Christopher J. Hough

Carbamazepine induction of apoptosis in cultured cerebellar neurons: effects ofN-methyl-d-aspartate, aurintricarboxylic acid and cycloheximide

We have previously demonstrated that carbamazepine (CBZ) at concentrations above the therapeutic range is toxic to cultured cerebellar granule cells. Here, we ask whether the effect of CBZ involves neuronal apoptosis or necrosis. Treatment of cultured cerebellar granule cells with CBZ for 3 days resulted in a concentration-dependent fragmentation of DNA revealed as a laddered pattern in agarose gel electrophoresis, a phenomenon characteristic of apoptosis. Pretreatment of cells with N-methyl-D-aspartate (NMDA) blocked CBZ-induced DNA fragmentation and neurotoxicity as assayed by loss of mitochondrial activity with MTT or by [3H]ouabain binding to Na+/K(+)-ATPase. Aurintricarboxylic acid (ATA), a polyanionic dye, also markedly suppressed DNA fragmentation and cell death detected by morphological examination. A considerable level of DNA ladder formation was detected in untreated cells and this basal DNA fragmentation was also blocked by NMDA and ATA. Moreover, NMDA and ATA prevented CBZ-induced chromatin condensation as revealed by DNA binding with the fluorescent dye Hoechst 33258. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, prevented CBZ-induced cell death detected morphologically and attenuated CBZ-induced neurotoxicity assessed by mitochondrial activity and [3H]ouabain binding assays. Taken together, our results suggest that CBZ-induces death of cerebellar granule cells by an apoptotic process that is sensitive to NMDA, ATA and cycloheximide.

β-Endorphin's modulation of lymphocyte proliferation is dose, donor, and time dependent

We find that beta-endorphin (Bend) can have, positive, negative, or neutral dose-dependent effects on the mitogen-stimulated proliferation of human peripheral blood lymphocytes. The distribution of positive, negative, or neutral responses was nonrandom. In studies carried out over a year, we show that an individuals mitogen-stimulated lymphocyte proliferative response to Bend can change with time. We show that the inhibition induced by cortisol can be, in part, relieved by Bend. On the basis of our results and those of others in the field, we put forward a model that can qualitatively account for many of the observations we and other investigators have made.

Mapping and isolation of large peptide fragments from bovine neurophysins and biosynthetic neurophysin-containing species by high-performance liquid chromatography

Abstract A peptide mapping procedure suitable for rapid analysis and peptide recovery was devised for the neurophysins. Tryptic fragments of performic acid-oxidized bovine neurophysins I and II were fractionated by reverse-phase high-performance liquid chromatography using γ-cyanopropyl-bonded columns. Elutions were achieved using a gradient from triethylammonium phosphate buffer to mixtures of this buffer with increasing proportions of acetonitrile. All tryptic fragments, except for dipeptides, were separated. Assignments of eluted peaks to particular authentic neurophysin peptides were achieved by collection of peaks and determination of their amino acid compositions. The use of this peptide mapping procedure to detect subpicomole amounts of neurophysin peptides in cell-free biosynthetic products was demonstrated.

β-Endorphin modulates T-cell intracellular calcium flux and c-myc expression via a potassium channel

To characterize the effect of beta-endorphin on T-lymphocyte activation, we examined its influence on membrane currents, intracellular calcium flux, and c-myc mRNA levels during mitogenic stimulation of Jurkat cells. While beta-endorphin weakly enhanced voltage-activated K+ currents of Jurkat cells by itself, it suppressed these currents in the presence of mitogen. Naloxone, by itself, also enhanced K+ current amplitude, but in the presence of mitogen partially reversed the suppressive effect of beta-endorphin. A 5-30 min exposure to beta-endorphin resulted in an increase in the rate of mitogen-stimulated intracellular calcium release and an increase in c-myc mRNA levels relative to controls. Longer exposure (1-2 h) to beta-endorphin retarded intracellular calcium release, and suppressed c-myc expression. The suppressive effects were reversed by naloxone and mimicked by the K+ channel blocker, tetraethylammonium ion. These data suggest that opiate receptors and K+ channels of Jurkat cells are functionally coupled in a way that modulates intracellular calcium release and c-myc expression - two key processes in T-cell mitogenesis.

