Application of a ring cavity surface emitting quantum cascade laser (RCSE-QCL) on the measurement of H2S in a CH4 matrix for process analytics.

The present work reports on the first application of a ring-cavity-surface-emitting quantum-cascade laser (RCSE-QCL) for sensitive gas measurements. RCSE-QCLs are promising candidates for optical gas-sensing due to their single-mode, mode-hop-free and narrow-band emission characteristics along with their broad spectral coverage. The time resolved down-chirp of the RCSE-QCL in the 1227-1236 cm-1 (8.15-8.09 µm) spectral range was investigated using a step-scan FT-IR spectrometer (Bruker Vertex 80v) with 2 ns time and 0.1 cm-1 spectral resolution. The pulse repetition rate was set between 20 and 200 kHz and the laser device was cooled to 15-17°C. Employing 300 ns pulses a spectrum of ~1.5 cm-1 could be recorded. Under these laser operation conditions and a gas pressure of 1000 mbar a limit of detection (3σ) of 1.5 ppmv for hydrogen sulfide (H2S) in nitrogen was achieved using a 100 m Herriott cell and a thermoelectric cooled MCT detector for absorption measurements. Using 3 µs long pulses enabled to further extend the spectral bandwidth to 8.5 cm-1. Based on this increased spectral coverage and employing reduced pressure conditions (50 mbar) multiple peaks of the target analyte H2S as well as methane (CH4) could be examined within one single pulse.

Analysis of ß-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives.

Valerenic acid (VA)-a ß2/3-selective GABA type A (GABAA) receptor modulator-displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed ß-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1ß1γ2S, α1ß2γ2S, or α1ß3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. ß2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1ß3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1ß2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1ß3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1ß2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA's methylamide (VA-MA) enhancing IGABA through ß3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1ß3γ2S: VA-TET: EC50 = 6.0 ± 1.0 µM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the ß2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg/kg), whereas impairment of locomotion was observed at doses ≥10 mg/kg.

Selective up-regulation of the vasodilator peptide apelin after dorsal root but not after spinal nerve injury.

Regeneration of sensory neurons is limited in response to lesion of their central axons when compared to lesion of their peripheral axons. To identify transcriptional changes underlying this differential regenerative response between dorsal root and spinal nerve axons, the L5 dorsal root ganglion (DRG) of adult rats was investigated three days after crushing the respective nerve branches by performing high density genome oligonucleotide microarrays. RT-PCR, in situ hybridization and immunohistochemistry confirmed the up-regulation of the vasodilator peptide apelin in non-neuronal cells of the DRG after dorsal root but not after spinal nerve lesion. Induction of apelin mRNA and peptide is accompanied by increased vascular permeability around neuronal cell bodies as demonstrated by Evans-blue albumin (EBA) leakage. Enhanced vasodilation and increased vascular permeability cause intraganglionic edema, which may play a key role in the reduced axonal regeneration rate after dorsal root injury.

Valerenic acid derivatives as novel subunit-selective GABAA receptor ligands - in vitro and in vivo characterization.

BACKGROUND AND PURPOSE: Subunit-specific modulators of gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a beta(2/3) subunit-specific modulator of GABA(A) receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABA(A) receptor modulators and to gain insight into the structure-activity relation of this molecule. EXPERIMENTAL APPROACH: The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABA(A) receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (I(GABA)) through GABA(A) receptors (EC(50)) and efficacies (maximal stimulation of I(GABA)) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test. KEY RESULTS: Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater I(GABA) enhancement than VA) and highest potency (EC(50)= 13.7 +/- 2.3 microM) on alpha(1)beta(3) receptors. Higher efficacy and potency of VA-A were also observed on alpha(1)beta(2)gamma(2s) and alpha(3)beta(3)gamma(2s) receptors. Anxiolytic effects were most pronounced for VA-A. CONCLUSIONS AND IMPLICATIONS: Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting beta(3) subunit containing GABA(A) receptors for development of anxiolytics.

