Mechanisms of action of endothelin 1 in maturing rabbit airway smooth muscle

1991 ◽  
Vol 260 (6) ◽  
pp. L434-L443 ◽  
Author(s):  
M. M. Grunstein ◽  
S. M. Rosenberg ◽  
C. M. Schramm ◽  
N. A. Pawlowski
Keyword(s):  
Smooth Muscle ◽  
Mechanisms Of Action ◽  
Maximal Response ◽  
Endothelin 1 ◽  
Kinase C ◽  
Adult Tissues ◽  
Age Dependent ◽  
Dose Dependent ◽  
Higher Doses ◽  
Pkc Activation

Maturational differences in the effects and mechanisms of action of endothelin 1 (ET-1) on airway contractility were investigated in tracheal smooth muscle (TSM) segments isolated from 2-wk-old and adult rabbits. In TSM under passive tension, ET-1 elicited dose-dependent contractions, with a potency of action that was significantly greater (P less than 0.001) in the 2-wk-old vs. adult tissues (i.e., mean +/- SE - log 50% of maximal response values: 8.59 +/- 0.17 vs. 7.79 +/- 0.15 - log M, respectively). In TSM half-maximally contracted with acetylcholine (ACh), however, ET-1 elicited dual and opposing dose-dependent effects. At lower doses (less than or equal to 10(-9) M), ET-1 induced TSM relaxation that was significantly greater in the adult vs. 2-wk-old TSM segments (i.e., approximately 100 vs. 26.5% decrease in active tension, respectively). The relaxant responses were associated with significantly enhanced (P less than 0.001) ET-1-induced release of prostaglandins E2 and I2 in the adult tissues. At higher doses (greater than 10(-9) M), ET-1 induced TSM contractions that were 1) attenuated to a relatively greater extent by the Ca2+ channel blocker, nifedipine (10(-5) M) in the 2-wk-old tissues and 2) associated with significantly (P less than 0.001) enhanced ET-1-stimulated accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the immature TSM. Moreover, the TSM contractions were inhibited by the protein kinase C (PKC) antagonist, H-7, and the latter effect was more potent in the immature TSM. Collectively, these findings demonstrate that ET-1 exerts a potent duality of action in rabbit TSM which varies significantly with maturation, wherein 1) age-dependent differences in airway relaxation are associated with changes in the evoked release of bronchodilatory prostaglandins and 2) maturational differences in airway contraction are associated with changes in Ins(1,4,5)P3 accumulation and extracellular Ca2+ mobilization, coupled to differences in PKC activation.

Role of protein kinase C in nonsensitized and passively sensitized human isolated bronchial smooth muscle

1995 ◽  
Vol 268 (6) ◽  
pp. L966-L971 ◽  
Author(s):  
M. Rossetti ◽  
J. P. Savineau ◽  
H. Crevel ◽  
R. Marthan
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Maximal Response ◽  
Bronchial Smooth Muscle ◽  
Kinase C ◽  
Passive Sensitization ◽  
Pkc Activation

To examine the role of protein kinase C (PKC) activation in the control of the mechanical activity of human isolated bronchial smooth muscle obtained at thoracotomy, the effect of the phorbol ester phorbol 12,13-dibutyrate (PDB) was evaluated. PDB produced slowly developing and sustained contractions that were reduced 1) by the PKC inhibitor staurosporine and 2) after long-term (12 h) exposure to PDB, which downregulates PKC. Moreover, the inactive phorbol ester 4 alpha-phorbol 12,13 didecanoate had no contractile effect. Removal of external Ca2+ or addition of the Ca(2+)-channel antagonist verapamil reduced the PDB-induced contraction. Passive sensitization of human isolated bronchial rings, i.e., incubation overnight of tissues in serum from atopic asthmatic patients, decreased the maximal response to PDB to 28.9 +/- 8% of the maximal response to acetylcholine (ACh) when compared with that of paired nonsensitized rings, i.e., tissues incubated overnight in serum from normal subjects (46.7 +/- 9.4% of the maximal response to ACh, n = 5, P < 0.05). The decrease in the response to PDB induced by either long-term preexposure to PDB or passive sensitization was reversed when both types of tissues were allowed to recover unstimulated for 3 h before PDB application. These results show that 1) PKC activation induces maintained contractions in human isolated airway smooth muscle that are largely dependent on extracellular calcium; 2) passive sensitization alters the PKC-mediated response in a way similar to that induced by prolonged stimulation of PKC.

