Rat Parotid Cells Research Articles

The inhibition of cyclic AMP-mediated exocytosis from rat parotid cells by calcium chelation is due to depression of cellular ATP

The role of second messenger calcium in cAMP-mediated exocytosis from parotid cell aggregates has been assessed following extracellular (ethylene glycol bis( β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA)) and intracellular (quin2) calcium chelation. Only in the former case was inhibition (60–70%) of isoproterenol- and N 6, O 2-dibutyryl cAMP-stimulated amylase release observed. This inhibition was accompanied by a 70% decrease in cellular ATP levels. Depression of ATP levels with mitochondrial inhibitors was also correlated with inhibition of amylase secretion. Overall, our results suggest that depletion of cellular Ca 2+ with EGTA leads to an inhibition of mitochondrial function and that these experimental conditions are therefore inappropriate for the evaluation of the role of cytosolic Ca 2+ transients during stimulus-secretion coupling.

Kinetics of inositol 1,4,5-trisphosphate and inositol cyclic 1:2,4,5-trisphosphate metabolism in intact rat parotid acinar cells. Relationship to calcium signalling.

Stimulation of rat parotid acinar cells by the muscarinic cholinergic receptor agonist methacholine results in the formation of inositol 1,4,5-trisphosphate [1,4,5)IP3) and inositol cyclic 1:2,4,5-trisphosphate [c1:2,4,5)IP3) which, after 40 min, accumulate to a ratio of 1:0.57. The turnover rates of these inositol trisphosphates have been determined in cholinergically stimulated rat parotid cells by measuring the degradation of the 3H-labeled compounds following receptor blockade. (1,4,5)IP3 is rapidly metabolized, with a half-time of 7.6 s; (c1:2,4,5)IP3 declines much more slowly with a half-time of almost 10 min. Because the formation and metabolism of (c1:2,4,5)IP3 are so slow, (c1:2,4,5)IP3 gradually accumulates upon prolonged receptor activation. Inositol trisphosphate turnover was compared to the receptor-mediated changes in cytoplasmic Ca2+ concentration, as measured by the fluorescent Ca2+ indicator, fura-2. The Ca2+ signal decays upon termination of inositol phosphate formation and returns to base line within 30 s. Thus, while (c1:2,4,5)IP3 may have some yet unknown biological effects on Ca2+ homeostasis, its metabolism seems far too slow to be the primary regulator of cytosolic Ca2+ levels under long term stimulatory conditions. The rate at which the Ca2+ signal decays is, however, somewhat slowed after prolonged agonist stimulation. Furthermore, the capacity of the cells to mobilize intracellular Ca2+ in response to a second agonist stimulation is slightly delayed when the duration of the first stimulus is prolonged. The results suggest that the regulation of cytoplasmic Ca2+ levels may be more complicated than initially realized and could depend on the combined actions of more than one inositol polyphosphate.

Modification of ATP-dependent Ca 2+ transport in rat parotid basolateral membranes during aging

Previous experiments have shown that senescent rat parotid acinar cells display marked reductions in Ca 2+ release following α 1-adrenoreceptor stimulation. We report here, that in this naturally occurring perturbation of exocrine secretion, ATP-dependent Ca 2+ transport in the parotid basolateral plasma membrane, the principal Ca 2+ extrusion pathway in the parotid, is also modified. ATP-dependent Ca 2+ transport in membrane vesicles isolated from senescent rats (approx. 24 months) is decreased approximately 30–50% as compared to that in vesicles isolated from younger rats (approx. 4 months). This alteration in Ca 2+ pump activity is not due to (i) non-specific effects of vesicle preparation in the two animal groups, (ii) increased leakiness to Ca 2+, or (iii) any apparent alteration in permeability of the membrane to K + and Cl −. Kinetic studies demonstrate that the ATP-dependent Ca 2+ transport activity in vesicles from senescent rats has similar maximal velocity to that of vesicles from young adult rats (27 vs. 31 nmol Ca 2+/mg protein per min), however, it exhibits an approximately 50% increase in K m for Ca 2+ (91 nM vs. 60 nM). Cytosolic free Ca 2+, measured by Quin 2 fluorescence, in parotid acini following α 1-adrenoreceptor stimulation was much less elevated in preparations from senescent rats. These results may account, at least in part, for the previously reported physiological alteration in Ca 2+ efflux seen in senescent rat parotid cells.

