Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter

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.     

Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells

Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from any of the known prostaglandin receptor types.     

Prostaglandin F2alpha, but not latanoprost, increases the Ca2+ sensitivity of the pig iris sphincter muscle

PURPOSE: To determine the mechanisms underlying prostaglandin (PG) F(2)alpha-(,) carbachol (CCh)-, or latanoprost (a PGF(2)alpha analogue)-induced contraction of the pig iris sphincter muscle. METHODS: Effects of these agents on myofilament Ca(2+) sensitivity were evaluated and compared with the use of receptor-coupled permeabilized preparations by alpha-toxin. The effects of PGF(2)alpha and CCh on the phosphorylation of myosin light chain (MLC) were also analyzed. RESULTS: In the intact strips, all three of these agents induced contractions. In permeabilized strips, PGF(2)alpha and CCh, but not latanoprost, caused an additional tension development at a fixed intracellular Ca(2+) concentration ([Ca(2+)](i)) and also shifted the [Ca(2+)](i)-tension curve to the left, thus indicating that PGF(2)alpha and CCh, but not latanoprost, induced increases in Ca(2+) sensitivity (Ca(2+) sensitization). This Ca(2+) sensitization could have been inhibited by Y27632, a rho kinase inhibitor, but not by GF109203X, a protein kinase C (PKC) inhibitor or by PD98059, a mitogen-activated protein (MAP) kinase inhibitor. PGF(2)alpha increased the level of MLC phosphorylation at a constant [Ca(2+)](i). CONCLUSIONS: PGF(2)alpha, but not latanoprost, induced Ca(2+) sensitization of the pig iris sphincter muscle in an MLC phosphorylation-dependent manner through the rho-rho kinase pathway. The effect of latanoprost on the Ca(2+) sensitization mechanism was different from that of PGF(2)alpha and was thought to play a beneficial role in glaucoma treatment.     

Effects of prostaglandin F(2alpha)and carbachol on MAP kinases, cytosolic phospholipase A(2)and arachidonic acid release in cat iris sphincter smooth muscle cells

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.     

Short-term desensitization of prostaglandin F2 alpha receptors increases cyclic AMP formation and reduces inositol phosphates accumulation and contraction in the bovine iris sphincter

The effect of short-term prostaglandin (PG) desensitization on PGF2 alpha receptor-mediated inositol phosphates accumulation, 1,2-diacylglycerol production, measured as phosphatidic acid (PA), myosin light chain (MLC) phosphorylation, cAMP formation and contraction was investigated in bovine iris sphincter smooth muscle. We have found that incubation of the sphincter with 25 microM PGF2 alpha for 45 min leads to: (a) significant loss in sensitivity of the tissue to PGF2 alpha receptor-stimulated inositol phosphates accumulation, PA production, MLC phosphorylation and contraction, and (b) significant increase in both basal and PGF2 alpha-stimulated cAMP formation. These changes are probably not due to reduction in phospholipid synthesis because there were no detectable differences in basal phospholipid labeling, either from 3H-inositol or from 32P, between normal and desensitized muscles. Preincubation of the sphincter in the absence of PGF2 alpha for 45 min did not lead to alterations in the biochemical-pharmacological responsiveness of the control muscle to PGF2 alpha. Our results suggest that desensitization of PG receptors in the iris sphincter occurs by a receptor-specific process. The PG receptor mediating contraction (IP3-Ca2+) is selectively susceptible to desensitization, in contrast with the receptor mediating smooth muscle relaxation (cAMP). These findings add further support to the developing hypothesis that there are functional and biochemical reciprocal interactions between the IP3-Ca2+ and cAMP messenger systems in the iris of the mammalian eye.     

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.     

Effect of latanoprost,prostaglandin F(2)alpha and nipradilol on isolated bovine ciliary muscle

PURPOSE: Ciliary muscle tone is considered to be an important factor for control of uveoscleral outflow. In an attempt to clarify the functional roles of the ciliary muscle in uveoscleral outflow, the effects of latanoprost, prostaglandin (PG)F(2)alpha or nipradilol, all of which are known to increase uveoscleral outflow, were investigated, using the bovine ciliary muscle.METHODS: We isolated longitudinal ciliary muscle from bovine eyes and investigated the effects of these three agents on the mechanical properties of this muscle using isometric tension recording methods.RESULTS: Latanoprost and PGF(2)alpha evoked small but discrete contractions at a concentration of 0.1 microM even during the sustained contraction evoked by 10 mM acetylcholine (ACh). However, nipradilol did not evoke any response at concentrations up to 0.1 mM. None of these agents had an effect on the amplitude of the ciliary muscle twitch contraction evoked by electrical field stimulation.CONCLUSIONS: Our findings indicate that these three agents have no relaxant effect on isolated bovine ciliary muscle even during the sustained contraction evoked by ACh. Further, these agents had no effect on the contraction evoked by field stimulation, which indicates that the drugs have no presynaptic effects. These results are inconsistent with the hypothesis that drugs that increase uveoscleral outflow relax the ciliary muscle with a consequent increase in uveoscleral outflow. Further investigation of the role of ciliary muscle contractility on uveoscleral outflow is warranted.     

