October 2001
Volume 42, Issue 11
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Physiology and Pharmacology  |   October 2001
Carbachol and Nitric Oxide Inhibition of Na,K-ATPase Activity in Bovine Ciliary Processes
Author Affiliations
  • Dorette Z. Ellis
    From the Neuroscience Center, Massachusetts General Hospital, Charlestown.
  • James A. Nathanson
    From the Neuroscience Center, Massachusetts General Hospital, Charlestown.
  • Jason Rabe
    From the Neuroscience Center, Massachusetts General Hospital, Charlestown.
  • Kathleen J. Sweadner
    From the Neuroscience Center, Massachusetts General Hospital, Charlestown.
Investigative Ophthalmology & Visual Science October 2001, Vol.42, 2625-2631. doi:https://doi.org/
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      Dorette Z. Ellis, James A. Nathanson, Jason Rabe, Kathleen J. Sweadner; Carbachol and Nitric Oxide Inhibition of Na,K-ATPase Activity in Bovine Ciliary Processes. Invest. Ophthalmol. Vis. Sci. 2001;42(11):2625-2631. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Nitric oxide (NO) donors and cholinergic agents decrease intraocular pressure, in part because they induce a decrease in aqueous humor production. Because Na,K-adenosine triphosphatase (ATPase) is involved in aqueous humor formation, this study was conducted to investigate the hypothesis that NO and cholinomimetics regulate its activity in bovine ciliary processes.

methods. Bovine tissue slices were incubated with agonists and antagonists in a physiological buffer in vitro. Na,K-ATPase activity was determined by assaying hydrolysis of adenosine triphosphate (ATP) in suspended permeabilized tissue slices.

results. Carbachol-induced inhibition of Na,K-ATPase activity correlated with increases in cGMP. This inhibition was abolished by the muscarinic blocker atropine, the NO inhibitor N w-nitro-l-arginine (l-NAME) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Sodium nitroprusside (SNP) mimicked the actions of carbachol. The SNP-induced decrease in Na,K-ATPase activity correlated with an increase in cGMP and was also abolished by ODQ. Both 8-bromo (Br)-cGMP and okadaic acid also inhibited Na,K-ATPase activity.

conclusions. Carbachol-induced inhibition of Na,K-ATPase activity involves muscarinic receptor activation. That SNP mimics and l-NAME reverses carbachol’s effect on Na,K-ATPase activity suggests that the actions of carbachol are mediated by NO. Carbachol’s and SNP’s effects on Na,K-ATPase activity involved soluble guanylate cyclase and cGMP. Inhibition of Na,K-ATPase activity by 8-Br-cGMP and okadaic acid indicates that protein phosphorylation events may mediate SNP-induced inhibition of Na,K-ATPase activity.

