Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina

Microprobes bearing immobilized antibodies to the carboxy-terminus of beta-endorphin were used to study the release of beta-endorphin in the urethane anaesthetized rat following electrical stimulation of the ipsilateral arcuate nucleus. The microprobes were inserted through the cerebral hemisphere, the superior colliculus and the midbrain periaqueductal grey. Since such microprobes detect extracellular molecules along their entire length they give information on the persistence and spread of compounds following release. Little immunoreactive-beta-endorphin was detected in the areas of brain sampled during electrical stimulation of arcuate nucleus but a remarkable spread throughout the midbrain and cerebral cortex occurred within 30 min of the cessation of stimulation. The results suggest that although beta-endorphin-containing fibres are absent in many parts of the brain, this neuropeptide can access receptors in these sites and it is not necessary for release to be directly adjacent to opiate receptors. As such it is important evidence supporting the hypothesis of volume transmission as a means of neuronal communication. The results also suggest that an important mechanism of the transport of beta-endorphin is the cerebrospinal fluid.     

Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.     

Exocytotic and nonexocytotic modes of glutamate release from cultured cerebellar granule cells during chemical ischaemia

We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.     

Adenosine A1 receptors in cultured cerebellar granule cells: role of endogenous adenosine

Adenosine A1 receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A1 receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of alpha-Gg and alpha-Gi, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A1 receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A1 receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A1 receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.     

Calcium influx via L-type voltage-gated channels mediates the delayed, elevated increases in steady-state c-fos mRNA levels in cerebellar granule cells exposed to excitotoxic levels of glutamate

The altered kinetics of steady-state c-fos mRNA production in cultured cerebellar granule cells under excitotoxic conditions was investigated in neurons subjected to depolarising stimuli, namely, high KCl and L-glutamate (Glu), in which Ca2+ influx occurs by differing routes. Increases in intracellular-free calcium levels ([Ca2+]i) stimulated by nontoxic or toxic levels of Glu were blocked by selective N-methyl-D-aspartate (NMDA) receptor antagonism; were blocked only partially by the L-type channel blocker, nifedipine; and were unaffected by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonists. Glu-induced cell death was prevented only by NMDA receptor blockade. Exposure of cells to nontoxic levels of Glu resulted in a transient increase in c-fos mRNA levels, whereas an excitotoxic dose produced a delay in the appearance of c-fos mRNA but a subsequent, progressive, and sustained (>4 hr) increase. An excitotoxic dose of Glu in combination with either nifedipine or selective NMDA receptor antagonists resulted in the normal, transient increase of c-fos mRNA levels. Chronic exposure to 55 mM KCl caused no cytotoxicity, although it resulted in a delayed, elevated increase in c-fos mRNA levels that was unaffected by NMDA receptor blockade but reverted to the normal, transient profile of c-fos mRNA formation when it was coadministered with nifedipine. The KCl-induced increase in [Ca2+]i levels was inhibited dramatically by nifedipine but was unaffected by any of the ionotropic Glu receptor antagonists. The results support the notion that the appearance of a delayed but elevated increase in steady-state c-fos mRNA levels following exposure to excitotoxic doses of Glu is mediated specifically by calcium influx via L-type voltage-gated channels.     

Effects of KT-362, a sarcolemmal and intracellular calcium antagonist, on calcium transients of cultured neonatal rat ventricular cells: a comparison with gallopamil and ryanodine

During the years 1990-92 in the Regional Poisons Control Center in Sosnowiec 42 epileptics (20 females and 22 males) were hospitalized because of suicide attempt. It amounted to 9% of all attempters, treated there in this period. The majority of patients were males of age range from 21 to 62 years. In 23 patients the suicide attempts were performed for the first time. the main reason for suicide was the family conflicted situation. Additionally, in 14 patients the poisoning attempts have been done during alcohol abuse. In the suicide attempts the antiepileptic drugs were most frequently used, mainly carbamazepine (23 cases).     

