Quantification of MUC5AC protein in human tears.

PURPOSE: MUC5AC has been identified as a major secretory mucin of conjunctival goblet cells and precorneal tear film. However, no method has been reported to quantify MUC5AC protein in human tears. The objective of this study was to establish a method to measure the amount of MUC5AC in human tears and to correlate the amount of MUC5AC with age, gender, and dry eye diseases. METHODS: A goat antibody was raised to synthetic peptides corresponding to nonglycosylated epitopes of human MUC5AC mucin. This antibody and a horseradish peroxidase-coupled second antibody were used to develop a quantitative immunoassay to measure the MUC5AC concentration of tear samples collected on Schirmer strips. Porcine stomach mucin was used as a standard for the assay. The chemiluminescent MUC5AC signal was digitized and quantified. Tear samples from 19 healthy volunteers and 31 clinically diagnosed dry eye patients were analyzed. RESULTS: MUC5AC concentration in human tears ranged from undetectable to more than 200 microg/mL porcine stomach mucin equivalent. In the healthy population, low, moderate, and high concentrations were found in the tear samples from younger and older persons and from both men and women. The mean MUC5AC content in tears was lower in the dry eye patients than in the age- and gender-matched healthy individuals. CONCLUSIONS: A method was established to quantify MUC5AC in human tear samples obtained on Schirmer strips. There was no correlation between the amount of MUC5AC and age or gender in the healthy population. Dry eye disease patients, however, typically showed reduced concentrations of soluble MUC5AC in the tear film.

[Presynaptic 5-hydroxytryptamine receptors enhance cholinergic neurosecretion in the human iris-ciliary body].

OBJECTIVE: To characterize presynaptic 5-hydroxytryptamine (5-HT) heteroreceptors which modulate 3H-acetylcholine (3H-ACh) release in isolated human iris-ciliary bodies (ICBs). METHODS: ICB tissue segments were perfused and incubated with 3H-choline, and electrically stimulated four times (S1, S2, S3 and S4) at 10 Hz for 1 min to elicit 3H-ACh secretion. Test agents, 5-HT agonists and antagonists, were added before S2, S3 and S4 and their effects were determined by the stimulation ratio (Sx/S1) of evoked 3H-ACh release. 3H-ACh in perfusate fractions was isolated by ion exchange chromatography and analyzed for radioactivity by liquid scintillation spectrometry. RESULTS: The evoked 3H-ACh release was enhanced in a concentration-dependent manner by 5-HT [10(-9)-10(-5) mol/L, 50% effective concentration values (EC50) = 3.36 x 10(-8) mol/L] as well as 5-methoaxytryptamine [5-MOT (agonist), 10(-8)-10(-5) mol/L, EC50 = 6.59 x 10(-7) mol/L]. The selective 5-HT4 antagonist, GR113808A (10(-8) mol/L) inhibited the 5-HT-inducing increase of the cholinergic response, producing parallel right shifts of the 5-HT concentration-response curves (t = 4.012, P < 0.01). The selective 5-HT3 antagonist ondersetron (5 x 10(-7) mol/L) and tropisetron (10(-9) mol/L) did not affect 5-HT inducing 3H-Ach release (t = 2.215, P > 0.05). CONCLUSION: Our results indicate that cholinergic terminals in the human ICB contain 5-HT heteroreceptors that may promote the release of 5-HT and belong to the 5-HT4 subtype.

Quantification of MUC2 and MUC5AC transcripts in human conjunctiva.