Na,K-adenosine triphosphatase (ATPase), which contributes to the transport of ions and the formation of aqueous humor in the ciliary processes, 1 2 3 4 catalyzes the transfer of 2K+ from the extracellular space into the cell and the extrusion of 3Na+, while hydrolyzing adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate (Pi). The resultant electrochemical gradient is harnessed by other cellular proteins, including ion transporters and cotransporters, and is thought to constitute the major energy source that drives the transepithelial transport of ions needed in the formation of aqueous humor. Na,K-ATPase is a plasma membrane protein, composed of two or three different polypeptides: the catalytic α-subunit and the β- and γ-subunits. The four known α isoforms of Na,K-ATPase are α1, α2, α3, andα 4; they are inhibitable by a class of natural toxins, the cardiac glycosides digitalis and ouabain. The three known β isoforms areβ 1, β2, and β3, and there are two splice variants of theγ -subunit, γa and γb. 5 Immunocytochemical studies have demonstrated that the ciliary processes contain the α1-, α2-,α 3-, β1-, β2-, and β3-subunits, but no γ subunit, in a distribution that differs between pigmented and nonpigmented epithelium. 6 7 The α1- and β1-subunits are found in pigmented epithelium, as they are in retinal pigmented epithelium, whereas the α2- and either β3- or β2-subunits predominate in nonpigmented epithelium. 
Prior reports have identified a cholinergic innervation of the ciliary processes, 8 9 and cholinomimetic agents have long been known to lower intraocular pressure. Although cholinergic agents, including carbachol, have been used for many decades as antiglaucoma drugs, very little is known about the mechanism of action of the cholinergic system in the ciliary processes. The ciliary processes are also enriched in nitric oxide synthase (NOS), as demonstrated by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and immunocytochemistry, 10 11 12 and topical application of NO donors lowers intraocular pressure. 13  
We have recently reported functional interactions between the cholinergic and nitric oxide systems and their ability to inhibit ouabain-sensitive Na,K-ATPase activity in the choroid plexus of the brain ventricle. 14 Because of the known functional, pharmacologic, and anatomic similarities between the choroid plexus and ciliary processes, we have in the present study tested the role of NO and carbachol in regulating Na,K-ATPase activity in bovine ciliary processes. 
Methods
Tissue Preparation
Bovine eyes were obtained from an abattoir within 2 hours after death and maintained on ice before dissection. We used standard ophthalmic microsurgery instruments to remove the cornea, iris, and lens, after which the exposed ciliary processes were dissected on ice. Tissue slices (0.4 × 0.4 × 1 mm) were prepared on a chopper (Brinkman Instruments, Westbury, NY) cooled to 4°C, and suspended (25–30 mg/ml wet weight) in microdissection buffer containing (in mM): NaCl, 137; KCl, 5; MgSO4, 0.8; CaCl2, 0.25; MgCl2, 1.0; HEPES, 10; and NaOH, 2, to adjust pH to 7.4 at 34°C. 
Na,K-ATPase and cGMP Measurements
Drugs used in these experiments were added to tubes that contained 1-ml aliquots of slice suspension. In studies using inhibitors, the inhibitors were added 3 minutes before addition of the drug. Tubes were incubated for 15 minutes at 34°C, and then frozen at− 80°C. Tubes were thawed and centrifuged (1700g for 15 minutes at 4°C), and supernatant (containing drug) was removed. The supernatant was heated for 5 minutes at 90°C, after which 75 mM sodium acetate was added, and the sample was dried and stored for cGMP assay by radioimmunoassay (RIA) or enzyme immunoassay (EIA). 
Na,K-ATPase activity was measured in suspended tissue slices using the colorimetric ATPase assay: ATP was hydrolyzed and the released Pi was measured by forming a complex with molybdate. The pelleted tissue slices were resuspended at the same concentration and refrozen for more than 20 minutes at −80°C in 1 ml resuspension buffer containing (in mM): NaCl, 85; KCl, 20; MgCl2, 4; EGTA, 0.2; and histidine, 30; adjusted to pH 7.2. Tubes were thawed in ice water. For further permeabilization of tissue slices, saponin (20 μg/ml) was added and slices were incubated for 10 minutes at 34°C. Aliquots of tissue slices (approximately 10–15 μg wet weight) were added to 400 μl ATPase buffer containing (in mM) ATP, 3; NaCl, 140; KCl, 20; MgCl2, 3; histidine, 30 (pH 7.2), with or without 3 mM ouabain. Because there was only a 5% reduction in the slope at 30 minutes compared with that at 20 minutes, Na,K-ATPase activity was measured at 30 minutes at 37°C. The reaction was terminated by the addition of a quenching solution (0.6 ml) containing 1 N H2SO4 and 0.5% ammonium molybdate. Formation of a phosphomolybdate complex was determined spectrophotometrically at 700 nm. 15 Na,K-ATPase activity was measured as the difference between ouabain-treated and untreated samples. Protein concentrations were determined by the Lowry method. 16  
Statistics
