Antisecretory factor peptide derivatives specifically inhibit [3H]-gamma-amino-butyric acid/36Cl- out-->in permeation across the isolated rabbit Deiters' neuronal membrane.

AIMS: Antisecretory factor (AF) is a 41-kDa protein, its main function being the regulation of intestinal ion/water transport, but it also inhibits chloride and gamma-amino-butyric acid transport across nerve cell membranes. The present experiments were designed to evaluate whether the same AF peptide sequence mediates the permeability effects seen at the nerve cell membrane and in the rat small intestine. METHODS: Four peptides were prepared by the solid phase technique with sequences derived from positions 1-51 of the full-length antisecretory factor AF and tested on nerve cell membranes isolated from rabbit Dieter cells. RESULTS: AF peptides containing the active 36-51 peptide exerted a blocking effect of the out-->in permeation of 36Cl- as well as of [3H]-gamma-amino-butyric acid. The minimal dose causing inhibition, however, varied between 10(-11) m (AF10) and 10(-7) m (AF13). The most potent peptides have been shown previously to be active in inhibiting experimental diarrhoea in vivo in small intestinal ligated loops in rats. The non-active sequence AF23-32 did not inhibit any of the two permeation markers in vitro, a result which supports the lack of activity found also in vivo. CONCLUSION: The results suggest that AF, or AF derivatives, counteract intestinal hypersecretion by blocking anion permeation across large anionic pores. Such a blocking effect could also influence the generation of action potentials in enteric nerve cells controlling the intestinal water and ion transport system.

A simple, inexpensive, and precise spectrophotometric method for evaluating the concentration of ascorbic acid in CSF samples: data from different neurological pathologies.

A method is presented for evaluating the concentration of ascorbic acid in 10 microliters samples of cerebrospinal fluid (CSF) with just ultraviolet (UV) scanning in the 220-300 nm range. The method assumes that the two main UV absorbing CSF components are proteins (with a peak at 280 nm) and ascorbic acid (with a peak at 264 nm). On this basis, the absorbances at those wavelengths are the starting points for a calculation that evaluates the sheer contribution of ascorbic acid to the absorbance at 264 nm. The rapidity of the UV analysis (just a few min), together with the precaution of keeping the samples under argon, along the short preparatory procedure, is especially suitable in the analysis of an air labile substance, such as ascorbic acid. The results of the UV procedure have been checked with parallel HPLC determinations for 19 test CSF samples and the results have shown an excellent correspondence. Finally, data are presented about the evaluation of ascorbic acid concentrations in CSF samples from various neurological pathologies in comparison with normal cases. These data show the interesting result of a significant reduction in the average ascorbic acid CSF level in patients suffering from Alzheimer's disease. However, a decrease in that parameter is found also for a few other pathologies.

GABA and chloride permeate via the same channels across single plasma membranes microdissected from rabbit Deiters' vestibular neurones.

The permeation of labelled gamma-aminobutyric acid (GABA) across single microdissected Deiters' membranes has been studied in a microchamber system. The GABA permeation is via pores which are blocked by 4,4'-diisothiocyanato stilbene-2-2'disulphonic acid (DIDS). As this substance blocks as well chloride permeation across these membranes we tested whether GABA and chloride permeate across the same pores. Membrane pre-treatment with different doses of corticotropin releasing factor (CRF), a membrane permeant cyclic AMP analogue and phalloidin parallelly block the permeation of the two substances. Thus, it is most probable that GABA and chloride pass across the same pores. These pores may be swelling activated ones, opened by the mechanical stress on the membranes in the microchamber system. The passage of GABA across these pores may be of physiological importance in the termination of GABA inhibitory action on the vestibular Deiters' neurones.

Holger Hydén's technique of preparation of single Deiters' neurons and study of permeability characteristics of their plasma membranes.

The protocols described here refer to Hydén's technique of isolation and microdissection of vestibular Deiters' neurons from adult mammals. The isolation of Deiters' cells from bovine is described and an example is given of the immunocytochemical visualization of their GABA(A) receptors by monoclonal antibodies against the beta(2/3) subunit. In addition, the protocol of the method for isolation of Deiters' cells from adult rabbit brain stem, the preparation of their plasma membranes and the study of their permeability characteristics is presented. Also in this case, examples of its application to the determination of chloride permeability and its modulation by GABA are given.

