Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Cloning of genes for a hemolytic factor of Leptospira interrogans serovar autumnalis strain Congo 21-543

A DNA fragment encoding a hemolytic factor was cloned from the parasitic spirochete Leptospira interrogans serovar autumnalis strain Congo 21-543. Initial clones were isolated by screening a genomic library in pBR322 in Escherichia coli for hemolytic activity. Hemolytic activity was coded by a 4.5 kilobase BamHI-HindIII fragment. Southern hybridization with DNAs from other strains of Leptospira using this gene as a probe showed that DNAs from non-parasitic strains failed to hybridize with the probe, whereas those from all parasitic strains tested had the sequence which hybridize to the probe.     

Localization of follicle regulatory protein in the porcine ovary

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.     

Intercellular CO2 concentration and water-use efficiency of temperate plants with different life-forms and from different microhabitats

Summary Photosynthesis and transpiration were measured simultaneously, under near-optimum and constant environmental conditions, in intact leaves of plants native to the temperate forest region. A linear relationship between photosynthetic rate and stomatal conductance was found in every species tested irrespective of leaf age or season, indicating that the calculated intercellular CO₂ concentration and water-use efficiency were fairly constant within a species. The values of intercellular CO₂ concentration and water-use efficiency ranged from 221 to 271 l l^–1 and 4.46 to 8.20 mol CO₂ mmolH₂O^–1 (6.24±0.90 mol CO₂ mmolH₂O^–1), respectively. The variations in intercellular CO₂ concentration and water-use efficiency were not directly related to photosynthetic capacities, life-forms, or microhabitat preferences. The intercellular CO₂ concentrations found in this study were close to values reported from cultivated plants and plants native to more arid regions, suggesting a common mechanism to maintain the stomatal conductance proportional to photosynthetic capacity over a wide variety of C₃ plants.     

Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937.     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.