Statistical comparisons were performed by ANOVA followed by the Fisher protected least-significant difference (PLSD) test and the Scheffé F-test for comparison of significant difference among different means. 
Chemicals
Routine reagents, sodium nitroprusside (SNP), ouabain, saponin, N w-nitro-l-arginine (l-NAME), carbachol, and atropine were purchased from Sigma Chemical Co. (St. Louis, MO). 8-bromo (Br)-cGMP sodium salt and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) where obtained from RBI/Sigma-Aldrich (Natick, MA). cGMP assay kits for RIA or EIA were purchased from Biomedical Technologies Inc. (Stoughton, MA) and Amersham Pharmacia Biotech (Piscataway, NJ), respectively. 
Results
Inhibition of Ouabain-Sensitive Na,K-ATPase Activity by Carbachol
Because of the known effects of cholinomimetic agents on intraocular pressure regulation and because Na,K-ATPase is involved in aqueous humor secretion and volume regulation, we wanted to determine the effects of the acetylcholine analogue carbachol on ouabain-sensitive Na,K-ATPase activity. Figure 1 shows that a 15-minute incubation of bovine ciliary process tissue slices with carbachol (100 μM) resulted in a significant inhibition of ouabain-sensitive Na,K-ATPase activity, compared with that of slices treated with vehicle. Ouabain-insensitive ATPase activity represented two-thirds of total ATPase activity, and there was no difference as a result of carbachol treatment. Addition of atropine (1 μM), a nonselective muscarinic receptor inhibitor, in the presence of carbachol, abolished the carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity (Fig. 1)
Mediation of Carbachol-Induced Inhibition of Na,K-ATPase Activity by NO and Soluble Guanylate Cyclase
The ciliary process epithelium is enriched in the NO synthetic enzyme, NOS. Because cholinergic stimulation causes release of NO in a variety of tissues, 17 18 it was of interest to determine whether the observed cholinergic-induced inhibition of Na,K-ATPase in ciliary processes is mediated by NO. Exposure of bovine ciliary process tissue slices to l-NAME (300 μM), a specific inhibitor of NOS, in the presence of carbachol largely blocked the carbachol-induced decrease in ouabain-sensitive Na,K-ATPase (Fig. 2A) . These data demonstrate that the action of carbachol is upstream from NO and predict that stimulation of the cholinergic system in the ciliary processes results in the release of NO. 
Stimulation of the cholinergic system in other tissues such as kidney medullary slices has been associated with activation of soluble guanylate cyclase and increases in cGMP. 19 To test whether soluble guanylate cyclase was activated in response to carbachol stimulation in ciliary processes, tissue slices were exposed to carbachol (100 μM) with or without ODQ (1 μM), an inhibitor selective for soluble guanylate cyclase. ODQ abolished the carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity (Fig. 2A) , suggesting that soluble guanylate cyclase mediates carbachol activity in ciliary process epithelium. 
We also wanted to determine whether there were alterations in cGMP levels in response to carbachol in ciliary processes. Addition of carbachol (100 μM) to tissue slices caused a significant increase in cGMP levels (more than twofold), which was abolished when ODQ (1 μM) was added (Fig. 2B)
Mimicking of Carbachol-Induced Inhibition of Ouabain-Sensitive Na,K-ATPase Activity by SNP
SNP as an artificial NO donor was used to bypass the normal cholinergic system to determine whether NO alone was sufficient to cause the observed effects. To determine the most effective dose of SNP on Na,K-ATPase activity in the ciliary processes, tissue slices were exposed for 15 minutes to various concentrations (1, 10, and 100 μM). There was a significant reduction of Na,K-ATPase activity in response to 10 and 100 μM SNP, whereas 1 μM SNP had no effect on basal levels of Na,K-ATPase activity (Fig. 3A) . Compared with the activity in control ciliary processes, Na,K-ATPase activity in slices treated with SNP (10 and 100 μM) was inhibited 40% to 45%. cGMP was measured in the supernatants of the same samples (treated with 1, 10, or 100 μM SNP) that were subsequently assayed for Na,K-ATPase. SNP at 10 μM caused a 44% increase in cGMP levels, whereas 100 μM SNP caused a 100% increase in cGMP levels (Fig. 3B) . Addition of 1 μM SNP had no effect on basal cGMP levels. 
Because NO, like muscarinic agonists, is known to stimulate soluble guanylate cyclase, it was important to determine whether the physiological action of SNP on Na,K-ATPase was also a result of activating this enzyme. Because inhibition of Na,K-ATPase was achieved with both 10 and 100 μM SNP, we used 100 μM in this study. Exposure of bovine ciliary process tissue slices to ODQ (1 μM) 20 blocked the SNP-induced inhibition of the Na,K-ATPase (Fig. 4A)
ODQ, in addition to blocking the SNP-induced alteration in Na,K-ATPase, caused decreased cGMP levels in tissue slices treated with SNP (Fig. 4B) . These results not only provide evidence for the possible involvement of cGMP in mediating the SNP- induced inhibition of Na,K-ATPase activity, but further support the involvement of soluble guanylate cyclase (versus membrane-bound guanylate cyclase) in mediating the SNP response in bovine ciliary processes. 
