Arteriosclerosis, Thrombosis, and Vascular Biology, 2013
Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. TAAD... more Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. TAAD can be inherited due to mutations in a number of genes, including MYH11, which encodes the smooth muscle myosin heavy chain. In non-familial TAAD patients, hypertension is the most common risk factor, but genetic contributors have not been explored. Exome sequencing has identified non-synonymous variants of unknown significance in MYH11 in the population, and these variants are enriched in TAAD patients without a family history. The goal of this study was to determine if rare MYH11 variants contribute to TAAD. A knock-in mouse model of one recurrent rare variant, Myh11 R247C, shows no structural or pathologic abnormalities of the aorta, but does have decreased aortic contractility. We sought to determine if the Myh11 R247C allele could modify aortic disease due to another gene mutation. The Myh11R247C allele was bred into Acta2-/- mice, which develop aortic aneurysms, and into Acta2+/- ...
Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. One-... more Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. One-fifth of TAAD patients have a first-degree relative with the disease, suggesting a genetic component to its pathogenesis. Missense mutations in a number of genes lead to TAAD, including MYH11 ,which encodes the smooth muscle isoform of myosin heavy chain. However, potential genetic contributors in the four-fifths of patients without affected relatives have not been explored. Variants of unknown significance in MYH11 are prevalent in the population, and are enriched in patients with TAAD and other vascular diseases. The goal of this study was to evaluate one of these variants as a potential contributor to TAAD. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11 R247C/R247C mice also have increased...
Highlights d Actin is post-translationally modified in the cell nucleus, similar to histones d Ar... more Highlights d Actin is post-translationally modified in the cell nucleus, similar to histones d Arginine 256 mono-methylation is a conserved mark mostly present in nuclear actin d A survey of actin R256me1 in yeast reveals a genome-wide landscape of nuclear actin d Actin R256me1 is implicated in transcription and linked to human diseases
Biochemical events associated with activation of smooth muscle contraction were studied in neural... more Biochemical events associated with activation of smooth muscle contraction were studied in neurally stimulated bovine tracheal smooth muscle. A latency period of 500 ms preceded increases in isometric force and myosin light chain phosphorylation. However, stimulation resulted in the rapid hydrolysis of inositol phospholipids as demonstrated by increases in inositol phosphates by 500 ms. Inositol trisphosphate increased 2-fold with no significant change in inositol tetrakisphosphate. The apparent activation state of myosin light chain kinase was assessed indirectly through measurements of the fractional activation of a second calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. The fractional activation of cyclic nucleotide phosphodiesterase increased after neural stimulation to a maximal extent by 500 ms and remained at this level for at least 4 s. The monophosphorylation of myosin light chain increased after 500 ms and reached a maximum value by 2 s. Diphosphorylation also occurred but to a much lesser extent. Fractional activation of cyclic nucleotide phosphodiesterase and myosin light chain phosphorylation both decreased after 10 min continuous stimulation, although the force response remained at a maximal level. These observations demonstrate that inositol trisphosphate formation and activation of cyclic nucleotide phosphodiesterase (and hence most likely myosin light chain kinase) by calmodulin precede myosin light chain phosphorylation and that these events are sufficiently rapid to mediate the contractile response of neurally stimulated tracheal smooth muscle.
Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phos... more Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca" concentration required for half-maximal phosphorylation was 500 n~ for MLCK and 250 n~ for myosin light chain. Addition of KN-62 or a synthetic peptide CK 11, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase I1 activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodaminecalmodulin in intact tracheal smooth muscle cells. An important step in the initiation of smooth muscle contraction is the phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2000
Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compar... more Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compared by quantifying contractile protein, myosin heavy chain (MHC) isoform contents, and contractile properties of aortas and bladders from female fetal ( n = 19) and postnatal ( n = 21) sheep. Actin, myosin, and protein contents rose progressively throughout development in both tissues ( P ≤ 0.003); however, expression patterns differed. During the last trimester, i.e., 101–130 days (term ∼145 days), bladder actin and MHC contents were approximately twofold greater ( P < 0.04) than those in the aorta. Although the fractional content of 204-kDa SM1 MHC in the bladder decreased from 74 ± 3% at midgestation to 48 ± 2% 3 mo postnatal, the aorta exhibited an increase from 30 ± 2% to 65 ± 2%. Bladder MHC (MHC-B) migrating at 200 kDa contained only SM2 throughout development. In contrast, 200-kDa MHC in the aorta was predominantly nonmuscle MHC-B at midgestation, which was gradually replaced b...