Evidence for Somatostatin receptor 2 in thyroid tissue.

Somatostatin receptor scintigraphy has found considerable interest for imaging thyroid tumours. Recently, also therapeutic application of Somatostatin analogues labelled with beta-emitting radionuclides has been suggested as treatment option for thyroid tumours with absent radioiodine uptake. Most of the radiolabelled analogues available show a predominant affinity for Somatostatin receptor subtype 2. This study reports on the in vitro characterisation of Somatostatin receptor subtype mRNAs in thyroid tumours and normal thyroid tissue by means of RT-PCR. Surgical samples of 21 patients were collected, and mRNA of 16 tumour and 17 control specimen was isolated. mRNA expression for Somatostatin, SSTR subtype 1-5, thyroid markers (NIS, TSH, Tg, TPO) and control markers (GAPDH, beta-actin) was determined. PCR results were correlated with immunohistochemistry staining using SSTR2 receptor specific antibodies. 94% of all samples expressed Somatostatin receptor mRNA with predominant expression of subtype 2, less predominant of subtype 5 and subtype 3. Somatostatin receptor subtype 2 mRNA expression correlated well with immunohistochemical staining pattern in 13/16 samples, SSTR2 immunohistochemistry was positive in 87% of the samples. Our results show that Somatostatin receptor 2 is predominantly expressed on thyroid tissue and is a valid target for treatment of thyroid tumours. Octreotide derivatives currently used in Nuclear medicine seem to be well suited to target receptors expressed in thyroid tumours.

Y1-receptors regulate the expression of Y2-receptors in distinct mouse forebrain areas.

Y-receptor-knockout mice have become an important tool to elucidate specific physiological roles of individual Y-receptors. However, their phenotypes are not always confirmatory to results obtained by pharmacological investigations in vivo or in vitro. These discrepancies may, at least in part, be due to compensatory changes in the expression of remaining Y-receptor types. To determine whether deletion of individual Y-receptors results in altered mRNA expression and/or binding toward other Y-receptor types, we applied in-situ hybridization and radioligand-binding studies on brain slices of Npy1r-, Npy2r- or Npy5r-knockout mice. Significant changes were seen in Y1-receptor-deficient mice. Thus, Y2-receptor mRNA and (125)I-peptide YY(3-36) binding in the hippocampus proper were increased by up to 55% and 89%, respectively. Similar increases in (125)I-peptide YY(3-36) binding were observed in the caudo-dorsal extension of the lateral septum, an area heavily targeted by hippocampal projections and involved in Y1-receptor-regulated anxiety. Increased (125)I-peptide YY(3-36) binding and Y2-receptor mRNA levels were also observed in the medial amygdaloid nucleus. In contrast, (125)I-peptide YY(3-36) binding was reduced in the central amygdaloid nucleus. Y2-receptor mRNA in the intermediate part of the lateral septum was reduced by 42%. Only minimal changes were observed in Y2- or Y5-receptor-deficient mice. Our results demonstrate that compensatory changes in the expression of Y2-receptors occur in Y1-receptor-deficient mice. These adaptations are likely to contribute to changed physiological function. Thus, alterations in Y2-receptors have to be taken in account upon discussion of Y1-receptor function, especially in emotional aspects like anxiety and aggression, but also alcoholism.

Altered expression of GABA(A) and GABA(B) receptor subunit mRNAs in the hippocampus after kindling and electrically induced status epilepticus.

