Exp.EyeRes. (1990) 51, 229-231
LETTER
Neuropeptide
TO THE EDITORS
Y Modulates Adenylate Ciliary Body and Ciliary
Neuropeptide Y (NPY), a 36-amino acid neuropeptide (Tatemoto, 1982) is one of numerous biologically active peptides that have been localized in nerve fibers innervating the anterior uvea (Stone, Kuwayama and Laties, 1987). The iris and ciliary body are richly supplied with NPY-containing sympathetic neurons originating in the superior cervical ganglia (Stone, Laties and Emson, 1986). Although the biological role of NPY in ocular tissues is unknown, NPY has been reported to potentiate noradrenergic vasoconstriction (Edvinsson et al., 1984; Ekblad et al., 1984) and contractions of the isolated rabbit iris dilator muscle (Piccone et al., 1988). To elucidate the possible biochemical basis for these effects, we have investigated the influence of NPY on basal and hormone-stimulated cyclic AMP (CAMP)accumulation in the rabbit iris-ciliary body, ciliary processes and ciliary epithelium. Adult male New Zealand White rabbits (2-2.5 kg) were used in accordance with NIH guidelines and the ARVO Resolution on the Use of Animals in Research. Rabbits were killed by injection of an overdosage of sodium pentobarbital. Anterior eye segments were removed, and iris-ciliary bodies (ICBs) dissected and maintained in oxygenated, bicarbonate-buffered Krebs solution containing 3 ,TLM indomethacin. For some experiments, individual ciliary processes (CP) were excised with fine dissection scissors and pooled. To isolate sheets of ciliary epithelium (CE), anterior segments were placed posterior side-up in a Petri dish, and the iris and lens were gently separated from the adjacent sclera. The iris was then transected from the pupillary margin to its outer edge, and gently peeled away from the lens, leaving a sheet of ciliary epithelium attached at the zonules. The epithelial sheet was dissected free of the lens capsule and cut into four to six approximately equal segments, Histological examination of the isolated ciliary epithelium by light and transmission electron microscopy revealed an intact cellular bilayer with no apparent contamination by vascular or stromal cells (B. Raphael, J. Jumblatt and M. Jumblatt, unpubl. res.) Cyclic AMP accumulation was assayed in situ by measurement of the conversion of [3~]~~~ to [3H]~AMP in tissues prelabeled with C3H]adenine (Mittag and Tormay, 1985). Isolated segments of ICB, CP or CE were incubated for 60 min at 37°C in Krebs solution containing 2.5 ,uCiml-’ [2,8-3H]adenine, rinsed briefly in non-radioactive Krebs solution, and placed in individual wells of 24-well culture plates containing 1 ml Krebs solution supplemented with 00144835/90/080229+03
$03.00/O
Cyclase in the Rabbit Epithelium
Iris
0.5 mM 3-isobutyl-1-methylxanthine (IMBX). After 10 min pretreatment, the solution was replaced with fresh Krebs/IBMX containing test agents, and the incubation continued for an additional 15 min at 37°C. The reaction was terminated by sonication of tissue segments in 1 ml 5% trichloracetic acid (4°C) containing 0.3 mM CAMP as carrier, followed by overnight extraction at 4°C and centrifugation at 10 000 g. Supernatant aliquots were chromographed sequentially on Dowex 50AG WX4 and neutral alumina columns (Salomon. Landos and Rodbell, 1974) to isolate [3H]cAMP and [“H]ATP fractions, which were subsequently analyzed for radioactivity by liquid scintillation spectrometry. [3H]~AMP fractions were corrected for recoveries of unlabeled carrier CAMP, as determined from absorbance measurements at 260 nm. [3H]cAMP was expressedas the percentage conversion of 3H-labeled ATP to CAMP: y0 Conversion = 100. [dpm cAMP/(dpm ATP + dpm CAMP)]. Drug-stimulated [3H]cAMP accumulation was determined by substraction of basal from total activity measured in drug-treated samples. Results are expressed as the mean + s.E.M., n = number of determinations. Statistical significant of drug effects was evaluated by Student’s t-test (unpaired data). NPY (lo-’ M) significantly (P < 0.05) inhibited basal as well as mediator-stimulated [3H]cAMP accumulation in ICB segments(Fig. 1). The magnitude of NPY-mediated inhibition varied with the mode of adenylate cyclase activation, as follows : vasoactive intestinal peptide (VIP, lo-’ M, 52.3% inhibition), forskolin ( 10e6, 