American Journal of Physiology-Heart and Circulatory Physiology, 1989
During pregnancy, sheep develop attenuated systemic and uterine vascular responsiveness to alpha-... more During pregnancy, sheep develop attenuated systemic and uterine vascular responsiveness to alpha-adrenergic stimulation. To determine whether this reflects altered vascular smooth muscle function, we studied the responsiveness of smooth muscle isolated from systemic and uterine arteries to KCl and phenylephrine. Uterine, renal, and carotid arteries were collected from nonpregnant, pregnant (131 +/- 2 days, +/- SD), and late postpartum (144 +/- 4 days) ewes; endothelium was removed and open rings were hung for measurement of isometric force. There were no differences in concentration-response relationships nor maximal stresses generated to phenylephrine between nonpregnant, pregnant, and late postpartum states for carotid or renal arteries. However, the 50% maximal concentration for phenylephrine of uterine arteries in the nonpregnant state (2.8 +/- 0.9 x 10(-6) M) was greater than the pregnant state (0.76 +/- 0.05 x 10(-6) M). Moreover, uterine arteries from pregnant sheep generated...
During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skel... more During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers or from endothelial cells (eNOS) may relax vascular smooth muscle contributing to functional hyperemia. To examine the relative importance of these pathways, smooth muscle myosin regulatory light chain (smRLC) phosphorylation was assessed as an index of vascular tone in isolated extensor digitorum longus (EDL) muscles from C57, nNOS−/−, and eNOS−/−mice. The smRLC phosphorylation (in mol phosphate per mol smRLC) in C57 resting muscles (0.12 ± 0.04) was increased 3.7-fold (0.44 ± 0.03) by phenylephrine (PE). Reversal of this increase with electrical stimulation (to 0.19 ± 0.03; P < 0.05) was partially blocked by Nω-nitro-l-arginine (NLA). In nNOS−/−EDL, the PE-induced increase in smRLC phosphorylation (0.10 ± 0.02 to 0.49 ± 0.04) was partially decreased by stimulation (0.25 ± 0.04). In eNOS−/−EDL, the control value for smRLC was increased (0.24 ± 0.04), and PE-induced...
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 1998
In fetal sheep umbilical responses to angiotensin II (ANG II) exceed those by systemic vasculatur... more In fetal sheep umbilical responses to angiotensin II (ANG II) exceed those by systemic vasculature. Two ANG II receptors (AT) exist, AT1 and AT2, but only AT1 mediates vasoconstriction in adult tissues. Thus differences in reactivity could reflect differences in subtype expression. Using competitive radioligand binding assays, we demonstrated AT1 predominance in umbilical arteries and AT2 in femoral arteries. Steady-state responses to intravenous ANG II (0.229–1.72 μg/min) were studied in 16 fetuses with umbilical and/or femoral artery flow probes without and with local AT1 (L-158,809) or AT2 (PD-123319) blockade. ANG II dose dependently ( P < 0.001) increased umbilical resistance more than arterial pressure (MAP) while decreasing umbilical blood flow. Femoral vascular resistance also increased dose dependently ( P = 0.02), but responses were less than umbilical ( P = 0.0001) and paralleled increases in MAP; blood flow was unaffected. Cumulative local doses of L-158,809 (125 μg) ...
Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of prematur... more Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of premature deaths if individuals at risk can be identified. Individuals with early-onset aortic dissections without a family history or syndromic features have an increased burden of rare genetic variants of unknown significance (VUSs) in genes with pathogenic variants for heritable thoracic aortic disease (HTAD). We assessed the role of VUSs in the development of disease using both in vitro enzymatic assays and mouse models. VUSs in LOX and MYLK identified in individuals with acute aortic dissections were assayed to determine whether they disrupted enzymatic activity. A subset of VUSs reduced enzymatic activity compared to the wild-type proteins but less than pathogenic variants. Additionally, a Myh11 variant, p.Arg247Cys, which does not cause aortic disease in either humans or mice, was crossed with the Acta2 mouse, which has aortic enlargement with age while Acta2 mice do not. Acta2Myh11 mice ...
Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase a... more Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not resul...
