Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 982-987
October 31, 1991
STRETCH INCREASES INOSITOL TRISPHOSPHATE AND INOSITOL TETRAKISPHOSPHATE IN CULTURED PULMONARY VASCULAR SMOOTH MUSCLE CELLS Thomas J. Kulik 1'2, Russell A. Bialecki 3, Wilson S. Colucci3, Abraham Rothman 4, Eileen T. Glennon 5, and Richard H. Underwood 5 1Department of Cardiology, Children's Hospital (Boston); 3The Cardiovascular Division of the Department of Medicine, Brigham and Women's Hospital (Boston); 4The Department of Pediatrics, The University of California, San Diego, CA; SThe EndocrineHypertension Unit, Brigham and Women's Hospital; and Harvard Medical School, Boston, MA Received September 19, 1991
There are no reports of the effect of stretch on inositol phosphates in smooth muscle. Phosphoinositide and inositol phosphate metabolism was studied in cultured rat vascular smooth muscle cells subjected to stretching. The masses of inositol trisphosphate and tetrakisphosphate increased (+ 34 + 7% and +58 + 12%, respectively; p < 0.001) after 25 s of a single 20% stretch and had returned to control levels by 45 s; phosphatidylinositol, phosphatidylinositol phosphate and bisphosphate did not change. Repetitive stretch did not alter the masses of any of the compounds. A single stretch also increased 45Ca2+ efflux (+52 + 5%, p< 0.01). These data suggest that stretch of cultured vascular smooth muscle can elicit a rapid, short-lived increase in inositol phosphates, which may subsequently affect Ca 2+. .1991 Aoademio~..... 1no.
Stretch of vascular smooth muscle cells (VSMC) in culture increases the rate of overall protein and collagen production (1,2) and causes the cells to change shape (3). How stretch is transduced into these and other effects is unknown, but mechanisms involved in transducing receptor-mediated stimuli may be involved: Stretching segments of blood vessels in vitro increases Ca 2÷ influx (4) and causes apparent activation of protein kinase C (5). Phosphoinositide metabolism results in production of second messengers (inositol phosphates and diacylglycerol) which mediate a variety of intracellular processes, including Ca 2+ mobilization and protein kinase C activation (6), 2To whom correspondence should be addressed at The Department of Cardiology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. The abbreviations used are: PI, phosphatidylinositol; PIP, phosphatidylinositol-4phosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol trisphosphate; IP4, 1,3,4,5-inositol tetrakisphosphate; PSS, physiological salt solution; VSMC, vascular smooth muscle ceils. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and may influence the cytoskeleton and cell shape (7). Generation of IP 3 is stimulated by chemical agonists (e.g., hormones) in cultured VSMC (8,9), but the effect of mechanical stimulation on phosphoinositide metabolism in these cells is unreported. We therefore measured the effect of stretch on the masses of PI, PIP, PIPz, IP3, and IP4 in cultured rat pulmonary VSMC. As a way of determining whether stretch-induced changes in IP3 and IP 4 may be of physiological significance, 45CaZ+ efflux was also measured, since IP3 (and perhaps IP4) can increase intracellular Ca 2÷ mobilization (6).
