Effects of training in the Morris water maze on the spatial learning acquisition and VAChT expression in male rats.

BACKGROUND AND THE PURPOSE OF THE STUDY: It has been well established that cholinergic pathway plays an important role in learning and memory processes. The present study was designed to evaluate the effects of Morris water maze (MWM) training on spatial memory acquisition and expression of the vesicular acetylcholine transporter (VAChT) in male rats. METHODS: In this study, training trials of all groups of animals were conducted in the MWM task. Rats received one training session consisting of four trials per day which continued for another four consecutive days. Controls received visible platform MWM training. The escape latency, the traveled distance and swimming speed for each rat were recorded and used to evaluate the performance of the animal during training period. For evaluation of expression of VAChT protein levels, brain tissues from animals in each experiment were obtained immediately after the last trial on the related experimental day and processed for immunohistochemistry staining and western blotting analysis. RESULTS: There was a significant difference between animals subjected to one day training and those receiving four days of training in escape latency and travel distance. There were an apparent increase in VAChT immunoreactivity in the medial septal area (MSA) and CA1 region of the hippocampus in one day and four day trained animals compared with controls (visible group). Quantitative immunostaining analysis by optical density measurements in the CA1 region and evaluation of immunopositive neurons in medial septal area of brain sections confirmed qualitative findings. Assessment of VAChT protein level expression in hippocampus by western blotting evaluation showed the same pattern of immunohistochemistry results. CONCLUSION: Overall, results of this study reveal changes in cholinergic neuron activity in different stages of training in the MWM task. Data suggest that there is a significant level of cholinergic neuronal activity during early stages of the training especially in the hippocampus region that may contribute to the apparent increase in VAChT expression.

Elevation of nerve growth factor and antisense knockdown of TrkA receptor during contextual memory consolidation.

We report here a series of experiments establishing a role for nerve growth factor and its high-affinity receptor TrkA in contextual memory consolidation. In all experiments, we trained rats in a novel chamber using tone and shock. Our first experiment revealed that endogenous nerve growth factor (NGF) increases in the hippocampus at a critical time during consolidation that occurs 1 week after training. NGF levels at other intervals (24 hr and 2 and 4 weeks after training) did not differ from those of naive control animals. In our second experiment, we blocked effects that NGF has at 1 week after training by infusing antisense TrkA phosphorothioate DNA oligonucleotide. Reduction of septohippocampal TrkA receptor expression selectively impaired memory consolidation for context but not for tone. Animals with antisense TrkA oligonucleotide infused into the medial septal area or CA1 of the hippocampus froze less when placed in the training chamber than did animals infused with inactive randomized oligonucleotide. At 4 weeks after training, antisense TrkA oligonucleotide had no effect on freezing. Third, we correlated levels of freezing with choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) immunohistochemistry. Antisense TrkA infused into CA1 of the hippocampus reduced cell body cross-sectional area for cholinergic cells in the medial septal area and decreased the density of hippocampal terminals labeled for ChAT and VAChT proteins. Cholinergic cell body measurements were significantly correlated with freezing. Taken together, these results indicate a role for nerve growth factor acting via the TrkA receptor on ChAT and VAChT proteins in contextual memory consolidation.

Distribution of the vesicular transporter for acetylcholine in the rat central nervous system.

In order to develop another selective marker for cholinergic cell bodies and fibres, we have raised a highly specific polyclonal antibody against a peptide derived from the C-terminus of a recently cloned putative vesicular acetylcholine transporter. This antibody recognizes the vesicular acetylcholine transporter protein on western blots of membranes from transfected monkey fibroblast COS cells as well as from various rat brain regions but not from untransfected COS cells or rat liver. In separate mapping studies, the antibody was found to stain cell bodies and fibres in all of the regions of the nervous system known to be cholinergic, including (i) the various nuclei of the basal nuclear complex and their projections to the hippocampus, amygdala, and cerebral cortex, (ii) the caudate-putamen nucleus, accumbens nucleus, olfactory tubercle, and islands of Calleja complex, (iii) the medial habenula, (iv) the mesopontine cholinergic complex and its projections to the thalamus, extrapyramidal motor nuclei, basal forebrain, cingulate cortex, raphe and reticular nuclei, and some cranial nerve nuclei, and (v) the somatic motor and autonomic nuclei of the cranial and spinal nerves. In many of these cholinergic neurons, it is possible to detect immunoreactivity for the vesicular acetylcholine transporter in proximal portions of processes and their branches, as well as in numerous puncta in close association with them. Some of these puncta are large and surround cell bodies and processes of neurons in several regions, including the somatic motor neurons of cranial nerve nuclei in the brainstem and in the ventral horn of the spinal cord. Double immunofluorescence studies indicated that neurons positive for the vesicular acetylcholine transporter also stained for the biosynthetic enzyme of acetylcholine, choline acetyltransferase. We conclude that antibody against the C-terminus of the putative vesicular acetylcholine transporter provides another marker for cholinergic neurons that, unlike in situ hybridization procedures, labels terminals as well as cell bodies. Therefore this antibody has the potential to reveal changes in number and morphology of cholinergic cell bodies and their terminal varicosities that occur in both physiologic and pathologic conditions.