American Journal of Physiology-Cell Physiology, 1991
Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to be an important mediator of my... more Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to be an important mediator of myometrial relaxation, and there is evidence to suggest that guanosine 3',5'-cyclic monophosphate (cGMP) is a mediator of smooth muscle relaxation in vascular and probably nonvascular tissues. To investigate the biochemical mechanisms involved in regulation of human myometrial contractility, we studied the effects of analogues of cAMP and cGMP, as well as activators of adenylate and guanylate cyclases, on uterine smooth muscle contractile activity. We found that myometrial smooth muscle cells in culture respond to analogues of cGMP and cAMP, as well as activators of guanylate cyclase, with a significant decrease in the resting and endothelin-induced increase in [Ca2+]i. Treatment of human uterine smooth muscle strips with sodium nitroprusside or isoproterenol results in diminished force and frequency of contraction as well as a decrease in the rate and extent of myosin light chain ...
American Journal of Physiology-Cell Physiology, 1983
Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. Howeve... more Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. However, peak values are not maintained and phosphorylation declines, while active stress increases monotonically to a sustained steady state. The aim of this study was to determine the reason(s) for the transient change in myosin phosphorylation. Four hypotheses were considered: 1) reduced substrate, i.e., ATP depletion, 2) altered access of either the myosin kinase or phosphatase to the cross bridge, 3) reduced myosin kinase activity secondary to its phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase, and 4) reduced myoplasmic [Ca2+] during the contraction. Our results suggest that the most likely explanation is that there are two Ca2+-dependent regulatory processes: 1) myosin phosphorylation and 2) a second, unidentified site allowing stress maintenance with reduced cross-bridge cycling rates. A higher cell Ca2+ concentration appears to be necessary to ...
American Journal of Physiology-Cell Physiology, 1985
Field stimulation of intrinsic nerves in bovine tracheal smooth muscle strips elicited atropine-s... more Field stimulation of intrinsic nerves in bovine tracheal smooth muscle strips elicited atropine-sensitive contractions that were more rapid than those obtained by addition of carbamylcholine to the bathing medium. These stimulus conditions were used to improve estimates of maximal rates of activation as indicated by myosin light chain phosphorylation, maximal shortening velocity (Vo), and isometric force. Maximal values of Vo [0.25 X optimal muscle length X s-1] and light chain phosphorylation (0.65 mol phosphate/mol light chain) were attained after 5 s of stimulation and preceded maximal force (60 s). Force gradually fell to 0.85 times maximal values during 30 min of stimulation, while both light chain phosphorylation and Vo declined to 0.3 times the maximal value. The temporal correlation between light chain phosphorylation and Vo supports the hypothesis that myosin phosphorylation in smooth muscle functions in regulating cross-bridge cycling rates. Myosin was dephosphorylated dur...
American Journal of Physiology-Cell Physiology, 1990
During pregnancy, the ovine uterine artery changes from a low- to a high-stress artery. We invest... more During pregnancy, the ovine uterine artery changes from a low- to a high-stress artery. We investigated the hypotheses that the increased stress reflects alterations in vessel wall cellularity, smooth muscle cell contractile protein contents, or activation properties. Uterine artery diameter increased during pregnancy, whereas the fractional cellular composition and thickness of the muscularis were unchanged. Results of morphometry suggest that vessel growth is associated with cell elongation. Uterine arteries from pregnant ewes had greater protein contents than those from nonpregnant ewes (104 vs. 69 mg/g, respectively); there were corresponding increases in the absolute cellular contents of actin and myosin. While the fraction of light chain phosphorylated in response to phenylephrine was unaltered, the total amount of myosin light chain phosphorylated per gram wet weight increased significantly during pregnancy. In addition, the distribution of myosin heavy chain isoforms was als...
Lau, Kim S., Robert W. Grange, Eiji Isotani, Ingrid H. Sarelius, Kristine E. Kamm, Paul L. Huang,... more Lau, Kim S., Robert W. Grange, Eiji Isotani, Ingrid H. Sarelius, Kristine E. Kamm, Paul L. Huang, and James T. Stull. nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle. Physiol. Genomics 2: 21–27, 2000.—Nitric oxide (NO) from Ca2+-dependent neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers may modulate vascular tone by a cGMP-dependent pathway similar to NO derived from NOS in endothelial cells (eNOS). In isolated fast-twitch extensor digitorum longus (EDL) muscles from control mice, cGMP formation increased ∼166% with electrical stimulation (30 Hz, 15 s). cGMP levels were not altered in slow-twitch soleus muscles. The NOS inhibitor Nω-nitro-l-arginine abolished the contraction-induced increase in cGMP content in EDL muscles, and the NO donor sodium nitroprusside (SNP) increased cGMP content ∼167% in noncontracting EDL muscles. SNP treatment but not electrical stimulation increased cGMP formation in muscles from nNOS...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2013
Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. TAAD... more Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. TAAD can be inherited due to mutations in a number of genes, including MYH11, which encodes the smooth muscle myosin heavy chain. In non-familial TAAD patients, hypertension is the most common risk factor, but genetic contributors have not been explored. Exome sequencing has identified non-synonymous variants of unknown significance in MYH11 in the population, and these variants are enriched in TAAD patients without a family history. The goal of this study was to determine if rare MYH11 variants contribute to TAAD. A knock-in mouse model of one recurrent rare variant, Myh11 R247C, shows no structural or pathologic abnormalities of the aorta, but does have decreased aortic contractility. We sought to determine if the Myh11 R247C allele could modify aortic disease due to another gene mutation. The Myh11R247C allele was bred into Acta2-/- mice, which develop aortic aneurysms, and into Acta2+/- ...
Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. One-... more Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. One-fifth of TAAD patients have a first-degree relative with the disease, suggesting a genetic component to its pathogenesis. Missense mutations in a number of genes lead to TAAD, including MYH11 ,which encodes the smooth muscle isoform of myosin heavy chain. However, potential genetic contributors in the four-fifths of patients without affected relatives have not been explored. Variants of unknown significance in MYH11 are prevalent in the population, and are enriched in patients with TAAD and other vascular diseases. The goal of this study was to evaluate one of these variants as a potential contributor to TAAD. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11 R247C/R247C mice also have increased...
Highlights d Actin is post-translationally modified in the cell nucleus, similar to histones d Ar... more Highlights d Actin is post-translationally modified in the cell nucleus, similar to histones d Arginine 256 mono-methylation is a conserved mark mostly present in nuclear actin d A survey of actin R256me1 in yeast reveals a genome-wide landscape of nuclear actin d Actin R256me1 is implicated in transcription and linked to human diseases
Biochemical events associated with activation of smooth muscle contraction were studied in neural... more Biochemical events associated with activation of smooth muscle contraction were studied in neurally stimulated bovine tracheal smooth muscle. A latency period of 500 ms preceded increases in isometric force and myosin light chain phosphorylation. However, stimulation resulted in the rapid hydrolysis of inositol phospholipids as demonstrated by increases in inositol phosphates by 500 ms. Inositol trisphosphate increased 2-fold with no significant change in inositol tetrakisphosphate. The apparent activation state of myosin light chain kinase was assessed indirectly through measurements of the fractional activation of a second calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. The fractional activation of cyclic nucleotide phosphodiesterase increased after neural stimulation to a maximal extent by 500 ms and remained at this level for at least 4 s. The monophosphorylation of myosin light chain increased after 500 ms and reached a maximum value by 2 s. Diphosphorylation also occurred but to a much lesser extent. Fractional activation of cyclic nucleotide phosphodiesterase and myosin light chain phosphorylation both decreased after 10 min continuous stimulation, although the force response remained at a maximal level. These observations demonstrate that inositol trisphosphate formation and activation of cyclic nucleotide phosphodiesterase (and hence most likely myosin light chain kinase) by calmodulin precede myosin light chain phosphorylation and that these events are sufficiently rapid to mediate the contractile response of neurally stimulated tracheal smooth muscle.
Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phos... more Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca" concentration required for half-maximal phosphorylation was 500 n~ for MLCK and 250 n~ for myosin light chain. Addition of KN-62 or a synthetic peptide CK 11, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase I1 activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodaminecalmodulin in intact tracheal smooth muscle cells. An important step in the initiation of smooth muscle contraction is the phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2000
Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compar... more Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compared by quantifying contractile protein, myosin heavy chain (MHC) isoform contents, and contractile properties of aortas and bladders from female fetal ( n = 19) and postnatal ( n = 21) sheep. Actin, myosin, and protein contents rose progressively throughout development in both tissues ( P ≤ 0.003); however, expression patterns differed. During the last trimester, i.e., 101–130 days (term ∼145 days), bladder actin and MHC contents were approximately twofold greater ( P < 0.04) than those in the aorta. Although the fractional content of 204-kDa SM1 MHC in the bladder decreased from 74 ± 3% at midgestation to 48 ± 2% 3 mo postnatal, the aorta exhibited an increase from 30 ± 2% to 65 ± 2%. Bladder MHC (MHC-B) migrating at 200 kDa contained only SM2 throughout development. In contrast, 200-kDa MHC in the aorta was predominantly nonmuscle MHC-B at midgestation, which was gradually replaced b...
