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Clathrin coated vesicles (CVs) purified from white matter of human or bovine brain contain amyloid precursor protein (APP), several C-terminal fragments encompassing the beta-amyloid domain (betaA), the alpha-secretase 11-12 kDa intermediate, ApoE and tau. The convergence of these components implicates CVs as potential sites for their interaction, yielding products linked to fibrillogenesis in Alzheimer's disease (AD). Analysis of components co-reactive with both anti-ApoE and betaA suggested presence of stable intravesicular conjugates. To evaluate these interactions in vitro, mixtures containing betaA(1-40), ApoE4 or E3 isoforms with and without lipid added as dymyristoyl phosphatidylcholine liposomes were co-incubated from 5 h to 7 days at 37 degrees C and analyzed on Western blots using a panel of antibodies recognizing betaA and ApoE. Data showed ApoE4 plus lipid induced betaA to form oligomers, conjugates and high Mr aggregates. The rates of formation for these products varied significantly with the ApoE isoform. E3 formed conjugates more rapidly, but these levels were exceeded by those of E4 at 7 days. ApoE4 plus lipid facilitated more rapid formation of higher Mr betaA aggregates which appeared in parallel with betaA oligomers containing up to seven molecules of betaA. Data suggest that the native ApoE, as found in CVs which contain lipid, can be an effective agent for promoting formation of betaA oligomers or other complexes that may be linked to formation of abnormal deposits in AD.

Rat adrenal chromaffin cells attached to either collagen-coated dextran (Cytodex 3) or glass bead microcarriers, both of 90-200 microns diameter, were used as dopamine-secreting implants in the caudate-putamen of rats with 6-hydroxydopamine-induced unilateral lesions of the substantia nigra. As controls, beads without cells and cells in suspension alone were implanted. Chromaffin cells adhered to microcarriers reduced apomorphine-induced rotation by 75% in lesioned animals. Animals that were lesioned but not receiving cell implants or receiving beads alone showed no reduction. Animals implanted with cells not attached to beads also showed a reduction in rotation but this effect lasted less than three months. Microcarrier-attached cells, however, maintained their effect in reducing rotation for at least eight months (rotations were reduced from a control mean of 10.9 +/- 1.4 to 3.6 +/- 1.1 turns/min) without any "drop-off" of the effect. Histological examination showed that eight months post-implant the cells pre-adhered to beads were still present and could be stained by anti-tyrosine hydroxylase antibody. Sections stained with hematoxylin-eosin showed no signs of an inflammatory response. In contrast to beads implanted into the striatum, Cytodex bead implants injected into the lateral ventricle induced a histopathological response appearing to involve the ependyma and choroid plexus. Results suggest that the striatal parenchyma but not the ventricle is amenable to studies using the microcarrier approach to transplantation.

Lysosomal cathepsin B but not L degraded rAPP751 to yield C-terminal 19-25 kDa fragments containing beta A4, reinforcing the view that acidic proteases participate in endosomal-lysosomal processing to yield amyloidogenic fragments in situ. This mechanism is consistent with fragmentation of endogenous APPs within clathrin-coated vesicles (CVs) by vesicular hydrolases, with the appearance of C-terminal amyloidogenic fragments following incubation at pH 6.5. A neutral endopeptidase resembling NEP 24.11 (PS-NEP) purified from detergent extracts of human brain degraded rAPP751; however, breakdown was not blocked robustly by metal chelators or phosphoramidon, suggesting the presence of an alternative processing enzyme. Effects of other inhibitors showed that breakdown was mediated by serine-protease-like component(s). A phosphoramidon-insensitive metalloendopeptidase (PI-NEP) partially purified from rat brain P2 using detergents, and resembling NEP 24.15, showed no activity towards rAPP751. Peptides containing putative beta- or gamma-secretase sites were synthesized for purposes of examining their metabolism by the brain enzymes. Those containing beta-secretase sites were hydrolysed at one or more sites by the four enzymes, but only PI- and PS-NEP acted at the Met-Asp site of Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg.NH2. In the case of substrates containing the gamma-site, these two categories of enzymes were the only ones degrading N-Ac-Ile-Ala.NH2. These data imply that the brain metalloendopeptidases, while inactive towards intact precursors, may be involved in turnover of intermediates containing beta- or gamma-sites.

