IL-1 beta alters beta-adrenergic receptor adenylyl cyclase system function in human airway epithelial cells.

Inflammatory cells release a variety of cytokines, including interleukin (IL)-1 beta, into the airway in asthma. This study examined the effects of human IL-1 beta on the function of the beta-adrenergic receptor (beta AR)-adenylyl cyclase (AC) system in BEAS-2B cells, a human airway epithelial cell line. IL-1 beta markedly increased beta AR density (Bmax; P < 0.001) primarily by increasing the percentage of the beta 2AR subtype (from 67 to 91%; P < 0.001). Bmax increased monotonically over time in response to 200 pM IL-1 beta and was approximately 2.5-fold greater than control cells between 36 and 42 h. In contrast, the concentration response of Bmax to IL-1 beta given for 18 h was biphasic. Bmax increased with IL-1 beta concentrations from 2 to 200 pM, but, at > 200 pM, it decreased progressively toward control values. IL-1 beta-induced increases in Bmax with IL-1 beta were associated with approximately threefold increases in beta 2 AR mRNA and were blocked by the protein synthesis inhibitor cycloheximide. Despite the marked increase in Bmax, however, IL-1 beta depressed adenosine 3',5'-cyclic monophosphate (cAMP) responses to isoproterenol and forskolin, a direct activator of AC (P < 0.001 by analysis of variance for both). The inhibitory effect of IL-1 beta on cAMP production appeared to be explained by increases in the activity of an inhibitory GTP binding protein because IL-1 beta treatment increased the activity of a pertussis toxin ADP-ribosylated Gi alpha protein by approximately 2.5-fold; and pretreatment of intact cells with pertussis toxin inhibited the effect of IL-1 beta on cAMP production. These data indicate that IL-1 beta-mediated changes in the beta AR-AC system function in airway epithelial cells are complex and involve expression of receptor protein, GTP binding protein, and possibly AC itself. Increases in IL-1 beta may contribute to abnormalities in airway function in subjects with asthma.

Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells.

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.

Expression of the beta-adrenergic receptor-adenylylcyclase system in basal and columnar airway epithelial cells.

Catecholamines acting through the beta-adrenergic receptor (beta AR) coupled-adenylylcyclase system stimulate a variety of responses by airway epithelial cells which affect airway caliber and the response to inflammatory stimuli. Although the tracheobronchial epithelium (TBE) is composed of several phenotypically differentiated cell types, surprisingly little is known about the expression of the beta AR system by the major subpopulations of TBE cells (i.e., basal and columnar). We, therefore, examined the function of the beta AR system in columnar and basal cell-enriched populations of rabbit tracheocytes. Cells were collected from 35 rabbits in 17 separate experiments and separated into basal and columnar cell-enriched fractions by centrifugal elutriation. The columnar fraction demonstrated a significantly greater (P < 0.005) adenosine 3',5'-cyclic monophosphate (cAMP) response to isoproterenol (10(-9)-10(-5) M) than the basal cell-enriched fraction (i.e., 74.7 +/- 5.1 and 49.4 +/- 2.8 pmol/10(6) cells, in columnar and basal cell-enriched fractions, respectively, P < 0.0001) as well as a higher beta AR density (i.e., 8,678 +/- 840 and 4,754 +/- 406 beta AR sites/cell, respectively, P < 0.0001). However, when corrected for differences in cell size assessed from measurements of total cell protein, cAMP production per milligram protein and beta AR density per milligram protein were similar in the two cell fractions (P > 0.50 for both comparisons). beta AR subtype assessed by beta 1AR and beta 2AR subtype selective antagonists demonstrated that the beta 2AR subtype predominated (i.e., > 90%) in both cell populations (P > 0.5).(ABSTRACT TRUNCATED AT 250 WORDS)

Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains.

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)

Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains.

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pulse-chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of proacrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N-glycosidase or O-glycosidase reduced the Mr by 3-7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N- and O-linked oligosaccharides.

Preparation and short-term culture of enriched populations of guinea pig spermatocytes and spermatids.

In order to study acrosome biogenesis in vitro, the authors developed techniques for the isolation and culture of enriched populations of guinea pig spermatogenic cells using a modification of the technique of Romrell et al (1976). The modifications include changes in the medium, enzyme concentrations, cell loads for gradient separations, and sedimentation times. Three major cell populations were pooled: Pachytene spermatocytes (PS: 2.0 x 10(7), 80-85% pure), round spermatids, (RS: 1.2 x 10(8), 80-85% pure), and condensing spermatids (CS: 2.0 x 10(8), 50-60% pure, contaminated with residual bodies). The ultrastructural properties of the isolated cells appeared similar to those of cells in situ. PS were 3-4 times more active than RS and 10 times more active than CS in synthesizing proteins. These experiments demonstrate that highly enriched populations of guinea pig spermatogenic cells can be isolate, and that these cells can be cultured in the presence of radioactive precursors for studies of protein synthesis during spermatogenesis.

