Ciliary Body Epithelial Cells Research Articles

Novel Needle for Intravitreal Drug Delivery: Comparative Study of Needle Tip Aspirates, Injection Stream and Penetration Forces.

PurposeTo study the efficacy of a novel needle for intravitreal injection (IVI) in comparison to the conventional needle under experimental conditions.MethodsThe newly designed 30-gauge (G) needle (NDN) (EP 18158 542.3, patent pending) with occluded outer orifice and a side port for drug delivery was compared to the conventional standard hypodermic 30 G needle for IVI (SHN). An animal study to obtain needle tip aspirates was performed on 10 albino rat eyes. During IVIs, cellular content, which was cut by the needle tip, was aspirated. Cellular material was studied in regard to cell types and their quantity. The injection stream was studied using trypan blue dye in vitro and pig cadaver eyes. The penetration force was tested on polyurethane Testing Foil Strips PU 04 (Melab, Leonberg, Germany) by applying a velocity of 100 mm/min. The results were analyzed using descriptive statistics, correlation matrices and t-test methods with p<0.05 as statistically significant.ResultsCytological analysis of the needle aspirates showed the presence of cellular content in each case. The amount of conjunctival, ciliary body epithelial cells and granulated basophilic protein sediments (sign of cellular damage) in the case of the NDN tips was significantly lower compared to the SHN. The average penetration force of the NDN was 0.791 N, and in the case of the SHN was 0.566 N. The injection stream study revealed a difference in the initial injection phase between the two needle types, although the diffuse filling of the vitreous area which surrounded the needle tip appeared to be similar.DiscussionThe NDN demonstrated superior performance with regard to a significantly reduced number of cells being captured by the needle tip. Delivery of the injected fluid into the vitreous cavity was comparable. In order to investigate superior properties of the NDN needle design, further studies with improved prototypes would be necessary.

Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension.

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R–MT1R and D3R–MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation

Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved.

TRPV4 Stimulation Induced Melatonin Secretion by Increasing Arylalkymine N-acetyltransferase (AANAT) Protein Level.

Melatonin is a molecule which has gained a great deal of interest in many areas of science; its synthesis was classically known to be in the pineal gland. However, many organs synthesize melatonin, such as several ocular structures. Melatonin is known to participate in many functions apart from its main action regulating the circadian rhythm. It is synthesized from serotonin in two steps, with a rate-limiting step carried out by arylalkymine N-acetyltransferase (AANAT). In this report, the role of TRPV4 channel present in human ciliary body epithelial cells in AANAT production was studied. Several experiments were undertaken to verify the adequate time to reach the maximal effect by using the TRPV4 agonist GSK1016790A, together with a dose–response study. An increase of 2.4 folds in AANAT was seen after 18 h of incubation with 10 nM of GSK1016790A (p < 0.001, n = 6). This increment was verified by antagonist assays. In summary, AANAT levels and therefore melatonin synthesis change after TRPV4 channel stimulation. Using this cell model together with human ciliary body tissue it is possible to suggest that AANAT plays an important role in pathologies related to intraocular pressure.

Sphere formation permits Oct4 reprogramming of ciliary body epithelial cells into induced pluripotent stem cells.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by defined sets of transcription factors. We previously described reprogramming of monolayer-cultured adult mouse ciliary body epithelial (CE) cells by Oct4 and Klf4, but not with Oct4 alone. In this study, we report that Oct4 alone is sufficient to reprogram CE cells to iPS cells through sphere formation. Furthermore, we demonstrate that sphere formation induces a partial reprogramming state characterized by expression of retinal progenitor markers, upregulation of reprogramming transcription factors, such as Sall4 and Nanog, demethylation in the promoter regions of pluripotency associated genes, and mesenchymal to epithelial transition. The Oct4-iPS cells maintained normal karyotypes, expressed markers for pluripotent stem cells, and were capable of differentiating into derivatives of all three embryonic germ layers in vivo and in vitro. These findings suggest that sphere formation may render somatic cells more susceptible to reprogramming.

Ocular stem cells: a status update!

