IL-15-deficient mice develop enhanced allergic responses to airway allergen exposure.

BACKGROUND: Interleukin-15 is a pleiotropic cytokine that is critical for the development and survival of multiple haematopoietic lineages. Mice lacking IL-15 have selective defects in populations of several pro-allergic immune cells including natural killer (NK) cells, NKT cells, and memory CD8+ T cells. We therefore hypothesized that IL-15-/- mice will have reduced inflammatory responses during the development of allergic airway disease (AAD). OBJECTIVE: To determine whether IL-15-/- mice have attenuated allergic responses in a mouse model of AAD. METHODS: C57BL/6 wild-type (WT) and IL-15-/- mice were sensitized and challenged with ovalbumin (OVA), and the development of AAD was ascertained by examining changes in airway inflammatory responses, Th2 responses, and lung histopathology. RESULTS: Here, we report that IL-15-/- mice developed enhanced allergic responses in an OVA-induced model of AAD. In the absence of IL-15, OVA-challenged mice exhibited enhanced bronchial eosinophilic inflammation, elevated IL-13 production, and severe lung histopathology in comparison with WT mice. In addition, increased numbers of CD4+ T and B cells in the spleens and bronchoalveolar lavage (BAL) were also observed. Examination of OVA-challenged IL-15Rα-/- animals revealed a similar phenotype resulting in enhanced airway eosinophilia compared to WT mice. Adoptive transfer of splenic CD8+ T cells from OVA-sensitized WT mice suppressed the enhancement of eosinophilia in IL-15-/- animals to levels observed in WT mice, but had no further effects. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that mice with an endogenous IL-15 deficiency are susceptible to the development of severe, enhanced Th2-mediated AAD, which can be regulated by CD8+ T cells. Furthermore, the development of disease as well as allergen-specific Th2 responses occurs despite deficiencies in several IL-15-dependent cell types including NK, NKT, and γδ T cells, suggesting that these cells or their subsets are dispensable for the induction of AAD in IL-15-deficient mice.

Regulatory B cells from hilar lymph nodes of tolerant mice in a murine model of allergic airway disease are CD5+, express TGF-ß, and co-localize with CD4+Foxp3+ T cells.

In a biphasic, ovalbumin (OVA)-induced murine asthma model where allergic airway disease is followed by resolution and the development of local inhalational tolerance (LIT), transforming growth factor (TGF)-ß-expressing CD5(+) B cells were selectively expanded locally in hilar lymph nodes (HLN) of LIT mice. LIT HLN CD5(+) B cells, but not LIT HLN CD5(-) B cells, induced expression of Foxp3 in CD4(+)CD25(-) T cells in vitro. These CD5(+) regulatory B cells (Breg) and CD4(+)Foxp3(+) T cells demonstrated similar increases in expression of chemokine receptors (CXCR4 and CXCR5) and co-localized in HLN B cell zones of LIT mice. The adoptive transfer of LIT HLN CD5(+) B cells, but not LIT HLN CD5(-) B cells, increased the number of CD4(+)Foxp3(+) T cells in the lung and inhibited airway eosinophilia in this OVA model. Thus, Breg in HLNs of LIT mice reside in a CD5(+) TGF-ß-producing subpopulation and co-localize with CD4(+)Foxp3(+) T cells.

Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin.

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.

Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease.

Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8(+) to CD4(+)TCRalphabeta lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-gamma and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-gamma and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.

Current concepts of respiratory complications of neuromuscular disease in children.

Chronic neuromuscular diseases affect the respiratory muscles in varying patterns and degrees. As a result, patients with these disorders develop restrictive pulmonary disease, ineffective cough, atelectasis and pneumonia, and chronic respiratory insufficiency leading to respiratory failure. Therapeutic strategies are evolving to augment cough and airway clearance, improve lung volumes, and support the patient with progressive ventilatory failure. These techniques have improved longevity and quality of life for many patients with neuromuscular disease.

Dexamethasone potentiates high-affinity beta-agonist binding and g(s)alpha protein expression in airway smooth muscle.

