Interactions between intracellular chloride concentrations, intracellular pH and energetic status in rat lactotrope cells in primary culture

Rat lactotrope cells in primary cultures have a higher intracellular Cl- concentration ([Cl-]i) than that predicted by a passive distribution across the membrane. This suggests that active cellular mechanisms ensure this ionic equilibrium. In this study, we examined the interactions between pHi, [Cl-]i regulation and cell energetics. We analyzed: 1. the interactions between extracellular Cl- concentrations, [Cl-]i and cellular energy; 2. the influence of [Cl-]i on respiratory chain function; 3. the correlation with glycolysis and; 4. the role played by pHi in these cellular mechanisms. We show that low [Cl-]i decreases ATP cell content, ATP/ADP ratio and modify phosphorylative oxidations. ATP production is rather due to the anaerobic pathway of the glucose metabolism than the aerobic one and depends also on other metabolic substrates among which glutamine probably has a special role. Finally, pHi appears as a determinant in the balance between aerobic and anaerobic pathways. These results are discussed in relation to the role of Cl- in normal and pathological (effect of hypoxia on mature and immature neurons) cell situations.     

Cell-type-specific function of the C-type natriuretic peptide gene promoter in rat anterior pituitary-derived cultured cell lines.

The promoter function of the human C-type natriuretic peptide (CNP) gene in various cultured cells was examined by transient transfection assays. The CNP promoter functioned very effectively in GH3 cells, which originated from the growth hormone-producing tumor of the rat anterior pituitary and somatomammotroph phenotype, but functioned much less effectively in GH1 cells, another type of rat pituitary-derived cell with a somatotroph phenotype, and rat primary cardiocytes. The CNP promoter did not function at all in other cells, including AtT20 cells of murine pituitary corticotroph origin. Functional analyses of the deleted promoters with various 5' deletion breakpoints revealed the existence of at least two negative and one positive regulatory regions. Within the positive regulatory region (positions -54 to -19), which conferred 90% of the promoter activity in GH3 cells, two equipotent GC-rich cis elements (positions -49 to -45 and -40 to -35) were identified. Both sites shared half of the promoter activity and binding properties to the nuclear protein in GH3 cells. Rat anterior pituitary tissue contained the binding protein of the identified cis element, which was identical or similar to that of GH3 cells. With Southwestern (DNA-protein) analysis, a 70-kDa specific binding protein distinct from known factors such as SP-1, AP-2, and Pit-1 was identified in the nuclear extract of GH3 cells.     

Tissue specific trans-acting factor interaction with proximal rat prolactin gene promoter sequences.

Using an exonuclease III protection assay, strong, reversible and tissue-specific binding of GH3 cell nuclear factors to proximal regions of the rat prolactin (rPrl) promoter (-31 to -77) has been detected. A second less prominent region of factor binding, that may have a correlate in HeLa cell extracts, was detected in the region (-155 to -180). The binding is eliminated in the presence of excess unlabelled rPrl promoter sequences (-423 to +38), excess unlabelled distal rPrl 5'-flanking sequences (-1960 to -1260) and SV40-enhancer/promoter sequences; it is largely unaffected by growth hormone (rGH) promoter and RSV-LTR sequences. A plasmid containing the proximal rPrl promoter sequences (-75 to +38) was also shown to be an avid inhibitor, at low concentrations, of rPrl promoter driven chloramphenicol acetyl transferase (CAT) gene expression in transient cotransfection competition studies; under these assay conditions distal rPrl 5'-flanking sequences and RSV and rGH promoter plasmids do not compete. The results emphasize the critical importance of proximal rPrl promoter sequences for prolactin gene expression in GH3 cells but recognize the related functional potential of more distal sequences.     

Interactions between neuropeptide Y and alpha 2-adrenoceptors in selective rat brain regions.

Neuropeptide Y significantly reduced the potassium-stimulated release of [3H]norepinephrine [( 3H]NE) from slices of rat hippocampus, hypothalamus and frontal cortex but not from slices of parieto-occipital cortex. The NPY-induced inhibition of [3H]NE release from frontal cortical slices was concentration dependent, reaching statistical significance at 10 nM. The alpha 2-adrenoceptor partial agonist, clonidine, also reduced the potassium-stimulated release of [3H]NE. The combination of NPY and clonidine in hippocampal slices produced a greater reduction of stimulated [3H]NE release than either of the two compounds alone, suggesting a potentiation of their activity, whereas in frontal cortical slices, the effect was additive. When NPY and clonidine were added to frontal cortical slices, they independently produced a significant concentration-dependent reduction in forskolin-stimulated cAMP accumulation. However, NPY and clonidine combined did not produce a further reduction in forskolin-induced cAMP accumulation than either compound when used alone. These results suggest that the ability of NPY to potentiate alpha 2-adrenoceptor-induced inhibition of [3H]NE release in discrete brain regions does not depend on the reductions in cAMP.     

