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.     

Peptide-amidating enzymes are expressed in the stellate epithelial cells of the thymic medulla

C-terminal amidation is a post-translational processing step necessary to convey biological activity to a large number of regulatory peptides. In this study we have demonstrated that the peptidyl-glycine alpha-amidating monooxygenase enzyme complex (PAM) responsible for this activity is located in the medullary stellate epithelial cells of the thymus and in cultured epithelial cells bearing a medullary phenotype, using Northern blot, immunocytochemistry, in situ hybridization, and enzyme assays. Immunocytochemical localization revealed a granular pattern in the cytoplasm of the stellate cells, which were also positive for cytokeratins and a B-lymphocyte-associated antigen. The presence of PAM activity in medium conditioned by thymic epithelial cell lines suggests that PAM is a secreted product of these cells. Among the four epithelial cell lines examined, there was a direct correlation between PAM activity and content of oxytocin, an amidated peptide. Taken together, these data provide convincing evidence that thymic epithelial cells have the capacity to generate amidated peptides that may influence T-cell differentiation and suggest that the amidating enzymes could play an important role in the regulation of thymic physiology.     

Two-headed, two-necked conjoined twin calf with Arnold-Chiari malformation in a Japanese shorthorn calf

Drugs having as their active substance aprotinin (trasylol, gordox, etc.) are widely indicated in the treatment of eye pathologies having as their basis an enhanced proteolytic enzyme activation. Our studies have exposed a decrease in proteolytic activity in both eye tissues and tear fluid in response to Gordox administration. Up to date there are no works concerning pharmacokinetics of aprotinin in the eye tissues. The aim of this work was to study the pharmacokinetics of 125I-aprotinin in rabbit eye tissues after instillation. A presence of the substance was registered in the cornea, aqueous humor, and ciliary body within a period of 10-90 minutes after administration, but not in other tissues. Calculation of the pharmacokinetic parameters of aprotinin penetration in the eye showed that its mean residence time in the cornea, aqueous humor and ciliary body was 50-60 min. The addition of polyvinyl alcohol to the instillation solution increased the level of the substance in the tissues, but did not change its mean residence time. The medicine should be instilled every 1-1.5 hrs to support high and stable levels of aprotinin in the eye.     

Post-translational N-glycosylation of a truncated form of a peptide processing enzyme

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the carboxyl-terminal amidation of bioactive peptides through a two-step reaction involving the monooxygenase and lyase domains. PAM undergoes endoproteolytic cleavage in neuroendocrine cells in the lyase domain. To determine which of the two possible paired basic sites is utilized, truncated PAM proteins ending at these sites were stably expressed in Chinese hamster ovary cells. While characterizing the truncation mutants, it became apparent that N-glycosylation occurred post-translationally at the single site localized near the carboxyl terminus of the lyase domain. The post-translational N-glycosylation of this site does not require the newly synthesized protein to remain tightly bound to membranes. Both malfolded, secretion incompetent proteins and fully active, secreted proteins were subject to post-translational N-glycosylation.     

Peptide amidating activity in human bronchoalveolar lavage fluid

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.     

Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye

Pituitary adenylate cyclase-activating peptide (PACAP)-like immunoreactivity was demonstrated by immunocytochemistry together with calcitonin gene-related peptide (CGRP)-like immunoreactivity in small to medium-sized neurons in the trigeminal ganglion and in nerve fibers in the iris, ciliary body, cornea, choroid and sclera of the rabbit eye. The regional distribution of PACAP-27- and PACAP-38-like immunoreactivity in the eye was studied by radioimmunoassay: the highest concentrations were found in the iris sphincter and ciliary body. The distribution pattern resembled that of CGRP-like immunoreactivity, which is a well-known constituent of sensory C-fibre neurons. Intravitreal injection of PACAP-27 or PACAP-38 induced conjunctival hyperemia, swelling of the anterior segment of the eye, miosis and breakdown of the blood-aqueous barrier, manifested as a marked aqueous flare response. Tetrodotoxin pretreatment inhibited the conjunctival hyperemia, the swelling of the anterior segment of the eye, and the miosis but not the aqueous flare response. The concentration of PACAP-like immunoreactivity in the aqueous humor was increased greatly following infrared irradiation of the iris, topical application of formaldehyde to the cornea, or intravitreal injection of endotoxin or bovine serum albumin. Also the concentration of CGRP-like immunoreactivity in the aqueous humor was increased greatly. Both in vivo and in vitro studies showed that capsaicin caused a parallel release of PACAP-like immunoreactivity and CGRP-like immunoreactivity from the uvea. Injection of PACAP-27 and PACAP-38 resulted in the release of CGRP-like immunoreactivity (and PACAP-like immunoreactivity) into the aqueous humor and PACAP-27 and PACAP-38 were also found to evoke tachykinin-mediated contractions of the isolated iris sphincter muscle, indicating that PACAP induces positive feedback on C-fibres. Thus, PACAP is a sensory neuropeptide in the eye. Since the PACAP-induced ocular responses mimicked the symptoms of inflammation, and since the PACAP-like immunoreactivity concentration in the aqueous humor was greatly increased following noxious stimulation, we suggest that it takes part in the inflammatory responses of the rabbit eye.     