American Journal of Physiology-Heart and Circulatory Physiology, 1989
During pregnancy, sheep develop attenuated systemic and uterine vascular responsiveness to alpha-... more During pregnancy, sheep develop attenuated systemic and uterine vascular responsiveness to alpha-adrenergic stimulation. To determine whether this reflects altered vascular smooth muscle function, we studied the responsiveness of smooth muscle isolated from systemic and uterine arteries to KCl and phenylephrine. Uterine, renal, and carotid arteries were collected from nonpregnant, pregnant (131 +/- 2 days, +/- SD), and late postpartum (144 +/- 4 days) ewes; endothelium was removed and open rings were hung for measurement of isometric force. There were no differences in concentration-response relationships nor maximal stresses generated to phenylephrine between nonpregnant, pregnant, and late postpartum states for carotid or renal arteries. However, the 50% maximal concentration for phenylephrine of uterine arteries in the nonpregnant state (2.8 +/- 0.9 x 10(-6) M) was greater than the pregnant state (0.76 +/- 0.05 x 10(-6) M). Moreover, uterine arteries from pregnant sheep generated...
During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skel... more During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers or from endothelial cells (eNOS) may relax vascular smooth muscle contributing to functional hyperemia. To examine the relative importance of these pathways, smooth muscle myosin regulatory light chain (smRLC) phosphorylation was assessed as an index of vascular tone in isolated extensor digitorum longus (EDL) muscles from C57, nNOS−/−, and eNOS−/−mice. The smRLC phosphorylation (in mol phosphate per mol smRLC) in C57 resting muscles (0.12 ± 0.04) was increased 3.7-fold (0.44 ± 0.03) by phenylephrine (PE). Reversal of this increase with electrical stimulation (to 0.19 ± 0.03; P < 0.05) was partially blocked by Nω-nitro-l-arginine (NLA). In nNOS−/−EDL, the PE-induced increase in smRLC phosphorylation (0.10 ± 0.02 to 0.49 ± 0.04) was partially decreased by stimulation (0.25 ± 0.04). In eNOS−/−EDL, the control value for smRLC was increased (0.24 ± 0.04), and PE-induced...
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 1998
In fetal sheep umbilical responses to angiotensin II (ANG II) exceed those by systemic vasculatur... more In fetal sheep umbilical responses to angiotensin II (ANG II) exceed those by systemic vasculature. Two ANG II receptors (AT) exist, AT1 and AT2, but only AT1 mediates vasoconstriction in adult tissues. Thus differences in reactivity could reflect differences in subtype expression. Using competitive radioligand binding assays, we demonstrated AT1 predominance in umbilical arteries and AT2 in femoral arteries. Steady-state responses to intravenous ANG II (0.229–1.72 μg/min) were studied in 16 fetuses with umbilical and/or femoral artery flow probes without and with local AT1 (L-158,809) or AT2 (PD-123319) blockade. ANG II dose dependently ( P < 0.001) increased umbilical resistance more than arterial pressure (MAP) while decreasing umbilical blood flow. Femoral vascular resistance also increased dose dependently ( P = 0.02), but responses were less than umbilical ( P = 0.0001) and paralleled increases in MAP; blood flow was unaffected. Cumulative local doses of L-158,809 (125 μg) ...
Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of prematur... more Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of premature deaths if individuals at risk can be identified. Individuals with early-onset aortic dissections without a family history or syndromic features have an increased burden of rare genetic variants of unknown significance (VUSs) in genes with pathogenic variants for heritable thoracic aortic disease (HTAD). We assessed the role of VUSs in the development of disease using both in vitro enzymatic assays and mouse models. VUSs in LOX and MYLK identified in individuals with acute aortic dissections were assayed to determine whether they disrupted enzymatic activity. A subset of VUSs reduced enzymatic activity compared to the wild-type proteins but less than pathogenic variants. Additionally, a Myh11 variant, p.Arg247Cys, which does not cause aortic disease in either humans or mice, was crossed with the Acta2 mouse, which has aortic enlargement with age while Acta2 mice do not. Acta2Myh11 mice ...
Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase a... more Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not resul...
