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 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.

Substance P-induced diacylglycerol formation in rat parotid acinar cells

1991 ◽  
Vol 207 (4) ◽  
pp. 329-335 ◽  
Author(s):  
Takao Komabayashi ◽  
Atsushi Yakata ◽  
Tetsuya Izawa ◽  
Kazuhiro Suda ◽  
Masamichi Noguchi ◽  
...  
Keyword(s):  
Substance P ◽  
Acinar Cells ◽  
Parotid Acinar Cells ◽  
Rat Parotid

scholarly journals Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells

1988 ◽  
Vol 253 (2) ◽  
pp. 459-466 ◽  
Author(s):  
H Sugiya ◽  
J F Obie ◽  
J W Putney
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Substance P ◽  
Acinar Cells ◽  
Control Mechanisms ◽  
Dependent Manner ◽  
Parotid Acinar Cells ◽  
Kinase C ◽  
Substance P Receptor ◽  
Rat Parotid

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.

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 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 Intracellular calcium signalling in rat parotid acinar cells that lack secretory vesicles

1998 ◽  
Vol 330 (2) ◽  
pp. 847-852 ◽  
Author(s):  
Peixin LIU ◽  
John SCOTT ◽  
Peter Matthew SMITH
Keyword(s):  
Parotid Gland ◽  
Calcium Signalling ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Vesicles ◽  
Apical Pole ◽  
Parotid Acinar Cells ◽  
Parotid Duct ◽  
Rat Parotid ◽  
In Cells

Secretory vesicles from pancreatic acinar cells have recently been shown to release Ca2+ after stimulation with Ins(1,4,5)P3 [Gerasimenko, Gerasimenko, Belan and Petersen, (1996) Cell 84, 473-480]. These observations have been used in support of the hypothesis that Ca2+ release from secretory vesicles could be an important component of stimulus secretion coupling in exocrine acinar cells. In the rat, ligation of the parotid duct causes a reversible atrophy of the parotid gland. Most notably, after atrophy the acinar cells are reduced in size and no longer contain secretory vesicles [Liu, Smith, and Scott (1996) J. Dent. Res. 74, 900]. We have measured cytosolic free-Ca2+ concentration ([Ca2+]i) in single, acutely isolated, rat parotid acinar cells, and compared Ca2+ mobilization in response to acetylcholine (ACh) stimulation in cells obtained from control animals to that in cells lacking secretory vesicles obtained after atrophy of the parotid gland. Application of 50-5000 nM ACh to control cells gave rise to a typical, dose-dependent, biphasic increase in [Ca2+]i, of which the later, plateau, phase was acutely dependent on the extracellular Ca2+ concentration. An identical pattern of response was observed with cells obtained from atrophic glands. Low concentrations of ACh (10-100 nM) occasionally produced [Ca2+]i oscillations of a similar pattern in cells from both control and atrophic glands. We were able to show that Ca2+ rises first in the apical pole of the cell and the increase then spreads to the rest of the cell in cells from control glands but not in cells from atrophic glands. However, at present we are unable to determine whether this is due to the lack of secretory vesicles or whether the separation is too small to measure in the smaller acinar cells obtained from atrophic glands. We conclude therefore, that secretory vesicles make no significant contribution to overall Ca2+ mobilization in rat parotid acinar cells, nor are they required for oscillatory changes in [Ca2+]i to occur. However we are unable to eliminate completely any role for secretory vesicles in initiating Ca2+ mobilization at the apical pole of the cell.

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