scholarly journals Isoprenaline-induced transcription of 4β-galactosyltransferase is inhibited by both cycloheximide and actinomycin D in rat parotid acinar cells

1988 ◽  
Vol 249 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M G Humphreys-Beher
Keyword(s):  
Steady State ◽  
Actinomycin D ◽  
Specific Activity ◽  
Acinar Cells ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Parotid Acinar Cells ◽  
Mrna Content ◽  
Polymerase Inhibitor ◽  
Rat Parotid

A cDNA clone for the Golgi enzyme 4 beta-galactosyltransferase (EC 2.4.1.38) was used to determine the steady-state mRNA content in cultured rat parotid acinar cells. Isoprenaline, a beta-adrenergic-receptor agonist, caused an increase in steady-state amounts of mRNA for 4 beta-galactosyltransferase in cultured acinar cells as well as in specific activity of the enzyme. The amount of 4 beta-galactosyltransferase-specific mRNA was dependent on transcription of the gene, as determined by incubation of cells with the RNA polymerase inhibitor actinomycin D, concomitant with the time of isoprenaline treatment. Transcription of the 4 beta-galactosyltransferase gene also required the active biosynthesis of additional cellular factors, since isoprenaline-induced increases in mRNA amounts were not observed on co-incubation with the protein-synthesis inhibitor cycloheximide.

scholarly journals Quinones increase γ-glutamyl transpeptidase expression by multiple mechanisms in rat lung epithelial cells

1998 ◽  
Vol 274 (3) ◽  
pp. L330-L336 ◽  
Author(s):  
Rui-Ming Liu ◽  
Michael Ming Shi ◽  
Cecilia Giulivi ◽  
Henry Jay Forman
Keyword(s):  
Specific Activity ◽  
Lung Epithelial Cells ◽  
Secondary Response ◽  
Protein Synthesis Inhibitor ◽  
Rat Lung ◽  
Synthesis Inhibitor ◽  
Lung Epithelial ◽  
Mrna Content ◽  
Gsh Metabolism ◽  
Glutamyl Transpeptidase

γ-Glutamyl transpeptidase (GGT) plays an important role in glutathione (GSH) metabolism. GGT expression is increased in oxidant-challenged cells; however, the signaling mechanisms involved are uncertain. The present study used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone that continuously produced H2O2in rat lung epithelial L2 cells. It was found that DMNQ increased GGT mRNA content by increasing transcription, as measured by nuclear run-on. This was accompanied by increased GGT specific activity. Cycloheximide, a protein synthesis inhibitor, blocked neither the increased GGT mRNA content nor the increased GGT transcription rate caused by DMNQ, suggesting that increased GGT transcription was a direct rather than secondary response. Previous data from this laboratory (R.-M. Liu, H. Hu, T. W. Robison, and H. J. Forman. Am. J. Respir. Cell Mol. Biol.14: 186–191, 1996) showed that tert-butylhydroquinone (TBHQ) increased GGT mRNA content by increasing its stability. TBHQ differs markedly from DMNQ in terms of its conjugation with GSH and H2O2generation. Together, the data suggest that quinones upregulate GGT through multiple mechanisms, increased transcription and posttranscriptional modulation, which are apparently mediated through generation of reactive oxygen species and GSH conjugate formation, respectively.

scholarly journals Regulation of phosphatidate synthesis by secretagogues in parotid acinar cells

1982 ◽  
Vol 204 (2) ◽  
pp. 587-592 ◽  
Author(s):  
S J Weiss ◽  
J S McKinney ◽  
J W Putney
Keyword(s):  
Steady State ◽  
Substance P ◽  
Specific Radioactivity ◽  
Acinar Cells ◽  
Half Time ◽  
Muscarinic Antagonist ◽  
Parotid Acinar Cells ◽  
Rat Parotid ◽  
In Cells

The metabolism of phosphatidate in rat parotid acinar cells was investigated, particularly with regard to the actions of agonists known to act by mobilizing Ca2+. When cells were incubated in medium containing 10 microM-[32P]Pi, phosphatidate was rapidly labelled, approaching an apparent steady-state with a half-time of approx. 20 min. Methacholine provoked a more than doubling of phosphatidate radioactivity, which was reversed by the muscarinic antagonist atropine. These results suggest that phosphatidate labels to near steady-state rapidly and that in cells prelabelled for 60 min the increase in radioactivity induced by agonists probably reflects net synthesis rather than an increase in specific radioactivity. Phosphatidate synthesis in response to methacholine was rapid and occurred, within the resolution of a few seconds, with no measurable latency. Adrenaline and substance P also stimulated phosphatidate synthesis but both agonists were less efficacious than methacholine. A Ca2+ ionophore, ionomycin, did not provoke phosphatidate synthesis. By using a protocol that eliminates the receptor-regulated Ca2+ pool, it was demonstrated that methacholine-induced phosphatidate formation does not come about as a consequence of Ca2+ influx nor of Ca2+ release. These results indicate that the phosphatidate synthesis response has characteristics compatible with its previously suggested role as a primary mediator of membrane Ca2+-gating.

scholarly journals Activation of calcium-dependent chloride channels in rat parotid acinar cells.

1996 ◽  
Vol 108 (1) ◽  
pp. 35-47 ◽  
Author(s):  
J Arreola ◽  
J E Melvin ◽  
T Begenisich
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Acinar Cells ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Channel Activation ◽  
Parotid Acinar Cells ◽  
Steady State Current ◽  
Rat Parotid ◽  
The Hill

The Ca2+ and voltage dependence of Ca(2+)-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp technique. Acinar cells were dialyzed with buffered free Ca2+ concentrations ([Ca2+]i) from < 1 nM to 5 microM. Increasing [Ca2+]i induced an increase in Cl- current at all membrane potentials. In cells dialyzed with [Ca2+]i > 25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential component. The steady-state current-voltage relationship showed outward rectification at low [Ca2+]i but became more linear as the [Ca2+]i increased because of a shift in Cl- channel activation toward more negative voltages. The Ca2+ dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparent Kd and Hill coefficient obtained from this analysis were both functions of membrane potential. The Kd decreased from 417 to 63 nM between -106 and +94 mV, whereas the Hill coefficient was always > 1 and increased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channel steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2+ binding steps before a final Ca(2+)-independent transition to the conducting conformation. Channel activation proceeds sequentially through three closed states before reaching the open state. The Ca2+ binding steps of this model have a voltage dependence similar to that of the Kd from the Hill analysis. The simplest interpretation of our findings is that these channels are directly activated by Ca2+ ions that bind to sites approximately 13% into the membrane electric field from the cytoplasmic surface.

scholarly journals β-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells

1985 ◽  
Vol 231 (2) ◽  
pp. 431-438 ◽  
Author(s):  
S R Grant ◽  
E E Kousvelari ◽  
D K Banerjee ◽  
B J Baum
Keyword(s):  
Half Life ◽  
Specific Radioactivity ◽  
Acinar Cells ◽  
Protein Glycosylation ◽  
Significant Enhancement ◽  
Adrenergic Stimulation ◽  
Parotid Acinar Cells ◽  
Secretory Glycoproteins ◽  
Rat Parotid ◽  
Stimulation Of

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by β-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after β-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.

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