Age-related impairment in rat parotid cell α1-adrenergic action at the level of inositol trisphosphate responsiveness

α 1-Adrenergic-stimulated calcium efflux from rat parotid cell aggregates declines approx. 40% between 3 and 24 months of age, with the bulk of the reduction occurring between 12 and 24 months. Intracellular free calcium levels following α 1-adrenoceptor stimulation are also reduced about 40% between 3 and 24 months. No significant age differences in stimulation of inositol mono-, bis- or trisphosphate production are observed. However, the ability of inositol trisphosphate to directly stimulate calcium efflux is reduced by about 50% with increasing age. Concentrations of this inositol phosphate required for maximal calcium release do not change between 3 and 24 months. Differences in response are not due to a reduction in uptake of inositol trisphosphate into older cells, but suggest an age-related defect in the ability of inositol trisphosphate to liberate calcium from intracellular stores. Such dysfunction may be at least partially responsible for impaired α 1-adrenergic responsiveness during aging.

Effect of NaF on cAMP accumulation, cAMP-dependent protein kinase activity in, and amylase secretion from, rat parotid gland cells.

Stimulation of amylase secretion from parotid glands by beta-adrenergic agonists is mediated by the activation of adenylate cyclase and the resultant increase in cellular cAMP. Since NaF is known to increase adenylate cyclase activity and cAMP accumulation in intact cells, we investigated whether it would stimulate amylase secretion from isolated rat parotid gland cells. The results provide evidence that the addition of NaF (0.01-10 mmol/L) increased cAMP concentration (1.5-2.8-fold) in, and amylase secretion (16-93%) from, isolated parotid gland acinar cells. NaF was found to increase cAMP-dependent protein kinase activity ratios (51-84%) in a concentration- and time-dependent manner. The data suggest that the stimulation of amylase secretion from parotid gland cells by NaF may be mediated by an increase in the cellular cAMP concentration, which exerts its effect, at least in part, by increasing the activity of cAMP-dependent protein kinase.

N-linked protein glycosylation in the rat parotid gland during aging

N-Linked protein glycosylation was examined in vitro in dispersed rat parotid acinar cells from young adult (3–6 months) and aged (22–24 months) rats. A small decrease in general protein production was observed with cells from aged animals (∼20% lower incorporation of [ 14C]leucine into 10% CCI 3 COOH insoluble protein during continuous pulse labeling). Incorporation of [ 3H]mannose into N-linked glycoproteins by aged cells was further reduced (∼35%). Similarly microsomal membranes from parotid glands of aged animals showed ∼50% reduction in the synthesis of mannosylphosphoryl dolichol, a key intermediate in the dolichol pathway of protein N-glycosylation. Man- P-Dol synthase, the microsomal enzyme responsible for production of this saccharide-lipid, displayed no change in apparent K m for GDP-mannose when preparations from aged animals were utilized, but did show ∼50% reduction in V max. Following β-adrenoreceptor activation, cells from both young adult and aged glands showed increased N-linked protein glycosylation almost to the same extent (∼2-fold). The data suggested that in aged rat parotid cells there is a basal reduction of activity in the pathway responsible for asparagine-linked protein glycosylation, but that following exocytotic stimuli this pathway responds in a manner comparable to cells from young adult glands.

High K + elevates cytosolic free Ca concentration due to mobilization from internal storage sites in rat parotid cells

1. 1. Effects of high K + on cytosolic free Ca concentration ([Ca 2+] i) in rat parotid cells were studied using quin2. 2. 2. High K + elevated [Ca 2+] i in a dose-dependent manner in normal and Ca-free media. The elevation of [Ca 2+], induced by high K + was less in the latter medium. 3. 3. High K + depolarized the membrane in a dose-dependent manner in normal and Ca-free media. 4. 4. Although monensin increased [Ca 2+] i, high K + did not affect 22Na uptake into cells. 5. 5. After treatment with oligomycin, high K + but not carbachol raised [Ca 2+] i. 6. 6. We suggest that high K + increases [Ca 2+] i due to mobilizing Ca 2+ from the intracellular storage site which does not need energy.

Modulation of oligosaccharide processing in an exocrine secretory glycoprotein of rat parotid cells by β-adrenoreceptor activation

Such stimulation of rat parotid acinar cells in vitro modulated the rate of processing of N-linked oligosaccharides in a high-molecular weight (220 kdalton) secretory glycoprotein. Conversion of polymannose-type oligosaccharides to complex-type oligosaccharides was evaluated by sensitivity to endoglucosaminidase H and α-mannosidase, and with a specific inhibitor of glucosidases I/II. Oligosaccharide maturation in the 220 kdalton glycoprotein required one-third to half less time in cells exposed to the β-adrenergic agonist isoproterenol than in controls.

