Crosstalk between PKCα and Notch-4 in endocrine-resistant breast cancer cells.

The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.

Inhibitory transmission, activity-dependent ionic changes and neuronal network oscillations.

Oscillatory network activity arises from interactions between synaptic and intrinsic membrane properties of neurons. In this review, we summarize general mechanisms of synchronous neuronal oscillations. In addition, we focus on recent experimental and computational studies which suggest that activity-dependent changes of ionic environment can affect both the synaptic and intrinsic neuronal properties and influence the network behavior. GABA(A) receptor (GABA(A)R)-mediated signaling, that is based on Cl(-) and HCO(3)(-) permeability, is thought to be important for the oscillogenesis and synchronization in cortical networks. A remarkable feature of GABAergic synapses is that prolonged GABA(A)R activation may lead to switching from a hyperpolarizing to a depolarizing response. This is partly due to a positive shift of the GABA(A) R reversal potential (E(GABA)) that is generated by GABA-induced Cl(-) accumulation in neurons. Recent studies suggest that activity-dependent E(GABA) changes may have important implications for the mechanisms of gamma oscillations and seizure-like discharges. Thus, a better understanding of the impact of intracellular Cl(-) dynamics on network behavior may provide insights into the mechanisms of physiological and pathological brain rhythms. Combination of experiments and simulations is a promising approach for elucidating which properties of the time-varying ionic environment can shape the dynamics of a given circuit.

Characterization of the spontaneous synaptic activity of amacrine cells in the mouse retina.

Amacrine cells are a heterogeneous class of interneurons that modulate the transfer of the light signals through the retina. In addition to ionotropic glutamate receptors, amacrine cells express two types of inhibitory receptors, GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs). To characterize the functional contribution of these different receptors, spontaneous postsynaptic currents (sPSCs) were recorded with the whole cell configuration of the patch-clamp technique in acutely isolated slices of the adult mouse retina. All amacrine cells investigated (n = 47) showed spontaneous synaptic activity. In six amacrine cells, spontaneous excitatory postsynaptic currents could be identified by their sensitivity to kynurenic acid. They were characterized by small amplitudes [mean: -13.7 +/- 1.5 (SE) pA] and rapid decay kinetics (mean tau: 1.35 +/- 0.16 ms). In contrast, the reversal potential of sPSCs characterized by slow decay kinetics (amplitude-weighted time constant, tau(w), >4 ms) was dependent on the intracellular Cl(-) concentration (n = 7), indicating that they were spontaneous inhibitory postsynaptic currents (sIPSCs). In 14 of 34 amacrine cells sIPSCs were blocked by bicuculline (10 microM), indicating that they were mediated by GABA(A)Rs. Only four amacrine cells showed glycinergic sIPSCs that were inhibited by strychnine (1 microM). In one amacrine cell, sIPSCs mediated by GABA(A)Rs and GlyRs were found simultaneously. GABAergic sIPSCs could be subdivided into one group best fit by a monoexponential decay function and another biexponentially decaying group. The mean amplitude of GABAergic sIPSCs (-42.1 +/- 5.8 pA) was not significantly different from that of glycinergic sIPSCs (-28.0 +/- 8.5 pA). However, GlyRs (mean T10/90: 2.4 +/- 0.08 ms) activated significantly slower than GABA(A)Rs (mean T10/90: 1.2 +/- 0.03 ms). In addition, the decay kinetics of monoexponentially decaying GABA(A)Rs (mean tau(w): 20.3 +/- 0.50), biexponentially decaying GABA(A)Rs (mean tau(w): 30.7 +/- 0.95), and GlyRs (mean tau(w) = 25.3 +/- 1.94) were significantly different. These differences in the activation and decay kinetics of sIPSCs indicate that amacrine cells of the mouse retina express at least three types of functionally different inhibitory receptors: GlyRs and possibly two subtypes of GABA(A)Rs.

Intracellular chloride and calcium transients evoked by gamma-aminobutyric acid and glycine in neurons of the rat inferior colliculus.