Plasmolipin is an 18-kDa proteolipid protein found in kidney and brain, where it is restricted to the apical surface of tubular epithelial cells and to mammalian myelinated tracts, respectively (Sapirstein, V.S., Nolan, C. E., Stadler, I.I., and Fischer, I. (1992) J. Neurosci. Res. 31, 96-102; Cochary, E. F., Bizzozero, O. A., Sapirstein, V. S., Nolan, C. E., and Fischer, I. (1990) J. Neurochem. 55, 602-610). Addition of plasmolipin to lipid bilayers induces the formation of ion channels, which are voltage-dependent and K(+)-selective (Tosteson, M. T., and Sapirstein, V. S. (1981) J. Membr. Biol 63, 77-84). The present study describes the isolation and cloning of plasmolipin cDNA that includes the sequence of the complete coding region of the protein, the analysis of plasmolipin mRNA expression and a proposed model for its membrane structure. Northern blot analysis 1) shows that plasmolipin is encoded by a 1.7-kilobase mRNA, 2) confirms that the distribution of plasmolipin is restricted to kidney and brain, and 3) indicates that the expression of plasmolipin mRNA in cultured oligodendrocytes increases with cell maturation consistent with changes in the level of the protein. Restriction enzyme digestion of DNA followed by Southern blot analysis indicates that plasmolipin is encoded by a single gene. Sequence analysis of plasmolipin cDNA shows an open reading frame encoding a 157-amino acid protein of 17.4 kDa. The deduced amino acid sequence confirms the hydrophobic nature and high helical content of the protein and predicts a structure with four transmembrane domains similar to several other small hydrophobic proteins implicated in ion movement. The proposed model for membrane topology shows an enrichment of hydroxyl groups within two of the transmembrane domains and places cysteine residues near the extracellular membrane surface. Examination of protein sequence data bases reveals little overall homology with other proteins including proteolipids; however, three of the four transmembrane segments of plasmolipin show strong similarity with known membrane transport proteins. These results indicate that plasmolipin is an unique proteolipid protein that may participate in ion transport events specific to select membrane domains.

This study reports the analysis of K+ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K+ channels were observed with amplitudes of 25-30, 45-55, and 80-100 pS, all of which exhibited mean open-times of 1-2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K+ channel activity was regulated by Ca2+. Little or no change in activity was observed when Ca2+ was added to the cis side of the bilayer. Addition of 10 microM total Ca2+ also resulted in little change in K+ channel activity. However, at 80 microM total Ca2+ all K+ channel activity was suppressed along with the activation of a 100 pS Cl- channel. The K+ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTP gamma S to the trans side of the bilayer resulted in a restriction of activity to the 45-50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K+ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the alpha subunits of G0, Gs alpha, and Gi alpha and the low molecular weight G protein, ras.(ABSTRACT TRUNCATED AT 250 WORDS)

The amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal-myelin from rat brain and have determined by Western blot that APPs, Mr 100-110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal-myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal-myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti-C-terminal APP antibodies but not with anti-N-terminal antibody, appears to be a membrane-associated C-terminal fragment. Western blots with antibodies to Alzheimer precursor-like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100-110 and the 10.5 kDa C-terminal peptide. The demonstration of APP in axolemma and periaxolemmal-myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin-coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal-myelin. In addition, these vesicles contain cysteinyl and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100-110 and Mr 10.5 when analyzed with antibodies to APP672-695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C-terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C-terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C-terminus and may be a useful tool for altering C-terminal APP function.

Amyloid precursor proteins (APPs) and C-terminal fragments were colocalized with cysteine proteinase-like enzymes in purified rat brain clathrin-coated vesicles. Vesicular extracts degraded beta A4(12-28), yielding a product profile similar to that of purified rat brain cathepsin B. Cathepsin B degraded this peptide sequentially, with initial cleavage occurring at Val18-Phe19 and Phe19-Phe20 followed by release of dipeptides. Enzyme also hydrolyzed beta A4(1-40) at Phe19-Phe20 bond but at lower rates, likely due to aggregate formation. An octapeptide analogue of the domain adjacent to beta A4 (N-Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2) was also hydrolyzed by brain cathepsins B and L, and metalloendopeptidase 24.11. Enzymes acted at multiple sites, but only 24.11 cleaved the Met-Asp bond, thus resembling a proposed beta-secretase. Data imply that clathrin-coated vesicles contain cysteine-like proteinases capable of initiating the processing of APP or its fragments.