PURPOSE: Transcripts of mucins 1, 4, and 5AC have been identified in human conjunctival tissue. Of these, only MUC5AC has been localized to goblet cells. MUC2 is a goblet cell mucin originally identified in the intestinal mucosa. The presence of MUC2 transcripts and levels of MUC2 and MUC5AC transcripts in normal human conjunctiva, as determined by quantitative polymerase chain reaction (PCR), is reported. METHODS: RNA from conjunctivae of six donors (3 men, 3 women, 44 to 69 years, all white) was isolated and subjected to competitive reverse transcription-PCR. Internal standards, which are dsDNA molecules with ends complementary to a given primer pair but containing nonhomologous central sequences, were prepared for each gene assayed. Titration of a constant amount of cDNA against serial dilutions of the internal standard allowed quantification of the template cDNA. MUC2 and MUC5AC levels were compared to levels of the "housekeeping" gene, beta2-microglobulin (beta2M). The identity of PCR products was confirmed by sequencing. RESULTS: In the six individual samples tested, beta2M mRNA is expressed, on average, at approximately 10(-20) moles per sample (1 microg RNA) or approximately 63.5x10(4) molecules. The mRNA encoding MUC5AC, a relatively abundant ocular mucin, exists at levels 10-fold lower than beta2M. In contrast to previous reports of MUC2 mRNA being absent at the ocular surface, these results show that MUC2 transcripts are present and expressed at levels 5900-fold lower than for MUC5AC. Apparently, MUC2 transcripts exist on the order of only approximately 100 to 1000 molecules/microg of RNA in the analyzed samples. CONCLUSIONS: MUC2 transcripts are present in human conjunctival tissue and their abundance is much lower than that of MUC5AC. This is the first application of competitive PCR to the quantitative analysis of mucin expression in human ocular tissue. The sensitivity of this method allows the detection of MUC2 transcripts that were not detected by Northern blot analysis or in situ hybridization in previous studies. It also makes possible the comparison of relative levels of expression for ocular mucins.

MUC5AC mucin is a component of the human precorneal tear film.

PURPOSE: Mucins are important structural and functional components of the precorneal tear film, yet little is known of their composition and synthesis. The mRNAs of MUC1, MUC4, and MUC5AC have previously been identified in human conjunctiva. Of these, only MUC5AC mRNA appears to be associated with goblet cells. The purpose of the this study was to quantify MUC5AC transcript levels, to identify MUC5AC protein in conjunctiva, tears, and goblet cells and to determine whether this mucin is secreted in response to the calcium ionophore ionomycin. METHODS: MUC5AC mRNA from normal human conjunctiva was identified, quantified, and compared with beta2-microglobulin levels using a competitive reverse transcription-polymerase chain reaction (RT-PCR) method. An antibody to a MUC5AC peptide was used to localize this mucin in conjunctival sections by immunohistochemistry. Anti-MUC5AC antiserum was used to label western blot analysis of conjunctiva and tears. Conjunctival tissues were incubated with ionomycin, and secreted mucins were detected with Helix pomatia agglutinin conjugated to horseradish peroxidase and with anti-MUC5AC antiserum. RESULTS: MUC5AC and beta2-microglobulin transcripts were expressed at a ratio of approximately 1:500. Immunochemical labeling showed that MUC5AC was localized in conjunctival goblet cells and at the apical surface of the conjunctival epithelium. MUC5AC protein was present in conjunctiva and in the tear film. Ionomycin stimulation of conjunctival secretion resulted in a fourfold increase in total mucin secretion and in a corresponding increase in secreted MUC5AC. CONCLUSIONS: MUC5AC is synthesized by goblet cells of the normal human conjunctiva, and this mucin is a component of conjunctival secretions and of normal human tears.

Regulation of ocular mucin secretion by P2Y2 nucleotide receptors in rabbit and human conjunctiva.

The effects of adenine analogues on secretion of high molecular weight, mucin-like glycoproteins (mucins) by conjunctival goblet cells were investigated using isolated rabbit and human conjunctiva. Mucin secretion was assayed using a quantitative dot-blot assay of Helix pomatia agglutinin-horseradish peroxidase binding to mucins absorbed to nitrocellulose filters. In rabbit conjunctiva, exogenous ATP (10(-7)-10(-3) m) induced a concentration-dependent, four-fold increase in mucin secretion that reached a plateau 15 min after drug addition. The rank order of potency of agonists was UTP>=ATPgammaS>ATP>ITP>ADP>>AMP. Adenosine, alpha,beta-methylene- ATP and beta,gamma-methylene-ATP were ineffective in stimulating mucin release. The response to ATP was unmodified by the putative P2 purinergic antagonists suramin or reactive blue (5x10(-5) m). In human conjunctiva, ATP and UTP were nearly equipotent in stimulating mucin secretion with an EC50 congruent with5x10(-6) m. These findings demonstrate that rabbit and human conjunctival cells contain functional P2Y2 (formerly designated as P2U) nucleotide receptors that govern mucin secretion. These receptors may provide useful pharmacological targets for therapeutic modulation of tear film mucins in dry-eye disorders and/or corneal wound healing.

