Rabbit Nonpigmented Ciliary Epithelial Cells Research Articles

Melatonin and Its Analog 5-Methoxycarbonylamino-N-Acetyltryptamine Potentiate Adrenergic Receptor-Mediated Ocular Hypotensive Effects in Rabbits: Significance for Combination Therapy in Glaucoma

Melatonin is currently considered a promising drug for glaucoma treatment because of its ocular hypotensive and neuroprotective effects. We have investigated the effect of melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine, 5-MCA-NAT, on β₂/α(2A)-adrenergic receptor mRNA as well as protein expression in cultured rabbit nonpigmented ciliary epithelial cells. Quantitative polymerase chain reaction and immunocytochemical assays revealed a significant β₂-adrenergic receptor downregulation as well as α(2A)-adrenergic receptor up-regulation of treated cells (P < 0.001, maximal significant effect). In addition, we have studied the effect of these drugs upon the ocular hypotensive action of a nonselective β-adrenergic receptor (timolol) and a selective α₂-adrenergic receptor agonist (brimonidine) in normotensive rabbits. Intraocular pressure (IOP) experiments showed that the administration of timolol in rabbits pretreated with melatonin or 5-MCA-NAT evoked an additional IOP reduction of 14.02% ± 5.8% or 16.75% ± 5.48% (P < 0.01) in comparison with rabbits treated with timolol alone for 24 hours. Concerning brimonidine hypotensive action, an additional IOP reduction of 29.26% ± 5.21% or 39.07% ± 5.81% (P < 0.001) was observed in rabbits pretreated with melatonin or 5-MCA-NAT when compared with animals treated with brimonidine alone for 24 hours. Additionally, a sustained potentiating effect of a single dose of 5-MCA-NAT was seen in rabbits treated with brimonidine once daily for up 4 days (extra IOP decrease of 15.57% ± 5.15%, P < 0.05, compared with brimonidine alone). These data confirm the indirect action of melatoninergic compounds on adrenergic receptors and their remarkable effect upon the ocular hypotensive action mainly of α₂-adrenergic receptor agonists but also of β-adrenergic antagonists.

Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5‐MCA‐NAT

We have previously demonstrated that melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduce intraocular pressure (IOP) in New Zealand rabbits. More recently, we have shown that 5-MCA-NAT can also regulate ciliary adrenoceptor gene expression. Like adrenoceptors, carbonic anhydrase (CA) enzymes are involved in aqueous humour secretion by the ocular ciliary epithelium. Moreover, CA enzymes have been reported to be regulated by melatonin. Hence, the aim of this study was to investigate whether the hypotensive effect of 5-MCA-NAT is also because of a regulation of CA genes and enzymes. Time course of 5-MCA-NAT effect on rabbit IOP was followed for 7 hr every day for up to 144 hr (6 days). 5-MCA-NAT reduced IOP, maximally by 51.30 ± 2.41% (at 3 hr), and the hypotensive effect was maintained for up to 96 hr with a single application. IOP studies with 5-MCA-NAT plus Trusopt(®) and immunohistochemical analysis confirmed that CA are molecular targets of 5-MCA-NAT. In addition, real-time quantitative PCR (qPCR) and immunocytochemical assays were performed to determine changes in CA2 (CAII) and CA12 (CAXII) expression in cultured rabbit nonpigmented ciliary epithelial cells (NPE) treated with 5-MCA-NAT. NPE cells showed a prominent decrease in both CA, at the mRNA and protein levels. These data confirm that the long-term hypotensive effect of 5-MCA-NAT is also due, to a down-regulation of CA2 (CAII) and CA12 (CAXII) expression.

Regulation of ocular adrenoceptor genes expression by 5-MCA-NAT

We have demonstrated that 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduces intraocular pressure (IOP) in rabbits. In addition, we have reported a link between hypotensive effect of 5-MCA-NAT and sympathetic nervous system. Moreover, it is known that aqueous humour production is controlled by the activation of adrenoceptors (ADRs) present in the ocular ciliary epithelium. Thus, the aim of this study is to investigate if the hypotensive effect of 5-MCA-NAT is due to a regulation of ciliary ADR genes expression. To confirm this we followed the effect of 5-MCA-NAT on rabbit IOP for 144 consecutive hours. A sustained IOP reduction for up to 72 h (P<0.01) was seen. In addition, changes in ADRB2 and ADRA2A mRNA were measured in cultured rabbit nonpigmented ciliary epithelial cells. After 5-MCA-NAT treatment, a significant downregulation of ADRB2 and upregulation of ADRA2A was observed. These results provide the regulation of ADRs mRNA by 5-MCA-NAT.

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.

