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Cholinergic and adrenergic signal transduction pathways in the lacrimal gland
Tuesday, Sep 22, 1992 La Palms/B
X ICER Abstracts 297
4 PROSTANOID RECE;TOR;hSIi Bhattacheriee. . Dept. of Ophthalmol/Vis University of Louisville
OCULAR TISSUES es. L. and Paterson. Sci, Kentucky Lions School of Medicine,
C.A. Eye Research Louisville,
Institute, KY USA
Prostanoid receptors were classified on the basis of the effects of prostaglandins (PGs) on smooth muscle preparations (Kennedy et al.: Prostaglandins, 24:667-689, 1982). Nomenclature for these receptors is EP-PGE,, FP=PGF,,, DP=PGD , IP=PGI and TP=thromboxane. EP receptors are known to be o P three sub types. Currently, we are investigating the PG receptors in the iris-ciliary body (ICB) of various specigs using binsding and second messenger assays. Binding studies with H-PGE, and H-PGF in the bovine lrls-sphincter, iris and ciliary body demonstrated t% e p resence of high affinity binding In the sites, the majority of which were in the iris-sphincter. rabbit ciliary body, the predominant binding sites were of EP subtype. We also examined stimulation of adenylyl cyclase (AC) by PL! receptor agonists including those with EP subtype selectivity in the intact ICB of rabbits, cows and cats. In all species AC stimulation, measured as CAMP formation by the agonists, was dose -dependent. The order of potency in stimulating AC in rabbits was 16,16-dimethyl PGE,(EP,/ EP,)>PGE,(EP)> 11-deoxy PGE,(EP,)=PGD (DP)>iloprost(IP). Cow ICB also has shown similar potency order excepi that PGD and iloprost were inactive. In the cat ICB, the order of agonis 5 potency was PGE,>ll-deoxy PGE z iloprost>l6,16-dimethyl PGE,. 17-phenyl trinor PGE PGF and su\prostone (EP ) did not stimulate AC in any species exc:bt at'high concentrations !>5 CM). The results show that in all species studied, most PG receptor agonists are coupled to AC; those without an effect are probably linked to the phosphoinositide pathway. Furthermore, it seems that in the rabbit and cow ICB, predominant receptors coupled to AC are of the EP subtype. Supported by NIH Grant EY06918 and Research to Prevent kindness, Inc.
298
5
CHOLINERGIC AND ADRENERGIC SIGNAL TRANSDUCTION PATHWAYS IN THE LACRIMAL GLAND Jhtt. D.A.. I&&s. R.R. and Dicker. D.M. Eye Research Institute and Harvard Medical School, Boston, MA, USA Cholinergic and alpha-1 adrenergic agonists use separate signal transduction pathways to stimulate lacrimal gland protein secretion. Cholinergic agonists act by increasing inositol &phosphates that release intmcellular Caz+ and by increasing diacylglycerol that translocates protein kinase C. Adrenergic agonists act by causing a small increase in intracellular Ca*+ and translocating protein kinase C. To determine the C!a*+ dependency of cholinergic and alpha-l admnergic agonistinduced protein secretion, the fluorescent dye Quin 2 was used to chelate and to measure the intracellular Caz+ concentration ([Ca2+]). Acini were prepared from rat exorbital lacrimal glands and incubated in buffer containing Quin 2 for 60 min at 370 C. Two aliquots of acini were incubated with 20 or 150 uM Quin 2 and stimulated with the cholinergic agonist carbachol (lO-5M). Fluorescence was measured at 340 nm. With 20 uM Quin 2, carbachol increased the [Ca2+I by 330+283 nM. With 150 UM Quin 2 carbachol decreased [Ca2+] by 109+69 nM. The remaining acini were incubated with 0 or 150 uM Quin 2 and stimulated with carbachol or the alpha-1 admnergic agonist phenylephrine (l@M). Peroxidase secretion, a marker of protein secretion, was measured specuophotometricaIIy. With 0 Quin 2 carbachol increased peroxidase secretion 13+4, 15~5 and 20+8 units/mg protein (n=6) at 1, 5 and 20 min, respectively. With 150 uM Quin 2 carbachol significantly decreased secretion 130% and 73% at 1 and 5 min and decreased secretion 31% at 20 min. With 0 Quin 2 phenylephrine increased secretion 2+3, 3ti and 9+6 units (n=6) at I, 5 and 20 min. With 150 uM Quin 2 phenylephrine-Induced secretion was unchanged. We conclude that the rapid phase of cholinergic agonist-induced secretion is dependent upon Ca2+. but the sustainedphaseofcholinergicandalpha-1 adrenergic agonist-induced secretionis not. Supported byNIH#EY06177.
