(Endo)cannabinoids mediate different Ca2+ entry mechanisms in human bronchial epithelial cells.

In human bronchial epithelial (16HBE14o(-)) cells, CB(1) and CB(2) cannabinoid receptors are present, and their activation by the endocannabinoid virodhamine and the synthetic non-selective receptor agonist CP55,940 inhibits adenylyl cyclase and cellular interleukin-8 release. Here, we analyzed changes in intracellular calcium ([Ca2+](i)) evoked by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), CP55,940, and virodhamine in 16HBE14o(-) cells. Delta(9)-THC induced [Ca2+](i) increase and a large transient [Ca2+](i) mobilization, the latter probably reflecting store-depletion-driven capacitative Ca2+ entry (CCE). In contrast, CP55,940 induced a rather moderate Ca2+ influx and a sustained [Ca2+](i) mobilization. CP55,940-induced Ca2+ influx was inhibited by Ni2+, indicating CCE, possibly mediated by transient receptor potential channel TRPC1, the mRNA of which is expressed in 16HBE14o(-) cells. CP55,940-induced calcium alterations were mimicked by virodhamine concentrations below 30 microM. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni2+, but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La3+ and Gd3+. Such pharmacological profile is supported by the presence of TRPV1, TRPV4, and TRPC6 mRNAs as well as TRPV1 and TRPC6 proteins in 16HBE14o(-) cells. Cannabinoid receptor antagonists increased virodhamine-induced Ca2+ entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A(2) inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca2+](i) increase similar to virodhamine. Collectively, these observations suggest that [Ca2+](i) alterations induced by Delta(9)-THC, CP55,940 and by low concentrations of virodhamine involve mobilization and subsequent CCE mechanisms, whereas such responses by high virodhamine concentrations involve NCCE pathways.

Angiotensin-(1-7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1-7) analog.

The in vivo efficacy of many therapeutic peptides is hampered by their rapid proteolytic degradation. Cyclization of these therapeutic peptides is an excellent way to render them more resistant against breakdown. Here, we describe the enzymatic introduction of a thioether ring in angiotensin [Ang-(1-7)], a heptapeptide that plays a pivotal role in the renin-angiotensin system and possesses important therapeutic activities. The lactic acid bacterium Lactococcus lactis, equipped with the plasmid-based nisin modification machinery, was used to produce thioether-bridged Ang-(1-7). The resulting cyclized Ang-(1-7) is fully resistant against purified angiotensin-converting enzyme, has significantly increased stability in homogenates of different organs and in plasma derived from pig, and displays a strongly (34-fold) enhanced survival in Sprague-Dawley (SD) rats in vivo. With respect to functional activity, cyclized Ang-(1-7) induces relaxation of precontracted SD rat aorta rings in vitro. The magnitude of this effect is 2-fold larger than that obtained for natural Ang-(1-7). The Ang-(1-7) receptor antagonist D-Pro(7)-Ang-(1-7), which completely inhibits the activity of natural Ang-(1-7), also abolishes the vasodilation by cyclized Ang-(1-7), providing evidence that cyclized Ang-(1-7) also interacts with the Ang-(1-7) receptor. Taken together, applying a highly innovative enzymatic peptide stabilization method, we generated a stable Ang-(1-7) analog with strongly enhanced therapeutic potential.

Insulin increases the expression of contractile phenotypic markers in airway smooth muscle.

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function.

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (E(max)) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in E(max) in BTSM strips and their proliferative responses in BSTM cells and for E(max) and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.

Rho-kinase as a drug target for the treatment of airway hyperrespon-siveness in asthma.

In asthma, inflammatory and structural cells contribute to increased bronchoconstriction acutely and more chronically to airway remodelling. Current asthma therapy doesn't inhibit these features satisfactorily. This review discusses Rho-kinase as a potential drug target, since increasing evidence suggests a central role for this pathway in acute and chronic airway hyperresponsiveness.

Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes.

