Src family kinase potentiates the activity of nicotinic acetylcholine receptor in rat autonomic ganglion innervating urinary bladder

Src family kinases (SFKs), one of the tyrosine kinase groups, are primary regulators of signal transductions that control cellular functions such as cell proliferation, differentiation, survival, metabolism, and other important roles of the cell. One of the crucial functions of SFKs is to regulate the activities of various neuronal channels. In this study, we investigated the modulatory action of SFK on nicotinic acetylcholine receptors (nAChRs) expressed in rat major pelvic ganglion (MPG) neurons innervating the urinary bladder. PP1 and PP2 (5 μM), selective Src-kinase inhibitors, attenuated ACh-induced ionic currents and [Ca2+](i) transients in MPG neurons, whereas PP3, an inactive analogue, had no effect. Blocking the tyrosine kinase activity of Src kinase by pp60 c-src inhibitory peptide also reduced the ACh-induced currents. Conversely, sodium orthovanadate (200 μM), a tyrosine phosphatase inhibitor, significantly augmented the ACh-induced currents. In the kinase assay, the activities of SFKs in MPG neurons were also inhibited by PP2, but not by PP3. These data suggests that SFKs may have a facilitative role on the synaptic transmission in rat pelvic autonomic ganglion.     

Prostaglandin E2 potentiates the excitability of small diameter trigeminal root ganglion neurons projecting onto the superficial layer of the cervical dorsal horn in rats

The aim of the present study was to investigate how prostaglandin E₂ (PGE₂) affects the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, using fluorescence retrograde tracing and perforated patch-clamp techniques. TRG neurons were retrogradely labeled with fluorogold (FG). The cell diameter of FG-labeled neurons was small (< 30 μm). Under the voltage-clamp mode, application of PGE₂ (0.01–10 μM) concentration-dependently increased the magnitude of the peak tetrodotoxin-resistant sodium current (TTX-R I _Na) and this current was maximal at a concentration of 1 μM. One micromolar PGE₂ application caused a hyperpolarizing shift of 8.3 mV in the activation curve for TTX-R I _Na. In the current-clamp mode, the PGE₂ (1 μM) application significantly increased the number of action potentials during the depolarizing step pulses as well as the level of overshoot but had no significant effect on the resting membrane potential. These results suggest that the excitability of small diameter TRG neurons seen after 1 μM PGE₂ application is involved in an increase in the TTX-R I _Na.     

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

Membrane currents and cytoplasmic sodium transients generated by glutamate transport in Bergmann glial cells

Effects of glutamate and kainate (KA) on Bergmann glial cells were investigated in mouse cerebellar slices using the whole-cell configuration of the patch-clamp technique combined with SBFI-based Na⁺ microfluorimetry. l-Glutamate (1 mM) and KA (100 μM) induced inward currents in Bergmann glial cells voltage-clamped at −70 mV. These currents were accompanied by an increase in intracellular Na⁺ concentration ([Na⁺]_i) from the average resting level of 5.2 ± 0.5 mM to 26 ± 5 mM and 33 ± 7 mM, respectively. KA-evoked signals (1) were completely blocked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA ionotropic glutamate receptors; (2) reversed at 0 mV, and (3) disappeared in Na⁺-free, N-methyl-D-glucamine (NMDG⁺)-containing solution, but remained almost unchanged in Na⁺-free, Li⁺-containing solution. Conversely, l-glutamate-induced signals (1) were marginally CNQX sensitive (∼10% inhibition), (2) did not reverse at a holding potential of +20 mV, (3) were markedly suppressed by Na⁺ substitution with both NMDG⁺ and Li⁺, and (4) were inhibited by d,l-threo-β-benzyloxyaspartate. Further, d-glutamate, l-, and d-aspartate were also able to induce Na⁺-dependent inward current. Stimulation of parallel fibres triggered inward currents and [Na⁺]_i transients that were insensitive to CNQX and MK-801; hence, we suggested that synaptically released glutamate activates glutamate/Na⁺ transporter in Bergmann glial cells, which produces a substantial increase in intracellular Na+ concentration.     

