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(Investigative Ophthalmology and Visual Science. 1999;40:1833-1837.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Isoproterenol, Forskolin, and cAMP-Induced Nitric Oxide Production in Pig Ciliary Processes

Rong Liu, Josef Flammer and Ivan O. Haefliger

From the Laboratory of Ocular Pharmacology and Physiology, University Eye Clinic, Basel, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate whether isoproterenol and forskolin, two adenylylcyclase activators, or 8-bromo-cAMP, an adenosine 3',5'-cyclic monophosphate (cAMP) analog, increase nitric oxide (NO) production in isolated porcine ciliary processes.

METHODS. Nitrite (an NO metabolite) was measured (Griess reaction) before and 2 hours after exposure to 0.1 to 100 µM isoproterenol (a ß-adrenoreceptor agonist), 0.01 to 100 µM forskolin, or 0.1 to 1000 µM 8-bromo-cAMP. Some experiments were conducted in the presence of 0.5 mM NG-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase [NOS] inhibitor), 10 µM propranolol (a ß-adrenoreceptor antagonist), or 1 µM KT 5720 (a cAMP-dependent protein kinase inhibitor). cAMP production was also measured (by immunoassay).

RESULTS. Nitrite production was increased by isoproterenol (maximum, 10 µM: 164%; P < 0.001), forskolin (maximum, 10 µM: 254%; P < 0.001), and 8-bromo-cAMP (maximum, 100 µM: 184%; P < 0.001), an effect prevented by L-NAME (P < 0.05–0.001). Propranolol inhibited only isoproterenol-induced (10 µM) nitrite production (P < 0.05), whereas KT 5720 (P < 0.05) inhibited isoproterenol- (10 µM) and 8-bromo-cAMP–induced (10 µM) nitrite production. Furthermore, cAMP production evoked by isoproterenol (10 µM, P < 0.05) but not by forskolin (10 µM, P < 0.001) was inhibited by propranolol (P < 0.05).

CONCLUSIONS. In isolated porcine ciliary processes, drugs activating adenylylcyclase or mimicking cAMP increase the production of NO by a mechanism that appears to involve both a cAMP-dependent protein kinase and NOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the eye, the primary site of aqueous humor formation is the epithelium of the ciliary processes. By a mechanism that still is unclear, drugs that decrease intracellular concentration of cyclic 3',5' adenosine monophosphate (cAMP), such as ß-adrenergic receptor antagonists (ß-blockers), also inhibit aqueous humor formation.1

Nitric oxide (NO) is a cellular mediator that can be produced by the enzyme nitric oxide synthase (NOS) from the amino acid L-arginine.2 Nitric oxide has a short half-life and is rapidly transformed in more stable compounds, such as nitrite (NO2-). In the kidney, the respiratory airway, and the colon, NO can modulate transepithelial ionic and/or fluid transport.3 4 5

The presence of an NOS activity6 that can be modulated by ß-adrenergic agents7 has recently been reported in porcine ciliary processes, raising the possibility that NO could be associated with the process of aqueous humor formation. This study investigated whether in isolated porcine ciliary processes NO production is increased by drugs that either activate adenylylcyclase, such as isoproterenol (a ß-adrenergic receptor agonist) and forskolin (an adenylylcyclase activator), or mimic cAMP, such as 8-bromo-cAMP (a stable lipophylic cAMP analog).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Preparation
In adherence to the ARVO Statement for the Use of Animals in Ophthalmology and Vision Research, porcine eyes (one eye/animal) were obtained from an abattoir immediately after animals were killed and were transported to the laboratory in ice-cold Krebs-Ringer’s physiologic solution. Within 3 hours after death of the animal, the ciliary processes were dissected in cold Krebs-Ringer’s solution. Tissues were then either stored at -70°C until nitrite measurements were conducted or used right away for cAMP determination.

Nitrite Measurements
Tissues were thawed at room temperature and washed with Hanks’ balanced salt solution (HBSS). Ciliary processes were placed in a 24-well plate (ciliary processes from one eye/well), covered with 700 µl HBSS–L-arginine medium (HBSS supplemented with 10 µM L-arginine), and incubated in a humidified incubator at 37°C.

