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Abstract: Biologically active peptides modulate pupillary responsiveness in many non-primate mammals. We examined the action of seven different peptides on iris sphincter and dilator muscles of rhesus monkey. Iris sphincter and dilator muscle preparations from rhesus monkeys were mounted in an organ bath, and tension changes were recorded by an isometric transducer. Electrical field stimulation (100Hz, 0·3msec, 10V) was applied through a pair of platinum plate electrodes. Monkey iris sphincter and dilator muscles produced simple cholinergic and adrenergic excitatory responses respectively to electrical field stimulation. Strong field stimulation did not elicit slow Substance P (SP) mediated contractions like those in rabbit iris sphincter. Exogenously applied pituitary adenylate cyclase-activating peptide (PACAP) enhanced in a concentration-dependent manner (0·3nM–0·1μm) the sphincter response to field stimulation, while neuropeptide Y (NPY) and somatostatin (SRIF) attenuated it. These three peptides did not affect sphincter contractions induced by acetylcholine, and therefore were acting at presynaptically. SP, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL) had no effect (at 0·1μm) on iris sphincter. None of seven exogenously applied peptides had an effect on monkey iris dilator muscle. The innervation of primate irises may be relatively simple compared to non-primates because each of the peptides in this study can modulate miosis or mydriasis in non-primate mammals. Future studies will be expected on the functional significance of species differences in iridial innervation. [Copyright &y& Elsevier]

The neuropeptides somatostatin and galanin are present in the iris and may modulate pupil diameter. We examined the effects of somatostatin and galanin on isolated rabbit iris dilator and sphincter smooth muscles that were mounted in an organ bath. An isometric transducer recorded changes in tension in response to electric field stimulation (100 Hz, 0.3 m sec in duration, 10 V in strength) delivered by a pair of platinum plate electrodes. The dilator muscle response to field stimulation was not changed by either peptide, even at the highest concentrations examined. The sphincter muscle response consisted of two components: a fast component mediated by acetylcholine and slow component mediated by substance P. Both somatostatin and galanin attenuated the cholinergic component in a dose-dependent manner (from 0.3 nm to 0.1 μm) but had no effect on responses mediated by substance P. Galanin was more effective (attenuation of 43% at 0.1 μm) compared with somatostatin (attenuation of 16% at 0.1 μm) in reducing the cholinergic response. Neither peptide affected the contraction induced by acetylcholine (1 mm). Therefore both peptides inhibited cholinergic transmission in the sphincter muscle, although the degree of inhibition by each was different. We conclude that somatostatin and/or galanin may induce mydriasis by attenuating cholinergic neurotransmitter release. [Copyright &y& Elsevier]

Nipradilol is a new antiglaucoma ophthalmic agent used in Japan. Topical application of nipradilol is reported to increase ocular blood flow. To investigate the action of this drug, we studied the effect of nipradilol on the isolated rabbit ciliary artery. Under the dissecting microscope, ciliary arteries were prepared from rabbit eyes and mounted on a myograph system. The effects of nipradilol on the isolated rabbit ciliary artery were investigated using isometric tension recording methods. Nipradilol provoked a dose-dependent (10 μM–1 m M) relaxation in ciliary arteries that were pre-contracted with high-K solutions (K+: 100.7 m M). It also inhibited the amplitude of smooth muscle contraction evoked by field stimulation. Nipradilol was more effective in relaxing phenylephrine-induced contraction (EC50: 21.6 ± 16.3 μM) compared to high-K solution-induced contractions (EC50: 230 ± 130 μM). Application of Nw-nitro- L -arginine methylester (300 μM), a nitric oxide (NO) synthase inhibitor, or denudations of endothelium by rubbing the inner surface with a scalp hair did not affect this relaxation. However, NO scavenger carboxy-PTIO (1 m M) or methylene blue (10 μM), a guanylate cyclase inhibitor, inhibited the nipradilol-induced relaxation. These results indicate that nipradilol relaxes the rabbit ciliary artery by two different mechanisms. First, the relaxation is due to the NO produced by denitrification of nipradilol itself. Second, nipradilol may act as an α-adrenergic antagonist. These actions of nipradilol may explain the mechanisms of increased ocular blood flow in vivo. [Copyright &y& Elsevier]