Acrosome biogenesis begins during meiosis: evidence from the synthesis and distribution of an acrosomal glycoprotein, acrogranin, during guinea pig spermatogenesis.

The biogenesis of the sperm-specific organelle, the acrosome, was investigated using an acrosomal glycoprotein as a marker of development. This component, which we have named acrogranin, was purified from an acid extract of guinea pig testes by standard chromatographic procedures. The molecular weight of reduced acrogranin was determined to be 67,000 by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunization of female rabbits with purified acrogranin produced an antiserum that recognized a single protein with Mr = 67,000 in an acid extract of guinea pig testes. By indirect immunofluorescence, acrogranin was found only in the acrosome of mature sperm. In haploid spermatids, acrogranin was localized in the developing acrosome and, weakly, in the cytoplasm. Acrogranin was also detected in the cytoplasm and juxtanuclear region in putative proacrosomal granules of meiotic cells (pachytene spermatocytes). Detergent extracts from different purified germ cell populations contained only the Mr = 67,000 form of acrogranin, but sperm extracts had four lower Mr immunoreactive forms not present in the testicular extracts. By two-dimensional gel electrophoresis, acrogranin was found to be an acidic glycoprotein. Analysis of glycosylated and trifluoromethanesulfonic acid-deglycosylated acrogranin indicated that the antibody recognized polypeptide determinants. After highly enriched germ cell populations were labeled overnight with [35S]methionine and extracted with detergent, anti-acrogranin immunoprecipitated a single protein of Mr = 67,000. The synthesis of acrogranin by pachytene spermatocytes and round spermatids was similar, but the synthesis of the glycoprotein by condensing spermatids was markedly reduced. These studies demonstrate that acrosome biogenesis, as determined by the synthesis of a specific acrosomal component, begins during meiosis and continues through the early stages of spermiogenesis.

Noncoordinate regulation of de novo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase in mouse Leydig cell cultures: relation to steroid production.

The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol side-chain cleavage (P-450scc) and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (P-450(17 alpha) was investigated in mouse Leydig cell cultures. In the absence of cAMP, the amount of immunoreactive P-450(17 alpha) decreased to less than 5% by day 4 and was undetectable between days 7 and 11. In contrast, the amount of immunoreactive P-450scc remained relatively constant throughout the same period. Treatment of Leydig cell cultures for 4 days with 0.05 mM 8-bromo-cAMP initiated on day 7 increased the amount of P-450(17 alpha) with relatively little effect on the amount of P-450scc. The rate of de novo synthesis of each of the P-450 enzymes was studied by determining [35S]methionine incorporation into newly synthesized protein. In the absence of cAMP, de novo synthesis of P-450(17 alpha) ceased while the rate of de novo synthesis of P-450scc increased with time in culture between days 2 and 11. Treatment with cAMP initiated on day 7 of culture caused a time-dependent increase in the rate of de novo synthesis of P-450(17 alpha) on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly isolated Leydig cells. The rate of de novo synthesis of P-450scc was increased 2-fold relative to untreated cultures on days 9 and 11. De novo synthesis of P-450(17 alpha) ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Catecholamine stimulation of androgen production by rat Leydig cells. Interactions with luteinizing hormone and luteinizing hormone-releasing hormone.

The mechanism(s) of the development of response to catecholamines (CA) by Leydig cells in culture was investigated with the use of primary culture of purified Leydig cells of adult rats. The interactions of a CA agonist, isoproterenol (ISOP), with luteinizing hormone (LH) and a luteinizing hormone-releasing hormone agonist analog (LHRHa) on production of androgen by the Leydig cells were also studied. Cells incubated with ISOP for 3 h increased release of cyclic adenosine 3',5'-monophosphate (cAMP) to similar extents at 0, 3, and 24 h of culture. The beta-agonist did not increase androgen release at 0 h but had a concentration-dependent effect at 3, 24, and 48 h of culture, with maximal effects at 24 h. LH stimulated high increases in production of cAMP and androgen by the cells at 0-24 h of culture. Leydig cell beta-receptors decreased with culture time. Low concentrations but not high levels of LH had additive effects with ISOP on androgen release. ISOP showed a complex interaction with LHRHa on androgen release. Chronic exposure of Leydig cells to LHRHa reduced basal androgen release as well as release of androgen stimulated by ISOP, forskolin, and LH. These studies suggest that the development of response to CA by rat Leydig cells is a postreceptor, postcAMP event and showed that CA can interact with LH or LHRH to regulate Leydig cell function.

Propranolol inhibits the compensatory increase in androgen secretion after unilateral orchidectomy in rats.

