Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes

Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 microM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death.     

Sp family of transcription factors is involved in iron-induced collagen alpha1(I) gene expression

The purpose of this study was to identify the cis-acting elements and the trans-acting factors involved in the iron-induced expression of the collagen alpha1(I) (COL1aI) gene. Rat hepatic stellate cells were cultured in the presence of 50 microM ferric chloride, 50 microM ascorbic acid, and 250 microM citric acid (Fe/AA/CA), and the effects on collagen gene expression and the binding of nuclear proteins to the COL1aI promoter were measured. The Fe/AA/CA treatment induced a time- and dose-dependent increase in the cellular levels of COL1aI mRNA that was abrogate by pretreating cells with cycloheximide, antioxidants, and inhibitors of aldehyde-protein adduct formation. Transient transfection experiments showed that Fe/AA/CA exerted its effect through regulatory elements located between -220 and -110 bp of the COL1aI promoter. Gel retardation assays showed that Fe/AA/CA increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the COL1aI promoter. These bindings were blocked by unlabeled consensus Sp1 oligonucleotide and supershifted with Sp1 and Sp3 antibodies. Finally, Fe/AA/CA increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Treatment with Fe/AA/CA stimulates COL1aI gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the COL1aI promoter.     

Divergent effects of phenothiazines on Leydig tumor cell steroidogenesis and adenylate cyclase activity

The dose and temporal (1-24 h) effects of two phenothiazines, chlorpromazine and trifluoperazine, on steroidogenesis and adenylate cyclase activity of gonadotropin-responsive Leydig tumor cells (M5480A) in primary culture were examined. At low doses (e.g. 0.1-1 microM) these antipsychotic drugs were slightly inhibitory (trifluoperazine) or without effect (chlorpromazine), while at 25 microM each drug was weakly stimulatory to basal testosterone production. Trifluoperazine was, in general, inhibitory to HCG-stimulated testosterone production, but chlorpromazine exhibited paradoxical effects. At 5 and 10 microM this neuroleptic agent increased HCG-stimulated steroidogenesis, while at 25 microM testosterone production was inhibited. In a particulate fraction prepared from the tumor the activity of adenylate cyclase was stimulated 3.4-fold in the presence of 10 microM 5'-guanylimidodiphosphate and 5-fold in the presence of HCG plus the non-hydrolyzable GTP analogue. Between doses of 1-100 microM neither drug altered the basal activity of adenylate cyclase. Trifluoperazine at doses of 1-100 microM inhibited 5'-guanylimidodiphosphate-stimulated adenylate cyclase activity both with and without added gonadotropin. At doses of 1-10 microM chlorpromazine had no effect on adenylate cyclase activity, but it stimulated activity in the dose range of 20-100 microM. Interestingly, in the presence of 5'-guanylimidodiphosphate this drug did not alter the stimulated enzymic activity achieved with a maximal dose of HCG. Therefore, these phenothiazines exhibit quite divergent dose-dependent effects and their actions must occur at multiple loci. Also, it seems unlikely that the effects of these agents on steroidogenesis and adenylate cyclase activity can be reconciled solely in terms of calmodulin-mediated processes.     

Pentobarbital inhibits hippocampal neurons by increasing potassium conductance

The effects of sodium pentobarbital were studied using intracellular recordings from CA1 and CA3 pyramidal cells in slices of guinea pig hippocampus. Drugs were applied either by perfusion or by pressure ejection at concentrations of 10(-6), 10(-5), and 10(-4) M. Pentobarbital at all concentrations caused neuronal hyperpolarization, decreased spontaneous activity, and sometimes decreased input resistance. Hyperpolarization also occurred in zero calcium perfusate or with tetrodotoxin in the perfusate. The postspike train long-lasting afterhyperpolarization, which is an intrinsic calcium-mediated potassium conductance, was increased at all doses. gamma-Aminobutyric acid induced depolarizing dendritic responses were augmented only at 10(-4) M pentobarbital. It is proposed that one of the important mechanisms of pentobarbital neuronal inhibition, particularly at lower doses, is an increase in potassium conductance.     

