Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

μ-Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine-Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

The receptors mediating the inhibition of D1 dopamine receptor-stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose-dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50 = 0.05 microM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 microM). SKF 38393 (1 microM)-stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective mu-agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO; EC50 = 0.006 microM) and high concentrations of the selective delta-agonist [D-Ser2(O-tert-butyl), Leu5]-enkephalyl-Thr6 (DSTBU-LET; EC50 = 0.13 microM) but not by the selective delta-agonist [D-penicillamine2, D-penicillamine5]enkephalin (DPDPE). D1 dopamine receptor-stimulated adenylate cyclase activity was also slightly reduced (by approximately 20%) by high concentrations of the kappa-agonist U50,488 (EC50 = 0.63 microM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the mu-opioid receptor-selective antagonist naloxone (EC50 of approximately 0.1 microM). Neither the irreversible delta-ligand fentanyl isothiocyanate (1 microM) nor the reversible delta-antagonist ICI 174864 (1 microM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (greater than 0.1 microM) of the kappa-opioid receptor-selective antagonist norbinaltorphimine. The effect of DAGO (1 microM) was already detectable after 1 day in culture, whereas DPDPE (1 microM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of mu-opioid receptors coupled as inhibitors to D1 dopamine receptor-stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.     

Hyaluronate-Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.     

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)     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

N-Methyl-D-Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca²⁺ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca²⁺ and 10 mM Mg²⁺. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca²⁺, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.     

Interleukin-1 Hyperproduction by In Vitro Activated Peripheral Macrophages from Cerebellar Mutant Mice

Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.     

Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase

Summary The Na⁺–H⁺ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V _e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA.These results indicate that limited trypsin digestion dissociates the activity of the renal Na⁺–H⁺ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na⁺–H⁺ exchanger by PKA-mediated, protein phosphorylation.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.