Stem cells are unspecialized cells that have been a major focus of the field of regenerative medicine, opening new frontiers and regarded as the future of medicine. The ophthalmology branch of the medical sciences was the first to directly benefit from stem cells for regenerative treatment. The success stories of regenerative medicine in ophthalmology can be attributed to its accessibility, ease of follow-up and the eye being an immune-privileged organ. Cell-based therapies using stem cells from the ciliary body, iris and sclera are still in animal experimental stages but show potential for replacing degenerated photoreceptors. Limbal, corneal and conjunctival stem cells are still limited for use only for surface reconstruction, although they might have potential beyond this. Iris pigment epithelial, ciliary body epithelial and choroidal epithelial stem cells in laboratory studies have shown some promise for retinal or neural tissue replacement. Trabecular meshwork, orbital and sclera stem cells have properties identical to cells of mesenchymal origin but their potential has yet to be experimentally determined and validated. Retinal and retinal pigment epithelium stem cells remain the most sought out stem cells for curing retinal degenerative disorders, although treatments using them have resulted in variable outcomes. The functional aspects of the therapeutic application of lenticular stem cells are not known and need further attention. Recently, embryonic stem cell-derived retinal pigment epithelium has been used for treating patients with Stargardts disease and age-related macular degeneration. Overall, the different stem cells residing in different components of the eye have shown some success in clinical and animal studies in the field of regenerative medicine.

Sphere Formation Permits Oct4 Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Sphere Formation Permits Oct4 Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Facile and Efficient Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells are attractive for cell replacement therapy, because they overcome ethical and immune rejection issues that are associated with embryonic stem cells. iPS cells have been derived from autonomous fibroblasts at low efficiency using multiple ectopic transcription factors. Recent evidence suggests that the epigenome of donor cell sources plays an important role in the reprogramming and differentiation characteristics of iPS cells. Thus, identification of somatic cell types that are easily accessible and are more amenable for cellular reprogramming is critical for regenerative medicine applications. Here, we identify ciliary body epithelial cells (CECs) as a new cell type for iPS cell generation that has higher reprogramming efficiency compared with fibroblasts. The ciliary body is composed of epithelial cells that are located in the anterior portion of the eye at the level of the lens and is readily surgically accessible. CECs also have a reduced reprogramming requirement, as we demonstrate that ectopic Sox2 and c-Myc are dispensable. Enhanced reprogramming efficiency may be due to increased basal levels of Sox2 in CECs. In addition, we are the first to report a cellular reprogramming haploinsufficiency observed when reprogramming with fewer factors (Oct4 and Klf4) in Sox2 hemizygous cells. Taken together, endogenous Sox2 levels are critical for the enhanced efficiency and reduced exogenous requirement that permit facile cellular reprogramming of CECs.

Facile and Efficient Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Facile and Efficient Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Functional Expression of MRP4 in the Ciliary Epithelium of the Eye

The multidrug‐resistance associated protein 4 (MRP4) is an efflux pump that transports organic anions, including para‐aminohippurate. Transepithelial transport of para‐aminohippurate by the ciliary epithelium was previously demonstrated (Pelis and Delamere, The FASEB Journal 23, Abstract #797.9, 2009). This led to the hypothesis that MRP4 contributes to organic anion transport by the ciliary epithelium. MRP4 expression was examined using reverse transcriptase PCR, Western blotting and functional assays. MRP4 mRNA and protein were detected in the freshly dissected human ciliary body and in a simian virus transformed cell line derived from human ciliary body epithelial cells (SVHCE cells). MRP4 protein levels (relative to total protein) in the native tissue and cultured cells were comparable. In SVHCE cells, the intracellular accumulation of the fluorescent MRP4 substrate bimane glutathione increased in a dose‐dependent manner in the presence of the MRP4 inhibitors, probenecid and indomethacin. These data indicate that MRP4 contributes to organic anion transport by the human ciliary epithelium. The functional expression of MRP4 in SVHCE cells makes them a potentially useful system for examining MRP4‐mediated organic anion transport by ciliary epithelia. Supported by NIH.

Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Recombinant AAV-Transduced Iris Pigment Epithelial Cell Transplantation May Transfer Vector to Native RPE but Suppress Systemic Dissemination

To determine whether adenoassociated virus (AAV) vectors transduced into iris pigment epithelial (IPE) cells and transplanted into the subretinal space of rats will transfer the AAV genome to the host cells and whether the vectors are disseminated systemically. Recombinant (r)AAV was transduced into rat IPE cells and transplanted into the subretinal space of rats. For the control, rAAVs alone were injected subretinally. The transplanted IPE cells were detected by LacZ staining. Immunohistochemistry, electron microscopy, electroretinography, and fluorescein-dextran angiography were performed. DNA was extracted from various organs and blood and examined for the AAV genome by polymerase chain reaction. No toxicity from rAAV transduction was observed in vitro. LacZ was expressed in the transplanted cells 1 and 2 weeks after transplantation. At 4 and 12 weeks, fewer transplanted cells were detected than at 1 week, and LacZ expression was occasionally detected at the level of host retinal pigment epithelial (RPE) cells. Expression was also detected in ciliary body epithelial cells. The electroretinograms and fluorescein-dextran angiography were only mildly altered. Significantly lower levels of AAV genome were detected in the organs and blood of rats receiving rAAV-IPE cell transplants than with direct intravenous injection of AAV vectors. AAV-mediated LacZ was expressed in the transplanted cells after subretinal transplantation, and the transplanted IPE cells may transfer the rAAV to host tissues, such as RPE cells, long after the transplantation. This method of gene delivery did not lead to systemic dissemination of the vectors.

Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells.

To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis were examined for RANTES expression by use of an ELISA and reverse transcription-polymerase chain reaction (RT-PCR). Expression of RANTES mRNA and protein content of primary cultures of equine ciliary pigmented epithelial cells (RT-PCR) and culture supernatant (ELISA) were measured 6 or 24 hours, respectively, after cultures were stimulated with interleukin-1beta and tumor necrosis factor-alpha. Strong reactions to intradermal hypersensitivity testing and significant chemotaxis of equine leukocytes to rh-RANTES wereas observed. Aqueous humor of eyes from horses with uveitis contained increased concentrations of rh-RANTES-like protein (mean +/- SD, 45.9+/-31.7 pg/ml), compared with aqueous humor from clinically normal horses (0 pg/ml). Ciliary body from horses with uveitis expressed RANTES mRNA, whereas ciliary body from clinically normal horses had low mRNA expression. Stimulated ciliary pigmented epithelial cells expressed increased amounts of rh-RANTES-like protein (506.1+/-298.3 pg/ml) and mRNA, compared with unstimulated samples. Ciliary epithelium may play a role in recruitment and activation of leukocytes through expression of RANTES.

Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells

We have examined the interaction between muscarinic and alpha(2)-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca(2+) concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca(2+) and IP formation, activation of alpha(2)-receptors does not significantly increase either intracellular Ca(2+) or IPs over basal levels. However, simultaneous activation of muscarinic and alpha(2)-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca(2+) increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and alpha(2)-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca(2+), because it is decreased but not prevented by blocking the entry of Ca(2+) with LaCl(3) or chelating intracellular Ca(2+) with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca(2+) resulting from simultaneous activation of muscarinic and alpha(2)-adrenergic receptors.

Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts

We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca2+ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca2+ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca2+ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell's cytoarchitecture.

Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells.