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

Involvement of three glutamate receptor epsilon subunits in the formation of N-methyl-D-aspartate receptors mediating excitotoxicity in primary cultures of mouse cerebellar granule cells

The N-methyl-D-aspartate receptors have been implicated in neuronal plasticity and their overactivation leads to neurotoxicity. Molecular cloning and co-expression of various glutamate receptor zeta and epsilon complementary DNAs support a heteromeric structural organization for N-methyl-D-aspartate receptors. In this study, we show that cerebellar granular neurons in primary culture of mouse express glutamate receptor zeta1 and at least three glutamate receptor epsilon (epsilon1, epsilon2, and epsilon3) protein subunits. In vitro, the temporal patterns of glutamate receptor epsilon1, epsilon2, and epsilon3 subunit expression depend on culture stages. By day 9, a somatic and neuritic immunolocalization for all N-methyl-D-aspartate subunits was clearly identified in most neuronal, but not glial cells. The role of particular subunits in N-methyl-D-aspartate-mediated excitotoxicity was probed by exposing the cerebellar granule cells to antisense oligodeoxynucleotides generated against specific N-methyl-D-aspartate receptor subunits. Antisense oligodeoxynucleotide treatments significantly down-regulated the amounts of the corresponding N-methyl-D-aspartate subunits. The decrease in N-methyl-D-aspartate subunit protein correlated with a reduction in N-methyl-D-aspartate-induced calcium influx and N-methyl-D-aspartate-mediated excitotoxicity in cerebellar cultures. In contrast, antisense oligodeoxynucleotide treatment failed to protect neurons from 1-methyl-4-phenylpyridinium-induced metabolic cell toxicity. Antisense oligodeoxynucleotide treatment targeted at N-methyl-D-aspartate glutamate receptor epsilon subunits demonstrate that glutamate receptor epsilon1, epsilon2, and epsilon3 proteins form N-methyl-D-aspartate receptors responsible for neurotoxic effects on cerebellar neurons. This study provides direct evidence for the existence of distinct N-methyl-D-aspartate receptor subunit proteins in cerebellar granule cells developing in vitro that may trigger N-methyl-D-aspartate-dependent excitotoxicity.     

Involvement of phosphatase activities in the run-down of GABA(A) receptor function in rat cerebellar granule cells in culture

PURPOSE: To compare the magnetic resonance (MR) imaging appearance of the anal sphincter in patients with fecal incontinence and scleroderma with that in patients with fecal incontinence alone, scleroderma alone, or neither. MATERIALS AND METHODS: The study population comprised 14 patients with fecal incontinence and scleroderma, four with scleroderma alone, 13 with incontinence alone, and six with neither. T1- and T2-weighted spin-echo, magnetization transfer contrast-weighted, and dynamic gadolinium-enhanced images were obtained and analyzed for the integrity, thickness, and length of sphincter components. Magnetization transfer contrast ratios and T2 were calculated to assess fibrosis of the internal sphincter. The percentage enhancement above baseline was calculated at 30-second intervals for the internal and the external sphincter. RESULTS: Eleven patients with incontinence and scleroderma showed descent of rectal air and feces into the anterior anal canal, with forward deviation of the significantly (P < .05) atrophied internal sphincter, which showed a slower gadolinium-enhancement pattern compared with that in other groups. Patients with incontinence alone showed no evidence of internal sphincter deviation or altered vascularity but had a significant reduction (P < .05) in deep external sphincter bulk. CONCLUSION: In patients with fecal incontinence and scleroderma, endoanal MR imaging helps delineate the anterior sphincter deformity and shows the slower gadolinium-enhancement pattern on dynamic studies of the internal sphincter.     

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.     