We have extended our studies on the content of white matter derived coated vesicles (WMCVs) to show that they are enriched in membrane-bound carbonic anhydrase. Within the myelin complex membrane-bound carbonic anhydrase is concentrated in the periaxolemmal domain; however, this protein is enriched almost sevenfold in the bilayer of coated vesicles even relative to this myelin membrane region. These data suggest that some vesicles are derived from a site at which this enzyme is highly localized. The enrichment observed for membrane-bound carbonic anhydrase is unique since other periaxolemmal proteins such as CNPase and plasmolipin are only present in equal amounts in periaxolemmal-myelin fractions and WMCVs. Based on their known localization, the presence of CNPase coupled with the absence of MAG in WMCVs suggest that these vesicles are derived from the paranodal region. The identification in WMCVs of periaxolemmal-myelin proteins associated with ion and fluid movement, such as carbonic anhydrase, Na+,K+ ATPase, and the putative K+ channel protein plasmolipin, prompted us to examine the status of these vesicles in triethyl tin (TET)-induced myelin edema. Coated vesicles and other membrane fractions were isolated from whole brains of control and TET-treated rats. Whole brains were used so we could compare the effects of TET on WMCV proteins with the effect on proteins enriched in gray matter coated vesicles. The results indicated that TET had no detectable effect on compact or periaxolemmal-myelin, however, Western blot analysis showed that WMCV proteins, such as carbonic anhydrase, CNPase, and plasmolipin, were virtually absent or greatly diminished from the whole brain coated vesicle fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.0M sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K+ ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.0M fraction was also highly enriched in Na+, K+ ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage-dependent Na+ channel, axon-specific fodrin, and MAP-1B. Based on these characteristics we concluded that the 0.8M and 1.0M subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipin10 was unique with equally high levels in myelin and in the 0.8M and 1.0M subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GA/1. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K+ and Cl- channels, which based on channel sizes and open times, are largely distinct from one another.

We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.

Plasmolipin is an hydrophobic plasma membrane proteolipid present in both kidney and brain. The protein consists of two subunits of 17-18.5 kD, which together form K+ selective voltage-dependent channels. In this report, we define the embryonic and postnatal expression of plasmolipin in the developing rat brain. Plasmolipin was found to be essentially restricted to the postnatal period increasing eight-fold between the first to fourth week after birth. A fetal plasmolipin immunoreactive protein (FPIP) was identified in embryonic brain and also during the early postnatal development of the cerebellum. The expression of FPIP was biphasic with an initial transient increase between E15-E20 followed by a decrease in its levels. FPIP was not detected in the developed rat CNS. FPIP was found in a variety of dividing and immature cells including cultured astrocytes and embryonic neurons, neuroblastoma cells, and rat thymus. In contrast, plasmolipin was restricted to oligodendrocytes of the neural cells tested and to renal tubular epithelial cells.

Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.

Single cell suspensions from 15-day embryonic rat hindbrain plated on collagen formed large clumps by day 1 in culture. Neurite outgrowth was visible within 2 days. By day 14, morphological synapses were observed in nearly all instances of contact of a neurite ending with another cell. At day 3 in culture, the Golgi apparatus consisted of relatively few, broad lamellae. By contrast, at day 7 in culture this organelle consisted of tightly packed lamellar stacks with a considerable increase in vesicles budding from lamellae. Electron-lucent vesicles, ranging in size from 60 to 180 nm, similar to those generated by the Golgi apparatus were noted in neurite shafts and growth cones, with fusion of these vesicles virtually exclusively at the growth cone leading edge. Monensin resulted in the loss of these vesicles in cell somata and neuritic profiles. The electron-dense marker horseradish peroxidase was not incorporated into these vesicles following its addition to the culture medium, indicating that the vesicles were exocytotic. The number of total vesicles increased during the first 7 days of neurite outgrowth with no further increase up to day 14. This increase was due entirely to vesicles not labeled with the impermeable electron-dense stain ruthenium red, indicating that this increase represents actual vesicular elements and not increased surface convolutions. These data suggest that the 60- to 180-nm electron lucent vesicles are derived from the Golgi apparatus and, by fusion with the growth cone plasmalemma, provide new membrane required for neuritic outgrowth and maintenance.