Effects of 8-Br-cGMP and Protein Phosphatase Inhibition
Further evidence for the role of cGMP in mediating the muscarinic and SNP responses was obtained from tissue slices that were exposed to 8-Br-cGMP (2 mM), a permeable derivative of cGMP. There was a significant inhibition of ouabain-sensitive Na,K-ATPase activity in bovine ciliary process tissue in response to treatment with 8-Br-cGMP (Fig. 5A)
cGMP is a potent activator of protein kinase G (PKG). Other evidence of the involvement of protein phosphorylation in regulating the Na,K-ATPase activity was obtained from tissue slices treated with okadaic acid. Okadaic acid concentrations (4 and 400 nM) known to inhibit protein phosphatases type 2A and 1, respectively, mimicked the actions of SNP in inhibiting Na,K-ATPase activity in bovine ciliary process specimens (Fig. 5A) . We also wanted to determine whether the cGMP system was regulated by protein phosphatase type 2A or 1. Addition of okadaic acid (400 nM) had no effect on basal cGMP levels nor on cGMP levels measured in SNP-treated tissue slices (Fig. 5B) . This supports a role for phosphorylation downstream of the elevation of cGMP. 
Discussion
The Na,K-ATPase in ciliary process epithelium plays a pivotal role in aqueous humor production, aqueous and vitreous humor turnover, and, subsequently, regulation of intraocular pressure. Of importance are observations that the cardiac glycosides ouabain and digitalis and 12(R)-hydroxyeicosatetraenoic acid inhibit Na,K-ATPase activity with subsequent decreases in intraocular pressure. 1 2 3 4 21 In addition to the Na,K-ATPase, Cl channels, aquaporin, ENaC, and bumetanide-sensitive Na+-K+-Cl exchanger also contribute to aqueous humor formation. 22 23 24 25  
In the present study, stimulation of the cholinergic system caused a significant decrease in ouabain-sensitive Na,K-ATPase activity in bovine ciliary process tissue. The carbachol-induced inhibition of Na,K-ATPase activity was abolished by atropine, demonstrating that carbachol’s response is mediated by muscarinic receptor(s). Although we did not identify the receptor subtype involved in the carbachol-induced inhibition of the Na,K-ATPase, other studies suggest that M1 receptors are activated in response to carbachol stimulation in ciliary process. 26 As with choroid plexus, 14 l-NAME blocked the cholinergic-induced inhibition of the Na,K-ATPase. This suggests that activation of the NOS system and subsequent formation of NO mediates the cholinergic response in ciliary process epithelium. Prior reports have demonstrated that cholinergic stimulation leads to increased[ Ca2+]i in the ciliary processes. 27 Other studies have shown that an increase in[ Ca2+]i is obligatory for activation of NOS. 28 NO release follows stimulation of cholinergic nerves in a number of tissues. 17 18  
We also demonstrated that addition of carbachol to ciliary process tissue results in stimulation of soluble guanylate cyclase and increases in cGMP levels. The presence of NOS and Na,K-ATPase in ciliary process epithelium and the known effects of cGMP in modulating its transepithelial ion transport 29 suggest that the activation of the NOS system, with subsequent cGMP synthesis, may regulate Na,K-ATPase. The addition of ODQ to tissue slices treated with carbachol obliterated the carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity. Whereas carbachol caused an increase in cGMP, ODQ partially abolished this effect. These results are consistent with other reports that showed that cholinergic regulation in kidney medullary slices 19 and choroid plexus 30 involves activation of soluble guanylate cyclase and stimulation of cGMP. Figure 6 diagrams the deduced regulatory pathway. 
The NO agonist SNP caused significant inhibition of the ouabain-sensitive Na,K-ATPase activity in the ciliary process tissue. Both 10 and 100 μM SNP inhibited the Na,K-ATPase 40% to 45%. The ability of NO to inhibit ouabain-sensitive Na,K-ATPase activity is corroborated by previous reports demonstrating NO-induced inhibition of ouabain-sensitive Na,K-ATPase activity in other tissues, including choroid plexus 14 and rat kidney medulla and cortex. 19 31 That increasing concentrations of SNP caused increases in cGMP levels is consistent with prior reports. 20 However, in our hands, increasing concentrations of cGMP did not cause further decreases in ouabain-sensitive Na,K-ATPase levels. 
Many of the actions of SNP are mediated by activation of soluble guanylate cyclase and subsequent formation of cGMP. 32 The ability of the specific soluble guanylate cyclase inhibitor, ODQ, to antagonize the actions of SNP on the Na,K-ATPase suggests that a direct consequence of NOS stimulation is activation of soluble guanylate cyclase. The inhibition of Na,K-ATPase by SNP was consistently associated with increases in cGMP levels that were determined from measurements of cGMP that were obtained concomitantly with measurements of Na,K-ATPase activity in SNP-exposed ciliary process tissue slices. Further evidence for the involvement of cGMP as a mediator in the action of SNP on Na,K-ATPase activity was obtained by treatment of bovine ciliary process tissue with 8-Br-cGMP. These observations suggest that cGMP may play a major role in regulating Na,K-ATPase activity (see Fig. 6 ). 
