2-Halopropionic acid-induced cerebellar granule cell necrosis in the rat: in vivo and in vitro studies.

Daily oral administration of 2.3 mmol/kg L-2-chloropropionic acid (L-2-CPA), DL-2-bromopropionic acid (2-BPA) or DL-2-iodopropionic acid (2-/PA) but not DL-2-fluoropropionic acid (2-FPA) produced cerebellar granule cell necrosis in the rat. Twenty four hours after three doses of L-2-CPA or two doses of 2-BPA, animals showed clinical signs of motor incoordination and reduced hindlimb function which was associated with marked cerebellar oedema and cerebellar granule cell necrosis. Biochemical analyses showed a marked increase in cerebellar water and Na+ content, and a reduction in cerebellar glutamate and aspartate. 2-IPA at this dose was toxic, the animals not surviving a second dose, histopathology showed hepatic and renal necrosis with mild cerebellar granule cell necrosis. 2-FPA was not neurotoxic after four daily doses. A marked decrease in hepatic and cerebellar non-protein sulphydryl (NP-SH) content was observed 4 h after a single dose of 2.3 mmol/kg L-2-CPA, 2-BPA and 2-IPA but not 2-FPA. Daily doses of 2-BPA for 3 days produced a sustained 50% depletion in cerebellar NP-SH. In vitro, L-2-CPA, 2-BPA and 2-IPA produced glutathione (GSH) depletion in the presence of rat liver cytosol, while 2-FPA did not. Depletion of GSH in the presence of cerebellar cytosol was only observed with 2-IPA. Studies using primary cultures of rat cerebellar granule cells, showed that all analogues produced a concentration dependent loss of cell viability. Mean EC50 values for 2-FPA, L-2-CPA, 2-BPA and 2-IPA toxicity were 1.7, >10, 0.5 and 0.3 mM, respectively, for 24 h continuous exposure. MK-801 and Vitamin E afforded protection against L-2-CPA-induced cytotoxicity but not against the other analogues. In summary, in addition to L-2-CPA, both 2-BPA and 2-IPA produce cerebellar granule cell necrosis in the rat. Depletion of GSH in the cerebellum may be contributory factor in the cascade of events leading to neurotoxicity.

Neurotoxic effect of L-2-chloropropionic acid on primary cultures of rat cerebellar granule cells.

L-2-Chloropropionic acid (L-CPA), when administered orally to rats, produces selective necrosis to the granule cell layer of the rat cerebellum which is delayed in onset, not appearing until 36-48 h after exposure. The present study was conducted to characterise the toxic effect of L-CPA in primary cell cultures of rat cerebellar granule cells in vitro. Exposure to L-CPA produced a time and concentration dependent loss in cerebellar granule cell viability. Mean 50% effective concentration (EC50) values for L-CPA toxicity were 18.3 +/- 0.3, 7.4 +/- 0.1, and 3.5 +/- 0.1 mM for 24, 48 and 72 h exposure respectively. Exposure for 24 h followed by a return to L-CPA free medium for 24 h was more toxic than exposure for 24 h alone. Cells maintained in culture for a longer duration were more susceptible to L-CPA-induced toxicity. The toxic effects of L-CPA could be partially or fully prevented by concomitant exposure of the cells to putative neuroprotective compounds. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (3 microM), afforded partial protection against L-CPA induced toxicity, whilst other glutamate receptor antagonists including, D(-)-2-amino-5-phosphopentanoic acid (D-AP5; 300 microM), D(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP; 300 microM), 5,7-dichlorokynurenic acid (10 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 microM) were ineffective. The antioxidant, vitamin E (10 microM), afforded significant but incomplete protection from L-CPA toxicity. However when both MK-801 (3 microM) and vitamin E (10 microM) were present during L-CPA exposure, a greater degree of protection was observed than with either compound alone, although the combination failed to provide complete protection. Cyclosporin A, an inhibitor of the mitochondrial transition pore, also provided partial protection. By contrast, the free radical trapping agent, N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) provided concentration (1-10 mM) dependent protection against the L-CPA-induced toxicity, which was complete at 10 mM. Our findings suggest that free radical production may be involved in the mechanism of L-CPA-induced toxicity.

Methyl iodide toxicity in rat cerebellar granule cells in vitro: the role of glutathione.

