Search

A potential role for myosin light chain kinase (MLCK) in regulating intraocular pressure and outflow function has recently been reported in living monkey eye and rabbit eye. There is little information about the effects of the ocular hypotensive agents, prostaglandin F2alpha (PGF2alpha) and latanoprost on this signaling pathway in ocular tissues. The aim of this study was to determine the agonist activity of PGF2alpha, latanoprost and carbachol (CCh) on the MLCK pathway in isolated bovine iris sphincter and furthermore to investigate the existence of the FP receptor in this tissue. In the present studies on the MLCK pathway four signal transduction mechanism assays were employed, phosphoinositide (PI) turnover, p42/p44 MAP kinase phosphorylation and activation, MLC phosphorylation and contraction. In the studies on the existence of the FP receptor in the bovine iris sphincter, the pharmacology and expression of the FP receptor protein, using a polyclonal anti-FP-receptor antibody and Western blot analysis, were determined. The data obtained on the MLCK pathway showed that the three agonists stimulated the biochemical and pharmacological responses in a concentration and time-dependent manner and that the order of potency and efficacy is PGF2alpha>latanoprost>CCh. The EC50 values in the PI turnover, MAP kinase phosphorylation, MLC phosphorylation and contraction assays were for PGF2alpha: 9, 42, 200 and 140 nM, respectively, for latanoprost: 13, 59, 250 and 828 nM, respectively, and for CCh: 22, 200, 630 and 910 nM, respectively. Wortmannin, a selective inhibitor of MLCK, dose-dependently inhibited MLC phosphorylation and contraction induced by PGF2alpha, demonstrating a close relationship between activation of the MLCK pathway and contraction. The pharmacological studies showed that in the concentration range of 1 nM to 10 microM, the FP-receptor agonists caused concentration-response curves with the following order of potencies: 17-phenyl trinor PGF2alpha (bimatoprost acid)>PGF2alpha>cloprostenol>latanoprost>latanoprost acid>bimatoprost amide>>fluprostenol. Immunoblot analysis of the FP receptor demonstrated expression of the prostaglandin FP receptor protein in this smooth muscle. These results clearly indicate that the MLCK signaling pathway is involved in the FP-receptor function of the bovine iris sphincter and furthermore demonstrate that functional FP receptors exist and are expressed in this tissue.

The signal transduction pathways initiated by Ca(2+)-mobilizing agonists, such as prostaglandin F(2alpha)(PGF(2alpha)) and carbachol (CCh), leading to activation of cytosolic phospholipase A(2)(cPLA(2)) 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 cPLA(2)and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [(3)H]AA for 24 hr and incubated in the absence or presence of the agonist for 5-10 min as indicated. MAP kinases activities and cPLA(2)phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA(2)antibodies. We found that: (a) PGF(2alpha)and 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 PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA(2)phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF(2alpha). (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF(2alpha)- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA(2)phosphorylation. We conclude from these results that in CISM cells PGF(2alpha)-induced cPLA(2)phosphorylation 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 PGF(2alpha)and 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 PGF(2alpha)and muscarinic receptors, and the stimulation of cPLA(2)and 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 PGF(2alpha)and its analog, latanoprost, in glaucoma therapy., (Copyright 2001 Academic Press.)

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 (t1/2=1.5 min) and concentration-dependent (EC50=5 nM) manner, which is primarily mediated through the ETA receptor 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 PLA2 inhibitors dexamethasone, mepacrine and manoalide in a concentration-dependent manner. Conversion of AA into PGE2 was 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 microg ml-1) attenuated the stimulatory effects of ET-1 on AA release and PGE2 formation. 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.