This study aims to investigate the effects of bradykinin, alone and in combination with growth factors on proliferation of cultured bovine tracheal smooth muscle cells. Bradykinin did not induce mitogenic responses by itself, but concentration-dependently augmented growth factor-induced [3H]thymidine incorporation and cell proliferation. The bradykinin effect was mediated by bradykinin B2 receptors, and not dependent on cyclo-oxygenase. Bradykinin-induced synergism with epidermal growth factor (EGF) could be suppressed by the protein kinase C (PKC) inhibitors GF 109203X (Bisindolylmaleimide I; specific for conventional and novel PKCs) and Gö 6976 (12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; specific for conventional PKCs). In addition, sole activation of PKC using Phorbol 12-myristate 13-acetate (PMA) was sufficient for a synergistic interaction with EGF. In contrast to bradykinin however, PMA was mitogenic by itself which was not at all affected by Gö 6976, but abolished by GF 109203X. Bradykinin transiently activated the p42/p44 MAP kinase pathway, whereas PMA-induced activation of p42/p44 mitogen activated protein (MAP) kinase was sustained. Neither the combination of bradykinin and EGF nor that of PMA and EGF induced synergistic activation of p42/p44 MAP kinase, however. These results show that bradykinin B2 receptor-stimulation augments growth factor-induced mitogenic responses of airway smooth muscle cells through activation of conventional PKC isozymes. In addition, the results show that PKC isozyme-specificity underlies stimulus-specific differences in mitogenic capacity for bradykinin and PMA.

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction.

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction.

BACKGROUND: In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction. METHODS: Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 microM), U-0126 (3 microM) and indomethacin (3 microM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2alpha (PGF2alpha) and prostaglandin E2 (PGE2) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 microM) and the selective EP1-antagonist AH-6809 (10 microM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2alpha-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay. RESULTS: Epidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2alpha-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2alpha-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 microM) significantly reduced (approximately 50 %) and the EP1-antagonist AH-6809 (10 microM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations. CONCLUSION: The results indicate that growth factors induce ASM contraction through contractile prostaglandins - not derived from the epithelium - which in turn rely on Rho-kinase for their contractile effects.

Differential desensitization of homozygous haplotypes of the beta2-adrenergic receptor in lymphocytes.

Single-nucleotide polymorphisms of the beta(2)-adrenergic receptor gene and its 5' promoter have been associated with differences in receptor function and desensitization. Linkage disequilibrium may account for inconsistencies in reported effects of isolated polymorphisms. Therefore, we have investigated the three most common homozygous haplotypes of the beta(2)-adrenergic receptor (position 19 [Cys/Arg] of the 5' leader cistron and positions 16 [Arg/Gly] and 27 [Gln/Glu] of the receptor) for putative differences in agonist-induced desensitization. Lymphocytes of well defined nonasthmatic, nonallergic subjects homozygous for the haplotype CysGlyGln, ArgGlyGlu, or CysArgGln were isolated. Desensitization of (-)-isoproterenol-induced cyclic adenosine monophosphate (cAMP) accumulation and beta(2)-adrenergic receptor sequestration and downregulation were measured in relation to beta(2)-adrenergic receptor-mediated inhibition of IFN-gamma and interleukin-5 production. We observed that lymphocytes of individuals bearing the CysGlyGln haplotype were more susceptible to desensitization of the beta-agonist-induced cAMP response than those of individuals with the ArgGlyGlu or CysArgGln haplotype. The haplotype-dependent desensitization of beta-agonist-induced cAMP response was not associated with haplotype-dependent beta(2)-adrenergic receptor sequestration or downregulation. In addition, our data suggest reduced inhibition, in lymphocytes of subjects with the CysGlyGln haplotype, of interleukin-5 production induced by treatment with antibodies to the T-cell receptor-CD3 complex and to costimulatory molecule CD28 (alphaCD3/alphaCD28). This is the first study demonstrating haplotype-related differences in agonist-induced beta(2)-adrenergic receptor desensitization in primary human cells. This haplotype-related desensitization of the beta(2)-adrenergic receptor in lymphocytes might have consequences regarding the regulation of helper T-cell type 2 inflammatory responses.

Microarray profiling of lymphocytes in internal diseases with an altered immune response: potential and methodology.

Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation.

Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction.

