Role and mechanism of thromboxane-induced of cultured airway smooth muscle cells
proliferation
JAMES P. NOVERAL AND MICHAEL M. GRUNSTEIN Division of Pulmonary Medicine, Joseph Stokes, Jr., Research Institute, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Noveral, James P., and Michael M. Grunstein. Role and mechanismof thromboxane-induced proliferation of cultured airway smooth muscle cells. Am. J. Physiol. 263 (Lung CeZZ. Mol. Physiol. 7): L555-L561,1992.-Thromboxane (Tx)A2 has been reported to play an important role in modulating airway contractility under various conditions associated with airways inflammation. To identify its potential role in contributing to airway smooth muscle(ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic effect and mechanism of action of TxA2 were investigated in cultured rabbit ASM cells. The stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619,elicited dose-dependent(lo-l2 to 10m6M) increasesin ASM cell number and induced acute augmentation of intracellular inositol 1,4,5-trisphosphateaccumulation. The latter action was blocked by neomycin, a phospholipaseC inhibitor; however, neomycin had no effect on the promitogenic action of the TxA2 mimetics. In contrast, TxA,-induced ASM cell proliferation was inhibited by inhibitors of phospholipase A, and 5-lipoxygenase, as well as blockade of the leukotriene (LT)D4 receptor. Moreover, in complementary studies, we found that exogenousadministration of LTD4 (10-l* to 10m6 M) potently induced ASM cell proliferation and that the TxA, mimetics evoked the enhanced releaseof endogenousleukotrienes from the cultured ASM cells. Taken together, these observations provide new evidence that 1) TxA2 stimulates ASM cell proliferation; 2) the promitogenic effect of TxA2 is associated with activation of phospholipaseA,; and 3) the latter mediates ASM cell proliferation via the releaseand autocrine mitogenic action of leukotrienes. The findings support a potential role for TxA, in contributing to the characteristic increasein ASM cell massobtained in asthma and other chronic airway diseases. airways; smooth musclehyperplasia; eicosanoids;inositol 1,4,5trisphosphate; phospholipaseC; phospholipaseA,; asthma VARIOUS PULMONARY pathophysiological
conditions are associated with the release and action of membranederived products of arachidonic acid metabolism including the prostaglandins, leukotrienes, and thromboxanes. Among these eicosanoids, thromboxane (TX) A2 has been shown to exert significant effects on the airways and pulmonary vasculature (5, 13, 16, 29, 30). In this regard, TxA2 has been implicated in the hyperresponsiveness of airway smooth muscle which accompanies inhaled allergen challenge (3) and ozone inhalation (I), as well as in the pathogenesis of airway hyperresponsiveness to cholinergic stimulation in asthmatic individuals, wherein TxA, synthetase inhibition was found to diminish the exaggerated airway responsiveness to acetylcholine (6). Moreover, apart from its airway and vascular smooth muscle constrictor properties, TxA2 has also been recently shown to exert a promitogenic action in cultured rat vascular smooth muscle cells (8, IO) and has been implicated in the pathogenesis of vascular smooth muscle hyperplasia which characterizes the spontaneously hypertensive rat (10). In light of its above effects in vascular smooth muscle, together with emerging evidence supporting a promito1040-0605/92
$2.00
Copyright
