Cat Iris Sphincter Smooth Muscle Research Articles

Cat Iris Sphincter Smooth-Muscle Contraction: Comparison of FP-Class Prostaglandin Analog Agonist Activities

The pharmacologic characteristics of a number of FP-class prostaglandin (PG) analogs were determined by using the cat iris sphincter smooth-muscle-contraction assay. Cumulative concentration-response curves were generated for each compound. The relative agonist potencies (EC(50)) of the compounds were: cloprostenol (0.0012 +/- 0.0004 nM) >> travoprost acid (0.46 +/- 0.13 nM) = bimatoprost acid (0.99 +/- 0.19 nM) > (+/-)-fluprostenol (15.8 +/- 2.6 nM) = PGF(2alpha) (18.6 +/- 1.8 nM) > latanoprost acid (29.9 +/- 1.6 nM) > bimatoprost (140 +/- 45 nM) > S-1033 (588 +/- 39 nM) > unoprostone (UF-021; 1280 +/- 50 nM; n = 4-14). The maximum response induced by travoprost acid (122% +/- 2.3% maximum response relative to PGF(2alpha)) was significantly greater than that induced by all the other PG compounds (P < 0.001 - P < 0.02). Interestingly, the FP-receptor antagonist, AL-8810, behaved as a moderate efficacy partial agonist (EC(50) = 2140 +/- 190 nM; 63 +/- 4.3% maximum response relative to PGF(2alpha)), indicating that the cat iris contains an extremely well-coupled FP-receptor population, and/or the tissue contains an extremely high density of the FP-receptor and/or spare receptors. The cat iris contraction data were well correlated with other FP-receptor-mediated signal-transduction processes, including FP-receptor binding in bovine corpus luteum (r = 0.86), FP-receptor binding in human iris (r = 0.61), phosphoinositide (PI) hydrolysis in human ciliary muscle and trabecular meshwork cells (r = 0.77 - 0.86), PI turnover in rat and mouse cells (r = 0.73 - 0.76) and via cloned human FP-receptor (r = 0.9), and rat uterus contraction (r = 0.84). These data confirm the presence of functional FP-receptors in the cat iris sphincter, which are exquisitely well coupled and which respond to a variety of FP-class PG analogs with differing potencies.

Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle

There is evidence from our own laboratory and that of others that EP-receptor ligands are strong contractile agonists in bovine iris sphincter and that FP-receptor agonists are strong contractile agonists in cat iris sphincter. Here, we have investigated the effects of prostaglandin (PG) receptor agonists of the FP-, EP-, TP- and DP-class on myosin light chain (MLC) phosphorylation, p42/p44 MAP kinase phosphorylation and contraction in the iris sphincter of bovine and cat. Using three signal transduction mechanism assays, namely MLC phosphorylation, MAP kinase phosphorylation and contraction, we demonstrated that in bovine iris sphincter the rank order of potency of the PG agonists in the contractile and MLC phosphorylation assays is as follows: E2>U46619>F2alpha>D2, and in cat F2alpha>D2>E2>U46619. In the MAP kinase assay, in bovine iris sphincter the rank order of potency is E2>F2alpha and in cat F2alpha>E2. These conclusions are supported by the following findings: (1) In the contractile assay, in the bovine sphincter the EC50s for PGF2alpha, PGE2, U46619 and PGD2 were found to be 1.4x10(-7), 5.0x10(-9), 9.0x10(-9) and 1.3x10(-6)M, respectively, and the corresponding values in the cat were 1.9x10(-8), 2.3x10(-7), 1.5x10(-6) and 6.9x10(-8)M, respectively. (2) In the MLC phophorylation assay, in the bovine sphincter PGF2alpha, PGE2, U46619 and PGD2 increased MLC phophorylation by 118%, 165%, 153% and 72%, respectively, and the corresponding values in cat were 175%, 99%, 90% and 95%, respectively. (3) In the MAP kinase assay, in the bovine iris sphincter PGF2alpha and PGE2, increased MAP kinase phosphorylation by 276% and 328%, respectively, and the corresponding values in cat were 308% and 245%, respectively. The data presented demonstrate pronounced species differences in the effects of the prostanoids on the MLC kinase signaling pathway in bovine and cat irides and furthermore confirm the existence of FP-receptors in that of the bovine.

Prostaglandin F 2α stimulates tyrosine phosphorylation of phospholipase C-γ1

In this study, we investigated the ability of prostaglandin F 2α (PGF 2α) to induce tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) in cat iris sphincter smooth muscle (CISM) cells. PGF 2 α (1 μM) stimulated PLC-γ1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5 min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF 2α, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF 2α. Furthermore, PGF 2α-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF 2α stimulates tyrosine phosphorylation of PLC-γ1 in CISM cells and indicate that PGF 2α-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.

Ocular hypotensive activity of prostaglandin F2 alpha (PGF2 alpha) analogs with neutral substituents at position 1 is predicted by the isolated cat iris sphincter smooth muscle preparation but not Ca+ signalling in Swiss 3T3 cells.

