A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells

OBJECTIVE: To evaluate the predictive value of clinical variables for the finding of a positive minor salivary gland biopsy (focus score > or = 2) in patients investigated for Sj?gren's syndrome (SS). METHODS: One hundred twenty-one patients with sicca symptoms were referred to a multidisciplinary SS clinic in a tertiary hospital. Each patient was evaluated on protocol and labial salivary gland (LSG) biopsy was obtained. Using the San Diego criteria as a model, patient data were subjected to a cross sectional analysis on an algorithm to determine when the LSG biopsy would be most useful for determining the diagnosis of SS in clinical practice. RESULTS: Eighty-four patients had sufficient data to be included in the study. Forty patients had LSG biopsy with focus score < 2 and 44 had focus score > or = 2. Twenty-three patients had objective evidence of sicca and positive serology according to criteria standards. Eighteen of these had a positive biopsy (78%). The remaining 5 patients had many extraglandular features suggestive of SS, and the biopsies appeared to add little practical information. Patients with incomplete criteria for sicca could be diagnosed as possible SS (3 of 4 criteria) with a positive biopsy in 14 of 18 cases. The finding of anti-Ro or anti-La positivity in patients with incomplete criteria for sicca predicted a positive LSG biopsy in 85.7% of such cases. Patients with incomplete sicca and negative anti-Ro and anti-La had a negative LSG biopsy in 82% of cases. CONCLUSION: The LSG biopsy is most necessary in patients who have partial San Diego criteria for sicca and positive anti-Ro or anti-La antibody. Where SS is not reasonably suspected, or where the diagnosis is clinically obvious, the LSG biopsy adds little useful clinical information.     

Histamine induced contraction of human ciliary muscle cells

In cultured human ciliary muscle cells we previously showed that histamine, via an H1 receptor, stimulates the production of inositol phosphates and mobilization of intracellular calcium. We further investigated in this study whether histamine would cause contraction of human ciliary muscle cells. Photomicrographs were taken of the ciliary muscle cells before and after exposure to histamine. Cross sectional surface area of the cells was quantified using image analysis software. A decrease in cross sectional surface area was interpreted as an indication of cell contraction. The results of this study indicated that histamine (10(-6) M-10(-4) M) caused contraction of human ciliary muscle cells in a concentration-dependent fashion. The effect of histamine was mediated by the H1 receptor subtype since the histamine effect was antagonized by 10(-6) M chlorphentramine (an H1 receptor subtype selective antagonist) but not by 10(-6) M cimetidine (H2 antagonist) or thioperamide (H3 antagonist). The phospholipase C (PLC) inhibitor, U73122 (10(-6) M) and the intracellular calcium store depleting agent thapsigargin (10(-6) M) both prevented the histamine induced contraction, demonstrating that the activation of PLC and the intracellular calcium release were the key steps necessary for contraction. Our data indicate that in ciliary muscle cells, histamine, via an H1 receptor, activates PLC and increases intracellular calcium, which subsequently causes contraction of the cells.     

Calmodulin antagonists increase the expression of membrane-type-1 matrix metalloproteinase in human uterine cervical fibroblasts

The treatment of human uterine cervical fibroblasts with concanavalin A (ConA), or a specific calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or trifluoperazine resulted in accumulation of an active form of matrix metalloproteinase 2 (MMP-2, gelatinase A). In contrast, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weaker antagonist of calmodulin, did not modulate the activation of proMMP-2. The activation of proMMP-2 was confirmed by the enhanced activity on gelatin and the conversion of proMMP-2 to a 62-kDa form by zymography and western blotting. The plasma membrane, but not the conditioned medium, of the W-7- or trifluoperazine-treated cells activated proMMP-2; this activation was blocked by membrane-type-1 MMP (MT1-MMP) antibody and EDTA. The plasma membrane from trifluoperazine- or ConA-treated cells contained MT1-MMP and tissue inhibitor of metalloproteinases 2. Both trifluoperazine treatment and ConA treatment increased the steady-state levels of MT1-MMP mRNA and proMMP-2 mRNA. These results, together with our previous observations on the production of proMMP-1 (interstitial procollagenase) and proMMP-3 (prostromelysin 1) [Ito, A., Sato, T., Ojima, Y., Chen, L.-C., Nagase, H. & Mori, Y. (1991) J. Biol. Chem. 266, 13598-13601], suggest that calmodulin negatively regulates the matrix turnover by suppressing the production of a number of proMMPs including proMMP-1, proMMP-3 and MT1-MMP, and the activation of proMMP-2 in human uterine cervical fibroblasts.     