Immunohistochemical localization of carboxypeptidases E and D in the human placenta and umbilical cord

Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins.     

Orchestration of neutrophil movement by intestinal epithelial cells in response to Salmonella typhimurium can be uncoupled from bacterial internalization

Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies of S. typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship between S. typhimurium invasion and epithelial promotion of PMN movement. First, we investigated whether S. typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. Blocking S. typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S. typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells and S. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by which S. typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-kappabeta activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilDelta) S. typhimurium mutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediate S. typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.     

Non-lysosomal cycling pathway for atrial natriuretic peptide activated by protein kinase C in human NPE cells

Atrial natriuretic peptide (ANP) stimulates aqueous humor formation in primates, but the membrane-bound receptors which mediate this effect have not been well studied in the eye. Endocytosis of [125I]ANP bound to natriuretic peptide C receptors was characterized in fetal human nonpigmented ciliary epithelial (NPE) cells. [125I]ANP which bound to cells at 4 degreesC was detected in the cell interior after a temperature shift to 37 degreesC. Appearance of ligand within the cell peaked at 5 min, and then declined towards zero over 20 min. The endocytosis inhibitor phenylarsine oxide blocked the appearance of internalized ligand, whereas the lysosomotropic drug chloroquine had no effect on internalization but blocked subsequent loss of internalized ligand. Chloroquine also blocked the accumulation of degraded ligand in the extracellular medium. Treatment with phorbol 12-myristate, 13-acetate accelerated the loss of internalized ligand from cells and increased the accumulation of ligand in the extracellular medium. Ligand in the medium was also increased by dioctanoylglycerol but not by 4alpha phorbol didecanoate, an isomer which does not activate protein kinase C. The protein kinase inhibitors staurosporine and bisindolylymaleimide blocked the increase in ligand. Phorbol ester-stimulated loss of internalized ligand occurred in the presence of chloroquine. TCA precipitation of ligand in the extracellular medium showed that both degraded and undegraded [125I]ANP were present. However, in the presence of chloroquine only, undegraded ANP was detected in the medium, and phorbol esters stimulated its rate of appearance by approximately 2 fold. A similar stimulation occurred when cells containing internalized ligand, but stripped of membrane-bound ligand, were exposed to phorbol esters. The data suggest that ANP bound to natriuretic peptide C receptors on NPE cells is endocytosed, and that protein kinase C activates a non-lysosomal pathway for ANP retroendocytosis in these cells.     

Possible new mechanism for the antiparkinsonian effect of amantadine

An obese mouse model (Cpefat/Cpefat) that has hyperproinsulinemia and late onset obesity has been described. Cpefat/Cpefat mice have a missense mutation in carboxypeptidase E (CPE), a processing enzyme essential for production of biologically active endocrine and neuroendocrine peptides. We have reported previously that CPE activity was absent in the antrum of the stomach and that processing of progastrin to the amidated biologically active form of gastrin is reduced. Since gastrin is a major secretagogue for gastric acid secretion, the purpose of the present experiments was to examine gastric acid secretion in Cpefat/Cpefat mice. In addition, secretion of amidated gastrin in response to inhibition of acid secretion was tested in Cpefat/Cpefat. Both gastric acid and challenged gastrin secretion are reduced in Cpefat/Cpefat mice. We conclude that stomach CPE activity is essential for gastric secretory activity and for challenged gastrin release.     