M2-type muscarinic receptors mediate prejunctional inhibition of norepinephrine release in the human iris-ciliary body.

Human muscarinic receptors comprise a family of five separate gene products, three of which (designated as M1, M2 and M3 subtypes) can be distinguished pharmacologically. Previous work indicates that sympathetic nerve terminals in the anterior uvea contain prejunctional muscarinic receptors that, upon activation by agonists, inhibit the neural release of norepinephrine. The aim of this study was to characterize the prejunctional effects of muscarinic agents on the electrically-evoked secretion of 3H-norepinephrine in isolated, superfused human iris-ciliary body tissue segments. Stimulation-evoked 3H-norepinephrine release was inhibited > 80% by carbachol and muscarine, but was unaffected by the M1-selective agonist McN A-343. Pilocarpine behaved as a partial agonist in this system, producing less than 40% of the maximum inhibition. The rank order of potency of selective antagonists at prejunctional muscarinic receptors was methoctramine (M2) > AF-DX 116 (M2) > pirenzepine (M1) > or = para-fluoro hexahydro-sila-definidol (M3). These data suggest that prejunctional muscarinic receptors in the human iris-ciliary body correspond to the M2 subtype. No evidence for age-related differences in prejunctional muscarinic receptor activity was seen in tissues obtained from 13 human donors, aged 10-83 years. Prejunctional muscarinic receptors may play a role in mediating the inhibitory effects of parasympathetic nerve stimulation or cholinomimetic drugs on ocular sympathetic neurotransmission in vivo.

Prejunctional alpha 2-adrenoceptors and adenylyl cyclase regulation in the rabbit iris-ciliary body.

Agents that elevate intracellular cyclic AMP (cAMP) have been found to enhance the synaptic discharge of norepinephrine (NE) from sympathetic nerve terminals in the rabbit iris-ciliary body and other peripheral tissues. We explored the hypothesis that prejunctional alpha 2-adrenergic receptors that mediate feedback inhibition of NE release may be coupled to adenylyl cyclase inhibition. To indirectly monitor cAMP changes in sympathetic axon terminals, we analyzed the cAMP-mediated activation of tyrosine hydroxylase, a sympathetic marker protein that undergoes acute phosphorylation and activation by cAMP-dependent protein kinase A. Tyrosine hydroxylase activity was assayed in situ by incubation of rabbit iris-ciliary body tissue segments in buffered Krebs-Ringer solution containing the substrate tyrosine (100 microM) and the DOPA decarboxylase inhibitor brocresine (30 microM). Intraneuronal DOPA accumulation was quantified by HPLC with electrochemical detection. Tyrosine hydroxylase activity was increased approximately 2 fold by incubation with forskolin (10 microM) plus IBMX (0.5 mM) or with 8-Bromo-cAMP (3 mM). Simultaneous addition of the alpha 2-adrenergic agonist clonidine (1 microM) attenuated the response to forskolin/IBMX, but had no effect on the response to 8-Br-cAMP. Clonidine-mediated inhibition of the forskolin/IBMX response was abolished by treatment of tissues with N-ethylmaleimide (NEM), an alkylating agent that inactivates pertussis toxin-sensitive G proteins (Gi) that couple receptors to adenylyl cyclase inhibition. These findings suggest that prejunctional alpha 2-adrenoceptors in the rabbit iris-ciliary body are negatively coupled to adenylyl cyclase. This mechanism may contribute to autofeedback regulation of NE biosynthesis and release.

Neuromodulatory effect of sulprostone on the circadian elevation of intraocular pressure in rabbits.

