Terbutaline, forskolin and cAMP reduce secretion of aqueous humour in the isolated bovine eye

Abstract

In order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 μM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.

Fig 1. The effect of terbutaline on AH formation rate in the bovine isolated perfused eye measured by fluorescein dilution technique.

AH formation rate was expressed as the rate constant (K_out.min^-1 × 10⁻⁴), defined as the slope of the regression line drawn on LN (Log_e) of the changes of fluorescence with time (min). Each value is a mean s.e.mean of the number (n) of experiments shown at the base of each column. In order to eliminate individual variation, control and treated data were obtained from the same eye. AH formation rate measured during the first 30 min prior to drug application was taken as the control value. AH formation rate for the subsequent 40–60 min after establishment of drug effects was taken as the treated value. Note that after addition of drug, 20 min stabilization period was allowed to establish drug effect. Significance of differences from controls; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig 2. The effect of forskolin on AH formation rate in the bovine isolated perfused eye measured by fluorescein dilution technique.

AH formation rate was expressed as the rate constant (K_out.min^-1 × 10⁻⁴), defined as the slope of the regression line drawn on LN (Log_e) of the changes of fluorescence with time (min). Each value is a mean s.e.mean of the number (n) of experiments shown at the base of each column. In order to eliminate individual variation, control and treated data were obtained from the same eye. AH formation rate measured during the first 30 min prior to drug application was taken as the control value. AH formation rate for the subsequent 40–60 min after establishment of drug effects was taken as the treated value. Note that after addition of drug, 20 min stabilization period was allowed to establish drug effect. Significance of differences from controls; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig 3. The effect of 8-Br-cAMP on AH formation rate in the bovine isolated perfused eye measured by fluorescein dilution technique.

AH formation rate was expressed as the rate constant (K_out.min^-1 × 10⁻⁴), defined as the slope of the regression line drawn on LN (Log_e) of the changes of fluorescence with time (min). Each value is a mean s.e.mean of the number (n) of experiments shown at the base of each column. In order to eliminate individual variation, control and treated data were obtained from the same eye. AH formation rate measured during the first 30 min prior to drug application was taken as the control value. AH formation rate for the subsequent 40–60 min after establishment of drug effects was taken as the treated value. Note that after addition of drug, 20 min stabilization period was allowed to establish drug effect. Significance of differences from controls; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig 4. Effect of KT-5720 (10nM) on the inhibitory effect of terbutaline (100μM) on AH formation in the bovine perfused eye.

Stock solution of the PKA inhibitor (KT-5720) in DMSO was added to the perfusate to obtain a final concentration of 10 nM. AH formation rate (shown as K_out.min^-1 10⁻⁴) was measured in separate groups of eyes for 90 min after injection of vehicle or drug. Each value is a mean s.e.mean of the number (n) of experiments shown at the base of each column. Control: injection of vehicle and PK inhibitor. Significance of difference from control; *** p < 0.001.

Fig 5. Increase in intracellular Ca²⁺ in bovine cultured ciliary epithelial cells in response to ATP (100 μM).

(A) Typical trace, obtained in the presence of 1.8mM extracellular calcium, shows a rapid increase in [Ca²⁺ⁱ]_i which is followed by a rapid decline and then a prolonged plateau above baseline. (B) Typical trace obtained in the absence extracellular calcium shows a rapid increase in [Ca²⁺ⁱ]_i which is followed by a rapid decline to baseline without forming the plateau.

Fig 6. Effect of increasing concentration of terbutaline on the Ca²⁺-mobilizing response of ATP (100μM) in bovine cultured CE, both at the peak of the Ca²⁺ transient and at selected time intervals thereafter.

Cultured cells were incubated for 6 min with appropriate concentration of terbutaline in HEPES bufererd Krebs’ solution containing 1.8mM calcium. The cells were then exposed to 100μM of ATP. Each point represents a mean±s.e.mean of 8–10 experiments. Significance of differences between incubations with or without terbutaline; ** p < 0.01 and *** p < 0.001, Bonferroni’s multiple comparison test. Base value indicates the [Ca²⁺]_i prior to addition of ATP.