genie action of TxA, in cultured rabbit airway smooth muscle (ASM) cells (23), the present study was designed to systematically evaluate the role of TxA2 in regulating ASM cell growth. In addition, because TxA2 has been associated with activation of the phospholipase C and/or phospholipase A2 signal transduction pathways in other cell types (7, 9, 17, ZO-22), we also examined whether the mitogenic actions of TxA~ in ASM are associated with activation of these transmembrane signaling pathways. Our findings provide new evidence that 1) TxA2 exerts a potent dose-dependent proliferative effect in cultured rabbit ASM cells; 2) the ASM cell proliferative response to TxA2 is mechanistically associated with pertussis toxin-insensitive activation of phospholipase A,; and 3) the latter induces ASM cell mitogenesis via the release and autocrine action of leukotrienes. MATERIALS AND METHODS MateriaZs. Ham’s F-12 culture medium and trypsin were obtained from Flow Laboratories (McLean, VA); tissue culture wells were purchased from Falcon (Lincoln Park, NJ); fetal bovine serum was from Hyclone (Logan, UT); carbocyclic thromboxane Az (CTA2), U-46619, leukotriene Dq, 4-nonylphenylketobutanoic acid (4-NPKB), neomycin, l-(5isoquinolinylsulfonyl)-2methylpiperazine (H-7)) and quinacrine were obtained from Biomol (Plymouth meeting, PA); nordihydroguaiaretic acid (NDGA), indomethacin, and pertussis toxin (PT) were purchased from Sigma Chemical (St. Louis, MO); MK-886 was generously provided by Merck Sharp and Dohme Research Laboratories (Rahway, NJ). ASM celZproliferation studies. Studies were conducted on cultured rabbit ASM cells that were previously characterized in detail with respect to their distinguishing morphological, histological, and immunological features (23). The cell isolation and subcultivation procedures used were also previously described (23). Briefly, smooth muscle cells were isolated from epithelium-denuded trachealis muscle from adult New Zealand White rabbits. After digestion in Ham F-12 culture medium (F-12) containing 30 mg/ml protease, 55 mg/ml type IV collagenase, and 100 mg/ml trypsin inhibitor, the dissociated cells were centrifuged and resuspended in F-12 containing 10% fetal bovine serum (FBS) and 100 mg/ml of gentamicin sulfate. The cells were then inoculated in lOO-mm tissue culture dishes and, after 4 wk, the cells had sufficiently proliferated to permit routine subcultivations. At weekly intervals, the subcultivated cells were suspended and then inoculated at a density of 1 X lo4 cells/cm2 in 25 cm2 tissue culture flasks containing F-12 with 20% FBS and incubated at 37°C in a humidified atmosphere of 5% CO,-95% air. Routine tests for mycoplasma contamination were negative. The ASM cell mitogenic effects of the stable TxA,
0 1992 the American
Physiological
Society
L555
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.
L556
THROMBOXANE-INDUCED
GROWTH
mimetics, CTA2, and U-46619, and leukotriene D4 (LTDJ were separately evaluated according to the following protocol. ASM cells were inoculated at 1 x lo4 cells/cm2 in 2 cm2 tissue culture wells in F-12 containing 20% FBS. At 24 h postinoculation, the original culture medium was replaced with serum-free (SF) F-12 containing either CTA2 (lo-l2 to 10v6 M), U-46619 (IO-l2 to 10e6 M), or LTD4 (10 -14 to 10e6 M). Control cells were refed with SF F-12 alone. The experimental and control cell numbers were then counted using a Coulter counter on day 4 postinoculation. In separate studies, we investigated whether the ASM cell proliferative responses to the thromboxane mimetics are modulated by pertussis toxin (100 rig/ml), phospholipase C inhibition with neomycin (10e6 M), phospholipase A2 inhibition with quinacrine ( 10m6 M), cyclooxygenase inhibition with indomethacin ( 10m5 M), lipoxygenase inhibition with NDGA (10m6 M) or MK886 ( 10d7 M), and LTD4 receptor blockade with 4-NPKB ( 10e5 M). In these studies, paired experiments were conducted in which cells were refed at 24 h postinoculation with SF F-12 containing CTA2 (10e6 M) alone and in combination with either of the above agents. ASM cell inositol1,4,5-trisphosphate [.ns(l,4,5)P,] accumulation. Mass changes in CTA2- and U-46619-induced intracellular Ins( I ,4,5)P3 were measured in confluent cells, initially inoculated at 1 X lo4 cells/cm2 in 25