Topical application of sulprostone, a preferential prostaglandin EP3 receptor agonist, caused a dose-dependent reduction of the circadian elevation of intraocular pressure (IOP) in New Zealand albino rabbits which were entrained to 12-hr/12-hr light-dark environment. Corresponding to the effect on IOP, 0.2 micrograms, 2 micrograms, and 20 micrograms sulprostone decreased the norepinephrine (NE) concentration in the aqueous humor in the dark phase. There was no breakdown of the blood-aqueous barrier. Bilateral applications of 2 micrograms and 20 micrograms sulprostone to entrained rabbits that had undergone unilateral, preganglionic transection of the cervical sympathetic trunk reduced the circadian IOP elevation in the intact eye, but caused little IOP change in the decentralized eye. These results indicate that the IOP-lowering effect of topical sulprostone in rabbits is dependent on sympathetic neural activity and that prejunctional inhibition of NE release may be an important mechanism of action.

Prejunctional receptors and second messengers for angiotensin II in the rabbit iris-ciliary body.

Angiotensin II has been shown to act prejunctionally to facilitate sympathetic neutrotransmission in various tissues including the iris-ciliary body. In the present study, we characterized the prejunctional angiotensin II receptor subtype and its signal transduction pathway in the rabbit iris-ciliary body. Angiotensin II caused concentration-dependent facilitation of electrically evoked [3H]-norepinephrine overflow from the isolated, superfused rabbit iris-ciliary body without affecting basal tritium efflux. Responses to angiotensin II were antagonized by saralasin and DuP753 but not by PD123177 indicating that prejunctional angiotensin II receptors of the AT1-subtype mediate the facilitation of evoked [3H]-norepinephrine release. The non-selective cyclic nucleotide phosphodiesterase inhibitor, isobutylmethyl xanthine enhanced the angiotensin II response whereas the cAMP-specific phosphodiesterase inhibitor, RO-20-1724 had no effect. In the presence of 8-bromo-cGMP, responses elicited by angiotensin II were significantly (P < 0.01) greater than that caused in the absence of 8-bromo-cGMP. In contrast, 8-bromo-cAMP had no effect on the angiotensin II-induced response. Guanylate cyclase inhibitors, methylene blue and LY83583 abolished angiotensin II-induced enhancement of [3H]-norepinephrine overflow without affecting basal tritium efflux. Taken together, these results suggest that cGMP could be involved in the angiotensin II response. Neither phospholipase C inhibitors (neomycin, 2-nitro-4-carboxyphenyl-N,N-diphenyl carbamate and phenylmethylsulfonyl fluoride) nor an inhibitor of protein kinase C (staurosporine) had any significant effect on the angiotensin II response, indicating that metabolites of inositol phospholipid metabolism or activation of protein kinase C are not involved in the response to this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Prejunctional modulation of norepinephrine release in the human iris-ciliary body.

PURPOSE: To characterize the prejunctional mechanisms that control the impulse-evoked release of norepinephrine in the isolated, superfused human iris-ciliary body. METHODS: Human iris-ciliary body tissue segments were preincubated with 3H-norepinephrine, superfused and electrically-stimulated in vitro to evoke the discharge of 3H-norepinephrine. The effects of prejunctional modulators on evoked 3H-norepinephrine overflow were evaluated. RESULTS: Stimulation-evoked (but not spontaneous) 3H-norepinephrine release was inhibited by alpha 2-adrenergic, muscarinic, dopaminergic, neuropeptide Y, and prostaglandin agonists and was enhanced by angiotensin II. Agonist-induced effects on 3H-norepinephrine overflow were blocked by selective antagonists, where available. Yohimbine and atropine alone enhanced 3H-norepinephrine output, suggesting that prejunctional alpha 2-adrenergic and muscarinic receptors undergo tonic activation by endogenously released neurotransmitters. CONCLUSIONS: Human ocular sympathetic nerves express inhibitory alpha 2-adrenergic, muscarinic, dopaminergic, prostaglandin, and neuropeptide Y receptors and facilitatory angiotensin II receptors that control the impulse-evoked release of 3H-norepinephrine. These receptors may be useful targets for pharmacologic manipulation of the adrenergic system in vivo.

Prejunctional prostaglandin receptors in the human iris-ciliary body.

