Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.

We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways.

Rat submandibular and parotid protein phosphorylation and exocytosis: effect of site-selective cAMP analogs.

A series of cAMP analogs that have different specificities for the two different binding sites on the regulatory subunit of type I and type II cAMP-dependent protein kinase (PKA) were used to determine whether selective activation of type I or type II PKA could link either or both isozyme forms of PKA with exocytosis and specific protein phosphorylation in salivary gland cells. Using dispersed rat submandibular or parotid cells, selective activation of either type I or type II resulted in a synergistic response for both rat submandibular mucin and parotid amylase secretion and the phosphorylation of a 26-kDa integral membrane phosphoprotein. These data suggest that the activation of either isozyme of PKA can elicit cellular exocytosis and specific protein phosphorylation in both of these cell types.

Purification and partial characterization of analogous 26-kDa rat submandibular and parotid gland integral membrane phosphoproteins that may have a role in exocytosis.

Rat submandibular and parotid gland exocytosis is primarily controlled by beta-adrenergic receptor stimulation. Although its precise role in the regulation of salivary gland exocytosis is not fully understood, protein phosphorylation, mediated by the activation of cAMP-dependent protein kinase, may be directly involved. Previous studies suggest that analogous 26-kDa integral membrane phosphoproteins may play a direct role in regulating exocytosis. Studies were here undertaken to purify and partially characterize both phosphoproteins. After endogenous phosphorylation with 32P, subcellular fraction and solubilization of the microsomal fraction in n-octyl beta-glucopyranoside, the 26-kDa integral membrane phosphoproteins were purified by high performance liquid chromatography (HPLC), followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroelution of the proteins. Amino acid analysis indicated a significant number of serine amino acids: N-terminal sequence data demonstrated a high level of homology; and trypsin digestion followed by reversed-phase HPLC indicated the possibility of multiple phosphorylation sites.

Activation and distribution of rat parotid cAMP-dependent protein kinase following beta-adrenergic receptor stimulation in vitro.

The extent of activation of parotid protein kinase A (EC 2.7.1.37) isozymes was determined using dispersed cells and an 8-N3-[32P]-cAMP photoprobe. Cold-trap studies indicated that 40% of type I protein kinase A was activated following maximal beta-adrenergic receptor stimulation, whereas type II activation was less than 20%. Both cytosolic and microsomal type I activation occurred rapidly after stimulation and both remain activated throughout the entire secretory period. The dose-response relationship for the isotypes following beta-adrenergic receptor activation demonstrated a greater extent of type I activation at maximal concentration of agonist. Although protein kinase A may not be the only kinase involved in rat parotid amylase release, these findings add further evidence of a direct regulatory role for this kinase, with type I having potentially a greater role than type II.

Evidence against a direct role for protein kinase C in rat submandibular salivary mucin secretion.

Calcium and protein kinase C may be directly involved in exocytosis. However, in the rat submandibular gland, cAMP-mediated events appear to be required for mucin secretion. Calcium may be involved, but a direct signal-transduction role for calcium and protein kinase C in regulating such secretion has yet to be established. With dispersed rat submandibular acinar-intercalated duct complexes, endogenous protein phosphorylation and mucin secretion studies were performed to determine if 12-O-tetradecanoylphorbol 13-acetate (PMA), a specific activator of protein kinase C, could act as an effective secretagogue for mucin secretion and if specific protein phosphorylation could be assigned to protein kinase C activation. PMA did not elicit such phosphorylation and it only slightly increased mucin secretion at high concentrations; these slight increases appeared to be non-specific. Therefore, protein kinase C activation may not be directly involved in regulating rat submandibular mucin secretion.

Subcellular distribution and activation of rat submandibular cAMP-dependent protein kinase following beta-adrenergic receptor stimulation.

The extent of activation of rat submandibular protein kinase A (EC 2.7.1.37) isozymes following beta-adrenergic receptor stimulation was determined in vitro using dispersed cells and an 8-N3-[32P]cAMP photoprobe. The half-maximal binding of the photoprobe for microsomal and cytosolic type I and cytosolic type II was 9 nM, 27 nM and 92 nM, respectively. 'Cold trap' studies indicated that 70% of type I protein kinase A was activated following maximal beta-adrenergic receptor stimulation, whereas type II activation was less than 40%. Both cytosolic and microsomal type I activation occurred rapidly following beta-adrenergic receptor stimulation and both remain activated throughout the entire secretory period. Type I inactivation occurred rapidly subsequent to beta-adrenergic receptor blockade. The dose-response relationship for the isotypes following beta-adrenergic receptor activation demonstrated a greater extent of type I activation at submaximal concentrations of agonist. Although protein kinase A may not be the only kinase involved in rat submandibular mucin release, these data add further support to a direct regulatory role for this kinase, with type I having potentially a greater role than type II.

The rate-determining step in cAMP-mediated exocytosis in the rat parotid and submandibular glands appears to involve analogous 26-kDa integral membrane phosphoproteins.