Different effects of depolarization and muscarinic stimulation on the Ca2+/force relationship during the contraction-relaxation cycle in the guinea pig ileum

The effects of K⁺ depolarization and of the muscarinic agonist carbachol on [Ca²⁺]_i and force were investigated in smooth muscle sheets of the longitudinal layer of the ileum loaded with Fura-2. K⁺ -rich solutions increased [Ca²⁺]_i and force to an initial peak value, which was determined by the concentration of [K⁺]_o. Thereafter, [Ca²⁺]_i and force declined to a lower maintained level. The Ca²⁺/force relationship observed during this contraction-relaxation cycle is represented by a clockwise hysteresis loop. At 140 mM [K⁺]_o, this loop consisted of three components while at lower [K⁺]_o a two-component loop was observed. The stimulation with 0.1 mM carbachol resulted in a transient increase of [Ca²⁺]_i and force followed by a continuous decline of these parameters despite the presence of the drug. Its EC₅₀ of relaxation was around 270 nM [Ca²⁺]_i. The Ca²⁺/force relationship proceeded along a counterclockwise hysteresis loop during the contraction-relaxation cycle. The extent of this loop decreased but remained unaltered in its direction during repeated stimulation with carbachol. These results suggest that (a) both agonists increase force and [Ca²⁺]_i during stimulation; (b) during depolarization with K⁺, desensitization to Ca²⁺ occurs resulting in a clockwise hysteresis loop; (c) during carbachol stimulation, a counterclockwise hysteresis is observed. This could be due to an increased sensitivity to Ca²⁺ mainly in tonic smooth muscle. These observations might be explained by a modulation of the Ca²⁺ sensitivity by sensitizing and desensitizing mechanisms. These modulations during different stimuli could be due to different myosin light-chain kinase/myosin light-chain phosphatase ratios.     

Mechanisms of intrinsic force in small human airways

We quantified the magnitude and investigated mechanisms regulating intrinsic force (IF) in human airway smooth muscle (hASM). IF was identified by reducing extracellular calcium (Ca2+) concentration to nominally zero in freshly isolated isometrically mounted 2mm human bronchi. Our results show: (1) the magnitude of IF is ～50% of the maximal total force elicited by acetylcholine (10(-5) M) and is epithelial independent, (2) IF can also be revealed by β-adrenergic activation (isoproterenol), non-specific cationic channel blockade (La3+) or L-type voltage gated Ca2+ channel blockade (nifedipine), (3) atropine, indomethacin, AA-861, or pyrilamine did not affect IF, (4) IF was reduced by the intracellular Ca2+ ([Ca2+]i) chelating agent BAPTA-AM, (5) ω-conotoxin had no effect on IF. In studies in cultured hASM cells nominally zero Ca2+ buffer and BAPTA-AM reduced [Ca2+]i but isoproterenol and nifedipine did not. Taken together these results indicate that rapid reduction of [Ca2+]i reveals a permissive relationship between extracellular Ca2+, [Ca2+]i and IF. However IF can be dissipated by mechanisms effecting Ca2+ sensitivity. We speculate that an increase of IF, a fundamental property of ASM, could be related to human airway clinical hyperresponsiveness and must be accounted for in in vitro studies of hASM.     

GABAergic synchronization in the limbic system and its role in the generation of epileptiform activity

GABA is the main inhibitory neurotransmitter in the adult forebrain, where it activates ionotropic type A and metabotropic type B receptors. Early studies have shown that GABA(A) receptor-mediated inhibition controls neuronal excitability and thus the occurrence of seizures. However, more complex, and at times unexpected, mechanisms of GABAergic signaling have been identified during epileptiform discharges over the last few years. Here, we will review experimental data that point at the paradoxical role played by GABA(A) receptor-mediated mechanisms in synchronizing neuronal networks, and in particular those of limbic structures such as the hippocampus, the entorhinal and perirhinal cortices, or the amygdala. After having summarized the fundamental characteristics of GABA(A) receptor-mediated mechanisms, we will analyze their role in the generation of network oscillations and their contribution to epileptiform synchronization. Whether and how GABA(A) receptors influence the interaction between limbic networks leading to ictogenesis will be also reviewed. Finally, we will consider the role of altered inhibition in the human epileptic brain along with the ability of GABA(A) receptor-mediated conductances to generate synchronous depolarizing events that may lead to ictogenesis in human epileptic disorders as well.     