GABA uptake by rabbit restiform body homogenates.

Restiform body (inferior cerebellar peduncle) preparations were obtained from rabbit brain stem slices and homogenized. When challenged with labelled GABA, these homogenates took it up briskly. We have characterized pharmacologically this uptake which resulted almost equally neuronal and glial. The neuronal component of the GABA uptake might be due to the adjacent cochlear nuclei coming along in the preparations, whereas the glial component probably belongs to the restiform body proper. Another possibility is that actually both components are due to the myelinated fibers and glia which make up the restiform body.

Modulation by acute stress of chloride permeation across microdissected vestibular neurons membranes: different results in two rabbit strains and CRF involvement.

Free hand isolation of adult rabbit vestibular Deiters' neurons and dissection of their single membranes allows the study of their ionic permeability characteristics in a microchambers device. In the case of hare-like rabbits, the dissection of such membranes presents evidence of a high basal permeation of labelled chloride, possibly related to mechanical disturbance of the plasma membrane-related cytoskeleton and activation of chloride channels. This did not apply to the laboratory strain of white New Zealand rabbits. However, membranes from hare-like rabbits which were stressed by being rotated on a platform before the experiment, behaved like those from the New Zealand strain. Vice versa, habituation to handling day after day of New Zealand rabbits resulted in a chloride permeation equal to that of unstressed hare-like rabbits. We propose that the stressful conditions result in the release of neurochemical messages to the vestibular Deiters' cells which influence their electrophysiological behavior. The corticotropin releasing factor (CRF), a stress-related peptide present in the climbing fibers, actually blocks the basal chloride permeation across the Deiters' membranes and this effect is partially reversed by its receptor antagonist, alpha-helical CRF [9-41].

Unraveling of important neurobiological mechanisms by the use of pure, fully differentiated neurons obtained from adult animals.

An important, and often overlooked, problem in the neurochemical approach to neurobiological problems is that analysis of tissue involves almost always a heterogeneous population of cells (neurons, glia and other types of tissue cells). The use of cell cultures has obvious limitations such as that they derive from embryonic or immediately postnatal animals; in addition, the cell culture conditions most certainly are quite different from the real tissue environment for the nerve cells. We underline here an alternative strategy, which is not new, but which, in our view, has already given formidable contributions to neurobiological studies and still is giving results of great importance. This is the technique proposed and used since the late fifties and early sixties by the senior author (H. Hydén). The method involves the isolation of the big vestibular neurons from the adult rabbit vestibular nucleus. The neurons, fully differentiated and performing a precisely defined function, are obtained rapidly and completely free from surrounding glial cells. The separate microbiochemical study of these cells and their surrounding glia has yielded already in 1962, the information that modifications in gene expression are associated with plastic modifications of the function of the relevant neurons, which take place in the behavioral event of learning. Another important concept was formulated in the same time period following determination of the activities of energy metabolism related enzymes separately in vestibular neurons and their glia under vestibular stimulation. This is the concept that, under increased functional activity glia increases its anaerobic metabolism and passes then on the resulting metabolites to the neurons for aerobic metabolism. Both these concepts (RNA and memory; metabolic cooperation between glia and neurons) are nowadays widely accepted. In addition, this approach with pure big nerve cells has allowed in recent years the discovery of a novel mechanism for chloride extrusion in these cells. This mechanism utilizes structures similar to GABA activated chloride channels in cyclic modifications resulting in the final extrusion of chloride ions. The energy for the process is provided by a protein phosphorylation step. Future approaches are warranted such as the possibility of recognizing by RT-PCR specific neuronal mRNAs and their modification in expression in relation to function and plastic modifications (learning). Another possible interesting application appears to be the recognition of the mRNAs for GABA(A) receptor subunits expressed here in these neurons in relation to the physiological and pharmacological characteristics of these native neuronal GABA(A) receptors.