Prostaglandins (PGs) of the E series have been shown to modulate sympathetic neurotransmitter release in a variety of peripheral tissues and organs, including the eye. In this study, we evaluated the inhibitory effects of a series of naturally-occurring and synthetic PGs on field stimulation-evoked release of 3H-norepinephrine (3H-NE) from isolated, superfused segments of human iris-ciliary body. Field-stimulated 3H-NE secretion was calcium-dependent, blocked by selective inhibitors of voltage-sensitive calcium and sodium channels, and originated from a desipramine-sensitive transmitter pool. Evoked 3H-NE release was inhibited in a concentration-dependent manner by PGE2 (EC50 = 45 nM) and several closely related compounds with the following rank order of potency: sulprostone greater than 16,16-dimethyl-PGE2 greater than PGE2 greater than 11-deoxy-PGE1. By contrast, PGF2 alpha was relatively inactive (EC50 greater than 10 microM) in this system. None of the above compounds significantly modified spontaneous 3H-NE efflux. PGE2-mediated inhibition was not antagonized by the selective prostanoid EP1-receptor antagonists AH 6809 (10 microM) or SC-19220 (30 microM), nor did these agents alone affect basal or field-stimulated 3H-NE release. The results suggest that human ocular sympathetic nerves possess inhibitory PG receptors which have the pharmacological properties of the EP3 subtype. These receptors may play a role in local feedback regulation of sympathetic transmission in the iris-ciliary body, and may contribute to symptoms of acute ocular inflammation, including vasodilation, miosis and hypotony.

Prejunctional inhibitory effects of prostanoids on sympathetic neurotransmission in the rabbit iris-ciliary body.

Both naturally occurring and synthetic prostaglandins (PGs) caused concentration-dependent inhibition of electrically evoked [3H]norepinephrine (NE) overflow from the isolated, superfused rabbit iris-ciliary body without affecting basal tritium efflux. The rank order of potencies of the agonists was: sulprostone greater than 16, 16-dimethyl-PGE2 greater than PGE2 greater than 11-deoxy-PGE1 greater than iloprost (stable PGl2 analog) greater than PGF2 alpha greater than or equal to PGD2. However, the Tx-mimetic, U-46619, was without effect on transmitter release at concentrations up to 1 microM. The selective EP1-receptor antagonists, AH 6809 (30 microM) or SC-19220 (10 microM) had no effect on basal or field-stimulated [3H]NE secretion, nor did they antagonize the PGE2-mediated reduction of evoked [3H]NE overflow. Indomethacin (3 microM) and the 5-lipoxygenase inhibitor, BW A4C (1 microM) were without effect on basal or evoked [3H]NE release, suggesting that endogenously formed arachidonic acid metabolites have no significant modulatory role in this in vitro system. Inhibitory effects of submaximal or maximal concentrations of PGE2 combined with corresponding concentrations of clonidine or carbachol were not additive, suggesting that prejunctional PGE2 receptors coexist with alpha-2 adrenergic and muscarinic receptors at neurotransmitter release sites. In the presence of yohimbine (100 nM) and/or atropine (100 nM), however, the inhibition produced by PGE2 was enhanced markedly, suggesting that tonic activation of prejunctional alpha-2 adrenergic or muscarinic receptors by endogenously released transmitters may impair the response to exogenous PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic cholinergic inhibition of adenylate cyclase in the rabbit iris-ciliary body and ciliary epithelium.

The effects of cholinergic agents on hormone-stimulated cyclic AMP (cAMP) accumulation were investigated in iris-ciliary body segments, excised ciliary processes, and isolated ciliary epithelium from albino rabbit eyes. In all three tissue preparations, the cholinergic agonist carbamylcholine markedly inhibited the stimulation of cAMP biosynthesis by vasoactive intestinal peptide VIP--a potent activator of nonpigmented ciliary epithelial adenylate cyclase. Carbamylcholine also attenuated cAMP increases mediated by isoproterenol, prostaglandin E2, and forskolin. The effects of carbamylcholine on VIP-induced cAMP synthesis were concentration dependent (EC50 = 23 nM), mimicked by selective muscarinic cholinergic agonists (oxotremorine, pilocarpine), and antagonized by atropine. Carbamylcholine- and clonidine-mediated inhibition of VIP-stimulated cAMP accumulation in ciliary processes were nonadditive, indicating that inhibitory muscarinic and alpha 2-adrenergic receptors coexist on VIP-responsive target cells. These findings suggest that the cholinergic system may have a direct role in modulation of ciliary epithelial adenylate cyclase and aqueous humor secretion.