Corticosteroids enhance beta-adrenergic responses by actions at both beta-adrenoceptor (beta-AR) and post-beta-AR sites. The present study investigated the effects of dexamethasone on beta-AR density, high-affinity beta-agonist binding, G(s)alpha and G(i)alpha protein expression, and cAMP responses in bovine tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM cells increased total beta-AR density 1.6- to 1.9-fold as assessed by the saturation binding of [(3)H]CGP-12177 and by displacement of radioligand binding with isoproterenol. Isoproterenol bound to the beta-AR at two sites, a high-affinity site with a density of 5.9 +/- 1.2 fmol/mg protein and a low-affinity site with a density of 16.9 +/- 1. 0 fmol/mg protein. Dexamethasone increased both high- and low-affinity isoproterenol binding sites to 11.1 +/- 2.2 and 25.9 +/- 2.1 fmol/mg protein, respectively, without influencing agonist binding affinities. Dexamethasone also selectively increased G(s)alpha protein levels from 0.99 +/- 0.14 to 1.46 +/- 0.17 microg/mg protein without affecting G(i)alpha levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP generation in dexamethasone-treated bTSM cells. These findings provide new insights into the corticosteroid regulation of beta-adrenergic signaling pathways in airway smooth muscle.

Proinflammatory roles of T-cell receptor (TCR)gammadelta and TCRalphabeta lymphocytes in a murine model of asthma.

The role of lymphocytes bearing alphabeta or gammadelta T-cell receptors (TCRs) was assessed during the acute allergic response in a mouse model of asthma. The inflammatory immune response to ovalbumin (OVA) was characterized in wild-type C57BL/6J mice and congenic TCRbeta(-/-) and TCRdelta(-/-) mice by evaluation of airway eosinophilia, histopathology, serum immunoglobulin (Ig)E levels, and in vivo airway responsiveness to methacholine. OVA-challenged wild-type mice demonstrated marked pulmonary inflammation, evidenced by airway eosinophilia (68 +/- 7 x 10(4) cells), peribronchial lympho-plasmocytic infiltration, and elevated serum IgE (4.9 +/- 0.6 microg/ml). These responses were markedly attenuated in TCRdelta(-/-) animals (5.0 +/- 1.0 x 10(4) eosinophils and 1.6 +/- 0. 3 microg/ml IgE) and were completely absent in TCRbeta(-/-) mice (< 1 x 10(3) eosinophils and 0.38 +/- 0.21 microg/ml IgE). Similar results were observed in mice treated with anti-TCRgammadelta or anti-TCRalphabeta monoclonal antibodies. Airway responsiveness to aerosolized methacholine was also reduced in challenged TCRdelta(-/-) animals relative to challenged wild-type mice. These results demonstrate that acute allergic airway responses are dependent upon intact TCRalphabeta and TCRgammadelta lymphocyte function and that TCRgammadelta cells promote acute airway sensitization.

beta-adrenergic relaxation of rabbit tracheal smooth muscle: a receptor deficit that improves with corticosteroid administration.

beta-Adrenergic agonists are potent relaxing agents of airway smooth muscle; however, they are often incapable of fully reversing agonist-mediated contractions. The present study was designed to quantitate the relationship between beta-adrenoceptor binding, signal transduction, and relaxation in rabbit tracheal smooth muscle (TSM). TSM segments contracted with acetylcholine to 25 to 75% maximal contraction were relaxed with cumulative administration of isoproterenol (ISO). A beta-adrenergic receptor "deficit" was found, such that incomplete relaxation was achieved with full receptor occupancy. Binding studies with [(3)H]dihydroalprenolol demonstrated a beta-adrenoceptor density of 33.1 +/- 8.6 fmol/mg protein in control TSM. Paired studies were performed in TSM from rabbits treated with dexamethasone. Relative to control tissues, dexamethasone-treated TSM displayed twice as much relaxation and cAMP production in response to ISO and twice the beta-adrenoceptor density (82.2 +/- 12.3 fmol/mg protein). Dexamethasone did not affect G(i) function, as assessed by the degree of functional antagonism exerted by acetylcholine on ISO-induced relaxations, or beta-adrenoceptor-G(s) coupling, as reflected in high-affinity beta-agonist binding. Collectively, these results demonstrate that corticosteroid administration exerts parallel potentiating effects on beta-adrenoceptor expression and function in rabbit airway smooth muscle.

