The AMPA receptor/Na(+) channel blocker BIIR 561 CL is protective in a model of global cerebral ischaemia.

In this study, we investigated whether the novel neuroprotective compound dimethyl-[2-[2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-phenoxy]-ethyl]-amine hydrochloride, BIIR 561 CL, a combined non-competitive antagonist of AMPA receptors and blocker of voltage-gated Na+ channels, is protective in a rat model of severe global ischaemia. BIIR 561 CL administered immediately after 10 min of ischaemia (occlusion of both carotid arteries plus reduction of arterial blood pressure to 38-40 mm Hg) significantly reduced hippocampal damage at 4 x 26.8 mg/kg (subcutaneous injections). The competitive AMPA receptor antagonist 2,3-dihydro-6-nitro-7-sulfamoyl-benz(F)quinoxaline, NBQX, was used as a reference compound and was protective at 3x30 mg/kg (intraperitoneal and/or subcutaneous administration). BIIR 561 CL significantly reduced the ischaemia-induced premature mortality from 33.6% in the controls to 14.3%, whereas NBQX treatment had no statistically significant effect.Thus, BIIR 561 CL could be shown to reduce hippocampal damage and premature mortality in a model of severe global ischaemia. A compound with these properties might be an interesting candidate for the treatment of disorders related to global cerebral ischaemia in man.

In vivo pharmacology of BIIR 561 CL, a novel combined antagonist of AMPA receptors and voltage-dependent Na(+) channels.

Glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype and voltage-gated Na(+) channels are associated with diseases of the central nervous system characterized by neuronal over-excitation as in epilepsy or cerebral ischaemia. In animal models, AMPA receptor antagonists and Na(+) channel blockers provide protection in these conditions. Dimethyl-[2-[2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-phenoxyl]-ethyl]-amine hydrochloride (BIIR 561 CL) combines both, AMPA receptor - and Na(+) channel blocking properties in one molecule. Here, BIIR 561 CL was investigated in vivo. BIIR 561 CL protected mice against AMPA-induced toxicity with an ED(50) value of 4.5 mg kg(-1) following subcutaneous (s.c.) administration. A 0.1% solution of BIIR 561 CL provided local anaesthesia in the corneal reflex test in rabbits. In mice, the compound prevented tonic seizures in the maximal electroshock (MES) model with an ED(50) value of 3.0 mg kg(-1) s.c. In amygdala-kindled rats, BIIR 561 CL inhibited seizures at doses of 3 and 11 mg kg(-1) following intraperitoneal (i.p.) injection. The data show that the combination of blocking AMPA receptor- and voltage-gated Na(+) channels in one molecule induces effective protection in animal models of neuronal over-excitation.

Pharmacodynamic profile of the M1 agonist talsaclidine in animals and man.

In functional pharmacological assays, talsaclidine has been described as a functionally preferential M1 agonist with full intrinsic activity, and less pronounced effects at M2- and M3 receptors. In accordance with this, cholinomimetic central activation measured in rabbits by EEG recordings occurred at a 10 fold lower dose than that inducing predominantly M3-mediated side effects. This pharmacological profile is also reflected in the clinical situation: Both in healthy volunteers and in Alzheimer patients--unlike after unspecific receptor stimulation through cholinesterase inhibitors--the mainly M3-mediated gastrointestinal effects (like nausea and vomiting) were not dose-limiting. Rather, sweating and hypersalivation, mediated through muscarinic receptors, occurred dose-dependently and were finally dose-limiting. In contrast to talsaclidine, sabcomeline had a less pronounced functional M1 selectivity in pharmacological assays. This was also shown in anaesthetized guinea pigs where sabcomeline alone induced bronchoconstriction, and in the rabbit EEG where central activation and cholinergic side effects occurred in the same dose range. Neither drug, however, showed convincing improvement of cognitive functions in patients with mild-to-moderate Alzheimer's disease. This asks for a reassessment of the muscarinic hypothesis for the treatment of this disease.