Recent evidence suggests that certain chemical NO donors may affect Na,K-ATPase activity directly by modification of sulfhydryl groups, 33 but we do not believe that this was the case in the current study. First, Sato et al. 33 demonstrated that SNP, unlike other NO donors, does not directly interact with Na,K-ATPase and modify its activity. Second, in studies with purified Na,K-ATPase from choroid plexus 14 and cerebellum (Ellis D, Sweadner K, unpublished data, 2001), SNP failed to change Na,K-ATPase activity. This suggests that modulation of the Na,K-ATPase by SNP in tissue slices is through a second-messenger system and that direct effects such as nitrosylation of sulfhydryl groups were not responsible for the observations. 
Our studies do not preclude the involvement of other second messengers, such as protein kinase A or C, 34 35 36 in modulating Na,K-ATPase activity in the ciliary processes. The present studies also do not rule out complementary regulation of Na+ transport through other mechanisms, such as the regulation of amiloride-sensitive cation channels, which, by regulating Na+ entry may alter Na,K-ATPase activity. Studies in kidney have suggested that there are such mechanisms for regulating Na+ transport through amiloride-sensitive channels (ENaC). 37 Of interest is the observation that the ciliary processes may also contain ENaC amiloride-sensitive channels. 24  
Because 8-Br-cGMP is known to be an activator of protein kinase G (PKG), the observation that 8-Br-cGMP mimics the actions of SNP suggests that PKG may be involved in the regulation of Na,K-ATPase by NO. Preliminary studies (data not shown) in ciliary processes and more extensive studies performed in choroid plexus 14 have demonstrated that the inhibitors used to study PKG involvement in NO regulation of Na,K-ATPase (Rp-8-pCPT-cGMP and KT5823) have a direct inhibitory effect on Na,K-ATPase activity. As such, they are not suitable reagents for assessing the participation of PKG in the NO pathway. 
We were able to provide other evidence for the role of protein phosphorylation in regulating Na,K-ATPase activity in the ciliary processes. The action of SNP in inhibiting ouabain-sensitive Na,K-ATPase was mimicked by okadaic acid, which is known to inhibit protein phosphatases type 2A and 1 (50% inhibitory concentration[ IC50] of 0.5–1.0 and 40–60 nM, respectively). The ability of 4 nM okadaic acid to inhibit ouabain-sensitive Na,K-ATPase activity in bovine ciliary process suggests that protein phosphatase type 2A is involved. When compared with Na,K-ATPase activity in tissue slices treated with 4 nM okadaic acid, addition of 400 nM okadaic acid did not induce greater inhibition of ouabain-sensitive Na,K-ATPase activity. We cannot however rule out involvement of other protein phosphatases. In the choroid plexus 38 and substantia nigra, 39 8-Br-cGMP causes phosphorylation of the proteins dopamine and cAMP-regulated phosphoprotein (DARPP32) and inhibitor-1, which in their phosphorylated forms are potent inhibitors of protein phosphatase type 1. These phosphoproteins are also enriched in the ciliary process epithelium. 40 Other studies in the kidney have shown that DARPP32 and inhibitor-1 are involved in the regulation of Na,K-ATPase activity. 41  
In the present studies, the protein phosphatase inhibitor okadaic acid mimicked the effects of SNP on Na,K-ATPase activity, without affecting either cGMP synthesis directly or the stimulation of cGMP synthesis by SNP. This suggests that okadaic acid acts at a point distal to the formation of cGMP. The fact that okadaic acid works also implies that there is a basal kinase activity in the absence of added agonists in our experimental protocols. 
The cholinergic-NO regulation of Na,K-ATPase activity in ciliary process epithelium may be more complex, due to a number of factors. The ciliary process epithelium contains multiple Na,K-ATPase isoforms. 6 Recent studies in mouse and rat have demonstrated that the relatively newly discovered β3 Na,K-ATPase isoform is a major component in ciliary process epithelium. 7 Consequently, in addition to α1, α2,α 3, β1, and β2, 6 bovine ciliary process epithelium may also contain β3. The ciliary epithelium is bilayered, comprising the inner pigmented epithelium and outer nonpigmented epithelium. Each layer contains different combinations of the functional Na,K-ATPaseα - and β-subunits. For example, α2 and β2 or β3 predominate in nonpigmented epithelium, whereas the α1 and β1 isoforms are detected in the pigmented epithelium, suggesting differences in functional roles. Whether cholinergic agonists and NO selectively inhibit the Na,K-ATPase of one or the other layer is an important question that remains to be answered. 
 