Shifts in lung lymphocyte profiles correlate with the sequential development of acute allergic and chronic tolerant stages in a murine asthma model.

T lymphocytes have a central regulatory role in the pathogenesis of asthma. We delineated the participation of lymphocytes in the acute allergic and chronic tolerant stages of a murine model of asthma by characterizing the various subsets of lymphocytes in bronchoalveolar lavage and lung tissue associated with these responses. Acute (10-day) aerosol challenge of immunized C57BL/6J mice with ovalbumin resulted in airway eosinophilia, histological evidence of peribronchial and perivascular airway inflammation, clusters of B cells and TCRgammadelta cells in lung tissue, increased serum IgE levels, and airway hyperresponsiveness to methacholine. In mice subjected to chronic (6-week) aerosol challenge with ovalbumin, airway inflammation and serum IgE levels were significantly attenuated and airway hyperresponsiveness was absent. The marked increases in lung B and T cell populations seen in the acute stage were also significantly reduced in the chronic stage of this model. Thus, acute ovalbumin challenge resulted in airway sensitization characteristic of asthma, whereas chronic ovalbumin challenge elicited a suppressed or tolerant state. The transition from antigenic sensitization to tolerance was accompanied by shifts in lymphocyte profiles in the lung and bronchoalveolar lavage fluid.

Tannin inhibits the cAMP-beta-adrenergic receptor pathway in bovine tracheal epithelium.

Tannin, isolated from cotton bracts, inhibits chloride secretion in airway epithelium. In bovine tracheal epithelial cells, tannin (25 micrograms/ml) blunted isoproterenol (Iso)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Inhibition was time and dose dependent, with 52 +/- 5% (mean +/- SE, n = 6) inhibition at 60 min and 82 +/- 9% (n = 3) inhibition at 8 h. Inhibition was reversible starting at 4 h. Low-molecular-mass tannin (1,000-5,000 Da) had no effect on Iso-stimulated cAMP accumulation, whereas N-acetylcysteine, which interacts with cysteine residues, blocked the effects of tannin on Iso-stimulated cAMP accumulation. Tannin exposure (25 micrograms/ml for 30 min) had no effect on the dissociation constant (Kd) for [3H]dihydroalprenolol (DHA) (0.41 +/- 0.03 nM, n = 3) but decreased maximal binding from 252 +/- 32 to 162 +/- 36 fmol/mg protein. Using single-point analysis and [3H]CGP-12177, we determined that tannin (25 micrograms/ml for 4 h) decreased surface beta-adrenergic receptor density from 26.4 +/- 4.3 (n = 12) to 11.9 +/- 3.0 fmol/mg protein and that the decrease was dose dependent. Agonist binding affinity by Iso displacement of DHA demonstrated a two-site model (Kd values = 27 +/- 9 and 2,700 +/- 600 nM) and a ratio of high- to low-affinity receptors of 1:1. Tannin (25 micrograms/ml) steepened the curve and shifted it to the right, as did Gpp(NH)p. Gpp(NH)p had no further effect on the shape or position of the displacement curve in the presence of tannin. In contrast, when polymer length was decreased by oxidation, tannin had no effect on the DHA displacement curve. These data demonstrate that tannin reversibly desensitizes bovine tracheal epithelial cells to Iso, decreases beta-adrenergic receptor density, and uncouples the receptor from its stimulatory G protein. These data also suggest that the polymer length of tannin and its interaction with cysteine residues are important for these effects. These studies provide additional evidence for the role of tannin in the occupational lung disease byssinosis.

Corticosteroid modulation of Na(+)-K+ pump-mediated relaxation in maturing airway smooth muscle.