Microfluorometric recordings showed that the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl- concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin-perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO3- and characterized by a continuous shift of their reversal potential (E(GABA/gly)) in the positive direction. In HCO3- -buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca2+ concentration. Ca2+ transients occurred only with large depolarizations and were blocked by Cd2+, suggesting an activation of voltage-gated Ca2+ channels. However, in the absence of HCO3-, only a small rise, if any, in the intracellular Ca2+ concentration could be evoked by GABA or glycine. We suggest that the activation of GABAA or glycine receptors results in an acute accumulation of Cl- that is enhanced by the depolarization owing to HCO3- efflux, thus shifting E(GABA/gly) to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca2+ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway.

On the mechanism of GABA-induced currents in cultured rat cortical neurons.

We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by gamma-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl- concentration ([Cl-]i). In current-clamp experiments in the presence of 26 mM HCO3- the application of 50 microM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50-200 microM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO3-. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO3--free saline, indicating a contribution from HCO3-. The reversal potential of the GABA-induced currents (EGABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO3- was -73 mV, while it was shifted to -59 mV in the presence of HCO3-. Combined patch-clamp and microfluorimetric measurements using the Cl--sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl-]i in 54% (n=13) of the neurons. We conclude that this increase of [Cl-]i in combination with the efflux of HCO3- results in a shift of EGABA above the resting membrane potential that gives rise to GABA-mediated depolarizations.

Glycine-activated currents are changed by coincident membrane depolarization in developing rat auditory brainstem neurones.

1. During early ontogeny, glycine receptors (GlyRs) exert depolarizing responses which may be of developmental relevance. We have used the gramicidin-perforated patch technique to elucidate the mechanism of glycine-activated currents in developing neurones of the rat lateral superior olive (LSO). 2. When the holding potential was set to -60 mV, perforated-patch recordings revealed glycine-induced inward currents in 59 %, outward currents in 5 % and biphasic currents in 34 % of the LSO neurones tested (n = 44). The biphasic currents were characterized by a transient outward phase which was followed by an inward phase. 3. Ion substitution experiments showed that both Cl- and HCO3- contributed to the glycine- induced biphasic current responses. 4. In the biphasic responses, the reversal potential of the glycine-induced current (Egly) depended on the response phase. A strong shift of Egly from a mean of -72 mV during the outward phase of the glycine response to a mean of -51 mV during the inward phase was observed, suggesting a shift of an ion gradient. 5. When the membrane potential was depolarized, 'tail' currents were induced in the presence of glycine. An increased duration or amplitude of the evoked depolarizations resulted in a proportional enlargement of these tail currents, indicating that they were produced by a shift of an ion gradient. Since changes of the HCO3- gradient are negligible, because of the carbonic anhydrase activity, we suggest that these tail currents were caused by a shift of the Cl- gradient. 6. We conclude that Cl- accumulates intracellularly during the activation of GlyRs and, consequently, Egly moves towards more positive values. 7. Coincident depolarizing stimuli enhanced intracellular Cl- accumulation and the shift of Egly, thereby switching hyperpolarizing to depolarizing action. This change could assist in an activity-dependent strengthening and refinement of glycinergic synapses during the maturation of inhibitory connectivity.

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration.

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca2+-dependent K+ channels via Ca2+-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba2+ and the reversal of its underlying current was significantly shifted towards EK+, suggesting that it is due to the activation of K+ channels. (2) The delayed second conductance increase disappeared in a Ca2+-free saline buffered with BAPTA, indicating that it depended on Ca2+-influx. (3) Co2+, Cd2+ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca2+ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca2+ stores with the Ca2+-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca2+ influx that could lead indirectly to the activation of Ca2+-dependent K+ channels.

Intracellular calcium transients and potassium current oscillations evoked by glutamate in cultured rat astrocytes.