Figure 1.
 
Carbachol (Carb) inhibits ouabain-sensitive Na,K-ATPase activity in ciliary process. Ouabain-sensitive Na,K-ATPase activity was determined as the difference between activity in ouabain-treated and untreated samples. Data for ouabain-sensitive Na,K-ATPase activity are mean ± SEM of triplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 1.
 
Carbachol (Carb) inhibits ouabain-sensitive Na,K-ATPase activity in ciliary process. Ouabain-sensitive Na,K-ATPase activity was determined as the difference between activity in ouabain-treated and untreated samples. Data for ouabain-sensitive Na,K-ATPase activity are mean ± SEM of triplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 2.
 
(A) The cholinergic and nitric oxide systems are linked in ciliary process tissue. Average normalized values for ouabain-sensitive Na,K-ATPase activity (± SEM); numbers above bars indicate number of experiments performed in triplicate. *Significantly different from the corresponding control at P < 0.005 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to cGMP increases. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.25 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments. *Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 2.
 
(A) The cholinergic and nitric oxide systems are linked in ciliary process tissue. Average normalized values for ouabain-sensitive Na,K-ATPase activity (± SEM); numbers above bars indicate number of experiments performed in triplicate. *Significantly different from the corresponding control at P < 0.005 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to cGMP increases. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.25 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments. *Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 3.
 
SNP mimicked the effects of carbachol on ouabain-sensitive Na,K-ATPase activity in bovine ciliary process. (A) Na,K-ATPase activity is expressed as mean ± SEM for three experiments. *Significantly different from the control group: P < 0.01 (by ANOVA). (B) SNP-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to increases in cGMP levels. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.45 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 3.
 
SNP mimicked the effects of carbachol on ouabain-sensitive Na,K-ATPase activity in bovine ciliary process. (A) Na,K-ATPase activity is expressed as mean ± SEM for three experiments. *Significantly different from the control group: P < 0.01 (by ANOVA). (B) SNP-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to increases in cGMP levels. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.45 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 4.
 
The effects of SNP on ouabain-sensitive Na,K-ATPase activity involved activation of soluble guanylate cyclase and increases in cGMP levels. (A) Ouabain-sensitive Na,K-ATPase activity is expressed as mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test); **significantly different from SNP-treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Levels of cGMP in bovine ciliary processes after incubation with SNP (100 μM), ODQ (1 μM), or SNP plus ODQ. Results are expressed as a percentage of control cyclic nucleotide levels (2.75 ± 0.2 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).** Significantly different from SNP treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 4.
 
The effects of SNP on ouabain-sensitive Na,K-ATPase activity involved activation of soluble guanylate cyclase and increases in cGMP levels. (A) Ouabain-sensitive Na,K-ATPase activity is expressed as mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test); **significantly different from SNP-treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Levels of cGMP in bovine ciliary processes after incubation with SNP (100 μM), ODQ (1 μM), or SNP plus ODQ. Results are expressed as a percentage of control cyclic nucleotide levels (2.75 ± 0.2 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).** Significantly different from SNP treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 5.
 
Inhibition of Na,K-ATPase with 8-Br-cGMP or phosphatase inhibitor. (A) Values for ouabain-sensitive Na,K-ATPase activity represent mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.01 (by ANOVA, and Fisher PLSD, and Scheffé F-test). (B) Inhibition of Na,K-ATPase by okadaic acid was not associated with increases in cGMP. Levels of cGMP in bovine ciliary process tissue after incubation with SNP (100 μM), okadaic acid (400 nM), ODQ (1μ M), or SNP (100 μM) plus okadaic acid (400 nM). Results are expressed as a percentage of control cyclic nucleotide levels (3 ± 0.4 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).
Figure 5.
 