Characterization of the anticonvulsant and neuroprotectant BIIR 561 CL in vitro: effects on native and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

BIIR 561 CL is a novel blocker of AMPA receptors and voltage-dependent sodium channels. In this study we further describe the effects of BIIR 561 CL on AMPA receptor-mediated membrane currents in rodent neurons, as well as in cells expressing recombinant human GluR1/2 receptors in more detail. BIIR 561 CL suppressed responses to kainate in neuronal cultures from rat cortex with an IC50 of 9.8 microM. Similar effects were observed using acutely dissociated neurons from the CA1 region of rat hippocampus (IC50 = 9.5 microM). Inhibition of kainate responses by BIIR 561 CL was prevented by preapplication of GYKI 53655, suggesting that both non-competitive inhibitors bind to a common site of the receptor. The effect of 10 microM BIIR 561 CL on kainate-induced currents was dependent on extracellular pH, with more pronounced block (84.1%) under acidic conditions (pHextern=6.4), compared to only 30.1% at a pHextern of 8.4. Thus, it can be hypothesized that BIIR 561 CL inhibits AMPA receptors in ischaemic brain regions more effectively than in healthy tissue. BIIR 561 CL inhibited responses to 1 mM glutamate in cells expressing recombinant human GluR1/2 receptors with similar potency, as compared to kainate responses in rat neurons (IC50=17.3 microM). The reference compound NBQX had an IC50 of 25.2 nM. None of the two compounds affected the glutamate-induced receptor desensitization at any tested concentration. The block by BIIR 561 CL was not use-dependent and had fast on- and off-kinetics (tauon=6.8 s; tauoff=1.3 s in hGluR1/2 receptors with 30 microM BIIR 561 CL). Thus, BIIR 561 CL can be anticipated to have a promising profile for the treatment of neurological disorders like brain ischaemia and head trauma.

Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus.

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

Treatment with the selective muscarinic agonist talsaclidine decreases cerebrospinal fluid levels of total amyloid beta-peptide in patients with Alzheimer's disease.

Brain amyloid load in Alzheimer's disease (AD) is, at least in genetic forms, associated with overproduction of amyloid beta-peptides (A beta). Thus, lowering A beta production is a central therapeutic target in AD and may be achieved by modulating such key enzymes of amyloid precursor protein (APP) processing as beta-, gamma-, and alpha-secretase activities. Talsaclidine is a selective muscarinic M1 agonist that stimulates the nonamyloidogenic alpha-secretase pathway in model systems. Talsaclidine was administered double-blind, placebo-controlled, and randomized to 24 AD patients and cerebrospinal fluid (CSF) levels of total A beta were quantitated before and after 4 weeks of drug treatment. We observed that talsaclidine decreases CSF levels of A beta significantly over time within the treatment group (n = 20) by a median of 16% as well as compared to placebo (n = 4) by a median of 27%. We conclude that treatment with selective M1 agonists may reduce A beta production and may thus be further evaluated as a potential amyloid-lowering therapy of AD.

BIIR 561 CL: a novel combined antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and voltage-dependent sodium channels with anticonvulsive and neuroprotective properties.

Antagonists of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, as well as of voltage-gated sodium channels, exhibit anticonvulsive and neuroprotective properties in vivo. One can postulate that a compound that combines both principles might be useful for the treatment of disorders of the central nervous system, like focal or global ischemia. Here, we present data on the effects of dimethyl-(2-[2-(3-phenyl-[1,2, 4]oxadiazol-5-yl)-phenoxy]ethyl)-amine hydrochloride (BIIR 561 CL) on neuronal AMPA receptors and voltage-dependent sodium channels. BIIR 561 CL inhibited AMPA receptor-mediated membrane currents in cultured cortical neurons with an IC50 value of 8.5 microM. The inhibition was noncompetitive. In a cortical wedge preparation, BIIR 561 CL reduced AMPA-induced depolarizations with an IC50 value of 10.8 microM. In addition to the effects on the glutamatergic system, BIIR 561 CL inhibited binding of radiolabeled batrachotoxin to rat brain synaptosomal membranes with a Ki value of 1.2 microM. The compound reduced sodium currents in voltage-clamped cortical neurons with an IC50 value of 5.2 microM and inhibited the veratridine-induced release of glutamate from rat brain slices with an IC50 value of 2.3 microM. Thus, BIIR 561 CL inhibited AMPA receptors and voltage-gated sodium channels in a variety of preparations. BIIR 561 CL suppressed tonic seizures in a maximum electroshock model in mice with an ED50 value of 2.8 mg/kg after s.c. administration. In a model of focal ischemia in mice, i.p. administration of 6 or 60 mg/kg BIIR 561 CL reduced the area of the infarcted cortical surface. These data show that BIIR 561 CL is a combined antagonist of AMPA receptors and voltage-gated sodium channels with promising anticonvulsive and neuroprotective properties.

The Royal College of Surgeons rat: an animal model for inherited retinal degeneration with a still unknown genetic defect.

The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an 'off' signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.

Activation of a Cl--conductance by protein kinase-dependent phosphorylation in cultured rat retinal pigment epithelial cells.