The monohalomethane methyl iodide (MeI) is toxic to a number of organ systems including the central nervous system. Clinical symptoms of neurotoxicity suggest that the cerebellum is the target within the brain, and we have now modelled the toxicity of MeI in cultured rat cerebellar granule cells. Cytotoxicity is maximal 24 h after a 5 min exposure to MeI, and the EC50 for MeI under these conditions was calculated to be 1.6 mM. The glutathione S-transferase (GST) dependent metabolism of MeI was investigated in these cultures. There was a marked decrease in intracellular glutathione (GSH) 15 min after exposure to MeI, and GSH concentrations then increased, reaching 130% of control levels 7 h after exposure. To investigate the role of conjugation with GSH in the toxicity of MeI, GSH levels were modulated prior to exposure. Depletion of GSH exacerbated the cytotoxicity of MeI while provision of a bioavailable source of GSH was protective. Inclusion of antioxidants [vitamin E, butylated hydroxytoluene (BHT) or desferrioxamine mesylate (DF)] also protected against the cytotoxicity of MeI. Our in vitro data suggest that MeI is conjugated with GSH in the cerebellum, and the resulting extensive depletion of GSH may be the first step en route to toxicity, rendering the tissue susceptible to methylation and/or oxidative stress.

Characterisation of kainate receptor mediated whole-cell currents in rat cultured cerebellar granule cells.

Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 microM) completely abolished inward currents evoked by AMPA (10-100 microM) in the presence of cyclothiazide (100 microM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35-140 pA, n = 13) resistant to blockade. The majority (73+/-7%, n = 5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 microM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 microM). (2S, 4R)-4-methylglutamate (SYM 2081, 10 microM) evoked inward currents in Con A treated cells (15-60 pA, n = 7), which were resistant to complete blockade by GYKI 53655 (50 microM) but antagonised by NBQX (100 microM). Kainate-evoked responses in the presence of GYKI 53655 (50 microM) had linear or slightly outwardly rectifying current-voltage (I-V) relationships in all cells examined (n = 5) and were resistant to blockade by Joro spider toxin (JsTx, 1 microM; n = 5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself.

L-2-chloropropionic acid inhibits glutamate and aspartate release from rat cerebellar slices but does not activate cerebellar NMDA receptors: implications for L-2-chloropropionic acid-induced neurotoxicity.

L-2-Chloropropionic acid (L-CPA), when orally administered at single high dose to rats produces a selective lesion in the cerebellum involving destruction of a high proportion of granule cells by a mechanism which involves N-methyl-D-aspartate (NMDA) receptors. Receptor binding studies demonstrated that L-CPA a had low affinity at the glutamate and glycine binding sites at NMDA receptors (530-660 microM), respectively, whereas L-CPA did not displace [3H]AMPA, [3H]NBQX or [3H]kainate from AMPA or kainate receptors. Whole cell-patch clamp experiments using cultured granule cells failed to demonstrate changes in membrane potential of cultured granule cells when either L-CPA (0.25 or 1 microM) was added alone to the bathing solution, or in combination with glycine (10 microM). Furthermore L-CPA did not alter the magnitude of the inward current produced by application of NMDA (100 microM)) to cultured granule cells, in the presence of glycine, as measured by patch clamp techniques. Experiments were also performed to discover whether L-CPA may alter the release of the excitatory amino acids from the cerebellum, which may then indirectly alter activity at glutamate receptors, leading to neuronal cell death. L-CPA (2 mM) did not affect either basal or stimulated (electrical or high potassium) endogenous aspartate release from superfused cerebellar slices nor did it alter the basal or stimulated release of [3H]aspartate from preloaded slices when introduced into the superfusion medium over 30 min. However, when cerebellar slices were preincubated with 2 mM L-CPA for 2 h at concentrations that are known to be neurotoxic to the brain in vivo, but not in vitro, the stimulated endogenous glutamate and aspartate net release was significantly attenuated, as compared to controls. Basal release was not significantly affected by the introduction of L-CPA-induced cerebellar neurotoxicity may be related to the inhibition of excitatory amino acid release from the cerebellum. In conclusion, although L-CPA does not appear to directly alter NMDA receptor activity the L-CPA-induced cerebellar neurotoxicity may be related to the inhibition of excitatory amino acid release from the cerebellum.

Inhibitory actions of ZENECA ZD7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells.