Glutamate responses in cultured rat astrocytes from cerebella of neonatal rats were investigated using the perforated-patch configuration to record membrane currents without rundown of intracellular messenger cascades, and microfluorometric measurements to measure the intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) with fura-2 AM and 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxy methylester respectively. In the perforated-patch mode, glutamate evoked single or multiple outward current transients in 82% of the cells, which disappeared when the recording technique was converted into a conventional whole-cell mode. The outward current transients were accompanied by [Ca2+]i transients, whereas pHi fell monophasically, without any sign of oscillation. Pharmacological analysis of the glutamate-induced responses indicated that ionotropic receptor activation evoked an inward current but no outward current transients, and metabotropic receptor activation (of the mGluR1/5 type) elicited outward current transients but no inward current. The outward current transients were reduced in frequency, or even abolished, after depletion of the intracellular Ca2+-stores by the Ca2+-ATPase inhibitor cyclopiaconic acid (10 microM). They reversed near -85 mV and were reduced by tetraethylammonium (10 mM), suggesting that they were caused by K+ channel activation. It is concluded that glutamate evoked these K+ outward current transients by oscillatory Ca2+ release mediated by mGluR activation. The corresponding membrane potential waves across the astroglial syncytium could provide spatial and temporal dynamics to the glial K+ uptake capacity and other voltage-dependent processes.

Blockade of AMPA receptors by nickel in cultured rat astrocytes.

The effect of Ni2+ on glial alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors was studied using the whole-cell patch-clamp technique in cultured rat cerebellar astrocytes. The application of kainate (10 mu M-5 mM) evoked inward currents at a holding potential of -70 mV. These currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and Evans Blue, and potentiated by cyclothiazide, suggesting that they were primarily mediated by the AMPA receptor subtype. Analysis of the kainate concentration-response relation in cultured astrocytes revealed a maximal current of 488 pA, a half-maximal effective concentration of 137 mu M and a Hill coefficient of 1.43, indicating more than one agonist binding site. Ni2+ inhibited the current activated by 300 mu M kainate in a concentration-dependent manner, displaying a half-maximal inhibition at 860 mu M Ni2+ and a Hill coefficient of 1.07. In the presence of 700 mu M Ni2+ the kainate-induced concentration-response curve was shifted towards higher concentrations, increasing the half-maximal effective concentration to 300 mu M, without significantly changing the Hill coefficient. The blocking effect of Ni2+ was counteracted by increasing kainate concentrations, suggesting a competitive mechanism.

Developmental variation of the permeability to Ca2+ of AMPA receptors in presumed hilar glial precursor cells.

Glial cells in the hilus of the dentate gyrus of the rat were investigated using the patch-clamp technique in acute slices of the hippocampal formation. According to their voltage-gated current patterns, two classes of glial cells--putative astrocytes and presumed glial precursor cells--were apparent. The glutamate receptor agonists kainate, glutamate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) evoked inward currents at a holding potential of -70 mV in astrocytes and presumed glial precursor cells. Inward currents could also be induced in nucleated patches, indicating a direct action on glial receptors. In presumed hilar glial precursor cells, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microM) blocked the kainate-induced current, while it was partially inhibited by Zn2+ (2 mM) and Evans Blue (10 microM). Cyclothiazide (100 microM), in contrast, potentiated this current, indicating the presence of AMPA receptors. In 90% of the presumed glial precursor cells the excitatory amino-acid-evoked current voltage (I/V) relations were linear or outwardly rectifying and reversed close to 0 mV, which is characteristic for non-specific cation channels. To determine the permeability to Ca2+, I/V relations were determined in a Na(+)-free solution containing 40 mM Ca2+ and showed reversal potentials of a wide variation ranging from -63 mV to +1 mV with corresponding PCa/PCs permeability ratios of between 0.09 and 2.10. Statistical analysis revealed that the permeability to Ca2+ significantly decreased with an advance in age (r = -0.596; n = 21; P < 0.01). These data suggest that the Ca2+ influx mediated by the activation of AMPA receptors expressed in presumed hilar glial precursor cells is dependent on the developmental stage.

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes.