39.4% inhibition), isoproterenol (10e6 M, 24.8% inhibition), and PGE, (low6 M, no significant inhibition). As shown in Fig. 2, NPY (O-l-100 nM) inhibited VIP-induced t3H]cAMP accumulation in a concentration-dependent manner, with maximal inhibition (52 %) attained at lo-’ M NPY; EC50z 3 nM. Comparable inhibition by 10m7M NPY was observed in excised ciliary processes(40.1% inhibition: Fig. 3) and in isolated sheets of ciliary epithelium (89.0% inhibition ; Fig. 3). despite the relatively high basal rate of CAMPaccumulation in the latter preparation. The above results demonstrate that NPY is a potent inhibitor of CAMP accumulation in the rabbit irisciliary body, ciliary processesand epithelium. Similar NPY-mediated inhibition of adenylate cyclase activity has been described in several tissues, including blood vessels (Fredholm. Jansen and Edvinsson, 1985), vas 0 1990 AcademicPressLimited
230
J. E. JUMBLATT
’ -
0 Activator DActlvotortNPY
(to-’
14, T M)
1
ii $5
6-
jd >t $i c” n
FIG. 1. Comparison of the effects of NPY on basal and mediator-stimulated [3H]~AMP accumulation in iris-ciliary body segments. Abbreviations and drug concentrations used: PGE, = prostaglandin E,, 10m6M: IS0 = isoproterenol, 10m6M; VIP = vasoactive intestinal peptide, 1O-7M: FSK = forskolin, 1O-6M. Vertical bars (and horizontal lines) represent meansf S.E.M. : n = number of determinations. * P < 0.05, **p < 0.001.
AND
1
txa8asal
0 I
J. M. GOOCH
VIP VIP+NPY
,171
FIG. 3. NPY-mediated inhibition of VIP-stimulated [3H]cAMP accumulation in excised ciliary processesand isolated ciliary epithelium. Vertical bars (and horizontal lines) represent meansrt:S.E.M. : n = number of determinations. *P < 0.05. ** P < 0.001.
observations remains to be established, several potential
roles for NPY in the anterior
uvea are
suggested. First, NPY may function as a feedback modulator of sympathetic neurotransmission, mediating prejunctional inhibition of norepinephrine release and postjunctional E’ a ,E “6 g
mediated reduction of intracellular
60-
of P-adrenergic CAMP might also
account for the potentiative effects of NPY on
40-
4 a >
/I 0
inhibition
(CAMP-dependent)responsesof target cells. The NPY-
EO-
Y
”
I -II
, -10
I
I
-9 log [NPY]
-8
I -7
I -6
CM)
FIG. 2. Concentration-dependence of NPY-mediated inhi-
bition of VIP (lo-’ M)-stimulated [3H]cAMP accumulation in iris-ciliary body segments. Data points (and vertical lines) represent means) S.E.M. : n = number of determinations. deferens (Haggblad and Fredholm, 198 7) and cardiac
cells (Kassis et al., 1987). We have recently reported evidence that NPY acts prejunctionally
to depress the
synaptic release of norepinephrine from intraocular sympathetic nerves (Ohia and Jumblatt, 1989). Although the biological significance of these
norepinephrine-induced (a-adrenergic) contractions of the dilator pupillae and vascular smooth muscle, since CAMP has been implicated in the relaxation of these muscles. The identification of inhibitory NPY
receptors in isolated ciliary
19 September
Acknowledgments This investigation was supported by USPHS Research Grant No. EYO-5246, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc. Special thanks go to MS Rita Hackmiller for expert technical assistance. J. E. JUMBLATT J. M. GOOCH
7989 and accepted in revised form 4 December
References Edvinsson, L., Ekblad, E., Hakanson, R. and Wahlestedt, C. (1984). Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels. Br. J. Pharmacol. 83, 5 19-2 5
(Fig. 3)
aqueous humor dynamics.
Department of Ophthalmology and Visual Sciences, University of Louisville, School of Medicine, Kentucky Lions Eye Research Institute, L 0 uis vile, KY 40202- 1511, U.S.A. (Received
epithelium
suggests, furthermore, that NPY may have a direct role in aqueous humor regulation. Clarification of any such role, however, will require a better understanding of the autonomic neurogenic mechanisms that govern
7989)
Ekblad, E., Fdvinsson, L., Wahlestedt, C., Uddman, R.. Hakanson, R. and Sundler, F. (1984). Neuropeptide Y co-exists and cooperates with noradrenaline in perivascular nerve fibers. Regul. Pept. 8, 225-35. Fredholm, B. B.. Jansen, I. and Edvinsson, L. (1985). Neuropeptide
Y is a potent inhibitor
of cyclic AMP
NPY
INHIBITS
accumulation
UVEAL
ADENYLATE
231
CYCLASE
in feline blood vessels. Acta Physiol.