1. ZENECA ZD7288 (4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride) is a sinoatrial node (SAN) modulating agent which produces a selective slowing of the heart rate. Its effects have been studied in single, freshly dissociated guinea-pig SAN cells, by standard patch clamp procedures. 2. Whole-cell inward currents were evoked by hyperpolarizing voltage clamp steps from a holding potential of -40 mV. ZD7288 inhibited the hyperpolarization activated cationic current (If) in a concentration-dependent manner. The 'selective bradycardic agents' alinidine and UL-FS 49 (zatebradine) both also inhibited If. 3. The activation of If was investigated by measuring tail current amplitudes at +20 mV after hyperpolarizing steps to different potentials to activate the current. The reduction in If resulted from both a shift in the If current activation curve in the negative direction on the voltage axis, and also a reduction in the activation curve amplitude. 4. ZD7288 did not affect the ion selectivity of the If channel, since the tail current reversal potential was unchanged in the presence of the drug. 5. With ZD7288 the inhibition of If was not use-dependent, whereas UL-FS 49 displayed use-dependence in the block of the If current. 6. Whereas ZD7288 had no significant effect on the delayed rectifier current (Ik) in these cells, both alinidine and UL-FS 49 significantly reduced Ik at the same concentrations which reduced If. 7. The data show that ZD7288 reduces If by affecting the activation characteristics of the If current; this inhibition may account for this agent's selective bradycardiac properties.

Voltage-activated currents in the CRI-G1 rat insulin-secreting cell-line.

1. The whole-cell configuration of the patch-clamp recording technique was used to characterize the electrophysiological properties of CRI-G1 insulin-secreting cells. 2. Current-clamp recordings demonstrated the excitable nature of these cells. 3. Voltage-clamp recordings revealed the presence of an inward Na+ current, an inward Ca2+ current and a delayed outward K+ conductance. 4. The electrophysiological properties of CRI-G1 closely resemble those of pancreatic beta-cells, thereby rendering this cell-line as a useful alternative to freshly isolated cells for the study of pancreatic beta-cell electrophysiology and pharmacology.

Steroid modulation of the GABAA receptor complex: electrophysiological studies.

The effect of some endogenous and synthetic steroids on the operation of inhibitory and excitatory amino acid neurotransmitter receptors was examined. Anaesthetic pregnane steroids (e.g. alphaxalone, 5 alpha-pregnan-3 alpha-ol-20-one, 5 alpha-pregnane-3 alpha,21-diol-20-one) potentiated GABAA receptor-mediated whole-cell currents recorded from bovine chromaffin cells. The threshold concentration for enhancement was 10-30 nM. Potentiation was stereoselective and was mediated by a steroid-induced prolongation of the burst duration of the GABA-activated channel. Additionally, the pregnane steroids directly activated the GABAA receptor. Both the potentiation and activation appear to be mediated through a site(s) distinct from the well-known barbiturate and benzodiazepine allosteric sites of the GABAA receptor. Intracellularly applied alphaxalone and 5 beta-pregnan-3 alpha-ol-20-one had no discernible effects on the GABAA receptor, suggesting that the steroid binding site can only be accessed extracellularly. Unlike behaviourally depressant barbiturates, which modulate GABAA receptor function in a manner similar to that of the pregnane steroids, alphaxalone and 5 beta-pregnan-3 alpha-ol-20-one show striking pharmacological selectivity. Voltage-clamp recordings from rat central neurons in culture indicate that pentobarbitone exerts its potentiating and GABA-mimetic effects over a range of concentrations which also depress currents mediated by glutamate receptor subtypes. In contrast, alphaxalone and several endogenous steroids greatly enhance responses to GABA, but have no direct effect on glutamate receptors. Such pharmacological selectivity, coupled with appropriate stereoselectivity of action, suggests that the GABAA receptor mediates some of the behavioural effects of synthetic and endogenous pregnane steroids.

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line.

1. The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulin-secreting cell line derived from a rat islet cell tumour. 2. Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40 microM and 0.2 microM, respectively. 3. Diazoxide (0.6-1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4. Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5. In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 microM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6. Diazoxide (0.6 mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 microM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7. Neither tolbutamide (1.0 mM) nor glibenclamide (10 microM) altered the open-state probability of the Ca2+-activated K+ channel or the Ca2+-activated non-selective cation channel which are present in this cell line. 8. It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.

Adenosine-5'-triphosphate-sensitive ion channels in neonatal rat cultured central neurones.