A functional interaction between the neuronal adhesion molecules TAG-1 and F3 modulates neurite outgrowth and fasciculation of cerebellar granule cells

F3 and TAG-1 are two closely related adhesion glycoproteins of the Ig superfamily that are both expressed by the axons of cerebellar granule cells. In an in vitro system in which cerebellar granule cells were cultured on monolayers of transfected Chinese hamster ovary (CHO) cells, we show that F3 and TAG-1 interact functionally. F3 transfectants have been shown to inhibit outgrowth and induce fasciculation of granule cell neurites. By contrast TAG-1 transfectants have no effect on these events. However, when TAG-1 is coexpressed with F3, the inhibitory effect of F3 is blocked. Two possible mechanisms may account for this functional interaction: (1) either TAG-1 and F3 compete for the same neuronal receptor, and in favor of this we observed that binding sites for microspheres conjugated with F3 and TAG-1 are colocalized on the granule cell growth cones, (2) or alternatively, F3 and TAG-1 associate in a multimolecular complex after their binding to independent receptors. Extensive co-clustering of F3 with TAG-1 can in fact be achieved by anti-TAG-1 antibody-mediated cross-linking in double-transfected CHO cells. Moreover, F3 coimmunoprecipitates with TAG-1 in Triton X-100-insoluble microdomains purified from newborn brain. These data strongly suggest that F3 and TAG-1 may associate under physiological conditions to modulate neurite outgrowth and fasciculation of the cerebellar granule cells.     

Parallin, a cerebellar granule cell protein the expression of which is developmentally regulated by Purkinje cells: evidence from mutant mice

In this paper we report on monoclonal antibody 3H6 with unique specificities for development of the cerebellum. Immunohistochemical studies on normal and mutant mice suggest that it is primarily located in or on granule cell parallel fibers in the cerebellum. The only other region showing immunoreactivity is a small region of the hippocampus. The antigen is detected immunohistochemically as early as postnatal day 11 in the molecular layer of the cerebellum. In adult wild-type mice parallin expression is seen in the molecular layer and to a lesser degree in the internal granular layer. In the cerebella of two neurological granule cell-deficient mutants, weaver (wv) and staggerer (sg), parallin is not detected. However, in two Purkinje cell-deficient mutants, Purkinje cell degeneration (pcd) and nervous (nr), a more complex and interesting pattern is observed. These two mutants do have granule cells and parallel fibers and 3H6 immunoreactivity is observed. However, in both of these Purkinje cell-deficient mutants the 3H6 immunoreactivity is drastically reduced in regions where Purkinje cells have degenerated. Furthermore, in nr mutants, the antigen appears to be concentrated in regions of the parallel fiber that are in close proximity to Purkinje cells, suggesting its possible association with synapses. Taken together these results suggest that parallin is a marker of granule cells and their parallel fibers, its onset correlates with the formation of granule cell synapses on developing Purkinje cells, and it requires Purkinje cells for the maintenance of expression.     

An analysis of centriolar orientation in cultured ESK cells under the action of the calcium ionophor A23187

Addition of 20 microM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells gave an increase of Ca2+ in cytosol by more than 10 times. The maximum of [Ca2+] was achieved in 1-2 min after introduction of the drug. Later on [Ca2+] gradually decreased, and after 30 min of incubation with A23187 [Ca2+] was 3-5 times above normal level. Immunofluorescent and electron microscope studies showed no alterations in the microtubule system of interphase cells after 1-30 min treatment. The electro microscope study showed that-following 1.5 min introduction of the drug the random orientation of material and daughter centrioles changed: most of them settled down at an angle more, than 74 degrees to the substrate surface. After 3 min of A23187 treatment more than half of maternal centrioles were oriented perpendicular to the substrate surface. After 5 min of A23187 treatment, the percentage of maternal centrioles with perpendicular orientation was the same and this orientation remained for 30 min. The percentage of perpendicular daughter centrioles decreased after 3 min of treatment, and after 30 min their orientation was random. We suggest that the perpendicular orientation of centrioles to the substrate surface is mediated through centrosome-associated calcium-binding proteins.     

