Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.     

Plasma membrane of the rat parotid gland: preparation and partial characterization of a fraction containing the secretory surface

A plasma membrane fraction from the rat parotid gland has been prepared by a procedure which selectively enriches for large membrane sheets. This fraction appears to have preserved several ultrastructural features of the acinar cell surface observed in situ. Regions of membrane resembling the acinar luminal border appear as compartments containing microvillar invaginations, bounded by elements of the junctional complex, and from which basolateral membranes extend beyond the junctional complex either to contact other apical compartments or to terminate as free ends. Several additional morphological features of the apical compartments suggest that they are primarily derived from the surface of acinar cells, rather than from the minority of other salivary gland cell types. Enzymatic activities characteristically associated with other cellular organelles are found at only low levels in the plasma membrane fraction. The fraction is highly enriched in two enzyme activities--K+ -dependent p-nitrophenyl phosphatase (K+ -NPPase, shown to be Na+/K+ adenosine triphosphatase; 20-fold) and gamma-glutamyl transpeptidase (GGTPase; 26-fold)--both known to mark plasma membranes in other tissues. These activities exhibit different patterns of recovery during fractionation, suggesting their distinct distributions among parotid cellular membranes. Secretion granule membranes also exhibit GGTPase, but no detectable K+ -NPPase. Since Na+/K+ adenosine triphosphatase and GGTPase, respectively, mark the basolateral and apical cellular surfaces in other epithelia, we hypothesize that these two enzymes mark distinct domains in the parotid plasmalemma, and that GGTPase, as the putative apical marker, may signify a compositional overlap between the two types of membranes which fuse during exocytosis.     

Rab3D redistribution and function in rat parotid acini

Rab3D is a low molecular weight GTP-binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane-associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co-localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc-tagged Rab3D and Rab3DQ81L, a GTP-binding mutant, were prepared and incubated with streptolysin O-permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D-binding to parotid membranes is guanine nucleotide-dependent. Moreover, wild-type and mutant Rab3D inhibited agonist-induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild-type Rab3D and the GTP-binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.     

Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides

Rat brain cortex membranes bind to a conjugate of substance P and 125I-labeled Bolton-Hunter reagent, and this binding can be inhibited by a low concentration of substance P (Kd = 1.2 +/- 0.4 X 10(-8) M). This binding is reversible and saturable (0.5 +/- 0.1 pmol of binding sites/mg of protein). Fragments of substance P as small as the carboxyl-terminal hexapeptide can inhibit the binding although their potency decreases with the decrease in the length of the peptides. The binding affinities of smaller peptides or peptides in which the carboxyl-terminal amide or amino acids are removed are drastically reduced. Biologically active analogs of substance P, physalaemin, eledoisin, substance P methyl ester, [D-Ala0]hepta(5-11)substance P, kassinin, and the eledoisin-related hexapeptide also can inhibit the binding. However, the binding is not inhibited by polypeptides structurally unrelated to substance P or by amine hormones/neurotransmitters. The binding affinities of biologically active peptides to rat brain cortex membranes are almost identical with their affinities for rat parotid cells which we previously determined. Furthermore, the recently described substance P antagonist, [D-Pro, D-Trp]substance P, inhibits the binding of the 125I-labeled substance P derivative to brain cortex membranes and to parotid cells equally well. These results suggest that the substance P receptors in the brain cortex and the parotid gland are similar. The brain cortex membrane binding of the 125I-labeled substance P derivative can be inhibited by micromolar concentrations of GTP, GDP, and their analogs. ITP and IDP were less active. Adenine and pyridine nucleotides were inactive.     

Rab3D redistribution and function in rat parotid acini

The original article to which this Erratum refers was published in J. Cell. Physiol. (2003) 197(3) 400–408 . Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition. J. Cell. Physiol. 199: 316, 2004© 2004 Wiley‐Liss, Inc.     

Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland

Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP)-mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTPS in PT-treated membranes was much larger than that in normal membranes. ANP dose-dependently inhibited adenylate cyclase stimulated by GTPS in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10^–7M) inhibited cAMP accumulation stimulated by forskolin (10^–6M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells, amylase release stimulated by isoproterenol (10^–6M) and forskolin (10^–6M) were also depressed by ANP (10^–7M) by 27 and 30%, respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP-ribosylated 41-KDa protein by incubation of parotid membranes with PT and [-³²P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi. (Mol Cell Biochem)139: 53–58, 1994)     

Lateral diffusion of luminal membrane components during secretion in parotid acinar cells of the rat

Summary The movements of the molecular components of the luminal plasma membrane during exocytotic secretion in parotid acinar cells were examined. For immunocytochemical study, we used an antiserum of dipeptidyl peptidase IV as a marker for the components of the luminal plasma membrane of acinar cells. In unstimulated acinar cells, dipeptidyl peptidase IV immunoreactivity is restricted to the luminal plasma membrane. However, after secretion was stimulated with a -adrenergic agonist, isoproterenol, immunostaining became detectable on the membrane of discharged granules. Freeze-fracture images showed that the density of intramembrane particles on the P-fracture leaflets of discharged granule membranes is much higher than that of undischarged granule membranes during secretion. These results suggest that in parotid acinar cells of the rat, the components of the luminal plasma membrane move laterally, during secretion, to the membranes of discharged granules.     