Fig 7. Effect of increasing concentration of forskolin on the Ca²⁺-mobilizing response of ATP (100μM) in bovine cultured CE, both at the peak of the Ca²⁺ transient and at selected time intervals thereafter.

Cultured cells were incubated for 6 min with appropriate concentration of forskolin in HEPES buffered Krebs’ solution containing 1.8mM calcium. The cells were then exposed to 100μM of ATP. Each point represents a mean±s.e.mean of 8–15 experiments. Significance of differences between incubations with or without forskolin; ** p < 0.01 and *** p < 0.001, Bonferroni’s multiple comparison test. Base value indicates the [Ca²⁺]_i prior to addition of ATP.

Fig 8. Effect of increasing concentration of 8-Br-cAMP on the Ca²⁺-mobilizing response of ATP (100μM) in bovine cultured CE, both at the peak of the Ca²⁺ transient and at selected time intervals thereafter.

Cultured cells were incubated for 6 min with appropriate concentration of 8-Br-cAMP in HEPES bufererd Krebs’ solution containing 1.8mM calcium. The cells were then exposed to 100μM of ATP. Each point represents a mean±s.e.mean of 8–15 experiments. Significance of differences between incubations with or without 8-BR-cAMP; ** p < 0.01 and *** p < 0.001, Bonferroni’s multiple comparison test. Base value indicates the [Ca²⁺]_i prior to addition of ATP.

Fig 9. The effect of the PKA inhibitor, KT-5720 (10nM), on the terbutaline-induced inhibition of [Ca²⁺]_i increase by ATP.

Cells were incubated with either KT-5720 (10nM), or terbutaline (100μM) alone or with KT-5720 (10nM) and terbutaline (100μM) together for 6 min before stimulating by ATP (100μM). KT-5720 largely reversed terbutaline-induced inhibition of the rapid phase of Ca2+ release by ATP. On the other hand, it completely abolished terbutaline-induced augmentation of the plateau phase of the ATP response. Each point is a mean±s.e.mean of 7–10 experiments. Significance of differences from 100μM terbutaline treated response; ** p < 0.01 and *** p < 0.001, Bonferroni’s multiple comparison test. Base value indicates the [Ca²⁺]_i prior to addition of ATP.

Fig 10. Effect of increasing concentration of terbutaline on the Ca²⁺-mobilizing response of ATP (100μM) in bovine cultured CE, in the absence of extracellular calcium.

Terbutaline’s effect on both the peak Ca²⁺ transient and at selected time intervals thereafter had been shown. Cultured cells were incubated for 6 min with appropriate concentration of terbutaline in HEPES buffered Krebs’ solution containing 40mM EGTA and no calcium. The cells were then exposed to 100μM of ATP. Each point represents a mean±s.e.mean of 6–11 experiments. Significance of differences between incubations with or without terbutaline; ** p < 0.01 and *** p < 0.001, Bonferroni’s multiple comparison test. Base value indicates the [Ca²⁺]_i prior to addition of ATP.

Fig 11. Effect of increasing concentration of terbutaline on the Ca²⁺-mobilizing response of ATP (100μM) in bovine cultured CE, in the present of extracellular calcium and NiCl₂ (4mM).

Terbutaline’s effect on both the peak Ca²⁺ transient and at selected time intervals thereafter had been shown. Cultured cells were incubated for 6 min with appropriate concentration of terbutaline in HEPES buffered Krebs’ solution containing 4mM NiCl₂ and1.8mM calcium. The cells were then exposed to 100μM of ATP. Each point represents a mean±s.e.mean of 8–12 experiments. Significance of differences between incubations with or without terbutaline; ** p < 0.01 and *** p < 0.001, Bonferroni’s multiple comparison test. Base value indicates the [Ca²⁺]_i prior to addition of ATP.