We have characterized, in C6 cells, an amiloride-sensitive Na+ entry pathway that can exchange for H+. In this report we demonstrate that this cation-exchange system can be induced within 24-36 h by either serum removal or by dibutyryl cyclic AMP; however, these modes of induction are not additive and are manifest only after activation by serum. In these glioma cells we found that activation by serum can be mimicked in part by specific serum factors, i.e., epidermal growth factor and bradykinin. We attempted to characterize this activation process further using several cell biologic probes. We had previously shown that that activation process involves a calcium-dependent step with full activation obtained in the presence of the calcium ionophore A23187. The activation by serum was inhibited by preincubation with colchicine but not with dihydrocytochalasin B, suggesting a cytoskeletal involvement in the activation process. Activation by epidermal growth factor and bradykinin was found to be unaffected by colchicine, suggesting that other factors must be present in serum that confer sensitivity to colchicine. Incubation of the cells with phorbol myristoyl acetate results in the activation of amiloride-sensitive transport, suggesting that stimulation of protein kinase C may be integral to the activation process. Unlike the effects of serum, activation by phorbol myristoyl acetate is not inhibited by colchicine, indicating that this drug works in a way that bypasses the cytoskeletal-dependent step. Since diacylglycerol is the presumed endogenous activator of protein kinase C, we studied the effects of dioleylglycerol. This intermediate of phospholipid turnover was found to increase specifically the amiloride-sensitive sodium pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Total and specific carbonic anhydrase activity was measured for 24 structures of the rat central and peripheral nervous system. In the CNS, white matter or regions containing largely white matter show a neuraxial distribution of enzyme activity; more cephalad structures display more activity. Gray matter regions do not show a rostrocaudal distribution and usually have lower activity than adjacent myelin-containing structures. PNS tissue shows neither the white-gray differences nor the rostrocaudal profile of CNS tissue. Subcellular fractionation of 18 regions of the CNS suggest that the predominance of membrane-bound carbonic anhydrase (60% of the total activity and independent of its magnitude) is a unique characteristic of all regions of the central nervous system.

The most reliable tool at present for the prenatal detection of open neural tube defects is the amniotic fluid alpha-fetoprotein assay. Nevertheless its nonspecificity and false-positive results produced by fetal blood admixture and other less well-defined causes have spurred the search for a more specific test. Increased amniotic fluid acetylcholinesterase activity has been reported in association with open neural tube defects. The authors have assayed acetylcholinesterase in second-trimester amniotic fluid samples with previously determined alpha-fetoprotein values from 200 pregnancies with 96.5% of the outcomes known. These pregnancies were selected because of known fetal abnormalities or difficulties in prenatal diagnosis. High alpha-fetoprotein and acetylcholinesterase activity was detected in all 66 cases of open neural tube defects. The authors were able to reclassify correctly 89% of normal pregnancies with spuriously high alpha-fetoprotein by demonstrating no acetylcholinesterase band after slab gel in electrophoresis. Nine cases with a normal outcome but high alpha-fetoprotein values and acetylcholinesterase activity constitute the remaining false-positive results. Notwithstanding the false-positive results, which may have been due to undetected fetal blood admixture, the acetylcholinesterase gel electrophoresis assay is clearly a valuable adjunctive test to the alpha-fetoprotein assay for the prenatal detection of neural tube defects. Although no specific for open neural tube defects, acetylcholinesterase analysis by gel electrophoresis of amniotic fluid is significantly less influenced by fetal blood than is alpha-fetoprotein and, furthermore, may prove as reliable a diagnostic test for open neural tube defect.

The activities of carbonic anhydrase and 2',3' cyclic nucleotide 3'-phosphohydrolase (CNPase) were measured in gray and white matter and in myelin from human brains obtained at autopsy. Carbonic anhydrase activity increased with age, and at all ages a major part of the activity was associated with membrane fractions. The percentage of membrane-bound carbonic anhydrase was lower than normal in white matter from Krabbe disease and adrenoleukodystrophy; isolated myelin had a low specific activity in these diseases. CNPase activity was decreased in both white matter and myelin, but was somewhat higher than normal in gray matter. Although the yield of myelin from a case of metachromatic leukodystrophy was markedly reduced, changes in the enzymes activities were minimal.

The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter-enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. This discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29 degrees C) than for the microsomal fraction (21 degrees C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions, Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10-3 M-strophanthidin. The evidence thus indicated that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggest that this is not a significant factor and that Na + K ATPase may be a myelin-associated enzyme.

In vitro assay of the adenylate cyclase of NB41A neuroblastoma cells in the presence of increasing concentrations of MnCl2 suggested that the enzyme is modulated by both high- and low-affinity sites for manganese. MnCl2 in a concentration of 1 microM significantly stimulated adenylate cyclase activity, but increasing the concentration of manganese to 3 microM or 10 microM had no further effect. Raising MnCl2 to 0.1 or 1 mM, however, further stimulated enzyme activity. In addition to differences in affinity for manganese, the two classes of binding sites may be distinguished by differences in their interaction with other agents that affect adenylate cyclase activity. Millimolar manganese and magnesium appeared to compete for a common site on the enzyme and the effect of manganese in this range and the effect of guanyl nucleotide were synergistic. In contrast, the stimulation of activity by micromolar manganese appeared to be additive to the effects of either increasing magnesium or the addition of guanyl nucleotide to the assay media. Comparison of the substrate dependency of the reaction measured in the presence and absence of manganese suggests that the stimulation of adenylate cyclase activity involves increases in both the apparent Vmax of the reaction and the affinity for ATP. The results raise the possibility that the interaction of Mn2+ may play a role in the modulation of adenylate cyclase in vivo.

