Presence Of TEA Research Articles

Paired pulse analysis of ATP and noradrenaline release from sympathetic nerves of rat tail artery and mouse vas deferens: effects of K+ channel blockers.

1. The paired pulse stimulus paradigm - two pulses of equal strength delivered at variable interpulse intervals was used to study the release of ATP and noradrenaline (NA) from post ganglionic sympathetic nerves of rat tail artery and mouse vas deferens. 2. Excitatory junction currents (EJCs) were used to measure the release of ATP, and differential pulse amperometry to measure that of NA. 3. At interpulse intervals of 0.1 - 1 s paired pulse stimulation caused an increase in the size of the second EJC, both in rat tail artery and mouse vas deferens. As the interpulse interval was increased to 10 s or more, the two EJCs became of equal size. 4. In both preparations the K+ channel blockers tetraethylammonium (TEA, 20 mM) and 4-aminopyridine (4-AP, 1 mM) prolonged the duration of the nerve terminal spike and greatly amplified the first EJC of the pair. 5. In the presence of TEA and 4-AP in rat tail artery paired pulse stimulation caused a dramatic depression of the second EJC without markedly affecting the nerve terminal spike. The depression of the second EJC decreased with increasing interpulse intervals, and also when external Ca2+ was reduced to 0.2 mM. In mouse vas deferens, TEA and 4-AP caused only a modest depression of the second EJC. 6. In rat tail artery in the presence of TEA and 4-AP paired pulse stimulation caused a depression of the NA oxidation current evoked by the second pulse, which was similar in magnitude and time course to that of the EJC. Similar TEA and 4-AP induced depression of the second pulse response was also observed when the purinergic and noradrenergic components of the contractile response were investigated. 7. The results show that in rat tail artery K+ channel blockers cause a dramatic paired pulse depression of the release of ATP and NA. The similarity in the depression of the EJC, the NA oxidation current, and the purinergic and noradrenergic components of the contractile response is compatible with the hypothesis that ATP and NA are released in parallel from the same neuronal sources.

Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries.

Gender differences in the incidence of stroke and migraine appear to be related to circulating levels of estrogen; however, the underlying mechanisms are not yet understood. Using resistance-sized arteries pressurized in vitro, we have found that myogenic tone of rat cerebral arteries differs between males and females. This difference appears to result from estrogen enhancement of endothelial nitric oxide (NO) production. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (Ovx), or ovariectomized with estrogen replacement (Ovx + Est). The maximal passive diameters (0 Ca2+ + 1 mM EDTA) of arteries from all four groups were identical. In response to a series of 10-mmHg step increases in transmural pressure (20-80 mmHg), myogenic tone was greater and vascular distensibility less in arteries from males and Ovx females compared with arteries from either untreated or Ovx + Est females. In the presence of NG-nitro-L-arginine methyl ester (L-NAME; 1 microM), an NO synthase inhibitor, myogenic tone was increased in all arteries, but the differences among arteries from the various groups were abolished. Addition of L-arginine (1 mM) in the presence of L-NAME restored the differences in myogenic tone, suggesting that estrogen works through an NO-dependent mechanism in cerebral arteries. To determine the target of NO-dependent modulation of myogenic tone, we used tetraethylammonium (TEA; 1 mM) to inhibit large-conductance, calcium-activated K+ (BKCa) channels. In the presence of TEA, the myogenic tone of arteries from all groups increased significantly; however, myogenic tone in arteries from males and Ovx females remained significantly greater than in arteries from either untreated or Ovx + Est females. This suggests that activity of BKCa channels influences myogenic tone but does not directly mediate the effects of estrogen. Estrogen appears to alter myogenic tone by increasing cerebrovascular NO production and/or action.

Semiconductor Photocatalysis. Part 22. Visible-Light Induced Photoreduction of CO2 with CdS Nanocrystallites — Importance of the Morphology and Surface Structures Controlled through Solvation by N,N-Dimethylformamide

Abstract Carbon dioxide (CO2) is reduced to CO on hexagonal CdS nanocrystallites (mean diameter of 4 nm) prepared in N,N-dimethylformamide (DMF) under visible-light irradiation with a quantum yield of 0.098 at λ = 405 nm in the presence of TEA as an electron donor. Solvation of the surface of CdS nanocrystallites by DMF or a related amide molecules should control the crystalline growth and stabilize the favorable morphology with a size quantization-effect. The effect of Cd2+ or H2S on CO production and the emission behavior, and the effect of electron donors suggest an important role of the surface structures of CdS–DMF for photo-induced electron transfer to CO2. The scope and limitation of the photoreduction of CO2 with semiconductor nanoparticles are discussed.