Differential distribution of the putative vesicular transporter for acetylcholine in the rat central nervous system.

The organization and distribution of the mRNA for the putative vesicular transporter for acetylcholine (VAChT) was studied in the rat brain by use of digoxigenin-labeled riboprobes and in situ hybridization technology. Signal was observed in all neural regions deduced to contain cholinergic somata on the basis of previous histochemical investigations employing choline acetyltransferase riboprobes and prior immunocytochemical studies with antibodies against choline acetyltransferase. It was absent in areas believed to contain no cholinergic neurons. Anti-sense riboprobes hybridized to the mRNA for the putative VAChT: (a) the projection neurons of the various nuclei of the basal nuclear complex, (b) the local circuit cells of the dorsal and ventral striata, (c) the projection neurons of the mesopontine complex, (d) perikarya in the ventral 2/3 of the medial habenula, (e) the somatic motor and autonomic cells of cranial nerves 3-7 and 9-12, as well as perikarya in the dorsal and ventral cochlear nuclei presumably giving rise to efferent fibers of cranial nerve 8, and (f) the alpha-motor and gamma-efferent motor neurons of the spinal cord. In addition, the mRNA for the VAChT was found in a few somata, probably ectopically located cells of the basal nuclear complex, in the internal capsule, central nucleus of the amygdala, entopeduncular nucleus, and zona incerta. It was also detected in some cell bodies in the reticular part of the substantia nigra, probably the rostral extension of the mesopontine complex, in the parabigeminal nucleus, and around the central canal in the spinal cord but not in cortical, hippocampal, and cerebellar perikarya. It is concluded that, like choline acetyltransferase, the mRNA for the putative acetylcholine vesicular transporter is another specific marker for neurons utilizing acetylcholine as a neurotransmitter. Further investigations of that transporter could have important implications for various diseases involving cholinergic systems, such as Alzheimer's disease.

Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles.

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C-terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT-immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.

Molecular cloning of a putative vesicular transporter for acetylcholine.

Classical neurotransmitters such as acetylcholine (ACh) require transport into synaptic vesicles for regulated exocytotic release. The Caenorhabditis elegans gene unc-17 encodes a protein with homology to mammalian transporters that concentrate monoamine neurotransmitters into synaptic vesicles. Mutations in unc-17 protect against organophosphorus toxicity, indicating a role in cholinergic neurotransmission. Using the relationship of unc-17 to the vesicular amine transporters, we first isolated a related sequence from the electric ray Torpedo californica [Torpedo vesicular ACh transporter (TorVAChT)] that is expressed by the electric lobe but not by peripheral tissues. Using the relationship of the Torpedo sequence to unc-17, we then isolated the cDNA for a rat homologue (rVAChT). Northern blot analysis shows expression of these sequences in the basal forebrain, basal ganglia, and spinal cord but not cerebellum or peripheral tissues. In situ hybridization shows expression of rVAChT mRNA in all cholinergic cell groups, including those in the basal forebrain, brainstem, and spinal cord that previously have been shown to express choline acetyltransferase mRNA. The human VAChT gene also localizes to chromosome 10 near the gene for choline acetyltransferase. Taken together, these observations support a role for rVAChT in vesicular ACh transport and indicate its potential as a novel marker for cholinergic neurons.

Intracellular early and late modifications of human apolipoprotein A-II. Effect of glutamine- +1 to leucine substitution.

It has been shown previously that apoA-II undergoes several intracellular modifications in HepG2 cells (Hussain & Zannis, 1990). In the present study, we have generated permanent cell lines in mouse C127 cells which express the normal apoA-II gene and a mutated form in which Gln+1 was substituted with Leu (Leu+1). This modification was designed to prevent cyclization of the N-terminal glutamine of apoA-II and thus identify the isoproteins which are precursors and products of the N-terminal cyclization reaction. The C127-expression cells were also utilized to study the cellular compartments where the apoA-II modifications occur as well as the importance of the modifications for apoA-II trafficking and secretion. We have found that apoA-II (Gln+1) synthesized by C127 and HepG2 cells had similar isoproteins. In both cell types, unmodified pro-apoA-II, designated isoprotein 3, had a similar isoelectric point as the cell-free translation product of apoA-II mRNA, suggesting that isoprotein 3 results from cleavage of the signal peptide. Isoprotein 3 represents an unmodified apoA-II isoprotein and undergoes an early modification into a more acidic isoprotein 1, which differs from isoprotein 3 by two negative charges. Brefeldin A treatment of the cells did not prevent the formation of isoprotein 1, suggesting that this modification occurs in a pre-Golgi compartment. Neuraminidase treatment of secreted apoA-II isoproteins did not affect isoprotein 1, indicating that it is not sialylated isoprotein. Isoprotein 1 undergoes further modifications which are consistent with cleavage of the propeptide, N-terminal cyclization and sialylation most likely resulting from O-glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosomal localization of the human vesicular amine transporter genes.