The possible direct involvement of protein phosphorylation in the regulation of exocytosis during beta-adrenergic receptor stimulation in rat parotid and submandibular salivary glands was investigated in vitro using dispersed cells. The dispersed cells were labeled with [32P]orthophosphate for 40 min prior to experimental manipulation. Subcellular fractions were isolated, the proteins were separated using sodium dodecylsulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE), and the phosphoproteins were detected by autoradiography. Changes in the extent of phosphorylation for each phosphoprotein were determined indirectly by densitometric analyses. The analogous parotid and submandibular 26-kDa membrane phosphoproteins had a rapid phosphate turnover rate (t 1/2 = 5-6 min) whereas the analogous 21-kDa membrane phosphoproteins had a much slower phosphate turnover rate (t 1/2 greater than 20 min). The results of Triton X-114 extraction indicated that the 26- and 21-kDa phosphoproteins were integral membrane proteins. The rate of phosphate turnover for the analogous 26-kDa phosphoproteins is compatible with a regulatory role in exocytosis, whereas the slower phosphate turnover rate for the analogous 21-kDa phosphoproteins suggests that these proteins may play a more subordinate role in secretion or they may coordinate secretion with other cellular metabolic events.

Effect of serum from normal and cystic fibrosis subjects on mucin secretion from dispersed rat submandibular cells.

The action of serum from cystic fibrosis patients, obligatory heterozygotes, siblings, and normal patients on mucin secretion from dispersed rat submandibular cells was investigated in an attempt to demonstrate the existence of cystic fibrosis specific factors affecting mucin secretion. No effects specific to cystic fibrosis serum were demonstrated using the following parameters for evaluation: (1) maximal stimulation of mucin release by a beta-adrenergic agonist, (-)-isoproterenol; (2) basal release (unstimulated secretion) of mucin material; and (3) the dose-response relationship for mucin release after beta-adrenergic receptor stimulation. No differences were observed in mucin secretion using media concentrations of serum of up to 10%. From these data, we conclude that serum from individuals with cystic fibrosis does not contain bioactive factors at concentrations that specifically alter mucin secretion in rat submandibular acinar cells.

Role of protein phosphorylation in regulating rat submandibular mucin secretion.

The possible involvement of protein phosphorylation during beta-adrenergic receptor stimulation in rat submandibular gland was investigated in vitro using a dispersed cell preparation. (-)-Isoproterenol, a beta-adrenergic agonist, or dibutyryl cAMP stimulation was associated with an enhanced phosphorylation of three protein bands having apparent molecular weights of 34,000, 26,000, and 21,000, respectively. (-)-Propranolol, a beta-adrenergic antagonist, inhibited the phosphorylation of the three proteins during beta-adrenergic stimulation but not during dibutyryl cAMP stimulation. The three proteins were not fragments of a higher-molecular-weight protein. Subcellular fractionation using differential centrifugation, fractionation in an aqueous two-phase polymer system, and discontinuous sucrose gradient ultracentrifugation coupled with marker enzyme analysis indicated that all three proteins were enriched in the same subfractions: a heavy plasma membrane fraction and a fraction containing plasma membrane and Golgi membrane material. The extent of protein phosphorylation for all three proteins increased as a function of time and dose after beta-adrenergic stimulation. After 20 min of maximal beta-adrenergic stimulation, the addition of a beta-adrenergic blocker caused a time-dependent decrease in the 32P content of all three proteins. Pure cholinergic or pure alpha-adrenergic receptor stimulation had no effect on the 32P content of the three proteins. These data are consistent with a role for cAMP-mediated protein phosphorylation during mucin secretion from rat submandibular cells.

Role of cyclic AMP-dependent protein kinase activation in regulating rat submandibular mucin secretion.

The extent of activation of rat submandibular gland cyclic AMP-dependent protein kinase (EC 2.7.1.37) was determined in vitro using dispersed cells to assess the involvement of this enzyme in submandibular mucin secretion. cAMP-dependent protein kinase activation, as determined by activity ratio method, was markedly increased following beta-adrenergic receptor activation. 0.5 M NaCl was required in the homogenization buffer for stabilization of the hormonally activated cAMP-dependent protein kinase. A role for cAMP-dependent protein kinase activation in regulating mucin secretion was strongly suggested by the following: (1) the kinase activity ratio increased rapidly after beta-adrenergic receptor stimulation; (2) dose-response relationship of the kinase activation following beta-adrenergic receptor activation correlated with isoproterenol induced mucin release; (3) termination of beta-adrenergic mediated mucin secretion caused a rapid decrease in the kinase activity ratio; (4) dibutyryl cyclic AMP stimulation caused an increase in the kinase ratio; whereas (5) pure cholinergic and pure alpha-adrenergic receptor stimulation had no effect on endogenous kinase activity. Although cAMP-dependent protein kinase activation may not be the only regulator of mucin secretion, these data suggest an important regulatory role for this kinase activation during rat submandibular mucin release.

Comparison in vitro of the incorporation of D-[2-3H(N)]-mannose and D-[1-14C]-glucosamine into glycoproteins of dispersed rat submandibular salivary gland cells.

The incorporation of two different radiolabelled sugars, D-[14C]-glucosamine and D-[2-3H(N)]-mannose, into cellular and secretory glycoproteins was compared using dispersed rat submandibular cells. Most of the de-novo biosynthesis appeared to be directed toward the synthesis of secretory material as the molecular profile of the 3H-labelled material released following sympathomimetic stimulation and the percentage of total 3H-labelled acid--precipitable material secreted following cholinergic- or adrenergic-receptor stimulation coincided with the data obtained from similar studies using [14C]-glucosamine. The [3H]-mannose label was found in the neutral sugars mannose, galactose, glucose and fucose, with trace amounts of radiolabel in the amino sugars, whereas the [14C]-glucosamine label was present in three different amino sugars; glucosamine, galactosamine and sialic acid.