After 30 minutes, 100 µl medium was taken from each well for the first nitrite measurement. Tissues were then exposed for 2 hours to different drugs (antagonists and/or agonists) before another 100-µl sample was collected in each well for a second nitrite measurement. For each experiment, quiescent controls were run in parallel in which tissues were not exposed to tested drugs. Nitrite production was defined as the amount of nitrite measured between the first and second measurements.

Nitrite was assessed by Griess reaction.7 In brief, samples were centrifuged at 1000g for 15 minutes. From each sample, 45 µl supernatant was mixed with 45 µl Griess reagent (0.75% sulfanilamide in 5% phosphoric acid and 0.075% N-1-naphthyl-ethylenediamine dihydrochloride in double-distilled water) and incubated at room temperature for 10 minutes. Optical density was then measured on a microplate reader at 540 nm. Nitrite concentrations were determined by comparisons with a standard sodium nitrite curve.

cAMP Measurements
In a 24-well plate, freshly dissected tissues (ciliary processes from one eye/well) were incubated in HBSS for 60 minutes at 37°C before exposure for 30 minutes to 0.5 mM isobutyl-methylxanthine, an inhibitor of cyclic nucleotide phosphodiesterase to prevent breakdown of accumulated nucleotides. Tissues were then further incubated for 30 minutes with the nonselective ß-adrenergic receptor antagonist propranolol (10 µM) before exposure for 10 minutes to isoproterenol (10 µM) or forskolin (10 µM). Similar experiments were also run in parallel without propranolol. At the end of the experimental protocol, tissues were rapidly frozen in liquid nitrogen and stored at -70°C until assayed for cAMP determination.

For cAMP measurement, each sample was homogenized at 4°C in the presence of 0.5 ml ice-cold 6% trichloroacetic acid (TCA) before they were centrifuged at 2000g for 20 minutes at 4°C. The supernatant was removed, placed in a test tube, and extracted four times with water-saturated ethyl ether. The extracted supernatant was further evaporated by a centrifuged vacuum pump at 40°C, and the cAMP content measured with a commercial enzyme immunoassay kit (Amersham, Amersham, UK). Experiments were repeated three times in duplicate. The amount of protein in each well was measured by dissolving the pellets in 1 ml of 0.1 N NaOH and assayed for protein concentration using a commercially available kit (Bio-Rad, Glattbrugg, Switzerland). The amount of cAMP was normalized to the amount of protein in each well.

Drugs
L-Arginine, 8-bromo-cAMP, dimethyl sulfoxide (DMSO), forskolin, isobutyl-methylxanthine, (-)isoproterenol, KT 5720, KT 5823, NG-nitro-D-arginine methyl ester (D-NAME), NG-nitro-L-arginine methyl ester (L-NAME), DL-propranolol, sodium nitrite, and TCA were purchased from Sigma (Buchs, Switzerland). Griess reagent was obtained from Merck (Darmstadt, Germany). HBSS was purchased from Gibco (Basel, Switzerland). All drugs were made up fresh the day of the experiment. The drugs were dissolved in HBSS except for forskolin, KT 5720, and KT 5823, which were dissolved in DMSO and isoproterenol for which ethanol was used. The final maximal concentration in a well for DMSO and ethanol was 0.1% and 0.01%, respectively.

Statistical Analysis
Nitrite production (the difference between the first and second nitrite measurements) was either expressed in micromolar per milligram of tissue or in the percentage of the mean nitrite production of quiescent controls (% of control) run in parallel with each experiment. Data of cAMP measurements were expressed in picomoles per milligram protein. Results are shown as mean ± SEM with n corresponding to the number of eyes assessed (ciliary processes from one eye per animal per well). Statistical comparisons were conducted using either an unpaired Student’s t-test or a one-way analysis of variance followed by Bonferroni’s multiple-comparison test with P < 0.05 considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basal Nitrite Production
In quiescent isolated ciliary processes, the basal production of nitrite (1.3 ± 0.2 µM/mg tissue; n = 20) was significantly (P < 0.05) inhibited by 0.5 mM L-NAME, an inhibitor of NO formation (0.7 ± 0.2 µM/mg tissue; n = 20). These results indicate the existence of basal NOS activity in isolated porcine ciliary processes.