The physiologic and behavioral effects of pharmacologic agents that interfere with the transport of monoamine neurotransmitters into vesicles suggest that vesicular amine transport may contribute to human neuropsychiatric disease. To determine whether an alteration in the genes that encode vesicular amine transport contributes to the inherited component of these disorders, we have isolated a human cDNA for the brain transporter and localized the human vesicular amine transporter genes. The human brain synaptic vesicle amine transporter (SVAT) shows unexpected conservation with rat SVAT in the regions that diverge extensively between rat SVAT and the rat adrenal chromaffin granule amine transporter (CGAT). Using the cloned sequences with a panel of mouse-human hybrids and in situ hybridization for regional localization, the adrenal CGAT gene (or VAT1) maps to human chromosome 8p21.3 and the brain SVAT gene (or VAT2) maps to chromosome 10q25. Both of these sites occur very close to if not within previously described deletions that produce severe but viable phenotypes.

The protooncogene bcl-2 inhibits apoptosis in PC12 cells.

During development, many neuronal populations undergo a process of normal, programmed cell death, or apoptosis. Trophic factors regulate this process, but the mechanism by which they suppress apoptosis remains unclear. In the immune system, recent studies have implicated the protooncogene bcl-2 in the lymphocyte survival response to growth factors. To determine whether a similar survival pathway exists in a neuroendocrine cell type, we have expressed bcl-2 in the rat pheochromocytoma PC12 cell line and found that it abrogates the requirement for stimulation by growth factors to survive. bcl-2 expression also substantially delays the onset of injury by the calcium ionophore A23187.

Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.

Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.

Gene transfer of a reserpine-sensitive mechanism of resistance to N-methyl-4-phenylpyridinium.

The toxin N-methyl-1,2,3,6-tetrahydropyridine produces a model of neural degeneration very similar to idiopathic Parkinson disease. To understand the cellular mechanisms that modulate susceptibility to its active metabolite N-methyl-4-phenylpyridinium (MPP+), we have transfected a cDNA expression library from the relatively MPP(+)-resistant rat pheochromocytoma PC12 cells into MPP(+)-sensitive Chinese hamster ovary (CHO) fibroblasts. Selection of the stable transformants in high concentrations of MPP+ has yielded a clone extremely resistant to the toxin. Reserpine reverses the resistance to MPP+, suggesting that a transport activity protects against this form of toxicity, perhaps by sequestering the toxin within an intracellular compartment. In support of this hypothesis, dopamine loaded into the CHO transformant shows a localized distribution that is distinct from the pattern observed in wild-type cells and is also reversed by reserpine.

A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter.

Classical neurotransmitters are transported into synaptic vesicles so that their release can be regulated by neural activity. In addition, the vesicular transport of biogenic amines modulates susceptibility to N-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin N-methyl-1,2,3,6-tetrahydropyridine that produces a model of Parkinson's disease. Taking advantage of selection in MPP+, we have used gene transfer followed by plasmid rescue to identify a cDNA clone that encodes a vesicular amine transporter. The sequence predicts a novel mammalian protein with 12 transmembrane domains and homology to a class of bacterial drug resistance transporters. We have detected messenger RNA transcripts for this transporter only in the adrenal gland. Monoamine cell populations in the brain stem express a distinct but highly related protein.

Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.

We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.

Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA.

Digoxigenin-labeled RNA probes and in situ hybridization histochemistry were used to examine choline acetyltransferase gene expression in the rat central nervous system. Hybridization signal was present only in brain sections processed with the antisense riboprobe. The sense probe did not yield labeling, further validating the specificity of tissue reactivity. Telencephalic neurons containing the mRNA for the cholinergic synthetic enzyme were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis. Some somata evincing hybridization signal were observed in the anterior amygdalar area, and an occasional such cell was seen in the basolateral and central amygdalar nuclei. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridocytochemically detectable amounts of choline acetyltransferase mRNA. Thalamic cells were devoid of reactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few somata labeled with riboprobe were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei were moderately reactive, whereas cells of the parabigeminal nucleus exhibited a very weak hybridization signal. No somata in the brainstem raphe nuclei, including raphe obscurus and raphe magnus, were observed to bind riboprobe. In contrast, motor neurons of the cranial nerve nuclei demonstrated relatively large amounts of choline acetyltransferase mRNA. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord were also labeled with riboprobe, as were a few cells around the central canal. We conclude that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons (i.e. those that synthesize and use acetylcholine as a neurotransmitter) in most of the neural regions deduced to contain them on the basis of previous histochemical and immunocytochemical data. Notable exceptions are the cerebral cortex and hippocampus, which do not possess neurons expressing detectable levels of choline acetyltransferase mRNA.