1. The ontogeny of the relaxant influence of the airway electrogenic Na(+)-K+ pump and its potential modulation by corticosteroids were examined in airway smooth muscle (ASM) segments isolated from newborn and adult rabbits. 2. Control and methylprednisolone-treated (MP) ASM segments were half-maximally contracted with methacholine in K(+)-free buffer and the ASM relaxant responses to Na(+)-K+ pump activation were subsequently evaluated. Relative to adult ASM, control newborn ASM showed significantly enhanced maximal relaxation (Rmax) to KCl (62.5 +/- 5.2% vs. 47.8 +/- 5.2%), but no difference in sensitivity (pC2 = -log concentration producing 50% Rmax: 2.18 +/- 0.12 vs. 2.29 +/- 0.09-log M). 3. Exposure of ASM segments to 500 microM methylprednisolone for 1 h potentiated the airway Na(+)-K+ pump activity. A more pronounced effect was obtained in newborn ASM, where both the Rmax and pC2 values were significantly enhanced. In mature ASM, only the Rmax response to KCl was increased in the presence of MP. 4. Collectively, these data demonstrate that: (i) the functional activity of the airway electrogenic Na(+)-K+ pump decreases with post-natal maturation in the rabbit: (ii) corticosteroid treatment potentiates Na(+)-K+ pump activity in rabbit ASM; and (iii) the latter effect of corticosteroids is enhanced in immature airways. 5. The above findings provide new evidence that the airway relaxant response to activation of the electrogenic Na(+)-K+ pump varies ontogenetically and that corticosteroids potentiate the Na(+)-K+ pump activity in an age-dependent manner.

Ontogeny of beta-adrenergic desensitization in rabbit tracheal smooth muscle.

Prolonged or repeated exposure to beta-agonist medications may result in a desensitization of the agonist-mediated response. Under certain conditions, such agonist-induced desensitization may limit the efficacy of administered beta-adrenergic agonists to elicit bronchodilation. Accordingly, the present study was designed to study the mechanism of acute beta-adrenergic desensitization in maturing rabbit tracheal smooth muscle (TSM). Isometric tension was measured in tracheal ring segments isolated from newborn and mature rabbits and half-maximally contracted with Methacholine (Meth) or KCl. TSM segments were serially relaxed with repetitive single doses of isoproterenol (ISO: 0.1, 1.0, 10, or 100 microM) or prostaglandin E2 (PGE2: 0.1 or 10 microM). Serial administration of ISO-elicited dose-dependent desensitization of relaxation in mature and newborn TSM, contracted with either Meth or KCl. In contrast, the relaxant response to PGE2 was retained in the ISO-desensitized tissue. Repeated administration of PGE2 elicited no desensitization of PGE2 responsiveness, but did induce some dose-dependent desensitization of the ISO response in mature TSM. Compared to mature tissues, newborn TSM developed subtotal desensitization to 100 microM ISO and no ISO desensitization in response to PGE2. Thus, these findings demonstrate that (1) beta-adrenoceptor responsiveness undergoes dose-dependent homologous and, to a lesser extent, heterologous desensitization in rabbit TSM; and (2) both beta-adrenergic desensitization mechanisms increase with postnatal maturation.

Methylprednisolone and isoproterenol inhibit airway smooth muscle proliferation by separate and additive mechanisms.

To determine whether glucocorticoids and beta-adrenoceptor agonists act independently to inhibit airway smooth muscle (ASM) proliferation, the present study investigated the effects of methylprednisolone (MP) and isoproterenol (ISO) alone and in combination on leukotriene D4-induced ASM proliferation. MP and ISO had no effect on unstimulated ASM cell growth. In contrast, MP and ISO demonstrated dose-dependent inhibition of LTD4-induced proliferation, and the inhibitory effect was additive for combinations of MP and ISO. The competitive cAMP receptor antagonist, Rp-cAMPS, ablated the ISO-induced inhibition but had no affect on the inhibitory response to MP. In cells exposed to both ISO and MP, Rp-cAMPS attenuated the growth inhibition to levels achieved by MP alone. Accordingly, these findings demonstrate that glucocorticoids and beta-adrenergic agonists inhibit LTD4-induced ASM proliferation, and that their inhibitory effects are mediated by different signaling pathways.