Cholinergic synaptic activation due to HCO 3 − in the superior cervical ganglion of the rat

Influence of acid-base change on synaptic transmission was studied in the isolated superior cervical ganglion of the rat. Effects of changes inP _CO2 [HCO ₃ ^– ], or pH of the superfusing solution were studied, using as an index of synaptic excitation the amplitude of the initial negative deflection of surface potential induced by preganglionic stimulation. An increase or decrease in the extracellular fluid (ECF) pH by changing [HCO ₃ ^– ] at a normalP _CO2 elicited respectively augmentation or suppression of the negative deflection. Similar shifts in the ECF pH with varyingP _CO2 at a normal [HCO ₃ ^– ] had small or almost negligible effects on the negative deflection. Simultaneous increase in both theP _CO2 and [HCO ₃ ^– ], which compensated for the pH change in the ECF, induced a consistent increase in the amplitude of the negative deflection. The amplitude of negative deflection in various acid-base conditions was positively correlated with the ECF [HCO ₃ ^– ] but not with ECF pH orP _CO2. These results suggest that an increase in the ECF [HCO ₃ ^– ] activates cholinergic (nicotinic) synaptic transmission in the ganglion.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Endothelial function in aorta segments of apolipoprotein E-deficient mice before development of atherosclerotic lesions

Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE^−/−) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca²⁺]_i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE^−/− and C57BL/6J (WT) mice were evoked by phenylephrine (3 × 10⁻⁹–3 × 10⁻⁵ M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca²⁺]_i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE^−/− mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca²⁺]_i in apoE^−/− strips with 10⁻⁶ M cyclopiazonic acid or 10⁻⁷ M Ca²⁺-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE^−/− and WT aorta segments. Basal [Ca²⁺]_i was significantly lower in apoE^−/− than in WT strips. Relaxation by ACh (3 × 10⁻⁹–10⁻⁵ M) was time- and dose-dependently related to [Ca²⁺]_i, but neither ACh-induced relaxation nor Ca²⁺ mobilization were diminished in apoE^−/− strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE^−/− mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca²⁺]_i. These findings further point to important differences between basal and stimulated eNOS activity.     

Ionic storm in hypoxic/ischemic stress: Can opioid receptors subside it?

Neurons in the mammalian central nervous system are extremely vulnerable to oxygen deprivation and blood supply insufficiency. Indeed, hypoxic/ischemic stress triggers multiple pathophysiological changes in the brain, forming the basis of hypoxic/ischemic encephalopathy. One of the initial and crucial events induced by hypoxia/ischemia is the disruption of ionic homeostasis characterized by enhanced K⁺ efflux and Na⁺-, Ca²⁺- and Cl⁻-influx, which causes neuronal injury or even death. Recent data from our laboratory and those of others have shown that activation of opioid receptors, particularly δ-opioid receptors (DOR), is neuroprotective against hypoxic/ischemic insult. This protective mechanism may be one of the key factors that determine neuronal survival under hypoxic/ischemic condition. An important aspect of the DOR-mediated neuroprotection is its action against hypoxic/ischemic disruption of ionic homeostasis. Specially, DOR signal inhibits Na⁺ influx through the membrane and reduces the increase in intracellular Ca²⁺, thus decreasing the excessive leakage of intracellular K⁺. Such protection is dependent on a PKC-dependent and PKA-independent signaling pathway. Furthermore, our novel exploration shows that DOR attenuates hypoxic/ischemic disruption of ionic homeostasis through the inhibitory regulation of Na⁺ channels. In this review, we will first update current information regarding the process and features of hypoxic/ischemic disruption of ionic homeostasis and then discuss the opioid-mediated regulation of ionic homeostasis, especially in hypoxic/ischemic condition, and the underlying mechanisms.     