ATP-sensitive channels were observed in isolated inside-out membrane patches from rat cultured central neurones. Two types of ATP-sensitive K+ channels were present in cortical neurones, one which had its open-state probability increased, the other its open-state probability decreased by application of ATP to the cytoplasmic membrane surface. Another, ATP-sensitive channel differing in ion conductance from all previously reported ATP-sensitive channels was also seen in patches from cortical neurones. This channel was nonselective with respect to Na+, K+ and Cl- ions and ATP produced a "flickery" type of block. The non-hydrolysable analogue, AMPPNP, did not mimic ATP and prevented ATP action. Preliminary experiments indicate that similar, but not, identical ATP-sensitive channels exist in cerebellar neurones.

Nucleotide-sensitive ion channels in human insulin producing tumour cells.

Cells from a human insulin producing tumour have been studied and single channel currents recorded. We have observed three main cation-selective channels in excised patch experiments. An ATP-sensitive potassium channel is present the activity of which can be inhibited by the oral hypoglycaemic drug, tolbutamide. A calcium-activated non-selective cation channel, which is inhibited by AMP could also be seen. In addition a large conductance potassium selective channel, possibly the "maxi" calcium-activated potassium channel is present in these cells.

Calcium and ATP regulate the activity of a non-selective cation channel in a rat insulinoma cell line.

A calcium-activated non-selective cation channel was observed in isolated plasma membrane patches from an insulin-secreting cell line (CRI-Gl). The conductance of the channel was approximately 25 pS with identical (140 mM KCl) solutions on either side of the membrane. However, some rectification was observed (smaller outward current) when sodium ions were present extracellularly. The channel was inactive on exposure to an intracellular calcium concentration of 10(-6) M and required high (greater than 10(-4) M) concentrations for a significant degree of activation. The open-state probability of the channel was voltage dependent, increasing with membrane depolarization. Analysis of single channel kinetics indicated that there were at least two open and two closed states. Application of ATP to the cytoplasmic membrane surface reduced the open state probability in a dose-dependent manner. The channel activity was blocked by quinine and 4-AP but was insensitive to TEA, TTX and amiloride. It is not clear what role this channel might play in the complex electrical activity of beta-cells.

Inhibition of a calcium-activated, non-selective cation channel, in a rat insulinoma cell line, by adenine derivatives.

The effects of adenosine and adenine nucleotides on a calcium-activated non-selective cation channel, present in the plasma membrane of an insulin-secreting cell line CRI-Gl were investigated. Single-channel currents were recorded from inside-out membrane patches and the adenine derivatives applied to the solution bathing the cytoplasmic aspect of the membrane surface. The activity of this channel is shown to be inhibited by all the derivatives tested. The potency sequence for inhibition was found to be AMP greater than ADP greater than ATP greater than adenosine.

Single channel recordings of potassium currents in an insulin-secreting cell line.

Using the patch-clamp technique we observed three distinct classes of K+ channels which were spontaneously active in excised 'inside-out' membrane patches from an insulin-secreting rat pancreatic islet cell line (CRI-G1). Two of these occurred infrequently, one with a conductance of approximately 7 pS, and the other a conductance of 220 pS. The activation of the 220 pS K+ channel was dependent upon the membrane voltage and was sensitive to the concentration of calcium ions at the cytoplasmic surface of the membrane. The third, and by far the most common class of K+ channel, was characterized by its sensitivity to ATP. Application of ATP to the cytoplasmic side of the membrane reversibly inhibited this K+ channel in a dose-dependent manner, but had no effect when applied to the external side. The properties of the ATP-sensitive K+ channel appear to be indistinguishable from those of a channel found in rat neonatal beta cells. Thus this insulin-secreting cell line should prove valuable in the investigation of the role of K+ channels in the regulation of insulin secretion.

The sulphonylurea receptor may be an ATP-sensitive potassium channel.

The stimulation of insulin secretion from the beta cells of the islets of Langerhans appears to be mediated by a decrease in the cell-membrane potassium-ion permeability. Tolbutamide reduced K+ movement through an ATP-sensitive K+ channel in patches of plasma membrane from an insulin-producing cell line when applied to the external surface of the membrane. The effect occurred at concentrations which exist in the serum of patients treated with tolbutamide and which stimulate insulin secretion from islets of Langerhans in vitro. Glibenclamide had a similar effect but, in keeping with its greater therapeutic potency, at concentrations one hundred times lower. These findings suggest that an ATP-sensitive K+ channel or a protein closely associated with it may be the receptor through which sulphonylureas act to stimulate insulin secretion in vitro.