Preparation of cultured smooth muscle cells from human myometrium for X-ray microanalysis

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer's solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer's solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer's solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer's solution.     

Confocal imaging of calcium release events in single smooth muscle cells

Filtrates of heat (54 degrees) treated day-5 Penicillium fellutanum cultures contained 70 mg of peptidophosphogalactomannan-II; an unheated control contained 30 mg. The polymer contained up to 60 phosphodiesters, and 5-O-beta-D-galactofuranosyl, mannopyranosyl, amino acyl and 2-aminoethanol residues. Its 13C NMR spectrum was nearly identical with that of the control polymer. The major 31P NMR signal was phosphocholine phosphodiester at delta 0.22 ppm: significant phosphodiester signals occurred at delta 1.15, 1.33 and 1.47. Dilute mineral acid released galactofuranosyl residues from the mannan. Signals at delta 1.15-1.47 ppm were associated with molecules of mass less than 3500 and contained galactose, 2-aminoethanol and peptide(s). After this acid treatment, signals at delta 1.0-0.22 remained associated with the mannan. Heat released up to 4-fold more of peptidophosphogalactomannan-III compared with the untreated control; carbohydrate and phosphate content, per mg polymer, were reduced by 4- and 2-fold, respectively. A galactofuranosyl-, phosphoryl- and amino acyl-containing polymer of Mr greater than 14,000 was solubilized by alkali treatment of P. fellutanum cell walls.     

Ambient pressure stimulates immortalized human aortic endothelial cells to increase DNA synthesis and matrix metalloproteinase 1 (tissue collagenase) production

In the present study, we investigated the effect of ambient pressure on [3H]-thymidine incorporation and on the production of matrix metalloproteinase 1 (tissue collagenase/proMMP-1) using human aortic endothelial cells immortalized with simian virus 40 (SE-1). Incubation of cells at ambient pressures of 50 and 100 mmHg for 24 h slightly increased [3H]-thymidine incorporation when directly compared with normal culture conditions. The amount of [3H]-thymidine incorporated in SE-1 reached a maximum at 150 mmHg, while a further increase in pressure to 200 mmHg decreased incorporation. The same ambient pressure slightly stimulated human aortic intimal smooth muscle cells (SMC) to increase [3H]-thymidine incorporation but not medial SMC. Immunoblot analysis also showed that ambient pressure, ranging from 50 to 200 mmHg, like 12-O-tetradecanoyl-phorbol-13-acetate stimulated SE-1 to produce proMMP-1, an effect not seen with either intimal or medial SMC. The amount of proMMP-1 produced also reached a maximum level at 150 mmHg. We postulate that human endothelial cells are ambient pressure sensitive and that relatively lower ambient pressures play an important role in the growth of endothelial cells, while higher pressures injure endothelial cells, resulting in the initiation of atherosclerosis. This cell line may prove useful in the investigation of both the physiological and pathological roles of blood pressure on endothelial cell function.     

Proliferation and extracellular matrix production by human infragenicular smooth muscle cells in response to interleukin-1 beta

PURPOSE: Atherosclerotic peripheral vascular disease commonly involves the infragenicular arterial tree. Our study evaluated the effect of interleukin (IL)-1 beta on the proliferation of vascular smooth muscle cells (VSMCs) derived from atherosclerotic infragenicular arteries of human subjects who underwent below-knee amputation, as well as the role of IL-1 beta in VSMCs' production of extracellular matrix components, substances that are important in the transformation of VSMCs from the contractile to the synthetic phenotype. This transformation to the synthetic phenotype is an important step in the formation of the atherosclerotic lesion. METHODS: Cultures were identified as being of smooth muscle origin through staining with the cytoskeletal marker, alpha-smooth muscle actin. Proliferation assays were performed by seeding confluent cultures of passages 4 to 7 into six-well plates at 10,000 cells per well. After serum starvation, samples were incubated with IL-1 beta (1 ng/ml). Cell number was determined on a daily basis. To study extracellular matrix production, cells were propagated in tissue culture chamber slides in the absence or presence of growth media containing IL-1 beta. After fixation with 100% methanol, each sample was stained with a primary antibody specific for an extracellular matrix component. After staining with the fluorescein-tagged secondary antibody, each sample was examined using immunofluorescent microscopic examination. RESULTS: The results of our proliferation assays showed that IL-1 beta caused a significant increase in the proliferation of VSMCs at 24, 48, 72, and 96 hours (p < or = 0.003 when comparing IL-1 beta-treated samples with control specimens at each time period using unpaired t test). The number of IL-1 beta-treated cells at 96 hours was double the number present in the control samples (16,033 +/- 238 vs 8102 +/- 824). When compared with control samples, IL-1 beta was found to affect the production of extracellular matrix proteins by infragenicular VSMCs. IL-1 beta caused an increase in the production of fibronectin, a decrease in the production of laminin, and no change in the production of collagen type IV. CONCLUSIONS: These results suggest that interleukin-1 beta acts as a potent stimulant of the proliferation of human infragenicular VSMCs. IL-1 beta also acts to augment the production of fibronectin by these cells. Fibronectin has been implicated in the phenotypic transformation of VSMCs from the contractile to the synthetic state. Therefore, IL-1 beta may serve as an important regulatory factor in the development of atherosclerosis by stimulating the proliferation of VSMCs and their transformation to the synthetic state, two important steps in the formation of the atherosclerotic lesion.     