(-)-Isoproterenol modulation of maxi-K+ channel in nonpigmented ciliary epithelial cells through a G-protein gated pathway

Purpose. Adrenergic agents decrease intraocular pressure by reducing aqueous humor secretion from ciliary epithelial cells. Since the ionic concentration of aqueous humor contributes to intraocular pressure, we have investigated the effect of (-)-isoproterenol, a ß-adrenergic agonist on the maxi-K + channel in rabbit nonpigmented ciliary epithelial (NPE) cells. Methods. Single-channel currents were recorded from the basolateral surface of acutely isolated NPE cells using patch clamp techniques. Results. A calcium dependent maxi-K + channel was identified in 31% of cell-attached patches. In the excised condition the channel was activated in presence of calcium. In symmetrical K + solution a linear current-voltage relationship and unitary conductance of 158 ± 15pS was observed. Replacing K + with Na + the current-voltage curve shifted to the right and approached a reversal potential for K + (~–80 mV). Barium (2 mM) from the intracellular side or iberiotoxin (50 nM) from the extracellular side blocked the channel activity. In cell-attached patches, the ß-receptor agonist (-)-isoproterenol (2.5 µM) increased channel open probability (P o) only when applied directly through the patch pipette. ß 2 -adrenoceptorantagonists (ICI-118, 551, l-timolol) blocked the channel activity more efficiently than the ß 1 -adrenoceptor antagonist betaxolol. In excised patches, (-)-isoproterenol increased baseline P o 5-fold (0.5±0.13) when GTP (100 µM) and GTP?S (100 µM) were present at the cytosolic surface of the pipette (control; P o, 0.12±0.006). GTP augmented baseline channel activity (0.1 ± 0.004) 7-fold (0.7 ± 0.03) when (-)-isoproterenol was included in patch pipette. Conclusions. Rabbit NPE cells expressed maxi-K + channels on their basolateral surface. The adrenergic agonist (-)-isoproterenol activated these channels via a ß 2 -adrenoceptor that was modulated by a direct G-protein gated pathway.

Studies on H(+)-ATPase in cultured rabbit nonpigmented ciliary epithelium.

Studies were conducted to examine the influence of the H(+)-ATPase inhibitor bafilomycin A(1) on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A(1) (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A(1)-treated cells as judged by a 50% increase of ouabain sensitive potassium ((86)Rb) uptake. In bafilomycin A(1)-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium ((86)Rb) uptake in a dose-dependent manner with an IC(50) of approximately 2 microm. DMA (10 microm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A(1). Added alone, DMA (10 microm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by approximately 0.4 pH units. In cells that first received 10 microm DMA, the subsequent addition of bafilomycin A(1) (100 nm) caused a further cytoplasmic pH reduction of approximately 0.3 pH units. Taken together, the results suggest H(+)-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A(1), Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.

Hyposmotically activated chloride channels in cultured rabbit non-pigmented ciliary epithelial cells.

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.

Elevation of intracellular Ca 2+ concentration in rabbit nonpigmented ciliary epithelial cells by allicin

A previous study has shown that allicin produces changes in aqueous humor dynamics, and this study was conducted to examine possible cellular mechanisms. In rabbit nonpigmented ciliary epithelial cells, basal levels of [Ca 2+], were determined to be 164 ± 34 nM. Allicin, a sulfhydryl-reactive agent, induced Ca 2+ transients at 0.01 mM and at 0.2 mM, the Ca 2+ transient peaked at 732 ± 35 nM. Allicin-induced Ca 2+ transients were prevented by pretreatment with dithiothreitol which did not affect the basal Ca 2+ levels. Allicin had only a slight, insignificant, effect on L-type Ca 2+ currents, and allicin-induced Ca 2+ transients were also present under extracellular Ca 2+-free conditions. These data suggest that intracellular Ca 2+ stores are the most probable source of allicin's effect. Pretreatment of cells with ryanodine, an inhibitor of Ca 2+-induced-Ca 2+-release, inhibited allicin-induced Ca 2+ transients, but the basal Ca 2+ levels were unaffected by ryanodine. Thus, allicin-induced Ca 2+ transients are most likely mediated through ryanodine-sensitive intracellular Ca 2+ stores.

Identification of Prostanoid Receptors in Rabbit Non-pigmented Ciliary Epithelial Cells