While chloride conductances are involved in signals of the electroretinogram generated by the retinal pigment epithelium (RPE), patch-clamp experiments of freshly isolated or cultured RPE cells have shown that potassium conductances predominate. The purpose of this study was to investigate mechanisms which activate Cl--conductances in RPE cells. Membrane currents of cultured rat RPE cells were measured using the whole-cell configuration of the patch-clamp technique under extra- and intracellular K+-free conditions. The bath solution was hyperosmolal to the pipette solution to prevent hypoosmotic swelling. Exchange of the physiological intracellular fluid by a pipette solution with physiological levels of ATP (2 mm) induced a continuous increase of membrane conductance. Conductance was blocked by DIDS (1 mm), and showed a reversal potential close to the Nernst potential for Cl-. When the experiments were carried out under conditions in which all cations, and not only potassium, were replaced by NMDG, the same responses could be observed. Current activation was independent of extracellular calcium. Chloride currents were also induced when ATPgammaS or AMP-PNP were used instead of ATP. In the presence of AMP-PNP currents were 10 times smaller than in the presence of ATP or ATPgammaS. In cells preincubated with staurosporine or chelerythrine no currents were induced. Establishing the whole-cell configuration with ATP and with myristoylated PKC substrate in addition, no voltage-dependent currents were activated. We conclude that ATP hydrolysis leads to activation of chloride currents via PKC in the whole-cell configuration. The perforated patch configuration, with the intracellular compartment intact, no currents were induced under otherwise identical experimental conditions. Inhibition of phosphatase by calyculin (10 nm) in the perforated-patch configuration did not change membrane conductance. In the intact cell, chloride conductance is possibly inhibited by a cytosolic factor which is washed out when the whole-cell configuration is established.

De novo acquisition of neuronal polarity in retinoic acid-induced embryonal carcinoma cells.

The mouse embryonal carcinoma cell line PCC7-Mz1 represents an advantageous model to study acquisition of polarity by neurons. During the first two days after differentiation is induced by the addition of retinoic acid, the neuronal derivatives develop extensions which for at least four more days do not differ from each other in growth characteristics, morphology, and marker expression. Beginning around differentiation day 6 and following the relocation of the nucleus from a central to a polar position in the cell soma, the morphology and marker expression changes dramatically: expression of MAP2 diminishes and eventually disappears in the thinner neurite (future axon), which originates at the nucleated pole, but remains strong in the branched, broad based neurite(s). The opposite changes in expression are observed for synaptophysin, together with a clustering of the vesicle protein in varicosity-like areas. Complete segregation of expression of the two markers is achieved around day 12, shortly followed by dendrite-specific location of MAP2 mRNA and the ability to generate and conduct action potentials. Our studies add several aspects to the process of neuronal polarity acquisition, as it was previously studied in primary cultures of embryonic neurons: (i) we monitored neuronal differentiation from the birth of neurons, rather than from later and less defined maturation stages, (ii) cell nucleus relocation may be associated with the induction of neuronal polarity, and (iii) functional competence of neurons is closely associated with previous acquisition of polarity. Acquisition of polarity by PCC7-Mz1 neuronal derivatives probably refers to de novo acquisition rather than to reestablishment of polarity.

The effects of copper ions on glutamate receptors in cultured rat cortical neurons.

Copper plays an important role in the function of many physiological processes and can affect different neurotransmitter systems. In this study, we used the patch-clamp technique to investigate the effect of copper ions on glutamate receptors in cultured rat cortical neurons. Cu2+ inhibited (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors with an IC50 of 4.3 +/- 0.6 microM (with 100 microM kainate, holding potential -60 mV). The concentration-response could be best described by a two-site binding model. Moreover, copper reduced the efficacy of kainate at the AMPA receptor: in the presence of 30 microM Cu2+, the EC50 of kainate was shifted from 100.3 +/- 2.0 microM to 329.9 +/- 31.4 microM. The block by copper ions was not use-dependent. Complete recovery only occurred after the application of a high agonist concentration, or in the presence of the antioxidant dithiotreitol (DTT). A high concentration of histidine, a physiological ligand for Cu2+, did not augment the recovery. The kinetics of block were compared to those induced by 2,3-dihydro-6-nitro-7-sulfamoyl-benz(F)quinoxaline (NBQX), a well-described competitive antagonist of AMPA receptors. The onset, as well as the offset of block by NBQX could be well approximated by single exponential functions with time constants of 0.28 +/- 0.02 and 0.87 +/- 0.09 s, respectively. Within seconds of wash-out of the antagonist, the response to kainate completely recovered. The kinetics of copper block were more complex: the block developed more slowly, and the onset, as well as the offset could be described by two exponential functions with quite different time constants (tau(on1), 0.8 +/- 0.13 s; tau(on2), 8.32 +/- 1.13 s; tau(off1), 0.17 +/- 0.01 s; tau(off2), 69 +/- 36.3 s). In addition to the described effects, Cu2+ also blocked currents induced by the application of N-methyl-D-aspartate (IC50: 15.0 +/- 2.6 microM with 50 microM NMDA). Based on these findings, a modulatory role of copper ions on the neurotransmission by excitatory amino acids is discussed.