Neuroendocrine cell type-specific and inducible expression of the secretogranin II gene: crucial role of cyclic adenosine monophosphate and serum response elements

Secretogranin II, an acidic protein in the chromogranin/secretogranin family, is widely distributed in neuroendocrine secretory granules. What factors govern such widespread, yet selective, expression? The 5' deletions localized neuroendocrine cell type-specific expression to the proximal mouse secretogranin II promoter: such expression was abolished after deletion past the cAMP response element (CRE; [-67 bp]TGACGTCA[-60 bp]), and transfer of the CRE to a neutral promoter conferred 3.4- to 5.3-fold neuroendocrine selectivity. Thus, the CRE is, at least partly, sufficient to confer tissue-specific expression. Substantial (48-59%) loss of cell type-specific expression also occurred upon deletion past the serum response element (SRE; [-302 bp]GATGTCC[-296 bp]), and transfer of the SRE to a neutral promoter also conferred neuroendocrine selectivity. Expression of both the endogenous gene and the transfected secretogranin II promoter was up-regulated after secretagogues, and the degree of trans-activation of the transfected promoter (2.2- to 5.4-fold) paralleled activation of the endogenous gene (1.8- to 3.2-fold). The 5' promoter deletions revealed complete loss of secretagogue responses after deletion past the CRE. Transfer of the CRE to a neutral promoter conferred secretagogue responses (by 2.2- to 18.6-fold). Substantial (59-74%) falls in secretagogue responses also occurred after deletion past the promoter's SRE. Transfer of the SRE to a neutral promoter conferred secretagogue responses (by 2.7- to 8.3-fold). We conclude that the CRE is a crucial determinant of cell type-specific constitutive and secretagogue-inducible expression of the secretogranin II gene and that the SRE also plays a substantial role in both processes.     

Neonatal piglet model of intraaortic balloon pumping: improved efficacy using echocardiographic timing

Secretoneurin (SN), a 33-amino acid neuropeptide, is derived from secretogranin II that is released from sensory afferent C-fibers by capsaicin. Described functions of secretoneurin include chemotaxis of monocytes and endothelial cells, and inhibition of endothelial cell proliferation. Inhibition of monocyte chemotaxis by staurosporine indicated involvement of specific signaling pathways. We have tested effects of SN, substance P (SP), and interleukin-8 (IL-8) on eosinophil migration in modified Boyden chambers including signaling mechanisms of neuropeptide and cytokine stimulation of human eosinophils. Experiments showed SN as eosinophil chemoattractant comparable in its potency to IL-8. Checkerboard analysis, usage of a specific anti-SN-antibody, and receptor desensitization experiments confirmed the chemotactic activity. Preincubation of the cells with effective concentrations of staurosporine or tyrphostin-23 showed no effect, whereas treatment with wortmannin (WTN) or 3-isobutyl-1-methylxantin (IBMX) completely blocked SN-induced migration. Additionally, experiments ruled out tyrphostin-23- and WTN-sensitive signaling pathways for SP-induced chemotaxis of eosinophils. We conclude that SN-stimulated human eosinophil chemotaxis is mediated via a unique and specific signal transduction pathway that involves activation of phosphodiesterases and WTN-sensitive enzymes, ie, phospholipase D and phosphatidylinositol-3-kinase. In contrast, we report that activation of the latter and tyrosine kinases is required for SP-induced chemotaxis of eosinophils.     

Phospholipase D mediates matrix metalloproteinase-9 secretion in phorbol ester-stimulated human fibrosarcoma cells

Phospholipase D (PLD) has been implicated in vesicle trafficking in the Golgi and hence secretion. In this study, we show that the secretion of matrix metalloproteinase-9 (MMP-9) from HT 1080 human fibrosarcoma cells was stimulated by phorbol 12-myristate 13-acetate in a time- and dose-dependent manner that involved protein kinase C. The phorbol ester also increased PLD activity in the cells. Evidence that PLD was involved in the stimulation of MMP-9 secretion was provided by the observations that the secretion of MMP-9 was stimulated by the introduction of short-chain phosphatidic acid (PA) into the growth medium and that inhibition of PA production by 1-propanol inhibited secretion. Using a short-chain diacylglycerol we excluded the possibility that MMP-9 secretion was induced by diacylglycerol formed from PA by phosphatidic acid phosphatase. Furthermore, propranolol, an inhibitor of this enzyme, had no effect on secretion induced by either phorbol 12-myristate 13-acetate or PA. The data presented here indicate that activation of protein kinase C increases MMP-9 secretion in HT 1080 cells and implicate PLD and PA formation in the effect.     

