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 [ 3 H]thymidine incorporation in response to platelet-derived growth factor (EC 50 ϭ 14 ng/ml), basic fibroblast growth factor (EC 50 ϭ 2.2 ng/ml), and epidermal growth factor (EC 50 ϭ 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-1  and TNF-␣ produced increases in
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 Qf 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 HC03-, 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 HC03-.6. Stopped-flow fluorimetric measurements were made of both BCECF and Fura-2 fluorescence in the presence of HCO3. Ii the presence and absence of external Ca2+,
1 The eects of the selective b 2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non-selective b adrenoceptor agonist isoprenaline on [ 3 H]-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 mg DNA/well 71 in a 6 well plate and 1.8 ml Transfectam/mg DNA. Expression was maximal at 48 ± 72 h. 3 Salbutamol (maximum response 19%, EC 50 0.6 mM), terbutaline (maximum response 38%, EC 50 2.3 mM) and salmeterol (maximum response 18%, EC 50 0.0012 mM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC 50 0.08 mM). However, all of the b 2 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 (EC 50 (cyclic AMP) /EC 50 (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 dierences exist in the ability of b 2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE-driven mechanism.
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 μM) and the increase in cell number (10 μM; 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.
Resolution of glomerular inflammation requires the removal of proliferating resident glomerular mesangial cells, but excessive loss of glomerular cells is a feature of postinflammatory scarring. Because apoptosis regulates mesangial cell number in glomerular inflammation, we have studied the exogenous control of apoptosis triggered in cultured mesangial cells by stimuli likely to be important in vivo. Apoptosis could be induced by serum deprivation to model decreased availability of survival factors, by etoposide as an example of DNA-damaging agents, by ligation of mesangial cell Fas, and by protein synthesis inhibition by cycloheximide. Insulin-like growth factor I (IGF-I), IGF-II, and basic fibroblast growth factor were each able to suppress apoptosis induced by serum deprivation, whereas TGF-beta 1, epidermal growth factor, and platelet-derived growth factor had no effect. IGF-I and IGF-II (but not basic fibroblast growth factor) were also able to protect cells from apoptosis induced by etoposide or cycloheximide. However, Fas-mediated apoptosis was resistant to suppression by all three cytokines. None of the cytokines tested caused a change in the levels of expression of Bcl-2, Bax, Bcl-x, or Bak proteins. The survival-promoting properties of serum-free medium conditioned by mesangial cells was abrogated by neutralizing IGF-I Ab. These experiments are the first to define cytokines that inhibit apoptosis and thereby promote survival of mesangial cells, and the data indicate a paracrine survival signaling role for IGF-I. Finally, the data show that Fas ligation can override cytokine survival signaling, emphasizing a candidate role for this molecule in the undesirable apoptotic loss of mesangial cells during the progression of glomerular scarring.
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