Arachidonic acid-induced calcium signalling in human airway smooth muscle cells

Until now computer-assisted parasite identification was based on database applications requiring data specification on an individual basis, thus limiting the ability of the system to handle rule-based knowledge as humans are used to do. A new Expert PArasite IdentificatiON (EPAION: Greek term for expert) system was developed to serve as an interface between the database and the user, where the database is a repository for bionomic and morphological facts about the parasites for the expert system. The system was developed by using a logic-based computer language which allows the definition of rules and facts to assist the creation of queries to the database. The components of the system are the knowledge base, the multimedia data base, the inference mechanism, and the graphical user interface. The operational modules of the system are the Parasite Identifier and the system Utilities. This expert system facilitates knowledge incorporation in a manner simulating the natural mental process, thus allowing the checking of the accuracy of the information that the user feeds to the computer and the creation of intelligent queries to the database. These characteristics accelerate focusing and optimize the parasite identification scheme regardless of the user's profile of competency.     

Shear stress-induced release of PGE2 and PGI2 by vascular smooth muscle cells

This study addresses the direct effect of fluid flow shear stress on production of the vascular mediators, PGE2 and PGI2 by vascular smooth muscle cells (SMC). Results indicate that shear stress increases PGE2 and PGI2 release in SMC. The production patterns, however, differ between PGE2 and PGI2. For PGE2, the rate of production is moderate for the first three hours after the onset of shear stress, then dramatically increases between the fourth and fifth hours, returning to basal levels in the sixth hour. On the other hand, the rate for PGI2 production is maximal right after the onset of shear and remains elevated for the first three hours. The rate then plateaus and remains at a moderate level during the next three hours. The results also indicate that SMC production of PGI2 is more sensitive to shear stress than PGE2 production since a level of 0.5 dynes/cm2 produces a maximal PGI2 release whereas 1 dyne/cm2 produces only 1/4 the response seen at 20 dynes/cm2 for PGE2. The physiological implications of fluid shear stress regulation of SMC are discussed.     

Membrane type-1 matrix metalloproteinase in human ocular tissues

PURPOSE: Membrane type-1 matrix metalloproteinase (MT1-MMP) (MMP-14) (EC 3.4.24.xx) is involved in the activation of progelatinase A (MMP-2) (EC 3.4.24.24). MMP-2 is present at least in the interphotoreceptor matrix and vitreous. The purpose of this study was to determine the distribution of MT1-MMP, and MMP-2, in human ocular tissues. METHODS: The distribution of MT1-MMP and MMP-2 was investigated in vitreous and in membrane extracts from eye tissues obtained from postmortem human eyebank eyes. Western blot analysis was performed using mouse monoclonal anti-MT1-MMP and anti-MMP-2 antibodies. RESULTS: MT1-MMP was found in sclera, cornea, lens, choroid, retinal pigment epithelium (RPE) and retina. MMP-2 was found in sclera, cornea, choroid, vitreous, RPE and retina but was absent from lens. CONCLUSION: We provide the first evidence for the presence and distribution of membrane-type-1 matrix metalloproteinase in human ocular tissues. MT1-MMP may be responsible for the activation of progelatinase A in many ocular extracellular matrices in a manner similar to that exhibited in other systems.     