Additive effect of latanoprost, a prostaglandin F2 alpha analogue, and timolol in patients with elevated intraocular pressure.

A randomised observer masked clinical study was conducted to assess the additive effect of latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinorprostaglandin F2 alpha-isopropylester) to timolol maleate in patients with elevated intraocular pressure (IOP). Patients were randomly assigned to two treatment groups. One group (n = 10) received timolol, the other group (n = 9) received latanoprost twice daily for 1 week. After 1 week all patients received both timolol and latanoprost. Eyes treated with timolol (mean diurnal IOP (SD) day 0, 24.2 (2.8) mm Hg) and latanoprost (mean diurnal IOP day 0, 28.5 (5.6) mm Hg) showed an IOP reduction of 5.9 (2.3) mm Hg (24%) and 8.9 (2.5) mm Hg (31%), respectively after the first week. Adding latanoprost to the eyes treated with timolol as well as timolol to the eyes receiving latanoprost gave a further reduction of 2.6 (1.1) mm Hg (13%) and 2.6 (2.2) mm Hg (14%), respectively. Only mild transient hyperaemia was observed in patients receiving latanoprost. The results indicate that latanoprost and timolol can be combined successfully and that complete or almost complete additivity is reached even at pressure levels below 20 mm Hg.     

Production of prostaglandin e(2) by iridial melanocytes exposed to latanoprost acid, a prostaglandin F(2 alpha) analogue.

Several prostaglandin analogues used for glaucoma treatment cause increased pigmentation of the iris. The purpose of the present study was investigate whether latanoprost, a PGF(2 alpha) analogue, has any effect on the production of endogenous prostaglandins in iridial melanocytes, which could be important in the mechanism leading to increased pigmentation. Bovine and human iridial melanocytes in culture were used for the experiments. Production of endogenous prostaglandins was measured by enzyme immunoassay, and the melanin content was measured spectrophotometrically. In bovine iridial melanocytes, latanoprost acid caused a significant increase of the PGE(2) production, which could be blocked by indomethacin and NS398, indicating an involvement of cyclo-oxygenase 2. In order to study the selectivity of the phenomenon other endogenous substances/drugs were tested, e.g., acetylcholine, carbachol, noradrenaline, neuropeptide Y, substance P and alpha-MSH, but none was found to have any significant effect. Human iridial melanocytes also responded to latanoprost acid with increased production of PGE(2) and in 1 out of 5 individuals increased melanogenesis coincided with increased PGE(2) production. In bovine iridial melanocytes, latanoprost acid did not stimulate melanogenesis. These results indicate that latanoprost acid cause enhanced formation of endogenous prostaglandins that may have auto- and/or paracrine effects in the melanocytes, possibly associated with melanogenesis.     

Prostaglandins E1 and E2, but not F2alpha or latanoprost, inhibit monkey ciliary muscle contraction

PURPOSE: To investigate the effects of prostaglandin (PG) E1, E2, F2alpha, and latanoprost acid on the electrically evoked contractile response of isolated rhesus monkey ciliary muscle. METHODS: Longitudinal ciliary muscle preparations from rhesus monkeys were mounted in an organ bath, and tension changes were recorded by an isometric transducer. Electrical field stimulation (100 Hz, 0.3 ms, 10 V) was applied through a pair of platinum plate electrodes. RESULTS: The ciliary muscle produced atropine-sensitive excitatory contraction in response to field stimulation. PGE1 and PGE2 (1 microM) attenuated the contraction to levels that were 68% and 65.1%, respectively, of the normal amplitude. However, PGF2alpha and latanoprost acid (1 microM) did not significantly change the response amplitude. CONCLUSIONS: Our results indicate that PGF2alpha and latanoprost acid do not interact with the prostanoid receptor involved at the pre- and/or postsynaptic site. Therefore, it is unlikely that the hypotensive action by these agents is due to relaxation of the ciliary muscle.     

Effect of prostaglandin E2 on the isolated rabbit iris sphincter

Summary Experiments on twenty-eight New Zealand albino rabbits have shown that prostaglandin E₂ causes contraction of the isolated iris sphincter and is about five times as effective as acetylcholine. Prostaglandin E₂ does not act on the cholinergic receptors; the effect of prostaglandin E₂ was not increased by eserine, and not abolished by atropine. In high doses indomethacin (3×10^–4 M) reversed the prostaglandin effect, but lower concentrations (3×10^–5 M) left it unaffected.