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse oligodendroglioma cells, G-26 clone 20 and 24, contain galactosylceramide (cerebroside) and sulfogalactosylceramide (sulfatide) as determined by an HPLC technique. The synthesis of both these lipids was stimulated by 10(-6) M hydrocortisone (cortisol) and also by the removal of serum from the culture medium. Forty-eight hours after the addition of cortisol the incorporation of H235SO4 into sulfatide, the level of sulfatide and the specific activity of the enzyme 3'-phosphoadenosine 5'-phosphosulfate:galactosylceramide sulfotransferase in the cells increased three- to fourfold. The level of cerebroside and the specific activity of UDP-galactose:hydroxyacyl sphingosine galactosyltransferase also increased threefold in the cells on treatment with cortisol. The effect of the hormone on the synthesis of cerebroside preceded the increase in sulfatide synthesis. Experiments with cycloheximide and actinomycin D showed that the effect of the hormone on glycolipid synthesis in these cells were mediated through de novo messenger RNA and protein synthesis. Removal of serum from the culture medium resulted in an approximately twofold enhancement of H235SO4 incorporation into sulfatide within 24 h. The levels of sulfatide and cerebroside and the specific activity of the galactosyltransferase and sulfotransferase also increased significantly after serum removal. However, in contrast to the effect of the steroid, the sulfotransferase activity and the level of sulfatide increased prior to elevations in galactosyltransferase and cerebroside. The effect of serum removal was also found to be mediated by de novo RNA and protein synthesis. The effects of cortisol and serum removal on the synthesis of cerebroside and sulfatide were strictly additive.

This chapter has described the characterization and biogenesis of soluble and membrane-bound CA in the central nervous system. The two forms of the enzyme appear to be quite similar in their molecular characteristics, however the data strongly indicate that they are synthesized on separate polysomal populations; the membrane-bound form resulting from synthesis on the RER. Our preliminary data suggest that the partitioning of mRNA for CA on the different polysomes results from the interaction of partial nascent chains with a specific receptor on the RER. We feel a function of membrane-associated synthesis is for the targeting of CA to sites in the cell where there are enzymes that can rapidly utilize the protons and bicarbonate produced by CA catalytic activity for ion exchange reactions. We have also presented arguments that CA may function as a bicarbonate source in the control of metabolism specifically in the acceleration of fatty acid synthesis in the oligodendrocyte.

Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane-bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p-nitrophenyl acetate. The membrane-bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane-bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane-bound form. The failure to observe any precursor-product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase-like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na+ influxes of approximately 25-30 nmol/mg protein min; less than 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na+ influx to 15-20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na+ influx, depending on the cell type, of 20-400% within 2 min. This increment in Na+ influx represented an increase in amiloride-sensitive Na+ transport with an apparent K'1 of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na+ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 micrograms/ml of cycloheximide blocked the increase in Na+ transport, indicating that de novo protein synthesis mediated this serum deprivation-induced increase in Na+ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na+ influx did not saturate with Na+ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na+ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.

The developmental patterns of membrane-bound carbonic anhydrase (MBCA) and glial fibrillary acid protein (GFA) were analyzed in high speed pellets from subcortical regions of quaking and control mice at ages ranging from 10 to 42 days. In control, MBCA was found to increase from approximately 1.5 unit/mg protein at 10 days to about 11 units/mg protein at 42 days. The percent increase was greatest between days 11 and 18 where the activity increased 2.5-fold while the largest absolute weekly increment was in the fourth postnatal week. By contrast, the activity in quaking mice was 1.0 units/mg protein at day 10 and increased to only 5 units/mg protein by 42 days. The activity, although reduced at 10 days, appeared to increase in parallel with the controls between day 11 and 18; however, the large increment in activity observed in the controls during the fourth week was greatly reduced in the mutant. The above findings were also observed when immune reactive material was determined by immuno-blotting using antisera raised against MBCA isolated from brain. GFA was also estimated using an immuno-blotting procedure. In contrast to previous studies on soluble GFA, we found GFA to increase about 5-fold in the third postnatal week and level off thereafter. The Q/C ratio was 1.5 at 10 days, but, although GFA increased over 5-fold between 11 and 42 days, the absolute difference between quaking and control increased very little. These data on GFA and MBCA suggest that a defect in the expression of oligodendroglial plasma membrane proteins may be expressed early in development which may correlate with a small but significant gliosis and/or an increase in GFA synthesis.