299
6 SHORT-TERM MECHANISMS Mito. T.. Department Eye Research Medicine,
MODULATION
OF SOLUTE TRANSPORT
IN CILIARY
Delamere. N.A. and Dona. J. of Ophthalmology and Visual Sciences, Institute, University of Louisville Louisville, KY USA
EPITHELIUM
Kentucky Lions School of
Na,K-ATPase in the ciliary epithelium is involved in the mechanism of aqueous humor formation. Other transport mechanisms such as the ascorbate transporter modify the composition of aqueous humor. Modulation of ciliary epithelium transport processes may therefore play a role in the regulation of aqueous humor secretion. We have determined that ciliary epithelium Na,K-ATPase activity is rapidly diminished by CAMP elevation. In studies with isolated cell membranes, CAMP-dependent protein kinase (protein kinase A) appears to suppress Na,K-ATPase activity. Activation of protein kinase C has a different effect; exposing cultured ciliary epithelium to phorbol esters or diacylglycerols activates Na,K-ATPase. Increasing cytoplasmic calcium also activates Na,K-ATPase. The protein kinase C effect appears to be mediated, in part, by activation of Na-Ii exchange since the effect can be blocked by amiloride. Cytoplasmic calcium appears to stimulate the sodium pump by a different mechanism. In experiments with cultured ciliary epithelium, activation of protein kinases A and C was found to inhibit the ciliary epithelium ascorbate transporter in an additive manner. These observations illustrate that activation of signal transduction pathways may bring about changes in membrane transport processes that could alter both the production and composition of aqueous humor. Supported
by NE1 Grant
No.
EY06915
s-92
X ICER Abstracts 297
4 PROSTANOID RECE;TOR;hSIi Bhattacheriee. . Dept. of Ophthalmol/Vis University of Louisville
OCULAR TISSUES es. L. and Paterson. Sci, Kentucky Lions School of Medicine,
C.A. Eye Research Louisville,
Institute, KY USA
Prostanoid receptors were classified on the basis of the effects of prostaglandins (PGs) on smooth muscle preparations (Kennedy et al.: Prostaglandins, 24:667-689, 1982). Nomenclature for these receptors is EP-PGE,, FP=PGF,,, DP=PGD , IP=PGI and TP=thromboxane. EP receptors are known to be o P three sub types. Currently, we are investigating the PG receptors in the iris-ciliary body (ICB) of various specigs using binsding and second messenger assays. Binding studies with H-PGE, and H-PGF in the bovine lrls-sphincter, iris and ciliary body demonstrated t% e p resence of high affinity binding In the sites, the majority of which were in the iris-sphincter. rabbit ciliary body, the predominant binding sites were of EP subtype. We also examined stimulation of adenylyl cyclase (AC) by PL! receptor agonists including those with EP subtype selectivity in the intact ICB of rabbits, cows and cats. In all species AC stimulation, measured as CAMP formation by the agonists, was dose -dependent. The order of potency in stimulating AC in rabbits was 16,16-dimethyl PGE,(EP,/ EP,)>PGE,(EP)> 11-deoxy PGE,(EP,)=PGD (DP)>iloprost(IP). Cow ICB also has shown similar potency order excepi that PGD and iloprost were inactive. In the cat ICB, the order of agonis 5 potency was PGE,>ll-deoxy PGE z iloprost>l6,16-dimethyl PGE,. 17-phenyl trinor PGE PGF and su\prostone (EP ) did not stimulate AC in any species exc:bt at'high concentrations !>5 CM). The results show that in all species studied, most PG receptor agonists are coupled to AC; those without an effect are probably linked to the phosphoinositide pathway. Furthermore, it seems that in the rabbit and cow ICB, predominant receptors coupled to AC are of the EP subtype. Supported by NIH Grant EY06918 and Research to Prevent kindness, Inc.