Role of muscarinic M2 receptors in regulating beta-adrenergic responsiveness in maturing rabbit airway smooth muscle.

Muscarinic M2 and M3 receptor subtypes have been pharmacologically distinguished in airway smooth muscle. Whereas M3 receptors have been associated with smooth muscle contraction, M2 receptors have been implicated in Gi protein-coupled inhibition of adenylyl cyclase. To determine whether the role of M2 receptors varies with age in tracheal smooth muscle (TSM), dose-dependent relaxation responses to isoproterenol were compared in TSM isolated from 3-day-old and adult rabbits precontracted with acetylcholine (ACh) in the absence (control) and presence of an M2 receptor antagonist (gallamine or methoctramine). From sustained half-maximal ACh contractions, adult TSM were 5.6-fold less sensitive than 3-day-old tissues to isoproterenol-induced relaxation. Furthermore, the magnitude of muscarinic functional antagonism of isoproterenol-mediated TSM relaxation, assessed by varying the initial degree of ACh-induced contraction, significantly increased with age. In gallamine- and methoctramine-treated tissues, the relaxation-response curves to isoproterenol were shifted to the left in both 3-day-old and adult TSM. In contrast, pretreatment with either M2 receptor antagonist had no significant effect on the magnitude of muscarinic functional antagonism at either age. Moreover, Western blot analysis of G alpha i common and specific subunit expression in TSM membranes demonstrated qualitatively similar levels in 3-day-old and adult TSM. Collectively, these findings provide new evidence that 1) there exist inherent age-dependent differences in both the airway relaxant responsiveness to beta-adrenoceptor stimulation and muscarinic functional antagonism of beta-adrenergic relaxation, and 2) the latter are attributed to mechanisms other than ontogenetic alteration in M2 receptor function or Gi protein expression in maturing rabbit TSM.

cAMP generation inhibits inositol 1,4,5-trisphosphate binding in rabbit tracheal smooth muscle.

Agonist-induced airway contraction involves the generation and subsequent binding of the phosphoinositide-derived second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to its Ca(2+)-mobilizing intracellular receptor. To the extent that regulatory cross-talk is known to exist between different signal transduction pathways, the present study examined whether activation of the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A (PKA) pathway induces altered binding of Ins(1,4,5)P3 to its receptor in membrane homogenates of rabbit tracheal smooth muscle (TSM). In control TSM, monophasic binding curves provided mean +/- SE values for Ins(1,4,5)P3 receptor density (Bmax) and binding affinity (Kd) amounting to 940 +/- 43 fmol/mg protein and 10.7 +/- 1.2 nM, respectively. Relative to control, binding of [3H]Ins(1,4,5)P3 was significantly reduced in paired TSM separately treated with isoproterenol, forskolin, or dibutyryl-cAMP. Ins(1,4,5)P3 binding was inhibited to a level averaging 60% of control binding by maximal concentrations of each agonist, an effect attributed to a reduction in Ins(1,4,5)P3 binding sites rather than altered ligand affinity. Collectively, these findings demonstrate that activation of the cAMP-dependent signaling pathway is associated with inhibition of Ins(1,4,5)P3 receptor binding and implicate a novel mechanism of action of beta-adrenergic agents in preventing and/or reversing airway contraction.

Restrictive lung disease following treatment for malignant brain tumors: a potential late effect of craniospinal irradiation.

