Beta-adrenergic stimulation of Na+, K+, Cl- cotransport in fetal nonpigmented ciliary epithelial cells.

PURPOSE: The effects of adrenergic agonists and antagonists on Na+, K+, Cl- cotransport in fetal human nonpigmented ciliary epithelial (NPE) cells were investigated. METHODS: 86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured NPE monolayers. Cyclic adenosine monophosphate (cAMP) formation in NPE cells was determined by radioimmunoassay. RESULTS: 1 microM isoproterenol caused a 1.65-fold stimulation in Na+,K+,Cl cotransport measured as bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake. The half-maximal concentration for this effect was 6.4 nM, with maximal stimulation at 100 nM isoproterenol. Epinephrine stimulated Na+, K+, Cl- cotransport similarly to isoproterenol, whereas norepinephrine stimulated at much higher concentrations (half-maximal effective concentration = 1.4 microM). Stimulation of Na+, K+, Cl- cotransport by 1 microM isoproterenol was inhibited completely by the beta 2-adrenergic antagonist ICI-118,551 at 100 nM, with a half-maximal inhibitory concentration of 5 nM. Neither atenolol, a beta 1-specific adrenergic antagonist, prazosin, an alpha 1-adrenergic antagonist, nor yohimbine, an alpha 2-specific antagonist, was as effective. These four antagonists inhibited isoproterenol-stimulated cAMP formation with potencies similar to those observed against stimulated Na+, K+, Cl- cotransport. The hypotensive adrenergic antagonist timolol, propranolol, and betaxolol also inhibited Na+, K+, Cl- cotransport stimulated by isoproterenol in the order timolol > propranolol > betaxolol. Na+, K+, Cl- cotransport could be maintained in a stimulated state for at least 2 hours in the presence of agonist, but activity returned to basal levels within 20 minutes of isoproterenol removal. Adrenergic stimulation of Na+, K+, Cl- cotransport was blocked 80% to 85% by 70 microM H-89, a protein kinase A inhibitor. CONCLUSIONS: These data suggest that beta 2-adrenergic receptor activation results in increased cAMP formation and sustained stimulation of Na+, K+, Cl- cotransport in fetal human NPE cells. Protein kinase A activation is required for maximal stimulation of Na+, K+, Cl- cotransport by adrenergic agonists.

Neurotransmitters and neuropeptides stimulate inositol phosphates and intracellular calcium in cultured human nonpigmented ciliary epithelium.

The effects of several neurotransmitters and neuropeptides on the inositol phosphate/diacylglycerol pathway were examined in human nonpigmented ciliary epithelial cells. Maximal stimulation of inositol phosphate formation by vasopressin (approximately 3-fold), carbachol (approximately 2-fold) and histamine (approximately 5-fold) was observed only after cells had been confluent for at least six days. In contrast, a response to bombesin (approximately 3-fold) declined with extended time in confluent culture. Inositol monophosphate, inositol bisphosphate, and inositol trisphosphate all were stimulated by these agonists. Dose-response studies showed a close correlation between the EC50s of the different agonists when elevation of inositol phosphates was compared to stimulation of intracellular Ca2+, with the exception of bombesin. Preliminary pharmacologic characterization of the receptors for vasopressin, carbachol, and bombesin provided rank order of potencies for selective agonists and antagonists. The data suggest that the muscarinic receptor on human NPE cells is the M3 subtype, whereas the vasopressin receptor, as defined by its linkage to the inositol phosphate/diacylglycerol pathway, is the V1 subtype.

Stimulation of Na+,K+,Cl- cotransport by forskolin-activated adenylyl cyclase in fetal human nonpigmented epithelial cells.