We have studied the regulation of lysosomal glycosidases during morphological differentiation of NB2a neuroblastoma cells. Cells treated with dibutyryl cAMP induced axon-like neuritis and showed a 2-4 fold increase in the activity of 6 lysosomal glycosidases, reaching their highest level after 5 days of treatment. Cells treated with retinoic acid, which induced dendrite-like neurites, did not show significant changes in the glycosidases activity although cell proliferation was also inhibited. There was no change in the pattern of the enzyme secretion during the dibutyryl cAMP treatment and morphological analysis using electron microscopy and cytochemical staining with acid phosphatase indicated the presence of lysosomes in the induced neurites.

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.

The present investigation focuses on the developmental changes in the activity and levels of carbonic anhydrase, adenylate cyclase and 3',5'-cyclic adenosine monophosphate (cAMP) in the Quaking mouse mutant in different regions of the brain. Carbonic anhydrase activity was found to be lower than normal in the forebrain of the C57/B Quaking mouse. The deficit in forebrain carbonic anhydrase was restricted to subcortical structures and was reflected equally in the membrane and soluble factions, indicating that neither pool was affected selectively. However, no difference in carbonic anhydrase activity was observed in purified myelin from Quaking and control mice. Investigation of the changes in carbonic anhydrase activity as a function of age showed a cessation in enzyme accumulation in Quaking mice at around 20 days postnatally, suggesting an abnormality in cellular development. A tritiated acetazolamide binding assay was used to quantitate the amount of enzyme present. The amount of carbonic anhydrase parallelled enzyme activity, suggesting that the defect in the Quaking animals was at the level of the control of enzyme synthesis. Similar studies on cyclic AMP metabolism showed a higher than normal adenylate cyclase activity in the upper brain stem region of Quaking mice of ages between 19 and 40 days. Adenylate cyclase activity was stimulated by norepinephrine in both control and Quaking animals. The increased adenylate cyclase activity in the Quaking mice was in contrast to a lower cyclic AMP level and could not be accounted for by an alteration in phosphodiesterase activity.

In this study we have characterized, in brain, the expression of a plasma membrane proteolipid protein (PM-PLP) complex that can form cation-selective channels in lipid bilayers. We isolated PLP fractions from synaptic plasma membrane and glial microsomes and found a high degree of similarity in both size and amino acid composition to the complex we had previously isolated from kidney. Antibodies specific to the kidney PM-PLP were prepared, and, on the basis of immunoblot and immunoprecipitation studies, the PM-PLP complex isolated from neural membranes was shown to be immunologically related to the kidney PM-PLP. These proteolipid proteins exhibited a molecular weight of approximately 14K and contained a high percentage of hydrophobic amino acids with an apparent absence of cysteine. The biogenesis of PM-PLP in brain was studied by in vitro translation of free and bound polysomes and total RNA in a rabbit reticulocyte lysate followed by immunoprecipitation of the translation products. From these studies it is concluded that the PM-PLP complex is synthesized on the rough endoplasmic reticulum. On the basis of the identical electrophoretic mobility of material isolated from plasma membranes and material immunoprecipitated after translation of bound polysomes and isolated RNA, it appears that the PM-PLP does not undergo detectable posttranslational processing between its site of synthesis and its incorporation into the plasma membrane.

We have characterized the effects of retinoic acid (RA) on the growth, morphology and biosynthesis of cytoskeletal proteins in NB2a mouse neuroblastoma cells. In addition, the morphological and biochemical changes were compared to those induced by dibutyryl cyclic AMP (db cAMP). Growth inhibition by RA was concentration-dependent and was first detected 24 h after addition of RA. The proliferation of RA-treated NB2a was more dependent on serum than was the proliferation of untreated cultures and RA decreased the saturation density of NB2a cells grown in serum. Morphological changes induced by RA include the formation of an elaborate network of branching neurites in NB2a cells. In contrast, neurites induced by db cAMP or serum deprivation were bipolar and unbranching. Ultrastructural observations of neurites induced by RA revealed dendritic characteristics such as polysomes, spines and absence of intermediate filaments, while neurites induced by db cAMP had axonal characteristics such as filament bundles, absence of ribosomes, and the formation of membrane densities when neurite endings contacted another cell body. These morphological differences were also reflected in a number of changes in the biosynthesis of cytoskeletal proteins. These results suggest that NB2a cells treated with RA and db cAMP are a model system for the study of distinct stages of differentiation.

