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1. beta-adrenoceptor activation leads to pulmonary vasodilatation. The increase in circulating catecholamines at birth may assist the postnatal fall in vascular resistance by their activation. To study beta(1)- and beta(2)-adrenoceptors during postnatal adaptation, we used [(125)I]-iodocyanopindolol (ICYP) binding to lung membranes and sections to quantify and locate the binding sites in piglets from birth to 14 days of age and compared them with those in adult pigs. In addition, pulmonary hypertension was induced in newborn piglets by hypobaric hypoxia. 2. In lung membranes the equilibrium dissociation constant (K(d)) did not change with age for total beta-adrenoceptors or for beta(2)-adrenoceptors, but there was a significant increase in maximum binding sites (B(max)) between birth and 3 days of age. On tissue sections, B(max) increased between 3 days and adulthood with no change in K(d). 3. Binding sites of beta(1)- and beta(2)-adrenoceptors were localized to the bronchial epithelium, to endothelium of extra- and intra-pulmonary arteries and to lung parenchyma. Total beta-adrenoceptor density increased with age at all locations (P<0.05 - 0.01). At birth intrapulmonary arteries showed no binding, beta(2)-adrenoceptors appeared on day 1 and increased up to 14 days of age. beta(1)-adrenoceptors appeared by 3 days of age and increased with age. 4. Hypobaric hypoxia from birth led to attenuation in the normal postnatal increase in receptor number, but hypoxia from 3 - 6 days did not decrease receptor density. 5. The normal postnatal increase in beta-adrenoceptors suggests a potential for catecholamine induced dilatation in the lung during adaptation which is attenuated in pulmonary hypertension.

1. The aim of the present study was to examine the effects of glucocorticoid dexamethasone on airway responsiveness to albuterol after intratracheal instillation of saline or IL-1beta in Brown-Norway rats in vivo and to elucidate the molecular mechanism of this effect. 2. IL-1beta caused a significant reduction in albuterol-mediated relaxation to protect against MCh-induced bronchoconstriction. Dexamethasone attenuated the IL-1beta-induced impaired relaxation while alone had no effect when compared to rats treated identically with saline. 3. The density of beta(2)-adrenoceptors was significantly reduced in lung membranes harvested from IL-1beta-treated rats, which was associated with impaired isoproterenol- and forskolin-stimulated cyclic AMP accumulation and adenylyl cyclase (AC) activity ex vivo. Dexamethasone did not prevent IL-1beta-induced down-regulation of beta(2)-adrenoceptors but completely blocked IL-1beta-induced impairment of cyclic AMP accumulation and AC activity stimulated by isoproterenol and forskolin. 4. The inhibitory G-protein subtypes, G(ialpha1), G(ialpha2) and G(ialpha3), were detected in lung membranes prepared from all groups of rats but the intensity of G(ialpha1) and G(ialpha2) was markedly increased in IL-1beta-treated rats, which were not prevented by dexamethasone. 5. The activity of cytosolic GRK and the expression of GRK2 and GRK5 were elevated in the lung of IL-1beta-treated rats, which were completely abolished by dexamethasone. 6. These results indicate that treatment of rats with IL-1beta results in desensitization of pulmonary beta(2)-adrenoceptors. In light of data obtained in this study, we propose that both the decrease in AC activity and the increase in GRK activity, which are reversed by dexamethasone, may underlie beta(2)-adrenoceptor desensitization.