O2 consumption,aerobic glycolysis and tissue phosphagen content during activation of the NA+/K+ pump in rat portal vein

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K⁺ to K⁺-depleted rat portal veins, in order to study the energy turnover associated with Na⁺/K⁺ pumping. During incubation in K⁺-free medium at 37° C spontaneous contractions disappeared in 10–20 min. Readdition of K⁺ (5.9 mM) after 40 min K⁺-free incubation caused hyperpolarization of the cell membrane for the first 5–10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10–20 min. During the first 4–6 min after K⁺ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O₂ consumption (in K⁺-free medium, 0.38 mol/min g) rose by about 10% when K⁺ was added and this increase lasted about 5 min. By 8 min after K⁺ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K⁺ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na⁺/K⁺ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.     

Human Th1 and Th2 lymphocytes are distinguished by calcium flux regulation during the first 10 min of lymphocyte activation

Preliminary data suggest different intracellular calcium handling of Th1 and Th2 lymphocytes that may contribute to distinct cytokine production patterns. In this study we explored the contribution of the main mechanisms in charge of the elevation and decrease of cytoplasmic free calcium levels, i.e., the endoplasmic calcium release, the calcium release activated calcium (CRAC) channel, the mitochondrial calcium uniporter (MCU), the sarco/endoplasmic reticulum calcium ATPase (SERCA), and the plasma membrane calcium ATPase (PMCA) during the first 10 min of activation in human Th1 and Th2 lymphocytes applying a kinetic flow cytometry approach. We isolated peripheral blood mononuclear cells from 10 healthy individuals. Cells were stained with CD4, CXCR3 and CCR4 cell surface markers to identify Th1 and Th2 cells, respectively and loaded with Fluo-3/AM calcium sensitive dye. Cells were activated with phytohemagglutinine and alterations of cytoplasmic free calcium levels were monitored for 10 min after specific inhibition of the above mechanisms. Our results revealed delicate differences in calcium flux kinetics of Th1 and Th2 lymphocytes. The lower activity of MCU, and therefore of CRAC channels, along with the higher activity of the SERCA pump account for the notion that Th2 cells go through a lower level of lymphocyte activation compared with Th1 cells upon identical activating stimuli. The observed differences in calcium flux of Th1 and Th2 cells may contribute to different calcium handling kinetics and, hence, to distinct cytokine production patterns by these subsets.     

Analysis of the variation in use-dependent inactivation of high-threshold tetrodotoxin-resistant sodium currents recorded from rat sensory neurons

This study addressed variation in the use-dependent inactivation (UDI) of high-threshold tetrodotoxin-resistant Na+ currents (TTX-R currents) and action potential firing behavior among acutely isolated rat dorsal root ganglion (DRG) cells. UDI was quantified as the percent decrease in current amplitude caused by increasing the current activation rate from 0.1-1.0 Hz for 20 s. TTX-R current UDI varied from 6% to 66% among 122 DRG cells examined, suggesting the existence of two or more levels of UDI. The voltage-dependency of the TTX-R currents was consistent with Na(V)1.8, regardless of UDI. However, TTX-R currents with more UDI had a more negative voltage-dependency of inactivation, a greater tendency to enter slow inactivation, and a slower recovery rate from slow inactivation, compared with those with less UDI. TTX-R currents with more UDI ran down faster than those with less UDI. However, UDI itself changed little over time, regardless of the initial UDI level observed in a particular DRG cell. Together, these two observations suggest that individual DRG cells did not express mixtures of TTX-R channels that varied regarding UDI. TTX-R current UDI was correlated with expression of a low-threshold A-current and whole-cell capacitance, suggesting that it varied among different nociceptor types. Whole-cell inward currents (WCI-currents), recorded without channel blockers, also exhibited UDI. WCI-current UDI varied similarly to TTX-R current UDI in magnitude, and relative to whole-cell capacitance and A-current expression, suggesting that the WCI-currents were carried predominantly by TTX-R channels. DRG cells with more WCI-current UDI exhibited a greater decrease in action potential amplitude and number, and a greater increase in action potential threshold over seven ramp depolarizations, compared with DRG cells with less WCI-current UDI. Variation in UDI of Na(V)1.8 channels expressed by different nociceptor types could contribute to shaping their individual firing patterns in response to noxious stimuli.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Effects of delta sleep-inducing peptide on pre-and postsynaptic glutamate and postsynaptic GABA receptors in neurons of the cortex, hippocampus, and cerebellum in rats