Effects of aging on parathyroid hormones stimulated ionic fluxes in rat parotid cell aggregates

The effects of aging on parathyroid hormone stimulated calcium and phosphate efflux from isolated rat parotid cell aggregates differed from that of epinephrine stimulated calcium efflux in magnitude, chronology, and ionic dependence. Parathyroid stimulated ionic fluxes were maximal at 1–3 months of age, declined until 12 months and remained constant thereafter. Epinephrine stimulated calcium efflux remained maximal between 3 and 12 months and declined until at least 24 months. Absolute stimulation of calcium efflux over basal levels was maximal at 4–8% while phosphate efflux was 20–30%.

The effect of calcium and cyclic AMP on amylase release in digitonin-permeabilized parotid gland cells

Rat parotid cells were permeabilized with digitonin to examine their secretory dynamics. Cells were isolated by a modification of the method previously described by Hootman ((1985). J. Biol. Chem. 260, 4186–4194) in which α-chymotrypsin was included. The final preparation consisted of approx. 40–60% single cells. The cells were 85–90% viable by trypan blue exclusion and secreted amylase when stimulated with isoproterenol. Digitonin (2 or 5 μM) was sufficient for permeabilization while 2 μM digitonin was somewhat more effective in maintaining cell integrity as indicated by lactate dehydrogenase release. Digitonin had minimal effects on intracellular granules in the whole cell and was, thus, relatively selective. The response of digitonin-permeabilized cells to calcium (without secretagogues) in the incubation medium was monitored by amylase release. For a wide range of applied free calcium concentrations (1·10 −7 M to 10 −4 M) a statistically significant increase in amylase secretion was observed. Control cells did not release amylase to a similar extent without secretagogue. Cyclic AMP (50 μM) significantly enhanced amylase secretion from digitonin-treated cells at all concentrations of free calcium tested. Neither calcium nor cyclic AMP alone was sufficient to stimulate maximal amylase release. Our results provide direct evidence for a model in which calcium and cyclic AMP work on separate pathways as interacting regulators of exocytosis.

Effects of sodium ions and monensin on amylase secretion from rat parotid cells

The role of sodium ions in amylase secretion from rat parotid cells was studied using various Na +-free media and monensin. In a sucrose medium, amylase secretion was not stimulated by isoproterenol but was significantly stimulated by dibutyryl cAMP. In choline chloride and LiCl media, both isoproterenol and dibutyryl cAMP clearly evoked amylase release. Monensin itself elicited amylase secretion slightly, but significantly inhibited the secretion stimulated by isoproterenol or dibutyryl cAMP. The inhibitory effect of monensin was detectable even in choline chloride, LiCl and KCl media. These results indicate that sodium ions are not essential for amylase secretion from rat parotid cells and that the inhibitory effect of monensin is independent of influx of sodium ions or efflux of potassium ions.

Effects of Ca 2+ agonist and antagonists on cytosolic free Ca 2+ concentration: Studies on Ca 2+ channels in rat parotid cells

1. Effects of Ca 2+ agonist and antagonists on cytosolic free Ca 2+ concentration ([Ca 2+] i) were studied using quin2. 2. Nicardipine (NIC), diltiazem (DIL) and verapamil (VER) had no effect on the rise in [Ca 2+] i evoked by carbachol. Methoxamine-elevated [Ca 2+] i was inhibited by VER but not by NIC and DIL. 3. All Ca 2+ antagonists tested produced a decline of [Ca 2+] i elevated by isoproterenol to the resting level. 4. The addition of 30 mM K + gradually elevated [Ca 2+] i in normal and Ca 2+-free media, but it did not increase 45Ca 2+ uptake into cells. BAY K 8644 did not increase [Ca 2+] i. 5. We suggest that voltage-sensitive Ca 2+ channels are lacking and that at least 2 distinct receptor-operated Ca 2+ channels exist in rat parotid cells.

P-85 - High K+ elevates cytosolic free Ca concentration due to the nobi1ization from storage sites in rat parotid cells

P-85 - High K+ elevates cytosolic free Ca concentration due to the nobi1ization from storage sites in rat parotid cells

O-302 - Modulation of rat parotid cell α1-adrenergic responsiveness during aging

O-302 - Modulation of rat parotid cell α1-adrenergic responsiveness during aging

Does cyclic AMP mobilize Ca 2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase are released from rat parotid cells incubated in Ca 2+-free medium containing 1 mM EGTA. Cells preincubated with 10 μM carbachol in Ca 2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 μM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.