MATERIALS AND METHODS Cell Culture Main and proximal branch pulmonary arteries from adult Sprague-Dawley rats were stripped of adventitia and enzymatically dissociated (10). The VSMC were grown (37°C, 95% air/5% CO2, in a humidified atmosphere) in medium 199 with 20% (v/v) fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. After multiple passages a clonal line was selected. By immunofluorescent staining, these cloned cells stably express (over many passages) smooth muscle specific myosin (antibody courtesy Dr. D.M. Larson [11]) and smooth muscle a-actin (Enzo Biochem Inc., N.Y., N.Y.). They also express mRNA for the a-smooth muscle actin isoform (by Northern blotting using a riboprobe derived from the 3' untranslated region of the a-smooth muscle actin isoform, courtesy Dr. J.Lessard) and predominantly express the smooth muscle atropomyosin mRNA isoform (12). Static Stretch Protocol, Inositol Phospholipid Studies Cells were grown on a 0.010 in. thick silicone sheet (Silastic, Dow Coming, Midland, MI) coated with type I collagen (Collaborative Research, Bedford, MA) as described (13). Preliminary experiments demonstrated that the cells remained attached to the surface with a 20% stretch for at least 1 hr. For initial studies, 4 strips (34 m m x 55 ram) submerged in culture media in a common chamber were used. In subsequent experiments (including all 4SCa2÷ efflux studies), silicone sheets were fashioned into "tubs" (34 mm wide x 40 mm long x 10 mm deep) which contained the media. Cells were plated and grown to confluence (in 2-4 d) in medium 199 or DME with 20% fetal calf serum and antibiotics. The results were not influenced by the type of medium. Eight hrs before study, the media was replaced by media containing 0.5% fetal calf serum and lacking antibiotics. Stretch was effected by elongating the membrane 20% in the direction of its long axis. After being stretched for 15 s, 25 s, or 45 s, 4°C methanol:chloroform (Me:Ch; 50:50 v/v) equal to the volume of media was rapidly added, aspirated, the cells quickly rinsed once in Me:Ch and scraped off the silicone into 10 ml of Me:Ch. Unstretched cells served as controls. In 2 experiments, pertussis toxin (Sigma), 20 ng/ml, was added to the culture medium 8 hrs before stretch. To determine the effect of repetitive stretch on the compounds of interest, a stretching device similar to that previously described (13) was used, excepi that it contained 20 wells, each 35ram in diameter. Confluent cells were stretched 20% at 0.5 hertz for 25 s, 120 s, and 300 s, cold Me:Ch added, and cellular material processed as noted above. Controls were obtained just prior to and immediately following the 5 min period of stretch. Ceils from 4 wells were used for each control and test time point. Measurement of Phosphoinositides and Inositol Phosphates PI, PIP, PIP2, IP 3 and IP4 were measured using a previously validated mass assay (14). Briefly, thin layer chromatography was used to separate the compounds, which were then extracted from the chromatography plates, hydrolyzed, and the amount of 983
Vol.
1 8 0 , N o . 2, 1 9 9 1
B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S
phosphorus was assayed by measuring optical absorbance at 600 nm with Malachite green-phosphomolybdate reagent. This system does not separate 1,4,5 IP 3 from 1,3,4 IP3, hence the reported IP 3 values include both. Measurement of 45Ca2* Efflux Measurement of 45Ca2÷ efflux was performed as previously reported (15). Briefly, confluent cells in tubs were equilibrated with 45CAC12 (1 uCi/ml, specific activity = 11.3 mCi/mg Ca2+; ICN Biomedicals, Inc., Irvine, CA) for 18-24 hr in D M E with 0.5% fetal calf serum. Just prior to the assay, the cells were rapidly washed 4 times with physiological salt solution ([PSS]; in mM, NaC1 = 130; KCI = 5, MgCI 2 = 1.0; CaC12 --1.5; glucose = 10; H E P E S = 20, buffered to pH 7.4 with TRIS) at 37°C. Efflux of 45Ca2+ was initiated by the addition of 6 ml of PSS to the tub and 5 s later the tubs were stretched 