Effects of a range of beta2 adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells.

1. The effects of the selective beta2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non-selective beta adrenoceptor agonist isoprenaline on [3H]-cyclic AMP formation and cyclic AMP response element (CRE) driven luciferase expression, assessed using the construct p6CRE/luc, were studied in primary cultures of human airway smooth muscle (HASM) cells. 2. Optimal transfection conditions for transient expression of pGL3 Control were 4 microg DNA/well71 in a 6 well plate and 1.8 microl Transfectam/microg DNA. Expression was maximal at 48 - 72 h. 3. Salbutamol (maximum response 19%, EC50 0.6 microM), terbutaline (maximum response 38%, EC50 2.3 microM) and salmeterol (maximum response 18%, EC50 0.0012 microM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC50 0.08 microM). However, all of the beta2 adrenoceptor agonists produced increases in CRE-driven luciferase activity, in cultured HASM transfected with the vector p6CRE/luc, which were equivalent or greater (salmeterol) than those seen with isoprenaline. 4. Both salbutamol and salmeterol were more potent at increasing luciferase expression than in elevating cyclic AMP levels in these cells. The potency ratios (EC50 (cyclic AMP)/EC50 (LUC)) for the agents studied were isoprenaline: 0. 2 fold, terbutaline: 3 fold, salbutamol: 24 fold, salmeterol: 38 fold. 5. These data suggest that important quantitative differences exist in the ability of beta2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE-driven mechanism.

Modulation of human airway smooth muscle proliferation by type 3 phosphodiesterase inhibition.

Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC50 2 microM) and the increase in cell number (10 microM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression.

Regulation of proliferation of human colonic subepithelial myofibroblasts by mediators important in intestinal inflammation.

An increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring. We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts. Using primary cultures of these cells, we have shown increases in [3H]thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml). Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II. In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in [3H]thymidine incorporation. IL-1beta and platelet-derived growth factor together produced an increase in [3H]thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers. Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4. 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.

Agonist-evoked changes in cytosolic pH and calcium concentration in human platelets: studies in physiological bicarbonate.

1. Cytosolic pH (pHi) and calcium concentration ([Ca2+]i) have been investigated in the presence and absence of physiological HCO3- in human platelets co-loaded with the fluorescent indicators BCECF and Fura-2. Basal pHi and changes evoked by butyrate, thrombin, platelet activating factor (PAF), ADP and phorbol ester were investigated, as were the effects of removing external Na+. 2. In the presence of physiological HCO3- and CO2, basal pHi was 7.02 +/- 0.04 compared with 7.15 +/- 0.05 in the absence of HCO3-. Estimated cytosolic buffering power was reduced from 35.6 +/- 3.0 to 14.5 +/- 0.4 mM/pH unit by the omission of HCO3-. 3. Thrombin evoked an immediate acidification of 0.03 +/- 0.01 pH units in the presence of HCO3- and 0.07 +/- 0.01 pH units in its absence. The acidifications were followed by a slow alkalinization. The final pHi was 0.10 +/- 0.01 units above basal in the presence of HCO3- and 0.08 +/- 0.02 units above basal in the absence of HCO3-. The initial acidification was significantly greater in the absence of HCO3-. The subsequent increase in pHi was similar in the presence and absence of this ion, but the calculated loss of proton equivalents was greater in the presence of HCO3-. 4. Replacement of extracellular Na+ with N-methyl-D-glucamine resulted in a fall in basal pHi and abolished recovery from thrombin-evoked acidification in both the presence and absence of HCO3-. 5. In the presence of HCO3-, PAF and ADP evoked an intracellular acidification similar to that caused by thrombin. However, with PAF and ADP, the subsequent recovery in pHi was slow and did not rise above basal levels. Phorbol dibutyrate, an activator of protein kinase C, evoked a similar elevation in pHi of 0.04 +/- 0.01 units over 3 min in the presence and absence of HCO3-. 6. Stopped-flow fluorimetric measurements were made of both BCECF and Fura-2 fluorescence in the presence of HCO3-. In the presence and absence of external Ca2+, thrombin-evoked rises in [Ca2+]i peaked before any cytoplasmic alkalinization occurred. ADP evoked rapid elevations in [Ca2+]i, but caused no alkalinization.(ABSTRACT TRUNCATED AT 400 WORDS)