TRPV channels mediate temperature-sensing in human corneal endothelial cells

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1–3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1–3 functional activity was evident based on capsaicin-induced Ca²⁺ transients and induction of these responses through rises in ambient temperature from 25 °C to over 40 °C. The currents underlying Ca²⁺ transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10–20 μM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 °C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La³⁺) (100 μM) and ruthenium-red (RuR) (10 μM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 μM) led to a reversible increase in intracellular Ca²⁺ indicating putative TRPV1–3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.     

Rapid increase of intracellular Ca2+ concentration caused by aminoadipic acid enantiomers in retinal Müller cells and neurons in vitro

The ability of the gliotoxic compounds D,L-, D- or L-2-aminoadipic acid (AAA) to increase selectively the intracellular concentration of free calcium ion ([Ca²⁺]_i) was examined in Müller cells cultured with or without retinal neurons. The monitoring of [Ca²⁺]_i following exposure to 0.06 to 6 mM AAA was performed by a microfluorometry using a fluorescent Ca²⁺ indicator, Fura-2 acetoxymethyl ester. A rapid increase of [Ca²⁺]_i occurred in the Müller cells following exposure to a relatively low concentration of the L-isomer. This is compatible with the known strong gliotoxicity of this isomer. The D,L- and D-forms of AAA activated neurons at low concentrations and activated the Müller cells at higher concentrations. The D-isomer appears to act selectively on retinal neurons and may be an agonist of an excitatory amino acid receptor. These results indicate that the ability of AAA to elevate cytosolic [Ca²⁺]_i depends on the stereospecificity of the AAA and on cell type.     

The mechanisms of calcium homeostasis and signalling in the lens

Excessive Ca²⁺ can be detrimental to cells and raised levels of Ca²⁺ in human lenses with cortical cataract have been found to play a major role in the opacification process. Ca²⁺ homeostasis is therefore, recognised as having fundamental importance in lens pathophysiology. Furthermore, Ca²⁺ plays a central role as a second messenger in cell signalling and mechanisms have evolved which give cells exquisite control over intracellular Ca²⁺ ([Ca²⁺]_i) via an array of specialised regulatory and signalling proteins. In this review we discuss these mechanisms as they apply to the lens. Ca²⁺ levels in human aqueous humour are approximately 1 mM and there is a large, 10,000 fold, inwardly directed gradient across the plasma membrane. In the face of such a large gradient highly efficient mechanisms are needed to maintain low [Ca²⁺]_i. The Na⁺/Ca²⁺ exchanger (NCX) and plasma membrane Ca²⁺-ATPase (PMCA) actively remove Ca²⁺ from the cells, whereas the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) sequesters Ca²⁺ in the endoplasmic reticulum (ER) Ca²⁺ store. In lens epithelial cells the dominant role is played by the ATPases, whilst in the fibre cells NCX activity appears to be more important. Usually, [Ca²⁺]_i can be increased in a number of ways. Ca²⁺ influx through the plasma membrane, for example, is mediated by an array of channels with evidence in the lens for the presence of voltage-operated Ca²⁺ channels (VOCCs), receptor-operated Ca²⁺ channels (ROCCs) and channels mediating store-operated Ca²⁺ entry (SOCE). Ca²⁺ signalling is initiated via activation of G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTK) of which the lens expresses a surprisingly diverse array responding to various neurotransmitters, hormones, growth factors, autocoids and proteases. Downstream of plasma membrane receptors are IP₃-gated channels (IP₃Rs) and ryanodine receptors (RYRs) located in the ER, which when activated cause a rapid increase in [Ca²⁺]_i and these have also been identified in the lens. Through an appreciation of the diversity and complexity of the mechanisms involved in Ca²⁺ homeostasis in normal lens cells we move closer to an understanding of the mechanisms which mediate pathological Ca²⁺ overload as occurs in the process of cataract formation.     

