Ahmed F. El-Yazbi

Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic response

Ca2+ sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure‐induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)‐potentiated protein phosphatase 1 inhibitor protein (CPI‐17) required to sustain this view is not currently available. Here, we determined whether Ca2+ sensitization pathways involving Rho kinase (ROK)‐ and PKC‐dependent phosphorylation of MYPT1 and CPI‐17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03–10 μmol l−1; H1152, 0.001–0.3 μmol l−1) and PKC inhibitors (GF109203X, 3 μmol l−1; Gö6976; 10 μmol l−1) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3‐step Western blot method was developed for detection and quantification of MYPT1 and CPI‐17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine‐855 (T855) and myosin light chain (LC20). Phosphorylation of MYPT1 at threonine‐697 (T697) and CPI‐17 were not affected by pressure. Pressure‐evoked elevations in MYPT1‐T855 and LC20 phosphorylation were reduced by H1152, but MYPT1‐T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC20 phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca2+ sensitization pathway involving ROK‐dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC20 phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca2+ sensitization mediated by CPI‐17 or MYPT1 phosphorylation.

Caveolae and calcium handling, a review and a hypothesis.

Caveolae are associated with molecules crucial for calcium handling. This review considers the roles of caveolae in calcium handling for smooth muscle and interstitial cells of Cajal (ICC). Structural studies showed that the plasma membrane calcium pump (PMCA), a sodium‐calcium exchanger (NCX1), and a myogenic nNOS appear to be colocalized with caveolin I, the main constituent of these caveolae. Voltage dependent calcium channels (VDCC) are associated but not co‐localized with caveolin 1, as are proteins of the peripheral sarcoplasmic reticulum (SR) such as calreticulin. Only the nNOS is absent from caveolin 1 knockout animals. Functional studies in calcium free media sugest that a source of calcium in tonic smooth muscles exists, partly sequestered from extracellular EGTA. This source supported sustained contractions to carbachol using VDCC and dependent on activity of the SERCA pump. This source is postulated to be caveolae, near peripheral SR. New evidence, presented here, suggests that a similar source exists in phasic smooth muscle of the intestine and its ICC. These results suggest that caveolae and peripheral SR are a functional unit recycling calcium through VDCC and controlling its local concentration. Calcium handling molecules associated with caveolae in smooth muscle and ICC were identified and their possible functions also reviewed.

Intravascular pressure augments cerebral arterial constriction by inducing voltage-insensitive Ca2+ waves

This study examined whether elevated intravascular pressure stimulates asynchronous Ca2+ waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20–100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (VM) were monitored using conventional techniques; Ca2+ wave generation and myosin light chain (MLC20)/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca2+ waves as well as event frequency. Ca2+ wave augmentation occurred primarily at lower intravascular pressures (<60 mmHg) and ryanodine, a plant alkaloid that depletes the sarcoplasmic reticulum (SR) of Ca2+, eliminated these events. Ca2+ wave generation was voltage insensitive as Ca2+ channel blockade and perturbations in extracellular [K+] had little effect on measured parameters. Ryanodine‐induced inhibition of Ca2+ waves attenuated myogenic tone and MLC20 phosphorylation without altering arterial VM. Thapsigargin, an SR Ca2+‐ATPase inhibitor also attenuated Ca2+ waves, pressure‐induced constriction and MLC20 phosphorylation. The SR‐driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca2+ waves facilitated pressure‐induced MLC20 phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca2+ wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca2+ required to directly control MLC20 phosphorylation.

Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

Blood flow to our organs is maintained within a defined range to provide an adequate supply of nutrients and remove waste products by contraction and relaxation of smooth muscle cells of resistance arteries and arterioles. The ability of these cells to contract in response to an increase in intravascular pressure, and to relax following a reduction in pressure (the ‘myogenic response’), is critical for appropriate control of blood flow, but our understanding of its mechanistic basis is incomplete. Small arteries of skeletal muscles were used to test the hypothesis that myogenic constriction involves two enzymes, Rho‐associated kinase and protein kinase C, which evoke vasoconstriction by activating the contractile protein, myosin, and by reorganizing the cytoskeleton. Knowledge of the mechanisms involved in the myogenic response contributes to understanding of how blood flow is regulated and will help to identify the molecular basis of dysfunctional control of arterial diameter in disease.

Pressure‐dependent contribution of Rho kinase‐mediated calcium sensitization in serotonin‐evoked vasoconstriction of rat cerebral arteries

Our understanding of the cellular signalling mechanisms contributing to agonist‐induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca2+ sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC‐potentiated protein phosphatase 1 inhibitor protein (CPI‐17) to serotonin (5‐HT)‐induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure‐induced myogenic and agonist‐induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI‐17 and 20 kDa myosin light chain subunit (LC20) phosphorylation were determined following treatment with 5‐HT (1 μmol l−1) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho‐associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5‐HT‐induced constriction and reduced phospho‐LC20 content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI‐17 phosphorylation. In contrast, an increase in MYPT1‐T697 and MYPT1‐T855 phosphorylation, but not phospho‐CPI‐17 content, was apparent at 60 mmHg following exposure to 5‐HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5‐HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross‐bridge cycling with blebbistatin (10 μmol l−1) did not affect phosphoprotein content. Taken together, the data indicate that Ca2+ sensitization owing to ROK‐mediated phosphorylation of MYPT1 contributes to 5‐HT‐evoked vasoconstriction only in the presence of pressure‐induced myogenic activation. These findings provide novel evidence of an interplay between myogenic‐ and agonist‐induced vasoconstriction in cerebral resistance arteries.