cAMP-Induced Nitrite Production
In a concentration-dependent manner, the basal production of nitrite was significantly increased by the ß-adrenergic receptor agonist isoproterenol (0.1–100 µM), the adenylylcyclase activator forskolin (0.01–100 µM), or the stable and lipophylic cAMP analog 8-bromo-cAMP (1–1000 µM). The maximal increase in nitrite production was observed at a concentration of 10 µM for isoproterenol (P < 0.001), 10 µM for forskolin (P < 0.001), and 100 µM for 8-bromo-cAMP (P < 0.001). These results indicate that in isolated porcine ciliary processes the production of nitrite can be enhanced by drugs known to activate adenylylcyclase (isoproterenol, forskolin) or to mimic cAMP (8-bromo-cAMP; Fig. 1 ).



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  		Figure 1. Effect of isoproterenol (a ß-adrenergic receptor agonist and adenylylcyclase activator), forskolin (an adenylylcyclase activator), and 8-bromo-cAMP (a stable and lipophylic cAMP analog) on the production of nitrite from isolated porcine ciliary processes. Nitrite production was significantly increased by isoproterenol and forskolin, which enhance intracellular production of cAMP, and by 8-bromo-cAMP, which mimics cAMP. Analysis of variance with Bonferroni’s comparison versus 0 µM: **P < 0.01, ***P < 0.001.

 
cAMP-Induced Nitrite Production and L-NAME
The increase in nitrite production evoked by isoproterenol (10 µM) was significantly (P < 0.01) blunted by the inhibitor of NO formation L-NAME (0.5 mM), but not by D-NAME (0.5 mM), the dextrorotatory nonmetabolizable isoform of L-NAME. Also, the nitrite production induced by forskolin (1 µM; P < 0.001) or 8-bromo-cAMP (10 µM; P < 0.05) was significantly inhibited by L-NAME (0.5 mM). These results demonstrate that in porcine ciliary processes the increase in nitrite production induced by isoproterenol, forskolin, or 8-bromo-cAMP is mainly caused by the activity of an NOS (Fig. 2) .



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  		Figure 2. Effect of the nitric oxide (NO) formation inhibitor L-NAME on the production of nitrite induced by isoproterenol (10 µM), forskolin (1 µM), or 8-bromo-cAMP (10 µM) from isolated porcine ciliary processes. The increase in nitrite production was significantly inhibited by L-NAME, showing the activity of an NOS. D-NAME is the dextrogyre nonmetabolized isoform of L-NAME. Analysis of variance with Bonferroni’s comparison versus 0 µM: **P < 0.01; unpaired Student’s t-test: {dagger}P < 0.05; {dagger}{dagger}{dagger}P < 0.001.

 
cAMP-Induced Nitrite Production and Isoproterenol
In a concentration-dependent manner, the increase in nitrite production induced by isoproterenol (10 µM) was significantly (P < 0.05) reduced by increasing concentrations of the nonselective ß-adrenergic receptor antagonist propranolol (0.1, 1, and 10 µM). In contrast, propranolol had no significant inhibitory effect on the nitrite production evoked by forskolin (1 µM) or 8-bromo-cAMP (10 µM). These results show that in porcine ciliary processes the increase in nitrite production induced by isoproterenol can be inhibited by a ß-blocker such as propranolol (Fig. 3) .