Role of the Phospholipase C Pathway and Calcium Mobilization in Oxytocin-Induced Contraction of Lacrimal Gland Myoepithelial Cells

PurposeWe reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP₃) which increases intracellular calcium concentration ([Ca²⁺]_i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC.MethodsTear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca²⁺]_i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software.ResultsOXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10⁻⁷ M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca²⁺]_i with a maximum response at 10⁻⁶ M. Furthermore, the activation of the IP₃ receptor to increase [Ca²⁺]_i is crucial for OXT-induced MEC contraction since blocking the IP₃ receptor with 2-APB completely abrogated this response.ConclusionsWe conclude that OXT uses the PLC/Ca²⁺ pathway to stimulate MEC contraction and increase lacrimal gland secretion.     

Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Müller cells

· Purpose: This study was conducted to detect the presence of muscarinic or nicotinic receptors in cultured retinal neurons and Müller cells. · Methods: Pure Müller cell cultures and cocultures of retinal neurons and Müller cells were used; the former, obtained from adult rabbit retinas, and the latter, retinal neurons from neonatal rats, were cocultured with Müller cells. Intracellular calcium ion concentration ([Ca²⁺]_i) following the administration of acetylcholine, a cholinesterase inhibitor (trichlorfon), nicotine or muscarinic agonist with or without a receptor antagonist was monitored using the calcium ion indicator, fura-2. · Results: Acetylcholine and trichlorfon induced rapid increase in [Ca²⁺]_i in half of either cell type. Trichlorfon induced positive response in coculture but not in the pure Müller cell cultures. This positive response was blocked only partially in the presence of atropine. Approximately 30–40% of neurons responded to nicotine at 5 μM, which was significantly blocked by α-bungarotoxin at 50 nM. No response to nicotine could be detected in Müller cells. Approximately 50% of neurons responded to muscarine at 50 μM, but 500 μM was required for the formation of calcium transients in 50% of Müller cells. The muscarine inducement of rapid increase in [Ca²⁺]_i was blocked by atropine. The agonist of M1 (a muscarinic receptor subtype), McN-A-343, at 0.5 μM induced the most significant and rapid increase in [Ca²⁺]_i both in neurons and Müller cells. McN-A-343 administration at 0.05 μM induced positive response in half the neurons, but only in approximately 10% of Müller cells. Such positive response was not observed following preincubation with the M1 antagonist, pirenzepine, at 50 μM. · Conclusions: Cocultured retinal neurons enhance the release of acetylcholine following anticholinesterase administration, and approximately half the neurons were found to possess muscarinic and nicotinic receptors. However, Müller cells appeared to possess only the less sensitive muscarinic receptor. Muscarinic receptor subtypes on either type of cell contained at least M1. Received: 17 November 1997 Revised version received: 20 April 1998 Accepted: 21 April 1998     

Expression profile of voltage-dependent Ca2+ channel subunits in the human retinal pigment epithelium

Background The secretion of a variety of factors by the retinal pigment epithelium (RPE) is essential for the structural integrity of the neuronal retina and choroid, but also plays a pivotal role in the etiology of diseases such as choroidal neovascularisation. A recent study showed that the secretory activity of the RPE is regulated by the activity of a certain type of voltage-dependent Ca²⁺ channels, the L-type channel. In order to provide a better base for the understanding of the underlying Ca²⁺ signalling in these cells, we investigated the expression profile of voltage-dependent Ca²⁺ channel subunits in RPE cells. Methods Using RT-PCR techniques with cDNA isolated from RPE cells, we investigated the expression pattern of Ca²⁺ channel subunits. Furthermore, we analysed Ba²⁺ currents through voltage-dependent Ca²⁺ channels in RPE cells by the patch-clamp technique. Results We detected the expression of two L-type channel subtypes and the expression of two different T-type channel subtypes. As accessory subunits, they expressed β2 and β4 and all known α₂δ subunits. In general, we were able to confirm these data with cDNA from the ARPE-19 cell line. They only showed some differences in their expression pattern of accessory subunits. Since the expression of T-type channels was so far unknown in RPE cells, we confirmed their expression in the RPE using cDNA isolated from freshly isolated human RPE cells. Furthermore, the patch-clamp analysis of Ba²⁺ currents showed a heterogeneous pattern of voltage-dependent inward currents in RPE cells. In some cells, typical slowly inactivating L-type currents were detected, whereas in other cells fast inactivating T-type currents could be detected. Conclusions These data indicate the expression of a so far not detected subtype of voltage-dependent Ca²⁺ channels, the T-type channels. Together with the expression of L-type channels, RPE cells show a comparable expression pattern to that of other secretory cells, such as β-islets of the pancreas.     