PURPOSE: To determine the stoichiometry of Na+,K+,Cl- cotransport in fetal human nonpigmented ciliary epithelial cells and the effect of forskolin, an adenylyl cyclase activator, on Na+,K+,Cl- cotransport. METHODS: 86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured human nonpigmented epithelial (NPE) monolayers. RESULTS: The dependence of ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake upon Na+, K+, and Cl- concentrations was determined. Maximal uptake was observed at about 12 mM, 20 mM, and 120 mM of these ions, respectively. Analysis by Hill plot suggested that the stoichiometry of Na+,K+,Cl- cotransport is 1:1:2, making this an electroneutral process. Na+,K+,Cl- cotransport was found to be stimulated approximately 1.5- to 2-fold after incubation of cells for 15 minutes with 1 microM forskolin. neither ouabain-sensitive 86Rb+ uptake nor bumetanide-insensitive, ouabain-insensitive uptake was affected. 8-Bromoadenosine cAMP and 8-chlorophenylthio cAMP at 1 mM stimulated Na+,K+,Cl- cotransport approximately 30% to 40%, whereas 1,9 dideoxyforskolin, a non-adenylyl cyclase-activating analogue of forskolin, had little effect. Stimulation of Na+,K+,Cl- cotransport by forskolin was blocked by prior exposure of cells to 10 microM H-89, a protein kinase A inhibitor. Stimulation by forskolin was also observed in the presence of either 1 mM DIDS, 30 microM NPPB, 3 mM DPC, or 5 mM BaCl2, although all four channel blockers inhibited Na+,K+,Cl- cotransport to various degrees. CONCLUSIONS: The data suggest that the human NPE Na+,K+,Cl- cotransporter transports Na+, K+, and Cl- in the ratio of 1:1:2. Activation of adenylyl cyclase stimulates Na+,K+,Cl(-)-cotransport via a mechanism involving protein kinase A. Reduction of Na+,K+,Cl- cotransport by chloride channel blockers raises the possibility that activities of some ion channels can influence the rate of ion influx via Na+,K+,Cl- cotransport.

Immunolocalization of the Na-K-Cl cotransporter in bovine ciliary epithelium.

PURPOSE: Recent evidence suggests that Na-K-Cl cotransport plays a major role in blood-to-aqueous anion transport across the ciliary body epithelium. The present study was undertaken to determine the location of the Na-K-Cl cotransporter in fixed sections of bovine eye. METHODS: Sections of paraformaldehyde-fixed adult and calf bovine eyes were treated with a monoclonal antibody to mammalian Na-K-Cl cotransporter and a fluorescent secondary antibody and examined under a fluorescent microscope. Na-K-Cl cotransporter protein was detected on immunoblots of dissected tissue and purified nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (PE) cells. RESULTS: Cotransporter immunofluorescence was most intense along the basolateral surfaces of the PE cells. Anterior pars plicata possessed the greatest PE immunofluorescence, and this diminished posteriorly toward the pars plana. Quantitation of immunofluorescence images indicated 7- to 10-fold more cotransporter protein in pars plicata PE than in pars plana PE. Diffuse cytoplasmic fluorescence was seen in the NPE cells, which was also brightest in anterior pars plicata. Immunoblots of separated PE and NPE cells from anterior pars plicata showed that PE contain four times more 170-kDa cotransporter protein than NPE. This confirmed fluorescence quantitation estimates. Cotransporter was barely detectable in cells isolated from pars plana in either cell layer. Immunoblots of the Na,K-ATPase catalytic (alpha) subunit in separated NPE and PE cells showed that NPE cells possessed approximately eight times more alpha subunit protein than PE. Immunofluorescence indicated a similar distribution of alpha subunits and indicated a basolateral membrane location for the subunit on both cell types. Na-K-Cl cotransporter fluorescence patterns showed more variability in adult animals than in calves, which may be related to aging and/or disease. Distinctive patterns of cotransporter fluorescence were also seen in the cornea, iris, and retina. CONCLUSIONS: Localization of the Na-K-Cl cotransporter to the plasma membrane on the blood side of the ciliary epithelium tight junctions supports a role for the Na-K-Cl cotransporter in ciliary epithelium as a chloride entry mechanism involved in blood-to-aqueous chloride transport. The concentration of Na,K-ATPase catalytic subunits on NPE basolateral membranes could provide net Na(+) efflux into the aqueous humor.

Dopamine D1 stimulation of Na+,K+,Cl- cotransport in human NPE cells: effects of multiple hormones.