1. Repeated allergen challenge has been shown to increase the role of Rho-kinase in airway smooth muscle (ASM) contraction. We considered the possibility that active allergic sensitization by itself, that is, without subsequent allergen exposure, could be sufficient to enhance Rho-kinase-mediated ASM contraction. 2. Guinea pigs were actively IgE-sensitized to ovalbumin (OA), using Al(OH)(3) as adjuvant. Contractile responsiveness to G(q)-coupled receptor agonists (methacholine, histamine or PGF(2alpha)) was investigated in tracheal rings. No effect of sensitization was observed on basal- and methacholine-induced myogenic tone. In contrast, potency of histamine and PGF(2alpha) increased, that is, EC(50) decreased, after OA-sensitization by 2.6- and 4.7-fold, respectively, without effect on maximal contraction (E(max)). 3. Basal tone in preparations from both control and OA-sensitized animals was strongly decreased in the presence of the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632) (1 microm). In control preparations, the E(max) and potency of histamine were unaffected by Y-27632, but were decreased for PGF(2alpha) (by 38.2% and 2.0-fold, respectively). However, in preparations from OA-sensitized animals, Y-27632 induced a significant reduction in E(max) (33.5%) and potency (2.3-fold) of histamine and of PGF(2alpha) (48.3% and 6.6-fold, respectively), normalizing the OA-sensitization-induced increase in sensitivity toward these agonists. 4. We also investigated the contribution of Rho-kinase in vivo by measuring airway responsiveness toward inhaled histamine in permanently instrumented, unanaesthetized control and OA-sensitized guinea pigs. Treatment with Y-27632 by inhalation (5 mm, nebulizer concentration) decreased airway responsiveness toward histamine both in control and OA-sensitized animals. However, the histamine PC(100) ratio pre/post Y-27632 inhalation was significantly smaller in OA-sensitized animals as compared to control animals, indicating an enhanced contribution of Rho-kinase. 5. Expression of RhoA, an upstream activator of Rho-kinase, was significantly increased (2.6-fold) in lung homogenates of OA-sensitized guinea pigs compared to control animals, as determined by Western analysis. 6. In conclusion, the results show a receptor-dependent role of Rho-kinase in agonist-induced ASM contraction. The contribution of Rho-kinase to contractile airway responsiveness, both in vivo and ex vivo, is augmented after active allergic sensitization, as a consequence of increased expression of RhoA presumably. Inhibition of the RhoA/Rho-kinase pathway may be considered a useful pharmacotherapeutical target in allergy and asthma.

Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent.

Growth factors have been implicated in the pathophysiology of asthma. However, the putative effects of these growth factors on human airway smooth muscle tone are still largely unknown. We performed contraction experiments using human bronchial smooth muscle ring preparations. The growth factor insulin-like growth factor-1 (IGF-1) induced a slowly developing sustained contraction, which was dependent on Rho-kinase, since contraction was almost completely inhibited by (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632; 1 microM). Angiotensin II, a G(q)-coupled receptor agonist which can act as a growth factor as well, induced a biphasic contraction, the sustained phase of which was also almost completely inhibited by Y-27632. We conclude that angiotensin II and IGF-1 induce a Rho-kinase-dependent sustained contraction of human bronchial smooth muscle. Since growth factors are associated with pathophysiological conditions such as asthma, inhibition of Rho-kinase could be effective under these conditions.

Modulation of beta2- and beta3-adrenoceptor-mediated relaxation of rat oesophagus smooth muscle by protein kinase C.

Although a prominent role for protein kinase C (PKC) in the cross-talk between the phosphoinositide pathway and beta2-adrenoceptor signalling has been indicated, modulation of beta3-adrenoceptor function by PKC has not been studied thus far. In the present study, we have compared the relative capacity of PKC in modulating beta2- and beta3-adrenoceptor-mediated relaxation of methacholine-contracted rat oesophagus smooth muscle. To this purpose the effects of the PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) on relaxation induced by fenoterol, formoterol, (-)-noradrenaline, BRL 35135 (4-[2-[(2-hydroxy-2-(chlorophenyl)ethyl)amino]-propyl]-phenoxyacetic-acidmethylester) and IBMX (3-isobutyl-1-methyl-xanthine) were studied, in the absence and presence of the selective beta2-adrenoceptor antagonist ICI 118,551 (erythro-1(7-methylindan-4-yloxy)-3-(isopropylamin)-butan-2-ol). Our results show that inhibition of PKC resulted in differential augmentation of both beta2- and beta3-adrenoceptor-mediated relaxation. In contrast, relaxation induced by IBMX was not influenced at all by GF 109203X. The beta2-adrenoceptor bears phosphorylation sites for several kinases, including PKC. Since the beta3-adrenoceptor lacks these consensus sites, the results may also indicate that PKC-mediated Galphas phosphorylation is involved in the cross-talk between the muscarinic receptor-mediated phosphoinositide pathway and beta2- and, particularly, beta3-adrenoceptor signalling.

Muscarinic M(3) receptor-dependent regulation of airway smooth muscle contractile phenotype.