X-ray diffraction was used to probe the effects of triethyl tin (TET) on the periodicity and amount of membrane disorientation in the lamellar myelin from respiring optic and sciatic nerves in vitro as well as from nerves of rats treated in vivo through their drinking water. The diffraction patterns show that in vitro TET at concentrations of 4-100 microM affects C.N.S. but not P.N.S. myelin structure. A planar, concentric membrane array with a 200 A period is detected in the C.N.S.; this ordered, swollen myelin contrasts with the vacuolar and vesicular structure seen in thin-sections in TET-induced oedema. No effects of short-term in vivo treatment with TET are observed in either the C.N.S. or P.N.S. The finding that carbonic anhydrase (CA) inhibitors have no effect on the TET-induced structural changes indicates that the swelling we observe is not related to a CA-dependent process. In comparison, the TET effect is prevented by replacing the mobile ions with isotonic sucrose. We conclude that TET-induced swelling in C.N.S. myelin arises from an increase in ion transport followed by obligatory fluid movement. Further, the ordered, swollen structure we detect may be an intermediate state that exists transiently in vivo in TET intoxication and that precedes the gross swelling and vacuolization usually observed.

Aldosterone (the 8,11-hemiacetal 0f 11beta,21-dihydroxy-3,20-dioxo-4-pregnen-18-al) markedly stimulates sodium transport in a number of epithelial tissues. We have attempted to determine whether aldosterone induces the synthesis of specific protein(s) in the course of its action upon the toad urinary bladder. Paired hemibladders were incubated in media containing either [3H]methionine or [35S]methionine; aldosterone in physiologic concentrations was added to one bath and, after incubation, the intact "mitochondria-rich" (MR) and "granular" (G) mucosal cells were isolated. The ratio (3H/35S) was used to identify proteins whose synthesis was induced in the mucosal cells of the steroid-treated bladders. Using exclusion gel chromatography and isoelectric focusing, we identified several aldosterone-induced proteins in the supernatant (105,000 x g) fraction of the MR cell. None was evident in this fraction of the G cell. These proteins have apparent molecular weights ranging from 17,000 to 38,000 and the isoelectric point of the major component is 4.5. Corticosterone (11beta,21-dihydroxy-4-pregnene-3,20-dione) induced the synthesis of proteins in the G cells, but none of these proteins was similar in molecular weight to the aldosterone-induced proteins in the MR cell. Our findings support the hypothesis that aldosterone induces the synthesis of specific proteins and indicate that, in this tissue, these proteins are synthesized by the MR cell.

Proteolipids extracted from bovine kidney plasma membrane induce irreversible changes in the electrical properties of lipid bilayers formed from diphytanoyl phosphatidylcholine. The interaction with the proteolipid produces channels which are cation selective. At low protein concentrations (i.e., less than 0.6 microgram/ml), the single-channel conductance is approximately 10 pS in 100 mM KCl and 3 pS in 100 mM NaCl. In the presence of protein concentrations above 1 microgram/ml, another population of channels appears. These channels have a conductance of about 100 pS in 100 mM KCl and 30 pS in 100 mM NaCl. Further, these channels are voltage dependent in KCl, closing when the voltage is clamped at values greater than or equal to 30 mV. The steady-state membrane conductance, measured at low voltages, was found to increase proportional to a high power (2-3) of the proteolipid concentration present in one of the aqueous phases. In 100 mM NaCl, the conductance increases at protein concentrations above 5 microgram/ml, whereas in 100 mM KCl it increases at protein concentrations above 0.6 microgram/ml. These measurements indicate that the higher steady-state conductance observed in KCl at a given proteolipid concentration in a multi-channel membrane presumably results because more channel incorporate in the presence of KCl than in the presence of NaCl. The two major fractions which comprise the proteolipid complex were also tested on bilayers. It was found that both fractions are required to produce the effects described.

The mucosal epithelium of the toad urinary bladder reabsorbs sodium, acidifies the urine, and is responsive to neurohypophyseal hormnones. Mucosal epithelial cells, consisting of two major morphologic cell types, "mitochondria-rich" and "granular," were removed from the bladder and separated by density gradient centrifugation. The mitochondria-rich cells contained three times as much carbonic anhydrase activity as the granular cells. Oxytocin caused a 235 percent increase in the adenosine 3',5'-monophosphate content of mitochondria-rich cells but had no effect on the granular cells. The evidence indicates that the mitochondria-rich cell, which accounts for only 15 percent of the mucosal cells, plays a major role in the mediation of sodium ion and hydrogen ion transport in the toad bladder and is a specific site of action of neurohypophyseal hormones.