Stand. 124. 467-9.
Haggblad,J. and Fredholm, B. B. (1987). Adenosineand neuropeptideY enhancealpha,-adrenoceptor-induced accumulation of inositol phosphatesand attenuate forskolin-inducedaccumulation of CAMP in rat vas deferens.Neurosci. I&t. 82, 211-16. Kassis,S.M., Glasman,L.. Terenius,L. and Fishman,P. H. (198 7). Neuropeptide Y inhibits cardiac adenylate cyclasethrough a pertussistoxin-sensitiveG protein. I. &of. Chem.262, 3420-31. Mittag. T. and Tormay. A. (1985). Drug responsesof adenylate cyclase in iris-ciliary body determinedby adenine labelling. Invest. Ophthalmol.Vis. Sci. 26, 396-9.
Ohia. S.E.andJumblatt,J. E.(1989). Effectof neuropeptideY on norepinephrinereleasein the rabbit iris-ciliary body.
Invest. Ophthalmol. Vis. Sci. 30 (Suppl.),21. Piccone,M., Littzi, J., Krupin, T., Stone,R. A., Davis,M. and Wax, M. B. (1988). FZTects of neuropeptideY on the isolatedrabbit iris dilator muscle. Invest. Ophthalmol. Vis. Sci. 29, 330-2.
Salomon,Y., Landos,C. and Rodbell.M. (1974). A highly sensitiveadenylate cyclase assay. Anal. Biochem. 58, 541-6. Stone, R. A., Kuwayama, Y. and Laties, A. M. (1987). Regulatorypeptidesin the eye. Elcperienta 43, 790-800. Stone. R. A., Laties, A. M. and Emson. P. C. (1986). NeuropeptideY and the ocular innervation of rat, guinea pig, cat, and monkey. Neuroscience17, 1207-16. Tatemoto, K. (1982). NeuropeptideY: the completeamino acidsequenceof the brain peptide.Proc. Natl. Acad. Sci. U.S.A. 79, 5485-9.
LETTER
Neuropeptide
TO THE EDITORS
Y Modulates Adenylate Ciliary Body and Ciliary
Neuropeptide Y (NPY), a 36-amino acid neuropeptide (Tatemoto, 1982) is one of numerous biologically active peptides that have been localized in nerve fibers innervating the anterior uvea (Stone, Kuwayama and Laties, 1987). The iris and ciliary body are richly supplied with NPY-containing sympathetic neurons originating in the superior cervical ganglia (Stone, Laties and Emson, 1986). Although the biological role of NPY in ocular tissues is unknown, NPY has been reported to potentiate noradrenergic vasoconstriction (Edvinsson et al., 1984; Ekblad et al., 1984) and contractions of the isolated rabbit iris dilator muscle (Piccone et al., 1988). To elucidate the possible biochemical basis for these effects, we have investigated the influence of NPY on basal and hormone-stimulated cyclic AMP (CAMP)accumulation in the rabbit iris-ciliary body, ciliary processes and ciliary epithelium. Adult male New Zealand White rabbits (2-2.5 kg) were used in accordance with NIH guidelines and the ARVO Resolution on the Use of Animals in Research. Rabbits were killed by injection of an overdosage of sodium pentobarbital. Anterior eye segments were removed, and iris-ciliary bodies (ICBs) dissected and maintained in oxygenated, bicarbonate-buffered Krebs solution containing 3 ,TLM indomethacin. For some experiments, individual ciliary processes (CP) were excised with fine dissection scissors and pooled. To isolate sheets of ciliary epithelium (CE), anterior segments were placed posterior side-up in a Petri dish, and the iris and lens were gently separated from the adjacent sclera. The iris was then transected from the pupillary margin to its outer edge, and gently peeled away from the lens, leaving a sheet of ciliary epithelium attached at the zonules. The epithelial sheet was dissected free of the lens capsule and cut into four to six approximately equal segments, Histological examination of the isolated ciliary epithelium by light and transmission electron microscopy revealed an intact cellular bilayer with no apparent