cm2 tissue culture flasks containing F-12 20% FBS. To examine the time course of TxA2 mimetic-induced Ins( 1,4,5)P3 accumulation, cells were exposed on day 7 postinoculation to the agonist for time periods ranging from 1 to 10 min. The mass levels of Ins(1,4,5)P3 were assayed as previously described (23, 26)) using a commercially available competitive binding assay (Amersham Corporation, Arlington, IL). After exposure to the TxA2 mimetic, cells were lysed with 6% perchloric acid, neutralized with K2C03, kept on ice for 1 h, centrifuged (20,000 g; 4” C; 25 min), and then loo-p1 aliquots of the supernatant were removed for Ins( 1,4,5)P3 determination. The measured Ins( 1,4,5)P3 levels were normalized to I x lo6 cells. Separate experiments were conducted to examine the effects of the phospholipase C inhibitor, neomycin ( low6 M), on CTA2-induced Ins( 1,4,5)P3 accumulation. In these studies, performed as above, ASM cells were incubated in the presence of neomycin (10v6 M) for 2 h before stimulation with CTA2. Measurement of ASM cell leukotriene release. The effect of CTA2 on ASM cell leukotriene production was assayed using a commercially available radioimmunoassay (Amersham) to quantify leukotriene accumulation in the cell culture medium. The assay utilized an antiserum that cross-reacts with and is specific for the peptidoleukotrienes C4, D4, and E4 and, accordingly, the measurements reflect a collective assay of the latter leukotrienes. In these experiments, cells were initially inoculated at 1 x lo4 cells/cm2 in F-12 20% FBS and then refed at 7 days postinoculation with either SF F-12 (control) or with SF F-12 containing lOa M CTA2 or U-46619. The leukotriene concentrations were measured in loo-p1 aliquots removed from the culture medium of both the control and CTA2- and U-46619-treated cells at 0, 2, 4, and 8 h after exposure to either TxA2 analogue. The measured leukotriene levels were then normalized to
OF AIRWAY
picograms
SMOOTH
lo6 cells. For each experimental condition, the cell numbers pertain to counts obtained in 4-8 tissue culture vessels, with each well counted in triplicate. The measurements of cell numbers, mass levels of Ins( 1,4,5)P3, and leukotriene accumulation are expressed as means t SE values. Statistical analyses were performed using the two-tailed Student’s t test and analysis of variance (ANOVA) with multiple comparisons of means, where appropriate. A P value of < 0.05 was considered significant. Statistical
per 1
MUSCLE
X
analyses.
RESULTS
Effect of thromboxane mimetics on ASM cell proliferation. Incubation of cells with SF F-12 alone (i.e., control
condition) had no significant effect on cell count for up to 7 days postinoculation. In contrast, incubation of cells in SF F-12 containing peak effective concentrations of the TxA2 mimetics, CTA2 (10m6 M) and U-46619 (10v6 M) induced increases in ASM cell count for up to 7 days postinoculation, with peak proliferative responses to the agonists obtained on day 4 (data not shown). Accordingly, the results below pertain to cell counts performed on day 4 postinoculation. Both CTA2 (Fig. IA) and U-46619 (Fig. 1B) elicited significant dose-dependent increases in ASM cell number within the range of 10 --12 to lo+ M (P < 0.01; ANOVA). The mean t SE peak ASM cell proliferative responses to CTA2 and U-46619 yielded cell counts that amounted to 46 -+ 12.5 and 37 t2.6% above their corresponding control levels, respectively. Of interest, the latter magnitudes of the mitogenic responses to CTA2 and U-46619 represent -45 and 37%, respectively, of the stimulated mitogenie response of cells cultured in medium containing 5%
B
control TxA2
-12 mimetic
-10 Concentration
-8
-6 (log
M)
Fig. 1. Dose-dependent effects of carboxylic thromboxane A 2 (CTA2) (A) and U-46619 (B) on airway smooth muscle (ASM) cell proliferation. Cell counts were determined on day 4 postinoculation for cells refed at 24 h postinoculation with either serum-free Ham’s F-12 (SF F-12) alone (control) or SF F-12 containing various concentrations of TxA2 mimetits (filled bars). Data represent means t SE from 8 experiments. Doseresnonse relationshins are statisticallv significant (P < 0.01: ANOVA).
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.