Epilepsy may result from altered transmission of the principal inhibitory transmitter GABA in the brain. Using in situ hybridization in two animal models of epileptogenesis, we investigated changes in the expression of nine major GABA(A) receptor subunits (alpha1, alpha2, alpha4, alpha5, beta1-beta3, gamma2 and delta) and of the GABA(B) receptor species GABA(B)R1a, GABA(B)R1b and GABA(B)R2 in 1) hippocampal kindling and 2) epilepsy following electrically-induced status epilepticus (SE). Hippocampal kindling triggers a decrease in seizure threshold without producing spontaneous seizures and hippocampal damage, whereas the SE model is characterized by spontaneous seizures and hippocampal damage. Changes in the expression of GABA(A) and GABA(B) receptor mRNAs were observed in both models, and compared with those seen in other models and in human temporal lobe epilepsy. The most prominent changes were a relatively fast (24 h after kindling and electrically-induced SE) and lasting (7 and 30 days after termination of kindling and SE, respectively) reduction of GABA(A) receptor subunit delta mRNA levels (by 43-78%) in dentate granule cells, accompanied by increases in mRNA levels of all three beta-subunits (by 8-79%) and subunit gamma2 (by 11-43%). Levels of the minor subunit alpha4 were increased by up to 60% in dentate granule cells in both animal models, whereas those of subunit alpha5 were decreased 24 h and 30 days after SE, but not after kindling. In cornu ammonis 3 pyramidal cells, downregulation of subunits alpha2, alpha4, alpha5, and beta1-3 was observed in the ventral hippocampus and of alpha2, alpha5, beta3 and gamma2 in its dorsal extension 24 h after SE. Similar but less pronounced changes were seen in sector cornu ammonis 1. Persistent decreases in subunit alpha2, alpha4 and beta2 transcript levels were presumably related to SE-induced cell loss. GABA(B) receptor expression was characterized by increases in GABA(B)R2 mRNA levels at all intervals after kindling and SE. The observed changes suggest substantial and cell specific rearrangement of GABA receptors. Lasting downregulation of subunits delta and alpha5 in granule cells and transient decreases in subunit alpha2 and beta1-3 mRNA levels in cornu ammonis 3 pyramidal cells are suggestive of impaired GABA(A) receptor-mediated inhibition. Persistent upregulation of subunits beta1-3 and gamma2 of the GABA(A) receptor and of GABA(B)R2 mRNA in granule cells, however, may result in activation of compensatory anticonvulsant mechanisms.

Seizure susceptibility and epileptogenesis are decreased in transgenic rats overexpressing neuropeptide Y.

Functional studies in epileptic tissue indicate that neuropeptide Y and some of its peptide analogs potently inhibit seizure activity. We investigated seizure susceptibility in transgenic rats overexpressing the rat neuropeptide Y gene under the control of its natural promoter. Seizures were induced in adult transgenic male rats and their wild-type littermates by i.c.v. injection of 0.3 microg kainic acid or by electrical kindling of the dorsal hippocampus. Transgenic rats showed a significant reduction in the number and duration of electroencephalographic seizures induced by kainate by 30% and 55% respectively (P<0.05 and 0.01). Transgenic rats were also less susceptible to epileptogenesis than wild-type littermates as demonstrated by a 65% increase in the number of electrical stimuli required to induce stage 5 seizures (P<0.01). This phenotype was associated with a strong and specific expression of neuropeptide Y mRNA in area CA1, a brain area involved in the seizure network. We conclude that endogenous neuropeptide Y overexpression in the rat hippocampus is associated with inhibition of seizures and epileptogenesis suggesting that this system may be a valuable target for developing novel antiepileptic treatments.

Changes in the GABA-ergic system induced by trimethyltin application in the rat.