The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain GM3, GM2, GM1, GD3, and GD1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GalNAc (beta 1 leads to 4) Gal(beta 1 leads to 4)Glc(beta 1 leads to 1)Cer (GgOse3Cer), and GalNAc(beta 1 leads to 3)Gal(alpha 1 leads to 4) Gal(beta 1 leads to 4)Glc(beta 1 leads to 1)Cer (GbOse4Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mM-EGTA showed a two-to threefold increase in GM3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mM-EGTA plus 0.4 mM-EDTA a similar increase in GM3 was observed but this change was now accompanied by decreases in GM2, GM1, GgOse3Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca2+ sufficient to bind all chelator. Mn2+ replacement reversed the chelator effects differentially; GM2 and GM1 levels were the most sensitive to increases in Mn2+ concentration; GgOse3Cer and GbOse4Cer were also sensitive, whereas GM3 was the least affected. These results suggest calcium serves an important regulatory role on GM3 levels and that manganese concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.

We examined the expression and distribution of microtubule-associated protein 2 (MAP2) during the differentiation in culture of both mouse NB2a neuroblastoma and primary embryonic rat neurons. The differentiation of NB2a cells was induced with retinoic acid (RA) which stimulated the extension of a highly branched neuritic network and dibutyryl cAMP which stimulated the outgrowth of long bipolar or monopolar processes. We found that although monoclonal antibodies to MAP2 stained the cell bodies of control and differentiated cells, only the RA-induced neurites were positive for this antigen. These data support our ultrastructural studies indicating that the RA-induced neurites were dendrite-like and that the dibutyryl cAMP-induced processes were axon-like. Studies on the biosynthesis of MAP2 indicated that RA induced a 2-3-fold increase in MAP2 synthesis in 24 h; however, this effect was transient, with the synthesis of MAP2 in RA-treated cells returning to control level by 72 h. Although biosynthetic studies suggested the synthesis of species at 250-300 kdalton, the major molecular weight form in the neuroblastoma cells was 230 kdalton. Immunocytochemical analysis of primary neurons showed staining of neuronal cell bodies and of short processes, but virtually no staining of the long axon-like processes. The staining of neuronal cell bodies and processes was evident at all stages of cell differentiation. This finding was corroborated by immunoblots which showed significant amounts of MAP2 throughout cell development. The molecular weight of the immunoreactive material was ca. 300 kdalton in both primary neurons and rat brain. Immunoblots also revealed that embryonic neurons expressed only MAP2B as they differentiated in culture for 14 days. Biosynthesis studies suggested that early in culture there was a modest increase in MAP2 synthesis, but no detectable change was observed thereafter. We concluded therefore that both neuroblastoma cells and primary neurons can differentiate neuritic processes, which show dendritic properties in terms of morphology and preferential distribution of MAP2.

We have utilized monoclonal antibodies to investigate the antigenic diversity of MAP2-immunoreactive proteins in the nervous system of vertebrates. We found that domains defined by the monoclonal antibodies differed in their conservation across vertebrate evolution, ranging from wide cross-reactivity with almost all vertebrates (mammals, birds, reptiles and amphibians) to a very limited cross-reactivity with only few mammalian species. However, we did not find MAP2-immunoreactive proteins in fish species with either of the monoclonal or polyclonal antibodies. There was also a significant divergence in the apparent molecular weight of MAP2, even in closely related species. For example, different species of wild mice and strains of laboratory mice showed variations of up to 30 kDa in their apparent molecular mass. Using alkaline phosphatase, under conditions that dephosphorylate neurofilaments, we showed that the observed heterogeneity was not the result of variations in the phosphate content. The heterogeneity in molecular weight of MAP2 may, therefore, be the result of changes in primary structure, transcriptional variations or different post-translational modifications. The heterogeneity of MAP2, as well as its specific distribution and implicated interactions with other molecules, underscore the complexity of MAP2 and its potential for structural and functional diversity. The phylogenic analysis of such a complex molecule also provides a method to establish the uniqueness of monoclonal antibodies and the degree of their conservation for their corresponding epitopes.

The effects of oxytocin upon tissue cAMP content and short-circuit current (SCC) were measured in the urinary bladder of the toad, Bufo marinus. Tissue cAMP levels doubled before any increment in SCC was observed, the two hormone responses were quantitatively related, and a threshold level for an effect of cAMP upon sodium transport was demonstrated. The period of time over which cAMP levels continued to rise after the threshold level had been attained seemed invariant with hormone concentration. The rate at which cAMP levels rose increased with hormone concentration yielding hormone concentration-dependent maximal levels. The decay of cAMP levels was delayed when sodium influx was curtailed, suggesting a sodium-regulatory effect upon tissue cAMP levels.