PURPOSE: To determine the effects of dopamine on Na+,K+,Cl- cotransport in human ciliary nonpigmented epithelial (NPE) cells. METHODS: The authors used 86Rb+ as a marker for K+ to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured fetal human NPE monolayers. RESULTS: Na+,K+,Cl- cotransport was stimulated 1.63-fold by 10 microM dopamine. Stimulation was dose dependent, with a maximum stimulation occurring at 10 microM dopamine and an EC50 of 0.5 microM. NaK-ATPase (measured as ouabain-sensitive, bumetanide-insensitive 86Rb+ uptake) and bumetanide-insensitive, ouabain-insensitive 86Rb+ uptake were not affected by dopamine. The D1-receptor-specific antagonist, SCH23390, inhibited stimulation by 10 microM dopamine more than 90% at 1 microM, with an IC50 of 4 nM, whereas the D2-receptor-specific antagonist, sulpiride, was over 250 times less effective. Similarly, a D1 agonist, SKF81297, was more potent than the D2 agonist bromocriptine in stimulation of Na+,K+,Cl- cotransport. The beta-adrenergic antagonists timolol and propranolol did not significantly inhibit stimulation of Na+,K+,Cl- cotransport by dopamine. Conversely, SCH23390, showed minimal inhibition of isoproterenol stimulation of Na+,K+,Cl- cotransport. Stimulation by maximally stimulating concentrations of isoproterenol and dopamine were not additive, but were similar to stimulation by 1 microM forskolin, suggesting that adenylyl cyclase may be close to maximally activated by either catecholamine. In vivo concentrations (stimulation approximately 25% over control) of dopamine, isoproterenol, and norepinephrine added together stimulated Na+,K+,Cl- cotransport 80% to 89% of stimulation by maximal concentrations of these drugs. The protein kinase A inhibitor N-[2-p-bromocinnamylaminoethyl]-5-isoquinolinesulfonamide (H-89) blocked dopamine stimulation of Na+,K+,Cl- cotransport by more than 75%, whereas phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, given with 10 microM dopamine inhibited Na+,K+,Cl- cotransport by 75% to 80%, similarly to inhibition by PMA given alone. CONCLUSIONS: Dopamine stimulates Na+,K+,Cl- cotransport in NPE through dopamine-D1-type receptors and activation of protein kinase A. Beta-adrenergic receptors do not appear to play a role. Inhibition of Na+,K+,Cl- cotransport by protein kinase C is dominant over stimulation of Na+,K+,Cl- cotransport through the cyclic adenosine monophosphate pathway.

Modification by cyclic adenosine monophosphate of basolateral membrane chloride conductance in chick retinal pigment epithelium.

PURPOSE: We investigated the hypothesis that cyclic adenosine monophosphate (cAMP) modifies the basolateral membrane chloride conductance of chick retinal pigment epithelium (RPE). METHODS: Placing freshly dissected chick retina-RPE-choroid tissues in a perfusion chamber that allows separate perfusion of its retinal and choroidal sides, the authors examined the effect of choroidal perfusion of forskolin (50 mM), an adenylate cyclase activator, on extracellular and intracellular RPE potentials, resistances, light-evoked responses, and chloride diffusion potentials. RESULTS: Forskolin hyperpolarized the RPE basolateral membrane, decreased the apical/basal membrane resistance ratio, increased the amplitude of the RPE membrane c-wave hyperpolarizations, decreased the amplitude of the transepithelial c-wave, and suppressed the light peak. In addition, forskolin decreased the basolateral membrane chloride diffusion potential by 31%. The effects of forskolin were diminished by pretreatment of the basolateral membrane with DIDS, a chloride channel blocker, or by pretreatment of the apical membrane with bumetanide, a blocker of NaK2Cl cotransport. Transepithelial potential, resistance, and c-wave amplitude were not changed by 1,9-dideoxyforskolin, which does not elevate cAMP levels. CONCLUSIONS: Elevation of cAMP results in diminished basolateral membrane chloride conductance in chick RPE. This could be due to a direct effect on the chloride channel or due to a decrease in intracellular chloride concentration secondary to inhibition of apical membrane NaK2Cl cotransport.

The role of NaKCl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium.

