Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

Short-term exposure to somatostatin or muscarinic agonists reduce acetylcholine-induced <Superscript>3</Superscript>H-MPP<Superscript>+</Superscript> release from bovine adrenal medullary cells

Summary The aim of this work was to investigate the effect of a short-term exposure to somatostatin (SS), its receptors (SSTR) selective agonists as well as muscarinic receptors agonists upon acetylcholine-induced release of ³H-MPP⁺ from bovine adrenal medullary cells. Acetylcholine (ACH, 100, 500 μM) was found to increase the release of ³H-MPP⁺ by these cells (to 175 and 171% of basal release, respectively). ACH-elicited ³H-MPP⁺ release was significantly reduced by hexamethonium (100 μM) and atropine (100 μM), selective nicotinic and muscarinic antagonists, respectively. Previous exposure to any of two muscarinic agonists, oxotremorine or pilocarpine, led to a significant reduction of ³H-MPP⁺ release in response to 100 μM ACH, to about a maximum of 51% and 78% of control, respectively. Somatostatin (SS, 0.01–0.1 μM), previously applied to the preparation, depressed ACH-elicited ³H-MPP⁺ release by 25–27%, but only when a 500 μM ACH concentration was used. The inhibition exerted by SS upon ACH-evoked ³H-MPP⁺ release appeared to be mediated by its SSTR: (1) SSTR2, 3 and 4 subtype agonists mimicked the effects seen with SS, and (2) the SSTR non-selective antagonist, cyclo-SS, counteracted the SS inhibitory effect. When SS was tested in the presence of any of the muscarinic agonists, oxotremorine or pilocarpine, its inhibitory effect on 500 μM ACH-induced ³H-MPP⁺ release was no longer detectable. These results, showing a somewhat similar effect of short-term exposure to SS and muscarinic agonists over ACH-induced release of ³H-MPP⁺, as well as the loss of effect of SS by the presence of the muscarinic agonists, suggest that these compounds may share signalling pathways.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

GPR39 up-regulation after selective antidepressants

Recent studies indicated that zinc activates neural transmission via the GPR39 Zn²⁺-sensing receptor. Preclinical and clinical studies demonstrated the antidepressant properties of zinc. To investigate whether the GPR39 receptor is involved in the mechanism of antidepressant action, we measured the expression of the GPR39 receptor (Western Blot) in the frontal cortex of mice treated intraperitoneally with imipramine (30 mg/kg), escitalopram (4 mg/kg), reboxetine (10 mg/kg) or bupropion (15 mg/kg) for 14 days. The present study shows the up-regulation of the GPR39 receptor protein level after escitalopram (by 290%), reboxetine (by 816%) and bupropion (by 272%), but not imipramine treatment. This is the first report to indicate the involvement of the GPR39 Zn²⁺-sensing receptor in the antidepressant effect of selective monoamine reuptake inhibitors.     

Inhibition of GABAA ligand-gated Cl− channels by zinc in adult rat brain: A regional study

Zinc (Zn²⁺) was shown to invariably inhibit muscimol-stimulated³⁶Cl⁻ uptake by synaptoneurosomes in the cerebral cortex, hippocampus and cerebellum. The Zn²⁺ sensitivity of the GABA_A receptor-gated³⁶Cl⁻ uptake in the cerebral cortex was comparable to that in the hippocampus, whereas the uptake in the cerebellum was less sensitive to Zn²⁺. Although diazepam-potentiation of muscimol-stimulated³⁶Cl⁻ uptake was unaltered by 100 μM Zn²⁺ in the cerebellum. Zn²⁺ inhibited [³H]diazepam binding significantly at 1 mM in the cerebral cortex and cerebellum, whereas Ni²⁺ increased the binding in a concentration-dependent manner in both regions. Although lower concentrations of Zn²⁺ did not affect [³H]Ro 15-4513 binding to diazepam-sensitive sites, higher concentrations of Zn²⁺ increased the binding in both regions. Unlike the diazepam-sensitive sites the diazepam-insensitive [³H]Ro 15-4513 binding was not affected by Zn²⁺ or Ni²⁺ at any of the tested concentrations. These results suggest that the GABA_A ligand-gated Cl⁻ flux and its diazepam-potentiation are heterogeneously modulated in various brain regions. It is also suggested that cerebellar diazepam-insensitive [³H]Ro 15-4513 binding sites are insensitive to Zn²⁺ and Ni²⁺.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

The Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus is Differentially Increased by Administration of Endobain E,a Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

Previous studies showed that endobain E, an endogenous Na⁺, K⁺-ATPase inhibitor, decreases dizocilpine binding to NMDA receptor in isolated membranes. The effect of endobain E on expression of NMDA receptor subunits in membranes of rat cerebral cortex and hippocampus was analyzed by Western blot. Two days after administration of 10 μl endobain E (1 μl = 29 mg fresh tissue) NR1 subunit expression enhanced 5-fold and 2.5-fold in cerebral cortex and hippocampus, respectively. NR2A subunit expression increased 2-fold in cerebral cortex and 1.5-fold in hippocampus. The level of NR2B subunit raised 3-fold in cerebral cortex but remained unaltered in hippocampus. NR2C subunit expression was unaffected in either area. NR2D subunit enhanced 1.6 and 2.1-fold for cerebral cortex and hippocampus, respectively. Results indicate that endogenous Na⁺, K⁺-ATPase inhibitor endobain E differentially modifies the expression of NMDA receptor subunits.     