Acute actions of marine toxin latrunculin A on the electrophysiological properties of cultured dorsal root ganglion neurones

The effects of latrunculin A, isolated from the nudibranch Chromodoris sp., on the excitability of neonatal rat cultured dorsal root ganglion neurones were investigated using patch-clamp recording and Ca(2+) imaging techniques. Under current-clamp conditions, acute application of latrunculin A (100 microM) reversibly induced multiple action potential firing and significantly increased action potential duration. No significant effects on action potential peak amplitude, threshold of action potential firing, resting membrane potential and input resistance were observed. Under voltage-clamp conditions, significant and dose-dependent suppression of K(+) current was seen with 10-100 microM latrunculin A. Additionally, a significant difference between inhibition of the current measured at the peak and the end of a 100 ms voltage step was seen with 100 microM latrunculin A. Fura-2 fluorescence Ca(2+) imaging revealed that latrunculin A (100 microM) significantly inhibited Ca(2+) transients evoked by KCl-induced depolarisation in all neurones. In 36% of DRG neurones, latrunculin A alone had no effect on intracellular Ca(2+). In 64% of neurones, latrunculin A alone evoked a transient rise in intracellular Ca(2+). Moreover, latrunculin A (10-100 microM) significantly inhibited the mean high voltage-activated Ca(2+) current. The effects of latrunculin A on action potential firing and K(+) currents were attenuated by intracellular phalloidin, an indication that these effects are mediated through actin disruption.     

Activity dynamics and behavioral correlates of CA3 and CA1 hippocampal pyramidal neurons

The CA3 and CA1 pyramidal neurons are the major principal cell types of the hippocampus proper. The strongly recurrent collateral system of CA3 cells and the largely parallel-organized CA1 neurons suggest that these regions perform distinct computations. However, a comprehensive comparison between CA1 and CA3 pyramidal cells in terms of firing properties, network dynamics, and behavioral correlations is sparse in the intact animal. We performed large-scale recordings in the dorsal hippocampus of rats to quantify the similarities and differences between CA1 (n > 3,600) and CA3 (n > 2,200) pyramidal cells during sleep and exploration in multiple environments. CA1 and CA3 neurons differed significantly in firing rates, spike burst propensity, spike entrainment by the theta rhythm, and other aspects of spiking dynamics in a brain state-dependent manner. A smaller proportion of CA3 than CA1 cells displayed prominent place fields, but place fields of CA3 neurons were more compact, more stable, and carried more spatial information per spike than those of CA1 pyramidal cells. Several other features of the two cell types were specific to the testing environment. CA3 neurons showed less pronounced phase precession and a weaker position versus spike-phase relationship than CA1 cells. Our findings suggest that these distinct activity dynamics of CA1 and CA3 pyramidal cells support their distinct computational roles.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Inhibition of Ca(2+)-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells.

To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K(+)-evoked catecholamine (CA) release. The IC50 for high K(+)-evoked CA release was 1 microM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca(2+)-dependent exocytosis was inhibited in a dose-dependent manner (IC50, 1 microM). These findings suggest the implication of MLCK in the Ca(2+)-triggered process in the machinery of exocytosis.     