P1-/P2-purinergic receptors on cultured rabbit retinal Müller cells

Adenosine 5′-triphosphate (ATP) and its metabolic products function as neurotransmitters or neuromodulators under the control of P₁/P₂-urinergic receptors. To determine the presence of these receptors on retinal Müller cells, spectrofluorometry was carried out on intracellular calcium mobilization, using Fura-2 images. Müller cells were cultured from adult rabbit retinas. Cytosolic calcium ([Ca²⁺]_i) increased dose dependently with the application of ATP. This response was not blocked when a calcium channel blocker, nifedipine, was present, but this response was blocked, for the most part, when a P₂ receptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) was present. Increase in [Ca²⁺]_i was noted by the A₁ or A₂ agonist, which was blocked completely by each antagonist. Response to the a₁ agonist was apparent only at high concentrations. Increase in [Ca²⁺]_i was seen in some cells following administration of the P_2x agonist, methylene ATP, only at a high concentration (100 μM) but not in the presence of PPADS (50 nM). The greatest increase in [Ca²⁺]_i was induced by a P_2y agonist, methyl thio ATP at 1 to 10 μm, which was completely blocked by PPADS. Cultured Müller cells are thus shown quite likely to possess the P₁-/P₂- purinergic receptors including A₂ and P_2y.     

Partial characterization of a putative new growth factor present in pathological human vitreous

Background Several growth factors have been implicated in the development of proliferative eye diseases, and some of those are present in human vitreous (HV). The effects of HV on cellular responses which modulate proliferative cell processes were studied. This study describes the partial characterization of a vitreous factor activity which does not correspond to any of the previously reported growth factors in pathological HV. Methods Vitreous humour was obtained from medical vitrectomies, from patients with PDR and PVR. The biological activity of the vitreous factor was determined by its ability to increase cytosolic calcium concentration ([Ca²⁺ _i]), increase production of inositol phosphates, and induce cell proliferation in the cell line EGFR T17. In some experiments other cell lines, such as NIH 3T3, 3T3-L1, FRTL5, A431, PC 12, Y79, and GH₃, were also employed. Measurement of [Ca²⁺]_i in cell suspensions was performed using the fluorescent Ca²⁺ indicator fura-2. The activity of the factor present in HV was compared with other growth factors by means of: (a) [Ca²⁺]_i mobilization pattern, (b) sequential homologous and heterologous desensitization of receptors, (c) effects of phorbol esters on their action, and (d) inactivation after treatment with different proteolytic enzymes. Results The HV-induced cell proliferation and increases in [Ca²⁺]_i concentration were characterized by a peculiar time pattern. The different approaches used ruled out its identity with PDGF, bFGF, EGF, TGF-, IGFs, TNF-, NGF, and other compounds such as ATP, angiotensin I, and bradykinin. Vitreous factor actions are mediated by specific receptors apparently regulated by PKC. This factor is able to induce [Ca²⁺]_i mobilization in most of the cell lines studied, indicating that its effects are not tissue specific. Conclusions These results suggest the presence of a growth factor activity in pathological HV which may be due to the presence of an undescribed growth factor in the eye.     