Atrionatriuretic peptide (ANP) lowers intraocular pressure in the eyes of humans and rabbits. We examined the effects of natriuretic peptides on cGMP formation and 125I-labelled-ANP binding to cultured cells derived from ciliary body epithelium, the site of aqueous humour formation in the eye. ANP, brain natriuretic peptide (BNP) and C-natriuretic peptide (CNP) at 1 microM stimulated cGMP formation 8.2(+/-1.2)-fold, 4.8(+/-0.6)-fold and 87.3(+/-12.1)-fold respectively. 125I-ANP bound to intact cells at a single site, with a dissociation constant KD=0.30+/-0.01 nM. BNP was as effective as ANP in displacing 125I-ANP, whereas CNP displaced label with a slightly higher IC50. 125I-ANP binding was displaced >95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of 125I-ANP to cells labelled predominantly a protein of Mr 62000. These data suggest that 125I-ANP binding was primarily to NPR-C, whereas cGMP stimulation occurred primarily via natriuretic peptide B receptors (NPR-B). Vasopressin and histamine, both activators of the inositol phosphate/diacylglycerol phosphate pathway in non-pigmented ciliary epithelial cells, inhibited CNP stimulation of guanylate cyclase (NPR-B) and 125I-ANP binding (NPR-C) by 30-38%. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4alpha phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of 125I-ANP binding and cGMP formation by PMA. These results suggest that protein kinase C (PKC) down-regulates both NPR-B and NPR-C. PKC down-regulation of NPR-B varied inversely with CNP concentration. Inhibition by 1 microM PMA was 30.6(+/-4.0)% with 500 nM CNP, but 83.4(+/-8.8)% with 10 nM CNP, indicating that increasing CNP could partially overcome inhibition by PMA. Since extracellular CNP levels were not affected by PKC activation, the effect of PKC on NPR-B is best explained as a reduction in NPR-B affinity for CNP. NPR-C measured as 125I-ANP binding was likewise reduced 36.4(+/-5.1)% by exposure to PMA. In contrast with NPR-B inhibition, however, inhibition of NPR-C was due largely to a reduction in the number of receptor binding sites per cell rather than a reduction in receptor affinity for ligand. The data therefore suggest that both NPR-B and NPR-C are down-regulated by PKC, but that the mechanisms of down-regulation of the two receptors are different.

Synergistic Rise in Ca2+Produced by Somatostatin and Acetylcholine in Ciliary Body Epithelial Cells

The purpose of these experiments was to demonstrate the presence of somatostatin receptors on the nonpigmented epithelial cells of the rabbit ciliary body and their link with intracellular Ca2+homeostasis. Freshly excised rabbit ciliary processes and nonpigmented cell layer explants were loaded with the fluorescent dye fura-2, and free-Ca2+concentration ([Ca2+]i) in the nonpigmented cells was measured with fluorescence ratio imaging. The cells were continuously perfused, and drugs were added to the perfusate. Somatostatin-14 (SS14, 0.1–1.0 μm) or acetylcholine (ACh, 10 μm) applied alone produced small increases in [Ca2+]i. However, SS14 (0.1 μm) in combination with ACh (10 μm) induced a massive increase in [Ca2+]i(25.7±3.3 times the baseline level,n=28). The dose-response curve for SS14 (in the presence of 10 μmACh) was sigmoidal with an EC50of 3.9 nmand Hill coefficient of 2.5, indicating the requirement for multiple SS receptor activation. Somatostatin-28 could mimic the effect of SS14, although a much higher concentration was required. Shifting the SS14 dose–response curve to the right by about two-orders of magnitude resulted in a fit to the SS28 data. The response to ACh+SS14 could not be blocked by the α2-adrenergic blocker yohimbine (Yoh, 10 μm) or the A1-specific adenosinergic antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μm). Incubation of the tissue with pertussis toxin (PTx, 1 μg ml−1) did not alter the response to ACh alone but eliminated the synergistic effect of somatostatin. We conclude that nonpigmented epithelial cells of the rabbit ciliary body possess a novel somatostatin receptor whose activation can synergistically potentiate the rise in [Ca2+]iproduced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through Gi.

Transient activation of K+ channels by carbachol in bovine pigmented ciliary body epithelial cells.