Contribution of non-inactivating Na+ current induced by oxidizing agents to the firing behavior of neuronal action potentials: experimental and theoretical studies from NG108-15 neuronal cells

The effects of chemical injury with oxidizing agents on voltage-gated Na+ current (I(Na)) in differentiated NG108-15 neuronal cells were investigated in this study. In whole-cell patch-clamp recordings, the challenge of these cells with t-butyl hydroperoxide (t-BHP; 1 mM) decreased the peak amplitude of I(Na) with no modification in the current-voltage relationship. It caused a slowing of current inactivation, although there was no alteration in the activation time course of I(Na). Cell exposure to t-BHP also increased a non-inactivating I(Na) (I(Na(NI)) elicited by long-lasting ramp pulses. The t-BHP-induced increase of I(Na(NI)) was reversed by a further application of riluzole (10 microM) or oxcarbazepine (10 microM). When I(Na) was elicited by simulated waveforms of action potentials (APs), during exposure to t-BHP, the amplitude of this inward current was diminished, accompanied by a reduction in inactivation/deactivation rate and an increase in current fluctuations. Under current-clamp recordings, addition of t-BHP (0.3 mM) enhanced AP firing in combination with clustering-like activity and sub-threshold membrane oscillations. In the simulation study, when the fraction of non-inactivating Na(v) channels was elevated, the simulated window component of I(Na) in response to a long-lasting ramp pulse was reduced; however, the persistent I(Na) was markedly enhanced. Moreover, when simulated firing of APs was generated from a modeled neuron, changes of AP firing caused by the increased fraction of non-inactivating Na(v) channels used to mimic the t-BHP actions were similar to the experimental observations. Taken together, it is anticipated that the effects of oxidizing agents on I(Na(NI)) could be an important mechanism underlying their neurotoxic actions in neurons or neuroendocrine cells occurring in vivo.     

Autofeedback effects of progressively rising oxytocin concentrations on supraoptic oxytocin neuronal activity in slices from lactating rats

Suckling stimuli induce somatodendritic oxytocin (OT) release from supraoptic nucleus (SON) neurons, which raises intranuclear OT concentrations and contributes to the effectiveness of the milk-ejection reflex. To clarify how such changes in OT concentrations modulate the activity of OT neurons, we examined OT effects using whole cell patch-clamp recordings from SON neurons in slices from lactating rats. Progressive increases from extremely low OT concentrations (0.1-10 fM) to high concentrations (0.1-10 nM) induced excitation and subsequent spike frequency reduction (SFR) in OT neurons. Significant effects of OT on firing rates were observed starting at 1 fM, reached peak level from 1 fM to 1 pM before SFR occurred in most neurons. The buildup of OT concentrations progressively promoted depolarization of membrane potential, spike broadening, decreases in spike amplitude, and increases in the rise time of spike afterhyperpolarizations, which were unrelated to firing rate. However, intermittent application of OT (1 fM, 1 pM, and 1 nM, each for 5 min) evoked dose-dependent excitation but not the SFR. Application of 1 pM OT for 40 min simulated the effects of progressively increasing OT concentrations. Vasopressin neurons were also activated by OT but did not show SFR. Consistent with presynaptic loci of OT action, ionotropic glutamate receptor antagonists reduced OT effects on firing rate, whereas bicuculline did not change the excitatory effects. These results suggest that the specific autoregulatory effects of OT, and perhaps other neuropeptides as well, are time and concentration dependent.     

ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases

Analgesia induced by certain tricyclic antidepressants has been largely used for decades, yet the mechanisms involved are incompletely understood. Starting from previously reported dual effects of amitriptyline on wild-type ENaC (Pena F, et al. J Pharm Pharmacol 54:1393-8: 2002), we extended our study to ASIC1a by performing a series of whole cell and single-channel recordings of proton-activated currents in HEK293 cells. Acid pulses were applied at 2 or 5 min intervals, and amitriptyline (1-500 microM) was applied at a holding pH of 7.4 or 8.4 between pulses. Dose-response plots were fitted with dual Hill type functions, yielding a half-activatory constant of 0.3 microM and a half-inhibitory constant of 382 microM at pH 7.4. At pH 8.4 both constants were shifted to higher values (0.5 and 444 microM, respectively). In whole-cell experiments, FMRF-amide increased the peak amplitude of ASIC1a transients at 0.1 microM and decreased it at 1 and 100 microM. Single-channel recordings were idealized and fitted using an 8-state linear connectivity model comprising four consecutive activation steps. Both amitriptyline (1 microM) and FMRF-amide (0.1 microM) increased the unitary current amplitude, and modified the opening and closing rates of the first gating mode. They also increased the transition rate from the second to the first gating mode, and the rate of final closure. The activatory effect of both compounds vanished after a mild trypsin pretreatment, suggesting the existence of activatory sites for FMRF-amide and amitriptyline in the outer vestibule of ASIC1a, which can be removed by exo- or endogenous serine-proteases.     

