Thiophosphorylation‐induced Ca<sup>2+</sup> sensitization of guinea‐pig ileum contractility is not mediated by Rho‐associated kinase

Pfitzer, Gabriele; Sonntag-Bensch, Dagmar; Brkic-Koric, D

doi:10.1111/j.1469-7793.2001.t01-2-00651.x

Cited by 20 publications

(13 citation statements)

References 49 publications

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“…In embryonic smooth muscle, our results demonstrating a dose dependence of the inhibition of GTP␥S-induced force enhancement by the Rho-kinase inhibitor Y-27632 (Fig. 3) are similar to those of others (8,24,26). The magnitude of force enhancement was not affected by inhibition of MLCK or by inhibition of PKC with a concentration of staurosporine known to inhibit contractions of smooth muscle produced by phorbol esters (18).…”

Section: Force Versus Casupporting

confidence: 89%

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Agonist-induced Force Enhancement

Richards

Ogut

Brozovich

2002

Journal of Biological Chemistry

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The magnitude of agonist-induced Ca 2؉ sensitization of force is tissue-dependent, but an explanation for this diversity is unknown. Ca 2؉ sensitization is thought to involve a G-protein-mediated inhibition of myosin light chain phosphatase activity by phosphorylation of the myosin-targeting subunit (MYPT). The MYPT has two isoforms that differ by a central insert, which lies near this phosphorylation site. Expression of MYPT isoforms is both developmentally regulated and tissue-specific. We hypothesized that the presence or absence of the central insert determines the magnitude of agonist-induced Ca 2؉ sensitization. Throughout development, the chicken aorta exclusively expresses the splice-in MYPT isoform, and guanosine 5-O-(thiotriphosphate) (GTP␥S) produces a significant force enhancement. Early during development, the chicken gizzard expresses the splice-in MYPT isoform, and GTP␥S produced a Ca 2؉ sensitization. In the gizzard coincident with the shift in expression from the splice-in to splice-out MYPT isoform, GTP␥S no longer produced force enhancement. In addition, adenosine 5-O-(thiotriphosphate) (ATP␥S) phosphorylated only adult gizzard tissue, the only tissue that did not demonstrate a Ca 2؉ sensitization. These results suggest that the relative expression of splice-in/ splice-out MYPT isoforms determines the magnitude of agonist-induced force enhancement and that MYPT phosphorylation is not required for Ca 2؉ sensitization.The importance of Ca 2ϩ -calmodulin-dependent myosin light chain kinase (MLCK) 1 for smooth muscle activation has been well described (1). However, recent evidence suggests that regulation of MLC phosphatase activity is physiologically important for force regulation in smooth muscle (reviewed in Refs. 2-6). This regulation of MLC phosphatase activity can produce a Ca 2ϩ sensitization, or an increase in force at a constant [Ca 2ϩ ], and a Ca 2ϩ desensitization, or a reduction in force at a constant [Ca 2ϩ ]. Agonist stimulation produces a Ca 2ϩ sensitization of the contractile filaments (7). The mechanism for the agonist-induced Ca 2ϩ sensitization, or force enhancement, is thought to depend on a G-protein-mediated inhibition of MLC phosphatase activity resulting in an increase in MLC 20 phosphorylation (8). The MLC phosphatase is a trimeric protein consisting of a 38-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a large (110 -133 kDa) myosin-targeting subunit (MYPT) (reviewed in Ref. 3)). G-protein-induced inhibition of MLC phosphatase activity has been suggested to be regulated by phosphorylation of the MYPT (9), possibly by either Rhokinase (10, 11) or Zip-like kinase (12). However, direct activation of the G-proteins (8), as well as agonist stimulation (7), does not produce the same magnitude of force enhancement in all smooth muscle tissues, and the mechanism to explain this diversity has yet to be elucidated.The MYPT has two isoforms that are produced by alternative splicing of a central exon (13). The alternatively spliced segment lies near a phosphoryl...

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Section: Force Versus Casupporting

confidence: 89%

“…It could be that the force enhancement produced by GTP␥S and ATP␥S are mediated by different pathways (26). If this is the case, the lack of thiophosphorylation of the MYPT in the aorta and embryonic gizzard would not preclude phosphorylation of the MYPT mediating G-protein stimulated Ca 2ϩ sensitization.…”

Section: Force Versus Camentioning

confidence: 99%

Agonist-induced Force Enhancement

Richards

Ogut

Brozovich

2002

Journal of Biological Chemistry

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“…1) can be taken as evidence for PKCmediated sensitization of pericytes to [Ca 2ϩ ] cyt , but this is not conclusive. PKC blockers are not entirely specific and might block other kinases including MLCK (32,38). The elevation of [Ca 2ϩ ] cyt by PKC stimulation (Figs.…”

Section: Discussionmentioning

confidence: 97%

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

Zhang

Rhinehart

Kwon

et al. 2004

American Journal of Physiology-Heart and Circulatory Physiology

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ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.