We studied the effect of delta sleep-inducing peptide on GABA receptors of hippocampal and cerebellar neurons in rats. It was shown that delta sleep-inducing peptide considerably and dose-dependently potentiates GABA-activated currents in these neurons and blocks NMDA-activated potentiation in cortical and hippocampal neurons. The peptide modulates activity of presynaptic NMDA receptors, which is seen from changes in ⁴⁵Ca²⁺ uptake into synaptosomes of the brain cortex after uptake stimulation with glutamate and NMDA. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 149–151, August, 2006     

Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

The mechanism of regulation of intracellular pH (pH_i) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2,7-bis(2-carboxyethyl)-5(and –6)-carboxyfluorescein. The pH_i in the TRIS/HEPES-buffered standard solution was 7.29±0.01. Addition of 1 mol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pH_i. These agents decreased pH_i in the absence of external Na⁺ or in the presence of amiloride. The rate of pH_i recovery from an acid load after NH ₄ ⁺ prepulse was a linear function of pH_i and increased as pH_i became more acidic. Addition of ACh shifted the relationship towards a more alkaline pH_i range. The increase in pH_i induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mol/l). Addition of 0.1–1 mol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pH_i within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pH_i. In Ca²⁺-free solution with 50 mol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pH_i and cytosolic Ca²⁺ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na⁺/H⁺ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na⁺/H⁺ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na⁺/H⁺ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca²⁺ concentration. The implication of the cytosolic acidification and cell volume change in pH_i regulation is discussed.     

Adenosine receptors control HIV-1 Tat-induced inflammatory responses through protein phosphatase

Recently, adenosine has been proposed to be a "metabolic" switch that may sense and direct immune and inflammatory responses. Inflammation and pro-inflammatory cytokine production are important in development of HIV-1 associated dementia, a devastating consequence of HIV-1 infection of the CNS. The HIV-1 protein Tat induces cell death in the CNS and activates local inflammatory responses partially by inducing calcium release from the endoplasmic reticulum. Because activation of adenosine receptors decreases production of the pro-inflammatory cytokine TNF-alpha in several experimental paradigms both in vitro and in vivo, we hypothesized that adenosine receptor activation would control both increased intracellular calcium and TNF-alpha production induced by Tat. Treatment of primary monocytes with Tat significantly increased the levels of intracellular calcium released from IP3 stores. Activation of adenosine receptors with CGS 21680 inhibited Tat-induced increases of intracellular calcium by 90 +/- 8% and was dependent on protein phosphatase activity because okadaic acid blocked the actions of CGS 21680. Tat-induced TNF-alpha production was inhibited 90 +/- 6% by CGS 21680 and concurrent treatment with okadaic acid blocked the inhibitory actions of CGS 21680. Using a model monocytic cell line, CGS 21680 treatment increased cytosolic serine/threonine phosphatase. Together, these data indicate that A2A receptor activation increases protein phosphatase activity, which blocks IP3 receptor-regulated calcium release and reduction of intracellular calcium inhibits TNF-alpha production in monocytes.