Inhibition of Na,K-ATPase with 8-Br-cGMP or phosphatase inhibitor. (A) Values for ouabain-sensitive Na,K-ATPase activity represent mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.01 (by ANOVA, and Fisher PLSD, and Scheffé F-test). (B) Inhibition of Na,K-ATPase by okadaic acid was not associated with increases in cGMP. Levels of cGMP in bovine ciliary process tissue after incubation with SNP (100 μM), okadaic acid (400 nM), ODQ (1μ M), or SNP (100 μM) plus okadaic acid (400 nM). Results are expressed as a percentage of control cyclic nucleotide levels (3 ± 0.4 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).
Figure 6.
 
Summary diagram of the pathway involving acetylcholine and NO regulation of Na,K-ATPase activity. Stimulation of cholinergic neurons causes the release of Ca2+, activation of NOS, and the subsequent formation of NO. Nitrovasodilators (NTVs) also cause the formation of NO, which then binds to and activates soluble guanylate cyclase (sGC), the synthetic enzyme of cGMP. cGMP and its analogue 8-Br-cGMP activate protein kinase G (PKG) which may, directly or indirectly through other proteins (dopamine and cAMP-regulated phosphoprotein [DARPP-32] or inhibitor-1 [I-1]), alter Na,K-ATPase activity.
Figure 6.
 
Summary diagram of the pathway involving acetylcholine and NO regulation of Na,K-ATPase activity. Stimulation of cholinergic neurons causes the release of Ca2+, activation of NOS, and the subsequent formation of NO. Nitrovasodilators (NTVs) also cause the formation of NO, which then binds to and activates soluble guanylate cyclase (sGC), the synthetic enzyme of cGMP. cGMP and its analogue 8-Br-cGMP activate protein kinase G (PKG) which may, directly or indirectly through other proteins (dopamine and cAMP-regulated phosphoprotein [DARPP-32] or inhibitor-1 [I-1]), alter Na,K-ATPase activity.
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Figure 1.
 
Carbachol (Carb) inhibits ouabain-sensitive Na,K-ATPase activity in ciliary process. Ouabain-sensitive Na,K-ATPase activity was determined as the difference between activity in ouabain-treated and untreated samples. Data for ouabain-sensitive Na,K-ATPase activity are mean ± SEM of triplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 1.
 
Carbachol (Carb) inhibits ouabain-sensitive Na,K-ATPase activity in ciliary process. Ouabain-sensitive Na,K-ATPase activity was determined as the difference between activity in ouabain-treated and untreated samples. Data for ouabain-sensitive Na,K-ATPase activity are mean ± SEM of triplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 2.
 
(A) The cholinergic and nitric oxide systems are linked in ciliary process tissue. Average normalized values for ouabain-sensitive Na,K-ATPase activity (± SEM); numbers above bars indicate number of experiments performed in triplicate. *Significantly different from the corresponding control at P < 0.005 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to cGMP increases. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.25 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments. *Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 2.
 
(A) The cholinergic and nitric oxide systems are linked in ciliary process tissue. Average normalized values for ouabain-sensitive Na,K-ATPase activity (± SEM); numbers above bars indicate number of experiments performed in triplicate. *Significantly different from the corresponding control at P < 0.005 (by ANOVA, Fisher PLSD, and Scheffé F-test). **Significantly different from carbachol-treated samples at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Carbachol-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to cGMP increases. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.25 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments. *Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 3.
 
SNP mimicked the effects of carbachol on ouabain-sensitive Na,K-ATPase activity in bovine ciliary process. (A) Na,K-ATPase activity is expressed as mean ± SEM for three experiments. *Significantly different from the control group: P < 0.01 (by ANOVA). (B) SNP-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to increases in cGMP levels. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.45 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 3.
 
SNP mimicked the effects of carbachol on ouabain-sensitive Na,K-ATPase activity in bovine ciliary process. (A) Na,K-ATPase activity is expressed as mean ± SEM for three experiments. *Significantly different from the control group: P < 0.01 (by ANOVA). (B) SNP-induced inhibition of ouabain-sensitive Na,K-ATPase activity corresponded to increases in cGMP levels. Data are from EIA measurements of cGMP present in supernatant from permeabilized tissue slices subsequently assayed for Na,K-ATPase activity. Results are expressed as a percentage of control cyclic nucleotide levels (2.45 ± 0.2 pmol/mg protein). Values for cGMP levels represent mean ± SEM for duplicate samples of three experiments.* Significantly different from the corresponding control at P < 0.001 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 4.
 