We have studied the regulation of intracellular pH (pHi), and HCO3(-)-dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pHi was 6.96 in both nominally CO2/HCO3(-)-free, HEPES-buffered saline (6.96 +/- 0.14; n = 48) and in a saline containing 5% CO2/24 mM HCO3- (6.96 +/- 0.18; n = 48) (at pH 7.4). Inhibition of the Na+/H+ exchange by amiloride (2 mM) caused a significant decrease of pHi in nominally CO2/HCO3(-)-free saline. Addition of CO2/HCO3- in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na+ also caused a fall of pHi, and addition of CO2/HCO3- in Na(+)-free saline evoked a further fall of pHi, while the outward current was reduced or even reversed. The stilbene 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM) reduced the pHi recovery from the CO2/HCO3(-)-evoked acidification, and blocked the prominent intracellular acidification upon removal of CO2/HCO3-. Removal of external Cl- had little effect on these pHi changes. Lowering the external pH from 7.4 to 6.6 in CO2/HCO3(-)-containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO2/HCO3-. The results suggest that the CO2/HCO3(-)-dependent current is partly due to a reversible bidirectional, electrogenic Na(+)-HCO3- cotransporter, which helps to regulate pHi in these cells. In addition, a prominent Na+/H+ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steady-state pHi.

Neuronal responses to purinoceptor agonists in the leech central nervous system.

Extracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous system, however, little is known about their role in neurotransmission. We have studied the response of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agonists depolarized the different neurons, but not the neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 microM) or ADP (100 microM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-gamma-S (5 microM) induced larger depolarizations than 100 microM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxious cells, ATP (100 microM) induced an inward current of 1.7 +/- 1.1 nA at a holding potential of -60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 mV. In a Na+-free extracellular solution, the ATP-induced inward current decreased and in a Na+- and Ca(2+)-free saline only a small residual current persisted. The possible P2 purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP > or = ADP > AMP. The results suggest that these cells express purinoceptors of the P2 type.

Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification.

Ligand-gated ion channels generally display a heterooligomeric subunit structure. The present report describes an electrophysiological method that provides criteria indicating the subunit stoichiometry of a recombinant GABAA receptor composed of alpha 3, beta 2 and gamma 2 subunits. Our results exclude the stoichiometries 3 alpha 1 beta 1 gamma, 1 alpha 3 beta 1 gamma, 1 alpha 1 beta 3 gamma and suggest that the possible subunit stoichiometries for this receptor are 2 alpha 1 beta 2 gamma, 2 alpha 2 beta 1 gamma or 1 alpha 2 beta 2 gamma, of which the alpha subunit composition 2 alpha 1 beta 2 gamma may be favoured. The method is based on the quantification of the outward rectification of the GABA-evoked current induced by point mutation of charged amino acids located near the ion channel pore.

Single-channel activity in cultured cortical neurons of the rat in the presence of a toxic dose of glutamate.

Rat cortical neurons grown in cell culture were exposed to 500 microM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca(2+-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K+ channel requiring intracellular calcium ([Ca2+]i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 +/- 6 pS; 'BK channel'). Another Ca(2+)-dependent channel permeable for Cl- (conductance for outward currents in cell-attached patches 72 +/- 17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K+ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca2+]i. Both phases of increasing channel activity required the presence of extracellular Ca2+ suggesting that [Ca2+]i was mainly increased by Ca2+ influx. The N-methyl-D-aspartate (NMDA) antagonists dizocilpine (MK-801, 10 microM) and DL-2-amino-5-phosphonovaleric acid (AP5; 100 microM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca2+ through NMDA receptor channels causes a strong activation of Ca(2+)-dependent K+ channels, which is likely to result in pronounced loss of intracellular K+. NMDA receptor channels seem to remain active for a long time (> 10 min) after the end of glutamate application.

Pharmacological and Electrophysiological Properties of Recombinant GABAA Receptors Comprising the alpha3, beta1 and gamma2 Subunits.