Ocular pharmacokinetics of orally administered azithromycin in rabbits

Azithromycin was orally administered to Dutch-belted rabbits following extracapsular lens extraction in one eye. At various times the animals were sacrificed, and serum and ocular tissues were obtained for drug level determination by HPLC-EC. Following a single dose, peak levels of drug in ocular tissues were measured within 8 hours (cornea > 0.5 micrograms/g [15mg/kg]; > 1.5 micrograms/g [3Omg/kg]). Highest levels were obtained in iris and ciliary body ( > 15 micrograms). Measurable tissue levels persisted for at least 120 hours. Trough levels increased proportionately during drug multiple dose administration. Five days following five daily 15mg/kg doses, corneal levels exceeded 0.5 micrograms/g, and iris and ciliary levels were higher than 15 micrograms/g. Aqueous humor and serum levels were equivalent. Vitreous humor levels, though higher than aqueous humor, were consistently < 1 microgram/ml. Extracapsular cataract extraction did not significantly affect drug uptake.     

Effects of HCO3- on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion

PURPOSE: To determine whether the Na+-K+-2Cl- symport or the parallel Na+/H+ and Cl-/HCO3- antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells, after which Na+ and Cl- diffuse across the gap junctions into the nonpigmented ciliary epithelial (NPE) cells and are released into the aqueous humor. METHODS: Rabbit iris ciliary bodies were preincubated in HCO3-/CO2-containing or HCO3-/CO2-free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis. RESULTS: The NPE and the PE cells contained more K and Cl when incubated with bicarbonate. Inhibition of carbonic anhydrase with 0.5 mM acetazolamide had little effect in HCO3--free medium but prevented the increase in Cl in both cell types in HCO3-/CO2 solution. Inhibition of the Na+-K+-2Cl- symport with 10 to 500 microM bumetanide caused Cl loss from both cell types in HCO3--free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3-/CO2 solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3--free solution and prevented the gains of Cl and Na in HCO3-/CO2 solution. CONCLUSIONS: The present results indicate that the dominant entry pathway of NaCl from the stroma into the ciliary epithelial syncytium is through an acetazolamide-inhibitable Cl-/HCO3 and a parallel Na+/H+ antiport. The dominant release pathways into the aqueous humor appear to be a Na+-K+-2Cl-symport, which can be outwardly directed under physiological conditions, together with the Na+/K+-exchange pumps and Cl- channels.     

Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor

PURPOSE: Ocular immune privilege is mediated in part by the activity of constitutively produced immunosuppressive cytokines and neuropeptides. Aqueous humor was examined for content of calcitonin gene-related peptide (CGRP), and the potential of CGRP to mediate immunosuppressive activity within aqueous humor was determined. METHODS: The concentration of CGRP in fresh, normal rabbit aqueous humor was assayed by competitive enzyme-linked immunosorbent assay. The ability of CGRP to suppress interferon (IFN)-gamma production by antigen-stimulated, primed lymph node cells was examined by assaying supernatants of stimulated CGRP-treated, primed T-cell cultures for IFN-gamma. The anti-inflammatory activity of aqueous humor and CGRP was assayed by treating IFN-gamma-lipopolysaccharide (LPS)-activated RAW 264.7 cells (macrophages) with aqueous humor, aqueous humor plus anti-CGRP antibody, or CGRP alone. Culture supernatants of the treated macrophages were examined for nitrite by Griess reagent. The production of inducible nitric oxide synthase (NOS2) protein was examined by immunoblotting cell lysates of treated activated macrophages. RESULTS: The constitutive level of CGRP in fresh, normal rabbit aqueous humor was 5+/-1 x 10(-5) M. At its ocular concentration, CGRP did not inhibit IFN-gamma production by stimulated effector T cells, but it suppressed nitric oxide generation by activated macrophages. Neutralization of CGRP in normal rabbit aqueous humor prevented the aqueous humor from suppressing nitric oxide generation by macrophages. Neither CGRP nor aqueous humor suppressed NOS2 protein synthesis in activated inflammatory macrophages. CONCLUSIONS: Calcitonin gene-related peptide is a constitutive neuropeptide in aqueous humor. Through CGRP, aqueous humor suppresses nitric oxide production by activated macrophages. This suppression appears to result from inhibiting NOS2 enzymatic activity, rather than from suppressing NOS2 synthesis. The results imply that the ocular microenvironment has diverse immunoregulatory mechanisms that suppress induction, activation, and mediation of immunogenic inflammation.