Human vascular smooth muscle responses mediated by α2 mechanisms in vivo and in vitro

1985 ◽  
Vol 68 (s10) ◽  
pp. 147s-150s ◽  
Author(s):  
S. Thom ◽  
J. Calvete ◽  
R. Hayes ◽  
G. Martin ◽  
P. Sever
Keyword(s):  
Smooth Muscle ◽  
Physiological Role ◽  
Smooth Muscle Contraction ◽  
Α1 Receptors ◽  
Dose Dependent ◽  
Higher Doses ◽  
Α2 Receptors ◽  
Autoregulatory Mechanism

1. The effects of compounds with α2-agonist and α2-antagonist properties on human forearm blood flow and on isolated human arterial segments have been studied. 2. The findings from these studies in vivo and in vitro did not provide evidence in support of the hypothesis that postsynaptic α2-receptors mediate smooth muscle contraction in the tissues under investigation. 3. The constriction of the forearm vascular bed in response to low intra-arterial doses of idazoxan (RX 781094), an α2-antagonist, provides evidence for a physiological role for a presynaptic α2 autoregulatory mechanism. 4. The variability of the forearm vascular responses to higher doses of idazoxan highlights the pitfalls that may have misled previous authors in their interpretation of the results of similar studies. A U-shaped dose-response curve to compounds with mixed α2-and α1-antagonist properties may be constructed, which emphasizes the importance of the dose-dependent selectivity of these antagonists at α2- and α1-receptors. 5. The effect of idazoxan on the responses of arterial segments in vitro to exogenous catecholamines was dependent on the integrity of the endothelium, and provides evidence that α2-receptors may mediate release of the endothelium-derived relaxing factor.

Activation of multiple mechanisms including phospholipase D by endothelin-1 in rat aorta

1992 ◽  
Vol 262 (4) ◽  
pp. C941-C949 ◽  
Author(s):  
Y. Liu ◽  
B. Geisbuhler ◽  
A. W. Jones
Keyword(s):  
Phospholipase D ◽  
Channel Blocker ◽  
Platelet Activating Factor ◽  
Rat Aorta ◽  
Cellular Mechanisms ◽  
Endothelin 1 ◽  
Kinase C ◽  
Arachidonate Cascade ◽  
Alpha Response ◽  
Pkc Activation

This study investigated the cellular mechanisms underlying the endothelin-1 (ET-1)-induced contraction of rat aorta with focus on the involvement of phospholipase D (PLD). Preincubating rat aorta in Ca(2+)-free solution reduced the contraction by 80%, whereas diltiazem (10 microM), a voltage-operated Ca2+ channel blocker, caused only a small reduction (27%, P less than 0.05) of the contraction. In myo-[3H]inositol-labeled aorta, ET-1 stimulated the formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate, indicating the activation of phospholipase C (PLC). In aorta labeled with 32PO4, [3H] myristic acid or [32P]lyso-platelet-activating factor followed by exposure to ethanol (0.5%), ET-1 stimulated phosphatidylethanol (PEt) production, suggesting that ET-1 activates PLD. The PEt response was not attenuated by staurosporine (ST, 0.1 microM), an inhibitor of protein kinase C (PKC) but was inhibited by removal of Ca2+. The ET-1-induced PEt response was at least additive to that induced by phorbol 12-myristate 13-acetate (1 microM). ET-1 also stimulated the release of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) into the tissue medium. Unlike the PEt responses, the 6-keto-PGF1 alpha response could be inhibited by ST. Removal of Ca2+ abolished the response. These results suggest that 1) ET-1 activates multiple cellular mechanisms including PLC, PLD, and the arachidonate cascade; 2) PKC activation may not be essential for the ET-1 activation of PLD but may play an important role in the ET-1 stimulation of 6-keto-PGF1 alpha release; and 3) Ca2+ is an important factor in the ET-1-induced PLD activity and 6-keto-PGF1 alpha release.

PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase

1999 ◽  
Vol 277 (3) ◽  
pp. C554-C562 ◽  
Author(s):  
J. R. Turner ◽  
J. M. Angle ◽  
E. D. Black ◽  
J. L. Joyal ◽  
D. B. Sacks ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Myosin Ii ◽  
Transepithelial Resistance ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
H 51 ◽  
Mlc Phosphorylation ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Pkc Activation

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 ( Gastrointest. Liver Physiol. 29): G214–G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 ( Cell Physiol. 42): C1378–C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.

scholarly journals Protein Kinase C-ε Induces Caveolin-Dependent Internalization of Vascular Adenosine 5′-Triphosphate–Sensitive K + Channels

Hypertension ◽  
2008 ◽  
Vol 52 (3) ◽  
pp. 499-506 ◽  
Author(s):  
Jundong Jiao ◽  
Vivek Garg ◽  
Baofeng Yang ◽  
Terry S. Elton ◽  
Keli Hu
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Cell Surface ◽  
Small Interfering Rna ◽  
Caveolin 1 ◽  
Kinase C ◽  
Interfering Rna ◽  
Pkc Activation

Vascular ATP-sensitive K + (K ATP ) channels are critical regulators of arterial tone and, thus, blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is known that protein kinase C (PKC) activation inhibits K ATP channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Here we report that caveolin-dependent internalization is involved in PKC-ε–mediated inhibition of vascular K ATP channels (Kir6.1 and SUR2B) by phorbol 12-myristate 13-acetate or angiotensin II in human embryonic kidney 293 cells and immortalized human saphenous vein vascular smooth muscle cells. We showed that Kir6.1 substantially overlapped with caveolin-1 at the cell surface. Cholesterol depletion with methyl-β-cyclodextrin significantly reduced, whereas overexpression of caveolin-1 largely enhanced, PKC-induced inhibition of Kir6.1/SUR2B currents. Importantly, we demonstrated that activation of PKC-ε caused internalization of K ATP channels, the effect that was blocked by depletion of cholesterol with methyl-β-cyclodextrin, expression of dominant-negative dynamin mutant K44E, or knockdown of caveolin-1 with small interfering RNA. Moreover, patch-clamp studies revealed that PKC-ε–mediated inhibition of the K ATP current induced by PMA or angiotensin II was reduced by a dynamin mutant, as well as small interfering RNA targeting caveolin-1. The reduction in the number of plasma membrane K ATP channels by PKC activation was further confirmed by cell surface biotinylation. These studies identify a novel mechanism by which the levels of vascular K ATP channels could be rapidly downregulated by internalization. This finding provides a novel mechanistic insight into how K ATP channels are regulated in vascular smooth muscle cells.

scholarly journals Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3378-3384 ◽  
Author(s):  
Beatriz Bellosillo ◽  
Mireia Dalmau ◽  
Dolors Colomer ◽  
Joan Gil
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent ◽  
Pkc Activation