Intracellular GABA-activated in-->out permeation of chloride across the Deiters' neuron membrane: modulation by phosphorylating activities.

The modulation of intracellular GABA activated 36Cl- in-->out permeation across single Deiters' neuron membranes has been studied in a microchamber system. Addition of Mg2+/ATP on the membrane cytoplasmic side reduces strongly the GABA effect as does ATP alone. However, the greatest inhibition of the GABA effect is given by the addition of Mg2+ to the intracellular side buffer: a complete block of the stimulation by GABA of 36Cl- in-->out permeation. This is interpreted as due to the presence in this case of a constant concentration of exogenous Mg2+ acting together with endogenous ATP in the small cytoplasmic layer on the membrane inner side. The addition of ADP to Mg2+/ATP increases the inhibitory effect of the latter. This is presumably due to an extra increase of ATP, locally under the membrane, due to phosphorylation of ADP by endogenous phosphocreatine. Overall, the data confirm that phosphorylating conditions impair the intracellular GABA action on 36Cl- in-->out permeation.

An electrogenic ionic pump derived from an ionotropic receptor: assessment of a candidate.

1. Data obtained studying permeability characteristics of single Deiters' membranes in a microchamber system show that intracellular GABA can activate chloride in-->out passage with a GABAA pharmacology. 2. The overall data suggest the presence of a chloride extrusion pump in these neurons based on intracellular GABA activated chloride channels. 3. This conclusion takes up a previous theoretical suggestion that ionic channels could work as ionic pumps provided an energy input modifies the energy profile along the permeation path. 4. According to our quantitative evaluation, this pumping mechanism works with a low yield and along a cycle with a strongly asymmetric behavior, being far from equilibrium due to powerful "leakage" pathways for chloride in these neurons.

Chloride permeation across the Deiters' neuron plasma membrane: activation by GABA on the membrane cytoplasmic side.

Single plasma membranes were microdissected from Deiters' neurons freshly obtained from the lateral vestibular nucleus of the rabbit and their chloride permeability was studied in a microchamber system. The basal in-->out 36Cl- permeation initially found was brought to zero by Zn2+, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and iodide. GABA on the membrane cytoplasmic side resulted in a measurable in-->out 36Cl- passage, which was blocked by the GABA(A) antagonists bicuculline and picrotoxin. This effect peaked at 1 microM GABA on the inner side of the membrane. At higher GABA concentrations, a strong desensitization of the effect was found. Stimulation of Cl- permeability by GABA on the extracellular side of the membrane peaked at much higher GABA concentrations, 10-100 microM. This excludes an effect due to passage of the neurotransmitter from the inner to the outer compartment in our microchamber device. Moreover, this possibility is also dismissed by the fact that 1 microM GABA on the membrane outside did not evoke any 36Cl- in-->out permeation. In addition, pentobarbitone by itself could also stimulate 36Cl- in-->out permeation when added on the cytoplasmic side of Deiters' membrane. On these bases and in agreement with our previous reports, we propose that structures behaving pharmacologically as GABA(A) receptors respond to low levels of GABA on the cytoplasmic side of these neurons' membranes. We suggest that these structures are devices that, at the expense of ATP consumed in their phosphorylation, extrude Cl- after postsynaptic GABA uptake into the Deiters' neuron.

Stimulation of chloride in-->out permeation across the Deiters' neuron membrane by pentobarbital on the cytoplasmic side: additional evidence of GABA(A) receptors acting as chloride extrusion pumps.

Pentobarbital stimulates 36Cl- permeation across single Deiters' membranes in a microchamber system, acting on classical, extracellularly facing, GABA(A) receptors. However, when applied on the membrane cytoplasmic side it activates per se labeled chloride in-->out permeation. No effect was found on chloride out-->in permeation. Similarly, at lower concentrations it facilitates the increase of 36Cl- in-->out permeation by application of GABA on the membrane inside, again via asymmetric chloride channels allowing in-->out but not out-->in passage. These data confirm that on the Deiters' membrane cytoplasmic side there are structures behaving pharmacologically as GABA(A) receptors whose function is that of a Cl- extrusion pump. This mechanism involves a cycle of activation-phosphorylation/desensitization-reactivation of the receptor complexes.