The role of gammadelta T cells in induction of bacterial antigen-specific protective CD8+ cytotoxic T cells in immune response against the intracellular bacteria Listeria monocytogenes

PURPOSE: To study the relation between abdominal aortic aneurysms and chronical obstructive pulmonary disease (COPD), in particular the suggested common elastin degradation caused by elastase and smoking. METHODS: A cross-sectional population study and a prospective cohort study of small abdominal aortic aneurysms was performed in a community setting. All previous diagnoses recorded in a hospital computer database were received for 4404 men 65 to 73 years of age who had been invited to a population screening for abdominal aortic aneurysm. One hundred forty-one men had AAA (4.2%). They were asked to participate in an interview, a clinical examination, and collection of blood sample. Men with an abdominal aortic aneurysm 3 to 5 cm in diameter were offered annual ultrasound scans to check for expansion. RESULTS: Among patients with COPD 7.7% had abdominal aortic aneurysms (crude odds ratio=2.05). The adjusted odds ratio, however, was only 1.59 after adjustment for coexisting diseases associated with abdominal aortic aneurysm (P=.13). The mean annual expansion was 2.74 mm per year among patients with COPD, 2.72 among patients without COPD, and 4.7 mm among patients who used oral steroids compared with 2.6 among patients who did not use steroids (P < .05). Concentration of serum elastin peptide and plasma elastase-alpha1-antitrypsin complexes correlated negatively with forced expiratory volume in the first second (FEV1) among patients with COPD. However, multivariate regression analysis showed that concentration of serum elastin peptide, therapy with beta-agonists, and FEV1 correlated positively with degree of expansion but that concentration of plasma elastase-alpha1-antitrypsin complexes and serum alpha1-antitrypsin did not influence expansion, suggesting that elastase plays an important role in the pathogenesis of COPD but not of abdominal aortic aneurysm. CONCLUSION: The high prevalence of abdominal aortic aneurysm among patients with COPD is more likely to be caused by medication and coexisting diseases rather than a common pathway of pathogenesis.     

C1q, a subunit of the first component of complement, enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37 degrees C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37 degrees C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 +/- 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 +/- 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 microg/ml of C1q significantly increased GMC-apoptosis up to 39.4 +/- 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab')2-ER4G (F(ab')2-ER4G-GMC), while ER14-GMC or F(ab')2-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse K-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.     

Induction of low-affinity GABAA receptors by the GABA-agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) in cultured rat cerebellar granule cells is prevented by inhibition of polyamine biosynthesis

GABAA agonist-induced formation of low-affinity GABAA receptors in cultured cerebellar granule cells was studied in the presence or absence of alpha-difluoromethylornithine (DFMO), a blocker of polyamine formation. High- and low-affinity GABAA receptors were monitored by Scatchard analysis of [3H]GABA binding to membranes from cells cultured for either 4 or 10 days in the presence or absence of the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Cultures grown for 4 days were exposed to THIP and DFMO for an additional period of 6 hr (acute exposure), whereas cultures grown for 10 days were exposed to the same agents during the entire culture period (chronic exposure). Regardless of the culture period or drug exposure protocol, control cells expressed only a high-affinity (KD 7 nM) binding site for GABA, whereas the cultures treated with THIP for either 6 hr or 10 days exhibited an additional low-affinity binding site (KD approximately 500 nM). Chronic exposure to DFMO prevented the THIP induction of low-affinity GABAA receptors, whereas acute exposure to DFMO had no effect on the ability of THIP to induce low-affinity GABAA receptors. Measurements of the intracellular polyamine concentration demonstrated a slight decrease in the putrescine level in the granule cells exposed to DFMO or THIP + DFMO for 6 hr. In contrast, granule cells chronically (10 days) exposed to DFMO or THIP + DFMO were depleted of putrescine and spermidine. Hence, the ability of THIP to induce low-affinity GABAA receptors was prevented by the simultaneous depletion of the cellular content of putrescine and spermidine, whereas inhibition of ornithine decarboxylase and of putrescine formation was not sufficient to prevent THIP-induced receptor formation.     