1. Airway smooth muscle (ASM) cells are known to switch from a contractile to a proliferative and synthetic phenotype in culture in response to serum and growth factors. Phenotype switching in response to contractile agonists, however, is poorly characterised, despite the possible relationship between ASM phenotype and airway remodelling in asthma. 2. To investigate the effects of muscarinic receptor stimulation on ASM phenotype, we used organ-cultured bovine tracheal smooth muscle (BTSM) strips, in which contractile responsiveness, contractile protein expression and proliferation were measured after pretreatment with methacholine. 3. Long-term methacholine pretreatment (8 days) decreased maximal contraction and sensitivity to methacholine as well as to histamine and KCl. This decrease was dose-dependent (pEC(50)=5.2+/-0.1). Pretreatment with the highest concentration of methacholine applied (100 microm) could suppress maximal histamine-induced contraction to 8+/-1% of control. In addition, contractile protein expression (myosin, actin) was downregulated two-fold. No concomitant increase in proliferative capacity was observed. 4. The M(3)/M(2) muscarinic receptor antagonist DAU 5884 (0.1 microm) completely inhibited the observed decrease in contractility. In contrast, the M(2)/M(3) muscarinic receptor antagonist gallamine (10 microm) was ineffective, demonstrating that M(2) receptors were not involved. 5. Pretreatment (8 days) with 60 mm KCl could mimick the strong decreases in contractility. This was completely prevented by pretreatment with verapamil (1 microm). 6. Regulation of contractility was not affected by protein kinase C inhibition, whereas inhibitors of phosphatidyl inositol 3-kinase and p42/p44 mitogen activated protein kinase were partially effective. 7. These results show that long-term methacholine pretreatment (8 days) induces an M(3) receptor-dependent decrease in BTSM contractility without increased proliferative capacity.

Role of Rho-kinase in maintaining airway smooth muscle contractile phenotype.

This study aims to investigate the role of Rho-kinase in phenotype switching and proliferation of bovine tracheal smooth muscle. To induce different phenotypic states, bovine tracheal smooth muscle strips were cultured (8 days) in 10% foetal bovine serum (foetal bovine serum, less contractile phenotype) or insulin (1 microM, hypercontractile phenotype) and compared to strips cultured in serum-free medium. In contraction experiments, the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632, 1 microM) decreased sensitivity to methacholine and KCl and lowered maximal responsiveness to KCl in all strips irrespective of the phenotype present. To investigate the effects of Rho-kinase bovine tracheal smooth muscle phenotypic regulation, strips were pretreated with Y-27632 (1 microM) for 8 days. This resulted in a decreased maximal contractility to both methacholine and KCl, quantitatively comparable to the decrease in contractility induced by platelet-derived growth factor (PDGF, 10 ng/ml). The combination of Y-27632 and PDGF responded additively. Y-27632 did not affect basal or PDGF-induced bovine tracheal smooth muscle cell proliferation, determined both as increases in [3H]thymidine incorporation and cell number. Inhibitors of the p42/p44 mitogen-activated protein kinase (MAPK) pathway, the p38 MAPK pathway and the phosphatidyl inositol (PI) 3-kinase pathway all inhibited PDGF-induced proliferation and phenotype changes. These results show that the functional contribution of Rho-kinase to bovine tracheal smooth muscle contraction is not dependent on phenotypic state. In addition, Rho-kinase is not involved in phenotypic modulation or proliferation induced by PDGF, whereas p42/p44 MAPK, p38 MAPK and PI 3-kinase are. Rho-kinase is, however, a major regulator involved in the basal maintenance of contractility in bovine tracheal smooth muscle.

Insulin induces a hypercontractile airway smooth muscle phenotype.

This study aims to investigate the effects of insulin on bovine tracheal smooth muscle phenotype in vitro. Contractility of muscle strips and DNA-synthesis ([3H]thymidine incorporation) of isolated cells were used as parameters for smooth muscle phenotyping. Insulin (1 microM) was mitogenic for bovine tracheal smooth muscle and potentiated DNA-synthesis induced by other growth factors. In contrast, after pretreatment of unpassaged bovine tracheal smooth muscle cells in culture, the mitogenic response induced by growth factors was strongly diminished, with no difference in the basal incorporation. Pretreatment of bovine tracheal smooth muscle strips in organ culture with insulin increased maximal contraction to methacholine and KCl. These results show that insulin acutely augments DNA-synthesis in the presence of other growth factors. In contrast, insulin pretreatment induces a hypercontractile phenotype with a decreased mitogenic capacity. This mechanism may be involved in the putative negative association between asthma and type I diabetes. In addition, these findings may have implications for the use of aerosolized insulin in diabetes mellitus.