Inhibitory effects of neuropeptide Y on sympathetic neurotransmission in the rabbit iris-ciliary body.

Neuropeptide Y (NPY, 1-300 nM) mediated a concentration-dependent inhibition of field stimulation-evoked [3H]norepinephrine (NE) overflow from the isolated, superfused rabbit iris-ciliary body. At equimolar concentrations (100 nM), the homologous neuropeptide peptide YY (PYY) mimicked the effects of NPY, whereas pancreatic polypeptide (PP) and the C-terminal fragment of NPY did not modify [3H]NE release. NPY-induced inhibition of [3H]NE release was unaffected by pretreatment of tissues with atropine (100 nM) plus yohimbine (100 nM) and was non-additive with the maximal prejunctional effects of carbamycholine or clonidine, indicating that NPY acts independently of prejunctional muscarinic or alpha 2-adrenergic receptor activity to reduce [3H]NE overflow. It is concluded that NPY is a specific, potent modulator of adrenergic neurosecretion in the rabbit iris-ciliary body. These findings confirm the role of NPY as a co-transmitter at ocular sympathetic neuroeffector junctions, either mimicking or augmenting the actions of endogenously released norepinephrine.

Potentiation of norepinephrine secretion by angiotensin II in the isolate rabbit iris-ciliary body.

The prejunctional effects of angiotensin II (AII) on stimulation-evoked secretion of 3H-norepinephrine (3H-NE) were investigated by in vitro methods in isolated, superfused rabbit iris-ciliary body preparations. AII (0.1-10 nM) concentration-dependently enhanced the field- stimulated release of 3H-NE (EC50 = 0.1 nM), nearly doubling evoked neurotransmitter release with no apparent effect on spontaneous 3H-NE efflux. The response to 1 nM AII was abolished by the selective AII receptor antagonist saralasin [( Sar1,Val5, Ala8]-angiotensin II; 500 nM), which alone did not modify 3H-NE overflow. AII-mediated effects on neurosecretion were partially additive to those of forskolin and were not potentiated by phosphodiesterase inhibition, suggesting that AII utilizes a mechanism other than increased cAMP synthesis to facilitate neurotransmitter release. AII also strongly enhanced calcium ionophore (A23187)-induced 3H-NE release in iris-ciliary body segments, indicating that AII can modulate calcium-dependent exocytosis at step(s) distal to calcium influx. These results demonstrate that sympathetic nerves in the rabbit eye contain prejunctional, facilitatory AII receptors, and support the possible involvement of the renin-angiotensin system in regulation of ocular sympathetic neurotransmission in vivo.

Benzodiazepine binding sites on PC12 cells: modulation by nerve growth factor and forskolin.

PC12 pheochromocytoma cells show increased binding of the peripheral type benzodiazepine Ro 5-4864 after treatment with nerve growth factor (NGF) in membrane preparations. Forskolin, an activator of adenylate cyclase, acts synergistically with NGF to produce further increases in binding, but by itself produces no effect. The increased binding appears to reflect increases in receptor number, since Kd remains unchanged. Binding shows a trend toward increase by day 6 after NGF treatment, and the increase is significant at days 9 and 12. The physiological roles of benzodiazepine binding sites on PC12 cells are unclear. Treatment of the cells with Ro 5-4864 produces no changes in basal or stimulated release of catecholamines, in activity of adenylate cyclase, or in cell proliferation.

Effects of calcium channel antagonists on the depolarization-evoked release of norepinephrine in the rabbit iris-ciliary body.

The effects of several representative calcium channel antagonists on depolarization-evoked release of [3H]-norepinephrine were investigated in isolated, superfused rabbit iris-ciliary bodies. Potassium (50 mM)-evoked neurosecretion was blocked by 5 mM CoCl2 and partially inhibited by 10(-6) M nitrendipine or verapamil. Electrically-evoked neurosecretion was similarly blocked by CoCl2, but was unaffected by nitrendipine or verapamil. It is concluded from these results that sympathetic terminals in the rabbit iris-ciliary body contain dihydropyridine- and verapamil-sensitive calcium channels which contribute, under conditions of prolonged depolarization, to neurotransmitter release.