Stromatoxin-sensitive, heteromultimeric Kv2.1/Kv9.3 channels contribute to myogenic control of cerebral arterial diameter

Cerebral vascular smooth muscle contractility plays a crucial role in controlling arterial diameter and, thereby, blood flow regulation in the brain. A number of K+ channels have been suggested to contribute to the regulation of diameter by controlling smooth muscle membrane potential (Em) and Ca2+ influx. Previous studies indicate that stromatoxin (ScTx1)‐sensitive, Kv2‐containing channels contribute to the control of cerebral arterial diameter at 80 mmHg, but their precise role and molecular composition were not determined. Here, we tested if Kv2 subunits associate with ‘silent’ subunits from the Kv5, Kv6, Kv8 or Kv9 subfamilies to form heterotetrameric channels that contribute to control of diameter of rat middle cerebral arteries (RMCAs) over a range of intraluminal pressure from 10 to 100 mmHg. The predominant mRNAs expressed by RMCAs encode Kv2.1 and Kv9.3 subunits. Co‐localization of Kv2.1 and Kv9.3 proteins at the plasma membrane of dissociated single RMCA myocytes was detected by proximity ligation assay. ScTx1‐sensitive native current of RMCA myocytes and Kv2.1/Kv9.3 currents exhibited functional identity based on the similarity of their deactivation kinetics and voltage dependence of activation that were distinct from those of homomultimeric Kv2.1 channels. ScTx1 treatment enhanced the myogenic response of pressurized RMCAs between 40 and 100 mmHg, but this toxin also caused constriction between 10 and 40 mmHg that was not previously observed following inhibition of large conductance Ca2+‐activated K+ (BKCa) and Kv1 channels. Taken together, this study defines the molecular basis of Kv2‐containing channels and contributes to our understanding of the functional significance of their expression in cerebral vasculature. Specifically, our findings provide the first evidence of heteromultimeric Kv2.1/Kv9.3 channel expression in RMCA myocytes and their distinct contribution to control of cerebral arterial diameter over a wider range of Em and transmural pressure than Kv1 or BKCa channels owing to their negative range of voltage‐dependent activation.

Cytoskeletal reorganization evoked by Rho-associated kinase- and protein kinase C-catalyzed phosphorylation of cofilin and heat shock protein 27, respectively, contributes to myogenic constriction of rat cerebral arteries.

Background: The myogenic response of cerebral arteries to intravascular pressure regulates blood flow to the brain. Results: Pressurization reduced smooth muscle G-actin and increased phospho-cofilin and -HSP27 content by a mechanism blocked by ROK or PKC inhibitors. Conclusion: ROK- and PKC-mediated control of cofilin and HSP27 contributes to actin polymerization in myogenic constriction. Significance: Knowledge of cytoskeletal dynamics is crucial for understanding myogenic control of cerebral arterial diameter. Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC20) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC20, calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.

Calcium extrusion by plasma membrane calcium pump is impaired in caveolin-1 knockout mouse small intestine

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells. PMCA4 is the predominant isoform operating in conditions of high intracellular calcium during contraction. PMCA appears to be localized in lipid rafts and caveolae. In this study we examined the effects of the PMCA4-selective inhibitor caloxin 1c2 (5 microM) in intestine of caveolin-1 knockout mice and in bovine tracheal smooth muscle after caveolae disruption on PMCA4 function. Small intestinal tissues from control mice treated with caloxin 1c2 showed a higher contractile response of the longitudinal smooth muscle to Carbachol (10 microM) when compared to control tissues treated with a similar concentration of a control peptide. This effect of caloxin 1c2 was not found in tissues from caveolin-1 knockout mice. Immunohistochemistry and Western blotting of membrane fractions showed that PMCA was co-localized with caveolin-1 in smooth muscle plasma membrane in control tissues. One of the PMCA4 splice variant bands was missing in the lipid raft-enriched fraction prepared from caveolin-1 knockout tissue. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin-1 and PMCA4b immunoreactivities, previously co-localized in the smooth muscle plasma membrane, and to the loss of the increase in Carbachol-induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.

Caveolin‐1 gene knockout impairs nitrergic function in mouse small intestine

1 Caveolin‐1 is a plasma membrane‐associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). 2 Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin‐1 knockout mice (Cav1−/−) were used to examine the effect of caveolin‐1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. 3 Immunohistochemical staining showed loss of caveolin‐1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1−/− mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. 4 Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1−/− mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N‐omega‐nitro‐L‐arginine (10−4 M), but relaxation of Cav1−/− animals was affected much less. Also, Cav1−/− mice tissues showed reduced relaxation responses to sodium nitroprusside (100 μM) compared to controls; yet there were no significant differences in the relaxation responses to 8‐bromoguanosine‐3′ : 5′‐cyclic monophosphate (100 μM). 5 Apamin (10−6 M) significantly reduced relaxations to EFS in NANC conditions in Cav1−/− mice, but not in controls. 6 The data from this study suggest that caveolin‐1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin‐sensitive inhibitory mediators.

PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization

Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.