Effect of dehydration of a silica surface on chemisorption of methanol

Using IR spectroscopy we investigate the chemisorption of methanol on surfaces of pyrogenous silica with different degrees of dehydration. We show that this dependence has an extremal character with minimum chemisorption of alcohol in silicas that have been thermally evacuated at 200–250°C. The presence of preadsorbed triethylamine molecules on the surface of thermally evacuated silica does not change the general degree of substitution of free silanol groups by methoxyl ones in the presence of TEA does not exceed 50% and also depends on the sequence in which the reagents are admitted.

Characterization and modulation of EDHF-mediated relaxations in the rat isolated superior mesenteric arterial bed.

1. We have used the isolated, buffer-perfused, mesenteric arterial bed of the rat (preconstricted with methoxamine or 60 mM K+) to characterize nitric oxide (NO)-independent vasorelaxation which is thought to be mediated by the endothelium-derived hyperpolarizing factor (EDHF). 2. The muscarinic agonists carbachol, acetylcholine (ACh) and methacholine caused dose-related relaxations in preconstricted preparations with ED50 values of 0.18 +/- 0.04 nmol (n = 8), 0.05 +/- 0.02 nmol (n = 6) and 0.26 +/- 0.16 nmol (n = 5), respectively. In the same preparations NG-nitro-L-arginine methyl ester (1-NAME, 100 microM) significantly (P < 0.05) decreased the potency of all the agents (ED50 values in the presence of L-NAME: carbachol, 0.66 +/- 0.11 nmol; ACh, 0.28 +/- 0.10 nmol; methacholine, 1.97 +/- 1.01 nmol). The maximal relaxation to ACh was also significantly (P < 0.05) reduced (from 85.3 +/- 0.9 to 73.2 +/- 3.7%) in the presence of L-NAME. The vasorelaxant effects of carbachol were not significantly altered by the cyclo-oxygenase inhibitor indomethacin (10 microM; n = 4). 3. The K+ channel blocker, tetraethylammonium (TEA, 10 mM) also significantly (P < 0.001) reduced both the potency of carbachol (ED50 = 1.97 +/- 0.14 nmol in presence of TEA) and the maximum relaxation (Rmax = 74.6 +/- 3.2% in presence of TEA, P < 0.05, n = 3). When TEA was added in the presence of L-NAME (n = 4), there was a further significant (P < 0.001) decrease in the potency of carbachol (ED50 = 22.4 +/- 13.5 nmol) relative to that in the presence of L-NAME alone, and Rmax was also significantly (P < 0.05) reduced (74.6 +/- 4.2%). The ATP-sensitive K+ channel inhibitor, glibenclamide (10 microM), had no effect on carbachol-induced relaxation (n = 9). 4. High extracellular K+ (60 mM) significantly (P < 0.01) reduced the potency of carbachol (n = 5) by 5 fold (ED50: control, 0.16 +/- 0.04 nmol; high K+, 0.88 +/- 0.25 nmol) and the Rmax was also significantly (P < 0.01) reduced (control, 83.4 +/- 2.7%; high K+, 40.3 +/- 9.2%). The residual vasorelaxation to carbachol in the presence of high K+ was abolished by L-NAME (100 microM; n = 5). In preparations preconstricted with high K+, the potency of sodium nitroprusside was not significantly different from that in preparations precontracted with methoxamine, though the maximal response was reduced (62.4 +/- 3.4% high K+, n = 7; 83.1 +/- 3.1% control, n = 7). 5. In the presence of the cytochrome P450 inhibitor, clotrimazole (1 microM, n = 5 and 10 microM, n = 4), the dose-response curve to carbachol was significantly shifted to the right 2 fold (P < 0.05) and 4 fold (P < 0.001) respectively, an effect which was further enhanced in the presence of L-NAME. Rmax was significantly (P < 0.01) reduced by the presence of 10 microM clotrimazole alone, being 86.9 +/- 2.5% in its absence and 61.8 +/- 7.8% in its presence (n = 6). 6. In the presence of the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (6 microM), the inhibitory effects of L-NAME on carbachol-induced relaxation were substantially enhanced (ED50: L-NAME alone, 0.52 +/- 0.11 nmol, n = 5; L-NAME + 8-bromo cyclic GMP, 1.42 +/- 0.28 nmol, n = 7, Rmax: L-NAME alone, 82.2 +/- 2.4%; L-NAME + 8-bromo cyclic GMP, 59.1 +/- 1.8%. P < 0.001). These results suggest that the magnitude of the NO-independent component of vasorelaxation is reduced when functional cyclic GMP levels are maintained, suggesting that basal NO (via cyclic GMP) may modulate EDHF activity and, therefore, on loss of basal NO production the EDHF component of endothelium-dependent relaxations becomes functionally greater. 7. The present investigation demonstrates that muscaranic receptor-induced vasorelaxation in the rat mesenteric arterial bed is mediated by both NO-dependent and independent mechanisms. The L-NAME-insensitive mechanism, most probably occurs via activation of a K+ conductance and shows the characteristics of EDHF-mediated responses. Finally, the results demonstrate that EDHF activity may become upregulated on inhibition of NO production and this may compensate for the loss of NO.