Phencyclidine actions measured intracellularly in hippocampal CA1 neurons

The electrophysiological effects of phencyclidine (PCP) were measured intracellularly in guinea pig hippocampal CA1 neurons in vitro. At all doses tested (0.2 microM - 10 mM), PCP increased the width of action potentials (APs). Doses of 10 microM and higher were associated with decreased action potential amplitude. PCP decreased inhibitory postsynaptic potentials and excitatory postsynaptic potentials but did not alter responses to focally applied GABA. At the lowest dose (0.2 microM), PCP decreased the input resistance (Rin), while at all other doses Rin was increased. PCP decreased post-spike train afterhyperpolarizations at low and medium doses. PCP effects persisted in low calcium medium and also in medium containing 10(-6) M tetrodotoxin. It is concluded that in these central neurons, PCP primarily blocks potassium conductances at all doses and, at anesthetic doses, depresses sodium-dependent spikes.     

Cav1.2 calcium channels modulate the spiking pattern of hippocampal pyramidal cells

Ca(v)1.2 L-type calcium channels support hippocampal synaptic plasticity, likely by facilitating dendritic Ca2+ influx evoked by action potentials (AP) back-propagated from the soma. Ca2+ influx into hippocampal neurons during somatic APs is sufficient to activate signalling pathways associated with late phase LTP. Thus, mechanisms controlling AP firing of hippocampal neurons are of major functional relevance. We examined the excitability of CA1 pyramidal cells using somatic current-clamp recordings in brain slices from control type mice and mice with the Ca(v)1.2 gene inactivated in principal hippocampal neurons. Lack of the Ca(v)1.2 protein did not affect either affect basic characteristics, such as resting membrane potential and input resistance, or parameters of single action potentials (AP) induced by 5 ms depolarising current pulses. However, CA1 hippocampal neurons from control and mutant mice differed in their patterns of AP firing during 500 ms depolarising current pulses: threshold voltage for repetitive firing was shifted significantly by about 5 mV to more depolarised potentials in the mutant mice (p<0.01), and the latency until firing of the first AP was prolonged (73.2+/-6.6 ms versus 48.1+/- 7.8 ms in control; p<0.05). CA1 pyramidal cells from the mutant mice also showed a lowered initial spiking frequency within an AP train. In control cells, isradipine had matching effects, while BayK 8644 facilitated spiking. Our data demonstrate that Ca(v)1.2 channels are involved in regulating the intrinsic excitability of CA1 pyramidal neurons. This cellular mechanism may contribute to the known function of Ca(v)1.2 channels in supporting synaptic plasticity and memory.     

Teratogenic effects of cyproterone acetate and medroxyprogesterone treatment during the pre- and postimplantation period of mouse embryos. I