Human anterior lens capsule epithelial cells contraction

Purpose: Human anterior lens epithelial cells, attached to surgically isolated capsules, were found to contract upon stimulation. The purpose of this study was to characterize these contractions, which create gaps between cells, and to assess the underlying physiological mechanisms and their possible association with cataract formation. Methods: Lens capsules obtained during cataract surgery were stained with fluorescent dye Fura‐2. Its fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in cell morphology and cytosolic free Ca²⁺concentrations ([Ca²⁺]_i) in response to pharmacological stimulation by acetylcholine (ACh) and to mechanical stimulation by flow of saline or direct contact. Results: Epithelial cells contracted in approximately a third of preparations when stimulated by either ACh application, fluid movement or direct mechanical contact. Contractions started either before or at best simultaneously with the rise in [Ca²⁺]_i. Contractions also occurred when there was hardly any change in [Ca²⁺]_i upon application of physiological saline alone. The probability of contractions occurring did not differ significantly among cortical, nuclear and combined cortical + nuclear cataract. Conclusions: This study provides the evidence that contractions of the anterior lens epithelial cells take place in significant portion of human lens anterior capsule postoperative preparations after non‐specific stimulation. Contractions are at least partially independent of changes in [Ca²⁺]_i. They can be mechanically induced, are localized and reversible and have a fast response and did not differ among different types of cataract. Physiological and clinical significance of this phenomenon remains to be elucidated.     

TRPV4在眼科疾病中的研究进展

瞬态电位受体香草醛4(TRPV4)是一组存在于细胞膜上的非选择性阳离子通道。它们是调节细胞功能和信号通路的感觉信号的重要介质。TRPV4在眼部各种组织中广泛表达,可参与多种生理功能,包括渗透压调节、Ca²⁺稳态、凋亡和自噬,在正常生理功能以及不同病理中起重要作用。最近研究发现TRPV4与角膜上皮损伤、青光眼、年龄相关性白内障、糖尿病视网膜病变、早产儿视网膜病变、视网膜脱离等疾病紧密联系,调控着相关眼科疾病的发生和发展过程。本文就TRPV4通路在眼科疾病中的研究进展做简要综述,为临床眼病治疗提供思路。     

Lens epithelial cell response to isoforms of platelet-derived growth factor

It has been reported previously that platelet derived growth factor (PDGF) may play an important role in the regulation of lens growth and differentiation. To evaluate PDGF-induced effects at the cellular level, we investigated the response of cultured bovine lens epithelial cells (BLEC) to PDGF-AB, -AA, and -BB isoforms at the cellular level. Stimulation of BLEC with PDGF isoforms showed no increase in cell proliferation under the culture conditions of this study. In contrast, measurement of cytosolic free calcium concentration ([Ca²⁺]_i), which has been shown to be an important second messenger for controlling multiple cellular processes in the lens, revealed a dose-dependent rise in [Ca²⁺]_i upon stimulation with PDGF-AB and -BB isoforms. PDGF-AA used in similar concentrations was not effective. Our data suggest that PDGF-AB and -BB may play a role in the regulation of cellular functions in BLEC via modulation of intracellular calcium homeostasis.Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 1991     

Increase in intracellular free calcium concentration of limulus photoreceptors caused by a metabolic inhibitor

Using a calcium indicator dye (arsenazo III), we detected a reversible rise in [Ca^2?]_i to about 2 mM in Limulus ventral photoreceptors when the cells were poisoned with the metabolic inhibitor DNP. Our results provide direct evidence for the hypothesis linking the action of metabolic inhibitors to an increase in [Ca^2?]_i in invertebrate photoreceptors.     

ETAReceptor Mediated Inhibition of Intracellular pH Regulation in Cultured Bovine Corneal Epithelial Cells