THROMBOXANE-INDUCED
GROWTH
OF AIRWAY
SMOOTH
L557
MUSCLE
FBS over the same time period. Effect of thromboxane mimetics on Ins(l,4,5)P3 accumulation. To the extent that TxA2 has been shown to exert
its varied physiological actions in different cell types through receptor/G protein-coupled activation of the phospholipase C and/or phospholipase A2 signal-transduction pathways (9, 17, 20, 22), the relative contributions of these transmembrane signaling mechanisms in mediating TxAz-induced ASM cell proliferation were separately investigated. Accordingly, in view of recent evidence (8, 20) that TxA2 elicits phospholipase C-mediated production of the second messenger, Ins( 1,4,5)P3, we examined whether CTA2 and U-46619 also induce Ins( 1,4,5)P3 accumulation in the cultured ASM cells. Although treatment with vehicle alone had no effect, both TxAz mimetics elicited time- and dose-dependent increases in Ins( 1,4,5)P3. As exemplified for CTA2 in Fig. 2, the time course of Ins(1,4,5)P3 accumulation in response to a maximally effective concentration of the agonist (i.e., 10e8 M) was characterized by a rise in Ins(1,4,5)P3 from a mean t SE control (i.e., basal) level of 10.9 t 1.1 to a peak value of 34.0 t 7.3 pmol/106 cells at 3 min. Thereafter, the level of Ins( 1,4,5)P3 gradually decreased, but tended to remain somewhat sustained above the initial control level for up to 10 min. U-46619 elicited a qualitatively similar time course of Ins( 1 ,4,5)P3 accumulation, however, the mean t SE maximal dose-dependent (lo-l2 to 10e6 M) increase in Ins(1,4,5)P3 obtained with U-46619 (IO --8 M) significantly exceeded that evoked with CTA2, i.e., 59.7 t 6.36 vs. 39.6 t 4.9 pmol/106 cells, respectively (P < 0.05). Of interest, both of the latter values of maximal Ins( 1 ,4,5)P3 accumulation elicited with the TxA2 mimetics significantly exceed that previously reported in response to the potent vasoactive and bronchoconstrictor peptide, endothelin-1, using the same cultured rabbit ASM cell preparation (23). In addressing the above mechanism of action of the TxA2 mimetics, we found that treatment of cells with the phospholipase C inhibitor, neomycin, blocked the Ins( l,4,5)P3 responses to the TxA2 mimetics. As exemplified in Fig. 3, relative to the Ins(1,4,5)P3 concentration obtained in control cells, ASM cells exposed to 10B8 M CTA2 for 3 min depicted significantly enhanced (P
proliferation
JAMES P. NOVERAL AND MICHAEL M. GRUNSTEIN Division of Pulmonary Medicine, Joseph Stokes, Jr., Research Institute, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Noveral, James P., and Michael M. Grunstein. Role and mechanismof thromboxane-induced proliferation of cultured airway smooth muscle cells. Am. J. Physiol. 263 (Lung CeZZ. Mol. Physiol. 7): L555-L561,1992.-Thromboxane (Tx)A2 has been reported to play an important role in modulating airway contractility under various conditions associated with airways inflammation. To identify its potential role in contributing to airway smooth muscle(ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic effect and mechanism of action of TxA2 were investigated in cultured rabbit ASM cells. The stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619,elicited dose-dependent(lo-l2 to 10m6M) increasesin ASM cell number and induced acute augmentation of intracellular inositol 1,4,5-trisphosphateaccumulation. The latter action was blocked by neomycin, a phospholipaseC inhibitor; however, neomycin had no effect on the promitogenic action of the TxA2 mimetics. In contrast, TxA,-induced ASM cell proliferation was inhibited by inhibitors of phospholipase A, and 5-lipoxygenase, as well as blockade of the leukotriene (LT)D4 receptor. Moreover, in complementary studies, we found that exogenousadministration of LTD4 (10-l* to 10m6 M) potently induced ASM cell proliferation and that the TxA, mimetics evoked the enhanced releaseof endogenousleukotrienes from the cultured ASM cells. Taken together, these observations provide new evidence that 1) TxA2 stimulates ASM cell proliferation; 2) the promitogenic effect of TxA2 is associated with activation of phospholipaseA,; and 3) the latter mediates ASM cell proliferation via the releaseand autocrine mitogenic action of leukotrienes. The findings support a potential role for TxA, in contributing to the characteristic increasein ASM cell massobtained in asthma and other chronic airway diseases. airways; smooth musclehyperplasia; eicosanoids;inositol 1,4,5trisphosphate; phospholipaseC; phospholipaseA,; asthma VARIOUS PULMONARY pathophysiological