Preliminary ligand binding studies demonstrated that the membrane preparations of the rabbit non-pigmented ciliary epithelial cell line have 3H-prostaglandin E 2binding sites. The binding sites were specific for 3H-prostaglandin E 2as demonstrated by competition with unlabeled prostaglandin E 2. The IC 50of prostaglandin E 2for the inhibition of 3H-prostaglandin E 2binding was 435n m. The stimulation of adenylyl cyclase and phospholipase C by prostanoid receptor agonists, in rabbit non-pigmented ciliary epithelial cells resulted in the formation of either cyclic AMP or inositol phosphates. Prostaglandin E 2and 16-16-dimethyl prostaglandin E 2(both are EP 1, EP 2, EP 3and EP 4receptor agonists), 11-deoxy prostaglandin E 1(EP 2, EP 3and EP 4receptor agonist), butaprost (EP 2receptor agonist), and prostaglandin D 2(DP receptor agonist) stimulated the formation of cyclic AMP in a dose-dependent manner. Maximal stimulation occurred between 1.25 and 2.5μ mfor prostaglandin E 2and 16,16-dimethyl prostaglandin E 2and between 10 and 20μ mfor 11-deoxy prostaglandin E 1and prostaglandin D 2. Prostaglandin E 2and 16,16-dimethyl prostaglandin E 2were more potent (EC 50of 0.25μ mand 0.42μ mrespectively) than 11-deoxy prostaglandin E 1, butaprost or prostaglandin D 2. The formation of cyclic AMP by prostaglandin D 2was inhibited by BW868C, a highly selective DP receptor antagonist. 17-phenyl trinor prostaglandin E 2, prostaglandin F 2αand U46619, the EP 1, FP and TP receptor agonists, respectively stimulated phospholipase C (as measured by the formation of total inositol phosphates) in a dose-dependent manner. The agonists 11-deoxy prostaglandin E 1and butaprost coupled to adenylyl cyclase via guanine nucleotide binding protein, G s, did not increase the turnover of inositol phosphates. The results of the present study suggest that rabbit non-pigmented ciliary epithelial cells express EP 1, EP 2, DP, FP and TP receptors.

Rilmenidine-induced ocular hypotension: role of imidazoline1 and alpha 2 receptors.

To examine ocular actions by rilmenidine, an imidazoline1 and alpha 2 adrenoceptor agonist. Intraocular pressure was measured in normal and sympathetically denervated rabbits by pneumatonometry. Electrically stimulated 3H-norepinephrine release from sympathetic nerves was determined in isolated, perfused rabbit iris-ciliary bodies. cAMP levels were evaluated in rabbit iris-ciliary bodies by radioimmunoassay. Ca2+ concentrations were measured in rabbit transformed nonpigmented ciliary epithelial cells by fluorescence ratio microscopy. Topical, unilateral administration of rilmenidine produced hypotensive responses in normal rabbits which were antagonized by either bilaterally administered efaroxan, an imidazoline receptor antagonist or rauwolscine, an alpha 2 receptor antagonist. Sympathectomy also eliminated the ocular hypotensive response. Rilmenidine (0.001, 0.01, 0.1, 1 microM) caused 5 +/- 1%, 18 +/- 5%, 35 +/- 10%, and 48 +/- 9% inhibition, respectively, of 3H-norepinephrine overflow whereas 10 microM efaroxan or rauwolscine caused enhancement of norepinephrine release by 102 +/- 23% or 86 +/- 25%, respectively. Furthermore, pretreatment with efaroxan or rauwolscine partially antagonized the inhibition of norepinephrine release induced by rilmenidine. In other experiments, rilmenidine (1 microM) inhibited isoproterenol-stimulated cAMP accumulation in rabbit iris-ciliary bodies by 43 +/- 9% which was antagonized by 10 microM efaroxan or rauwolscine. Rilmenidine induced large increases in [Ca2+]i in rabbit nonpigmented ciliary epithelial cells which were effectively antagonized by efaroxan or rauwolscine. These in vivo and in vitro data suggest that the ocular hypotensive activity induced by rilmenidine is due, in part, to suppression of sympathetic neuroeffector function in the rabbit ciliary body and that alpha 2 adrenergic receptors and/or imidazoline1 receptors are involved.

Oxymetazoline: potential mechanisms of inhibitory effects on aqueous humor dynamics.

Oxymetazoline, an alpha 2 agonist, was active in lowering intraocular pressure in normal and sympathetically denervated rabbit eyes. Ocular hypotension was accompanied by decreased aqueous humor inflow. Topical pretreatment with rauwolscine, an alpha 2 antagonist, reduced the oxymetazoline-induced hypotensive effect more in contralateral than in ipsilateral eyes indicating the possible involvement of central alpha 2 adrenoceptors. Efaroxan, a relatively selective imidazoline antagonist, and diclofenac, a cyclooxygenase inhibitor, failed to inhibit the oxymetazoline-induced ocular hypotensive response. Oxymetazoline induced mydriasis in treated eyes at all doses. In in vitro studies, oxymetazoline inhibited isoproterenol-stimulated cAMP production in rabbit iris-ciliary bodies and cultured rabbit nonpigmented ciliary epithelial cells. The inhibition of cAMP accumulation induced by oxymetazoline was antagonized by rauwolscine or by BRL-44408, a relatively selective alpha 2A-adrenoceptor antagonist. These data indicate that oxymetazoline lowered intraocular pressure by activating alpha 2A receptors (ciliary epithelium) and that the ocular hypotensive effect was not totally dependent on intact sympathetic nerves. Results suggest that mechanisms involving centrally mediated effects of oxymetazoline are probable and this possibility is currently under investigation.