American Journal of Physiology-Cell Physiology, 1991
Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to be an important mediator of my... more Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to be an important mediator of myometrial relaxation, and there is evidence to suggest that guanosine 3',5'-cyclic monophosphate (cGMP) is a mediator of smooth muscle relaxation in vascular and probably nonvascular tissues. To investigate the biochemical mechanisms involved in regulation of human myometrial contractility, we studied the effects of analogues of cAMP and cGMP, as well as activators of adenylate and guanylate cyclases, on uterine smooth muscle contractile activity. We found that myometrial smooth muscle cells in culture respond to analogues of cGMP and cAMP, as well as activators of guanylate cyclase, with a significant decrease in the resting and endothelin-induced increase in [Ca2+]i. Treatment of human uterine smooth muscle strips with sodium nitroprusside or isoproterenol results in diminished force and frequency of contraction as well as a decrease in the rate and extent of myosin light chain ...
American Journal of Physiology-Cell Physiology, 1983
Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. Howeve... more Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. However, peak values are not maintained and phosphorylation declines, while active stress increases monotonically to a sustained steady state. The aim of this study was to determine the reason(s) for the transient change in myosin phosphorylation. Four hypotheses were considered: 1) reduced substrate, i.e., ATP depletion, 2) altered access of either the myosin kinase or phosphatase to the cross bridge, 3) reduced myosin kinase activity secondary to its phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase, and 4) reduced myoplasmic [Ca2+] during the contraction. Our results suggest that the most likely explanation is that there are two Ca2+-dependent regulatory processes: 1) myosin phosphorylation and 2) a second, unidentified site allowing stress maintenance with reduced cross-bridge cycling rates. A higher cell Ca2+ concentration appears to be necessary to ...
American Journal of Physiology-Cell Physiology, 1985
Field stimulation of intrinsic nerves in bovine tracheal smooth muscle strips elicited atropine-s... more Field stimulation of intrinsic nerves in bovine tracheal smooth muscle strips elicited atropine-sensitive contractions that were more rapid than those obtained by addition of carbamylcholine to the bathing medium. These stimulus conditions were used to improve estimates of maximal rates of activation as indicated by myosin light chain phosphorylation, maximal shortening velocity (Vo), and isometric force. Maximal values of Vo [0.25 X optimal muscle length X s-1] and light chain phosphorylation (0.65 mol phosphate/mol light chain) were attained after 5 s of stimulation and preceded maximal force (60 s). Force gradually fell to 0.85 times maximal values during 30 min of stimulation, while both light chain phosphorylation and Vo declined to 0.3 times the maximal value. The temporal correlation between light chain phosphorylation and Vo supports the hypothesis that myosin phosphorylation in smooth muscle functions in regulating cross-bridge cycling rates. Myosin was dephosphorylated dur...
American Journal of Physiology-Cell Physiology, 1990
During pregnancy, the ovine uterine artery changes from a low- to a high-stress artery. We invest... more During pregnancy, the ovine uterine artery changes from a low- to a high-stress artery. We investigated the hypotheses that the increased stress reflects alterations in vessel wall cellularity, smooth muscle cell contractile protein contents, or activation properties. Uterine artery diameter increased during pregnancy, whereas the fractional cellular composition and thickness of the muscularis were unchanged. Results of morphometry suggest that vessel growth is associated with cell elongation. Uterine arteries from pregnant ewes had greater protein contents than those from nonpregnant ewes (104 vs. 69 mg/g, respectively); there were corresponding increases in the absolute cellular contents of actin and myosin. While the fraction of light chain phosphorylated in response to phenylephrine was unaltered, the total amount of myosin light chain phosphorylated per gram wet weight increased significantly during pregnancy. In addition, the distribution of myosin heavy chain isoforms was als...
Lau, Kim S., Robert W. Grange, Eiji Isotani, Ingrid H. Sarelius, Kristine E. Kamm, Paul L. Huang,... more Lau, Kim S., Robert W. Grange, Eiji Isotani, Ingrid H. Sarelius, Kristine E. Kamm, Paul L. Huang, and James T. Stull. nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle. Physiol. Genomics 2: 21–27, 2000.—Nitric oxide (NO) from Ca2+-dependent neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers may modulate vascular tone by a cGMP-dependent pathway similar to NO derived from NOS in endothelial cells (eNOS). In isolated fast-twitch extensor digitorum longus (EDL) muscles from control mice, cGMP formation increased ∼166% with electrical stimulation (30 Hz, 15 s). cGMP levels were not altered in slow-twitch soleus muscles. The NOS inhibitor Nω-nitro-l-arginine abolished the contraction-induced increase in cGMP content in EDL muscles, and the NO donor sodium nitroprusside (SNP) increased cGMP content ∼167% in noncontracting EDL muscles. SNP treatment but not electrical stimulation increased cGMP formation in muscles from nNOS...
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