Parathyroid Hormone Stimulates Phosphate Efflux through an Apparently Adenosine 3′,5′-Monophosphate-Independent Process in Rat Parotid Cell Aggregates

The effects of PTH on phosphate fluxes and cAMP and protein secretion in rat parotid cell aggregates were studied. PTH-(1-34) as well as PTH-(1-84) stimulated phosphate efflux in a dose-dependent [5 X 10(-7) to 7 X 10(-6) M for human (h) PTH-(1-34), 2 X 10(-7) to 5 X 10(-6) M for bovine (b) PTH-(1-34), and 3.8-114 U/ml for bPTH-(1-84)] and time-dependent [detectable by 15-30 min after incubation with N-terminal PTH and 30-45 min after incubation with bPTH-(1-84)] manner. The effect of PTH on phosphate efflux was saturable, with maximal stimulation at 10(-6) M above for N-terminal PTH and at 57.8 U/ml and above for bPTH-(1-84). Half-maximal stimulation of phosphate efflux by hPTH-(1-34), bPTH-(1-34), and bPTH-(1-84) was achieved at 5.7 X 10(-7) M, 10(-7) M, and 40 U/ml, respectively. Neither bPTH-(1-34) nor bPTH-(1-84) had any effect on phosphate uptake by parotid cell aggregates at the concentrations examined. bPTH-(1-34) and bPTH-(1-84) also stimulated amylase secretion, with a significant increase in cAMP production (70-90% over basal). However, (BU)2cAMP did not stimulate phosphate efflux. Isoproterenol (10(-5) M) markedly stimulated cAMP production and amylase secretion without causing a significant increase in phosphate efflux. Oxidized bPTH-(1-84) and the C-terminal fragment of hPTH had no stimulating effect on phosphate efflux. The present results suggest, therefore, that PTH may play an important role in regulating rat parotid cell phosphate metabolism through an apparently cAMP-independent process.

Impaired adrenergic stimulation of rat parotid cell glucose oxidation during aging: the role of calcium.

Adrenergic stimulation of glucose oxidation by epinephrine is reduced by 30 to 40% in isolated rat parotid cell aggregates from aged rats compared with young adults. Such impairment affects both the external calcium dependent and independent components of the response. Age-related differences in glucose oxidation are obliterated if adrenergic receptors are bypassed and stimulation achieved by exposing cells to various concentrations of calcium in the presence of the ionophore, A23187. Thus, impaired calcium mobilization may play, in part, a role in reduced rat parotid adrenergic metabolic responsiveness during aging.

Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca 2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca 2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.

P-224 - Effects of Ca antagonists on cytosolic free Ca concentration elevated by autonomic agents in dispersed rat parotid cells

P-224 - Effects of Ca antagonists on cytosolic free Ca concentration elevated by autonomic agents in dispersed rat parotid cells

Involvement of cyclic AMP and calcium in exocrine protein secretion induced by vasoactive intestinal polypeptide in rat parotid cells

The effects of vasoactive intestinal polypeptide (VIP) on exocrine protein secretion were studied in enzymatically dispersed cell aggregates from rat parotid glands. VIP (0 −9–10 −7 M) stimulated secretion of α-amylase in a dose-dependent manner. The VIP-induced release of α-amylase was potentiated in the presence of a phosphodiesterase inhibitor. Basal levels of cyclic AMP of the dispersed cells were increased 6.7-fold after stimulation for 10 min by VIP (10 −7 M). The VIP-induced release of α-amylase was reduced by 40% when cells were incubated in a Ca 2+-free medium in the presence of ethylene glycol bis( β-aminoethyl ether)- N, N′-tetraacetic acid (EGTA). Efflux of 45Ca 2+ was significantly increased over basal levels by stimulation with VIP (10 −8 and 10 −7 M), but this increased efflux was approximately only half the increased efflux induced by carbachol (10 −5 M). VIP had no effect on the incorporation of [ 14C]leucine into protein by parotid cells, whereas incorporation was reduced to 30% of the control value by carbachol (10 −5 M). Thus, the VIP-ergic secretory response in the rat parotid gland is associated with a raised intracellular cyclic AMP level and the mobilisation of a different intracellular Ca 2+ pool than that mobilised by carbachol. It is, therefore, closely analogous to the β-adrenergic response.