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H⁺-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pH_i). Furthermore, we characterized the effects on pH_iregulation of exposure to 1 μmET-1 under control and acid loaded conditions. With the pH sensitive dye, 2′,7′ Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pH_iwas 7.1 in NaCl (nominally HCO₃-free) Ringers. Inhibition of NHE with 100 μmdimethylamiloride (DMA) rapidly decreased pH_iby 0.37 units. Similarly, selective inhibition of V-type H⁺-ATPase with 10 μmbafilomycin A₁decreased pH_iby 0.22 units. Following acid loading in NaCl Ringers with a 20 mmNH₄Cl prepulse, pH_irecovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 μmDMA or 20 μmbafilomycin A₁. Based on decreases in H⁺efflux resulting from selective inhibition of NHE and V-type H⁺pump activity, NHE activity accounts for 76% of the pH_irecovery following acid loading. Under control conditions, ET-1 (1 μm) had no effect on pH_iwhereas ET-1 completely suppressed pH_irecovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ET_Abecause in the presence of BQ-123 (10 μm), a selective ET_Areceptor antagonist, pH_irecovery was completely restored. Suppression of pH_irecovery also occurred following stimulation of protein kinase C (PKC) with 10⁻⁷mphorbol myristate (PMA) whereas 10⁻⁷m4 α phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pH_irecovery after inhibition of PKC with 0.5 μmcalphostin C suggesting that the inhibition of pH_irecovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca²⁺-dependent calmodulin stimulated CaM II kinase with KN-62 (10 μm) reversed the suppression of pH_irecovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 μmphenylarsine oxide, 10 nmokadaic acid, 10 μmcyclosporin A₁or 20 μmBAPTA, also obviated the suppression of pH_irecovery by ET-1. Therefore ET_Areceptor mediated inhibition of pH_iregulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H⁺pump. This alteration may prevent them from becoming adequately stimulated to elicit pH_irecovery in response to acid loading.     

Effect of bestrophin-1 on L-type Ca channel activity depends on the Ca channel beta-subunit

Best’s vitelliforme macular degeneration is an inherited retinal degeneration associated with a reduction of the light-peak in the patient’s electro-oculogram. Bestrophin-1, the product of the disease-promoting/forming gene can function as regulator of voltage-dependent L-type Ca²⁺ channels in the retinal pigment epithelium (RPE). Since mice deficient for either β4-subunits or Ca_V1.3 subunits show reduced light-peaks, the regulatory function of bestrophin-1 on heterologously expressed Ca²⁺ channels composed of the pore-forming Ca_V1.3 and the auxiliary β4-subunit was analyzed. Precipitation of β4-subunits led to co-precipitation with bestrophin-1 and subsequent analysis of subcellular localization showed co-localization of bestrophin-1, Ca_V1.3 and β4-subunit in the cell membrane. Ca_V1.3 currents in the presence of β4-subunits and bestrophin-1 showed accelerated time-dependent activation and decreased current density compared to currents measured in the absence of bestrophin-1. In the presence of the β3-subunit, which is not expressed in the RPE bestrophin-1 did not modulate Ca_V1.3 activity. Deletion of a cluster of proline-rich motifs in the C-terminus of bestrophin-1 reduced its co-immuno precipitation with the β4-subunit and strongly reduced the Ca_V1.3 activity. Cells co-expressing bestrophin-1 lacking the proline-rich motifs and Ca_V1.3 subunits showed less efficient trafficking of bestrophin-1 into the cell membrane. In summary, we conclude that bestrophin-1 modulates L-type channels of the RPE via proline-rich motif-dependent interaction with β4-subunits. A disturbed interaction reduces the currents of the Ca_V1.3 subunits. This mechanism could open new ways to understand changes in the patient’s electro-oculogram and functional alterations of the RPE leading to retinal degeneration.     

Histological protection by cilnidipine, a dual L/N-type Ca channel blocker, against neurotoxicity induced by ischemia-reperfusion in rat retina

Although a blockade or lack of N-type Ca²⁺ channels has been reported to suppress neuronal injury induced by ischemia-reperfusion in several animal models, information is still limited regarding the neuroprotective effects of a dual L/N-type Ca²⁺ channel blocker, cilnidipine. We histologically examined the effects of cilnidipine on neuronal injury induced by ischemia-reperfusion, intravitreous N-methyl-d-aspartate (NMDA) (200 nmol/eye) and intravitreous NOC12 (400 nmol/eye), an nitric oxide donor, in the rat retina, and compared its effects with those of ω-conotoxin MV IIA, an N-type Ca²⁺ channel blocker and amlodipine, an L-type Ca²⁺ channel blocker. Morphometric evaluation at 7 days after ischemia-reperfusion showed that treatment with cilnidipine (100 μg/kg, i.v. or 0.5 pmol/eye, intravitreous injection) prior to ischemia dramatically reduced the retinal damage. Treatment with ω-conotoxin MV IIA before ischemia (0.1 pmol/eye, intravitreous injection) significantly reduced the retinal damage. However, amlodipine (30-100 μg/kg, i.v. or 0.1-1 pmol/eye, intravitreous injection) did not show any protective effects. Treatment with cilnidipine (100 μg/kg, i.v.) reduced the retinal damage induced by intravitreous NMDA, but not NOC12. These results suggest that cilnidipine reduces Ca²⁺ influx via N-type Ca²⁺ channels after NMDA receptors activation and then protects neurons against ischemia-reperfusion injury in the rat retina in vivo. Cilnidipine may be useful as a therapeutic drug against retinal diseases which cause neuronal cell death, such as glaucoma and central retinal vessel occlusion.     