Ca<Superscript>2+</Superscript> Buffering Capacity of Mitochondria After Oxygen-Glucose Deprivation in Hippocampal Neurons

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca²⁺ ([Ca²⁺]_c) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca²⁺influx. The [Ca²⁺]_c level increased sharply, followed by a rapid increase in mitochondrial Ca²⁺ [Ca²⁺]_m. The increase in the [Ca²⁺]_m level accompanied a reduction in the [Ca²⁺]_c level. After reaching a peak, the [Ca²⁺]_c level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.     

Biosorption of Pb<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> by Non-Living Biomass of <Emphasis Type="Italic">Spirulina</Emphasis> sp.

Removal of heavy metals (Pb²⁺, Zn²⁺) from aqueous solution by dried biomass of Spirulina sp. was investigated. Spirulina rapidly adsorbed appreciable amount of lead and zinc from the aqueous solutions within 15 min of initial contact with the metal solution and exhibited high sequestration of lead and zinc at low equilibrium concentrations. The specific adsorption of both Pb²⁺ and Zn²⁺ increased at low concentration and decreased when biomass concentration exceeded 0.1 g l⁻¹. The binding of lead followed Freundlich model of kinetics where as zinc supported Langmuir isotherm for adsorption with their r ² values of 0.9659 and 0.8723 respectively. The adsorption was strongly pH dependent as the maximum lead biosorption occurred at pH 4 and 10 whereas Zn²⁺ adsorption was at pH 8 and 10.     

External Ni<Superscript>2+</Superscript> and ENaC in A6 Cells: Na<Superscript>+</Superscript> Current Stimulation by Competition at a Binding Site for Amiloride and Na<Superscript>+</Superscript>

In cultured A6 monolayers from distal Xenopus kidney, external Ni²⁺ stimulated active Na⁺ uptake via the epithelial Na⁺ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni²⁺ effect. Na⁺ current noise analysis was performed using the neutral Na⁺-channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni²⁺ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni²⁺ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni²⁺ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na⁺—known to compete with amiloride and so producing the self-inhibition phenomenon—and Ni²⁺ exerted mutually exclusive analogous effects on amiloride kinetics. Na⁺ current kinetics revealed that Ni²⁺ prevents ENaC to be downregulated by self-inhibition. Co²⁺ behaved similarly to Ni²⁺, whereas Zn²⁺ did not. Attempts to disclose the chemical nature of the site reacting with Ni²⁺ suggested cysteine but not histidine as reaction partner.     

K<Superscript>+</Superscript> Channels in Cardiomyocytes of the Pulmonate Snail <Emphasis Type="Italic">Helix</Emphasis>

We used the patch-clamp technique to identify and characterize the electrophysiological, biophysical, and pharmacological properties of K⁺ channels in enzymatically dissociated ventricular cells of the land pulmonate snail Helix. The family of outward K⁺ currents started to activate at –30 mV and the activation was faster at more depolarized potentials (time constants: at 0 mV 17.4 ± 1.2 ms vs. 2.5 ± 0.1 ms at + 60 mV). The current waveforms were similar to those of the A-type family of voltage-dependent K⁺ currents encoded by Kv4.2 in mammals. Inactivation of the current was relatively fast, i.e., 50.2 ± 1.8% of current was inactivated within 250 ms at + 40 mV. The recovery of K⁺ channels from inactivation was relatively slow with a mean time constant of 1.7 ± 0.2 s. Closer examination of steady-state inactivation kinetics revealed that the voltage dependency of inactivation was U-shaped, exhibiting less inactivation at more depolarized membrane potentials. On the basis of this phenomenon, we suggest that a channel encoded by Kv2.1 similar to that in mammals does exist in land pulmonates of the Helix genus. Outward currents were sensitive to 4-aminopyridine and tetraethylammonium chloride. The last compound was most effective, with an IC₅₀ of 336 ± 142 µmol l^–1. Thus, using distinct pharmacological and biophysical tools we identified different types of voltage-gated K⁺ channels. Present address for S.A.K.: Brigham and Womens Hospital, Cardiovascular Division, Harvard Medical School, 75 Francis St., Thorn 1216, Boston, MA 02115.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Alteration of some toxic aflatoxin responses in Syrian hamsters fed zinc carbonate-supplemented diets