The action of carbachol (CCh) on isolated pigmented ciliary epithelial cells was examined using whole cell patch-clamp recording. Application of 100 microM CCh caused transient, occasionally oscillatory, increases in the inward and outward currents, followed by a long-term decrease in both currents. Caffeine produced transient responses similar to those of CCh. The responses to CCh were blocked by the muscarinic receptor antagonist atropine and the inositol 1,4,5-trisphosphate receptor blocker heparin (200 micrograms/ml in patch pipette). Manipulation of the internal ionic concentrations indicated that only K+ conductances were affected by CCh. Changing intracellular Ca2+ concentration ([Ca2+]i) with the calcium ionophore ionomycin demonstrated that both the inward rectifier K+ current and the outward current exhibited Ca2+ dependence. There was no Cl- current stimulated either directly by CCh or indirectly by modulators of [Ca2+]i, and any Cl- currents present arose from osmotic effects. In the short term, muscarinic stimulation will activate K+ channels by causing a transient increase in [Ca2+]i. This effect only lasts for 1-5 min, however, and, in the long term, the conductance decreases below its original level. The effect of such a transient increase in [Ca2+]i on secretion would be complex, involving effects on gap junction communication between the pigmented and nonpigmented cell layers and the activation state of Cl- channels in the nonpigmented cells. This complexity probably accounts for the variable reports of the effects of muscarinic stimulation of the ciliary body in vivo.

Cell-attached patch clamping of the intact rabbit ciliary epithelium.

Following thorough removal of adhering aqueous humor, we have succeeded in patch clamping the intact rabbit ciliary epithelium in the cell-attached and inside-out excised-patch modes. Rapidly fluctuating currents ("chatter activity') were observed during recordings conducted for as long as 1 h. Chatter activity did not reflect seal instability since interconversion was noted between chatter activity and transitions between stable open and closed states, excision of patches into the bath was associated with substantial shifts in the reversal potential, and chatter activity could be triggered by sustained hyperpolarization, but was insensitive to stretch. The chatter channel was identified as cation-nonselective from the reversal potentials both in the cell-attached and excised-patch modes. The channel's kinetics were similar to those of the cGMP-activated phototransduction channel. The results of PCR amplifications of fragments in cDNA libraries from both human ciliary body and human nonpigmented ciliary epithelial (NPE) cells indicated that human ciliary epithelial cells transcribe message for the retinal phototransduction channel. The possible role of the phototransduction channel in expressing chatter activity was further explored by perfusing preparations with a known activator of that channel (cGMP) and with a known inhibitor (L-cis-diltiazem). Neither agent significantly affected chatter behavior. We conclude that: (1) this is the first demonstration of the feasibility of patch-clamping the intact ciliary epithelium; (2) the NPE cells display chatter activity arising from rapidly fluctuating transitions of a cation-nonselective channel; (3) NPE cells can transcribe message for the cation-nonselective phototransduction channel; and (4) if the observed chatter activity is from a homologue of the photo-transduction channel, the homologue is pharmacologically distinct.

Swelling activates chloride current and increases internal calcium in nonpigmented epithelial cells from the rabbit ciliary body

Membrane current and [Ca]i in rabbit nonpigmented ciliary body epithelial cells (NPE cells) were monitored with combined patch-clamp and fura-2 measurements during cell swelling induced by anisosmotic conditions. In the presence of K-channel blockers, cell swelling produced an increase in membrane current, accompanied by an increase in [Ca]i. Structural changes in the cell, associated with membrane deformation, may be the cause of the increase in [Ca]i during swelling. The conductance activated by swelling was permeable to Cl: it was dependent on the Cl concentration gradient across the cell membrane, and it was blocked by the Cl-channel blockers DIDS, SITS, NPPB, and DIOA. Although swelling increased both Cl current and [Ca]i, there was no evidence that Ca was involved in the regulation of the Cl conductance. Cell swelling activated the current even when [Ca]i was strongly buffered at an elevated level (500 nM) or at a low level (approximately 0) with internal Ca-BAPTA/Cs-BAPTA mixtures. In addition, Cl conductance was unaffected when [Ca]i was increased with a Ca ionophore. There was also no evidence that cAMP participates in the regulation of the Cl conductance: swelling activation of the current occurred in the presence of cAMP inhibitor (Rp-cAMP-S) and cAMP mimic (Sp-cAMP-S). The data suggest independent involvement of Cl conductance and internal Ca in the regulation of cell volume in NPE cells.