scholarly journals Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle

2001 ◽  
Vol 280 (3) ◽  
pp. G381-G388 ◽  
Author(s):  
K. S. Murthy ◽  
H. Zhou ◽  
J. R. Grider ◽  
G. M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Clostridium Botulinum ◽  
Dominant Negative ◽  
Calphostin C ◽  
Kinase C ◽  
C3 Exoenzyme ◽  
Sequential Activation ◽  
Adp Ribosylation Factor ◽  
Dependent Pathway

The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated Gq/11, G13, and G12, and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G13 and inhibited by Gα13 antibody. CCK-stimulated PLD activity was partly mediated by RhoA and could be inhibited to the same extent (47 ± 2% to 53 ± 6%) by 1) a dominant negative RhoA mutant, 2) RhoA antibody or Gα13 antibody, and 3) Clostridium botulinum C3 exoenzyme. PLD activity was also inhibited by ARF antibody, and the effect was additive to that of RhoA antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C, bisindolylmaleimide I, and a selective protein kinase C (PKC)-α inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G12 was not coupled to RhoA or ARF, and Gα12 antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G13-dependent RhoA and by ARF and PKC-α and is modulated by an inhibitory G12-dependent pathway.

Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of ϵ-protein kinase C-dependent pathway

2003 ◽  
Vol 285 (6) ◽  
pp. C1454-C1463 ◽  
Author(s):  
Andrea Dallas ◽  
Raouf A. Khalil
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Muscle Contraction ◽  
Smooth Muscle Contraction ◽  
Channel Blockers ◽  
Cell Contraction ◽  
Coronary Smooth Muscle ◽  
Calphostin C ◽  
Gf 109203X ◽  
Dependent Pathway

Certain angina and coronary artery disease forms do not respond to Ca2+ channel blockers, and a role for vasoactive eicosanoids such as PGF2α in Ca2+ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF2α-induced coronary smooth muscle contraction is Ca2+ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca2+ concentration ([Ca2+]i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca2+), PGF2α (10-5 M) caused transient [Ca2+]i increase followed by maintained [Ca2+]i increase and 34% cell contraction. Ca2+ channel blockers verapamil and diltiazem (10-6 M) abolished maintained PGF2α-induced [Ca2+]i increase but only partially inhibited PGF2α-induced cell contraction to 17%. Verapamil-insensitive PGF2α contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and ϵ-PKC V1-2. PGF2α caused Ca2+-dependent α-PKC and Ca2+-independent ϵ-PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF2α-induced α-but not ϵ-PKC translocation. PMA (10-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca2+]i increase and ϵ-PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca2+ influx, caused 36% cell contraction and [Ca2+]i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause α- or ϵ-PKC translocation. Thus a significant component of PGF2α-induced contraction of coronary smooth muscle is Ca2+ antagonist insensitive, involves Ca2+-independent ϵ-PKC activation and translocation, and may represent a signaling mechanism of Ca2+ antagonist-resistant coronary vasospasm.

scholarly journals Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

2008 ◽  
Vol 411 (3) ◽  
pp. 543-551 ◽  
Author(s):  
Wimolpak Sriwai ◽  
Huiping Zhou ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Lysophosphatidic Acid ◽  
Light Chain ◽  
Rho Kinase ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Sustained Contraction ◽  
C3 Exoenzyme ◽  
In Cells

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA3 receptors, but not LPA1 and LPA2, receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the Gαq, but not the Gαi, minigene. Initial contraction and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-β (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC20 and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IκBα were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr696 was masked by phosphorylation of MYPT1 at Ser695 by PKA derived from IκB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-β and MLCK and phosphorylation of MLC20, and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC20. Although Rho kinase was activated, phosphorylation of MYPT1 at Thr696 by Rho kinase was masked by phosphorylation of MYPT1 at Ser695 via cAMP-independent PKA derived from the NF-κB pathway.

Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

1998 ◽  
Vol 274 (5) ◽  
pp. C1253-C1260 ◽  
Author(s):  
Dorothee H. Bremerich ◽  
Tetsuya Kai ◽  
David O. Warner ◽  
Keith A. Jones
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Isometric Force ◽  
Calphostin C ◽  
Regulatory Myosin Light Chain ◽  
Kinase C ◽  
The Relationship

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19—31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.

scholarly journals The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis inDictyostelium discoideum

1999 ◽  
Vol 10 (2) ◽  
pp. 393-406 ◽  
Author(s):  
David J. Seastone ◽  
Linyi Zhang ◽  
Greg Buczynski ◽  
Patrick Rebstein ◽  
Gerald Weeks ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Protein Tyrosine Kinase ◽  
Dominant Negative ◽  
Wild Type ◽  
Cortical Actin ◽  
Calphostin C ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes inDictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

Role of protein kinase C in calcium sensitization during muscarinic stimulation in airway smooth muscle

1997 ◽  
Vol 273 (4) ◽  
pp. L775-L781 ◽  
Author(s):  
Dorothee H. Bremerich ◽  
David O. Warner ◽  
Robert R. Lorenz ◽  
Robin Shumway ◽  
Keith A. Jones
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Muscarinic Receptor ◽  
Receptor Stimulation ◽  
Calcium Sensitization ◽  
Calphostin C ◽  
Kinase C ◽  
Chelerythrine Chloride