Typically, the C-1 carboxylic acid (CO2H) has been considered an essential pharmacophoric group for ligand binding to the FP-receptor. Historically, in drug design and development, a formal negatively charged carboxylic acid can be replaced with a group of similar pKa, such as tetrazole or an acyl sulfonamide. This change sometimes provides better selectivity and/or increased potency due to better metabolic stability. We investigated replacement of the C-1 carboxylic acid with a variety of uncharged C-1 substituents. A diverse series of Cl-analogs was synthesized and certain compounds, especially AGN-191129 (PGF2aOCH3), exhibited an apparent unique pharmacology, thus confiming the importance of the Cl-CO2H for binding to the FP-receptor.KeywordsIntraocular PressureRadioligand Binding StudyHuman RecombinantProstanoid ReceptorDiverse SeriesThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effects of Prostaglandin F2αand Carbachol on MAP Kinases, Cytosolic Phospholipase A2and Arachidonic Acid Release in Cat Iris Sphincter Smooth Muscle Cells

The signal transduction pathways initiated by Ca2+-mobilizing agonists, such as prostaglandin F2α(PGF2α) and carbachol (CCh), leading to activation of cytosolic phospholipase A2(cPLA2) and arachidonic acid (AA) release in a wide variety of tissues remain obscure. To further define the role of protein kinases in receptor mediated stimulation of cPLA2and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF2α- and CCh-induced cPLA2phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [3H]AA for 24hr and incubated in the absence or presence of the agonist for 5–10min as indicated. MAP kinases activities and cPLA2phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA2antibodies. We found that: (a) PGF2αand CCh increased p38 MAP kinase activity by 197 and 215%, respectively, and increased p42/p44 MAP kinase activity by 200 and 125%, respectively. (b) SB202190, a p38 MAP kinase specific inhibitor, inhibited PGF2α- and CCh-induced cPLA2phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA2phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF2α. (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF2α- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA2phosphorylation. We conclude from these results that in CISM cells PGF2α-induced cPLA2phosphorylation and AA release is mediated through p38 MAP kinase, but not through p42/p44 MAP kinases, whereas that of CCh is mediated through both p38 MAP kinase and p42/p44 MAP kinases. These effects of PGF2αand CCh are regulated by the MAP kinases in a PKC-dependent manner. Studies aimed at elucidating the role of protein kinases in the coupling mechanism between the activation of PGF2αand muscarinic receptors, and the stimulation of cPLA2and AA release in the smooth muscles of the iris-ciliary body will provide important information about the role of protein kinases signaling pathways in smooth muscle function, as well as about the mechanism of the intraocular pressure-lowering effects of PGF2αand its analog, latanoprost, in glaucoma therapy.

Mitogen-activated protein kinase inhibitors suppress prostaglandin F 2α-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F 2α, latanoprost, a prostaglandin F 2α analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2′-Amino-3′-methoxyflavone (PD98059) (10 μM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F 2α- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F 2α increased MAP kinase phosphorylation in a concentration-dependent manner with EC 50 value of 1.1×10 −8 M and increased contraction with EC 50 of 0.92×10 −9 M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1,4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F 2α-induced contraction in a concentration-dependent manner with IC 50 values of 2.4, 3.0 and 4.8 μM, respectively. PD98059 had no effect on prostaglandin F 2α- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP 3) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F 2α-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-( N-(4-Chloro-cinnamyl)- N-methylaminomethyl)phenyl]- N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 μM), a Ca 2+-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 μM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F 2α- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in the mechanism of prostaglandin F 2α-, but not in that of carbachol, induced contraction. In addition, these data clearly indicate that the stimulation of the iris sphincter with prostaglandin F 2α and carbachol activate two distinct pathways, the MAP kinase pathway and the Ca 2+ mobilization pathway.

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- t 1/2 = 3.5 min and concentration- EC 50 = 200 nM dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1.6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18.9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM 26-52, but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC 50 value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Giα antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.

Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

Ocular hypotensive activity of prostaglandin F 2α (PGF 2α) analogs with neutral substituents at position 1 is predicted by the elated cat iris sphincter smooth muscle preparation but not Ca 2+ signaling in Swiss 3T3 cells

Ocular hypotensive activity of prostaglandin F 2α (PGF 2α) analogs with neutral substituents at position 1 is predicted by the elated cat iris sphincter smooth muscle preparation but not Ca 2+ signaling in Swiss 3T3 cells

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells

We have shown previously that cytosolic phospholipase A2 (cPLA2) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA2 in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA2 phosphorylation, cPLA2 activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA2. Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [3H]arachidonic acid release and cPLA2 activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA2. Activation of cPLA2 leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.

