Changes in the content and distribution of sialic acid on the basal surface of isoproterenol-stimulated mouse parotid acinar cells

The presence, distribution and content of sialic acid on the cell surface in collagenase-dispersed acini obtained both from unstimulated as well as from in vivo isoproterenol-stimulated mouse parotid have been studied. To this end, sialic acid residues have been qualitatively and quantitatively analyzed by 1) cytochemical labeling by wheat germ agglutinin (WGA), 2) biochemical procedures and 3) isotopic labeling by [3H]WGA (WGA-N-[acetyl-3H]-acetylated). Electron microscopy revealed striking differences in the binding of ferritin-conjugated WGA at the basal, lateral and apical cell surface. Unstimulated acinar cells showed a heavy patch-distributed binding of ferritin-conjugate on the basal cell surface while it was homogeneous and very scarce on the lateral one and absent on the apical cell surface. During the first few hours after isoproterenol, the WGA binding sites at the basal cell surface became homogeneously distributed. This fact was coincident with a loss of about 60 to 70% both in the content of neuraminidase-releasable sialic acid and in the binding of [3H]WGA to the acinar surface. These findings suggest that the release of sialic acid as free residues, which has been involved in the isoproterenol-triggered cell proliferation-inducing mechanism in the mouse parotid, would occur at the glycocalyx corresponding to the basal plasma membrane of the acinar cells.     

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.     

The relationship between synthesis of secretory products and reducing capacity in pancreas and parotid acinar cells

The secretory products in exocrine pancreas acinar cells in utero were found to reduce osmium tetroxide. This reducing capacity was also exhibited by adult pancreas and parotid glands in different phases of synchronized secretion, and after single or chronic administration of a secretagogue, pilocarpine or isoprenaline. In utero, the reducing capacity appeared in the pancreas concomitantly with the synthesis of secretory products, and was limited to the transitional vesicles on the cis Golgi side. After birth, osmium staining occurred in the cis Golgi vesicles and cisternae of both glands. In the chronically-treated parotid gland, where the occupational programme for secretory proteins had been altered, the reducing capacity was diminished, resembling that in embryonic exocrine pancreas.     

Localization of 5′-nucleotidase activity in the parotid acinar cells of a rat treated with isoproterenol

Summary Cytochemical localization of 5- nucleotidase (AMPase) has been investigated in the parotid acinar cells of rats at various stages of exocytic secretion induced by an administration of isoproterenol (IPR).In the resting stage, the acinar cells show AMPase activity located on the baso-lateral and luminal plasmalemma, and in the earliest secretory stage the luminal plasma membranes are devoid of the enzymatic activity. However, these particular regions exhibit AMPase activity during the advanced stages of secretion, and the AMPase positive membranes become absorbed into the cytoplasm by endocytic activity. The absorbed membrane components then seem to be degraded by the action of lysosomes.The intracellular fate of the endocytic vacuoles has been examined by the aid of ferritin particles introduced retrogradely through ductal lumina. Ferritin containing vacuoles are distributed in the cytoplasm, and these droplets change into secondary lysosomes. No tracer particles are recognized in the internal space of the Golgi lamella and its associated vesicles.The results suggested that in the exocytic secretion of parotid acinar cells, AMPase originating from plasma membrane intermingles with the membranes derived from secretion granules, and is translocated into cytoplasm by an endocytic mechanism. The internalized membrane components are, at least partly, degraded by lysosome action.     

Function of coated membranes and multivesicular bodies during membrane regulation in stimulated exocrine pancreas cells

Summary Stimulation of secretion by pilocarpine results in a 70% loss of zymogen granules from pancreatic acinar cell during the first hr after injection of the drug. In previous work (Geuze and Poort, 1973), we found that the amount of membrane stored in the surface of the microvilli and of the numerous infoldings present in highly stimulated cells, increases during the first 2 hr and then decreases again during the 3rd hr after stimulation, concurrently with maximal endocytosis of sorbitol-[su14C].Further observations on the fine structure of stimulated cells at various time intervals after injection of pilocarpine showed that during the first hr numerous smooth vesicles and multivesicular bodies (mvb's) appear in the apical cytoplasm, while the number of coated vesicles and their relative total volume increase significantly 3 hr after stimulation.By infusion of ferritin in the pancreatic duct system in vivo and application of cytochemical techniques (osmium impregnation, electron microscope autoradiography and acid phosphatase cytochemistry) it could be established that after stimulated exocytotic secretion, redundant apical cell membrane is withdrawn by at least two routes: 1) During the initial rapid increase of the amount of apical cell membrane, withdrawal is accomplished by interiorization of luminal invaginations into smooth endocytotic vesicles, which in turn give rise to mvb's by infolding and subsequent fission of their limiting membrane. 2) Once the bulk of stored secretion granules has been discharged, endocytotic coated vesicles become gradually more prominent as carriers for redundant cell membrane. The contents of endocytotic structures ultimately become incorporated in residual bodies, suggesting lysosomal degradation of cell membrane prior to eventual reutilization.Coated vesicles also originate by pinching off from mature Golgi cisternae and condensing vacuoles. A possible function of the coated membranes in the concentration of exportable protein within forming secretory granules is discussed.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse

Summary Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal -N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate -galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20–50% of these cells in all glands contained terminalN-acetylglucosamine residues. In contrast, terminal -N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.     