Secretion of lipid-poor nascent human apolipoprotein apoAI, apoCIII, and apoE by cell clones expressing the corresponding genes.

The human apolipoprotein apoAI, apoCIII, and apoE genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector that also contained the human metallothionein 1A gene. Following transfection of mouse C127 cells with the expression vector, cell clones resistant to Cd2+ were selected and found to express in high abundance specific apolipoprotein genes. Individual cell clones expressing apoAI, apoCIII, or apoE genes were used further to study the isoprotein composition and the flotation properties of the corresponding nascent apolipoproteins. It was found that the lipoproteins secreted by cell clones expressing the apoAI, apoCIII, and apoE genes consisted of the proapoAI disialylated form of apoCIII (apoCIIIS2) and mainly sialylated forms of apoE. Separation of the secreted apolipoproteins by density gradient ultracentrifugation resulted in limited flotation of nascent apoAI, apoE and apoCIII in the high density lipoprotein (HDL) fraction. Similar analysis in the presence of human serum increased the flotation of apoAI, apoE, and apoCIII to 6.5-, 4.5-, and 5.5-fold, respectively, and resulted in their redistribution to various lipoprotein fractions. HDL increased the flotation of apoAI to 12-fold and very low density lipoprotein (VLDL) increased the flotation of apoCIII and apoE to 6.5- and 5.5-fold, respectively. These findings suggest that in the cell system used, the majority of nascent apoAI, apoCIII and apoE is secreted in the lipid-poor form, which then associates extracellularly with preexisting lipoproteins.

Mutagenesis of the glycosylation site of human ApoCIII. O-linked glycosylation is not required for ApoCIII secretion and lipid binding.

We have used site-directed in vitro mutagenesis to alter the codon ACT of human apoCIII gene, specifying Thr-74, to GCT (Ala-74). The normal and mutant apoCIII genes were then placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector and were used for cell transfection and selection of stable cell lines. Blotting analysis of RNA isolated from several independent cell clones showed that both the normal and mutant genes produced apoCIII mRNA in amounts larger than that found in human fetal liver. Pulse-chase analysis of cell clones expressing the normal and mutant apoCIII genes showed that only the normal apoCIII is modified intracellularly to produce a disialated form (apoCIIIs2). Cell clones expressing the normal apoCIII gene secrete exclusively the disialated form, whereas those expressing the mutant gene secrete the unmodified form. The amount of mutant apoCIII protein produced by C127 cell clones expressing the mutant gene was reduced as compared to that produced by the control cells. Density gradient ultracentrifugation analysis of the secreted apoCIII showed that the flotation properties of the secreted normal and mutant proteins were similar. These findings suggest that the intracellular glycosylation of apoCIII is not required for its intracellular transport and secretion. Furthermore, lack of glycosylation has no effect on the relative affinities of apoCIII for plasma very low density lipoproteins and high density lipoproteins.

Alterations of the glutamine residues of human apolipoprotein AI propeptide by in vitro mutagenesis. Characterization of the normal and mutant protein forms.

We have used site-directed mutagenesis to independently alter the Gln residues at positions -1 and -2 of the human apoAI propeptide to Arg residues. The normal and mutated genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus (BPV) vector which also carries a copy of the human metallothionein 1A gene. Following transfection of mouse C127 cells [corrected] with the vectors, cell clones resistant to CdCl2 were selected and analyzed for production of apoAI mRNAs and protein. The RNA blotting analysis showed that the steady-state apoAI mRNA levels of cell clones expressing either the normal or the mutant apoAI gene are 3-5-fold higher than that of the liver or HepG2 cells. Two-dimensional gel electrophoresis of radiolabeled apoAI showed that the apoAI-expressing clones secreted mainly the proapoAI form. Furthermore, both mutant proapoAI's differed by one positive charge from the normal apoAI. Secretion of apoAI into the culture medium follows apparent first-order kinetics and gives similar rate constants for the normal and mutant apoAI forms. Separation of secreted apoAI by density gradient ultracentrifugation in the presence of human plasma or HDL shows identical distribution of plasma and nascent (normal and mutant) apoAI. The findings indicate that in the cell system used the modification of either of the two glutamines of the apoAI prosegment does not affect the intracellular transport and secretion of apoAI, and its ability to associate with HDL.