PURPOSE: To evaluate the role of NaKCl cotransport in short-circuit current (Isc) and chloride fluxes across rabbit ciliary epithelium mounted in a Ussing-type chamber. METHODS: Bilayered intact ciliary epithelium free of stroma was obtained after perfusion and dissection of rabbit eyes and mounted in an Ussing-type chamber. The effects of bumetanide and other drugs on Isc and transepithelial 36Cl fluxes in bicarbonate-containing Ringer's were determined. Immunoblot analysis was performed by standard techniques. RESULTS: Bumetanide (100 microM) applied to the blood (pigmented epithelium [PE]) side of the ciliary bilayer caused a dose-dependent decrease in Isc from 18.2 +/- 2.2 to 10.4 +/- 1.4 microA/cm2 (43%). Bumetanide applied to the aqueous (nonpigmented epithelium [NPE]) side of the tissue inhibited Isc by only 12%. Immunoblots of dissected NPE and PE tissue probed with an antibody to mammalian NaKCl cotransporter detected approximately 10 times more NaKCl cotransporter protein in PE than in NPE. 36Cl flux studies revealed a PE-to-NPE chloride flux of 180.3 +/- 37.2 microEq/cm2 per hour and an NPE-to-PE flux of 72.3 +/- 22.9 microEq/cm2 per hour, indicating a net PE-to-NPE flux of 108.0 +/- 31.3 microEq/cm2 per hour across rabbit ciliary epithelium. Bumetanide inhibited the PE-to-NPE chloride flux by 52% but did not inhibit the NPE-to-PE flux. Isoproterenol (10 microM) added to the PE side of the bilayer increased Isc by a dose-dependent 53%. Prior addition of bumetanide to the PE side blocked the increase due to isoproterenol by 37%. Isoproterenol (10 microM) stimulated the PE-to-NPE chloride flux by 75% but had no stimulatory effect on the NPE-to-PE chloride flux. 4,4'Diisothiocyanatostilbene-2,2'disulfonic acid (DIDS) inhibited Isc when added to either side of the bilayer but was more potent at low concentrations (<100 microM) when added to the NPE side and more potent at higher concentrations (>100 microM) when added to the PE side. Prior addition of 1 mM DIDS to the NPE side decreased isoproterenol stimulation of Isc by 56%. CONCLUSIONS: NaKCl cotransporters located primarily on the blood side of rabbit ciliary epithelium contribute to aqueous-negative Isc and to blood-to-aqueous chloride transport across the tissue in bicarbonate-containing medium. DIDS-inhibitable mechanisms, possibly including HCO3-Cl exchange and Cl channels, also play a role. Isoproterenol stimulation of Isc involves coordinate upregulation of PE-side NaKCl cotransport and an NPE-side DIDS-inhibitable mechanism(s).

Vasoactive intestinal peptide stimulates cyclic AMP metabolism in choroid plexus epithelial cells.

Some peptides of the glucagon-secretin family were found to stimulate intracellular cyclic AMP accumulation in cultured bovine choroid plexus epithelial cells. Vasointestinal peptide and porcine intestinal peptide at concentrations of 30 and 300 nM, respectively, evoked 50-fold elevations of cyclic AMP; half-maximal responses were obtained with concentrations of 15 and 102 nM for the two peptides, respectively. Secretin and glucagon each produced 25- to 50-fold elevations of cyclic AMP at 330 microM, but showed no effect below 3 microM. Gastric inhibitory peptide and prealbumin had little or no response at any concentration tested. Experiments measuring the cellular cyclic AMP accumulation in response to pairs of peptides suggested that vasointestinal peptide, porcine intestinal peptide and secretin act through a common receptor. Studies with antagonists to isoproterenol and histamine indicated that this receptor is distinct from the beta-adrenergic and H2-histamine receptors known to exist on choroidal cells.

Stimulation of inositol phosphate formation in cultured human retinal pigment epithelium.