Muscarinic M3-receptors mediate cholinergic synergism of mitogenesis in airway smooth muscle.

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [(3)H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M(3)-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 microM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1-10 microM), but only in cells having functional muscarinic M(3)-receptors. This synergism could be suppressed significantly by the selective M(3)-receptor antagonists DAU 5884 (0.1 microM) and 4-DAMP (10 nM), but not at all by the M(2)-subtype selective antagonist gallamine (10 microM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M(3)-receptors in bovine tracheal smooth muscle cells.

Functional characterization of serum- and growth factor-induced phenotypic changes in intact bovine tracheal smooth muscle.

1. The present study aims to investigate whether phenotypic changes, reported to occur in cultured isolated airway smooth muscle (ASM) cells, are of relevance to intact ASM. Moreover, we aimed to gain insight into the signalling pathways involved. 2. Culturing of bovine tracheal smooth muscle (BTSM) strips for up to 8 days in the presence of 10% foetal bovine serum caused a time-dependent (t(1/2)=2.8 days) decrease in maximal contraction (E(max)) to methacholine compared to serum-deprived controls (E(max)=74+/-4% at day 8). A reduced E(max) was also found using insulin-like growth factor-1 (30 ng ml(-1)) and platelet-derived growth factor (30 ng ml(-1)), but not using epidermal growth factor (10 ng ml(-1)) (E(max)=83+/-3, 67+/-8, 100+/-4%, respectively). Similar serum and growth factor-induced changes in E(max) were found for KCl-induced contraction (65+/-9, 80+/-7, 64+/-11% and 107+/-2%, respectively). 3. Strong correlations were found between the growth factor-induced reductions in E(max) and their proliferative responses, assessed by [(3)H]-thymidine-incorporation, in BTSM cells. (r=0.97, P=0.002 for methacholine and r=0.93, P=0.007 for KCl). 4. The PDGF-induced reduction in E(max) was inhibited completely by combined treatment with either PD 98059 (30 micro M) or LY 294002 (10 micro M). 5. These results indicate that serum and growth factors may cause a functional shift towards a less contractile phenotype in intact BTSM, which is associated with their proliferative response and dependent on signalling pathways involving the mitogen-activated protein kinase pathway and the phosphatidylinositol-3-kinase pathway.

Normalization of aortic function during arousal episodes in the hibernating ground squirrel.

Hypothermia is commonly used to restrict organ damage during preservation of tissue, but does not offer complete protection. Organ damage after reperfusion/rewarming is amongst others caused by an impairment of vascular properties, particularly endothelium-dependent vasodilatation. We hypothesized that hibernating small animals, which frequently cycle through periods of deep cooling (torpor) and full rewarming (arousal), employ specific mechanisms to preserve vascular function after cooling and reperfusion. Therefore we measured contraction of aortic tissue of hibernating European ground squirrels after 24 h and 7 days of torpor, arousal (1.5 h) and in non-hibernating animals. To assess the role of nitric oxide (NO), experiments were performed in the absence and presence of the NO-synthesis inhibitor, L-NMMA (10(-4) M). Maximum contraction to phenylephrine and angiotensin II was doubled in 7-days torpid animals without a shift in EC50, compared to the other 3 groups. Maximum contraction to KCl was doubled in 7-days torpid animals compared to the arousal group and non-hibernating animals. Relaxation to acetylcholine (ACh) and sodium nitrite in phenylephrine precontracted rings did not differ between groups. In the presence of L-NMMA, the maximum of concentration-response curves for all three vasoconstrictors was increased by about 30% in the arousal group, but unaffected in other groups. L-NMMA completely inhibited ACh-induced relaxation in 24-h torpid animals and non-hibernating animals, but only partially in 7-days torpid animals and in the arousal group. From this we conclude that vascular adaptation proceeds during torpor. Further, increased contractility of aortic tissue during long torpor returns to normal within 1.5 hours of arousal, which is associated with an increased basal NO synthesis. In addition, involvement of NO in agonist-mediated relaxation differs between the various stages of hibernation.Thus, hibernating animals have effectively developed mechanisms to preserve vascular function after cooling and rewarming.