Modulation by calcium of Deiters' neuron GABAA receptors in the rabbit.

The function of classical GABAA receptors of the rabbit Deiters' neurons has been studied at the single membrane level by a biochemical micromethod involving the study of labelled chloride permeation. In particular, labelled chloride permeation across microdissected fresh single membranes was studied in a microchamber system. The stimulation of 36Cl- out-->in permeation by "extracellular" GABA was determined under different conditions in the respect of Ca++. When the conditions were such that "intracellular" Ca++ was 0.02 microM there appeared to be an optimal effect by GABA on chloride passage. Conditions presumably resulting in an increase of [Ca++]i beyond the level reported above led to a decreased GABA effect, especially at the highest GABA concentrations used (> or = 10(-4)M). However, complete removal of Ca++ by a high (12 mM) intracellular EGTA concentration erased completely the GABA effect. These results indicate that in these neurons an optimal GABAA receptor function requires [Ca++]i levels well below micromolar. The high [EGTA]i effect seems to imply that too low a [Ca++]i is also harmful to the proper function of these GABAA receptors. However, an alternative explanation is possible.

Nitric oxide and GABAA receptor function in the rat cerebral cortex and cerebellar granule cells.

The aim of the present work was to investigate the mechanism by which the diffusible factor nitric oxide regulates GABAA receptor function in the brain. The effect of nitric oxide on GABAA receptor function has been studied in two different neuronal preparations: rat cerebral cortex microsacs and rat cerebellum granule cells in culture. In the first case, GABA-stimulated 36Cl-accumulation was studied as an index of GABAA receptor function. The maximal rate of GABA-stimulated 36Cl- accumulation (Vmax) was reduced by treatment of microsacs with nitric oxide chemical donors such as sodium nitroprusside (-26%) and S-nitroso-acetyl-penicillamine (-11%). The greater effect of the former agent is due to an additional interference by its breakdown products. The biochemical precursor L-arginine (1 mM) produced the same Vmax decrease as S-nitroso-acetyl-penicillamine. This effect was reversed by a nitric oxide synthase blocker and appears truly nitric oxide mediated. The action of nitric oxide in this system does not seem to imply cyclic GMP formation. GABAA receptor function was studied by whole-cell patch-clamp in rat cerebellum granule cells in culture. In this case, L-arginine (100 microM) profoundly reduced the Cl- current elicited by 10 microM GABA and its effect subsided following washing out. The effect of L-arginine was observed almost exclusively on the rapidly desensitizing component of the GABA-activated current. The action of L-arginine was blocked by a protein kinase G inhibitor and mimicked by its activators. Thus, it appears that this effect in these cells involves nitric oxide formation, cyclic GMP accumulation and protein kinase G-catalysed phosphorylation of GABAA receptor.

Screening of the macromolecular component of CSF from various pathologies for its interference with GABAA receptor function.

Cerebrospinal fluid (CSF) samples from patients undergoing lumbar puncture for suspected neurological diseases were fractioned into macromolecular (MW > 3000 Daltons) and small molecule components (MW < 3000 Daltons). The macromolecular component was tested for its interference with GABA stimulation of 36Cl- accumulation in rat cerebral cortex microsacs. For many pathologies, no interference was apparent; an exception was two patients with Parkinson's disease in whom there were clear signs of stimulated GABA activity. This last result seems worthy of further investigation.

Modulation by nitric oxide of rat brain GABAA receptors.

The effect of nitric oxide (NO) on the function of GABAA receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABAA receptors were studied by preparing microsacs and evaluating 36Cl- accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor precursor arginine there was a 15-25% reduction in the Vmax for GABA-stimulated 36Cl- accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor N omega-nitro-L-arginine. GABAA receptor mediated Cl- currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and L-arginine reduced the Cl- current elicited by 10 microM GABA. The L-arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABAA receptor function in cerebellar granule cells.