Use of reverse-phase high-performance liquid chromatography in structural studies of neurophysins, photolabeled derivatives, and biosynthetic precursors

The neurophysins are a class of hypothalamo-neurohypophyseal proteins that function as carriers of the neuropeptide hormones oxytocin and vasopressin. Currently, we are using reverse-phase high-performance liquid chromatography for structural characterization of the neurophysins, their chemically modified derivatives, and biosynthetic precursors. A cyanopropylsilyl (Zorbax CN) matrix has been found to be efficient and convenient for separation of major tryptic peptides of performic acid, oxidized or reduced, and alkylated neurophysins. Using this peptide mapping system we have studied the site of modification of a photoaffinity-labeled derivative of bovine neurophysin II by separation and identification of covalently modified peptides. In addition, this system has been used for mapping subfemtomole amounts of radioactively labeled biosynthetic precursors of the neurophysins. This procedure has allowed identification of neurophysin sequences within both pre-pro-neurophysins produced by in vitro translation and rat pro-neurophysins produced by in vivo pulse labeling.

In Vitro Synthesis of Hypothalamic Neurophysin Precursors

Biochemical study of the neurophysins, hypothalamo-neurohypophyseal protein carriers of the neuropeptide hormones oxytocin and vasopressin (Breslow, 1979), has focused on fundamental questions about the biosyn-thetic and molecular events by which these species are formed and stabilized into noncovalent complexes (see Fig. 1). While interacting complexes can be formed between native protein and hormone in their isolated forms, it has become evident from several properties of these molecules that bio-synthetic assembly is more complex. For example, unlike most proteins that remain close to their intact biosynthesized states after translation, neurophysins do not themselves contain sufficient amino acid sequence information to code for spontaneous folding to functional forms. When these proteins, which are highly disulfide cross-linked (seven disulfides per protein molecule of 10,000 daltons), are treated with “catalytic” (substoichio-metric) amounts of disulfide-reducing agent, they are inactivated readily, due to scrambling of the disulfides to nonnative combinations (Chaiken et al., 1975). Further, this process is speeded up by the inclusion of microsomal disulfide interchange enzyme.

ON THE BIOSYNTHETIC ORIGIN OF NEUROPHYSIN-NEUROHYPOPHYSEAL PEPTIDE HORMONE COMPLEXES

Characterization of products of cell-free translation of bovine hypothalamic mRNA, in both wheat germ extracts and rabbit reticulocyte lysates, has allowed identification of two pre-pro-neurophysins, each unique for one of the major bovine neurophysin species. The combined use of specific, purified anti-neurophysins and two peptide mapping procedures has provided rigorous biochemical evidence for establishing these as neurophysin-containing species. Data have been obtained for the presence of two size classes of polyA + mRNA which code for neurophysin biosynthetic products. While preliminary studies indicate that the biosynthesized products become immunochemically reactive with vasopressin antisera after partial proteolysis, rigorous chemical evidence is still lacking for the presence of intact hormone sequences within the bovine pre- proneurophysins.

Antagonists have a greater selectivity for muscarinic receptor subtypes in intact cerebellar granule cells than in membranes

A comparison of muscarinic acetylcholine receptor (mAChR) antagonist binding properties was made between intact cerebellar granule cell cultures and membranes prepared from these cells. [3H]quinuclidinyl benzylate (QNB) binding displacement by four mAChR antagonists was measured and the selectivities for m2- or m3-mAChRs estimated by curve fitting. For each antagonist, the preparation of membranes caused a subtype selective decrease in receptor affinity, as compared to intact cell binding. The m2-selective antagonists had lower affinities in membranes for m2- but not for m3-mAChR, while the m3-selective antagonists had lower affinities for m3- but not for m2-mAChR. As a result, the m2-mAChR selectivity of AF-DX 116 and methoctramine in membranes was 66- and 1.7-fold less than in intact cells, and the m3-mAChR selectivity of 4-DAMP and pFHHSiD was 2.4- and 3.9-fold less in membranes than in intact cells. The m3-mAChR selectivity of 4-DAMP in intact cells was unaffected by cytoskeletal depolymerization with cytochalasins and colchicine. We suggest that the changes in selectivity seen with cell disruption may be due to a loss of cellular factors which regulate receptor properties. Antagonists binding to receptors on intact cells may cause subtype-specific changes in the interaction of the mAChR with these factors. These data suggest that mAChR antagonist binding selectivity needs to be re-examined in intact cell systems.