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“…Metabolic syndrome has been shown to be associated with alterations in vascular reactivity. Indeed, blunted endothelial-dependent vasodilation and augmented vasoconstriction have been demonstrated in both obese humans (3,14,26,43,47) and animals (10, 17-20, 30, 34, 37, 51 (11,24,27,36). MLCP consists of a 37-to 38-kDa catalytic subunit (PP1c), an associated 110-to 130-kDa targeting subunit (MYPT1), and a third 20-kDa subunit of unknown function.…”

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confidence: 99%

Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats

Naik¹,

Xiang²,

Hester³

2006

American Journal of Physiology-Regulatory, Integrative and Comp

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Obesity, insulin resistance, dyslipidemia, and hypertension are components of the pathophysiological state known as metabolic syndrome. Adrenergic vasoconstriction is mediated through increases in cytosolic Ca 2ϩ and the myofilaments' sensitivity to Ca 2ϩ . In many pathophysiological states, there is an enhanced role for Rho kinase (ROK)-mediated increases in Ca 2ϩ sensitivity of the contractile apparatus. Thus we hypothesized that there is a greater role for ROK-mediated increases in Ca 2ϩ sensitivity in ␣1-adrenergic vasoconstriction in arteries from obese Zucker (OZ) rats. Therefore, small gracilis muscle arteries from 11-to 12-wk-old and 16-to 18-wk-old lean and OZ rats were isolated, cannulated, and pressurized to 75 mmHg. For some experiments, vessels were loaded with fura 2-AM. Changes in luminal diameter and vessel wall Ca 2ϩ concentration ([Ca 2ϩ ]) were measured in response to phenylephrine (PE), the thromboxane mimetic U-46619, and KCl. ␣1-Adrenergic vasoconstriction was similar between 11-to 12-wk-old lean and obese animals and greater in older obese animals compared with controls. PE-induced increases in vascular smooth muscle cell [Ca 2ϩ ] were blunted in OZ animals compared with lean controls in both age groups of animals. KCl and U-46619 elicited similar vasoconstriction and vascular smooth muscle cell [Ca 2ϩ ] in both groups. ROK inhibition attenuated PE vasoconstriction to a greater degree in arteries from 11-to 12-wk-old OZ rats compared with lean animals; ROK inhibition in arteries from older rats right shifted both concentration-response curves to the same point. Total RhoA and ROK␣ protein expressions were similar between groups. These results suggest an enhanced role for the ROK pathway in ␣1-adrenergic vasoconstriction in metabolic syndrome. vascular smooth muscle; vascular reactivity; isolated vessel; fura 2; phenylephrine; Type 2 diabetes METABOLIC SYNDROME IS A COHORT of pathophysiological conditions that include obesity, insulin resistance, dyslipidemia, and hypertension. Epidemiological reports suggest that ϳ24% of adults in the United States over 20 yr of age meet the criteria for metabolic syndrome established by the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults III (15). On the basis of year 2000 census data, ϳ47 million Americans have metabolic syndrome (16). Metabolic syndrome has been shown to be associated with alterations in vascular reactivity. Indeed, blunted endothelial-dependent vasodilation and augmented vasoconstriction have been demonstrated in both obese humans (3,14,26,43,47) and animals (10, 17-20, 30, 34, 37, 51 (11,24,27,36). MLCP consists of a 37-to 38-kDa catalytic subunit (PP1c), an associated 110-to 130-kDa targeting subunit (MYPT1), and a third 20-kDa subunit of unknown function. Phosphorylation of the MYPT1 subunit of MLCP inhibits its activity, increasing the Ca 2ϩ sensitivity of the contractile apparatus for a given [Ca 2ϩ ]. It is well established that MLCP can be i...

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Thiophosphorylation‐induced Ca²⁺ sensitization of guinea‐pig ileum contractility is not mediated by Rho‐associated kinase

Cited by 20 publications

References 49 publications

Agonist-induced Force Enhancement

Agonist-induced Force Enhancement

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats

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Thiophosphorylation‐induced Ca2+ sensitization of guinea‐pig ileum contractility is not mediated by Rho‐associated kinase

Cited by 20 publications

References 49 publications

Agonist-induced Force Enhancement

Agonist-induced Force Enhancement

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats

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Thiophosphorylation‐induced Ca²⁺ sensitization of guinea‐pig ileum contractility is not mediated by Rho‐associated kinase