contamination by vascular or stromal cells (B. Raphael, J. Jumblatt and M. Jumblatt, unpubl. res.) Cyclic AMP accumulation was assayed in situ by measurement of the conversion of [3~]~~~ to [3H]~AMP in tissues prelabeled with C3H]adenine (Mittag and Tormay, 1985). Isolated segments of ICB, CP or CE were incubated for 60 min at 37°C in Krebs solution containing 2.5 ,uCiml-’ [2,8-3H]adenine, rinsed briefly in non-radioactive Krebs solution, and placed in individual wells of 24-well culture plates containing 1 ml Krebs solution supplemented with 00144835/90/080229+03
$03.00/O
Cyclase in the Rabbit Epithelium
Iris
0.5 mM 3-isobutyl-1-methylxanthine (IMBX). After 10 min pretreatment, the solution was replaced with fresh Krebs/IBMX containing test agents, and the incubation continued for an additional 15 min at 37°C. The reaction was terminated by sonication of tissue segments in 1 ml 5% trichloracetic acid (4°C) containing 0.3 mM CAMP as carrier, followed by overnight extraction at 4°C and centrifugation at 10 000 g. Supernatant aliquots were chromographed sequentially on Dowex 50AG WX4 and neutral alumina columns (Salomon. Landos and Rodbell, 1974) to isolate [3H]cAMP and [“H]ATP fractions, which were subsequently analyzed for radioactivity by liquid scintillation spectrometry. [3H]~AMP fractions were corrected for recoveries of unlabeled carrier CAMP, as determined from absorbance measurements at 260 nm. [3H]cAMP was expressedas the percentage conversion of 3H-labeled ATP to CAMP: y0 Conversion = 100. [dpm cAMP/(dpm ATP + dpm CAMP)]. Drug-stimulated [3H]cAMP accumulation was determined by substraction of basal from total activity measured in drug-treated samples. Results are expressed as the mean + s.E.M., n = number of determinations. Statistical significant of drug effects was evaluated by Student’s t-test (unpaired data). NPY (lo-’ M) significantly (P < 0.05) inhibited basal as well as mediator-stimulated [3H]cAMP accumulation in ICB segments(Fig. 1). The magnitude of NPY-mediated inhibition varied with the mode of adenylate cyclase activation, as follows : vasoactive intestinal peptide (VIP, lo-’ M, 52.3% inhibition), forskolin ( 10e6, 39.4% inhibition), isoproterenol (10e6 M, 24.8% inhibition), and PGE, (low6 M, no significant inhibition). As shown in Fig. 2, NPY (O-l-100 nM) inhibited VIP-induced t3H]cAMP accumulation in a concentration-dependent manner, with maximal inhibition (52 %) attained at lo-’ M NPY; EC50z 3 nM. Comparable inhibition by 10m7M NPY was observed in excised ciliary processes(40.1% inhibition: Fig. 3) and in isolated sheets of ciliary epithelium (89.0% inhibition ; Fig. 3). despite the relatively high basal rate of CAMPaccumulation in the latter preparation. The above results demonstrate that NPY is a potent inhibitor of CAMP accumulation in the rabbit irisciliary body, ciliary processesand epithelium. Similar NPY-mediated inhibition of adenylate cyclase activity has been described in several tissues, including blood vessels (Fredholm. Jansen and Edvinsson, 1985), vas 0 1990 AcademicPressLimited
230
J. E. JUMBLATT
’ -
0 Activator DActlvotortNPY
(to-’
14, T M)
1
ii $5
6-
jd >t $i c” n
FIG. 1. Comparison of the effects of NPY on basal and mediator-stimulated [3H]~AMP accumulation in iris-ciliary body segments. Abbreviations and drug concentrations used: PGE, = prostaglandin E,, 10m6M: IS0 = isoproterenol, 10m6M; VIP = vasoactive intestinal peptide, 1O-7M: FSK = forskolin, 1O-6M. Vertical bars (and horizontal lines) represent meansf S.E.M. : n = number of determinations. * P < 0.05, **p < 0.001.
