Calcium- Versus G Protein-Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0-1.0 mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-D-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 microM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C

Summary Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (3 pS). Prior to complete electrical uncoupling, varius discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC. isolated from rat brain, also caused electrical uncoupling. The presence of 0.1mm dibutyryl cyclic AMP and 5mm ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed.     

Characteristics of the volume- and chloride-dependent K transport in human erythrocytes homozygous for hemoglobin C

Summary In human red cells homozygous for hemoglobin C (CC), cell swelling and acid pH increase K efflux and net K loss in the presence of ouabain (0.1mm) and bumetanide. We report herein, that K influx is also dependent on cell volume in CC cells: cell swelling induces a marked increase in the maximal rate (from 6 to 18 mmol/liter cell × hr) and in the affinity for external K (from 77±16mm to 28±3mm) of K influx. When the external K concentration is varied from 0 to 140mm, K efflux from CC and normal control cells is unaffected. Thus, K/K exchange is not a major component of this K movement. K transport through the pathway of CC cells is dependent on the presence of chloride or bromide; substitution with nitrate, acetate or thiocyanate inhibits the volume- and pH-dependent K efflux. When CC cells are separated according to density, a sizable volume-dependent component of K efflux can be identified in all the fractions and is the most active in the least dense fraction. N-ethylmaleimide (NEM) markedly stimulates K efflux from CC cells in chloride but not in nitrate media, and this effect is present in all the fractions of CC cells separated according to density. The persistence of this transport system in denser CC cells suggests that not only cell age, but also the presence of the positively charged C hemoglobin is an important determinant of the activity of this system. These data also indicate that the K transport pathway of CC cells is not an electrodiffusional process and is coupled to chloride.     

Uptake of Exogenous PhosphatidyJserine by Human Neuroblastoma Cells Stimulates the Incorporation of [methyl-l4C]Cholne into Phosphatidylcholine

The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C.     

Antiproliferative Action of Benzodiazepines in Cultured Brain Cells Is Not Mediated Through the Peripheral-Type Benzodiazepine Acceptor

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.     

Interferon inhibits the accumulation of glycerol phosphate dehydrogenase mRNA in oligodendrocytes and C6 cells

We investigated the effect of rat interferon-/ (IFN) on the expression of glycerol phosphate dehydrogenase (E.C.1.1.1.8; GPDH), in both C6 cells and pure cultures of oligodendrocytes. IFNs are naturally produced inhibitors of cell growth that can also affect differentiated cell functions. GPDH is a biochemical marker for oligodendrocytes and is known to be developmentally regulated and steroid inducible. GPDH activity is induced by hydrocortisone (HC) 3.5 fold in C6 cells and 5 fold in oligodendrocytes compared to untreated cultures. A pretreatment of these cells with 75 U/ml of rat IFN-/ resulted in an inhibition of the HC induction of GPDH enzymatic activity by 50% and 40% in C6 cells and oligodendrocytes respectively. We also found that IFN impaired the accumulation of GPDH mRNA in both cell types. These results demonstrate that IFNs are capable of modifying the cellular response to hormones in cells of neuroepithelial origin, and suggest the possibility that IFNs may be able to influence the development and function of the brain.Special issue dedicated to Dr. Paola S. Timiras     

The perennial taxa ofCrucianella (Rubiaceae) in SW. Asia and their eco-geographical differentiation

The perennial taxa ofCrucianella in Asia form a coherent group, apparently diploid (x = 11) and outbreeding throughout, and should be placed into sect.Roseae. This Irano-Turanian group has its centre of diversity in the mountain systems south of the Caspian Sea and reaches with outposts NE. and E. Anatolia, NE. Iraq, S. Iran and C. Asia. Four species and 13 subspecies (within the polymorphicC. gilanica) are recognized, described (partly as new), and illustrated (Figs. 1–6). Conspectus, keys and distribution maps (Figs. 7 and 8) as well as plesio- and apomorphic character states and data on size of areas are provided (Table 1). There is an obvious correlation between more plesiomorphic taxa with smaller areas in the distribution centre of the group, and more apomorphic taxa with larger areas towards its periphery (Fig. 9). These findings are linked to a working hypothesis on the evolution of the group.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Induction of ?2 frameshift mutations within alternating GC sequences by carcinogens that bind to the C8 position of guanine residues Development of a specific mutation assay

Summary Using a forward mutation assay we have previously found that N-2-acetylaminofluorene (AAF), a strong chemical carcinogen, induces a majority of frameshift mutations located at specific sequences called mutation hot spots. Among these hot spot sequences, the NarI sequence (GGCGCC), is specific for –2 frameshifts (GGCGCC) GGCC). Interestingly, these frameshift mutations occur independently of a functional umuDC locus. Being interested in elucidating this mutation pathway we have developed a reversion assay that is specific for this class of mutations. The assay is based on the reversion of a +2 frameshift mutant of plasmid pBR322 from tetracycline sensitivity to tetracycline resistance. It is shown that only true reversion events lead to tetracycline resistance. The carcinogen AAF induces this reversion event at a frequency that is increased four- to fivefold over the background frequency. A series of chemical carcinogens which, like AAF, bind covalently to the C8 position of guanine, are compared for their efficiency to induce this specific mutation event. Large variations in the mutagenic efficiency of these chemicals are observed and discussed in terms of the anti/syn conformation of the carcinogen-modified guanine residue. Based on this test, we describe a convenient spot assay that this presently used in our laboratory to isolate Escherichia coli mutants affected in this mutation pathway.