Loss of hyperpolarization-activated Cl(-) current in salivary acinar cells from Clcn2 knockout mice

ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl(-) current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl(-) current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of the Clcn2 gene was generated. The resulting homozygous Clcn2(-/-) mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal in Clcn2(-/-) mice following in vivo stimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2(-/-) mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl(-) channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.     

Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice

Multiple Na(+)/H(+) exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na(+)/H(+) exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1(-/-)). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1(-/-) and Nhe2(-/-) mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na(+)/K(+)/2Cl(-) cotransporter mRNA increased dramatically in Nhe1(-/-) parotid glands but not in those of Nhe2(-/-) or Nhe3(-/-) mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2(-/-) or Nhe3(-/-) mice were comparable with those of wild-type mice. In contrast, Nhe1(-/-) mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na(+) absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na(+)/K(+)/2Cl(-) cotransporter activity.     

cAMP and Ca(2+)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton

The potential involvement of actin and fodrin (brain spectrin) in secretory events has been assessed in primary cultured guinea pig parotid acinar cells, using as a tool affinity purified anti-alpha-fodrin antibody, phalloidin, and immunofluorescence techniques. In resting parotid acinar cells fodrin and actin appeared as a continuous ring under the plasma membrane of most of the cells. Upon stimulation with secretagogues fodrin and actin labeling at the level of the plasma membrane disappeared almost completely. To establish a correlation between secretion and cytoskeletal changes at the individual cell level, anti-alpha-amylase-antibodies were used to label secreted amylase exposed at the surface of secreting cells. The number of cells expressing alpha-amylase on their surface followed bulk secretion of alpha-amylase. A strict correlation between secretion and alteration of the actin-fodrin labeling was observed at the individual cell level. The cytoskeletal changes occurred in parallel with secretion independently of the secretagogue used (carbamoylcholine in the presence of Ca2+, isoproterenol in presence or absence of Ca2+, forskolin, or dibutyryl-cyclic-AMP). The changes were reversible upon removal of the secretagogue. Since Ca2+, as well as cAMP-mediated secretion, was associated with the same kind of cytoskeletal changes, a reorganization of the cytoskeleton may play an essential part in regulated secretion.     

Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size,and for reductions in membrane surface area and immature granule volume during granule maturation

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 unit progranules of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures.     

Down-regulation of cellular proto-oncogenes during inhibition of rat parotid acinar cell proliferation.

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, alpha-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into beta-agonist-treated rats prevented acinar cell proliferation as determined by [3H]thymidine incorporation after 96 h of treatment. However, [3H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/alpha-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimulated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.     

No evidence for calpain I involvement in fodrin rearrangements linked to regulated secretion.

Stimulation of secretion in chromaffin and parotid acinar cells is associated with dramatic rearrangements of the subplasmalemmal cytoskeleton, notably of fodrin and F-actin. It has been proposed that a proteolytic cleavage of fodrin resulting from an activation of the neutral calcium activated protease (calpain) could be responsible for these changes. Using an affinity-purified anti-alpha-fodrin antibody, several cleavage products of fodrin could clearly be detected following incubation of total cell homogenates from chromaffin and parotid acinar cells with purified calpain I. On the other hand, maximum stimulation of secretion of chromaffin cells by nicotine, and of parotid acinar cells by carbachol plus isoproterenol, was not associated with an increased appearance of cleavage products of fodrin. This result is not compatible with the 'proteolytic cleavage' hypothesis.     

Characterization of cysteine string protein in rat parotid acinar cells

Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP1_1–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP1_1–82, containing the J-domain only, nor GST-CSP1_83–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.     

Spatiotemporal analysis of exocytosis in mouse parotid acinar cells

Exocrine cells of the digestive system are specialized to secrete protein and fluid in response to neuronal and/or hormonal input. Although morphologically similar, parotid and pancreatic acinar cells exhibit important functional divergence in Ca²⁺ signaling properties. To address whether there are fundamental differences in exocytotic release of digestive enzyme from exocrine cells of salivary gland versus pancreas, we applied electrophysiological and optical methods to investigate spatial and temporal characteristics of zymogen-containing secretory granule fusion at the single-acinar cell level by direct or agonist-induced Ca²⁺ and cAMP elevation. Temporally resolved membrane capacitance measurements revealed that two apparent phases of exocytosis were induced by Ca²⁺ elevation: a rapidly activated initial phase that could not be resolved as individual fusion events and a second phase that was activated after a delay, increased in a staircaselike fashion, was augmented by cAMP elevation, and likely reflected both sequential compound and multivesicular fusion of zymogen-containing granules. Optical measurements of exocytosis with time-differential imaging analysis revealed that zymogen granule fusion was induced after a minimum delay of 200 ms, occurred initially at apical and basolateral borders of acinar cells, and under strong stimulation proceeded from apical pole to deeper regions of the cell interior. Zymogen granule fusions appeared to coordinate subsequent fusions and produced persistent structures that generally lasted several minutes. In addition, parotid gland slices were used to assess secretory dynamics in a more physiological context. Parotid acinar cells were shown to exhibit both similar and divergent properties compared with the better-studied pancreatic acinar cell regarding spatial organization and kinetics of exocytotic fusion of zymogen granules. membrane capacitance; differential imaging; zymogen; gland slice; exocrine cells     

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.     

THE FINE STRUCTURE OF VON EBNER''S GLAND OF THE RAT

The fine structure of von Ebner's gland was studied in untreated rats and rats stimulated to secrete by fasting-refeeding or injection of pilocarpine. Cytological features were similar to those reported for pancreas and parotid gland. Abundant granular endoplasmic reticulum filled the basal portion of the cell, a well-developed Golgi complex was located in the vicinity of the nucleus, and the apical portion of the cell was filled with dense secretory granules. Dense heterogeneous bodies resembling lysosomes were closely associated with the Golgi complex. Coated vesicles were seen in the Golgi region and also in continuity with the cell membrane. Granule discharge occurred by fusion of the granule membrane with the cell membrane at the secretory surface. Successive fusion of adjacent granules to the previously fused granule formed a connected string of granules in the apical cytoplasm. Myoepithelial cells were present within the basement membrane, and nerve processes were seen adjacent to acinar and myoepithelial cells. Duct cells resembled the intercalated duct cells of the major salivary glands.     

Local and global calcium signals and fluid and electrolyte secretion in mouse submandibular acinar cells

Polarized Ca(2+) signals that originate at and spread from the apical pole have been shown to occur in acinar cells from lacrimal, parotid, and pancreatic glands. However, "local" Ca(2+) signals, that are restricted to the apical pole of the cell, have been previously demonstrated only in pancreatic acinar cells in which the primary function of the Ca(2+) signal is to regulate exocytosis. We show that submandibular acinar cells, in which the primary function of the Ca(2+) signal is to drive fluid and electrolyte secretion, are capable of both Ca(2+) waves and local Ca(2+) signals. The generally accepted model for fluid and electrolyte secretion requires simultaneous Ca(2+)-activation of basally located K(+) channels and apically located Cl(-) channels. Whereas a propagated cell-wide Ca(2+) signal is clearly consistent with this model, a local Ca(2+) signal is not, because there is no increase in intracellular Ca(2+) concentration at the basal pole of the cell. Our data provide the first direct demonstration, in submandibular acinar cells, of the apical and basal location of the Cl(-) and K(+) channels, respectively, and confirm that local Ca(2+) signals do not Ca(2+)-activate K(+) channels. We reevaluate the model for fluid and electrolyte secretion and demonstrate that Ca(2+)-activation of the Cl(-) channels is sufficient to voltage-activate the K(+) channels and thus demonstrate that local Ca(2+) signals are sufficient to support fluid secretion.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Immunocytochemical demonstration of cyclic AMP-dependent protein kinases in duct cells of the rat parotid gland

The cyclic AMP-dependent protein kinases were immunolocalized in the rat parotid gland using a monospecific antiserum against their catalytic subunit. The kinases were found to be primarily located in the cytoplasm of the parotid duct cells with a preference for the apical cell region. The result questions the traditional view of the control of parotid gland secretion and suggests a role of cyclic AMP not only in the acinar protein secretion but also in ductal functions like fluid and electrolyte transport.     

Evaluation of hydroxyl radical-scavenging property of garlic

While antibiotics are broadly used in dental and medical therapy, little attention has been directed towards the potential toxic side effects of antibiotics on tissue regeneration. Here we examined the effect of a quinolone antibiotic, pefloxacin (Rhone Poulenc) on rat parotid gland responses to chronic isoproterenol treatment. Groups of rats received injections of isoproterenol to induce glandular growth, saline (controls), pefloxacin, or isoproterenol and pefloxacin in combination. Parotid gland weight decreased significantly after pefloxacin treatment for 7 days as well as inhibiting glandular enlargement provoked by isoproterenol. The same trend was observed for the rates of DNA synthesis, with the incorporation of [³H]-thymidine in isoproterenol/pefloxacin-treated rats reduced to 49% of isoproterenol treatment alone levels. Saline-treated animals were 42% of the rate of [³H]-thymidine incorporation into DNA observed in isoproterenol treated rats. While isoproterenol treatment increased steady-state mRNA levels for fos, jun, myc, src, c-erbB-2, ras and topo II, inclusion of pefloxacin with the isoproterenol regimen blocked these increases. Pefloxacin treatment by itself did not alter proto-oncogene mRNA levels in the parotid gland. Glandular amylase activity was decreased in the pefloxacin treated group, while the combination of isoproterenol with pefloxacin did not decrease glandular amylase levels to the extent of that observed with -agonist treatment alone. In acute experiments, pefloxacin significantly decreased the volume of saliva secreted by the parotid gland. These results suggest that quinolone-based antibiotics disturb the secretory function of the parotid gland and can inhibit cell proliferation and regeneration. (Mol Cell Biochem 165: 55–63, 1996)