Adrenergic antagonists were administered to rats by intratesticular injection at the time of unilateral orchidectomy and 5 h before autopsy, 24 h after surgery. Injections of the beta-receptor antagonist DL-propranolol (0.5 or 1.0 mg/injection) significantly inhibited the increase in the concentration of androgens in testicular vein plasma or interstitial fluid that occurred in unilaterally orchidectomized animals injected with vehicle. DL-Propranolol injections in animals with both testes did not reduce testicular or peripheral androgen concentrations or their increase after hCG administration. Injections of the less potent isomer (+)-propranolol or the alpha-receptor antagonist phentolamine did not inhibit the response to unilateral orchidectomy. It is concluded that the compensatory increase in androgen secretion induced by unilateral orchidectomy is, at least in part, the result of beta-adrenergic stimulation of steroidogenesis.

Characterization of beta-adrenergic binding sites on rodent Leydig cells.

A radioligand binding technique was used to study beta-adrenergic binding sites on rodent Leydig cells. Beta-Adrenergic binding sites were found on Leydig cells in both the rat and mouse. Binding of [3H]CGP-12177 [4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazole-2-one] to purified rat Leydig cells was found to be saturable, temperature and time dependent, stereospecific, and readily reversible by the beta-adrenergic antagonist propranolol. Scatchard analysis revealed the presence of high-affinity sites with an apparent dissociation constant (Kd) of 0.79 +/- 0.22 nM and maximal binding capacity (Bmax) of 1716 +/- 245 sites per rat Leydig cell. Competition of various beta-adrenergic agonists and antagonists with [3H]CGP indicates an order of potency of L-isoproterenol greater than epinephrine = salbutamol greater than norepinephrine greater than D-isoproterenol and dl-propranolol = ICI 118,551 much greater than atenolol, respectively. These observations suggest that the binding sites are predominantly of the beta 2-receptor subtype. Incubation of freshly isolated rat Leydig cells with luteinizing hormone (100 ng/ml) caused consistent stimulation of androgen production, but only occasional stimulation by the beta-agonist isoproterenol (10 microM) was observed. However, these cells consistently responded to the beta-agonist after 3 h in primary cultures. These findings indicate that rodent Leydig cells possess beta-adrenergic binding sites and point out a possible dissociation between receptor recognition and physiologic response.

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells.

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.

Ontogeny of rodent testicular androgen production in response to isoproterenol and luteinizing hormone in vitro.

The ontogeny of rodent testicular androgen production in response to the catecholamine agonist L-isoproterenol (Isop) and luteinizing hormone (LH) was studied in vitro. Isop (10(-4) M) significantly increased rat androgen production by 1.2- 3.3-fold on Days 18.5 to 21.5 of gestation and on Days 1 to 60 of postnatal life during the 3-h incubation in Medium 199. Similarly, Isop augmented mouse androgen production by 1.4- to 4.3-fold on Days 16.5 to 19.5 of gestation and on 1, 10, 20 40 and 60 days of postnatal life. No effect of the beta-agonist was observed in postnatal rats aged 6 and 10 days, and mice aged 2, 5, 15 and 30 days. In both species, LH (100 ng/ml) induced large increases (2.8- to 265.6-fold) in androgen production at all fetal and postnatal ages. Isop was 0.3-22.9% as effective as LH in the two species. The minimum concentration of Isop to significantly increase androgen production by testes from 20.5-day rat fetuses was 10(-8) M, while 10(-5) M was required to stimulate 17.5-day mouse fetuses. Isop significantly increased androgen production by 20.5-day rat and 17.5-day mouse fetuses after a time lag of 30 and 180 min, respectively. In both species, a beta-adrenergic antagonist, propranolol (10(-5) M), abolished the increased androgen production attributed to Isop. Propranolol alone and the D-isomer of Isop had no effect. The results indicate that although LH is the key stimulant of testicular androgen production, catecholamines may play an important role(s) in testicular development, especially at fetal ages.

Effects of forskolin on androgen production by mouse interstitial cells in vitro. Interactions with luteinizing hormone and isoproterenol.

Micromolar concentrations of the diterpene forskolin stimulated androgen production by collagenase-dispersed mouse testicular interstitial cells. With maximum stimulatory concentrations, forskolin and luteinizing hormone (LH) increased androgen production with similar time courses and to similar extents. The concentration of LH required for half-maximum stimulation (EC50) was reduced approximately 10-fold in the presence of forskolin whereas maximum androgen production was unaffected. Likewise, LH reduced the EC50 for forskolin approximately 5-fold. The observed synergism between LH and forskolin was most likely at the level of cAMP generation as forskolin did not alter the EC50 for dibutyryl cAMP activation of androgen production. When cells were allowed to attach to the wells of tissue culture plates for 3 h prior to stimulation, isoproterenol treatment induced a small increase in androgen production when, and only when, submaximum concentrations of forskolin were also present. The increase in androgen production attributable to isoproterenol was blocked by simultaneous exposure to the beta-antagonist propranolol. When cells were immediately (O h) exposed to forskolin and isoproterenol, no interaction was observed. These results demonstrate the ability of forskolin treatment to reveal the presence of "latent" beta-adrenergic receptors. They also indicate that isolated adult mouse Leydig cells may not contain such receptors.