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+sensitivity is in part mediated by protein kinase C (PKC). With the use of a β-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors {calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19—31)], and staurosporine} on Ca2+sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl- sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

scholarly journals Phosphorylated Pleckstrin Induces Cell Spreading via an Integrin-Dependent Pathway

2000 ◽  
Vol 150 (6) ◽  
pp. 1461-1466 ◽  
Author(s):  
Richard L. Roll ◽  
Eve Marie Bauman ◽  
Joel S. Bennett ◽  
Charles S. Abrams
Keyword(s):  
Chimeric Protein ◽  
Cytoplasmic Tail ◽  
Cell Spreading ◽  
Competitive Inhibitor ◽  
Dominant Negative ◽  
Kinase C ◽  
Dominant Negative Inhibitor ◽  
Β Chain ◽  
Dependent Pathway

Pleckstrin is a 40-kD phosphoprotein containing NH2- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, αIIbβ3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with αIIbβ3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant αIIbβ3 Ser753Pro, or β3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that αIIbβ3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of β3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of β3 Ser753Pro or αIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin β-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of Gi-1 and Gi-2

2000 ◽  
Vol 279 (4) ◽  
pp. C925-C934 ◽  
Author(s):  
K. S. Murthy ◽  
J. R. Grider ◽  
G. M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
G Proteins ◽  
Muscle Activation ◽  
Somatostatin Receptors ◽  
Muscle Cells ◽  
Calphostin C ◽  
Heterologous Desensitization ◽  
Phosphatase 2A ◽  
Clearance Receptor

This study examined the ability of protein kinase C (PKC) to induce heterologous desensitization by targeting specific G proteins and limiting their ability to transduce signals in smooth muscle. Activation of PKC by pretreatment of intestinal smooth muscle cells with phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, or the phosphatase 1 and phosphatase 2A inhibitor, calyculin A, selectively phosphorylated Gαi-1 and Gαi-2, but not Gαi-3 or Gαo, and blocked inhibition of adenylyl cyclase mediated by somatostatin receptors coupled to Gi-1 and opioid receptors coupled to Gi-2, but not by muscarinic M2 and adenosine A1 receptors coupled to Gi-3. Phosphorylation of Gαi-1 and Gαi-2 and blockade of cyclase inhibition were reversed by calphostin C and bisindolylmaleimide, and additively by selective inhibitors of PKCα and PKCɛ. Blockade of inhibition was prevented by downregulation of PKC. Phosphorylation of Gα-subunits by PKC also affected responses mediated by βγ-subunits. Pretreatment of muscle cells with cANP-(4–23), a selective agonist of the natriuretic peptide clearance receptor, NPR-C, which activates phospholipase C (PLC)-β3 via the βγ-subunits of Gi-1 and Gi-2, inhibited the PLC-β response to somatostatin and [d-Pen2,5]enkephalin. The inhibition was partly reversed by calphostin C. Short-term activation of PKC had no effect on receptor binding or effector enzyme (adenylyl cyclase or PLC-β) activity. We conclude that selective phosphorylation of Gαi-1 and Gαi-2 by PKC partly accounts for heterologous desensitization of responses mediated by the α- and βγ-subunits of both G proteins. The desensitization reflects a decrease in reassociation and thus availability of heterotrimeric G proteins.

Agonist-induced redistribution of calponin in contractile vascular smooth muscle cells

1994 ◽  
Vol 267 (5) ◽  
pp. C1262-C1270 ◽  
Author(s):  
C. A. Parker ◽  
K. Takahashi ◽  
T. Tao ◽  
K. G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Single Cells ◽  
Physiological Saline ◽  
Smooth Muscle Contraction ◽  
Cellular Distribution ◽  
Resting Cells ◽  
Kinase C ◽  
Physiological Saline Solution ◽  
Dependent Pathway ◽  
Activate Protein Kinase

Calponin is a thin filament-associated protein that has been implicated in playing an auxiliary regulatory role in smooth muscle contraction. We have used immunofluorescence and digital imaging microscopy to determine the cellular distribution of calponin in single cells freshly isolated from the ferret portal vein. In resting cells calponin is distributed throughout the cytosol, associated with filamentous structures, and is excluded from the nuclear area of the cell. The ratio of surface cortex-associated calponin to cytosol-associated calponin (R) was found to be 0.639 +/- 0.021. Upon depolarization of the cell with physiological saline solution containing 96 mM K+, the distribution of calponin did not change from that of a resting cell (R = 0.678 +/- 0.025, P = 0.369). Upon stimulation with an agonist (10 microM phenylephrine) that is known to activate protein kinase C (PKC) in these cells, the cellular distribution of calponin changed from primarily cytosolic to primarily surface cortex associated (R = 1.24 +/- 0.085, P < 0.001). This agonist-induced redistribution of calponin was partially inhibited by the PKC inhibitor calphostin, overlapped in time with PKC translocation, and preceded contraction of these cells. These results suggest that the physiological function of calponin may be to mediate agonist-activated contraction via a PKC-dependent pathway.

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