The increase in Cl- permeation across the Deiters' neuron membrane by GABA on its cytoplasmic side is abolished by protein kinase C (PKC) activators.

1. Cl- ion outward permeation across microdissected Deiters' neuron plasma membranes is augmented by GABA on the membrane cytoplasmic side. When these neurons are preincubated with a PKC activator, phorbol-12,13-dibutyrate (PdBu), there is a complex pattern of effects on basal and GABA-activated 36Cl- in-->out permeation. A distinct fact is an increase in basal Cl- passage and a disappearance of the 10(-6) M GABA effect at [PdBu] = 0.1 microM. 2. Likewise, 0.1 microM oleylacetylglycerol (OAG) treatment erases the effect completely, further supporting a role for PKC in modulating GABA-stimulated Cl- in-->out permeation. 3. The inactive ester, phorbol-12,13-didecanoate (Pdd), at 0.1 microM, does not affect GABA stimulation of Cl- passage. 4. High concentration (15-20 microM) of OAG and PdBu block the "intracellular" GABA efefct. However, the 20 microM PdBu effect is reversed by 30 microM H7. 5. These results indicate a role of endogenous PKC in Cl- extrusion by GABAA receptors on the cytoplasmic side of the Deiters' neuron membrane.

Modulation by intracellular Ca++ of GABA activated Cl- extrusion from Deiters' neuron.

The increase in 36Cl- in --> out permeation across plasma membranes from the Deiters' neurons by GABA on the cytoplasmic side is modulated by Ca++. The GABA stimulation was maximal with Ca++ on the intracellular side at the physiologically likely concentration of 3 x 10(-8) M. A lower effect was found in the 10(-7) - 10(-5) M [Ca++]i range with a total disappearance of it at [Ca++] of .2 mM. This Cl- extrusion mechanism is likely to be involved in the establishment of a Cl- out --> in electrochemical gradient. Modulation of it by intracellular Ca++ transients may be of physiological importance.

Evaluation of the mechanisms by which gamma-amino-butyric acid in association with phosphatidylserine exerts an antiepileptic effect in the rat.

The i.p. injection in rats of GABA (740 mg/Kg) after sonication with an equal amount of phosphatidylserine (PS) has an antiepileptic effect. The injection of plain GABA has no such an effect. Blood, brain and synaptosomal accumulation of exogenous labeled GABA under the two circumstances are evaluated. In the case of GABA/PS injection there is a higher passage of the exogenous labeled neurotransmitter into the blood and brain nerve endings (synaptosomes). A higher synaptosomal accumulation of the exogenous labeled neurotransmitter is found even when GABA and PS are injected separately. Since these accumulation increases occur at a time when there is the antiepileptic effect, they seem relevant to it. Our interpretation of the chain of the events resulting in the antiepileptic action is that the phospholipid facilitates from the beginning the first passage of the exogenous neurotransmitter form the peritoneum to the blood. Then a higher passage to the brain tissue and eventually to the GABA-ergic nerve endings ensues. The brisker accumulation of the exogenous neurotransmitter in the nerve endings could be at the basis of a more efficient GABA-ergic inhibitory control in the brain.

Further evidence for the presence of gamma-aminobutyric acidA (GABAA) receptors on the cytoplasmic side of Deiters' membrane.

1. The permeation of labeled Cl- across single nerve membranes microdissected from rabbit Deiters' neurons was studied in a microchamber system. The in----out permeation of the ions was evaluated under control conditions and in the presence of either 10(-6) M GABA or 10(-6) M GABA plus 10(-5) M bicuculline methiodide (BMI) on the membrane cytoplasmic side. 2. In 32 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied with the addition of GABA. Within each experiment all the membranes were obtained from the same animal. 3. In an additional 10 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied in the presence of GABA plus bicuculline methiodide on the membrane cytoplasmic side. 4. The data show that 10(-6) M GABA on the Deiters' membrane cytoplasmic side stimulates Cl- permeation in----out by 42% (P = 0.0000001). When 10(-5) M BMI was present together with GABA, no stimulation of Cl- in----out permeation occurred.

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection.

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5-30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that compartment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.