Calcium-activated potassium channel-mediated arteriolar relaxation during endotoxic shock.

The role of calcium-activated potassium (KCa) channels in the in vivo relaxation of arterioles was investigated before endotoxin shock (Pre-ENDT) and during endotoxin shock at 180 min (Post-ENDT). Diameters of 2nd and 3rd order (A2 and A3) arterioles in the left cremaster muscle of male Sprague-Dawley rats anesthetized with pentobarbital sodium were measured using videomicroscopy. Adenosine (ADO) at 534 micrograms intraarterially, topical ADO at 10(-3) M, and the endothelium-dependent agonist topical acetylcholine (ACH) at 10(-4) M significantly dilated both A2 and A3 arterioles Pre-ENDT and Post-ENDT. Topical tetraethylammonium chloride (TEA) at 1 mM blocked ADO (intraarterially and topical)-induced A2 and A3 arteriolar dilations Pre-ENDT and Post-ENDT. Arteriolar dilation to ACH was maintained Pre-ENDT, but was blocked by TEA in A2 and A3 arterioles Post-ENDT. The endothelium-independent agonist sodium nitroprusside (10(-5) M), when topically applied, caused maximal arteriolar dilation Pre-ENDT and Post-ENDT in the presence of TEA. The data show that vascular smooth muscle KCa channels are a significant factor in ADO-induced relaxation of cremaster microvessels and are not significantly affected by ENDT. The results also suggest that the mechanism for endothelium-dependent ACH vasodilation changes from a non-KCa channel-mediated mechanism Pre-ENDT to a KCa-mediated mechanism Post-ENDT.

Effect of metabolic inhibitors on membrane potential and ion conductance of rat astrocytes.

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin. 2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13-20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique. 3. After 2.0 min of incubation ATP decreased from the control level to 43% with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 microM), and 69% with oligomycin (10 microM). 4. Under normal conditions V was -74.4 +/- 1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2 +/- 1.3 and 19.7 +/- 3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve. 5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1 +/- 2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8 +/- 3.5 mV and increased g by 45.5 +/- 9.6%. The action of FCCP was not affected by the presence of TEA. 6. Perfusion of the astrocytes with a Cl- free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2 +/- 1.5 mV in DNP and 3.7 +/- 0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl-]. 7. Block of tentative KATP channels with tolbutamide (1 mM) or Cl- channels with Zn2+ (1 mM) did not inhibit the depolarization caused by DNP or FCCP. 8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl- channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.

Effects of tea and chlorophyllin on the mutagenicity of N-hydroxy-IQ: studies of enzyme inhibition, molecular complex formation, and degradation/scavenging of the active metabolites.