Pregnant mice were treated with a single subcutaneous injection of either cyproterone acetate (CA) or medroxyprogesterone acetate (MPA). In the first experiment the animals received 5-900 mg/kg of the hormone before implantation (day 2 of pregnancy). CA treatment on day 2 caused a dose-dependent decrease in fetal weight and a significant dose-dependent increase in the rates of cleft palate and urinary tract abnormalities. Exencephaly and heart abnormalities were also significantly more frequent, but this increase was not dose-dependent. MPA treatment on day 2 was followed by sporadic increases in dead and resorbed fetuses, a decrease in fetal weight and an increase in the rates of cleft palate, and malformed or abnormally developed fetuses. None of these effects, however, was dose-dependent. In the second experiment the mice were given one single injection (30 mg/kg) of CA or MPA on any one of days 1-12 of gestation. Treatment with CA on one day between days 1 and 12 revealed that the specific sensitivity for abnormalities of the urinary tract was on days 5 and 6, for the respiratory tract on days 8 and 9, and for cleft palate on days 10 and 11. Treatment with MPA on one day between days 1 and 12 only revealed a high rate of respiratory and urinary tract abnormalities on day 9. After treatment with MPA cleft palate was again significantly more frequent in all treated groups, however, days of peak sensitivity were not detected. The long half-life of CA (60 hours) explains the teratogenic effect of high doses of this progestin after treatment on day 2 and also the pattern of abnormal development found after treatment with a single dose of CA on one of the days between day 1 and day 12.     

Activity-dependent feedback modulation of spike patterning of supraoptic nucleus neurons by endogenous adenosine

Neuropeptide secretion from the dendrites of hypothalamic magnocellular supraoptic nucleus (SON) neurons contributes to the regulation of neuronal activity patterning, which ultimately determines their peptide output from axon terminals in the posterior pituitary gland. SON dendrites also secrete a number of other neuromodulators, including ATP. ATP degrades to adenosine in the extracellular space to complement transported adenosine acting on pre- and postsynaptic SON A1 receptors to reduce neuronal excitability, measured in vitro. To assess adenosine control of electrical activity in vivo, we made extracellular single-unit recordings of the electrical activity of SON neurons in anesthetized male rats. Microdialysis application (retrodialysis) of the A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT) increased phasic vasopressin cell intraburst firing rates progressively over the first 5 s by 4.5 +/- 1.6 Hz (P < 0.05), and increased burst duration by 293 +/- 64% (P < 0.05). Hazard function plots were generated from interval interspike histograms and revealed that these effects were associated with increased postspike excitability. In contrast, CPT had no effect on the firing rates and hazard function plot profiles of continuously active vasopressin and oxytocin cells. However, CPT significantly increased clustering of spikes, as quantified by the index of dispersion, in oxytocin cells and continuously active vasopressin cells (by 267 +/- 113% and 462 +/- 67%, respectively, P < 0.05). Indeed, in 4 of 5 continuously active vasopressin cells, CPT induced a pseudophasic activity pattern. Together, these results indicate that endogenous adenosine is involved in the local control of SON cell activity in vivo.     

GABA(B) presynaptically modulates suprachiasmatic input to hypothalamic paraventricular magnocellular neurons

This study used whole cell patch clamp recordings in rat hypothalamic slice preparations to evaluate the effects of GABA(B) receptor activation on GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) in paraventricular nucleus magnocellular neurons evoked by electrical stimulation in the suprachiasmatic nucleus (SCN). Baclofen induced a dose-dependent (1-10 microM) and reversible reduction in SCN-evoked IPSC amplitude (11/11 cells), blockable with 2-hydroxysaclofen (300 microM; 3/3 cells). IPSCs displayed paired-pulse depression (PPD), attenuated by both baclofen and 2-hydroxysaclofen, but neither altered resting membrane conductances or IPSC time constants of decay. Baclofen induced a significant dose-dependent (1-100 microM) reduction in frequency, but not amplitude, of spontaneous IPSCs and miniature IPSCs, reversible with 2-hydroxysaclofen pretreatment. Baclofen effects and PPD persisted in slices pretreated with pertussis toxin (PTX) and N-ethylmaleimide, implying that these GABA(B) receptors are coupled to PTX-insensitive G proteins. Responses were unaltered by barium (2 mM) or nimodipine, ruling out involvement of K(+) channels and L-type Ca(2+) channels. Thus pre- and postsynaptic GABA(B) and GABA(A) receptors participate in SCN entrainment of paraventricular neurosecretory neurons.     