conditions are associated with the release and action of membranederived products of arachidonic acid metabolism including the prostaglandins, leukotrienes, and thromboxanes. Among these eicosanoids, thromboxane (TX) A2 has been shown to exert significant effects on the airways and pulmonary vasculature (5, 13, 16, 29, 30). In this regard, TxA2 has been implicated in the hyperresponsiveness of airway smooth muscle which accompanies inhaled allergen challenge (3) and ozone inhalation (I), as well as in the pathogenesis of airway hyperresponsiveness to cholinergic stimulation in asthmatic individuals, wherein TxA, synthetase inhibition was found to diminish the exaggerated airway responsiveness to acetylcholine (6). Moreover, apart from its airway and vascular smooth muscle constrictor properties, TxA2 has also been recently shown to exert a promitogenic action in cultured rat vascular smooth muscle cells (8, IO) and has been implicated in the pathogenesis of vascular smooth muscle hyperplasia which characterizes the spontaneously hypertensive rat (10). In light of its above effects in vascular smooth muscle, together with emerging evidence supporting a promito1040-0605/92
$2.00
Copyright
genie action of TxA, in cultured rabbit airway smooth muscle (ASM) cells (23), the present study was designed to systematically evaluate the role of TxA2 in regulating ASM cell growth. In addition, because TxA2 has been associated with activation of the phospholipase C and/or phospholipase A2 signal transduction pathways in other cell types (7, 9, 17, ZO-22), we also examined whether the mitogenic actions of TxA~ in ASM are associated with activation of these transmembrane signaling pathways. Our findings provide new evidence that 1) TxA2 exerts a potent dose-dependent proliferative effect in cultured rabbit ASM cells; 2) the ASM cell proliferative response to TxA2 is mechanistically associated with pertussis toxin-insensitive activation of phospholipase A,; and 3) the latter induces ASM cell mitogenesis via the release and autocrine action of leukotrienes. MATERIALS AND METHODS MateriaZs. Ham’s F-12 culture medium and trypsin were obtained from Flow Laboratories (McLean, VA); tissue culture wells were purchased from Falcon (Lincoln Park, NJ); fetal bovine serum was from Hyclone (Logan, UT); carbocyclic thromboxane Az (CTA2), U-46619, leukotriene Dq, 4-nonylphenylketobutanoic acid (4-NPKB), neomycin, l-(5isoquinolinylsulfonyl)-2methylpiperazine (H-7)) and quinacrine were obtained from Biomol (Plymouth meeting, PA); nordihydroguaiaretic acid (NDGA), indomethacin, and pertussis toxin (PT) were purchased from Sigma Chemical (St. Louis, MO); MK-886 was generously provided by Merck Sharp and Dohme Research Laboratories (Rahway, NJ). ASM celZproliferation studies. Studies were conducted on cultured rabbit ASM cells that were previously characterized in detail with respect to their distinguishing morphological, histological, and immunological features (23). The cell isolation and subcultivation procedures used were also previously described (23). Briefly, smooth muscle cells were isolated from epithelium-denuded trachealis muscle from adult New Zealand White rabbits. After digestion in Ham F-12 culture medium (F-12) containing 30 mg/ml protease, 55 mg/ml type IV collagenase, and 100 mg/ml trypsin inhibitor, the dissociated cells were centrifuged and resuspended in F-12 containing 10% fetal bovine serum (FBS) and 100 mg/ml of gentamicin sulfate. The cells were then inoculated in lOO-mm tissue culture dishes and, after 4 wk, the cells had sufficiently proliferated to permit routine subcultivations. At weekly intervals, the subcultivated cells were suspended and then inoculated at a density of 1 X lo4 cells/cm2 in 25 cm2 tissue culture flasks containing F-12 with 20% FBS and incubated at 37°C in a humidified atmosphere of 5% CO,-95% air. Routine tests for mycoplasma contamination were negative. The ASM cell mitogenic effects of the stable TxA,
0 1992 the American
Physiological
Society
L555
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.