Prostaglandin F2 alpha (PGF 2 alpha) and its analog latanoprost are effective in lowering intraocular pressure (IOP) in both animal and human subjects. There is mounting experimental evidence now which indicates that the IOP-lowering effect of these PGs occurs through an increased uveoscleral outflow of aqueous humor. The ciliary muscle constitutes the main resistance in this pathway. Work from several laboratories, including our own, has shown that in this smooth muscle PGF 2 alpha has little effect on cAMP accumulation or on Ca2+ mobilization. In the present study, we hypothesized that some of the effects of PGF2 alpha and its analogs may be mediated through the release of endogenous PGs. The purpose of this work was to determine whether or not PGF2 alpha and its analogs can enhance the release of endogenous PGs in iris and ciliary muscles isolated from different species. This report documents for the first time that exogenous PGF2 alpha and its analogs, PhXA85 and latanoprost, stimulate the formation of PGE2, PGD2 and PGF2 alpha in iris and ciliary muscles isolated from cat, bovine, rabbit, dog, rhesus monkey and human. PG-induced PG release was demonstrated by means of both radioimmunoassay and radiochromatography. Kinetic studies on cat iris revealed that PGF2 alpha-induced PGE2 release is time (t 1/2 = 1.7 min) and dose-dependent (EC50 = 45 nM). The increase in PGE2 release was blocked by indomethacin (Indo) and by dexamethasone in a dose-dependent manner with IC50 s of 9.2 nM and 2.6 microM, respectively. Furthermore, dexamethasone inhibited arachidonic acid (AA) release, suggesting the involvement of phospholipase A2 in PGF2 alpha-induced PG release. The data presented demonstrate that PGF2 alpha and its analogs interact with the PG receptor to stimulate phospholipase A2 and release AA for PG synthesis. Relaxation of ciliary muscle by PGF2 alpha and its analogs, via release of endogenous PGE2, a potent activator of the adenylate cyclase system, could in part explain how these PGs may increase uveoscleral outflow and consequently lower IOP.

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.

Previously, we have established that treatment of iris sphincter smooth muscle with carbachol (CCh) results in increased phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) and in muscle contraction. To throw more light on the mechanism of muscarinic stimulation of PLC in this tissue we have investigated the properties of this enzyme and its regulation by GTP analogs and protein phosphorylation in bovine iris sphincter membranes. The data obtained can be summarized as follows: (1) the presence of PLC-beta 1 and a GTP-binding protein, Gq alpha, was detected in the microsomal (membrane) fraction by anti-PLC beta 1 and anti-Gq alpha antibodies, respectively. The membrane PLC hydrolysed exogenously added PIP2 and this hydrolysis was increased dose-dependently by Ca2+ (1-10 microM) but the enzyme activity was inhibited by Mg2+. (2) Addition of guanosine 5'-O-thiotriphosphate (GTP gamma S, 0.1 microM) to the membrane fraction increased PIP2 hydrolysis by 30%, whereas addition of CCh (10 microM) was without effect. However, when added together, CCh and GTP gamma S increased PIP2 hydrolysis by 46%. This effect was significantly inhibited by atropine and by the anti-PLC-beta 1 and anti-Gq alpha antibodies. (3) Removal of PLC-beta 1 from the membranes with 2 M KCl resulted in a significant reduction of the CCh-induced PIP2 hydrolysis, and this effect of the muscarinic agonist was restored when the membrane fraction was supplemented with PLC-beta 1 purified from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study it is demonstrated that in bovine ciliary muscle, muscarinic stimulation results in: (a) release of 14C-arachidonic acid (14C-AA) and 14C-labeled prostaglandins (PGs) from muscle prelabeled with 14C-AA; (b) release of endogenous PGs, measured by means of radioimmunoassay; (c) enhanced IP3 production and (d) muscle contraction. In addition, PGs, such as PGE2 and PGD2, increased cAMP formation and induced muscle relaxation. The studies on the kinetics of 14C-AA metabolism revealed that incorporation of 14C-AA into glycerolipids and its conversion into PGs by the ciliary muscle were rapid and time-dependent. The amounts of 14C-radioactivity recovered in the major PGs decreased in the following order: PGD2 > PGE2 < PGF2 alpha > 6-keto-PGF1 alpha. The rate of endogenous PGF2 alpha synthesis by iris-ciliary body tissues from different mammalian species was found to be in the following order: ciliary muscle < ciliary processes < sphincter muscle. The EC50s for muscarinic-stimulated release of 14C-AA, 14C-labeled PGs, and endogenous PGF2 alpha and PGE2, and for IP3 production and contraction of the ciliary muscle indicate that CCh is 2-16 times as potent as pilocarpine in eliciting these responses, with the greatest difference being for contraction. The maximal increase in ciliary muscle tension due to CCh was 48% greater than that evoked by pilocarpine. All PGs tested, including PGE2, 17-phenyl trinor PGE2, 11-deoxy PGE1, PGF2 alpha and PGD2 had no effect on IP3 production and contraction in the ciliary muscle. However, PGE2 and PGD2 stimulated cAMP formation and inhibited CCh-induced IP3 production in a dose-dependent manner. In addition, PGE2 and PGD2 induced relaxation in ciliary muscle precontracted by CCh. In presence of indomethacin (1 microM), the CCh-induced contraction was greater than that observed in absence of the cyclo-oxygenase inhibitor. It is suggested that in the ciliary muscle certain PGs, such as PGE2 and PGD2, may function to modulate, via cAMP, the responses to muscarinic stimulation in this tissue.