Interactions between prolactin and dopaminergic neurons

The secretion of prolactin from the adenohypophysis is tonically inhibited by dopamine that is released into the hypophysial portal blood from terminals of tuberoinfundibular neurons located in the external layer of the median eminence. These tuberoinfundibular neurons are unique among other dopaminergic neurons in the brain (including the well-characterized nigrostriatal neurons) in that they are not directly regulated by dopaminergic receptor-mediated mechanisms, but instead are selectively responsive to changes in prolactin concentrations in blood and cerebrospinal fluid. In the rat, the activity of the tuberoinfundibular dopaminergic neurons is higher in the female than in the male, exhibits a characteristic cyclical pattern during the first half of pregnancy and is constantly high as a result of stimulation by placental lactogen during the last 9 days of pregnancy, and is reduced in lactating animals and acutely inhibited during suckling.     

Interactions between proteins bound to the duck β-globin gene promoter and enhancer detected by the DNaseI footprinting

The duck β^A-globin (β^AGLB) enhancer was closely linked to the duck β^A-GLB promoter, and the construct was used to study binding of nuclear proteins to specific sites of these regulatory elements. DNaseI-footprint analysis showed that the presence of the enhancer induced binding of proteins to additional sites on the promoter. The results are consistent with the looping-out model, based on specific interactions of enhancer-bound and promoter bound-proteins.     

Optimizing gene expression in BPV-transformed cells: effects of cell type on enhancer/promoter interaction.

We have compared several combinations of enhancers and promoters in expressing the chloramphenicol acetyl transferase gene in transient assays, in mouse C127, the most widely used host cell for the bovine papilloma virus (BPV) expression vector. Of the various combinations tested, the unit comprised of the SV40 enhancer and adenovirus type 2 major late promoter (MLP) was the most active in BPV transformed C127 cells. We further demonstrate that untransformed and BPV transformed C127 cells respond differently to the various enhancer/promoter combinations tested. Moving the SV40 enhancer closer to the cap site of a complete MLP (from -414 to -107) reduced potentiation to less than half in BPV transformed cells. The level of potentiation with enhancer at either site was similar in human HeLa cells. In BPV transformed C127 cells, the SV40 enhancer and the MLP (at the -414 site) supports 4-5 fold greater levels of expression than the murine sarcoma virus (MSV) enhancer/mouse metallothionein (MT) promoter which has previously been extremely effective in BPV vectors. Our findings provide a basis for the improvement of the BPV vector system in supporting increased levels of expression of proteins of important therapeutic application.     

The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.     

A two-component nodule-specific enhancer in the soybean N23 gene promoter.

The two positive cis elements in the soybean nodulin N23 gene promoter were investigated in transgenic Lotus corniculatus plants and shown to constitute a two-component nodule-specific enhancer. Equal quantitative contributions from the two components were suggested by the similar expression level of chimeric N23-chloramphenicol acetyltransferase genes after deletion of either the distal positive element (PE-A, -320 to -298) or the proximal positive element (PE-B, -257 to -165). A combined effect of the two elements was indicated by orientation-dependent effects in the N23 promoter, and by the observation that neither PE-A nor PE-B separately was able to confer any activity to the cauliflower mosaic virus 35S minimal promoter. Reactivation of the minimal N23 and the minimal cauliflower mosaic virus 35S promoters by the inverted complete element (PE-AB) further suggested that PE-AB is a nodule-specific enhancer containing two equally strong enhancer components. Two 12-bp sequence motifs, InvA and InvB, constituting an inverted repeat, were identified as the core of the enhancer components PE-A and PE-B, respectively. Point mutations in InvA or InvB resulted in lower expression levels and mutations in both abolished enhancer activity. Point mutations in two nodulin consensus sequences, 5'-CTCTT and 5'-AAAGAT located downstream of PE-AB, resulted in a decreased level of expression, confirming the involvement of these two motifs in nodulin gene expression. The binding site for the nodule-specific trans-acting factor, NAT2, present in the PE-A segment, was removed without affecting expression significantly. This interaction is, therefore, dispensable for enhancer activity.     

UV inducibility of rat proliferating cell nuclear antigen gene promoter.

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase delta, is required for eukaryotic cell DNA synthesis and nucleotide excision repair. Expression of PCNA gene is growth-regulated and UV inducible. In our previous study, we have observed that the rat PCNA promoter has the serum responsiveness. In this study, we demonstrate its UV inducibility in CHO.K1 cells. The UV induction of the rat PCNA promoter activity was dose-dependent in the cells synchronized at different phases. In addition, the sequences of the promoter responsible for the UV inducibility were delimited to the region between nucleotides -70 and +125, which contains an AP-1 site and a downstream proximal ATF/CRE site. While mutation of the AP-1 site abrogated the UV inducibility, mutation of the ATF/CRE site enhanced the UV inducibility, suggesting that the two sites play different roles in the UV induction of the promoter. In addition, the role of p53 in the UV induction of rat PCNA promoter was investigated. We found that exogenous p53 was unable to mimic the UV irradiation to induce rat PCNA promoter and that the UV induction of the rat PCNA promoter was seen in p53 deficient cells. Therefore, it is unlikely that the UV induction of the rat PCNA promoter is p53 dependent.