Several hormones, neurotransmitters, and neuropeptides were screened for the ability to stimulate inositol phosphate formation in cultured human retinal epithelial (RPE) cells. Carbachol, vasopressin and thrombin were found to be effective. Treatment of RPE cells with all three agents produced increases in inositol monophosphate, inositol bisphosphate and inositol trisphosphate in the presence of 10 mM LiCl. Carbachol stimulated a 4-fold increase in the total of inositol phosphates at 1 mM. Studies with cholinergic antagonists showed a rank order of 4 DAMP greater than QNX greater than pirenzepine greater than methoctramine, suggesting the presence of M3 muscarinic receptors. Vasopressin gave a 2.5-fold stimulation at 10 microM. Agonists of vasopressin were also tested and gave differential responses. Studies using a V1 agonist (PIOVP) and a V2 agonist (DAVP) showed DAVP matching the level of stimulation elicited by vasopressin whereas treatment with PIOVP only reached 50% of the vasopressin response. These data suggested the presence of V2 receptors in the RPE cells. Several proteases were tested for their ability to stimulate RPE inositol phosphates. Thrombin caused a 7-fold increase in inositol phosphate formation at 1 U/ml, whereas trypsin and plasmin elicited smaller responses (approximately 2-fold). The thrombin effect was blocked by the thrombin-specific inhibitor, hirudin, but not by other protease inhibitors. Several mediators of inflammation such as bradykinin, histamine and serotonin were also tested, and they were ineffective in stimulating inositol phosphate turnover in the RPE cells.

Histamine H1 receptor occupancy triggers inositol phosphates and intracellular calcium mobilization in human non-pigmented ciliary epithelial cells.

Agonists and antagonists of histamine were used to characterize the stimulation of inositol phosphates formation and elevation of intracellular Ca2+ by histamine in cultured non-pigmented epithelial (NPE) cells from human ciliary body. Agonists specific for the H1 histamine receptor subtype were 20- to 200-fold more potent than the H2-specific agonists tested, and 5-16% as potent as histamine in inositol phosphates stimulation. An H1 antagonist was 10,000-fold more potent than an H2 antagonist in blocking histamine stimulation of inositol phosphates. H1 agonists also mimicked and H1 antagonists inhibited the elevation of intracellular Ca2+ by histamine. The first phase of the Ca2+ response to histamine was largely independent of extracellular Ca2+ while the second phase required extracellular Ca2+. Dose-response curves for histamine elevation of intracellular Ca2+ (EC50 = 10 microM, maximum at 100 microM) and inositol phosphates (EC50 = 2 microM, maximum at 100 microM) were similar. These data support the characterization of the NPE histamine receptor as an H1 receptor linked to elevation of inositol phosphates and intracellular Ca2+.

Natriuretic peptide receptors on human trabecular meshwork cells.

The effects of natriuretic peptides on cGMP formation and [125I]ANP binding in human trabecular meshwork cells were investigated. CNP at 1 microM stimulated cGMP formation approximately 18-25 fold, with a half maximal effective concentration approximately 20-30nM. BNP at 1 microM stimulated approximately 7 fold, while ANP stimulated cGMP formation 2-fold at 1 microM but had little or no effect at concentrations below 1 microM. Displacement binding of [125I]ANP to intact TM cells in the presence of unlabeled ANP indicated a single binding site with a dissociation constant approximately 0.15nM.c-ANP, which binds specifically to natriuretic peptide C receptors, displaced > 95% [125I]ANP binding to surface receptor sites with a half-maximal effective concentration comparable to that of ANP or BNP. c-ANP had no inhibitory effect on CNP stimulation of cGMP formation. The data suggest that human TM cells possess natriuretic peptide B receptors as the primary guanylyl cyclase-containing subtype and C receptors as the numerically predominant subtype of natriuretic peptide receptors.

Histamine stimulation of inositol phosphate metabolism in cultured human non-pigmented ciliary epithelial cells.

Treatment of cultured human non-pigmented ciliary epithelial cells with 100 microM histamine for 30 minutes resulted in a 3-5 fold increase in intracellular inositol phosphates. The stimulation by histamine was dose-dependent, with a half-maximal concentration of 3 microM and a maximal concentration of 100 microM. In response to histamine, inositol monophosphate increased approximately linearly for 30 min in the presence of 10 mM LiCl2, while inositol bisphosphate and inositol trisphosphate showed rapid rises complete within a few minutes. Treatment of cells with the H1 antagonist diphenhydramine resulted in a complete inhibition of the histamine effect at 1 microM, with a half-maximal inhibition at 56 nM, whereas cimetidine, an H2 antagonist, had little effect at any concentration tested. Schild analysis of the diphenhydramine/histamine receptor interaction gave an apparent dissociation constant of 7.1 nM. The data suggest that human non-pigmented ciliary epithelial cells possess H1 histamine receptors.