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  		Figure 3. Effect of the nonselective ß-adrenergic receptor antagonist propranolol on the production of nitrite induced by isoproterenol (10 µM), forskolin (1 µM), or 8-bromo-cAMP (10 µM) from isolated porcine ciliary processes. Only the increase in nitrite production induced by the ß-adrenergic receptor agonist isoproterenol was significantly inhibited by propranolol. Analysis of variance with Bonferroni’s comparison versus 0 µM: *P < 0.05.

 
cAMP-Induced Nitrite Production and KT 5720
The increase in nitrite production evoked by isoproterenol (10 µM) was significantly (P < 0.05) inhibited by different concentrations of the specific cAMP-dependent protein kinase A (PKA) inhibitor KT 5720 (0.1 and 1 µM), but not by the specific cGMP-dependent protein kinase G (PKG) inhibitor KT 5823 (1 µM). In a similar manner, the nitrite production induced by 8-bromo-cAMP (10 µM) was significantly (P < 0.05) inhibited by the PKA inhibitor KT 5720 (1 µM) but not by the PKG inhibitor KT 5823 (1 µM). Therefore, in porcine ciliary processes, it appears that the increase in nitrite production evoked by isoproterenol and 8-bromo-cAMP is linked to the activity of PKA (Fig. 4) .



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  		Figure 4. Effect of KT 5720, a cAMP-dependent PKA inhibitor, and KT 5823, a cGMP-dependent PKG inhibitor on the production of nitrite evoked by isoproterenol (10 µM) and 8-bromo-cAMP (10 µM) from isolated porcine ciliary processes. The increase in nitrite production was significantly reduced by KT 5720, suggesting a link with the activity of PKA. Analysis of variance and Bonferroni’s comparison versus 0 µM: *P < 0.05).

 
cAMP-Production in Porcine Ciliary Processes
In isolated porcine ciliary processes, when compared with control experiments (16 ± 1.8 picomoles/mg protein), isoproterenol (10 µM: 30 ± 2.7 picomoles/mg protein) and forskolin (10 µM: 74 ± 8.5 picomoles/mg protein) significantly increased cAMP concentration (n = 5; P < 0.05 and P < 0.001, respectively). Furthermore, the increase in cAMP production induced by isoproterenol, but not by forskolin, was significantly (P < 0.05) inhibited by propranolol (10 µM, 14 ± 2.8 picomoles/mg protein). These results indicate that drugs such as isoproterenol and forskolin that enhance nitrite production in isolated porcine ciliary processes also increase cAMP production in this particular tissue.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that drugs that activate adenylylcyclase (isoproterenol, forskolin) or mimic cAMP (8-bromo-cAMP) increase the production of nitrite in isolated porcine ciliary processes. The effect appears to be linked to the activity of a cAMP-dependent protein kinase (PKA) and an NOS, because the increase in nitrite production could be blunted by KT 5720 (a PKA inhibitor) or L-NAME (an inhibitor of NO formation).

Stimulation of ß-adrenergic receptors can lead to the activation of a membrane-bound adenylylcyclase and to an increase of intracellular cAMP.1 Evidence of such a mechanism, which is known to occur in ciliary processes, could also be observed in our porcine ciliary process preparations: the ß-adrenergic receptor agonist isoproterenol increased cAMP production, an effect prevented by the nonselective ß-adrenergic receptor antagonist propranolol.

Recently, we reported that ß-adrenergic receptor activation increases NO production in isolated porcine ciliary processes.7 The present study further showed that the second-messenger cAMP was involved in this process. Indeed, not only the ß-adrenergic receptor agonist isoproterenol (which enhances cAMP production) but also forskolin (an adenylylcyclase activator) or 8-bromo-cAMP (a stable and membrane-permeable cAMP analog) increased NO production in this tissue. This effect appears to reflect the activity of PKA, because the increase in NO production evoked by isoproterenol or 8-bromo-cAMP could be blunted by the PKA inhibitor KT 5720, but not by the PKG inhibitor KT 5823. Consistent with these results are several observations made in the literature of the modulation of NOS activity and/or expression by cAMP or PKA.8 9

Isoproterenol, forskolin, and 8-bromo-cAMP increased nitrite production in a dose-dependent manner until a maximum level of production was reached. When higher concentrations of these drugs were used, for a reason we cannot yet explain the increase in nitrite production was reduced.

Until now, several NOS isoforms have been identified that are responsible for the production of NO in many different types of cells (e.g., vascular endothelial cells, neurons, epithelial cells, macrophages).2 In the present study we did not investigate which NOS isoform or which type of cells (ciliary epithelial cells, smooth muscle cells, endothelial cells, or neuronal cells) were involved in the NO production observed in porcine ciliary processes.