L556
THROMBOXANE-INDUCED
GROWTH
mimetics, CTA2, and U-46619, and leukotriene D4 (LTDJ were separately evaluated according to the following protocol. ASM cells were inoculated at 1 x lo4 cells/cm2 in 2 cm2 tissue culture wells in F-12 containing 20% FBS. At 24 h postinoculation, the original culture medium was replaced with serum-free (SF) F-12 containing either CTA2 (lo-l2 to 10v6 M), U-46619 (IO-l2 to 10e6 M), or LTD4 (10 -14 to 10e6 M). Control cells were refed with SF F-12 alone. The experimental and control cell numbers were then counted using a Coulter counter on day 4 postinoculation. In separate studies, we investigated whether the ASM cell proliferative responses to the thromboxane mimetics are modulated by pertussis toxin (100 rig/ml), phospholipase C inhibition with neomycin (10e6 M), phospholipase A2 inhibition with quinacrine ( 10m6 M), cyclooxygenase inhibition with indomethacin ( 10m5 M), lipoxygenase inhibition with NDGA (10m6 M) or MK886 ( 10d7 M), and LTD4 receptor blockade with 4-NPKB ( 10e5 M). In these studies, paired experiments were conducted in which cells were refed at 24 h postinoculation with SF F-12 containing CTA2 (10e6 M) alone and in combination with either of the above agents. ASM cell inositol1,4,5-trisphosphate [.ns(l,4,5)P,] accumulation. Mass changes in CTA2- and U-46619-induced intracellular Ins( I ,4,5)P3 were measured in confluent cells, initially inoculated at 1 X lo4 cells/cm2 in 25
cm2 tissue culture flasks containing F-12 20% FBS. To examine the time course of TxA2 mimetic-induced Ins( 1,4,5)P3 accumulation, cells were exposed on day 7 postinoculation to the agonist for time periods ranging from 1 to 10 min. The mass levels of Ins(1,4,5)P3 were assayed as previously described (23, 26)) using a commercially available competitive binding assay (Amersham Corporation, Arlington, IL). After exposure to the TxA2 mimetic, cells were lysed with 6% perchloric acid, neutralized with K2C03, kept on ice for 1 h, centrifuged (20,000 g; 4” C; 25 min), and then loo-p1 aliquots of the supernatant were removed for Ins( 1,4,5)P3 determination. The measured Ins( 1,4,5)P3 levels were normalized to I x lo6 cells. Separate experiments were conducted to examine the effects of the phospholipase C inhibitor, neomycin ( low6 M), on CTA2-induced Ins( 1,4,5)P3 accumulation. In these studies, performed as above, ASM cells were incubated in the presence of neomycin (10v6 M) for 2 h before stimulation with CTA2. Measurement of ASM cell leukotriene release. The effect of CTA2 on ASM cell leukotriene production was assayed using a commercially available radioimmunoassay (Amersham) to quantify leukotriene accumulation in the cell culture medium. The assay utilized an antiserum that cross-reacts with and is specific for the peptidoleukotrienes C4, D4, and E4 and, accordingly, the measurements reflect a collective assay of the latter leukotrienes. In these experiments, cells were initially inoculated at 1 x lo4 cells/cm2 in F-12 20% FBS and then refed at 7 days postinoculation with either SF F-12 (control) or with SF F-12 containing lOa M CTA2 or U-46619. The leukotriene concentrations were measured in loo-p1 aliquots removed from the culture medium of both the control and CTA2- and U-46619-treated cells at 0, 2, 4, and 8 h after exposure to either TxA2 analogue. The measured leukotriene levels were then normalized to
OF AIRWAY
picograms
SMOOTH
lo6 cells. For each experimental condition, the cell numbers pertain to counts obtained in 4-8 tissue culture vessels, with each well counted in triplicate. The measurements of cell numbers, mass levels of Ins( 1,4,5)P3, and leukotriene accumulation are expressed as means t SE values. Statistical analyses were performed using the two-tailed Student’s t test and analysis of variance (ANOVA) with multiple comparisons of means, where appropriate. A P value of < 0.05 was considered significant. Statistical
per 1
MUSCLE
X
analyses.