Ultrastructural observations of the human uterine smooth muscle cells during gestation

At term pregnancy, the myometrium consists of bundles of smooth muscle cells bound together by varying amounts of connective tissue. Each bundle contains both dark and light muscle cells. During uterine contractions it is believed that the smooth muscle cells become darker, decrease in volume, and exhibit changes in diameter. This is accompanied by widening of the interspaces and by a decrease in the areas of cellular contact. Between contractions, there are more light cells which become arranged closer to each other and exhibit large areas of interdigitation. The significance of these observations in the mechanism of uterine contraction and retraction is discussed. Cell believed to be modified smooth muscle cells occupy the myoendometrial junction and the decidua basalis. They are irregular in shape, poor in myofilament content, and rich in other cytoplasmic organelles and form a loosely arranged layer of cells between the myometrium and the trophoblast. The possible functional significance of these cells is also discussed.     

Ion channels in freshly isolated and cultured human bronchial smooth muscle cells

Voltage-gated ion currents were studied in human bronchial airway smooth muscle (ASM) cells. Proliferating or growth-arrested cells in culture were compared with freshly isolated cells. Three types of charybdotoxin (ChTX)-sensitive K+ channel were observed in all cell types, with conductances in symmetrical 140 mM KCl solutions ([Ca2+]i < 0.1 nM) of 206 +/- 14 pS (n = 32), 144 +/- 11 pS (n = 27) and 109 +/- 5 pS (n = 25). The relative proportion of each channel type differed substantially between the three groups of cells. In freshly isolated ASM cells large conductance K+ channels were represented almost entirely by a conductance of 206 pS, which was found in all twenty-three patches studied. In contrast, in most patches from proliferating cells the majority of channels had conductances of either 144 pS (14 of 21 patches) or 109 pS (8 of 21 patches). Cultured cells that had been growth arrested by serum depletion revealed the same set of channels as the proliferating cells, but the occurrence of the 109 pS channel was much more frequent (16 of 19 patches). As has been shown previously, 206 pS channels were active at a physiological membrane potential (-60 to -20 mV) even at a very low free [Ca2+]. The 144 pS channels could be recorded only at depolarized potentials (+80 to +100 mV), whereas 109 pS channels were active over a wide range of potentials (-60 to +100 mV), but only in the presence of GTP. In a proportion of cultured cells a tetrodotoxin-sensitive Na+ current and a hyperpolarization-induced inwardly rectifying K+ current were also observed (15 and 21%, respectively, of all cells examined). Neither of these currents were observed in freshly isolated cells. Whole-cell outward current in all groups was sensitive to tetraethylammonium, ChTX, and iberiotoxin, but not to 4-aminopyridine. In summary, it is clear that during proliferation there are considerable changes in the expression of ionic channels in ASM that have profound functional significance. In particular, these changes would tend to make the tissue more excitable, and may be of relevance to the proliferative process itself.     

The effect of radiographic contrast media on human vascular smooth muscle cells

The relation between intravascular radiographic contrast media (RCM) and myointimal hyperplasia after percutaneous transluminal angioplasty is not known. We have investigated the cytotoxic effects of RCM on human vascular smooth muscle cells (VSMCs) and their effect on the growth of these cells. The cytotoxic effects of RCM were studied using human VSMCs. The cells after being grown to confluency were exposed for 60 min to 250 mgI ml-1 of diatrizoate, ioxaglate, iopromide, iotrolan and saturated mannitol solutions. The control group was treated with only 15% fetal calf serum (FCS) containing medium. The viability of the cells was examined using the trypan blue exclusion test. The effect of RCM on growth was assessed by exposing the VSMCs after growth arrest, for either 15 or 60 min to 250 mgI ml-1 of diatrozoate, ioxaglate, iopromide, iotrolan and saturated mannitol solution. There was no significant change in the viability of the VSMCs after 60 min exposure to iopromide, iotrolan, saturated mannitol solution, and after 15 min exposure to diatrizoate or ioxaglate. After exposure to diatrizoate or ioxaglate for 60 min, 16.5 +/- 2.2% or 9.2 +/- 2.6% dead cells were found, respectively (p < 0.05 versus control). In the growth assay of VSMCs, diatrizoate, ioxaglate and saturated mannitol solutions reduced the growth rate (p < 0.05 versus control). No significant change was observed with iopromide and iotrolan. In conclusion, ionic RCM have cytotoxic and cytostatic effects on VSMCs while non-ionic media have no effects. There is no direct stimulatory effect of contrast media on the growth of VSMCs. The cytotoxic and cytostatic effects of contrast media seems to be both osmolality and chemotoxicity dependent. Low osmolar non-ionic RCM are not likely to contribute to the mechanisms responsible for myointimal hyperplasia after angioplasty.     

Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone-metastasizing cancer cells

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases are implicated in various steps of development of metastasis, through their ability to degrade the extracellular matrix. Increased matrix metalloproteinase activity of tumor cells has been associated with a higher metastatic potential. Inhibitors of metalloproteinases have been shown to effectively reduce or prevent the formation of metastases. The family of tetracyclines is able to inhibit matrix metalloproteinase activity through chelation of the zinc ion at the active site of the enzyme. Using tumor cell lines relevant to bone metastases, i.e. PC-3, MDA-MB-231, Hs696, B16/F1, we showed that tetracycline and derivatives of tetracycline, namely doxycycline and minocycline, also induced cytotoxicity. The effective concentrations are relatively high for plasma, but are clinically achievable in the bone, since tetracyclines are osteotropic. All four bone-metastasizing tumor cells produced and secreted various matrix metalloproteinases. Doxycycline was able to inhibit the activity of 72- and 92-kDa type IV collagenase secreted by bone-metastasizing cells by 79-87%. These characteristics could make tetracycline a unique candidate as a therapeutic agent to prevent bone metastases in cancer patients with a high likelihood for development of bone metastasis. Studies using animal models of experimental bone metastasis will be necessary to confirm this.     

Role of extracellular signal-regulated kinases in angiotensin II-stimulated contraction of smooth muscle cells from human resistance arteries

BACKGROUND: We assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries. METHODS AND RESULTS: VSMCs derived from resistance arteries (<300 microm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4+/-0.36, Emax=541+/-55 nmol/L), pHi (pD2=9. 4+/-0.29, Emax=7.19+/-0.01), and contraction (pD2=9.2+/-0.21, Emax=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses. CONCLUSIONS: These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.     

Identification of myoglobin in human smooth muscle

Myoglobin (Mb) has been believed to be absent generally from mammalian smooth muscle tissue. Examination of human rectal, uterine, bladder, colon, small intestine, arterial, and venous smooth muscle by immunohistochemical techniques shows that each of these tissues is immunopositive for both smooth muscle myosin and human Mb. Mb-specific primers were used for the polymerase chain reaction to generate cDNA from smooth muscle tissues. Southern hybridization with a Mb-specific probe gave a very strong signal with the cDNA from rectum, weaker signals from small intestine and uterus, a faint signal from colon, and no signal from bladder tissue. High performance liquid chromatography analysis coupled with sequence determination has shown that contaminating heme-binding serum albumin as well as hemoglobin in extracts of smooth muscle seriously compromise any heme-based or spectrophotometric assay of Mb. Combined affinity and size exclusion chromatography, however, provide the necessary resolution. The cDNA-derived amino acid sequence of human smooth muscle Mb was found to be identical to that of Mb from striated muscle.     

Effect of glycated collagen on proliferation of human smooth muscle cells in vitro

While non-enzymatic glycation of long-lived tissue proteins such as collagen has been implicated in chronic complications of diabetes mellitus, its role in the aetiology of diabetic macroangiopathy has not been elucidated. To test the hypothesis that glycation of collagen abolishes the inhibitory effect of native collagen on the proliferation of human smooth muscle cells, we obtained smooth muscle cells from human gastric arteries and cultured them on dishes coated with glycated or non-glycated collagen. The proliferation of human smooth muscle cells in the presence of 10% fetal calf serum or platelet derived growth factor-BB (10 ng/ml) was inhibited by type 1 collagen coated on the dishes. Glycation of collagen with glucose 6-phosphate for 7 days abolished the growth-inhibitory effect of native collagen. Succinylation of collagen, which like glycation blocked the lysyl residues in collagen, also abolished the growth-inhibitory effect. Adhesion of human smooth muscle cells to collagen-coated dishes was not affected by glycation of collagen. Addition of glycated albumin to the medium did not affect the growth of human smooth muscle cells on plastic dishes. The inhibition of human smooth muscle cell proliferation by collagen was not reversed by the glycation of collagen in the presence of aminoguanidine. Results suggest that early glycation abolishes the inhibitory effect of collagen on human smooth muscle cell proliferation and may thus participate in the progression of macro-angiopathy in diabetes.     

A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl--channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.