Alpha-Adrenergic Agonists Stimulate Fluid Secretion in Lacrimal Gland Ducts

PurposeThe role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms.MethodsFluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca²⁺ level [Ca²⁺_i] were investigated with microfluorometry.ResultsBoth norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α_1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca²⁺-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [Cai2+].ConclusionsOur results prove the direct role of α₁-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of β-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α_1D receptor. Ca²⁺ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.     

Effects of Rho-associated protein kinase inhibitors Y-27632 and Y-39983 on isolated rabbit ciliary arteries

Purpose

In normotensive eyes, reduced ocular blood flow can lead to glaucoma pathogenesis. Drugs that reduce intraocular pressure (IOP) often cause vasodilation of the ciliary arteries and improve blood flow to the eye. A novel class of drugs called Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors can lower IOP. Therefore, we tested the ability of two ROCK inhibitors, Y-27632 and Y39983, to relax rabbit ciliary arteries.

Methods

We measured in vitro ciliary artery smooth muscle contractions by isometric tension recordings and changes of intracellular free calcium concentration ([Ca²⁺]_i) by fluorescence photometry.

Results

Both Y-27632 and Y-39983 induced a concentration-dependent relaxation in rabbit ciliary arteries precontracted with a high-potassium (high-K) solution. The amplitude of relaxation induced by Y-27632 and Y-39983 was not affected by either 100 ??M N ^G-nitro-l-arginine methyl ester (l-NAME) or 10 ??M indomethacin. In Ca²⁺-free solution, Y-27632 and Y-39983 significantly inhibited the transient contraction of ciliary arteries induced by 10 ??M histamine. However, neither Y-27632 nor Y-39983 affected the elevation of [Ca²⁺]_i induced by high-K solution and histamine.

Conclusions

We concluded that Y-27632 and Y-39983 relaxed isolated rabbit ciliary artery segments in vitro. The mechanism of relaxation was not dependent on endothelial-derived factors such as nitric oxide (NO) or prostacyclin, nor was it dependent on changes in intracellular Ca²⁺ concentration.     

Wakayama Symposium: Dependence of Corneal Epithelial Homeostasis on Transient Receptor Potential Function

Transient receptor potential (TRP) protein expression in the corneal epithelial layer contributes to the maintenance of tissue transparency. These proteins are members of a superfamily that form nonselective cation channels. This superfamily is a product of 28 different genes that are subdivided into six different subfamilies according to differences in amino acid sequence homology. The six subfamilies have very diverse functions. They are: 1) canonical (C); 2) vanilloid (V); 3) melastatin (M); 4) ankyrin (A); 5) polycystin (PP); 6) mucolipin (ML). TRP channels are composed of four monomeric subunits that are either members of the same or different subfamilies. In the corneal epithelium, C, V, and A subfamily subtype expression was identified. These include TRPV1-4, TRPC4, and TRPA1, which upon activation by either environmental stresses or selective ligands induce adaptive responses to stresses through transient increases in Ca²⁺ influx. Even though TRPs' Ca²⁺ permeability is variable relative to other cations, TRP activation is sufficient to stimulate mitogen-activated protein kinase cascade signaling through epidermal growth factor receptor transactivation. The host of TRP-mediated responses includes stimulation of cell proliferation, migration, regulatory volume behavior, and the release of a host of proinflammatory cytokines and chemoattractants. This review describes the multiple roles of these different channel subtypes in eliciting responses underlying maintenance of corneal epithelial function in health and disease.