Atrial Natriuretic Peptide Provokes a Dramatic Increase in Cyclic GMP Formation and Markedly Inhibits Muscarinic-Stimulated Ca 2+ Mobilisation in SV-40 Transformed Cat Iris Sphincter Smooth Muscle (SV-CISM-2) Cells

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca 2+] i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca 2+] i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 μM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca 2+] i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca 2+] i mobilisation with ic 50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with ec 50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>>CNP>>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant ( K d) and total receptor density ( B max) values of the natriuretic peptide receptor for [ 125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic responses in the iris sphincter smooth muscle cells, and SV-CISM-2 cells can serve as an excellent model for investigating the cross talk between cGMP and the Ca 2+ signalling system.

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

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 (EC 50 = 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 (IC 50 = 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 (IC 50 = 66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca 2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca 2+ 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 Ca 2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca 2+-mobilizing agonists in the iris sphincter smooth muscle.

Tyrosine kinase inhibitors suppress prostaglandin F 2α-induced phosphoinositide hydrolysis, Ca 2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F 2α- and carbachol-induced inositol-1,4,5-trisphosphate (IP 3) production, [Ca 2+] i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F 2α and carbachol induced contraction in a concentration-dependent manner with EC 50 values of 0.92×10 −9 and 1.75×10 −8 M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F 2α, but not those evoked by carbachol, on IP 3 accumulation, [Ca 2+] i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F 2α analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 μM) markedly reduced (by 67%) prostaglandin F 2α-stimulated increase in [Ca 2+] i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC 50 of 82 μM and increased IP 3 generation in a concentration-dependent manner with an EC 50 of 90 μM. The effects of vanadate were abolished by genistein (10 μM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F 2α- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca 2+] i evoked by prostaglandin F 2α. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F 2α receptor activation and increases in intracellular Ca 2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.

Role of protein kinase C α in endothelin-1 stimulation of cytosolic phospholipase A 2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells

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 (EC 50=8 nM) and time-dependent ( t 1/2=1.2 min) manner. Cytosolic phospholipase A 2 (cPLA 2), 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 AACOCF 3, quinacrine and manoalide, PLA 2 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 PKC α 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 PKC α and β specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC 50 value of 8 nM. Thymeatoxin (0.1 μM), a specific activator of PKC α, β, and γ induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKC α, but not PKC β, from cytosol to the particulate fraction. These results suggest that PKC α plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA 2, p42 mapk and p44 mapk in the CISM cells. The data presented are consistent with a role for PKC α, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA 2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA 2 phosphorylation and cPLA 2 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 PKC α, which activates cPLA 2, which liberates AA for prostaglandin synthesis.

Inhibition of Muscarinic-Stimulated Polyphosphoinositide Hydrolysis and Ca 2+ Mobilization in Cat Iris Sphincter Smooth Muscle Cells By cAMP-Elevating Agents

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP 3) production and intracellular calcium ([Ca 2+] i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP 3 production; the t 1/2 and EC 50 values were 68 s and 0.5 μM, respectively. The muscarinic agonist provoked a transient increase in [Ca 2+] i which reached maximum within 77 s, and increased [Ca 2+] i mobilization in a concentration-dependent manner with an EC 50 of 1.4 μM. Thapsigargin, a Ca 2+-pump inhibitor, caused a rapid rise in [Ca 2+] i and subsequent addition of CCh was without effect. Both CCh-induced IP 3 production and CCh- induced [Ca 2+] i mobilization were more potently antagonized by 4-DAMP, an M 3 muscarinic receptor antagonist, than by pirenzepine, an M 1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP 3 production and [Ca 2+] i mobilization. These data indicate close correlation between enhanced IP 3 production and [Ca 2+] i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca 2+] i could be mediated through increased IP 3 production. Isoproterenol (ISO) inhibited CCh-induced IP 3 production (IC 50 = 80 nM) and [Ca 2+] i mobilization (IC 50 = 0.17 μM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 μM) and GTPγS (0.1 μM). Pretreatment of the cells with ISO or forskolin, 5 μM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTPγS-stimulated IP 3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M 3 muscarinic receptors results in stimulation of PLC-mediated PIP 2 hydrolysis, generating IP 3 which mobilizes [Ca 2+] i. Furthermore, elevation of cAMP may inhibit IP 3 production and [Ca 2+] i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP 3 receptor and/or IP 3 metabolizing enzymes.

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

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 A 2 (PLA 2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol triphosphate (IP 3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP 3 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 (EC 50 = 1.2 nM) than in activating PLC (EC 50 = 1.5 nM) or PLA 2 (EC 50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA 2 was stimulated first ( t 1 2 = 12 s ), followed by PLC ( t 1 2 = 48 s ) and lastly PLD ( t 1 2 = 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 A 2, C and D in a temporal sequence.

Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: Growth, morphological, biochemical and pharmacological characteristics

The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-adrenergic, peptidergic (substance P and endothelin). Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphorylation (118%), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as an important model system for investigations on the biochemical and pharmacological properties of receptors and their signal transduction pathways in smooth muscle. The advantage of these cells over normal iris sphincter cells is that they can be propagated over many generations without alterations in their morphological, biochemical and physiological characteristics.