Green tea and black tea inhibit the formation of carcinogen-DNA adducts and colonic aberrant crypts in rats given 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), a mutagen from cooked meat. The Salmonella mutagenicity assay was used in the present study to test individual constituents of tea as inhibitors of 2-hydroxyamino-3-methylimidazo[4, 5-f]quinoline (N-hydroxy-IQ), a direct-acting metabolite of IQ. Testing of pure compounds at doses relevant to their levels in tea identified epigallocatechin (EGC) and epigalocatechin-3-gallate (EGCG) as the primary antimutagens. Studies of the inhibitory mechanisms established that the rate of degradation of N-hydroxy-IQ under aqueous conditions was not increased significantly in the presence of tea, in contrast to the results obtained with the complexing agent chlorophyllin (CHL), which rapidly degraded the mutagen. Interaction between N-hydroxy-IQ and several tea constituents was detected in spectrophotometric studies, but the binding constants were only on the order of 1 x 10(3) M-1, suggesting that mechanisms other than complex formation might prevail under the conditions of the Salmonella assay. Comparison of the results in two different strains of Salmonella typhimurium, TA98 and TA98/1,8-DNP6, indicated that the antimutagenic activity of EGCG was dependent, at least in part, on a functional O-acetyltransferase activity in the bacteria. These studies suggest that tea constituents inhibit the enzyme(s) which generate the aryl nitrenium ion and directly scavenge the reactive electrophile, whereas CHL complexes with heterocyclic amines and facilitates the degradation of active metabolites.

Effects of Anion Channel Inhibitors on Light-Induced Potential Changes in the Liverwort Conocephalum conicum

The effect of three different anion channel inhibitors, namely (5-nitro-2-3-phenylpropyl-amino)benzoic acid (NPPB), Zn 2+ and anthracene-9-carboxylic acid (A-9-C) on the action potential in the liverwort Conocephalum conicum were tested. All three caused an increase of the excitability threshold and a decrease of action potential amplitudes. This confirms the involvement of anion channels in the action potentials in Conocephalum. In plants treated with 1 or 2 mM A-9-C but not with NPPB (50 or 100 //M) and Zn 2+ (100 or 500 //M), a light-induced transient depolarization occurred. In contrast to action potentials, the amplitude of this voltage transient depended on the light intensity and on the duration of preceding dark period. Also in contrast to action potentials, which are blocked by TEA, when applied together with A-9-C, TEA even increased the amplitudes of the light-induced voltage transients to up to 170 mV. The depolarization was obviously limited by the voltage-dependent opening of K + channels in the absence of TEA. The amplitude of the light-induced voltage transients (in the presence of TEA) increased in elevated CaCI2 concentrations pointing to a Ca 2+ permeability giving rise to the depolarization. However, none of the Ca 2+ channel blockers tested, La 3+ , Gd 3+ , nifedipine, verapamil or diltiazem, had an effect. The light-induced voltage transients in A-9-C treated plants are quite different from light- and electrically triggered action potentials but share some similarities with light-induced generator potentials.

Ionic Currents Underlying Developmental Regulation of Repetitive Firing inAplysiaBag Cell Neurons

We have investigated the developmental regulation of the ability to fire repetitively in the bag cell neurons of Aplysia californica, a neuronal system in which the behavioral effects of repetitive firing are well characterized. Adult bag cell neurons exhibit an afterdischarge, consisting of prolonged depolarization and repetitive firing, which causes the release of several peptides from these neurons that induce egg-laying behaviors. Afterdischarge can be triggered in vitro by a variety of stimuli, including electrical stimulation and exposure to the potassium channel blocker tetraethyl ammonium chloride (TEA). In contrast to adults, juvenile neurons did not exhibit afterdischarge in response to pleural-abdominal connective shock or TEA. Juvenile neurons did exhibit, however, prolonged depolarizations in the presence of TEA, perhaps reflecting the anlage of the mechanism responsible for afterdischarge in the adult. To investigate developmental mechanisms underlying the regulation of repetitive firing, we compared ionic currents in adult and juvenile bag cell neurons. We found that during the period in which these neurons acquire the capacity to fire repetitively, a number of currents are regulated: (1) three K+ currents decrease (Ca2+)-dependent K+ and two components of voltage-dependent delayed-rectifier K+ current); (2) A-type K+ current increases; and (3) two Ca2+ currents increase (basal and PKC-activated). This pattern is consistent with the increase in the ability to fire repetitively that we observe during maturation: our results indicate that developmental control of repetitive firing in this system is accompanied by selective regulation of specific ionic currents which, after maturation, play important roles in generating the afterdischarge and triggering egg-laying behaviors.