Interactions of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine, with (S)- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured rat cortical neurons: electrophysiological evidence for at least two different binding sites for non-competitive antagonists.

The effects of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine, on (S)- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors in primary cultured rat cortical neurons were investigated using the patch-clamp technique. Evans Blue and GYKI 52466 reduced the currents induced by the application of 100 microM kainate with IC50 values of 10.6 +/- 1.4 microM and 12.1 +/- 0.4 microM, respectively. In contrast to the similar potencies of the two compounds, their kinetics of block were quite different with those of Evans Blue being more complex. The on-, as well as the off-reaction of the block by GYKI 52466 could be described by single exponential functions, whereas two different time-constants for binding and one time-constant for the unbinding of Evans Blue were found. The block of AMPA receptors by Evans Blue was not completely reversible under the experimental conditions applied in this study. GYKI 52466 was not able to augment the recovery after inhibiting AMPA receptors with Evans Blue. Moreover, preapplication of a high concentration of GYKI 52466 did not prevent the inhibition of AMPA receptors by Evans Blue. We therefore conclude that GYKI 52466 and Evans Blue bind to two different sites at AMPA receptors in primary cultured cortical neurons.

Activation of Cl- currents in cultured rat retinal pigment epithelial cells by intracellular applications of inositol-1,4,5-triphosphate: differences between rats with retinal dystrophy (RCS) and normal rats.

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl--currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 microM inositol-1, 4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl- currents, blockable by 1 mm 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). IP3 activated Cl- currents in the presence of a high concentration of the calcium chelator BAPTA (10 mM) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10(-5)M Ca2+ via the patch pipette also led to a transient activation of Cl- currents. When the cells were preincubated in a bath solution containing thapsigargin (1 microM) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca2+ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl--current activation induced by IP3 which could be reduced by nifedipine (1 microM). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3.

Investigations on the mechanism of action of the antiproliferant and ion channel antagonist flufenamic acid.

The compound flufenamic acid has been previously described as an inhibitor of chloride- and non-selective cation channels. Moreover, this compound showed antiproliferative effects in the mouse fibroblast cell line LM(TK-). In this study, we investigated the effects of this compound on cell proliferation and membrane currents induced by mitogens (such as fetal calf serum, FCS) or platelet-derived growth factor (PDGF) in LM(TK-) cells. After a brief application of FCS or PDGF (5-15 s), the electrical response of the cells was biphasic: First, a transient potassium conductance was activated, which appeared 8.3 +/- 0.7 s after the onset of stimulation and lasted for 30.1 +/- 2.9 s. The corresponding single channel currents in cell-attached patches had an amplitude of 3-4 pA (at a holding potential of +60 mV). The second effect of serum or PDGF was the occurrence of a cation conductance for monovalent ions (sodium, potassium and cesium) and calcium. In contrast to the potassium current, this conductance activated later (11.8 +/- 1.6 s after onset of fetal calf serum stimulation) and remained activated for minutes. Flufenamic acid inhibited the proliferation of LM(TK-) cells reversibly and in a concentration-dependent manner. This effect can be correlated with the inhibitory effects of flufenamic acid on mitogen-induced membrane currents: The compound inhibited the non-selective cation current with an IC50 of 38 microM, whereas 135 microM were necessary for halfmaximal inhibition of the potassium current; this is very close to the concentration for halfmaximal inhibition of cell proliferation (120 microM). Hence, on the grounds of this comparison the blockade of the non-selective cation current appears to be of only minor importance for the blockade of cell proliferation.

Potassium currents in cultured cells of the rat retinal pigment epithelium.

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than -30 mV, a time-dependent outward current was activated. Inhibition by Ba2+ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba2+ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than -70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.

Extracellular matrix proteins as substrate modulate the pattern of calcium channel expression in cultured rat retinal pigment epithelial cells.

We investigated the effect of different culture substrates on the expression of membrane conductances for calcium in cultured rat retinal pigment epithelial (RPE) cells using the perforated patch technique and barium as charge carrier. In younger cultures (up to 12 days old) the RPE cells expressed L-type calcium channels, in older cultures (more than 12 days old) LVA-type channels. The LVA-type channels have been characterized as a tetrodotoxin sensitive Ca2+ channels. Coating the culture substrate with laminin, shifted the culture age for expression of LVA-type channels to 7 days. When collagen type 4 was used as substrate LVA-type channels and L-type channels were expressed simultaneously in 7 days old cultures. We concluded that proteins of the extracellular matrix which are known to enhance cell differentiation in culture, enhance the expression of LVA-type channels in RPE cells.