Fluticasone propionate reduced the production of GM-CSF, IL-6 and IL-8 generated from cultured nasal epithelial cells

The stages of mental rotation of complicated three-dimensional figures were studied using the intracortical interaction mapping. The role of the parietal areas in the mental rotation was shown. The frontal, central and the right temporal areas are involved in the prerotation setup. Both frontal areas and the left temporo-parieto-occipital areas participate in decision making and verbal response. In the case of unsuccessful task solving the process stops at the prerotation setup stage. During the verbal control the zones of the predominant connections at the first 3 stages are close to those in the background activity.     

Evidence for Mg2, ATP-dependent accumulation of Ca2+ by the intracellular non-mitochondrial calcium store in uterine smooth muscle cells

Using carbachol contracture as the experimental model for testing the properties of the intracellular calcium store in intact tissue and 45Ca2+ accumulation in the chemically skinned by digitonin smooth muscle cells isolated from oestrogen-dominated rat uterus the evidence for the presence of Mg2+, ATP-dependent Ca2+ pump in the non-mitochondrial store has been found which is supposed to play a key role in the process of refilling' of the store on the cytoplasmic level. The experiments performed on intact muscle showed that the functional activity of the carbachol-releasable Ca2+ store is critically dependent on Ca2+ entry. It is found that Ca2+ entry via voltage operated Ca2+ channels or on the Na(+)-Ca2+ exchange was needed to refill the store in this tissue. However, when Ca2+ extrusion systems located in the plasma membrane were inhibited by La3+, the store retained its ability to discharge and reaccumulate Ca2+ released on the regular basis suggesting the presence of the energy-dependent Ca2+ accumulating system in the store. The process of the store refilling was totally inhibited by cyclopiazonic acid. Chemically skinned uterine smooth muscle cells demonstrated the presence of Mg2+, ATP-dependent accumulation of Ca2+ in the non-mitochondrial (ruthenium red insensitive) intracellular store(s) potentiated by Ca(2+)-precipitating anions (potassium oxalate and phosphate), in a time- and concentration dependent way which was inhibited by Ca(2+)-ionophore A 23187 (5 microM) and cyclopiazonic acid with Ki = 0.4 microM. It is suggested that in the uterine smooth muscle of the oestrogen-dominated rats, nonmitochondrial receptor-operated intracellular calcium store is represented by endoplasmic reticulum.     

Contraction and intracellular calcium-ion elevation of cultured human aortic smooth muscle cells by endothelin-1, vasoactive intestinal contractor (VIC) and the derivatives

Effects of endothelin (ET) family peptides and their derivatives on cellular contraction and calcium-ion level were examined by using cultured human vascular smooth muscle cells (VSM). Contraction of cultured human VSM, isolated from human fetal aortic segments, was induced within 1 min after the treatment with ET-1 (100 nM) as seen in the changes of cytosolic calcium-ion localization. In parallel with the cell contraction, cytosolic calcium-ion level in the human VSM increased very rapidly and then dropped with some oscillation as determined by Anchorage Cell Analyzing System. It was noted that transient calcium-ion mobilization rather than sustained calcium-ion influx was significant in the contraction of cultured human VSM. Vasoactive intestinal contractor (VIC), three amino acids different from ET-1, had less activity in increase of intracellular calcium-ion level and in percent of response cells than ET-1, ET-2, and VIC-S4L6 (one amino acid different from ET-1). EC50 of ET-1, VIC-S4L6, ET-2, and VIC were 0.5 nM, 0.6 nM, 2.0 nM, and 20 nM, respectively. VIC-like peptide (VIC-LP), 16 amino acids fragment of VIC precursor protein, had no effect with a single administration of up to 10 microM. However, the increase in calcium-ion level by VIC was suppressed with a prior treatment of cells with high concentration (10 microM) of VIC-LP. The establishment of cultured human VSM for the simultaneous examination of the contraction and calcium-ion level will provide a new system to study signal transduction of vasocontractor peptides.