The interphotoreceptor matrix (IPM) is a highly organized structure with interconnected domains surrounding cone and rod photoreceptor cells and extends throughout the subretinal space. Based on known roles of the extracellular matrix in other tissues, the IPM is thought to have several prominent functions including serving as a receptor for growth factors, regulating retinoid transport, participating in cytoskeletal organization in surrounding cells, and regulation of oxygen and nutrient transport. In addition, a number of studies suggest that the IPM also may play a significant role in the etiology of retinal degenerative disorders. In this review, we describe the present knowledge concerning the structure and function of the IPM under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the effects of prostaglandin F2α (PGF2α) analogues on the endothelin-1 (ET-1)-induced impairment of optic nerve head (ONH) blood flow and on ET-1-induced contraction in isolated ciliary artery segments. In male rabbits, one of four PGF2α analogues [0.0015% tafluprost, 0.0015% 15-hydroxyl tafluprost (15-OH tafluprost), 0.005% latanoprost, or 0.004% travoprost] was topically administered at various pretreatment times before intravitreal ET-1 injection. ONH blood flow was estimated by the laser speckle method, which expresses blood velocity as a quantitative index, the squared blur rate (SBR). SBR was measured just before (baseline value) and at 30, 60, and 120 min after ET-1 injection. SBR was significantly decreased from 4.47 ± 0.20 to 3.50 ± 0.10 (78.6 ± 2.4% of baseline) at 120 min after intravitreal ET-1 injection (5 pmol/eye). The ET-1-induced decrease was almost completely prevented by tafluprost and significantly inhibited by the other three analogues. The inhibitory effect lasted longest with tafluprost, as indicated by the effective pretreatment times (tafluprost: 90, 120, or 240 min; 15-OH tafluprost: 90, but not 120 or 240 min; latanoprost and travoprost: 120, but not 240 min). In vitro, the effects of PGF2α analogues on ET-1-induced contractions in male rabbit ciliary arteries were evaluated using an isometric tension recording system. Tafluprost, latanoprost, travoprost, and 15-OH tafluprost concentration-dependently relaxed the 10 nM ET-1-induced ciliary artery contraction. Improvement of the ocular circulation may be superior with tafluprost than with the other PGF2α analogues. The underlying mechanism may involve relaxation of ocular resistance vessels. [ABSTRACT FROM AUTHOR]

Abstract: Vascular peripheral resistance is increased during hypertension, and endothelium-derived hyperpolarizing factor (EDHF) is an important for regulating vasodilation in small arteries. Therefore we characterized the role of EDHF in regulating vascular resistance of ocular ciliary arteries from spontaneous hypertensive rats (SHR) and age-matched Wistar Kyoto rats (WKY). Isometric tension recordings were used in isolated ocular ciliary artery segments from SHR and WKY. Ocular ciliary arteries pre-contracted with 100 μM norepinephrine exhibited a concentration-dependent relaxation to acetylcholine, and the effect on SHR arteries was smaller than that on WKY arteries (P < 0.05). The EDHF-mediated component of this relaxation, determined in the presence of 100 μM l-NAME plus 10 μM indomethacin, was also smaller in SHR than in WKY arteries (P < 0.05). Apamin (1 μM), a blocker of small-conductance calcium-activated K+ (KCa) channels, had no effect on EDHF-mediated relaxation in either preparation. However, charybdotoxin (0.1 μM), which blocks intermediate- and large-conductance KCa channels, and iberiotoxin (0.1 μM), which blocks large-conductance KCa channels, almost completely suppressed EDHF-mediated relaxation in both preparations. The tension of ciliary arteries from both SHR and WKY was increased above baseline by 100 μM l-NAME plus 10 μM indomethacin. In these preparations, apamin had no effect on the tension in arteries from either SHR or WKY. However, both charybdotoxin and iberiotoxin further increased tension above that induced by l-NAME and indomethacin. The increase was smaller for SHR than WKY (P < 0.05). In summary, the ability of EDHF to relax ocular ciliary artery vascular tone in SHR is smaller than in WKY. The large-conductance calcium-activated K+ channel is utilized in EDHF-signaling pathway. [Copyright &y& Elsevier]