298
5
CHOLINERGIC AND ADRENERGIC SIGNAL TRANSDUCTION PATHWAYS IN THE LACRIMAL GLAND Jhtt. D.A.. I&&s. R.R. and Dicker. D.M. Eye Research Institute and Harvard Medical School, Boston, MA, USA Cholinergic and alpha-1 adrenergic agonists use separate signal transduction pathways to stimulate lacrimal gland protein secretion. Cholinergic agonists act by increasing inositol &phosphates that release intmcellular Caz+ and by increasing diacylglycerol that translocates protein kinase C. Adrenergic agonists act by causing a small increase in intracellular Ca*+ and translocating protein kinase C. To determine the C!a*+ dependency of cholinergic and alpha-l admnergic agonistinduced protein secretion, the fluorescent dye Quin 2 was used to chelate and to measure the intracellular Caz+ concentration ([Ca2+]). Acini were prepared from rat exorbital lacrimal glands and incubated in buffer containing Quin 2 for 60 min at 370 C. Two aliquots of acini were incubated with 20 or 150 uM Quin 2 and stimulated with the cholinergic agonist carbachol (lO-5M). Fluorescence was measured at 340 nm. With 20 uM Quin 2, carbachol increased the [Ca2+I by 330+283 nM. With 150 UM Quin 2 carbachol decreased [Ca2+] by 109+69 nM. The remaining acini were incubated with 0 or 150 uM Quin 2 and stimulated with carbachol or the alpha-1 admnergic agonist phenylephrine (l@M). Peroxidase secretion, a marker of protein secretion, was measured specuophotometricaIIy. With 0 Quin 2 carbachol increased peroxidase secretion 13+4, 15~5 and 20+8 units/mg protein (n=6) at 1, 5 and 20 min, respectively. With 150 uM Quin 2 carbachol significantly decreased secretion 130% and 73% at 1 and 5 min and decreased secretion 31% at 20 min. With 0 Quin 2 phenylephrine increased secretion 2+3, 3ti and 9+6 units (n=6) at I, 5 and 20 min. With 150 uM Quin 2 phenylephrine-Induced secretion was unchanged. We conclude that the rapid phase of cholinergic agonist-induced secretion is dependent upon Ca2+. but the sustainedphaseofcholinergicandalpha-1 adrenergic agonist-induced secretionis not. Supported byNIH#EY06177.
299
6 SHORT-TERM MECHANISMS Mito. T.. Department Eye Research Medicine,
MODULATION
OF SOLUTE TRANSPORT
IN CILIARY
Delamere. N.A. and Dona. J. of Ophthalmology and Visual Sciences, Institute, University of Louisville Louisville, KY USA
EPITHELIUM
Kentucky Lions School of
Na,K-ATPase in the ciliary epithelium is involved in the mechanism of aqueous humor formation. Other transport mechanisms such as the ascorbate transporter modify the composition of aqueous humor. Modulation of ciliary epithelium transport processes may therefore play a role in the regulation of aqueous humor secretion. We have determined that ciliary epithelium Na,K-ATPase activity is rapidly diminished by CAMP elevation. In studies with isolated cell membranes, CAMP-dependent protein kinase (protein kinase A) appears to suppress Na,K-ATPase activity. Activation of protein kinase C has a different effect; exposing cultured ciliary epithelium to phorbol esters or diacylglycerols activates Na,K-ATPase. Increasing cytoplasmic calcium also activates Na,K-ATPase. The protein kinase C effect appears to be mediated, in part, by activation of Na-Ii exchange since the effect can be blocked by amiloride. Cytoplasmic calcium appears to stimulate the sodium pump by a different mechanism. In experiments with cultured ciliary epithelium, activation of protein kinases A and C was found to inhibit the ciliary epithelium ascorbate transporter in an additive manner. These observations illustrate that activation of signal transduction pathways may bring about changes in membrane transport processes that could alter both the production and composition of aqueous humor. Supported
by NE1 Grant
No.
EY06915
s-92