High affinity vasoactive intestinal peptide receptors on fetal human nonpigmented ciliary epithelial cells.

The effect of vasoactive intestinal peptide (VIP) on stimulation of adenylyl cyclase in fetal human nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (CPE) cells was studied. 1 microM VIP elicited a 5-10 fold increase in intracellular cAMP in NPE cells from three fetal donors, but caused little or no response in CPE from two fetal donors and other ocular cell types employed as controls. Appearance of cAMP in the extracellular medium was stimulated in NPE but not in CPE in response to VIP. Both NPE and CPE gave similar cAMP responses (8-13 fold) to the beta-adrenergic agonist, isoproterenol. Binding studies of [125I]VIP to intact NPE and CPE revealed that VIP bound to NPE cells at a high affinity site (KD = .33 nM and a low affinity site (KD = 16 nM), whereas VIP bound to CPE cells only at the low affinity site (KD = 18 nM). In NPE cells, VIP stimulated cAMP formation with an EC50 of approximately 0.6-1 nM, similar to the high affinity binding site KD, with maximal stimulation at 10 nM. Four peptides with various degrees of sequence homology to VIP were also studied. Of these, PHM and PHI stimulated cAMP with EC50s of 50 and 300 nM, respectively, while secretin and glucagon stimulated only at concentrations above 0.1 microM. These results suggest that in fetal human ciliary epithelium, as in rabbit ciliary epithelium (Mittag et al., J Pharm Exp Ther 241: 230, [1987]), VIP stimulation of adenylyl cyclase is a characteristic of NPE but not CPE cells.

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.

Effect of glutamine on the degradation of glutamine synthetase in hepatoma tissue-culture cells.

In certain lines of hepatoma tissue-culture cells, the extracellular glutamine concentration regulates the specific activity of glutamine synthetase. By quantifying the radioactivity in immunoprecipitated glutamine synthetase on polyacrylamide gels, we found that the rate of degradation, but not of synthesis, of glutamine synthetase is a sensitive function of extracellular glutamine. The activiy that degrades this enzyme appears to be labile.

Calcitonin gene-related peptide stimulates intracellular cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells.

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.

Down-regulation of vasoactive intestinal peptide receptors by protein kinase C in fetal human non-pigmented ciliary epithelial cells.

Stimulation of cAMP formation in fetal human non-pigmented ciliary epithelial cells by 10 microM prostaglandin E1 was inhibited by 30-50% by 15 min prior exposure to 1 microM phorbol 12-myristate, 13-acetate. Evidence that this inhibition was due to activation of protein kinase C is the following. First, inhibition was also caused by 10 microM dioctanoylglycerol, a diacylglycerol analog. Second, no inhibition was observed using 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, whereas phorbol didecanoate was effective. And third, prior exposure of cells to staurosporine, an inhibitor of protein kinase C, blocked phorbol ester-induced inhibition of cAMP stimulation. Phorbol esters also inhibited stimulation of cAMP formation by 10 nM vasoactive intestinal peptide and by 1 microM isoproterenol. Stimulation of cAMP formation by either 1 microM cholera toxin or 10 microM forskolin was not inhibited by prior exposure of cells to phorbol esters. This suggests that protein kinase C acts neither at the level of GS activation of adenylyl cyclase, nor by inhibiting adenylyl cyclase directly. The possibility that protein kinase C acts on adenylyl cyclase-linked receptors was assessed by measuring the effect of phorbol esters on specific binding of [125I]vasoactive intestinal peptide to intact cells. Treatment of cells with either 1 microM phorbol 12-myristate,13-acetate or phorbol didecanoate resulted in a 25-40% reduction in the number of binding sites for [125I]vasoactive intestinal peptide, with little change in dissociation constants.(ABSTRACT TRUNCATED AT 250 WORDS)

Catecholaminergic regulation of Na-K-Cl cotransport in pigmented ciliary epithelium: differences between PE and NPE.

