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Mechanism of polylysine stimulation of smooth muscle
J Mol Cell Cardiol23 (Supplement III) (1991) p-4-32
MECHANISM OF POLYLYSINE STIMULATION OF SMOOTH MUSCLE. Pawel T. Szymanski, Eugene Almer, Donald G. Ferguson & Richard J. Paul. Depts. of Physiology and Biophysics, and Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576. Phosphorylation of the 20 kDa myosin light chain is a generally accepted regulator of smooth muscle contractility, but the underlying mechanism(s) is unclear. Thus it was of interest that polylysine (lo- 13 kDa) increased isometric force in permeabilized taenia coli fibers, and activated bovine aortic actomyosin and turkey gizzard myosin ATPase in the absence of phosphorylation. We further investigated the effects of polylysine with sedimentation, electron microscopy and light scattering techniques. At 0.2 M KCl, myosin is soluble whereas in the presence of polylysine (10 PM), it forms a precipitate. Electron microscopy indicates that under conditions in which myosin is monomeric, polylysine induces the formation of non-filamentous aggregates. Light scattering experiments indicate that aggregation rate decreases two-fold as [KCI] is increased from 0.05 to 0.25M. Increasing [KC11 decreased the activation of ATPase elicited by addition of polylysine. However after preincubation with polylysine, addition of KC1 (to 0.25M) did not substantially alter the ATPase. Our results indicate that the increases smooth muscle ATPase induced by polylysine are correlated with a formation of nonfilamentous myosin aggregates. The physiological significance is unclear, but polylysine may be binding to a negatively charged region important for regulation. As polylysine does not stimulate skeletal muscle myosin ATPase, it may be an important too1 in probing smooth muscle specific regulatory mechanisms. Supported by NIH 22619.
P-4-33 BASIC FIBROBLAST GROWTH FAC-HJR IN DEWELOPMHN’I’ INDUCED
INJURY
AND AFIXR
JSOPROTHRHNO~
IN THE RAT IIEART
Raymond R. Padua, Bradley W. Doble, Lei Liu and Elissavet Kardami. St. Boniface General Hospital Research Centre and Department of Physiology, University of Manitoba, Winnipeg, Canada RZH 2A6. To investigate a potential role for endogenousbasic fibroblast growth factor @FGF) in the control of cardiac proliferative and regenerative potential we examined its expression during development and after injury, using immunolocalization with bFGF-specific antibodies and Western blotting analysis of bFGF-like peptides in heart extracts. Here we report that immature phenotype and early response to isoproterenol-induced injury are accompanied by increased levels of a 22-24 kDa bFGF species. Differentiated phenotype and late responseto isoproterenol-induced injury are characterized by increases in the 18 kDa species of bFGF. Augmented anti-bFGF labelling is seen in myocyte nuclei from newborn compared to adult rats, accompanied by increased levels of a 25 kDa bFGF in corresponding nuclear protein extracts. Intense nuclear anti-bFGF labelling is also seen in some myocytes adjacent to the site of injury. Finally, degenerative lesions in the heart are characterized by a tremendous increase in cytoplasmic bFGF, which precedes and likely promotes cellular infiltration. Our data show a participation of bFGF in developmental and injury-related processesin the heart and point to different roles for the 22-24 kDa or 18 kDa bFGF species, related to a proliferative
P-5-1
or hypertrophic
phenotype, respectively.
RAT FETUS VENTRICULAR Ca2+ CURRENT IN DIHYDROPYRIDINE-RESISTANT CARDIOMYOCYTES Noritsugu Tohse, Nicholas Sperelakis. Department of Physiology & Biophysics, University of Cincinnati, College of Medicine, Cincinnati, OH 45267, USA. To investigate developmental changes in cardiac Ca 2+ channels, single cardiomyocytes of 18-day-old rat fetus were freshly isolated using 0.1% collagenase. Currents were recorded (at 25OC) by whole-cell voltaee clam0 in Na. K-free externa1 and K-free internal solutions. The Ca2+ current (1~~) was elicited by depolarizing test .pulses above -57 mV from a holding potential (HP) of -87 mV. &-was maximal around -7 mV: 8.4 + 0.7 pA/pF (M + SE, n = 44). It-. was blocked by 2 mM Co2+, but not completely by the dihydropyridine (DHP) Ca 2+-antagonist nife&$ne. Nifedipine (3 GM) decreased the amplitude of ICa at -7 mV by 65.9 + 3.4% (n = 20); 10 nM showed no further decrease (66.4 5 5.1%, n = IO). The DHP resistance of the ICa was not due to a voltage/dependence, becauseICa was decreasedby 77.1 + 4.7% with HP of -47 mV. The half-inactivation potentials of the DHP-sensitive and -resistant currents were -42.0 and -58.4 mV, respectively. The DHP-resistant current was not completely blocked bv 40 uM Ni2+ (48.6 + 7.2%) and 1 nM tetramethrine (28.1 + 16.8%). These results indicate that the DHP-resistant current mav be a different tvoe of 1~. from T-tvpe and L-type currents. In l2-day rat fetal cardiomyocytes, ICa-current density was 6.4 g-1.6 pA/pF (n = 19) at-7 mV. 3 nM nifedipine decreased 1~~ by 32.5 5 5.7% (n = 18). The current density of the DHP-resistant current in 12-day-old and II-day-oldfetus heart cells was 3.5 + 0.7 pA/pF (n = IS) and 2.4 + 0.3 pA/pF (n = 20), respectively. In conclusion, rat fetal cardiomyocytes may have a unique type of ICa, which decreases and becomes less prominent during development. s.90