Ingestion of trimethyltin (TMT) produces mental confusion and temporal lobe seizures in humans. In rats, it causes increased seizure susceptibility, hyperactivity, aggression, learning impairment, and neuronal loss especially of hippocampal CA3c pyramidal cells and in the piriform cortex. As some of these symptoms may be due to impaired inhibitory neurotransmission, mRNA levels of the nine major GABA(A) receptor subunits, of GABA(B) receptors 1 and 2, and the 65- and 67-kD glutamate decarboxylase (GAD) variants were investigated by in situ hybridization 2, 5, and 16 days after TMT administration. GAD-65 mRNA levels were enhanced in hippocampal interneurons by up to 46% 5 days after TMT application, suggesting increased activity of respective neurons. In the granule cell layer, only the GABA(A) receptor subunit delta mRNA was altered (decreased by 48%). In the hippocampal sector CA3c and in the piriform cortex, mRNA levels of GABA(A) receptor subunits alpha1, alpha5, beta1, beta2, beta3, gamma2 and of both GABA(B) receptors declined (by 46-72%) after 5-16 days, being consistent with the extensive cell loss. In contrast, subunit alpha2 mRNA levels decreased already after 2 days at an extent exceeding the cell loss in CA3. Subunit alpha4 mRNA levels increased (about two-fold) in surviving CA3 neurons. In sector CA1, mRNA levels of subunits alpha1, alpha5, beta2, beta3, and gamma2 decreased by 35-54% in spite of only a minor (9%) cell loss. The data indicate neurodegeneration related decreases in mRNA levels in sector CA3 and piriform cortex, whereas decreases in sector CA1 may be a consequence of impaired excitatory input to this area.

Distribution of the major gamma-aminobutyric acid(A) receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat.

Within the basal ganglia, gamma-aminobutyric acid (GABA) exerts a fundamental role as neurotransmitter of local circuit and projection neurons. Its fast hyperpolarizing action is mediated through GABA(A) receptors. These ligand-gated chloride channels are assembled from five subunits, which derive from multiple genes. Using immunocytochemistry, we investigated the distribution of 12 major GABA(A) receptor subunits (alpha1-5, beta1-3, gamma1-3, and delta) in the basal ganglia and associated limbic brain areas of the rat. Immunoreactivity for an additional subunit (subunit alpha6) was not observed. The striatum, the nucleus accumbens, and the olfactory tubercle displayed strong, diffuse staining for the subunits alpha2, alpha4, beta3, and delta presumably located on dendrites of the principal medium spiny neurons. Subunit alpha1-, beta2-, and gamma2-immunoreactivities were apparently mostly restricted to interneurons of these areas. In contrast, the globus pallidus, the entopeduncular nucleus, the ventral pallidum, the subthalamic nucleus, and the substantia nigra pars reticulata revealed dense networks of presumable dendrites of resident projection neurons, which were darkly labeled for subunit alpha1-, beta2-, and gamma2-immunoreactivities. The globus pallidus, ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata, all areas receiving innervations from the striatum, displayed strong subunit gamma1-immunoreactivity compared to other brain areas. In the substantia nigra pars compacta and in the ventral tegmental area, numerous presumptive dopaminergic neurons were labeled for subunits alpha3, gamma3, and/or delta. This highly heterogeneous distribution of individual GABA(A) receptor subunits suggests the existence of differently assembled, and presumably also functionally different, GABA(A) receptors within individual nuclei of the basal ganglia and associated limbic brain areas.

Gadolinium as an opener of the outwardly rectifying Cl(-) channel (ORCC). Is there relevance for cystic fibrosis therapy?

There is indirect evidence that the plasmalemma-integrated eukaryotic porin (the voltage-dependent anion-selective channel, VDAC) functions as the outwardly rectifying chloride channel (ORCC). The channel, which is believed to play a role in cell volume regulation, appears to be relevant for cystic fibrosis (CF) therapy, in that it may function as an alternative Cl(-) channel. In the present study we showed first that Gd(3+) altered the voltage dependence of human type-1 porin incorporated into artificial planar lipid bilayers. Next, using a light-scattering approach on transformed normal or CF human B-lymphocytes in hypotonic Ringer solution, we found slightly differing regulatory volume decrease (RVD) curves for the cell lines under study. Addition of 15-60 microM GdCl3 in hypotonic Ringer increased light scattering, pointing to cell swelling beyond normal values. RVD was not observed in those experiments. A corresponding effect was seen in isotonic Ringer containing GdCl3. In either osmotic situation Gd(3+)-induced cell swelling was abolished by monoclonal mouse anti-human type-1 porin antibodies. Agonist and antibody effects were dose dependent. Finally, videocamera-monitored control experiments with adherent HeLa cells verified the direct effect of the agonist on cell swelling in hypo- or isotonic situations and its prevention by the antibodies. We conclude that GdCl3 opens plasmalemma-integrated porin channels, allowing ions to following their gradients, resulting in cell swelling. Since respiratory epithelium expresses porin channels in the apical membrane, the use of gadolinium to activate ORCC may represent a new therapeutic approach in CF.

GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain.

GABA(A) receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABA(A) receptor subunits deriving from 13 different genes (alpha1-alpha6, beta1-beta3, gamma1-gamma3 and delta) in the adult rat brain. Subunit alpha1-, beta1-, beta2-, beta3- and gamma2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit alpha2-, alpha3-, alpha4-, alpha5-, alpha6-, gamma1- and delta-immunoreactivities were more confined to certain brain areas. Thus, alpha2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit alpha6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit gamma1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The alpha5-subunit-immunoreactivity was strongest in Ammon's horn, the olfactory bulb and hypothalamus. In contrast, alpha4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit alpha3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit gamma3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit delta-immunoreactivity was frequently co-distributed with alpha4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit alpha2-, alpha4-, beta1-, beta3- and delta-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the alpha1-, beta2-, gamma1- and gamma2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits alpha3, beta1, beta3 and gamma2, most thalamic nuclei were rich in alpha1-, alpha4-, beta2- and delta-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits alpha4, beta2 and delta, the ventral lateral geniculate nucleus was predominantly labeled for subunits alpha2, alpha3, beta1, beta3 and gamma2; subunit alpha1- and alpha5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits alpha1, alpha2, beta1, beta2 and beta3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit alpha1, alpha2, beta2, and gamma2 antibodies was contrasted by perykarya labeled for alpha5-, beta1- and delta-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit beta2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit alpha1-, beta2- and gamma2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits beta1, gamma1 and gamma2. In this study, a highly heterogeneous distribution of 13 different GABA(A) receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABA(A) receptor subunits support the assumption of multiple, differently assembled GABA(A) receptor subtypes and their heterogeneous distribution within the adult rat brain.

Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes.

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.

Altered hippocampal expression of neuropeptide Y, somatostatin, and glutamate decarboxylase in Ihara's epileptic rats and spontaneously epileptic rats.

By in situ hybridization and immunocytochemistry, expression of neuropeptide Y (NPY), somatostatin and glutamate decarboxylase 65 (GAD65) was studied in the hippocampus of two different epileptic mutant rats, Ihara's epileptic rat (IER) and the spontaneously epileptic rat (SER). GAD65 mRNA expression was enhanced in interneurons of the hippocampus in young IER, that had not yet developed generalized seizures. In older IER and older SER that both showed spontaneous seizures, marked increases of NPY mRNA in hippocampal granule cells and interneurons were found, as well as elevated GAD65 mRNA levels in interneurons. NPY immunoreactivity was enhanced in hilar interneurons and the dentate gyrus of older IER. In addition, some older IER stained heavily for NPY in mossy fibers. These findings suggest that up-regulation of NPY and GAD65 synthesis may be important in epileptogenesis.

Studies on human porin XXII: cell membrane integrated human porin channels are involved in regulatory volume decrease (RVD) of HeLa cells.

Cell volume regulation receives increasing attention not only as the basis of regulatory volume increase or regulatory volume decrease (RVD) of cells in surroundings of changing osmolarity, but also appears to be relevant in cell proliferation, differentiation, and apoptosis. A central event in RVD is the opening of a volume-sensitive chloride/anion channel(s), and blocking this pathway would abolish RVD. This is shown here with monoclonal mouse anti-human type-1 porin antibodies, proving that porin is involved in this process. HeLa cells preincubated with these antibodies dramatically increase their volume within about 1 min after a hypotonic stimulus by 70 mM NaCl Ringer solution, but do not move back toward their starting volume, thus indicating abolished RVD. Corresponding effects are induced by the established anion channel inhibitor DIDS. Video camera monitoring of cell size over time was used as a direct and noninvasive approach. We had already accumulated evidence that plasmalemma integrated eukaryotic porin channels form chloride/anion channels in this cell compartment and that they are involved in cell volume regulation. Finally, the present data again demonstrate the suitability of our anti-porin antibodies in physiological studies.