Nerve Growth Factor and Dexamethasone Modulate Synthesis and Storage of Catecholamines in Cultured Rat Adrenal Medullary Cells: Dependence on Postnatal Age

Catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and noradrenaline N-methyltransferase (NMT) were measured in cultures of isolated adrenal medullary cells from newborn and young postnatal rats to study the effects of the differentiation factors glucocorticoids and nerve growth factor (NGF). During the 4-day culture period the cellular catecholamine (CA) content and TH activity remained stable, whereas NMT activity dropped to about half of the initial level. In cells from 2- and 10-day-old rats 10 microM dexamethasone specifically prevented this loss in NMT activity. Furthermore, this glucocorticoid treatment increased, in a dose-dependent manner, the total CA content by 50-100% over control levels without changes in the adrenaline (A) proportion or TH activity. In contrast, NGF did not affect NMT activities at all. In cells from 10-day-old rats 100 ng/ml NFG elevated TH activity and total CA content to about 160% of controls and did not change the proportion of A. This increase in total CA content was linear with the NGF dose and required greater than 5 ng/ml NGF. In chromaffin cells from 2-day-old rats 100 ng/ml NGF affected neither TH activity nor the total content, whereas it significantly reduced the proportion of A by about 25%.     

Modulation of the bursting properties of single mouse pancreatic beta-cells by artificial conductances

Glucose triggers bursting activity in pancreatic islets, which mediates the Ca2+ uptake that triggers insulin secretion. Aside from the channel mechanism responsible for bursting, which remains unsettled, it is not clear whether bursting is an endogenous property of individual beta-cells or requires an electrically coupled islet. While many workers report stochastic firing or quasibursting in single cells, a few reports describe single-cell bursts much longer (minutes) than those of islets (15-60 s). We studied the behavior of single cells systematically to help resolve this issue. Perforated patch recordings were made from single mouse beta-cells or hamster insulinoma tumor cells in current clamp at 30-35 degrees C, using standard K+-rich pipette solution and external solutions containing 11.1 mM glucose. Dynamic clamp was used to apply artificial KATP and Ca2+ channel conductances to cells in current clamp to assess the role of Ca2+ and KATP channels in single cell firing. The electrical activity we observed in mouse beta-cells was heterogeneous, with three basic patterns encountered: 1) repetitive fast spiking; 2) fast spikes superimposed on brief (<5 s) plateaus; or 3) periodic plateaus of longer duration (10-20 s) with small spikes. Pattern 2 was most similar to islet bursting but was significantly faster. Burst plateaus lasting on the order of minutes were only observed when recordings were made from cell clusters. Adding gCa to cells increased the depolarizing drive of bursting and lengthened the plateaus, whereas adding gKATP hyperpolarized the cells and lengthened the silent phases. Adding gCa and gKATP together did not cancel out their individual effects but could induce robust bursts that resembled those of islets, and with increased period. These added currents had no slow components, indicating that the mechanisms of physiological bursting are likely to be endogenous to single beta-cells. It is unlikely that the fast bursting (class 2) was due to oscillations in gKATP because it persisted in 100 microM tolbutamide. The ability of small exogenous currents to modify beta-cell firing patterns supports the hypothesis that single cells contain the necessary mechanisms for bursting but often fail to exhibit this behavior because of heterogeneity of cell parameters.     

