Establishment and neurite outgrowth properties of neonatal and adult rat olfactory bulb glial cell lines

Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.     

Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes

We have studied the inhibitory action of long- and short-chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2-3H] and [6-3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the 'cycling' between glucose and glucose-6-phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 mumol/min/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 mumol/min/g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.     

Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes

We have shown recently that the bovine corpus luteum (CL) possesses specific luteal cell surface membrane binding sites for progesterone. We have now confirmed and extended these observations to compare the subcellular distribution of these binding sites in developing, mature and regressing CL. The median buoyant densities of luteal progesterone binding sites from early-, mid- and late-luteal phase CL were similar (though three of five density profiles for late-luteal phase CL showed association of steroid binding with a fraction with a lower density), and clearly resolved from nuclear, mitochondrial, lysosomal, peroxisomal, Golgi-endoplasmic reticulum-lysosomal and smooth endoplasmic reticulum markers. Specific binding of [3H]progesterone overlapped with the distributions of 5'-nucleotidase and luteinizing hormone receptor (luteal cell surface membrane markers) in both control and digitonin-treated gradients at all stages of the luteal phase. Since steroidogenic 'large luteal' and 'small luteal' cells of the CL are derived from the granulosa cells (GC) and theca of the preovulatory follicle, we also investigated whether similar receptors were present in the follicle, and describe for the first time specific membrane binding sites for progesterone in purified GC and thecal membranes from healthy bovine follicles of different sizes. Specific binding increased linearly with GC and thecal membrane protein concentration; however, it was detectable only when digitonin was included in the binding incubation. Binding sites were specific for progesterone; unlabelled progesterone competed for [3H]progesterone binding at low concentrations (IC50, 35 and 31 nmol/l) compared with testosterone (IC50, 905 and 870 nmol/l) and delta4-androstenedione (IC50, 1050 and 660 nmol/l) for GC and thecal receptors respectively. In contrast, oestradiol, oestrone, pregnenolone, cortisol, cholesterol, and a genomic progesterone receptor antagonist, RU486, competed poorly. Steroid binding was present in GC and thecal membranes of follicles of all sizes, but [3H]progesterone binding to GC membranes decreased significantly with increasing follicle size (P<0.02), perhaps indicating developmental regulation of GC membrane non-genomic progesterone receptors in the preovulatory bovine follicle. We suggest that these membrane steroid receptors may be involved in the autocrine/paracrine regulation of follicular function by progesterone.     

Congenic rats reveal three independent Copenhagen alleles within the Mcs1 quantitative trait locus that confer resistance to mammary cancer

It has previously been shown that the Copenhagen (COP) rat contains several genetic loci that contribute to its mammary tumor-resistant phenotype after 7,12-dimethylbenz(a)anthracene (DMBA) administration. One of these loci, mammary carcinoma susceptibility 1 (Mcs1), is located on the centromeric end of chromosome 2 and appears to act in a semidominant fashion. To confirm the existence and independent action of this locus and also aid in the identification of the physical location of the Mcs1 gene, congenic lines were generated by transferring the Mcs1 COP allele onto a Wistar Furth (WF) genetic background. Male carriers were genotyped using microsatellite markers spanning 20-30 cM of the Mcs1 locus. One of the congenic lines minimally retained the COP allele at D2Mit29 on the centromeric end of chromosome 2 and extended distally to D2Rat201. Heterozygous Mcs1 carrier rats were interbred, and the female offspring were treated with DMBA. The female rats from the Mcs1 congenic line that carried one or two COP alleles of the Mcs1 region had a significantly reduced (65 and 85%, respectively) tumor development (P < 0.001) compared with rats carrying zero COP alleles at this locus. A WF.COP-D2Mit29/D2Rat201 homozygous congenic strain derived at the N10 generation was treated with DMBA, and the COP homozygous rats developed 1.5 +/- 0.3 carcinomas/rat versus 6.3 +/- 0.5 in WF control rats (P < 0.0001). Fine mapping of this congenic interval using several recombinant lines identified three genetic loci within the Mcs1 congenic region that independently supported a tumor resistance phenotype. These genetic loci have been termed Mcs1a, Mcs1b, and Mcs1c. In rats for which each locus was homozygous for the COP allele, tumor development was reduced by approximately 60% compared with littermate controls. The identification of these independent loci within the Mcs1 COP allele provide a model of the genetic complexity of cancer.