20%. Unstretched cells in tubs served as controls. The assay was terminated after 6 rain by washing 4 times with 4°C PSS containing 10 mM LaCl3, which removes extracellular Ca 2÷ and prevents further Ca 2÷ influx or efflux. Intracellular 45CaZ+ was quantitated by standard scintillation techniques. Statistical Analysis All data are expressed as mean + standard error of the mean. Comparisons between control (non-stretched) and experimental values of phosphoinositides, inositol phosphates, and 45Ca2+ efflux were made using a one way analysis of variance with post hoc comparisons by a 2-tailed Student's paired t test with the Bonferroni correction. Differences were taken to be significant when p
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 982-987
October 31, 1991
STRETCH INCREASES INOSITOL TRISPHOSPHATE AND INOSITOL TETRAKISPHOSPHATE IN CULTURED PULMONARY VASCULAR SMOOTH MUSCLE CELLS Thomas J. Kulik 1'2, Russell A. Bialecki 3, Wilson S. Colucci3, Abraham Rothman 4, Eileen T. Glennon 5, and Richard H. Underwood 5 1Department of Cardiology, Children's Hospital (Boston); 3The Cardiovascular Division of the Department of Medicine, Brigham and Women's Hospital (Boston); 4The Department of Pediatrics, The University of California, San Diego, CA; SThe EndocrineHypertension Unit, Brigham and Women's Hospital; and Harvard Medical School, Boston, MA Received September 19, 1991
There are no reports of the effect of stretch on inositol phosphates in smooth muscle. Phosphoinositide and inositol phosphate metabolism was studied in cultured rat vascular smooth muscle cells subjected to stretching. The masses of inositol trisphosphate and tetrakisphosphate increased (+ 34 + 7% and +58 + 12%, respectively; p < 0.001) after 25 s of a single 20% stretch and had returned to control levels by 45 s; phosphatidylinositol, phosphatidylinositol phosphate and bisphosphate did not change. Repetitive stretch did not alter the masses of any of the compounds. A single stretch also increased 45Ca2+ efflux (+52 + 5%, p< 0.01). These data suggest that stretch of cultured vascular smooth muscle can elicit a rapid, short-lived increase in inositol phosphates, which may subsequently affect Ca 2+. .1991 Aoademio~..... 1no.
Stretch of vascular smooth muscle cells (VSMC) in culture increases the rate of overall protein and collagen production (1,2) and causes the cells to change shape (3). How stretch is transduced into these and other effects is unknown, but mechanisms involved in transducing receptor-mediated stimuli may be involved: Stretching segments of blood vessels in vitro increases Ca 2÷ influx (4) and causes apparent activation of protein kinase C (5). Phosphoinositide metabolism results in production of second messengers (inositol phosphates and diacylglycerol) which mediate a variety of intracellular processes, including Ca 2+ mobilization and protein kinase C activation (6), 2To whom correspondence should be addressed at The Department of Cardiology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. The abbreviations used are: PI, phosphatidylinositol; PIP, phosphatidylinositol-4phosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol trisphosphate; IP4, 1,3,4,5-inositol tetrakisphosphate; PSS, physiological salt solution; VSMC, vascular smooth muscle ceils. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
982
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and may influence the cytoskeleton and cell shape (7). Generation of IP 3 is stimulated by chemical agonists (e.g., hormones) in cultured VSMC (8,9), but the effect of mechanical stimulation on phosphoinositide metabolism in these cells is unreported. We therefore measured the effect of stretch on the masses of PI, PIP, PIPz, IP3, and IP4 in cultured rat pulmonary VSMC. As a way of determining whether stretch-induced changes in IP3 and IP 4 may be of physiological significance, 45CaZ+ efflux was also measured, since IP3 (and perhaps IP4) can increase intracellular Ca 2÷ mobilization (6).