The possible role of ATP and PACAP as mediators of apaminsensitive NANC inhibitory junction potentials in circular muscle of guinea-pig colon.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM), and the selective tachykinin NK1 and NK2 receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 microM each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal colon, recorded by the modified single sucrose gap technique. 2. The P2 purinoceptor agonist, alpha, beta methylene ATP (alpha, beta mATP, 100 microM) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 microM) both produced hyperpolarization (11 +/- 0.8 mV, n = 14 and 10.2 +/- 0.8 mV, n = 19, respectively) and relaxation (1.1 +/- 0.2 mV, n = 14 and 1.5 +/- 0.2 mN, n = 19, respectively) of the circular muscle. 3. Apamin (0.1 microM) nearly abolished (about 90% inhibition) the NANC i.j.p. and the alpha, beta mATP-induced hyperpolarization, markedly reduced the alpha, beta mATP-induced relaxation (73% inhibition) and the PACAP-induced hyperpolarization (65% inhibition), while the PACAP-induced relaxation was unaffected. 4. Tetraethylammonium (TEA, 10 mM) increased the EFS-evoked i.j.p. and revealed an excitatory junction potential (e.j.p.). In the presence of TEA, alpha, beta mATP induced a biphasic response: transient depolarization and contraction followed by hyperpolarization and relaxation. The hyperpolarization to PACAP was reduced by TEA (45% inhibition) but the relaxation was unaffected. 5. The combined application of apamin (0.1 microM) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times longer than that of the i.j.p. In the majority of strips tested, alpha, beta mATP and PACAP elicited a biphasic response : depolarization and small contraction followed by hyperpolarization and relaxation. 6. The P2 purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) inhibited the NANC i.j.p. in concentration-dependent manner and inhibited the alpha, beta mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P2 purinoceptor antagonist, suramin (100 microM) inhibited to a similar extent (60-80%) the NANC i.j.p. and the hyperpolarization and relaxation induced by alpha, beta mATP or PACAP. 7. PPADS and suramin reduced the NANC e.j.p. evoked by a single pulse EFS in the presence of apamin and TEA (100 microM of PPADS and 300 microM of suramin inhibited the e.j.p. by about 40%). 8. We conclude that ATP, but not PACAP, mediates the apamin-sensitive NANC i.j.p. in the circular muscle of the guinea-pig colon. After blockade of the NANC i.j.p., ATP may act as an excitatory transmitter by activating excitatory P2 purinoceptors. The subtypes of P2 purinoceptor involved in the inhibitory and excitatory responses remain to be established. The data suggest that excitatory P2 purinoceptors may be located extrajunctionally.

Synchronization of rat hippocampal neurons in the absence of excitatory amino acid-mediated transmission

Extracellular and intracellular recordings and measurements of extracellular K + concentration ([K +] o) were performed in the adult rat hippocampus in an in vitro slice preparation. Excitatory amino acid receptor antagonists, as well as the K +-channel blockers 4-aminopyridine (4AP, 50 μM) and/or tetraethylammonium (TEA, 5 mM), were added to the bath. Synchronous, negative-going field potentials were recorded in the CA3 stratum radiatum during application of 4AP and excitatory amino acid receptor antagonists. Each of these events was associated with an intracellular long-lasting depolarization and a concomitant rise in [K +] o that attained peak values of 4.3 + 0.1 mM (mean ± S.E.M., n = 6 slices) and lasted 29 ± 3 s. These field potentials were still recorded in CA3 stratum radiatum after addition of TEA. Under these conditions, prolonged field potentials (40.2 ± 4.5 s, n = 18) characterized by a prominent positive component; discharge of population spikes also occurred. [K +] o, increases associated with these prolonged field-potential discharges had a considerable variability in magnitude (peak value = 3.8–14.1 mM, 6.1 ± 0.7 mM, n = 5) and duration (14–210 s; 48 ± 13 s, n = 5). In 8% of the cases spreading depression-like episodes were observed. [K +] o increases during spreading depression-like episodes attained peak values of 11–27 mM (22.8 ± 0.2 mM, n = 2) and had a duration of 160–396 s (244 ± 29 s, n = 2). All types of synchronous activity were abolished by the GABAA receptor antagonist bicuculline methiodide (t0 μM) ( n= 11). A similar effect was obtained by applying Ca 2+-free/high-Mg 2+ medium ( n = 5). Simultaneous field-potential recordings in CA3, CAI, dentate area and subiculum demonstrated that negative-going potentials and prolonged field-potential discharges occurred in all areas in a synchronous fashion. Spreading depression-like episodes were more frequently recorded in the CAI than in the CA3 area and were not seen in the subiculum or dentate area. These experiments indicate that a glutamatergic-independent, synchronous GABA-mediated potential which is elicited by 4AP in the adult rat hippocampus continues to occur in the presence of TEA. In addition, concomitant application of these K +-channel blockers induces a novel type of prolonged field-potential discharge as well as spreading depression-like episodes. Since all synchronous potentials (including spreading depression-like episodes) were abolished by bicuculline methiodide, we conclude that their occurrence is presumably dependent upon the post-synaptic activation of GABA A receptors located on neuronal and glial elements. As excitatory synaptic transmission was nominally blocked under our experimental conditions, we also propose that rises in [K +] o and consequent redistribution processes are per se sufficient to make all types of synchronous activity propagate.