It has been reported that in the kidney, the trachea, or the colon NO is involved in transepithelial ionic and/or fluid transport.3 4 5 The present observation that activation of the ß-adrenoreceptor–cAMP pathway increases NO production, raises the possibility that NO could, to a certain extent, be involved in the process of aqueous humor formation in ciliary processes. An action that would be different from the effect of NO at the level of the iridocorneal angle where it increases the excretion of aqueous humor from the eye.10

In conclusion, it appears that the second-messenger cAMP can modulate NO production in isolated porcine ciliary processes. With references made to the role played by NO in transepithelial fluid transport (kidney, trachea, colon)3 4 5 and by cAMP as a common second messenger of different ocular hypotensive drugs (ß-blockers, {alpha}2-adrenergic receptor agonists),1 the present observation raises the possibility that NO could be involved in the regulation of aqueous humor formation.


   Acknowledgements
 
The authors thank Andreas Schötzau for his help and advice in the statistical analysis of the data.


   Footnotes
 
Reprint requests: Ivan O. Haefliger, Laboratory of Ocular Pharmacology and Physiology, University Eye Clinic Basel, Mittlere Strasse 91, PO Box, CH-4012 Basel, Switzerland.

Supported by Grant 32-52783.97 from the Swiss National Science Foundation, Bern; the Velux Foundation, Zurich; the Schwickert Foundation, Basel; and the Roche Research Foundation, Basel, Switzerland.

Submitted for publication November 25, 1998; revised March 3, 1999; accepted March 16, 1999.

Proprietary interest category: N.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mittag, TW (1996) Adrenergic and dopaminergic drugs in glaucoma Ritch, R Shields, MG Krupin, T eds. The Glaucomas 2nd ed. ,1409-1424 Mosby Year Book St. Louis.
  2. Nathan, C, Xie, QW (1994) Nitric oxide synthases: roles, tolls, and controls Cell 78,915-918[Medline][Order article via Infotrieve]
  3. Gabbai, FB, Thomson, SC, Peterson, O, Wead, L, Malvey, K, Blantz, RC (1995) Glomerular and tubular interactions between renal adrenergic activity and nitric oxide Am J Physiol 268,F1004-F1008[Abstract/Free Full Text]
  4. Takemura, H, Tamaoki, J, Tagaya, E, Chiyotani, A, Konno, K. (1995) Isoproterenol increases Cl diffusion potential difference of rabbit trachea through nitric oxide generation J Pharmacol Exp Ther 274,584-588[Abstract/Free Full Text]
  5. Wilson, KT, Xie, Y, Musch, MW, Chang, EB (1993) Sodium nitroprusside stimulates anion secretion and inhibits sodium chloride absorption in rat colon J Pharmacol Exp Ther 266,224-230[Abstract/Free Full Text]
  6. Haufschild, T, Nava, E, Meyer, P, Flammer, J, Lüscher, TF, Haefliger, IO (1996) Spontaneous calcium-independent nitric oxide synthase activity in porcine ciliary processes Biochem Biophys Res Commun 222,786-789[Medline][Order article via Infotrieve]
  7. Liu, R, Flammer, J, Lüscher, TF, Haefliger, IO (1998) ß-adrenergic agonist-induced nitrite production in isolated pig ciliary processes Graefes Arch Clin Exp Ophthalmol 236,613-616[Medline][Order article via Infotrieve]
  8. Oddis, CV, Simmons, RL, Hattler, BG, Finkel, MS (1995) cAMP enhances inducible nitric oxide synthase mRNA stability in cardiac myocytes Am J Physiol 269,H2044-H2050[Abstract/Free Full Text]
  9. Tamaoki, J, Kondo, M, Takemura, H, Chiyotani, A, Yamawaki, I, Konno, K. (1995) Cyclic adenosine monophosphate-mediated release of nitric oxide from canine cultured tracheal epithelium Am J Respir Crit Care Med 152,1325-1330[Abstract]
  10. Nathanson, JA (1992) Nitrovasodilators as a new class of ocular hypotensive agents J Pharmacol Exp Ther 260,956-965[Abstract/Free Full Text]



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