MATERIALS AND METHODS Cell Culture Main and proximal branch pulmonary arteries from adult Sprague-Dawley rats were stripped of adventitia and enzymatically dissociated (10). The VSMC were grown (37°C, 95% air/5% CO2, in a humidified atmosphere) in medium 199 with 20% (v/v) fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. After multiple passages a clonal line was selected. By immunofluorescent staining, these cloned cells stably express (over many passages) smooth muscle specific myosin (antibody courtesy Dr. D.M. Larson [11]) and smooth muscle a-actin (Enzo Biochem Inc., N.Y., N.Y.). They also express mRNA for the a-smooth muscle actin isoform (by Northern blotting using a riboprobe derived from the 3' untranslated region of the a-smooth muscle actin isoform, courtesy Dr. J.Lessard) and predominantly express the smooth muscle atropomyosin mRNA isoform (12). Static Stretch Protocol, Inositol Phospholipid Studies Cells were grown on a 0.010 in. thick silicone sheet (Silastic, Dow Coming, Midland, MI) coated with type I collagen (Collaborative Research, Bedford, MA) as described (13). Preliminary experiments demonstrated that the cells remained attached to the surface with a 20% stretch for at least 1 hr. For initial studies, 4 strips (34 m m x 55 ram) submerged in culture media in a common chamber were used. In subsequent experiments (including all 4SCa2÷ efflux studies), silicone sheets were fashioned into "tubs" (34 mm wide x 40 mm long x 10 mm deep) which contained the media. Cells were plated and grown to confluence (in 2-4 d) in medium 199 or DME with 20% fetal calf serum and antibiotics. The results were not influenced by the type of medium. Eight hrs before study, the media was replaced by media containing 0.5% fetal calf serum and lacking antibiotics. Stretch was effected by elongating the membrane 20% in the direction of its long axis. After being stretched for 15 s, 25 s, or 45 s, 4°C methanol:chloroform (Me:Ch; 50:50 v/v) equal to the volume of media was rapidly added, aspirated, the cells quickly rinsed once in Me:Ch and scraped off the silicone into 10 ml of Me:Ch. Unstretched cells served as controls. In 2 experiments, pertussis toxin (Sigma), 20 ng/ml, was added to the culture medium 8 hrs before stretch. To determine the effect of repetitive stretch on the compounds of interest, a stretching device similar to that previously described (13) was used, excepi that it contained 20 wells, each 35ram in diameter. Confluent cells were stretched 20% at 0.5 hertz for 25 s, 120 s, and 300 s, cold Me:Ch added, and cellular material processed as noted above. Controls were obtained just prior to and immediately following the 5 min period of stretch. Ceils from 4 wells were used for each control and test time point. Measurement of Phosphoinositides and Inositol Phosphates PI, PIP, PIP2, IP 3 and IP4 were measured using a previously validated mass assay (14). Briefly, thin layer chromatography was used to separate the compounds, which were then extracted from the chromatography plates, hydrolyzed, and the amount of 983
Vol.
1 8 0 , N o . 2, 1 9 9 1
B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S
phosphorus was assayed by measuring optical absorbance at 600 nm with Malachite green-phosphomolybdate reagent. This system does not separate 1,4,5 IP 3 from 1,3,4 IP3, hence the reported IP 3 values include both. Measurement of 45Ca2* Efflux Measurement of 45Ca2÷ efflux was performed as previously reported (15). Briefly, confluent cells in tubs were equilibrated with 45CAC12 (1 uCi/ml, specific activity = 11.3 mCi/mg Ca2+; ICN Biomedicals, Inc., Irvine, CA) for 18-24 hr in D M E with 0.5% fetal calf serum. Just prior to the assay, the cells were rapidly washed 4 times with physiological salt solution ([PSS]; in mM, NaC1 = 130; KCI = 5, MgCI 2 = 1.0; CaC12 --1.5; glucose = 10; H E P E S = 20, buffered to pH 7.4 with TRIS) at 37°C. Efflux of 45Ca2+ was initiated by the addition of 6 ml of PSS to the tub and 5 s later the tubs were stretched 20%. Unstretched cells in tubs served as controls. The assay was terminated after 6 rain by washing 4 times with 4°C PSS containing 10 mM LaCl3, which removes extracellular Ca 2÷ and prevents further Ca 2÷ influx or efflux. Intracellular 45CaZ+ was quantitated by standard scintillation techniques. Statistical Analysis All data are expressed as mean + standard error of the mean. Comparisons between control (non-stretched) and experimental values of phosphoinositides, inositol phosphates, and 45Ca2+ efflux were made using a one way analysis of variance with post hoc comparisons by a 2-tailed Student's paired t test with the Bonferroni correction. Differences were taken to be significant when p