Summary Chronic exposure to aflatoxins (AFTs) below the LD₅₀ can result in reduced weight gain, hepatocellular necrosis and bile duct cell proliferation. Here, we report whether dietary zinc (Zn²⁺) protects against both aflatoxicosis and precancer in male weanling hamsters fed either 14.6 mg/kg AFTs, 3000 mg/kg zinc carbonate, or both for 17 weeks. The AFTs (either alone or with Zn²⁺) reduced weight gains but not feed consumption. Whereas controls possessed 172.7±21.7 mg/100 ml plasma glucose, the AFTs and Zn²⁺ groups had 132.1±19.5 and 122.7 mg/100 ml, respectively. For plasma cholesterol, the AFTs plus Zn²⁺ group's was 26.5±4.3 compared to 32.3±3.0, 31.5±4.8 and 36.0±2.1 mg/100 ml for control, Zn²⁺ and AFTs groups, respectively. The latter exhibited bile duct cell hyperplasia, focal liver necrosis and hemorrhage but the AFTs plus Zn²⁺ group's livers had less damage. Meglahepatocytes indicated precancerous changes. These data suggest a trend toward Zn²⁺-induced reduction for AFTs-promoted liver damage.     

Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>2+</Superscript>-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca²⁺–ATPase-dependent increase in nuclear Ca²⁺-influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca²⁺-influx by pretreatment with clonidine, an inhibitor of high affinity Ca²⁺–ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n = 4), hypoxic (Hx, n = 4), and hypoxic treated with clonidine (100 mg/kg) (Hx–Cl, n = 5). Anesthetized, ventilated animals were exposed to an FiO₂ of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (μmoles/g brain) was 4.6 ± 0.3 in Nx, 1.7±0.4 in Hx (P < 0.05 vs. Nx), and 1.5 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). PCr (μmoles/g brain) was 3.6 ± 0.4 in Nx, 1.1 ± 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 ± 0.0642 in Nx and increased to 0.808 ± 0.080 (P < 0.05 vs. Nx and Hx–Cl) in the Hx and 0.562 ± 0.050 in the Hx–Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 ± 1.3 in Nx and 32 ± 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 ± 3.2 in the Hx–Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca²⁺–ATPase-dependent increased nuclear Ca²⁺ during hypoxia results in increased caspase-9 and caspase-3 activity.     

Distribution of [3H]GR65630 Binding in Human Brain Postmortem

We investigated the distribution of serotonin (5-HT) receptors of type 3 (5-HT₃) in human brain areas, by means of the the specific binding of [³H]GR65630. The brains were obtained during autoptic sessions from 6 subjects. Human brain membranes and the binding of [³H]GR65630 were carried out according to standardized methods. The highest density (Bmax ± 6 SD, fmol/mg protein) of [³H]GR65630 binding sites was found in area postrema (13.1 ± 9.7), followed at a statistically lower level, by nucleus tractus solitarius (6.7 ± 3.4), nervus vagus (5.5 ± 2.1), striatum (4.8 ± 2.4) with a progressive decrease in amygdala, olivar nuclei, hippocampus, olfactory bulbus and prefrontal cortex, and then by the other cortical areas and the cerebellum, where no binding was detected. These observations extend previous findings on the distribution of 5-HT₃ receptors and confirm interspecies variations that might explain the heterogeneous properties of 5-HT₃ receptors in different animals.     

Ligand Binding to CNS Muscarinic Receptor Is Transiently Modified by Convulsant 3-Mercaptopropionic Acid Administration

The administration of convulsant drugs has proven a powerful tool to study experimental epilepsy. We have already reported that the administration of convulsant 3-mercaptopropionic acid (mp) at 150 mg/kg enhances binding affinity of muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to certain rat CNS membranes during seizure and postseizure without affecting site number. Results obtained with a 100-mg/kg dose of mp have shown reversible increases in [³H]QNB binding to cerebellum and hippocampus, whereas a delayed response has been found in striatum. Neither a subconvulsant dose nor in vitro addition modifies binding. In order to evaluate preseizure, seizure as well as early (30 min) and late (24 h) postseizure stages, we employed a 50 mg/kg dose and tested [³H]QNB binding to CNS membranes. Changes in binding were as follows (in %): in cerebellum, +37, +86, and +40 at preseizure, seizure and early postseizure stages, respectively, but there was a decrease at late postseizure; in hippocampus, +27 at pre- and seizure stages, but a decrease at early and late postseizure. No changes were found in striatum or cerebral cortex membranes at any stage studied. Saturation curves analysed by Scatchard plots indicated that changes in [³H]QNB binding to cerebellar membranes are attributable to an increase in ligand affinity at seizure, followed by a decrease in binding site number at postseizure. A similar profile was observed for hippocampus except that the decrease in binding site number, though lower than at postseizure, was already evident at seizure stage. Results confirm a region-specific response to the convulsant and transient changes provide an example of neuronal plasticity.