Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells

doi:10.1016/0929-7855(96)00520-2

Journal of Lipid Mediators and Cell Signalling

Volume 14, Issues 1–3, September 1996, Pages 147-155

Journal of Lipid Med...

https://doi.org/10.1016/0929-7855(96)00520-2 Get rights and content

Abstract

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A₂ (PLA₂), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol triphosphate (IP₃), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP₃ production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC₅₀ = 1.2 nM) than in activating PLC (EC₅₀ = 1.5 nM) or PLA₂ (EC₅₀ = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA₂ was stimulated first ( $t_{12} = 12 s$ ), followed by PLC ( $t_{12} = 48 s$ ) and lastly PLD ( $t_{12} = 106 s$ ). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ET_B receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ET_A receptor subtype to activate, via G proteins, phospholipases A₂, C and D in a temporal sequence.

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Cited by (8)

Endothelin-induced prostacyclin production in rat aortic endothelial cells: Role of calcium
1999, Prostaglandins Leukotrienes and Essential Fatty Acids
Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca²⁺) in ET-1-induced prostacyclin (PGI₂) release. ET-1 (10⁻9 M) produced a significant increase in PGI₂release. Pretreatment of rat aortic endothelial cells with different doses (10⁻9 M and 10⁻⁶M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI₂release. Inhibition was first noted at 10⁻⁹M and was complete at 10⁻⁶M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10⁻⁹M and 10⁻⁶M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI₂release. These results provide further support for the concept that PKC mediates ET-induced PGI₂release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.
Endothelin induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C
1999, Prostaglandins Leukotrienes and Essential Fatty Acids
Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI₂) by radioimmunoassay. ET (10⁻⁹M) produced a 10-fold increase in PGI₂release. Pretreatment with 10⁻⁹M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI₂release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A₂(7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10⁻⁹M) or cyclooxygenase (indomethacin) (10–9M). We conclude that ET activates PKC which activates phospholipase A₂which liberates arachidonic acid which increases PGI₂production and release.
Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells
1998, Life Sciences
We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC₅₀ = 98nM), which was 2.5 times higher than that of ET-1. The ET_B receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET_B receptor antagonist BQ 788, but not the ET_A receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC₅₀ = 10nM). The phorbol ester, Phorbol 12, 13 — dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC₅₀ = 66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca²⁺ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca²⁺ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET_B receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca²⁺ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca²⁺-mobilizing agonists in the iris sphincter smooth muscle.
Role of protein kinase C α in endothelin-1 stimulation of cytosolic phospholipase A<inf>2</inf> and arachidonic acid release in cultured cat iris sphincter smooth muscle cells
1998, Biochimica et Biophysica Acta - Lipids and Lipid Metabolism
We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC₅₀=8 nM) and time-dependent (t_1/2=1.2 min) manner. Cytosolic phospholipase A₂ (cPLA₂), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF₃, quinacrine and manoalide, PLA₂ inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCα and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. Gö-6976, a compound that inhibits PKCα and β specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC₅₀ value of 8 nM. Thymeatoxin (0.1 μM), a specific activator of PKCα, β, and γ induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCα, but not PKCβ, from cytosol to the particulate fraction. These results suggest that PKCα plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA₂, p42^mapk and p44^mapk in the CISM cells. The data presented are consistent with a role for PKCα, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA₂ activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA₂ phosphorylation and cPLA₂ activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCα, which activates cPLA₂, which liberates AA for prostaglandin synthesis.
Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in cultured human ciliary muscle cells: Activation of phospholipase A<inf>2</inf>
1997, Experimental Eye Research
In the present study we have examined the effects and mechanisms of endothelin-1 (ET-1) on arachidonic acid (AA) release and prostaglandin (PG) synthesis in human ciliary muscle (HCM) cells. ET-1 stimulated AA release in a time (t_1/2=1.5 min) and concentration-dependent (EC₅₀=5 nM) manner, which is primarily mediated through the ET_Areceptor subtype. The AA liberated by ET-1 appears to derive mainly from the phosphoinositides and phosphatidylcholine. Our data show that phospholipase A₂(PLA₂), but not phospholipase C (PLC), plays an important role in ET-1-induced AA release. This conclusion is supported by the following findings: (1) ET-1-evoked AA release was inhibited by the PLA₂inhibitors dexamethasone, mepacrine and manoalide in a concentration-dependent manner. Conversion of AA into PGE₂was inhibited by the cyclooxygenase inhibitors in the following order: Indomethacin>naproxen >ibuprofen>NS-398>aspirin. (2) The phorbol ester, PDBu, an activator of protein kinase C, potentiated ET-1-induced AA release by 39%, but inhibited that of inositol phosphates formation by 62%. (3) Pretreatment of the labeled cells with isoproterenol lowered ET-1-induced inositol phosphates production, but had no effect on AA release. (4) U71322, a PLC inhibitor, inhibited ET-1-induced inositol phosphates production, but had no effect on that of AA release. (5) Pretreatment of the cells with pertussis toxin (0.1 μg ml⁻¹) attenuated the stimulatory effects of ET-1 on AA release and PGE₂formation. These data demonstrate that ET-1 is a potent agonist for AA release and PG synthesis in HCM cells, and that PLA₂, but not PLC, plays an important role in ET-1-induced AA release and PG synthesis. In ciliary muscle, AA and its metabolites play important roles in intracellular signalling, modulation of physiological processes, and regulation of intraocular pressure.
Mediation by prostaglandins of the stimulatory effect of substance P on cyclic AMP production in dog iris sphincter smooth muscle
1996, Biochemical Pharmacology
The purpose of the present study was to examine the mechanism of the stimulatory effect of substance P (SP) on cyclic AMP (cAMP) accumulation in dog iris sphincter. We found that: (1) SP increased cAMP accumulation in a time- and concentration-dependent manner, the $T_{12}$ and ec₅₀ values being 1.2 min and 44 nM, respectively. SP has no effect on inositol trisphosphate and muscle contraction in this tissue. (2) SP-stimulated cAMP formation was inhibited by quinacrine, a non-specific phospholipase A₂ inhibitor (ic₅₀ = 9.5 μM), and by indomethacin (Indo), a cyclooxygenase inhibitor (ic₅₀ = 3.5 nM), in a concentration-dependent manner, suggesting that SP induces cAMP accumulation via an Indo-sensitive pathway. (3) SP-induced arachidonic acid release and SP-induced prostaglandin E₂ (PGE₂) release were inhibited concentration dependently by quinacrine and Indo, with ic₅₀ values of 11 μM and 0.8 nM, respectively. (4) PGE₂ (1 μM) increased cAMP formation in the sphincter muscle by 94%, and, furthermore, the PG, but not SP, stimulated the activity of adenylyl cyclase in membrane fractions isolated from this tissue. (5) Indo (1 μM) blocked the relaxing effect of SP (1 μM) in iris sphincter precontracted with carbachol (1 μM). (6) The inhibitory effect of Indo on SP-induced cAMP accumulation was species specific. Increases in cAMP represent a mechanism by which extracellular SP can regulate smooth muscle function. Thus, we conclude from these studies that in dog iris sphincter SP-induced cAMP accumulation is mediated through PGs, and that in this cholinergically innervated muscle SP via cAMP could function, in part, to modulate the physiological responses to muscarinic receptor stimulation. BIOCHEM PHARMACOL 52;8:1261–1269, 1996.

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Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells

Abstract

J. Biol. Chem.

Exp. Eye Res.

Biochem. Biophys. Res. Commun.

Cell Signal.

Species differences in the effects of endothelin-1 on myo-inositol trisphosphate accumulation, cAMP formation and contraction of isolated iris sphincter of rabbit and other species

Invest. Ophthalmol. Vis. Sci.

Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle; activation of phospholipase A2

Curr. Eye Res.

Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle; activation of phospholipase A₂