In vitro evaluation of the cytotoxic and mutagenic potential of the 5-aminolevulinic acid hexylester-mediated photodynamic therapy

Photodynamic therapy (PDT) of tumors with 5-aminolevulinic acid hexylester (h-ALA) causes photo-oxidative reactions in treated tissues. In order to study cytotoxic and/or mutagenic effects, cells of the tumor cell line RPMI 2650 as well as fibroblasts of the cell line WS 1 were given photodynamic treatment in vitro. The cells were photosensitized with a 1mM h-ALA-medium solution for 5h and illuminated with different light doses (0.5, 1.0, 1.5 and 2.0 J/cm2) using red light (633+/-20 nm). PDT-induced cytotoxic effects were determined by measurement of the mitotic index (MI) and the nuclear division index (NDI). Chromosome aberrations (CA) and micronuclei (MN) were recorded to study mutagenicity. After treatment of the photosensitized RPMI 2650 cells with a light dose of 2.0 J/cm2, the MI was significantly decreased to 16.9 per thousand in comparison with that of the h-ALA control (33.8 per thousand ). In photosensitized WS 1 cells, light doses up to 2.0 J/cm2 showed no significant effect. The NDI of photosensitized RPMI 2650 cells was significantly decreased by light doses from 1.0 to 2.0 J/cm2, whereas no significant effect was seen in WS 1 cultures. Thus, h-ALA-PDT only induced desirable cytotoxic effects in tumor cells, but not in the fibroblasts. After application of light doses from 0.5 to 2.0 J/cm2, photosensitized RPMI 2650 cultures showed CA in 7.0-7.5% of the metaphases, which was not a significant increase (h-ALA control: 5.5%). In WS 1 cultures metaphases containing CA varied non-significantly from 5.0 to 7.5%. The MN rates were approximately the same in illuminated RPMI 2650 cultures and in the corresponding h-ALA control (4.4-4.9 per thousand ). The MN rates of the illuminated WS 1 cultures also varied non-significantly from 4.5 to 5.0 per thousand in comparison with the h-ALA control (5.5 per thousand ). In the mutagenicity tests the h-ALA-PDT had no significant effect, neither on the tumor cells nor on the fibroblasts. In addition to the cytogenetic analysis, spectral karyotyping (SKY) was used to characterize the cell lines and gain more detailed information on possibly PDT-induced CA. The SKY evaluation also showed no significant increase of the CA rate, but confirmed the result of the CA test. Thus, within the scope of the experiments performed, a mutagenic potential of the h-ALA-PDT can be excluded.     

Isatin, an endogenous monoamine oxidase inhibitor, triggers a dose- and time-dependent switch from apoptosis to necrosis in human neuroblastoma cells

Isatin is an endogenous indole that is increased in stress, inhibits monoamine oxidase (MAO) B and improves bradykinesia and striatal dopamine levels in rat models of Parkinson's disease. Consequently, it has been suggested that isatin might be a possible treatment for Parkinson's disease although little is known about its effects on neural cell growth and survival. The aim of this study was to investigate the survival of dopaminergic human neuroblastoma (SH-SY5Y) cells following treatment with increasing concentrations of isatin. SH-SY5Y cells were exposed to isatin for defined time points, after which cell survival was determined by MTT assay. A combination of Annexin V binding and propidium iodide (PI) exclusion was used to distinguish apoptosis from necrosis in flow cytometry experiments and FACS profiles of permeabilised PI-labelled cells were employed for the assessment of cell cycle distribution. Isatin treatment (1-400 microM) for 24h induced a significant dose-dependent increase in MTT metabolism by SH-SY5Y cells in culture, but this was not due to an increase in cell division. At the higher concentrations (200-400 microm) isatin triggered cell death, although MTT metabolism was still increased in the culture, suggesting that surviving cells were hypermetabolic. Following a longer (48 h) exposure, isatin was found to cause cell death in a dose-dependent manner; at lower concentrations (50 microM), the predominant mode of cell death was apoptosis while at the highest concentration (400 microm) increasing numbers of necrotic cells were also evident. Thus, in dopaminergic SH-SY5Y cells isatin induces cell death in dose- and time-dependent manner. This death occurred as a continuum of survival, apoptosis and necrosis. Our results re-emphasise that caution should be exercised when considering high doses of isatin as a putative anti-Parkinson's disease therapeutic.