Studies on human porin XXI: gadolinium opens Up cell membrane standing porin channels making way for the osmolytes chloride or taurine-A putative approach to activate the alternate chloride channel in cystic fibrosis.

We recently proposed that cell-membrane-integrated vertebrate porin/voltage-dependent anion-selective channel (VDAC) forms part of the outwardly rectifying chloride channel (ORCC) complex that may be involved in volume regulation. The results we present here support this thesis. According to light scattering measurements micromolar concentrations of Gd(3+) induce cell swelling of human healthy and cystic fibrosis (CF) B-lymphocyte cell lines in isotonic Ringer solution. In high-potassium Ringer solution additional swelling is observed. Gd(3+) induces excessive cell swelling of cell lines in hypotonic Ringer solutions, containing 70 mM NaCl or 135 mM taurine, respectively. The gadolinium effect is lost when NaCl is replaced by Na-gluconate. Using video camera monitoring we show that HeLa cells also swell in micromolar concentrations of Gd(3+) in isotonic taurine Ringer solution. The dose-dependent effect of the agonist was always blocked by extracellular application of anti-human type-1 porin antibodies. Together with data on a decreasing effect of micromolar amounts of gadolinium on the voltage dependence of reconstituted human porin the results prove the involvement of porin channels in the swelling behavior in different cell lines. As a mechanism we propose that ionic gadolinium opens up plasmalemma-integrated porin channels, chloride or taurine then following their concentration gradients into the cells. Furthermore, our data argue for a single pathway for inorganic and organic osmolytes during regulatory volume decrease after cell swelling. There is indirect evidence that porin forms part of the cystic fibrosis relevant ORCC channel. Gadolinium thus may work to open the alternate chloride channel in CF.

The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels.

Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins.

Trimethyltin-induced expression of neuropeptide Y Y2 receptors in rat dentate gyrus.

Trimethyltin (TMT) causes prominent neuronal damage and enhanced expression of neuropeptide Y in the hippocampus. We investigated expression of neuropeptide Y Y2 receptors after TMT intoxication. Markedly elevated (by 470%) concentrations of Y2 receptor mRNA were found in the suprapyramidal blade of the dentate granule cell layer after 5 days. Increases in the infrapyramidal blade were less prominent (by 198%). After 16 days, mRNA levels in both blades of the granule cell layer showed no significant difference from those in controls. Quantification of Y2 receptor-specific binding revealed no significant change at both 5 and 16 days after TMT intoxication. It is suggested, together with a previous report describing a similar increase of neuropeptide Y expression, that a transient expression of Y2 receptors in the dentate gyrus in the initial phase of TMT intoxication may be involved in mediating TMT-induced hippocampal damage.

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures.

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.

Metabotropic glutamate receptors mediate activation of NPY-Y2 receptor expression in the rat dentate gyrus.

Neuropeptide Y-Y2 receptor mRNA and binding were investigated after local injection of excitatory amino acid receptor agonists into the rat hippocampus. The general metabotropic glutamate receptor (mGluR) agonist (1S,3R)ACPD (200 and 400 nmol) and the group I mGluR agonist DHPG (50 nmol) enhanced Y2 receptor mRNA levels in granule cells (by up to 470%) and [125I]PYY(3-36) binding in mossy fibers. The group I mGluR antagonist 4-CPG (200 nmol) inhibited the action of (1S,3R)ACPD. On the other hand, AMPA and NMDA enhanced Y2 receptor expression only at neurodegenerative doses (> 0.3 and 3 nmol, respectively). It is suggested that seizure-induced Y2 receptor expression in granule cells may be mediated by group I mGluRs.