AND
1
txa8asal
0 I
J. M. GOOCH
VIP VIP+NPY
,171
FIG. 3. NPY-mediated inhibition of VIP-stimulated [3H]cAMP accumulation in excised ciliary processesand isolated ciliary epithelium. Vertical bars (and horizontal lines) represent meansrt:S.E.M. : n = number of determinations. *P < 0.05. ** P < 0.001.
observations remains to be established, several potential
roles for NPY in the anterior
uvea are
suggested. First, NPY may function as a feedback modulator of sympathetic neurotransmission, mediating prejunctional inhibition of norepinephrine release and postjunctional E’ a ,E “6 g
mediated reduction of intracellular
60-
of P-adrenergic CAMP might also
account for the potentiative effects of NPY on
40-
4 a >
/I 0
inhibition
(CAMP-dependent)responsesof target cells. The NPY-
EO-
Y
”
I -II
, -10
I
I
-9 log [NPY]
-8
I -7
I -6
CM)
FIG. 2. Concentration-dependence of NPY-mediated inhi-
bition of VIP (lo-’ M)-stimulated [3H]cAMP accumulation in iris-ciliary body segments. Data points (and vertical lines) represent means) S.E.M. : n = number of determinations. deferens (Haggblad and Fredholm, 198 7) and cardiac
cells (Kassis et al., 1987). We have recently reported evidence that NPY acts prejunctionally
to depress the
synaptic release of norepinephrine from intraocular sympathetic nerves (Ohia and Jumblatt, 1989). Although the biological significance of these
norepinephrine-induced (a-adrenergic) contractions of the dilator pupillae and vascular smooth muscle, since CAMP has been implicated in the relaxation of these muscles. The identification of inhibitory NPY
receptors in isolated ciliary
19 September
Acknowledgments This investigation was supported by USPHS Research Grant No. EYO-5246, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc. Special thanks go to MS Rita Hackmiller for expert technical assistance. J. E. JUMBLATT J. M. GOOCH
7989 and accepted in revised form 4 December
References Edvinsson, L., Ekblad, E., Hakanson, R. and Wahlestedt, C. (1984). Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels. Br. J. Pharmacol. 83, 5 19-2 5
(Fig. 3)
aqueous humor dynamics.
Department of Ophthalmology and Visual Sciences, University of Louisville, School of Medicine, Kentucky Lions Eye Research Institute, L 0 uis vile, KY 40202- 1511, U.S.A. (Received
epithelium
suggests, furthermore, that NPY may have a direct role in aqueous humor regulation. Clarification of any such role, however, will require a better understanding of the autonomic neurogenic mechanisms that govern
7989)
Ekblad, E., Fdvinsson, L., Wahlestedt, C., Uddman, R.. Hakanson, R. and Sundler, F. (1984). Neuropeptide Y co-exists and cooperates with noradrenaline in perivascular nerve fibers. Regul. Pept. 8, 225-35. Fredholm, B. B.. Jansen, I. and Edvinsson, L. (1985). Neuropeptide
Y is a potent inhibitor
of cyclic AMP
NPY
INHIBITS
accumulation
UVEAL
ADENYLATE
231
CYCLASE
in feline blood vessels. Acta Physiol.
Stand. 124. 467-9.
Haggblad,J. and Fredholm, B. B. (1987). Adenosineand neuropeptideY enhancealpha,-adrenoceptor-induced accumulation of inositol phosphatesand attenuate forskolin-inducedaccumulation of CAMP in rat vas deferens.Neurosci. I&t. 82, 211-16. Kassis,S.M., Glasman,L.. Terenius,L. and Fishman,P. H. (198 7). Neuropeptide Y inhibits cardiac adenylate cyclasethrough a pertussistoxin-sensitiveG protein. I. &of. Chem.262, 3420-31. Mittag. T. and Tormay. A. (1985). Drug responsesof adenylate cyclase in iris-ciliary body determinedby adenine labelling. Invest. Ophthalmol.Vis. Sci. 26, 396-9.
Ohia. S.E.andJumblatt,J. E.(1989). Effectof neuropeptideY on norepinephrinereleasein the rabbit iris-ciliary body.
Invest. Ophthalmol. Vis. Sci. 30 (Suppl.),21. Piccone,M., Littzi, J., Krupin, T., Stone,R. A., Davis,M. and Wax, M. B. (1988). FZTects of neuropeptideY on the isolatedrabbit iris dilator muscle. Invest. Ophthalmol. Vis. Sci. 29, 330-2.
Salomon,Y., Landos,C. and Rodbell.M. (1974). A highly sensitiveadenylate cyclase assay. Anal. Biochem. 58, 541-6. Stone, R. A., Kuwayama, Y. and Laties, A. M. (1987). Regulatorypeptidesin the eye. Elcperienta 43, 790-800. Stone. R. A., Laties, A. M. and Emson. P. C. (1986). NeuropeptideY and the ocular innervation of rat, guinea pig, cat, and monkey. Neuroscience17, 1207-16. Tatemoto, K. (1982). NeuropeptideY: the completeamino acidsequenceof the brain peptide.Proc. Natl. Acad. Sci. U.S.A. 79, 5485-9.
