction by anion exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography. By use of this new method 2.5 l culture yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. The enzyme did not contain stoichiometric amounts of copper. The basal activities of the purified enzyme were 153 and 1259 nmol min^–1 mg^–1 in the presence of Mg²⁺ and Mn²⁺, respectively. The nitric oxide releasing agent 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fold with Mg²⁺, whereas the NO-independent activator 3-(5’-hydroxymethyl-2’-furyl)-1- benzylindazole (YC-1) induced an increase in the activity of 101-fold at a concentration of 300 μM. The combination of DEA/NO (10 μM) and YC-1 (100 μM) elicited a dose-dependent synergistic stimulation with a maximum of a 792-fold increase over the basal activity in the presence of Mg²⁺, resulting in a specific activity of 121 μmol min^–1 mg^–1. The synergistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 μM) by 94%. In a different experimental setup a saturated carbon monoxide solution in the absence of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme spectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 424 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme spectra were not affected by YC-1 in the absence or in the presence of DEA/NO or of carbon monoxide, which reflects the fact that YC-1 does not interact directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The described method should help to facilitate the investigation of the new therapeutic principle of NO-independent guanylyl cyclase activators. Received: 20 May 1998 / Accepted: 5 October 1998     

α1-Adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium

 The effects of adrenoceptor agonists on the transepithelial Cl^– conductance (G ^Cl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively and reversibly inhibited the voltage-activated G ^Cl in toad skin and the short-circuit G ^Cl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands, since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations of Epi were variable among individual tissues. G ^Cl was reduced to 34±17% (n=46) with 1 μmol/l Epi, but in some tissues 0.1 μmol/l inhibited more than 80% of G ^Cl, whereas some preparations were little influenced at >3 μmol/l Epi. The affected receptor type was identified by the use of the α₁-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 μmol/l, whereas the α₂-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific α₁-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The α₂-antagonist yohimbine, at concentrations ≤0.3 μmol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations of 1–10 μmol/l. This might indicate affinity to α₁-adrenoceptors in amphibian skin. Activation of β-adrenoceptors by isoproterenol (0.1–5 μmol/l) led to a transient increase of the baseline inactivated component of G ^Cl with a slight reduction of the voltage-activated G ^Cl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented nor reversed the induction of a voltage-insensitive G ^Cl in toad skin caused by application of cAMP at supramaximal concentrations (>100 μmol/l CPT-cAMP). Preincubation of the serosal medium with Ca²⁺-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It is concluded that α₁-adrenoceptors participate in the physiological control of voltage-activated Cl^– conductance in amphibian skin epithelium via modulation of intracellular Ca²⁺, presumably by efflux from intracellular stores. Received: 11 March 1998 / Received after revision: 2 June 1998 / Accepted: 8 June 1998     

The cellular mechanisms of Cl– secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias

 We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [I _sc=transepithelial voltage/transepithelial resistance (R _te)] as well as basolateral membrane voltage (V _bl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a ”stimulation cocktail” (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in I _sc (–31.6±7.7 to –316±82.2 μA/cm², n=15). CNP significantly depolarized the luminal membrane from –87.4±1.0 to –82.3±2.6 mV (n=12). V _bl was not changed (n=12) but the fractional conductance for K⁺ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on I _sc were not additive (n=5). The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02±0.05 to 1.32±0.05 (n=8) with a time constant in the 1–2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in I _sc in the rectal gland. The concomitant fall in R _te corresponds to increases in the luminal membrane Cl^– conductance and in the basolateral membrane K⁺ conductance. The latter effect is probably due to an increase in [Ca²⁺]_i. Received: 21 December 1998 / Received after revision: 5 February 1999 / Accepted: 9 February 1999     

Effect of antibodies against AMPA glutamate receptors on brain neurons in primary cultures of the cerebellum and hippocampus

Rabbit antibodies against GluR₁ subunit of AMPA glutamate receptors in a concentration of 1 μg/ml significantly increased intracellular Ca²⁺ concentration and decreased mitochondrial potential in hippocampal neurons, i.e. produced changes typical of the influence of glutamate in toxic concentrations. In cerebellar neurons rabbit antibodies potentiated glutamate-induced increase in intracellular Ca²⁺ concentration and significantly decreased the mitochondrial potential (compared to the level observed after application of glutamate alone). The exposure of cultured cerebellar neurons to antibodies in a concentration of 0.1 μg/ml for 24 h was followed by a 50% decrease in ATP concentration and development of neuronal necrosis. Our results attest to an important role of autoimmune damage to neurons during hyperstimulation of glutamate receptors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 59–62, July, 2006     

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

 The effect of adenosine triphosphate (ATP) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca²⁺]_i increase, which was dose dependent in the 2.5–50 μM range (EC₅₀=4.3 μM). The ATP-evoked [Ca²⁺]_i rise was not modified during the first minute following the removal of external Ca²⁺. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca²⁺]_i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca²⁺]_i rise. The pituicytes responded to various P₂ purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P₂ purinoceptor antagonists blocked the ATP-evoked [Ca²⁺]_i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca²⁺]_i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca²⁺]_i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca²⁺]_i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P_2Y receptor. Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998     

Lack of involvement of G proteins in the activation of cardiac CFTR Cl– current by genistein

 The involvement of guanine nucleotide-binding proteins (G proteins) in the activation of cardiac adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– current (I _Cl) by the tyrosine kinase inhibitor genistein (GST) was investigated in guinea-pig ventricular myocytes. Pertussis toxin (PTX) and intracellular application of 1 mM non-hydrolysable guanosine-5′-0-(2-thiodiphosphate) (GDPβS) and guanosine-5′-0-(3-thiotriphosphate) (GTPγS) were used to modify G protein activity, and the efficacy of the treatments determined by examining the activation of I _Cl by isoproterenol (ISO) and forskolin (FSK), and its inhibition by 1 μM acetylcholine (ACh). GDPβS inhibited ISO-activated I _Cl by 80–90%, but had little effect on I _Cl activated by different GST regimens (50 μM; 100 μM; 50 μM plus 0.1 μM FSK). GTPγS had little effect on the amplitude of I _Cl activated by 1 μM ISO, whereas it increased the amplitude of the current activated by 50 and 100 μM GST and rendered it insensitive to 1 μM ACh (inhibition of 2±2% versus (PTX-sensitive) inhibition of 94±3% in control myocytes). Unlike I _Cl activated by ISO in GTPγS-dialysed myocytes, I _Cl activated by GST deactivated on removal of the drug. GST (50 μM) reversibly increased I _Cl by nearly 50% in myocytes with G_s selectively activated by 1 μM ISO, and also reversibly increased the I _Cl that was persistently activated after withdrawal of ISO from GTPγS-dialysed myocytes. These results indicate that G proteins are not involved in the pathway between GST binding and CFTR opening, and suggest that enhanced adenylate cyclase activity in GTPγS-dialysed myocytes mediates the potentiated responses to GST. Received: 11 September 1998 / Received after revision: 6 January 1999 / Accepted: 12 January 1999