Secretion of lactoferrin and lysozyme by cultures of human airway epithelium

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.     

Lipoxin A4 stimulates a cytosolic Ca2+ increase in human bronchial epithelium

Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A(4) (LXA(4)) on intracellular [Ca(2+)] ([Ca(2+)](i)) of human bronchial epithelial cell. Exposure of the cells to LXA(4) produced a dose-dependent increase in [Ca(2+)](i) followed by a recovery to basal values in primary culture and in 16HBE14o(-) cells. The LXA(4)-induced [Ca(2+)](i) increase was completely abolished after pre-treatment of the 16HBE14o(-) cells with pertussis toxin (G-protein inhibitor). The [Ca(2+)](i) response was not affected by the removal of external [Ca(2+)] but completely inhibited by thapsigargin (Ca(2+)-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (R(p))-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca(2+) response to LXA(4). However, the response was not affected by chelerytrine chloride (protein kinase C inhibitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA(4) receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o(-) cells. The functional consequences of the effect of LXA(4) on intracellular [Ca(2+)](i) have been investigated on Cl(-) secretion, measured using the short-circuit techniques on 16HBE14o(-) monolayers grown on permeable filters. LXA(4) produced a sustained stimulation of the Cl(-) secretion by 16HBE14o(-) monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca(2+)](i) increase by LXA(4) through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca(2+)-dependent Cl(-) secretion by human airway epithelial cells.     

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.     

Biochemical interactions among intercellular adhesion molecules expressed by airway epithelial cells

Intercellular adhesion between adjacent airway epithelial cells plays a critical role in maintaining the barrier function of the respiratory mucosa. In the current study, we examined the expression and interaction of cell surface adhesion molecules (E-cadherin, ICAM-1, and MUC1) and their intracellular binding partners (alpha-catenin, beta-catenin, gamma-catenin, and ezrin) in 16HBE14o-, HBE1, 1HAEo-, BEAS-2B, A549, and NCI-H292 human airway epithelial cells. Expression of E-cadherin and MUC1, both in whole cell lysates and biotinylated surface proteins, was observed in 16HBE14o-, HBE1, A549, and NCI-H292 cells, while ICAM-1 was detected only in NCI-H292. In contrast, alpha-, beta-, and gamma-catenin and ezrin were expressed in all of the cells. E-cadherin formed coimmunoprecipitation complexes with beta- and gamma-catenin, whereas MUC1 only associated with beta-catenin. ICAM-1, but not MUC1, coimmunoprecipitated with ezrin in NCI-H292 cells. We conclude that airway epithelial cell-cell adhesion involves a complex network of protein-protein interactions mediated by a diverse array of membrane-bound and cytosolic protein partners.     

S. aureus haemolysin A‐induced IL‐8 and IL‐6 release from human airway epithelial cells is mediated by activation of p38‐ and Erk‐MAP kinases and additional,cell type‐specific signalling mechanisms

Soluble virulence‐associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o‐, S9 or A549 cells with recombinant Hla (rHla). In a dose‐dependent manner, rHla induced secretion of IL‐8 in all three cell types, but IL‐6 release only in 16HBE14o‐ and S9 cells. rHla‐mediated secretion of IL‐8 and IL‐6 was suppressed by pre‐incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL‐8 release in all three cell types and for IL‐6 release in 16HBE14o‐ and S9 cells. The rHla‐mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca²⁺]_i, an early response in rHla‐treated cells. Inhibitors of calmodulin or calcium/calmodulin‐dependent kinase II attenuated rHla‐mediated release of IL‐8 in 16HBE14o‐ and A549 cells and of IL‐6 in 16HBE14o‐ cells. This indicates that rHla may mediate simultaneous activation of calmodulin‐dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin‐dependent signalling did not affect rHla‐induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

Smooth Brucella strains invade and replicate in human lung epithelial cells without inducing cell death

Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar (A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5–3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity.     