Presence of the voltage-gated potassium channels sensitive to charybdotoxin in inhibitory presynaptic terminals of cultured rat hippocampal neurons

To determine whether the charybdotoxin-sensitive subtypes of voltage-gated K + channels (Kv1.2 and Kv1.3) exist in inhibitory presynaptic terminals, effects of K + channel blockers including TEA, charybdotoxin (ChTX), iberiotoxin (IbTX), kaliotoxin (KTX) and margatoxin (MgTX) on the inhibitory transmission were examined with cultured rat hippocampal neurons. Monosynaptic inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of single presynaptic neurons were recorded from the whole-cell clamped postsynaptic neurons. In the presence of TEA, application of ChTX greatly increased the amplitude of IPSCs. A specific maxi-K + channel blocker IbTX failed to augment IPSCs. KTX and MgTX, both of which block Kv1.3 but not Kv1.2, mimicked the facilitating effect of ChTX. In the absence of TEA, application of ChTX increased the IPSC amplitude significantly, while IbTX was without effect. These results indicate that the ChTX-sensitive subtypes of voltage-gated K + channels, most likely Kv1.3, contribute to the repolarization of action potentials at presynaptic terminals of hippocampal inhibitory neurons, and that the ChTX-induced facilitation of the transmission can be explained by its effects on the Kv channels rather than maxi-K + channels.

Relaxant actions of isoprenaline on guinea-pig isolated tracheal smooth muscle.

1. The effects of isoprenaline on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in guinea-pig isolated tracheal muscle were studied by use of intracellular micro-electrodes and fura-2 signals respectively. Measurements of membrane potential were carried out in the presence of spontaneously-generated muscle tone, whereas fura-2 signals were measured during contraction produced by exogenous prostaglandin E2 (100 nM). The potency of isoprenaline in causing relaxation was the same in these two different situations. 2. Isoprenaline (0.01 microM) produced relaxation accompanied by 5 mV hyperpolarization. A combination of tetraethylammonium (TEA, 10 mM) and verapamil (3 microM) did not alter the effects of isoprenaline. Removal of external K+ did not increase the degree of hyperpolarization produced by isoprenaline. 3. In the presence of TEA (10 mM) and verapamil (3 microM), isoprenaline (0.03-1 microM) reduced [Ca2+]i concentration-dependently. A similar degree of inhibition was observed when isoprenaline was applied during the maintained contraction induced by prostaglandin E2 and against the contraction evoked by the addition of Ca2+ to tissues bathed in a Ca(2+)-free medium and pretreated with both isoprenaline and prostaglandin E2. 4. It is concluded that activation of TEA-sensitive Ca(2+)-dependent K+ channels does not play a significant role in isoprenaline-induced relaxation. We propose that, in the guinea-pig tracheal muscle, isoprenaline may produce relaxation mainly by inhibiting a receptor-operated pathway for Ca2+ influx across the plasma membrane which is normally activated by prostaglandins.