Characterization of high affinity GTPase activity correlated to beta-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes.

beta-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The beta-adrenergic agonist isoproterenol (10 microM) increased the Vmax of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its Km value (approximately 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), isoproterenol increased cAMP formation to the same extent as that observed with AlF-4. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (approximately 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyl cyclase activation seen following beta-adrenergic stimulation of rat parotid glands.     

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.     

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.     

The Effect of Isoproterenol upon the Chemical Composition of Plasma Membranes in the Mouse Parotid Gland

A single intraperitoneal injection of isoproterenol induces resting cells from the acini of the mouse parotid gland to enter the proliferative cycle. Parotid plasma membrane from non-stimulated and isoproterenol-treated mice were prepared by differential centrifugation of the homogenates. Comparing the chemical composition of plasma membranes from non-stimulated and isoproterenol-treated mice, no variation in the phospholipid/protein ratio was observed. However, the levels of neutral sugars, hexosamines and sialic acid falls drastically in the early prereplicative phase. The decrease in neutral sugars and hexosamines in plasma membranes caused by isoproterenol is imitated by pilocarpine, which induces secretion but little or no increase in DNA synthesis. However, pilocarpine does not mobilize sialic acid from the plasma membrane. Moreover, dosis of isoproterenol that elicits secretion but not mitosis in the acinar cells, does not induce the movement of sialic acid from the plasma membrane. The mobilization of sialic acid from plasma membranes caused by isoproterenol was also demonstrated in an in vitro system. Treatment of the plasma membrane with chloro-form/methanol shows that around 60% of the sialic acid is present in the less polar phase. We conclude that the separation of sialic acid from the plasma membrane is one of the early steps in the sequence of events leading to DNA synthesis and cell division in the isoproterenol-stimulated parotid gland of mice.     

Adenylate cyclase activity in the parotid gland of the mouse after isoproterenol stimulation

Previous studies have described a decrease in the activity of adenylate cyclase in the parotid gland of isoproterenol-treated rats. In the present studies, a similar decrease was observed in mice treated with isoproterenol. Studies on the subcellular distribution of adenylate cyclase after isoproterenol stimulation of the parotid gland showed that enzyme activity was increased in the lysosomal fraction and decreased in the cellular membrane fractions. Cytochemical studies on the localization of adenylate cyclase in stimulated gland showed an increase in vesicles which contained enzyme activity and a decrease in activity at the luminal and plasma membranes. It is suggested, based on the present findings and results reported by other investigators, that after isoproterenol stimulation of the parotid gland, adenylate cyclase (along with excess membrane) is degraded by lysosomes. If this suggestion is true, then the observed decrease in adenylate cyclase would have a molecular explanation.     

Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.     

Alkaline phosphatase and myoepithelial cells in the parotid gland of the rat

Synopsis Alkaline phosphatase activity has been studied in the parotid glands of rats at the light and electron microscopical levels. Reaction product was found to outline the plasma membranes of myoepithelial cells. It was also found in the walls of many capillaries and on the luminal surface and between apposing cells in some intercallary ducts.The distribution of myoepithelial cells in the rat parotid is unusual. The cells run longitudinally around intercalary ducts and send processes on to the bases of adjoining acini but do not embrace the acini. The possible functions of these myoepithelial cells are discussed.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodi-phosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

Absence of a direct role of phospholipid methylation in stimulus-secretion coupling and control of adenylate cyclase in guinea-pig and rat parotid gland.

The present study was undertaken to investigate a possible involvement of phospholipid methyltransferases in the coupling of receptor-mediated stimulation to secretion. Phospholipid methyltransferases were assayed in isolated parotid acini in the presence of carbamoylcholine or isoprenaline. Carbamoylcholine reduced the incorporation of methyl groups into phospholipids, whereas isoprenaline showed no effect. Amylase secretion stimulated either by carbamoylcholine or by isoprenaline could not be affected by inhibitors of methyltransferases (3-deaza-adenosine alone or plus homocysteine thiolactone) under conditions where phospholipid methylation was strongly inhibited. The activity of adenylate cyclase in isolated parotid microsomal membranes was not inhibited or stimulated by S-adenosyl-homocysteine or -methionine respectively. These results indicate that phospholipid methylation does not play an essential role in stimulus-secretion coupling in the parotid gland.