PURPOSE: To examine the effects of lomustine (CCNU), a commonly used nitrosourea, and craniospinal radiation therapy on the subsequent development of restrictive lung disease (RLD) following treatment for malignant brain tumors. PATIENTS AND METHODS: Pulmonary function testing with measurement of lung volume, spirometry, and diffusion capacity was performed in 28 patients who had received CCNU and/or radiation therapy as treatment for a malignant brain tumor. The median age at the time of treatment was 11.4 years (range, 3.9 to 36.7) and radiation therapy was completed 6 months to 11.6 years (median, 2.6 years) before testing. Patients were divided into four groups based on prior therapy. Group 1 received involved-field irradiation and a CCNU-containing chemotherapy regimen (n = 7); group 2, craniospinal irradiation with a boost to the primary tumor site and a CCNU-containing chemotherapy regimen (n = 6); group 3, craniospinal irradiation with a boost to the primary tumor site and a non-CCNU-containing chemotherapy regimen (n = 7); and group 4, craniospinal irradiation with a boost to the primary tumor site without chemotherapy (n = 8). RESULTS: Fourteen patients (50%) had findings consistent with RLD. One of seven patients (14.3%) who received CCNU without spinal irradiation had RLD, whereas 13 of 21 (61.9%) who received spinal irradiation with or without CCNU had RLD (P = .038), including four of eight patients treated with craniospinal irradiation alone. Logistic regression analysis showed that only spinal irradiation was a significant predictor for RLD. Patients who received spinal irradiation were 4.3 times more likely to have RLD than those who did not receive spinal irradiation. CONCLUSION: Spinal irradiation may be a risk factor for the development of RLD.

Mechanism of protein kinase C potentiation of airway beta-adrenergic relaxation.

To evaluate the regulatory action of protein kinase C (PKC) on airway beta-adrenergic function, the relaxant effects of isoproterenol (ISO) and 8 bromo-cyclic AMP (BrcAMP) were examined in tracheal smooth muscle (TSM) segments half-maximally contracted with acetylcholine in the absence (control) and presence of PKC activation with the phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB). Relative to control tissues, TSM treated with 0.1 microM DPB depicted significantly enhanced maximal relaxation and sensitivity to ISO but not to BrcAMP. The enhancing effect of DPB on ISO responsiveness was completely inhibited in the presence of the PKC antagonist H-7. Inhibition of the Na(+)-K+ pump with either ouabain or K(+)-free buffer diminished the TSM relaxant response to ISO but not to BrcAMP. Inhibition of the Na(+)-K+ pump also ablated the DPB-induced potentiation of beta-adrenoceptor responsiveness. Collectively, these data demonstrate that: 1) PKC activation enhances TSM relaxant responsiveness to beta-adrenoceptor stimulation; 2) inhibition of the airway Na(+)-K+ pump markedly blunts the relaxant response to beta-adrenoceptor stimulation; and 3) inhibition of the Na(+)-K+ pump abolishes the above potentiating effect of DPB on beta-adrenoceptor-mediated relaxation of rabbit TSM. Thus, the above findings provide new evidence that PKC activation enhances the airway relaxant response to beta-adrenoceptor stimulation, and that the latter effect is dependent on potentiated stimulation of the airway electrogenic Na(+)-K+ pump.

Maturation of catecholamine response and extraneuronal uptake in rabbit trachea.

The present study investigated whether maturational changes in extraneuronal catecholamine clearance accounted for the ontogenetic attenuation of isoproterenol responsiveness in rabbit tracheal segments. Following half-maximal contraction with acetylcholine (ACh) or KCl, tracheal ring segments (TS) isolated from newborn, 1-mo, and adult rabbits were relaxed with cumulative administration of isoproterenol. With postnatal maturation, there occurred a significant decrease in both maximal relaxation and sensitivity to isoproterenol in both ACh- and KCl-contracted TS. Inhibition of extraneuronal catecholamine uptake with methylprednisolone resulted in enhanced sensitivity to isoproterenol in 1-mo and adult, but not in newborn tracheal segments. In separate studies, extraneuronal catecholamine uptake was directly assayed in tracheal segments isolated from rabbits of similar age and pretreated with neuronal uptake and monoamine oxidase inhibitors. Maximal extraneuronal uptake of [3H]norepinephrine significantly decreased with age in tracheal tissues. In contrast, the sensitivity of the uptake process to norepinephrine increased with age. The relative catecholamine reserve markedly decreased with age in rabbit tracheal segments and was directly related to the maximal degree of relaxation elicited with the beta-adrenergic catecholamine, isoproterenol, in age-matched TS precontracted with either ACh or KCl. These findings suggest that, with maturation, extraneuronal uptake more effectively competes with beta-adrenoreceptor stimulation and, accordingly, may contribute to the ontogenetic attenuation of beta-adrenergic responsiveness in rabbit tracheal tissues.