Abstract B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived B lymphocytes that express high levels of Bcl-2. We examined the involvement of CED-3/ICE-like proteases in the apoptosis of B-CLL cells. One of the substrates of these proteases is poly(ADP [adenosine 5′-diphosphate]-ribose) polymerase (PARP). The effect of different factors that induce the apoptosis of B-CLL cells on the proteolytic cleavage of PARP has been studied. Treatment of B-CLL cells with different concentrations of dexamethasone (1 to 1,000 μmol/L) induced in a dose-dependent manner the cleavage of PARP. Dexamethasone induced PARP cleavage after 12 hours of incubation, which was almost complete at 48 hours. PARP cleavage during apoptosis of B-CLL cells was studied in cells from eight patients and a correlation was found between cell viability and the degree of PARP cleavage. Incubation in vitro of B-CLL cells with fludarabine for 48 hours induced PARP cleavage in all the cases studied. Protein kinase C (PKC) activation with 100 nmol/L TPA (12-O-tetradecanoylphorbol 13-acetate) or incubation with interleukin-4 (10 ng/mL) prevented either dexamethasone- or fludarabine-induced proteolysis of PARP. Incubation of B-CLL cells with the CED-3/ICE–like protease inhibitor Z-VAD.fmk inhibited spontaneous and dexamethasone-induced PARP cleavage and DNA fragmentation in a dose-dependent manner. Furthermore, Z-VAD.fmk prevented the cytotoxic effect of dexamethasone. These results indicate that CED-3/ICE–like proteases play an important role in the apoptosis of B-CLL cells.

Modulation of ventricular action potential by alpha 1-adrenoceptors and protein kinase C

1990 ◽  
Vol 258 (3) ◽  
pp. H907-H911 ◽  
Author(s):  
R. T. Dirksen ◽  
S. S. Sheu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Guinea Pig ◽  
Action Potential ◽  
Kinase C ◽  
Adrenoceptor Agonist ◽  
Lower Temperature ◽  
Cardiac Excitation ◽  
Dose Dependent ◽  
Pkc Activation

The effects of the alpha 1-adrenoceptor agonist methoxamine (MTX) and the direct protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBU) on action potentials from guinea pig papillary muscles were studied. Measured with conventional microelectrodes, MTX (1 x 10(-7) to 3 x 10(-4) M) and PDBU (1 x 10(-9) to 1 x 10(-6) M) both caused a dose-dependent and reversible decrease in the action potential duration measured at 90% repolarization (APD90) at 36.5 degrees C. The MTX-mediated response was blocked by both prazosin (3 x 10(-6) M) and phentolamine (1 x 10(-6) M) and mimicked by phenylephrine. Maximal concentrations of the two agents together resulted in only a partial (50%) additive decrease in the APD90. At a lower temperature (27.5 degrees C), PDBU no longer produced a shortening in the APD90 and MTX produced a prolongation in the APD90. These results demonstrate that although alpha 1-adrenoceptor stimulation and PKC activation in guinea pig papillary muscle both lead to a decrease in the APD90, the differences in their effects as related to the magnitude, partial additivity, and temperature dependence suggest that the mechanism of action are not identical. These subtle differences may help to delineate the exact physiological implications of alpha 1-adrenoceptors in cardiac excitation and contraction.

scholarly journals Ganglioside GQ1b-induced terminal differentiation in cultured mouse keratinocytes. Phosphoinositide turnover forms the onset signal

1991 ◽  
Vol 279 (3) ◽  
pp. 665-670 ◽  
Author(s):  
Y Yada ◽  
Y Okano ◽  
Y Nozawa
Keyword(s):  
Terminal Differentiation ◽  
Keratinocyte Differentiation ◽  
Dependent Manner ◽  
Transglutaminase Activity ◽  
Phosphoinositide Turnover ◽  
Kinase C ◽  
Pre Treatment ◽  
Mouse Keratinocytes ◽  
Dose Dependent ◽  
Pkc Activation