Abstract: Our objective was to determine if tafluprost, a newly synthesized antiglaucoma drug, can relax precontracted rabbit ciliary arteries, and if so, to elucidate the underlying mechanism. We used isometric tension recordings of smooth muscle contractions and fluorescence photometry to monitor the change of intracellular free calcium concentration ([Ca2+]i) in isolated rabbit ciliary artery segments. Tafluprost induced a concentration-dependent relaxation in rabbit ciliary arteries precontracted with a high-K solution. The amplitude of relaxation induced by tafluprost was unchanged by 100μM l-NAME, 10μM indomethacin, denudation of vascular endothelium, 30μM thapsigargin, or 100μM ouabain. In Ca2+-free solution, 30μM tafluprost did not decrease the amplitude of contraction induced by 1μM histamine or the amplitude of the [Ca2+]i increase induced by 10μM histamine. The mechanism of tafluprost-induced relaxation was different from diltiazem, a voltage-dependent Ca2+ channel blocker. However, in thapsigargin-pretreated preparations incubated in Ca2+-free solution, tafluprost attenuated the capacitative increase of [Ca2+]i upon Ca2+ reintroduction to the extracellular medium. Thus, we conclude that tafluprost relaxed isolated rabbit ciliary artery segments precontracted with a high-K solution. The relaxing mechanism was not dependent on endothelial-derived factors, and not affected by the intracellular Ca2+ cycles or the Ca2+ extrusion component of the extracellular Ca2+ cycles. Relaxation of rabbit ciliary artery smooth muscle by tafluprost may be due, at least in part, to inhibition of capacitative Ca2+ entry from the extracellular space. [Copyright &y& Elsevier]

Abstract: Topical application of levobunolol hydrochloride, a β-adrenergic antagonist used for treatment of glaucoma, is reported to increase ocular blood flow. We studied the mechanism of levobunolol-induced vasodilation in arterial smooth muscle. The effects of levobunolol or other agents on isolated, pre-contracted rabbit ciliary artery were investigated using an isometric tension recording method. The effects of the same agents on intracellular free calcium ([Ca2+]i) in cultured human aortic smooth muscle cells were also studied by fluorophotometry. Levobunolol relaxed ciliary artery rings that were pre-contracted with either high-K solution, 1μM histamine, 10μM phenylephrine, or 100nM endothelin-1. The relaxation induced by levobunolol was concentration-dependent over the range of 10μM to 0.3mM. Inhibition of endothelial nitric oxide synthase or denudation of the endothelium did not affect this relaxation. Histamine-induced contractions were inhibited by the histamine H1 antagonist pyrilamine. Radioligand binding experiments showed that levobunolol did not bind to the H1 receptor. Further, histamine induced transient contraction in Ca2+-free solution, and levobunolol inhibited this contraction by 74.6±11.0%. In cultured smooth muscle cells in the presence of extracellular Ca2+, levobunolol significantly inhibited the histamine-induced elevation of [Ca2+]i. However, it did not inhibit the increase of [Ca2+]i in histamine-stimulated cells incubated in Ca2+-free solution. These results indicate that levobunolol may relax rabbit ciliary artery by two different mechanisms. First, the relaxation could be due to the blockade of Ca2+ entry through non-voltage-dependent Ca2+ channels. Second, levobunolol may change the Ca2+ sensitivity of vascular smooth muscle cells. [Copyright &y& Elsevier]

Abstract: Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5mg/100g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2+ cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II+ cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2+ cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2+ cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC class II antigen. These findings may have important implications for our understanding of the characteristics of limbal slow-cycling cells. [Copyright &y& Elsevier]