It is well established that production of prostaglandins by ocular tissues is dependent upon the species. The rabbit iris-ciliary body produces greater amounts of prostaglandins than that of the bovine. To throw more light on the biochemical basis underlying these differences we have compared the fatty acid composition and phospholipases A2 and C activities in rabbit and bovine irides. When the concentration of arachidonic acid is expressed as % of total fatty acids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, separated from rabbit iris phospholipids, contained 56, 38 and 18% more arachidonic acid, respectively, than those of the bovine. The total lipid phosphorus in rabbit and bovine iris-ciliary body were found to be 13.74 and 9.34 mumol g-1 wet tissue, respectively. Subcellular fractions, prepared from rabbit iris, contained 30-230% more phospholipase C activity than those of the bovine, and about 5-41 times higher phospholipase A2 activity than those of the bovine. In the rabbit iris microsomal fraction, phospholipase C activity is 33 times higher than that of phospholipase A2. However, the data presented suggest that phospholipase A2, rather than phospholipase C, is the enzyme which is more involved in arachidonic acid release for eicosanoid biosynthesis. These findings suggest that the high contents of arachidonic acid and phospholipases A2 and C in the rabbit iris could contribute to its unique capacity to synthesize and release prostaglandins in the anterior segment as compared to that of other mammalian species.

The effects of substance P (SP) on inositol trisphosphate (IP3) accumulation, myosin light chain (MLC) phosphorylation, cAMP formation and contraction were studied in iris sphincter smooth muscle of different mammalian species. SP receptor density was also examined in membrane fractions from this tissue. The data obtained can be summarized as follows. (1) In the iris sphincters of rabbit, bovine and pig, SP receptors are coupled to the phospholipase C system, whereas in dog, cat and human these receptors are coupled to the adenylate cyclase system. (2) In those species which employ the phospholipase C system, SP induced IP3 accumulation, MLC phosphorylation and contraction in a dose-dependent manner; in contrast, in those species in which SP induced the formation of cAMP we found the neuropeptide to cause muscle relaxation. The findings on cAMP formation in intact tissue were confirmed in iris sphincter membranes. Both the effect of SP on IP3 accumulation in rabbit and bovine sphincters and its effect on cAMP formation in the dog were blocked by the SP antagonist, (D-Pro2, D-Trp7, 9)-SP. (3) The density of SP receptors in rabbit, bovine and dog were found to be 227, 110.9 and 13.6 fmol mg-1 protein, respectively, and the Kd values were 1.9, 1.8 and 1.3 nM, respectively. (4) Of the neuropeptides investigated SP, neurokinin A and neurokinin B had significant stimulatory effects on IP3 accumulation and on contraction in the rabbit iris sphincter; however, neither neurokinin Y nor the calcitonin gene-related peptide (CGRP) had any effect on these responses. In addition, none of the neuropeptides studied had any effect on IP3 or on contraction in the dog iris sphincter. While it is possible that SP may have dual actions, with the predominant action dependent on the species, the data presented could suggest the presence of two SP receptor subtypes, one coupled to phospholipase C and the other to adenylate cyclase. The results of this investigation indicate major species differences in biochemical and functional responsiveness to SP and in SP receptor density in the iris sphincter of the mammalian eye, and support a modulatory role for the neuropeptide in muscle response in this tissue.

We have investigated: (a) synthesis and release of prostaglandin E2 (PGE2) and the effects of norepinephrine (NE) and other pharmacological agents thereon in rabbit and bovine irides; (b) the role of Ca2+ and the type of adrenergic receptors involved in PGE2 release by rabbit iris; (c) the structural requirement for catecholamine stimulation of PGE2 release by rabbit iris. Irides were incubated in Krebs-Ringer buffer (pH 7.4) in the absence and presence of pharmacological agents at 37 degrees C for 20 min. PGE2 in the medium was quantitated by radioimmunoassay; or by means of radiometric and chromatographic methods when [1-14C]-arachidonic acid was employed as precursor. Analysis of tissue PGE2 at the start of incubation revealed that rabbit and bovine irides contained about 185 and 18 ng/g tissue, respectively. During 15 min of incubation the release of PGE2 from rabbit and bovine irides was approximately 1100 and 14 ng/g tissue, respectively. We found that the rabbit iris and iris microsomes synthesize PGE2, both from endogenous and exogenous arachidonate pools, at a rate several times as high as that of bovine. Release of PGE2 by irides from both species is time-dependent; the stimulatory effects of NE on PGE2 release by irides from rabbit are more pronounced than those of the bovine; and the NE-induced release of PGE2 was abolished by low concentrations of indomethacin (1.5 microM). Synthesis and the effect of NE on PGE2 release by irides from albino and pigmented rabbit eyes are similar. It is concluded that the differences observed in the synthesis of PGE2 and the effect of NE on its release by rabbit and bovine irides are due to species differences. NE stimulation of PGE2 release by rabbit iris is probably mediated through alpha-adrenoceptors and maximal adrenergic stimulation requires the presence of Ca2+. Ca2+-ionophore A23187 significantly increased PGE2 release. These data suggest that the control step in PG synthesis, that is the release of free arachidonate from membrane phospholipids, could be involved in the NE-induced PG synthesis in this tissue. The structural studies revealed that both the catechol nucleus and the ethylamine polar-side chain are required for catecholamine activation of PG release by the rabbit iris. Thus normetanephrine significantly stimulated PGE2 release by the rabbit iris and iris microsomes; 3-methoxy, 4-hydroxy mandelic acid had no effect; and catechol at 50 microM inhibited PGE2 release by more than 50%. Catecholamines and adrenergic drugs are routinely employed therapeutically to lower intraocular pressure in the eye, and a catecholamine-induced increase in PG synthesis may play a significant role in mediating the pharmacological effects of these therapeutic agents.

Addition of substance P (10(-7) to 10(-6) M) to rabbit iris sphincter muscle induced: (a) a rapid phosphodiesteratic breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DG), measured as phosphatidic acid, and inositol triphosphate (IP3), measured by anion-exchange chromatography; (b) a rapid and strong contractile response, and (c) a rapid release of prostaglandin E2 (PGE2), measured by radioimmunoassay, and rapid release of 14C-labeled arachidonic acid, measured by radiochromatography. These substance P actions are concentration and time-dependent, and are blocked by substance P antagonist, [D-Pro2, D-Trp7,9]SP. The effects of substance P on arachidonic acid release and PG synthesis are not mediated through the cyclo-oxygenase and lipoxygenase pathways. Substance P exerted little effect on PGE2 release by the iris dilator muscle. We conclude that substance P, which is liberated from the sensory nerves that innervate the sphincter region of the iris and plays a role in miosis, may function as a Ca2+-mobilizing agonist in this tissue. Thus, a substance P-induced release of IP3 and formation of DG, a source for arachidonic acid in PG synthesis, followed by Ca2+ mobilization could underlie the mechanism for the biological actions, such as muscle contraction, of the neuropeptide reported in the eye. However, the precise relationship remains to be established.