Pigmented (PE) and nonpigmented (NPE) ciliary epithelial cells comprise the ciliary epithelium, the site of aqueous humor formation in the eye. In man, catecholamines increase the rate of aqueous humor formation, but the mechanism underlying these effects is not understood. Recent evidence suggests that Na-K-Cl cotransport plays a central role in blood-to-aqueous chloride transport across ciliary epithelium in cow and rabbit. We therefore investigated whether catecholamines stimulate Na-K-Cl cotransport in human PE cells. Na-K-Cl cotransporter protein was detected as a 170 kDa protein band on immunoblots. Immunofluorescence microscopy detected cotransporter on the basolateral membranes of the PE layer of ciliary epithelium from a human donor. Cotransporter immunofluorescence was also detected in cultured PE cells. Na-K-Cl cotransport activity measured as ouabain-insensitive bumetanide-sensitive(86)Rb uptake was stimulated by isoproterenol 1.6-fold, with an EC(50) = 28 n M and maximal stimulation at 1 microM. Other transport mechanisms involved in(86)Rb uptake were not affected. Stimulation by 1 microM isoproterenol was blocked by 10 n M ICI 118,551, a beta(2)-specific receptor antagonist, whereas the receptor subtype-specific antagonists yohimbine (alpha(2)), prazosin (alpha(1)) and atenolol (beta(1)) were ineffective. Norepinephrine stimulation (EC(50) = 280 n M) was also blocked by ICI 118,551. Dopamine stimulated Na-K-Cl cotransport 1.6-fold with an EC(50) = 14 microM. The dopamine effect could not be blocked by 10 microM SCH 23390, a D1-antagonist, but was abolished by ICI 118,551. Forskolin and CPT-cAMP stimulated Na-K-Cl cotransport 1.79- and 1.71-fold, respectively, whereas the inactive forskolin analogue 1,9-dideoxyforskolin had no effect. However, high concentrations of the PKA inhibitors PKI amide 14-22 and KT 5720 were needed to inhibit both PKA activity in cell lysates and isoproterenol stimulation of cotransport. This finding may indicate the presence of a novel PKA isoform in PE cells. Inhibitors of other protein kinases, including myosin light chain kinase, protein kinase G, calmodulin-dependent kinase and tyrosine kinase, were without effect on stimulated Na-K-Cl cotransport. When EC(50)s for catecholaminergic stimulations of Na-K-Cl cotransport in PE were compared to those in NPE, values within five-fold of one another were seen for isoproterenol and norepinephrine. In contrast, dopamine was 28-fold more potent in NPE than in PE. The data suggest that both PE and NPE possess beta(2)adrenergic receptors, but only NPE cells possess dopamine D1 receptors linked to Na-K-Cl cotransport.

Thrombin stimulates inositol phosphate formation, intracellular calcium fluxes and DNA synthesis in cultured fetal human non-pigmented ciliary epithelial cells.

Thrombin at concentrations as low as 20 pM (0.002 U ml-1) was found to stimulate inositol phosphate levels in cultured human non-pigmented ciliary epithelial cells. Several other proteases, including trypsin and plasmin, had little or no effect, of several protease inhibitors tested, only those with specificity for thrombin blocked the effect. Studies with active site-blocked thrombin suggested that the esterolytic active site of thrombin is required for inositol phosphate stimulation, while gamma-thrombin, which has reduced binding affinity to fibrinogen also showed reduced effectiveness in stimulating inositol phosphates. In the presence of 10 mM LiCl, thrombin stimulated inositol monophosphate, inositol bisphosphate and inositol trisphosphate formation, with a prolonged rise of the first and transient early rises in the latter two species. Thrombin also elevated intracellular Ca2+ levels as measured with the fluorescent calcium probe, indo-1-AM. This elevation could be blocked by prior addition to cells of the thrombin inhibitor, hirudin, and was dependent upon extracellular Ca2+ for the maintenance of an elevated level in the presence of thrombin. Incorporation of thymidine into DNA in confluent cultures was also stimulated by thrombin, with a four-fold increase in incorporation at 35 hr in thrombin-treated cells compared to controls. The half-maximal concentration for this process was 0.25 U ml-1. Pretreatment with 100 ng ml-1 pertussis toxin greatly reduced the thrombin effect, which is consistent with a role for a G-protein in stimulation of DNA synthesis by thrombin.