To assess the role of subunits for channel function and drug modulation in recombinant GABAA receptors, the alpha3beta1gamma2 subunits and the dual combinations alpha3beta1, beta1gamma2 and alpha3gamma2 were expressed by transfection of human embryonic kidney cells and by RNA injection in Xenopus oocytes (alpha3beta1gamma2 combination). GABA-induced chloride currents were recorded using the whole-cell configuration of the patch-clamp technique (transfected cells) or the voltage-clamp technique (oocytes). The currents recorded from the alpha3beta1gamma2 subunit combination in transfected cells were reduced by bicuculline and picrotoxin, enhanced by flunitrazepam in a flumazenil-sensitive manner and reduced by beta-carboline-3-carboxylic acid methyl ester (beta-CCM). The GABA-induced current was reduced by beta-CCM in all combinations containing the gamma2 subunit, but potentiation by flunitrazepam was only obtained when the gamma2 subunit was coexpressed in the presence of the alpha3 subunit (alpha3beta1gamma2 or alpha3gamma2). The GABA sensitivities of the receptors were similar when the alpha3beta1gamma2 combination was expressed in oocytes (half-maximum effective concentration=240 microM) or in the kidney cell line (270 microM). However, the currents were less potentiated by flunitrazepam in oocytes (129% of controls) than in transfected cells (189%). These results suggest that the alpha3beta1gamma2 subunit combination, which is coexpressed in various brain regions as shown by in situ hybridization histochemistry, may represent a building block of functional GABAA receptors in situ.

Use of etretinate for treatment of primary keratinization disorders (idiopathic seborrhea) in cocker spaniels, west highland white terriers, and basset hounds.

An open clinical trial was used to evaluate the synthetic retinoid, etretinate, for treatment of idiopathic seborrhea in Cocker Spaniels, West Highland White Terriers, and Basset Hounds. Clinical and histologic improvement was seen in the Cocker Spaniels. Etretinate had no beneficial effect on the skin disease of the West Highland White Terriers or the Basset Hounds. Etretinate treatment did not alter the type or degree of otitis externa. Clinical side effects were minimal. Relevant laboratory abnormalities were not detected.

Action of diazepam on the voltage-dependent Na+ current. Comparison with the effects of phenytoin, carbamazepine, lidocaine and flumazenil.

The influence of diazepam, an agonist, and flumazenil (Ro 15-1788), an antagonist of the benzodiazepine receptor, on repetitive firing of action potentials in cultured spinal neurons and on voltage-dependent Na+ currents in cultured N2A neuroblastoma cells was examined. The effects were compared to those of the antiepileptics phenytoin and carbamazepine and the local anesthetic lidocaine. The whole-cell configuration of the patch-clamp technique was used for potential and current recording. Diazepam (10 microM) or phenytoin (10 microM) reduced the duration of repetitive action potential discharges in 50 or 67% of the spinal neurons, respectively. At a concentration of 100 microM repetitive firing was completely blocked. Flumazenil (100 microM) had no effect. In N2A neuroblastoma cells diazepam, phenytoin, carbamazepine and lidocaine, but not flumazenil, at a concentration of 100 microM reduced the Na+ current to 60-67% of control. At 10 microM no or only a weak depression was seen with any drug. In the presence of diazepam (100 microM) the Na+ channel inactivation curve was shifted in the hyperpolarizing direction by -4.8 +/- 0.5 mV. Phenytoin, carbamazepine and lidocaine (all 100 microM) caused stronger shifts of -17.4 +/- 2.1, -10.6 +/- 0.9 and -17.0 +/- 2.1 mV, respectively. Inhibition of the Na+ current by diazepam increased use-dependently over 9 depolarizing pulses repeated at high frequency (200 Hz), whereas use-dependent effects of the other compounds developed less rapidly. At a low stimulation rate (7 Hz) use-dependent block was pronounced with lidocaine, but weak or absent with diazepam and carbamazepine.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of neurokinin receptors modulates K+ and Cl- channel activity in cultured astrocytes from rat cortex.

Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP-dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch-clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential.

Pharmacological characterization of the glutamate receptor in cultured astrocytes.

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.