MECHANISM OF POLYLYSINE STIMULATION OF SMOOTH MUSCLE. Pawel T. Szymanski, Eugene Almer, Donald G. Ferguson & Richard J. Paul. Depts. of Physiology and Biophysics, and Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576. Phosphorylation of the 20 kDa myosin light chain is a generally accepted regulator of smooth muscle contractility, but the underlying mechanism(s) is unclear. Thus it was of interest that polylysine (lo- 13 kDa) increased isometric force in permeabilized taenia coli fibers, and activated bovine aortic actomyosin and turkey gizzard myosin ATPase in the absence of phosphorylation. We further investigated the effects of polylysine with sedimentation, electron microscopy and light scattering techniques. At 0.2 M KCl, myosin is soluble whereas in the presence of polylysine (10 PM), it forms a precipitate. Electron microscopy indicates that under conditions in which myosin is monomeric, polylysine induces the formation of non-filamentous aggregates. Light scattering experiments indicate that aggregation rate decreases two-fold as [KCI] is increased from 0.05 to 0.25M. Increasing [KC11 decreased the activation of ATPase elicited by addition of polylysine. However after preincubation with polylysine, addition of KC1 (to 0.25M) did not substantially alter the ATPase. Our results indicate that the increases smooth muscle ATPase induced by polylysine are correlated with a formation of nonfilamentous myosin aggregates. The physiological significance is unclear, but polylysine may be binding to a negatively charged region important for regulation. As polylysine does not stimulate skeletal muscle myosin ATPase, it may be an important too1 in probing smooth muscle specific regulatory mechanisms. Supported by NIH 22619.
P-4-33 BASIC FIBROBLAST GROWTH FAC-HJR IN DEWELOPMHN’I’ INDUCED
INJURY
AND AFIXR
JSOPROTHRHNO~
IN THE RAT IIEART
Raymond R. Padua, Bradley W. Doble, Lei Liu and Elissavet Kardami. St. Boniface General Hospital Research Centre and Department of Physiology, University of Manitoba, Winnipeg, Canada RZH 2A6. To investigate a potential role for endogenousbasic fibroblast growth factor @FGF) in the control of cardiac proliferative and regenerative potential we examined its expression during development and after injury, using immunolocalization with bFGF-specific antibodies and Western blotting analysis of bFGF-like peptides in heart extracts. Here we report that immature phenotype and early response to isoproterenol-induced injury are accompanied by increased levels of a 22-24 kDa bFGF species. Differentiated phenotype and late responseto isoproterenol-induced injury are characterized by increases in the 18 kDa species of bFGF. Augmented anti-bFGF labelling is seen in myocyte nuclei from newborn compared to adult rats, accompanied by increased levels of a 25 kDa bFGF in corresponding nuclear protein extracts. Intense nuclear anti-bFGF labelling is also seen in some myocytes adjacent to the site of injury. Finally, degenerative lesions in the heart are characterized by a tremendous increase in cytoplasmic bFGF, which precedes and likely promotes cellular infiltration. Our data show a participation of bFGF in developmental and injury-related processesin the heart and point to different roles for the 22-24 kDa or 18 kDa bFGF species, related to a proliferative
P-5-1
or hypertrophic
phenotype, respectively.
RAT FETUS VENTRICULAR Ca2+ CURRENT IN DIHYDROPYRIDINE-RESISTANT CARDIOMYOCYTES Noritsugu Tohse, Nicholas Sperelakis. Department of Physiology & Biophysics, University of Cincinnati, College of Medicine, Cincinnati, OH 45267, USA. To investigate developmental changes in cardiac Ca 2+ channels, single cardiomyocytes of 18-day-old rat fetus were freshly isolated using 0.1% collagenase. Currents were recorded (at 25OC) by whole-cell voltaee clam0 in Na. K-free externa1 and K-free internal solutions. The Ca2+ current (1~~) was elicited by depolarizing test .pulses above -57 mV from a holding potential (HP) of -87 mV. &-was maximal around -7 mV: 8.4 + 0.7 pA/pF (M + SE, n = 44). It-. was blocked by 2 mM Co2+, but not completely by the dihydropyridine (DHP) Ca 2+-antagonist nife&$ne. Nifedipine (3 GM) decreased the amplitude of ICa at -7 mV by 65.9 + 3.4% (n = 20); 10 nM showed no further decrease (66.4 5 5.1%, n = IO). The DHP resistance of the ICa was not due to a voltage/dependence, becauseICa was decreasedby 77.1 + 4.7% with HP of -47 mV. The half-inactivation potentials of the DHP-sensitive and -resistant currents were -42.0 and -58.4 mV, respectively. The DHP-resistant current was not completely blocked bv 40 uM Ni2+ (48.6 + 7.2%) and 1 nM tetramethrine (28.1 + 16.8%). These results indicate that the DHP-resistant current mav be a different tvoe of 1~. from T-tvpe and L-type currents. In l2-day rat fetal cardiomyocytes, ICa-current density was 6.4 g-1.6 pA/pF (n = 19) at-7 mV. 3 nM nifedipine decreased 1~~ by 32.5 5 5.7% (n = 18). The current density of the DHP-resistant current in 12-day-old and II-day-oldfetus heart cells was 3.5 + 0.7 pA/pF (n = IS) and 2.4 + 0.3 pA/pF (n = 20), respectively. In conclusion, rat fetal cardiomyocytes may have a unique type of ICa, which decreases and becomes less prominent during development. s.90