The effects of SNP on ouabain-sensitive Na,K-ATPase activity involved activation of soluble guanylate cyclase and increases in cGMP levels. (A) Ouabain-sensitive Na,K-ATPase activity is expressed as mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test); **significantly different from SNP-treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Levels of cGMP in bovine ciliary processes after incubation with SNP (100 μM), ODQ (1 μM), or SNP plus ODQ. Results are expressed as a percentage of control cyclic nucleotide levels (2.75 ± 0.2 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).** Significantly different from SNP treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 4.
 
The effects of SNP on ouabain-sensitive Na,K-ATPase activity involved activation of soluble guanylate cyclase and increases in cGMP levels. (A) Ouabain-sensitive Na,K-ATPase activity is expressed as mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test); **significantly different from SNP-treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test). (B) Levels of cGMP in bovine ciliary processes after incubation with SNP (100 μM), ODQ (1 μM), or SNP plus ODQ. Results are expressed as a percentage of control cyclic nucleotide levels (2.75 ± 0.2 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).** Significantly different from SNP treated group: P < 0.05 (by ANOVA, Fisher PLSD, and Scheffé F-test).
Figure 5.
 
Inhibition of Na,K-ATPase with 8-Br-cGMP or phosphatase inhibitor. (A) Values for ouabain-sensitive Na,K-ATPase activity represent mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.01 (by ANOVA, and Fisher PLSD, and Scheffé F-test). (B) Inhibition of Na,K-ATPase by okadaic acid was not associated with increases in cGMP. Levels of cGMP in bovine ciliary process tissue after incubation with SNP (100 μM), okadaic acid (400 nM), ODQ (1μ M), or SNP (100 μM) plus okadaic acid (400 nM). Results are expressed as a percentage of control cyclic nucleotide levels (3 ± 0.4 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).
Figure 5.
 
Inhibition of Na,K-ATPase with 8-Br-cGMP or phosphatase inhibitor. (A) Values for ouabain-sensitive Na,K-ATPase activity represent mean ± SEM for triplicate samples of three experiments.* Significantly different from control group: P < 0.01 (by ANOVA, and Fisher PLSD, and Scheffé F-test). (B) Inhibition of Na,K-ATPase by okadaic acid was not associated with increases in cGMP. Levels of cGMP in bovine ciliary process tissue after incubation with SNP (100 μM), okadaic acid (400 nM), ODQ (1μ M), or SNP (100 μM) plus okadaic acid (400 nM). Results are expressed as a percentage of control cyclic nucleotide levels (3 ± 0.4 pmol/mg protein). Values shown are the mean ± SEM for duplicate samples each assayed for cGMP in triplicate. *Significantly different from control group: P < 0.05 (by ANOVA and Fisher PLSD).
Figure 6.
 
Summary diagram of the pathway involving acetylcholine and NO regulation of Na,K-ATPase activity. Stimulation of cholinergic neurons causes the release of Ca2+, activation of NOS, and the subsequent formation of NO. Nitrovasodilators (NTVs) also cause the formation of NO, which then binds to and activates soluble guanylate cyclase (sGC), the synthetic enzyme of cGMP. cGMP and its analogue 8-Br-cGMP activate protein kinase G (PKG) which may, directly or indirectly through other proteins (dopamine and cAMP-regulated phosphoprotein [DARPP-32] or inhibitor-1 [I-1]), alter Na,K-ATPase activity.
Figure 6.
 
Summary diagram of the pathway involving acetylcholine and NO regulation of Na,K-ATPase activity. Stimulation of cholinergic neurons causes the release of Ca2+, activation of NOS, and the subsequent formation of NO. Nitrovasodilators (NTVs) also cause the formation of NO, which then binds to and activates soluble guanylate cyclase (sGC), the synthetic enzyme of cGMP. cGMP and its analogue 8-Br-cGMP activate protein kinase G (PKG) which may, directly or indirectly through other proteins (dopamine and cAMP-regulated phosphoprotein [DARPP-32] or inhibitor-1 [I-1]), alter Na,K-ATPase activity.
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