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Zusammenfassung Versuche an 28 neuseeländischen Albinokaninchen haben gezeigt, daß Prostaglandin E₂ eine Kontraktion des isolierten Irisschließmuskels bewirkt und daß es ca. fünfmal so wirksam ist wie Acetylcholin. Prostaglandin E₂ wirkt nicht auf die cholinergischen Rezeptoren, seine Wirkung wurde durch Eserin nicht gesteigert und durch Atropin nicht aufgehoben. Hohe Indomethacindosen (3×10^–4 M) hatten eine Umkehr des Prostaglandineffektes zur Folge, niedrigere Dosen aber blieben wirkungslos.

     

Pharmacological effects of latanoprost, prostaglandin E2, and F2 α on isolated rabbit ciliary artery

BACKGROUND: Latanoprost is a prostaglandin (PG)F2alpha analogue widely recognized in the treatment of glaucoma. To investigate the action of this drug on the ocular circulation, we have studied its effects on isolated rabbit ciliary artery. The data obtained on this drug are compared with the data from PGE2 and PGF2alpha. METHODS: Under the microscope, ciliary artery specimens were prepared from rabbit eyes and mounted in a myograph system. The effects of latanoprost, PGE2, and PGF2alpha on the isolated rabbit ciliary artery were investigated in vitro using isometric tension recording methods. RESULTS: Exogenously applied PGF2alpha, but not latanoprost, evoked contraction in the rabbit ciliary artery. After precontraction with excess-[K]0 solution, latanoprost evoked relaxation dose-dependently. Latanoprost at a concentration of 100 microM induced maximum relaxation, which was not blocked by 10 microM L-nitro-L-arginine methylester (L-NAME), 1 microM 8-37 calcitonin gene-related peptide (CGRP) or 10 microM indomethacin. Moreover, latanoprost induced relaxation even in preparations without endothelium. The maximum relaxation obtained with PGE2 was somewhat less than 50% of that with latanoprost. CONCLUSIONS: These results indicate that latanoprost and PGE2 relaxed rabbit ciliary artery to different degrees. The relaxation provoked by latanoprost was not dependent on endothelium and was not caused by intrinsic PG, CGRP or nitric oxide. The mechanism of this relaxation is not yet clear.     

A comparison of intraocular pressure-lowering effect of prostaglandin F2 -alpha analogues, latanoprost, and unoprostone isopropyl.

PURPOSE: To compare the intraocular pressure-lowering effect of unoprostone isopropyl (unoprostone) 0.12% and latanoprost 0.005%. A correlation between the intraocular pressure-lowering effect of unoprostone and latanoprost was also evaluated. METHODS: A single-masked randomized study included 18 patients between 49 and 68 years (mean, 60.7 +/- 5.1 years) with an intraocular pressure of both eyes from 21 to 27 mm Hg. The patients were prospectively randomized to receive latanoprost in the right eye and unoprostone in the left eye, or unoprostone in the right eye and latanoprost in the left eye. The patients were followed up for 8 weeks. This study evaluated the intraocular pressure-lowering effect and incidence of drug-related side effects. RESULTS: Mean baseline intraocular pressure was 22.8 +/- 1.2 mm Hg in latanoprost-treated eyes and 22.4 +/- 1.0 mm Hg in unoprostone-treated eyes; there was no statistically significant difference between these groups. Mean intraocular pressure at 8 weeks after the start of the administration was 16.7 +/- 2.0 mm Hg in latanoprost-treated eyes and 19.0 +/- 1.5 mm Hg in unoprostone-treated eyes. Patients in the latanoprost-treated group showed a greater intraocular pressure reduction compared with those in the unoprostone-treated group. Mean intraocular pressure changes in latanoprost-treated eyes were significantly greater at every visit (P < 0.0001). A change of intraocular pressure at 8 weeks in the latanoprost-treated eyes was significantly correlated with that in the contralateral unoprostone-treated eyes (r = 0.665, P = 0.0013) (Figure). There was no significant difference in the rate of ocular side effects between latanoprost- and unoprostone-treated eyes. CONCLUSIONS: Latanoprost appears to have a more beneficial effect for intraocular pressure control compared with unoprostone. An intraocular pressure reduction in the latanoprost-treated eyes was significantly correlated with that in the contralateral unoprostone-treated eyes. There was no significant difference in the incidence of ocular side effects between both drugs. Further investigation using more cases and longer follow-up periods are needed.     