RESULTS
Effect of thromboxane mimetics on ASM cell proliferation. Incubation of cells with SF F-12 alone (i.e., control
condition) had no significant effect on cell count for up to 7 days postinoculation. In contrast, incubation of cells in SF F-12 containing peak effective concentrations of the TxA2 mimetics, CTA2 (10m6 M) and U-46619 (10v6 M) induced increases in ASM cell count for up to 7 days postinoculation, with peak proliferative responses to the agonists obtained on day 4 (data not shown). Accordingly, the results below pertain to cell counts performed on day 4 postinoculation. Both CTA2 (Fig. IA) and U-46619 (Fig. 1B) elicited significant dose-dependent increases in ASM cell number within the range of 10 --12 to lo+ M (P < 0.01; ANOVA). The mean t SE peak ASM cell proliferative responses to CTA2 and U-46619 yielded cell counts that amounted to 46 -+ 12.5 and 37 t2.6% above their corresponding control levels, respectively. Of interest, the latter magnitudes of the mitogenic responses to CTA2 and U-46619 represent -45 and 37%, respectively, of the stimulated mitogenie response of cells cultured in medium containing 5%
B
control TxA2
-12 mimetic
-10 Concentration
-8
-6 (log
M)
Fig. 1. Dose-dependent effects of carboxylic thromboxane A 2 (CTA2) (A) and U-46619 (B) on airway smooth muscle (ASM) cell proliferation. Cell counts were determined on day 4 postinoculation for cells refed at 24 h postinoculation with either serum-free Ham’s F-12 (SF F-12) alone (control) or SF F-12 containing various concentrations of TxA2 mimetits (filled bars). Data represent means t SE from 8 experiments. Doseresnonse relationshins are statisticallv significant (P < 0.01: ANOVA).
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.
THROMBOXANE-INDUCED
GROWTH
OF AIRWAY
SMOOTH
L557
MUSCLE
FBS over the same time period. Effect of thromboxane mimetics on Ins(l,4,5)P3 accumulation. To the extent that TxA2 has been shown to exert
its varied physiological actions in different cell types through receptor/G protein-coupled activation of the phospholipase C and/or phospholipase A2 signal-transduction pathways (9, 17, 20, 22), the relative contributions of these transmembrane signaling mechanisms in mediating TxAz-induced ASM cell proliferation were separately investigated. Accordingly, in view of recent evidence (8, 20) that TxA2 elicits phospholipase C-mediated production of the second messenger, Ins( 1,4,5)P3, we examined whether CTA2 and U-46619 also induce Ins( 1,4,5)P3 accumulation in the cultured ASM cells. Although treatment with vehicle alone had no effect, both TxAz mimetics elicited time- and dose-dependent increases in Ins( 1,4,5)P3. As exemplified for CTA2 in Fig. 2, the time course of Ins(1,4,5)P3 accumulation in response to a maximally effective concentration of the agonist (i.e., 10e8 M) was characterized by a rise in Ins(1,4,5)P3 from a mean t SE control (i.e., basal) level of 10.9 t 1.1 to a peak value of 34.0 t 7.3 pmol/106 cells at 3 min. Thereafter, the level of Ins( 1,4,5)P3 gradually decreased, but tended to remain somewhat sustained above the initial control level for up to 10 min. U-46619 elicited a qualitatively similar time course of Ins( 1 ,4,5)P3 accumulation, however, the mean t SE maximal dose-dependent (lo-l2 to 10e6 M) increase in Ins(1,4,5)P3 obtained with U-46619 (IO --8 M) significantly exceeded that evoked with CTA2, i.e., 59.7 t 6.36 vs. 39.6 t 4.9 pmol/106 cells, respectively (P < 0.05). Of interest, both of the latter values of maximal Ins( 1 ,4,5)P3 accumulation elicited with the TxA2 mimetics significantly exceed that previously reported in response to the potent vasoactive and bronchoconstrictor peptide, endothelin-1, using the same cultured rabbit ASM cell preparation (23). In addressing the above mechanism of action of the TxA2 mimetics, we found that treatment of cells with the phospholipase C inhibitor, neomycin, blocked the Ins( l,4,5)P3 responses to the TxA2 mimetics. As exemplified in Fig. 3, relative to the Ins(1,4,5)P3 concentration obtained in control cells, ASM cells exposed to 10B8 M CTA2 for 3 min depicted significantly enhanced (P