Transient outward current in opossum esophageal circular muscle

The whole cell patch-clamp technique was used to record a transient outward K+ current (ITO) from single smooth muscle cells isolated from opossum esophageal circular muscle. The threshold for its activation was -50 mV from holding potentials negative to -70 mV. The current peaked within 10 ms and decayed completely in 200 ms between test depolarization of -40 and -10 mV. ITO was recorded at room temperature in the presence of 5 mM internal ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid. Both activation and inactivation kinetics of ITO were markedly changed when recordings were made at higher temperatures (32 degrees C). 4-Amino-pyridine (4-AP, 3 mM) abolished the fast component of the outward current. Tetraethylammonium ion (TEA, 1-30 mM) reduced the sustained component but did not affect ITO. In the presence of TEA and nifedipine, the voltage dependence of the steady-state inactivation data was well fitted by a Boltzmann distribution with a half-inactivation potential of -57 mV. The half-inactivation potential was shifted to a more positive potential in the presence of Cd2+ (-35 mV). The steady-state inactivation and activation data overlap between -50 and -30 mV, suggesting the presence of a "window" current in this potential range. In current-clamp mode, 4-AP depolarized single esophageal cells by approximately 8 mV and shifted the upstroke of the action potential to the left. These results indicate that, in the esophageal circular muscle, ITO is involved in the resting membrane potential and modulation of the onset of action potential.

Copper bioavailability from breakfasts containing tea. Influence of the addition of milk

AbstractThe influence of drinking tea on copper bioavailability is unclear, particularly when tea is consumed with food. A breakfast meal containing white bread, margarine, strawberry jam, cheese and tea, with or without milk, was digested in; vitro and the dialysis of copper investigated. Reference breakfasts were prepared with water and water with milk. Copper dialysability (percentage of copper dialysed) was markedly increased by tea. The influence of adding milk to tea was not significant. A second study was carried out in rats using the breakfasts containing tea and water. They were spiked with 64Cu and given orally to the animals in a single dose. Whole‐body 64Cu retention was measured over a 4‐day period after administration. In order to determine the true absorption of 64Cu a control group of rats received the dose intraperitioneally. The results in vivo indicate a tendency to higher absorption and retention of 64Cu with tea. Liver 64Cu retention was significantly elevated by tea. Therefore, the presence of tea in a breakfast meal favours the formation of soluble low‐molecular‐weight ligands which can be absorbed and retained by rats. The concomitant increase in liver copper retetition may be associated with higher bioavailability and/or lower copper mobilisation.

Photobleaching of xanthene dyes in a poly(vinyl alcohol) matrix

Photobleaching of three different xanthene dyes namely, Erythrosin B (ERB), Eosin Y (EOY) and Rose Bengal (ROB) in Poly(Vinyl Alcohol) (PVA) matrix has been studied. Quantum yield values of photobleaching using an argon-ion laser (514.5 nm) were determined in presence of an electron donor (TriEthAnolamine, TEA) and/or Cr(VI). Evaluated quantum yield values suggest that erythrosin B undergoes faster photobleaching than eosin Y or rose bengal in presence of TEA. Presence of Cr(VI) in addition to TEA drastically decreases the quantum yield. Possible reaction pathways of photobleaching are discussed.

Sulphonylureas reduce the slowly inactivating D-type outward current in rat hippocampal neurons.

1. Using intracellular recording in hippocampal slices, we have examined, in CA3 pyramidal neurons, the effects of sulphonylureas (blockers of ATP-sensitive K+ channels) on the slowly inactivating D-type K+ current (ID). 2. In the presence of TTX (1 microM) to block Na+ currents, ID had the following characteristics: activation by large depolarizing pulses from membrane potentials negative to -75 mV, slow inactivation kinetics, high sensitivity to 4-aminopyridine (4-AP, 3-40 microM), insensitivity to tetraethylammonium (TEA, 10 mM), Cs+ (3 mM) and carbachol (50 microM). 3. Applications of glibenclamide (10 microM) did not modify the input conductance of the cell, but reduced the amplitude of ID by 31.2 +/- 5.6% (n = 16), without altering its voltage dependence and inactivation kinetics. The effects were usually reversible. 4. Glibenclamide also reduced ID in the presence of TEA (10 mM), Cs+ (3 mM) and carbachol (50 microM), to block several K+ currents (IK, IC, IQ, IM), as well as kynurenate (1 mM) and bicuculline (10 microM) to block on-going synaptic currents mediated by activation of non-NMDA (N-methyl-D-aspartate) and GABA (gamma-aminobutyrate)-A receptors, respectively. 5. Comparable depressions of ID were produced by two other sulphonylureas: gliquidone (10 microM), 42.6 +/- 7.9% (n = 13) and tolbutamide (500 microM), 39.1 +/- 12.8 (n = 8). 6. It is concluded that, in the central nervous system, sulphonylureas can modulate K+ currents which are not generated by ATP-sensitive K+ channels.