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation of hormone-sensitive airway smooth muscle adenylate cyclase from dissociated canine trachealis cells

Conventional homogenizing methods produced membrane preparations of canine trachealis airway smooth muscle which contained adenylate cyclase activity that was stimulated by fluoride but not by isoproterenol. We have devised methods using collagenase digestion of minced trachealis which destroy most of the tough connective tissues but leave dissociated canine trachealis cells in suspension. Gentle homogenization of these cells permitted preparation of a particulate fraction containing adenylate cyclase that was readily stimulated by beta-adrenergic agonist of prostaglandin E2. Isoproterenol stimulation was 2.34 +/- 0.58 (S.E.) times basal and 122 +/- 25% of the stimulation induced by NaF. The beta-adrenergic blocking agent propranolol prevented isoproterenol-induced stimulation of the cyclase but had no effect on prostaglandin E2 stimulation. Catecholamine order of potency was isoproterenol greater than epinephrine greater than norepinephrine. These methods enable demonstration of stimulatory effects of hormones in broken cell preparations of airway smooth muscle that are comparable to those when hormone-stimulated cyclic AMP formation is measured in intact muscle strips.     

Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.     

Membrane reserves and hypotonic cell swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.     

Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni

Adenosine, acting via A1 adenosine receptors, can inhibit adenylate cyclase activity in adipocytes. To assess the effects of chronic adenosine agonist exposure on the A1 adenosine receptor system of adipocytes, rats were infused with (-)-phenylisopropyladenosine or vehicle for 6 days and membranes were prepared. Basal as well as isoproterenol-, sodium fluoride-, and forskolin-stimulated adenylate cyclase activities were significantly increased (approximately 2-fold) in membranes from treated animals. (-)-Phenylisopropyladenosine-mediated inhibition of forskolin-stimulated adenylate cyclase activity was significantly (p = 0.0001) attenuated in membranes from treated rats (20.1 +/- 2.1% inhibition) versus controls (31.6 +/- 2.3% inhibition). Prostaglandin E1-induced inhibition of forskolin-stimulated adenylate cyclase activity was also attenuated: 11.7 +/- 3.6 versus 23.2 +/- 4.6% (p = 0.001). Using the A1 adenosine receptor agonist radioligand (-)-N6-(3-[125I]iodo-4-hydroxyphenylisopropyl)adenosine, 32% fewer high affinity binding sites were detected in membranes from treated animals (p less than 0.04). Photoaffinity labeling with N6-2-(3-[125I]iodo-4-azidophenyl)ethyladenosine revealed no gross difference in receptor structure. The number of beta-adrenergic receptors as well as the percentage of receptors in the high affinity state as assessed by (-)-3-[125I]iodocyanopindolol binding were the same in both groups. In membranes from treated rats, the amount of [alpha-32P]NAD incorporated by pertussis toxin into the alpha subunit of the inhibitory guanine nucleotide regulatory protein (Ni) was decreased by 37 +/- 11%. Concurrently, the quantity of label incorporated by cholera toxin into the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Ns) was increased by 44 +/- 14% in treated membranes. Finally, the capacity of Ns solubilized from treated membranes to stimulate adenylate cyclase activity when reconstituted into cyc- S49 lymphoma cell membranes was enhanced by approximately 50% compared to control. Thus, heterologous desensitization, manifested by a diminished capacity to inhibit adenylate cyclase and an enhanced responsiveness to stimulatory effectors, can be induced in the A1 adenosine receptor-adenylate cyclase system of adipocytes. A decrease in Ni alpha subunit concomitant with an increase in Ns alpha subunit quantity and activity may represent the biochemical mechanism of desensitization in this system.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

Expression and functional analysis of beta-adrenoceptor subtypes in rabbit submandibular gland

beta-Adrenoceptors (beta-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of beta-AR subtypes in rabbit submandibular gland (SMG). Both beta(1)- and beta(2)-ARs, but not beta(3)-AR, were strongly expressed in rabbit SMG. beta(1)-AR proteins were widely expressed in acinar and ductal cells whereas beta(2)-AR proteins were mainly detected in ductal cells. A [(3)H]-dihydroalprenolol binding assay revealed that beta-AR B(max) was 186+/-11.9 fmol/mg protein and K(d) was 2.71+/-0.23 nM. A competitive binding assay with CGP 20712A, a beta(1)-AR antagonist, indicated that the proportion of beta(1)-AR and beta(2)-AR was 71.9% and 28.1%, respectively. Gland perfusion with the beta-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective beta-AR antagonist propranolol. Pretreatment with beta(1)- or beta(2)-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of alpha-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased alpha-amylase protein storage in SMG and increased alpha-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both beta(1)- and beta(2)-ARs are expressed in rabbit SMG. beta(1)-AR is the predominant subtype and may play an important role in regulating saliva and alpha-amylase secretion.