Abstract: Impairment of the ocular circulation induced by diabetes mellitus has not been fully defined, but is thought to be related to hemodynamic changes in the ocular circulation. The purpose of the present study is to investigate the functional and morphological changes occurring in the ciliary artery wall of rabbits with alloxan-induced diabetes mellitus. A single intravenous bolus injection of alloxan (100mg/kg) was given to each of 26 10-week-old rabbits and 16 sham-injected control rabbits. Twenty weeks later, control rabbits and diabetic rabbits were sacrificed, and their ciliary arteries were mounted in a myograph system. The responses of these arteries to high K+ solution (K-Krebs solution), phenylephrine and carbachol were investigated using isometric tension recording. L-NAME (NG-nitro-l-arginine methyl ester; 100μM) and indomethacin (1μM) were also used to test the mechanism causing the carbachol induced relaxation. The arteries were also examined morphologically. The maximum tensions induced by K-Krebs solution in this tissue were not significantly different: 17.2±0.8mN (n =16) in the control rabbits and 17.6±0.8mN (n =23) in the diabetic rabbits (P =0.36). Phenylephrine caused dose-dependent contraction with EC50 values of 1.3±0.4μM (n =6) in the control and 5.1±2.3μM (n =6) in the diabetic rabbits, but there was no significant difference between the two (P =0.36). Carbachol induced dose-dependent relaxations in segments precontracted with K-Krebs solution. These relaxations were significantly reduced in the diabetic rabbits. The maximum relaxation induced by carbachol was 77.0±2.4% (10μM) and 66.4±2.5% (100μM) in the control and diabetic rabbits, respectively. These values were significantly different (P =0.0076). The IC50 value for carbachol was 396.3±58.4nM (n =16) in the control, and 443.6±141.1nM (n =23) in the diabetic rabbit (P =0.87). Application of a 100μM nitric oxide synthase inhibitor, L-NAME, significantly inhibited the amplitude of relaxations evoked by carbachol (P =0.0066). However, these relaxations were not inhibited by pretreatment with 1μM indomethacin (P =0.60). Histologically, the frequency of invaginations was less in the diabetic arterioles with a flattening of the lamina in the diabetic rabbits than in the controls. The cytoplasm of endothelial cells contained large vacuoles, indicating weak adhesion to the lamina. Some endothelial cells even showed vacuolar degeneration due to breakdown of the cell membranes. However, the smooth muscle cells were well preserved in the diabetic rabbit. These results suggest that the mechanism of impairment of ocular circulation induced by diabetes mellitus is mainly the reduction of NO synthase due to endothelial cell dysfunction. Furthermore, the characteristics of rabbits with alloxan-induced diabetes mellitus probably make them a useful model for investigating ocular complications induced by diabetic mellitus. [Copyright &y& Elsevier]

We have investigated functional and morphological changes occurring in the wall of the hypercholesterolemic rabbit ciliary artery. A mutant rabbit with hypercholesterolemia and atherosclerosis was created by serial breeding. Ciliary arteries from hypercholesterolemic, age-matched control and young control rabbits'' eyes were mounted in a myograph system. The effects of phenylephrine (PE), carbachol and electrical field stimulation on this artery were investigated using isometric tension recording methods. The arteries were also examined morphologically. PE caused dose-dependent contraction in young control, age-matched control and hypercholesterolemic rabbits. The EC50 values were 1·0 μm (0·2–2·1, n=6), 1·4 μm (0·4–2·4, n=6) and 4·7 μm (1·8–7·7, n=8) in the young, age-matched controls and in the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits, respectively. The EC50 values of the KHC rabbit were significantly different from those of control.Electrical field stimulation evoked contraction only in the control rabbits. On the other hand, electrical field stimulation evoked relaxation when the ciliary artery was pre-contracted by 10 μm histamine in each type of rabbit equally. Carbachol also induced approximately equal dose-dependent relaxation after pre-contraction. The morphological findings of KHC rabbit ciliary artery revealed irregular contours on the internal elastic lamina and deformation of the shape of the medial smooth muscle cells with irregularity in size and widening of the intercellular spaces. However, the endothelial cells were well preserved. Compared with the ciliary artery, typical atherosclerotic changes existed in the intima, not the media, in the KHC rabbit aorta. In the hypercholesterolemic rabbit ciliary artery, the vasoconstricting function was reduced but vasodilatation was well preserved. Morphological findings supported this. The pharmacological vascular reactivity in the hypercholesterolemic rabbit ciliary artery is quite different from that of the large arteries. [Copyright &y& Elsevier]