Sardar Y.K. Yousufzai

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

We have investigated the effects of endothelin-1 (ET1) on phospholipid hydrolysis and 3H-arachidonic acid (AA) release and prostaglandin synthesis in the rabbit iris sphincter smooth muscle. ET1 actions are concentration- and time dependent with an EC50 for AA release of 1 nM and t1/2 value of 1.5 min. We have identified the AA metabolites released by ET1, employing HPLC, as both cyclooxygenase and lipoxygenase products. The AA released by ET1 appears to derive mainly from the phosphoinositides through phospholipase A2, rather than phospholipase C activation. A key role for phospholipase A2 in AA release in the sphincter muscle is supported by the following observations. (1) Pretreatment of the labeled sphincter with the phorbol ester, PDBu (100 nM) inhibited ET1-stimulated IP3 formation, but it potentiated ET1-stimulated AA release. (2) Pretreatment of the labeled tissue with isoproterenol (5 M) inhibited ET1-stimulated IP3 production without altering AA release. (3) The potency for ET1-stimulated AA release (EC50 = 1 nM) was much higher than that for IP3 formation (EC50 = 45 nM). (4) There were considerable increases, rather than decreases, in 1, 2-diacyl-glycerol formation (1.2-folds) and its phosphorylated product, phosphatidic acid (2.6-folds) by ET1. It is concluded that in the rabbit iris sphincter ET1 is a potent agonist for AA release and eicosanoid synthesis and that AA is released from phosphoinositides mainly through activation of phospholipase A2.

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.

Effects of norepinephrine and other pharmacological agents on prostaglandin E2 release by rabbit and bovine irides

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.

Species differences in the effects of substance P on inositol trisphosphate accumulation and cyclic AMP formation, and on contraction in isolated iris sphincter of the mammalian eye: differences in receptor density.

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.

Tyrosine kinase inhibitors suppress prostaglandin F2α-induced phosphoinositide hydrolysis, Ca2+ 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 F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]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 F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]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 EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ 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.

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.

Carbachol stimulates adenylate cyclase and phospholipase C and muscle contraction-relaxation in a reciprocal manner in dog iris sphineter smooth muscle

In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscarinic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mammalian species were found to be coupled to phospholipase C and contraction at all concentrations of carbachol investigated (1-100 microM). In the dog sphincter, lower concentrations (less than 5 microM) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP3) production, inhibited cAMP formation and induced contraction, and higher concentrations (greater than 5 microM) enhanced cAMP formation, inhibited IP3 production and induced relaxation. The mechanisms for the stimulatory effects on cAMP formation through muscarinic receptors were investigated. Carbachol (25 microM) increased both basal and isoproterenol- and forskolin-stimulated cAMP levels. Atropine inhibited the carbachol-stimulated increase in cAMP levels in a dose-dependent manner with an IC50 of 9 nM. Intracellular Ca2+, derived from IP3-induced Ca2+ release and/or from muscarinic receptor-operated Ca2+ influx, and protein kinase C may mediate the muscarinic receptor-linked rise in intracellular cAMP. This conclusion is supported by the following findings. (1) At short time intervals (less than 1 min) carbachol (25 microM) increased IP3 production and contraction and this was followed (between 1 and 20 min) by cAMP formation and muscle relaxation. (2) Carbachol-stimulated IP3 production was detected at a concentration of the agonist 26-fold lower than that required for cAMP formation, and it was completely blocked by the phorbol ester, phorbol 12,13-dibutyrate (50 nM). (3) A Ca(2+)-calmodulin stimulated adenylate cyclase was demonstrated in membranes from dog iris sphincter but not in that from rabbit and bovine. (4) Trifluoperazine (0.1 microM), a calmodulin antagonist, inhibited the carbachol-stimulated cAMP accumulation. (5) The Ca2+ ionophore A23187 and the phorbol ester increased cAMP production in a dose-dependent manner. A23187 potentiated cAMP production induced by either carbachol or by the phorbol ester. (6) Muscarinic stimulation of cAMP production persisted even after the tissue was pretreated with the phorbol ester or staurosporine. (7) Nifedipine (0.01-0.5 microM), a Ca2+ channel antagonist, inhibited carbachol stimulation of cAMP production, suggesting the presence of a muscarinic receptor-operated Ca2+ influx pathway in this tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-term desensitization of prostaglandin F2α 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.

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 A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (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 (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB 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 ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.

In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5‐bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo‐[3H]Inositol or [l‐14C]ara‐chidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H‐Iabeled inositol phosphates were analyzed by either ion‐exchange chromatography or HPLC. The aqueous humor was analyzed for 14C‐labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo‐[3H]inositol into phosphoinositides and accumulation of 3H‐labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo‐inositol 1‐phosphate. 36% in myo‐inosi‐tol bisphosphate, 0.95% in myo‐inositol 1,3,4‐trisphosphate (1,3,4‐IP3), and 2.6% in 1,4,5‐IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for α1‐adrenergic receptors in this tissue, (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus. We conclude that IP3 accumulation and arachidonate release are produced in the iris muscle under in vivo conditions that produce contraction, i.e., electrical stimulation of the sympathetic nerve. These observations add further support to the concept that PIP2 and its derived second messengers are involved in excitation‐contraction coupling in the smooth muscle cells of the iris.