Investigations were undertaken to see whether mouse keratinocyte differentiation was elicited by gangliosides. Among the gangliosides tested only GQ1b, a tetrasialoganglioside containing two disialosyl residues, induced keratinocyte differentiation, as indicated by the formation of cornified envelopes, enhancement of transglutaminase activity and suppression of DNA synthesis. Upon stimulation with GQ1b the mass content of Ins(1,4,5)P3 and the intracellular Ca2+ levels were markedly enhanced in a time- and dose-dependent manner, whereas no significant changes were observed with other gangliosides, thereby indicating activation of phospholipase C for the onset of keratinocyte differentiation. Furthermore, only GQ1b promoted the translocation of protein kinase C (PKC) from cytosol to membrane. Inhibition of PKC with H-7 or down-regulation of the enzyme by prolonged pre-treatment with phorbol 12,13-dibutyrate greatly suppressed transglutaminase activity and formation of cornified envelopes induced by GQ1b. These results demonstrate that the tetrasialoganglioside GQ1b generates the initial differentiation signal in mouse keratinocytes through phosphoinositide turnover, and also suggest that PKC activation may act at certain, as yet unidentified, stages of differentiation processes.

Sevoflurane Inhibits Angiotensin II–induced, Protein Kinase C–mediated but Not Ca2+-elicited Contraction of Rat Aortic Smooth Muscle

2004 ◽  
Vol 100 (4) ◽  
pp. 879-884 ◽  
Author(s):  
Jingui Yu ◽  
Yasuyuki Tokinaga ◽  
Koji Ogawa ◽  
Shizue Iwahashi ◽  
Yoshio Hatano
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Isometric Force ◽  
Force Transducer ◽  
Aortic Smooth Muscle ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Dose Dependent

Background Whether volatile anesthetics attenuate angiotensin II-mediated vascular tone has not been determined. The current study was designed to investigate the effects of sevoflurane on the angiotensin II-stimulated, Ca2+- and protein kinase C (PKC)-mediated contraction of rat aortic smooth muscle. Methods The dose-dependent effects of sevoflurane on angiotensin II (10 m)-induced contraction, the increase in intracellular Ca2+ concentration, and PKC phosphorylation of rat aortic smooth muscle were measured using an isometric force transducer, a fluorometer, and Western blotting, respectively. Results Angiotensin II induced a transient increase in intracellular Ca2+ concentration, phosphorylation of Ca2+-dependent PKC (cPKC)-alpha, and consequently, a transient contraction of rat aortic smooth muscle. Phosphorylation of the Ca2+-independent PKC-epsilon was not detected. The angiotensin II-induced contraction was almost completely abolished by removing extracellular Ca2+ and was significantly inhibited by the selective cPKC inhibitor Gö 6976 (10 M) but was not inhibited by the nonselective PKC inhibitor Ro 31-8425 (10 M). Sevoflurane dose-dependently inhibited the angiotensin II-induced contraction, with reductions of 14.2 +/- 5.2% (P &gt; 0.05), 26.7 +/- 8.9% (P &lt; 0.05), and 38.5 +/- 12.8% (P &lt; 0.01) (n = 10) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively. The angiotensin II-elicited increase in intracellular Ca2+ concentration was not significantly influenced by 3.4, 5.1, or 8.5% sevoflurane. However, cPKC-alpha phosphorylation induced by angiotensin II was inhibited dose dependently by 1.7, 3.4, and 5.1% sevoflurane, with depressions of 20.5 +/- 14.2% (P &gt; 0.05), 37.0 +/- 17.8% (P &lt; 0.05), and 62.5 +/- 12.2% (P &lt; 0.01) (n = 4), respectively. Conclusion The current study indicates that Ca2+ and cPKC-alpha are involved in angiotensin II-induced vascular contraction. Sevoflurane dose-dependently inhibited the angiotensin II-stimulated, cPKC-mediated but not Ca2+-elicited contraction of rat aortic smooth muscle.

Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1295-C1301 ◽  
Author(s):  
K. Janakidevi ◽  
M. A. Fisher ◽  
P. J. Del Vecchio ◽  
C. Tiruppathi ◽  
J. Figge ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Dna Synthesis ◽  
Synthetic Activity ◽  
Proliferative Potential ◽  
Endothelin 1 ◽  
Kinase C

Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.

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