Ca(2+)-conductances in cultured rat retinal pigment epithelial cells.

Membrane conductances for Ca2+ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamil- and diltiazem-sensitive Ba2+ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than -30 mV and blocked by nifedipine (1 microM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 microM) insensitive current was activated by depolarization to potentials more positive than -55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 microM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba2+ currents indicating the presence of L-type Ca2+ channels. In older primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels.

Cultured retinal pigment epithelial cells from RCS rats express an increased calcium conductance compared with cells from non-dystrophic rats.

The Royal College of Surgeon (RCS) rats suffer from a retinal dystrophy that is caused by a malfunction of the retinal pigment epithelium (RPE). We compared the membrane currents of cultured RPE cells from non-dystrophic and RCS rats by using the whole-cell configuration of the patch-clamp technique. Cultured RPE cells from RCS rats showed voltage-dependent, barium- and 4-aminopyridine-sensitive outward currents, which had characteristics of the delayed-rectifier and voltage-dependent, barium- and 4-aminopyridine-sensitive inward currents, which had characteristics of the inward rectifier. Differences between RPE cells from RCS rats and normal rats were as follows. (a) Cultured RCS rat RPE cells showed a resting potential and an activation threshold for the voltage-dependent outward current significantly more positive than that found in cells from non-dystrophic rats. (b) In the presence of 10 mM barium, the voltage-dependent outward current was reduced in both types of cells; in cells from RCS rats, an additional voltage-dependent inward current was observed. (c) This additional inward current had characteristics of L-type calcium channels and was reduced by verapamil (30 microM) and diltiazem (30 microM). In summary, we conclude that the membrane conductances of RPE cells from normal and RCS rats are dominated by potassium conductances. In contrast to cells from non-dystrophic rats, cells of RCS rats expressed an increased membrane conductance for calcium.

Voltage-dependent potassium currents in cultured human retinal pigment epithelial cells.

Membrane currents in primary cultures of human retinal pigment epithelial cells were studied using the whole-cell configuration of the patch-clamp technique. Two types of voltage-dependent whole-cell currents were observed. First, a time- and voltage-dependent outward current, which was activated by depolarizing the cell to potentials more positive than -30mV, was sensitive to Ba2+ (10mM), 4-aminopyridine (10mM) and TEA+ (30mM). Tail-current analysis indicated that the current was mainly carried by K(+)-ions. Second, hyperpolarization of the cell to potentials more negative than -70mV led to a time- and voltage-dependent inward current which was blocked by Ba2+ (10mM) and 4-aminopyridine (10mM), but not by TEA+ (30mM). In summary, human retinal pigment epithelial cells in primary culture express currents which indicate the presence of a delayed rectifier K(+)-channel and an inward rectifier K(+)-channel.

Effects of receptor-selective neurokinin agonists and a neurokinin antagonist on the electrical activity of spinal cord neurones in culture.

1. Rat spinal cord neurones grown in tissue culture were used to examine the electrophysiological effects of the neurokin in (NK)-selective agonists (pGlu6, Pro9) substance P(6-11) (septide; NK1, 10(-6)M) and (pGlu5, MePhe8, MeGly9)SP(1-7) (DiMe-C7; NK3, 10(-6)M). In addition, the effect of the neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP (10(-5)M) on the neurokinin-evoked responses was investigated. 2. Neurokinin-evoked responses consisted of an increase in neuronal activity with or without long-lasting (mean: 50s) depolarizations of the membrane potential of up to 25mV. The latter also occurred in the presence of tetrodotoxin (10(-7)M) (direct response). 3. In a number of spinal cord neurones (n = 17) only septide induced a membrane depolarization while DiMe-C7 elicited no response. On the other hand, in 2 neurones a response was exclusively evoked by DiMe-C7. 4. The neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP had no effect of its own but blocked the septide- and DiMe-C7-induced depolarizations. It had no effect on the glutamate (10(-5)M)-evoked depolarization. 5. It is concluded that by the use of neurokinin receptor-selective agonists, subpopulations of spinal cord neurones in primary dissociated cell culture can be differentiated which express the NK1 or the NK3 receptor. Cells expressing only the NK1 receptor outnumber those expressing only the NK3 receptor subtype. Both receptors can be blocked by the neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP.