Bacterial cell wall-induced hepatic granulomas. An in vivo model of T cell-dependent fibrosis.

In vitro studies implicate a molecular link between inflammatory mononuclear cells and alterations in fibroblast growth and function. We have extended these observations in an experimental animal model in which we document the T cell-dependence of fibrosis that occurs after activation of the cell-mediated immune system by specific antigen. Chronic granulomatous lesions were induced in the livers of susceptible rats by the intraperitoneal injection of group A streptococcal cell walls (SCW). The development of granulomas that are composed primarily of lymphocytes and macrophages was associated with the recruitment and proliferation of connective tissue cells. Furthermore, this expanded population of fibroblasts generated a collagenous structure consisting primarily of types I and III collagen around the granuloma. The progression of these chronic inflammatory lesions leads to the formation of fibrotic nodules throughout the livers of the treated animals. Intact granulomas, as well as mononuclear cells derived from the granulomas, spontaneously elaborated a soluble factor(s) that stimulates fibroblast proliferation. Physicochemical analysis revealed that the primary granuloma-derived peak of fibroblast growth activity corresponded to an apparent Mr of 40,000, which is consistent with a previously described T lymphocyte--derived fibroblast-activating factor (FAF) in guinea pig and human. Furthermore, the fibrosis that occurs in the granuloma is apparently T cell--dependent, since no fibrotic lesions developed in SCW-injected athymic nude rats nor in SCW-injected animals treated with the T cell inhibitor, cyclosporin A (CsA). Mononuclear cells from neither of these functionally T cell--deficient animals could generate FAF activity. These data show a role for T lymphocyte--derived cytokines in the development of hepatic fibrosis in SCW-injected rats.

Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta.

After intraarticular injection of TGF-beta 1 or TGF-beta 2, marked swelling and erythema of the injected joints were apparent within 12-24 h. On a scale of 0 to 4, by day 3, the TGF-beta-treated joints had articular indices (AI) of 3.6 +/- 0.5 to 4.0 +/- 0.0 compared with no response for the vehicle-injected contralateral joints. Histopathologic evaluation revealed a predominantly mononuclear phagocyte infiltrate with some neutrophils and T lymphocytes, consistent with active inflammation. The monocytic pattern of leukocyte infiltration at 2-3 d was comparable to that seen in animals with antigen-induced arthritis after 2-3 wk. Extensive synovial fibroblast hyperplasia became apparent within 48 h, likely as a result of TGF-beta induction of growth factor synthesis by the accumulating monocytes. TGF-beta 2, a homologue of TGF-beta 1, was found to induce a similar level of synovitis and synovial hyperplasia consistent with its parallel monocyte and fibroblast chemotactic properties and ability to induce transcription and translation of monocyte/macrophage-derived growth factors. These data suggest that TGF-beta, released by platelets and activated inflammatory cells, may play a direct role in leukocyte recruitment and activation in arthritic and other chronic inflammatory lesions.

Immunocytochemical localization of MG1 mucin in human bulbourethral glands.

Salivary mucins MG1 and MG2 have been found in the oral cavity where they perform several functions such as the formation of the mucous layer covering the oral mucosa and teeth. Recent studies have demonstrated their presence in other organs and tissues. The aim of this study was to determine their expression in human bulbourethral (Cowper's) glands. Normal bulbourethral glands were obtained at surgery and fixed in a mixture of 1% paraformaldehyde-1.25% glutaraldehyde in 0.1 M cacodylate buffer and embedded in Epon resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells were intensely reactive with anti-MG1, whereas no labeling was detected for MG2. These results indicate that MG1 is not exclusively a salivary component and furthermore show that bulbourethral glands represent a significant source of the MG1 detected in human seminal plasma.

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.

The structural organization of the septate and gap junctions of Hydra.

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50-60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80-100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15-0.5 micro in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.

Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells.

The formation and fate of apical endocytic vesicles in resting and isoproterenol-stimulated rat parotid acinar cells were studied using luminally administered horseradish peroxidase (HRP) to mark the vesicles. The tracer was taken up from the lumen by endocytosis in small, smooth-surfaces "c"- or ring-shaped vesicles. About 1 h after HRP administration the vesicles could be found adjacent to the Golgi apparatus. At later times HRP reaction product was localized in multivesicular bodies and lysosomes; in isoproterenol-stimulated cells it was also present in autophagic vacuoles. HRP reaction product was never localized in any structure associated with secretory granule formation. These results suggest that the apical endocytic vesicles play a role in membrane recovery, but that they are degraded and not reutilized directly in secretory granule formation. Additionally, it was found that when isoproterenol was injected before HRP administration, the apical junctional complexes became permeable to the tracer, allowing it to gain access to the lateral and basal intercellular spaces. This permeability may provide an additional route whereby substances in the extracellular fluid could reach the saliva.

Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland.

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.

Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation.

The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland. Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min. Secretion was induced in some animals by intraperitoneal injection of isoproterenol. In resting glands, the tracers microperoxidase , cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts. In glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase , cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands. Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells. The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution. Freeze-fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight greater than or equal to 1,900; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight less than or equal to 34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights. Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution.

Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes.

Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study.

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.

Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.

The sphingolipid metabolite sphingosine-1-phosphate (SPP) has been implicated as a second messenger in cell proliferation and survival. However, many of its biological effects are due to binding to unidentified receptors on the cell surface. SPP activated the heterotrimeric guanine nucleotide binding protein (G protein)-coupled orphan receptor EDG-1, originally cloned as Endothelial Differentiation Gene-1. EDG-1 bound SPP with high affinity (dissociation constant = 8.1 nM) and high specificity. Overexpression of EDG-1 induced exaggerated cell-cell aggregation, enhanced expression of cadherins, and formation of well-developed adherens junctions in a manner dependent on SPP and the small guanine nucleotide binding protein Rho.

Changes in organization of the endoplasmic reticulum during Xenopus oocyte maturation and activation.

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.

Histological and biochemical characterization of von Ebner's glands in the Syrian hamster; comparison with rat von Ebner's glands.

We report here for the first time a morphological description and observations on some of the secretory proteins of the von Ebner's lingual salivary glands (VEG) of the Syrian hamster. Hamster VEG were macroscopically less distinct, but histologically similar to rat VEG. VEG extracts of hamster and rat were assayed for lipase, alpha-amylase and peroxidase activities. Unlike rat VEG, which is rich in lipase activity, hamster VEG extract had no detectable lipase activity and did not react with antibodies to either rat lingual lipase or human gastric lipase in Western blots. Immunohistochemical reactions with the anti-rat lingual lipase antibody were very weak in hamster VEG and strong in rat VEG. Moderate alpha-amylase enzyme activities and immunohistochemical reactions were demonstrated in both hamster and rat VEG. Peroxidase activity was negligible in the VEG, unlike the high activity in the submandibular glands of both species. An 18 kDa von Ebner's gland protein (VEGP), a member of the lipocalin superfamily of hydrophobic ligandbinding proteins, was abundant in rat VEG, but not detected in hamster VEG. Thus, hamster VEG differs from rat VEG in macroscopic appearance and the absence of lipase and VEGP. It is similar to rat VEG histologically and with regard to the presence of alpha-amylase and absence of peroxidase.

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells.

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.

The sld mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression.

NFS/N-sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187-191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in "atypical" exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. "Atypical" cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

Cyclic AMP-receptor protein activity in rat preputial cells.

Cells from the rat preputial gland--a type of sebaceous gland--exhibited specific responsiveness of cyclic 3',5'-adenosine monophosphate (AMP) dependent protein kinase to stimulation by agents that elevate intracellular cyclic AMP. Electron microscopy shows that the rat preputial gland resembles the human sebaceous gland, not only in terms of containing a sebocyte-like population of cells in an acinar arrangement at different maturational stages, but also in the morphology of its organelles such as abundant and sometimes atypical mitochondria, many perinuclear lysosomes with crystalline inclusions, lipid droplets of various sizes, and peroxisomes. Other cell types, among them duct and inflammatory cells, were evident in the tissue sections, but constituted a minor component. Responses to stimulation of the adenylate cyclase-protein kinase pathways were determined using preputial cells that had been both freshly dispersed and grown in monolayer culture. Stimulation with isoproterenol (IPR) or forskolin (FS) resulted in both cases in an increase of cyclic AMP binding of the regulatory (R) subunits of cyclic AMP-dependent protein kinase, as determined by photoaffinity labeling of R subunits with an azido analog of cyclic AMP ([32P]-8-azido cyclic AMP). Cells from the epidermis under comparable conditions responded to a lesser degree and with a different distribution of R subunit isoforms. There are, therefore, differences in receptor activity as well as in the transduction pathways between the two types of epithelial cell populations. These results indicate that the preputial gland contains precursor cells that differentiate in culture to retain specific molecular mechanisms of action mediated via cyclic AMP.

Prostaglandins regulate the expression of fibroblast growth factor-2 in bone.