1. The aim of the present study was to examine the effects of chronic infusion of the long-acting agonist salmeterol on pulmonary beta(2)-adrenoceptor function in Sprague-Dawley rats in vivo and to elucidate the molecular basis of any altered state. 2. Systemic administration of rats with salmeterol for 7 days compromised the ability of salmeterol and prostaglandin E(2) (PGE(2)), given acutely by the intravenous route, to protect against ACh-induced bronchoconstriction when compared to rats treated identically with vehicle. 3. beta(1)- and beta(2)-adrenoceptor density was significantly reduced in lung membranes harvested from salmeterol-treated animals, which was associated with impaired salmeterol- and PGE(2)-induced cyclic AMP accumulation ex vivo. 4. Three variants of G(s alpha) that migrated as 42, 44 and 52 kDa peptides on SDS polyacrylamide gels were detected in lung membranes prepared from both groups of rats but the intensity of each isoform was markedly reduced in rats that received salmeterol. 5. The activity of cytosolic, but not membrane-associated, G-protein receptor-coupled kinase was elevated in the lung of salmeterol-treated rats when compared to vehicle-treated animals. 6. The ability of salmeterol, administered systemically, to protect the airways of untreated rats against ACh-induced bronchoconstriction was short-acting (t(off) approximately 45 min), which contrasts with its long-acting nature when given to asthmatic subjects by inhalation. 7. These results indicate that chronic treatment of rats with salmeterol results in heterologous desensitization of pulmonary G(s)-coupled receptors. In light of previous data obtained in rats treated chronically with salbutamol, we propose that a primary mechanism responsible for this effect is a reduction in membrane-associated G(s alpha). The short-acting nature of salmeterol, when administered systemically, and the reduction in beta-adrenoceptor number may be due to metabolism to a biologically-active, short-acting and non-selective beta-adrenoceptor agonist.

We have investigated the role of protein kinase C (PKC) in the desensitization of histamine H1-receptors and in the expression of the histamine H1-receptor gene in airway smooth muscle. Prolonged 4beta-phorbol 12,13 dibutyrate (PDBu) pretreatment (4 h, 100 nM-1 microM) of bovine trachealis caused a concentration-dependent loss of contraction in response to histamine H1-receptor stimulation, which was associated with a concentration-dependent decrease in histamine-induced total [3H]-inositol phosphates accumulation. In contrast, the responses to sodium fluoride, a direct G-protein activator, were unalterd by PDBu (100-300 nM) pre-incubation and only slightly reduced following incubation with 1 microM PDBu. A selective PKC inhibitor, GF 109203X, partially blocked the PDBu (1 microM)-induced desensitization and completely blocked the effect of 100 nM PDBu, confirming the involvement of PKC. Binding experiments using [3H]-pyrilamine revealed a class of high-affinity binding sites within the range for the histamine H1 receptor in airway smooth muscle. PDBu (1 microM) pretreatment for 4 h did not change the number of histamine H1 receptors. PDBu (1 microM) exposure caused a time-dependent reduction in the steady-state levels of histamine H1-receptor mRNA, which was inhibited by pre-incubation with GF 109203X and by cycloheximide, a protein synthesis inhibitor. Nuclear run-on assays revealed a 50% reduction in the rate of histamine H1-receptor gene transcription after 17 h PDBu pretreatment, whereas mRNA stability was not affected by PDBu pretreatment (17 h). In conclusion, we have shown a PKC-mediated desensitization of the histamine H1-receptor in BTSM and a transcriptional down-regulation of the histamine H1-receptor gene expression, which requires new protein synthesis.

1. Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1-100 microM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72 +/- 8% (n = 5) following pre-exposure to 100 microM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation. 2. The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 microM) but not by cimetidine (an H2-receptor antagonist, 10 microM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors. 3. Indomethacin (10 microM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R. 4. GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization. 5. The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 microM, 0.5, 1, 2, 4, 16 and 24 h). 6. These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase.

1. The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells). 2. Saturation studies performed with the non-selective hydrophilic ([3H]-N-methyl-scopolamine, [3H]-NMS) and lipophilic (3H]-quinuclidinyl benzilate, [3H]-QNB) muscarinic antagonists revealed a single class of high affinity binding sites. 3. Carbachol (1 mM) induced a rapid down-regulation of [3H]-NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h. 4. The loss of [3h]-QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [3H]-NMS binding sites as a result of receptor sequestration. 5. Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin-induced cyclic AMP accumulation was lost following 24 h carbachol stimulation. 6. The steady state level of muscarinic m2 mRNA measured by Northern blot analysis was not affected by carbachol had no effect on the stability of m2 mRNA. 7. The rate of transcription of m2 muscarinic receptor gene as measured by nuclear RNA run-on assay was unaltered by carbachol stimulation. 8. These results suggest that homologous sequestration, desensitization, and down-regulation of M2 modifications of m2 muscarinic receptor mRNAs.