Effects of acetylcholine and carbachol on the iris sphincter

We compared the effects of acetylcholine and carbachol on iris sphincters. The muscle strips (mostly bovine tissues) were isolated and incubated in a 0.2ml organ bath. Results revealed an approximately ten-thousandfold difference in potency between carbachol and acetylcholine, in the bovine iris. Acetylcholine and/or endogenous chemical agents were spontaneously released from bovine tissue. Acetylcholinesterase activity in this tissue strongly inhibited the acetylcholine action, in order to protect the nerve terminals from random depolarization due to the released acetylcholine. Among several species (bovine, rabbit, hamster), Ach action was the least in the bovine iris sphincter muscle.     

The different contributions of prostaglandins (E1, E2, F2alpha,D2) to the tone and neurogenic response of bovine iris sphincter muscle

The function and sites of action of prostaglandins (PGs) vary in different animal species and tissues. In this study the influence of PGs (E₁, E₂, F_2alpha, D₂) on muscle tone and nerve-mediated contraction was investigated in isolated bovine iris sphincter muscles.None of these PGs exogenously applied influenced the neuromuscular transmission. By contrast, after treatment with indomethacin, all PGs tested contracted the muscle much more than in the absence of indomethacin and under these conditions the PGs potentiated responses to cholinergic nerve stimulation. Their ED₅₀ were (2.2 ± 0.2) × ^–7 M for PGE₁, (6.7 ± 0.3) × ^–8M for PGE₂, and (7.3 ± 0.4) × ^–8 M for PGF_2alpha. PGE₁ acted both on nerves and the muscle cell. PGE₂ had its influence mostly via nerves. Whereas PGF_2alpha was less potent in the absence of indomethacin, PGF_2alpha had much more potent action primarily on nerves and partly on muscles after treatment with indomethacin. High concentrations of PGD₂ had both pre- and post-junctional action with accompanying weak contraction of the muscle. Thus the degrees of pre- and post-junctional involvement were different from one another.There is a possibility that the application of these PGs alone masked the role of such endogenous agents. In order to understand and clarify the site and action of PGs, pretreatment with indomethacin may be useful in the iris muscle. In conclusion, PGs modulate cholinergic activity in the bovine iris sphincter muscles, as well as regulate the muscle tone.     

Effects of forskolin, prostaglandin F2 alpha, and Ba2+ on the short-circuit current of the isolated rabbit iris-ciliary body

To gain information on the role of cyclic AMP (cAMP), ion transport and cell membrane permeability on aqueous humor formation, agents with well-known effects on transport properties in other epithelia were tested on the isolated rabbit iris-ciliary body. Forskolin stimulated the short-circuit current (SCC) by 37.5% when added to the aqueous-side solution. Forskolin was ineffective when added to the blood-side solution or when HCO3- was absent from the bathing solutions. The effect of forskolin confirms the presence of adenylate cyclase in the ciliary epithelium and the involvement of cAMP in ion transport. In HCO3- -rich media, 5 X 10(-5) M prostaglandin F2 alpha (PGF2 alpha), produced a prompt 25% increase in the SCC when added to the aqueous-side, and a small stimulatory SCC response when added to the blood-side. No change in SCC occurred when PGF2 alpha was added to either side of a HCO3- -free bathing solution. It is implied that cAMP acts on a HCO3- dependent transport system. These results are consistent with the previously observed stimulation of the SCC by 8Br-cAMP. BaCl2, 2.5 mM, on the aqueous-side increased the SCC by 240.5%, but reduced the SCC by 26% when added to the blood-side solution. The Ba2+ effects indicate the presence of high conductance K+ channels in the basolateral membranes of both the pigmented and non-pigmented cell layers.     

Localisation of prostaglandin F2 alpha and E2 binding sites in the human eye.

Prostaglandin F2 alpha reduces intraocular pressure possibly by increasing uveoscleral outflow. To further understand the mechanism of its action binding sites for prostaglandin F2 alpha and, for comparison, prostaglandin E2 were localised in sections of human cadaveric eyes using an in vitro ligand-binding technique and autoradiography. Specific binding sites for both prostaglandin F2 alpha and E2 were co-localised at a high level in the areas of the ciliary muscles and iris sphincter muscles, and at a lower level in the iris epithelium and the retina. The results suggest that prostaglandin F2 alpha and also prostaglandin E2, could modulate uveoscleral outflow by binding to their receptors located on the ciliary muscles and inducing their relaxation.     

Effects of substance P on inositol triphosphate accumulation, on contractile responses and on arachidonic acid release and prostaglandin biosynthesis in rabbit iris sphincter muscle

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.     

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells

PURPOSE: Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. METHODS: Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. CONCLUSIONS: Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.