We examined the effect of PGs, particularly PGF2alpha, on basic fibroblast growth factor-2 (FGF-2) messenger RNA (mRNA) and protein in the rat osteoblastic cell line Py1a and in fetal rat calvariae. Py1a cells expressed multiple FGF-2 mRNA transcripts. PGF2alpha dose-dependently increased the 6-kb transcript at 6 h. The selective PGF2alpha agonist, fluprostenol (Flup), was more potent than PGF2alpha. Phorbol myristate acetate (10(-6) M) also increased a 6-kb mRNA at 6 h. By immunofluorescence microscopy, Flup increased perinuclear staining for FGF-2 protein at 6 h and nuclear labeling at 24 h. Immunogold labeling of calvariae revealed that treatment with Flup for 3 h caused a transition of FGF expression from matrix to cells and an increase in cytoplasmic labeling for FGF-2 protein in periosteal cells and in osteoblasts. After treatment with Flup for 24 h, nuclear labeling was marked in periosteal cells and in osteoblasts, and a further increase in cytoplasmic labeling for FGF-2 was noted in osteocytes, periosteal cells, and osteoblasts. We conclude that PGs can increase FGF-2 mRNA and protein in bone cells. Because the effect of Flup was mimicked by phorbol myristate acetate, we hypothesize that PGs' regulation of FGF-2 is mediated by a PGF2alpha-selective receptor acting through protein kinase C. Hence, effects of PGs on bone remodeling may be mediated, in part, by endogenous FGF-2.

Cytochemical differentiation of the Golgi apparatus from GERL.

GERL exhibits a number of structural properties, such as its location at the trans face of the Golgi apparatus, thick limiting membranes with occasional coated regions, and close relationship to the ER, which are similar in all cells wehre it has been identified. Acid hydrolase activity and the formation of lysosomes, also considered characteristic of GERL, have been shown to be less consistent properties of GERL in some cells. Finally, GERL and the Golgi saccules of certain cells undergo significant changes in their cytochemical and structural properties in response to specific alterations in cellular activity. These variations are important for at least two reasons: first, they indicate the care required in differentiating GERL from the Golgi saccules based on limited cytochemical observations; second, and most important, they may yield information regarding the biogenetic and functional relationships between the ER, Golgi saccules, and GERL, such as the origin of GERL in various cells and the role of each organelle in the processing of lysosomal and secretory proteins.

Ultrastructural localization of catalase and L-alpha-hydroxy acid oxidase in microperoxisomes of Hydra.

The ultrastructural localization of catalase and L-alpha-hydroxy acid oxidase (LalphaHAO) was studied in two species of Hydra. Diaminobenzidine reaction product of catalase activity was present in small round or elongated bodies resembling microperoxisomes in the epitheliomuscular, digestive and gland cells. They were closely related to the endoplasmic reticulum, and were often found in proximity to deposits of lipid and glycogen. Reaction product of LalphaHAO activity was also associated with the microperoxisomes. With rapidly oxidized substrates, such as L-lactic acid, reaction product diffused into the cytoplasm around the microperoxisomes. With slowly oxidized substrates, such as DL-alpha-hydroxyisovaleric acid, reaction product was restricted to the matrix of the microperoxisomes. No reaction product was present in the microperoxisomes in the absence of substrate or with D-lactic acid. The rate of substrate oxidation measured biochemically roughly paralleled the amount of cytochemical reaction product deposited with different substrates. Microperoxisome-like bodies reactive for LalphaHAO were also found in the epidermal cnidoblasts; however, catalase could not be demonstrated in them. This study provides the first cytochemical evidence for the presence of an H2O2-producing oxidase in microperoxisomes.

Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery.

We utilized quantitative electron microscopic immunogold labeling procedures to follow changes in the intragranular content of five secretory proteins of the rat submandibular gland (SMG) during and after chronic treatment with the beta-adrenergic agonist isoproterenol (IPR). Labeling intensities (gold particles/microns2) of acinar cell secretory granules for mucin and glutamine/glutamic acid-rich proteins, major secretory proteins of the normal SMG, showed opposite responses to IPR. Labeling intensities increased for mucin and decreased for glutamine/glutamic acid-rich proteins immediately after IPR injections began, then rapidly returned to control levels after cessation of IPR treatment. SMG Protein C immunoreactivity, found in both acinar and intercalated duct granules, was less affected by IPR. However, opposite changes in labeling intensity were observed between acinar and intercalated duct granules. Labeling intensities for proline-rich proteins, IPR-inducible secretory proteins, increased only after 10 days of stimulation and maintained a high level even after cessation of drug treatment. Type 2 cystatin, another IPR-inducible protein, increased gradually with chronic IPR treatment and decreased slowly during the recovery phase. These results suggest that chronic beta-adrenergic stimulation affects the expression of genes for several rat SMG secretory proteins in a different manner.