K+ currents in rabbit esophageal muscularis mucosae.

Single cells were obtained from esophageal muscularis mucosae of the rabbit using enzymatic dispersion. Their electrophysiological properties were studied with both conventional whole cell and nystatin-perforated patch techniques. The latter technique was used to prevent "washout" of intracellular constituents and to maintain endogenous buffering of Ca2+. The average resting potential of these cells was -54 +/- 3.2 mV in the conventional recording and -51 +/- 4.4 mV in perforated patch recordings. In the current-clamp mode, regenerative responses were consistently observed in perforated patch recordings, but not when conventional whole cell gigaseal methods were used. Conventional whole cell voltage-clamp methods revealed outward currents on depolarization from a holding potential of -70 mV. These currents were inhibited by extracellular tetraethylammonium (TEA, 5-10 mM) and CoCl2 (4 mM), indicating that the predominant outward current is a Ca(2+)-activated K+ current. In the presence of TEA, inward Ca2+ currents were unmasked. In contrast, when the nystatin-perforated patch technique was used, depolarizations resulted in a net inward current followed by an outward current. The outward current was inhibited by CoCl2 (2 mM) and TEA (5 mM) to the same extent as in conventional recordings. A second component of K+ current was observed in both types of recordings when extracellular Ca2+ influx was abolished and also in the presence of TEA. This slowly activating persistent K+ current resembled a delayed rectifier K+ current. These studies show that the rabbit tunica muscularis mucosal cells possess voltage-activated Ca2+ and K+ channels as well as the capability to elicit action potentials.

Effects of manipulations of cytoplasmic pH on the mechanical responses of isolated porcine coronary arteries.

Isolated porcine coronary arteries contracted transiently when exposed to 20 mM Na salts of organic acids (acetate, propionate, butyrate and lactate) at constant external pH (pH0 = 7.4). The peak of these phasic contractions, completely inhibited by 1 mM amiloride, amounted to about 10% of that elicited by depolarization with 50 mM K0. The slope of the relaxing branch of the phasic contractions induced by 20 mM acetate in HEPES buffered physiological salt solution decreased from 1.05 +/- 0.12 to 0.49 +/- 0.07 mN/min (S.E.M.; n = 14), when Na0 was reduced from 137 to 20 mM. In Na free HEPES-buffered PSS only sustained contractions were obtained. The EC50 values for the concentration-response curves for contractions induced by K-propionate (2-20 mM) in Tris-PSS were 8.2 +/- 0.5 mM, n = 6. After total isoosmotic replacement of external Na+ by sucrose in bicarbonate-buffered PSS, contractions induced by 20 mM K-propionate had the same height as those induced with 50 mM K0+. Longlasting exposure to 0 Ca0 PSS did not affect significantly contractions induced by 20 mM propionate. During an exposure to procaine (10(-4) M) acetate-induced contractions were transiently and significantly enhanced, when induced in bicarbonate buffered PSS, but not in HEPES buffered PSS. In normally polarized PCA preparations 20 mM NH4+ induced both sustained relaxations and polyphasic responses; in depolarized PCA preparations only polyphasic responses were evoked. The time course of the polyphasic responses to application and removal of weak bases (NH(4+)-pulse technique) or to removal and reintroduction of CO2/HCO3- paralleled exactly the expected pHi perturbations, as they have been described in a great variety of cells and tissues, alkalinization leading however to relaxation, acidification to contraction. In presence of NH4+, a transient relaxation (phase 1) was immediately followed by an overshooting contraction (phase 2); at removal of NH4+ a rebound contraction (phase 3) was observed, reaching a peak and changing thereafter to a relaxation (phase 4), the slope of which was sensitive to variation of the external Na0 concentration. These mechanical effects were abolished by 1 hr exposure to 0 Ca PSS. Phase 2 was enhanced 2-5 fold in presence of TEA, Ba ions, Na3 VO4 or beta-methyldigoxin. Contractile responses of PCA preparations activated by various agonists (acetylcholine, histamine and serotonin) to pHi manipulations were similar and generally amplified when compared to unactivated preparations.(ABSTRACT TRUNCATED AT 400 WORDS)