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Agonist Response of Human Isolated Posterior Ciliary Artery

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Agonist Response of Human Isolated Posterior Ciliary Artery Investigative Ophthalmology & Visual Science, Vol. 33, No. 1, January 1992 Copyright © Association for Research in Vision and Ophthalmology Agonist Response of Human Isolated Posterior Ciliary Artery Doo-Yi Yu, Valerie A. Alder, Er-Ning Su, Edward M. Mele, Stephen J. Cringle, and William H. Morgan The isometric responses of isolated human posterior ciliary artery to adrenergic agonists, histamine (HIS), and 5-hydroxytryptamine (5-HT) were studied in passively stretched ring segments mounted in a myograph bath. Cumulative dose response curves were measured for nine agonists: HIS, 5-HT, dopamine (DOPA), epinephrine (A), norepinephrine (NA), tyramine (TYR), phenylephrine (PHE), isoproterenol (ISOP), and xylazine (XYL), and the log(molar concentration) at which one half of the maximum active tension was developed (EQo) was estimated. The ring segments were unresponsive to DOPA and XYL; HIS and ISOP produced biphasic responses with a mild relaxation for low concentra- tions and small contractions for high concentrations of the agonist. The remaining agonists caused contractile responses of magnitude listed in the rank order following compared with the maximum active tension in response to 0.124 M K+-Krebs: Kmax > A > 5-HT = PHE > NA > TYR It was concluded that functional HIS, a,-adrenergic, and 5-HT receptors were present on human posterior ciliary artery but that there are no a2-adrenergic receptors. Invest Ophthalmol Vis Sci 33:48-54,1992 The regulation of ocular blood flow to ensure that may affect many aspects of ocular function involving all regions receive an adequate supply despite continu- the outer retina and the optic nerve head, the iris, and ously changing local tissue demands is a complex and the ciliary body. There is still disagreement whether hierarchic task, requiring continuous interaction be- vascular disease, particularly of the optic nerve head tween local and global mechanisms. The isolated ves- circulation, plays a role in the etiology of glaucoma. sel preparation in which a small segment of the artery Information about the pharmacologic control of in question is challenged pharmacologically is a pow- smooth muscle in the posterior ciliary artery would be erful approach to understanding the location and type helpful in understanding its normal and pathologic of controlling mechanisms of vascular beds. Because function. There currently have been only two pre- it is probable that there are species differences in the vious reports describing the function of human poste- pharmacologic sensitivity of ocular vessels, donated rior ciliary arteries using an in vitro technique.34 One3 human tissue is an invaluable source for such data if describes the effect of age on spontaneous myogenic we are to extrapolate experimental results to clinical activity, and the other4 used a constant flow technique situations to treat retinal vascular diseases. to ascertain the vascular responses to putative recep- The posterior ciliary arteries have autonomic in- tor agonists. We used ring segments of donated hu- nervation12 and supply blood to the optic nerve head, man posterior ciliary artery to test the pharmacologi- the choroid, the iris, and the ciliary body. Any impair- cal response to adrenergic agonists, histamine (HIS), ment of their ability to control blood flow therefore and 5-hydroxytryptamine (5-HT). From the Lions Eye Institute and University of Western Austra- Materials and Methods lia, Nedlands, Western Australia, Australia. Supported by the Juvenile Diabetes Foundation International, Thirteen eyes from seven donors were used, and the New York, New York, Medical Research Fund of Western Austra- lia, Perth, Australia, TVW Telethon, Perth, Australia, Clive and experiments were done on 18 ring segments. Human Vera Ramaciotti Foundations, Sydney, Australia, The Ophthalmic donor eyes were obtained either postmortem or post- Research Institute of Australia, The Medical Faculty of the Univer- operatively after consent for use of the tissue for re- sity of Western Australia, Perth, Australia, and the Australian Na- search was obtained according to the guidelines of the tional Health and Medical Research Council, Canberra, Australia. National Health and Medical Research Council and Submitted for publication: May 7,1991; accepted July 25,1991. Reprint requests: Dr. Valerie A. Alder, Lions Eye Institute and under the supervision of the Human Ethics Commit- Department of Surgery, University of Western Australia, Nedlands, tee of the University. Patient identity was not revealed WA 6009, Australia. to the experimenter, but patient history, diagnosis, 48 Downloaded from iovs.arvojournals.org on 10/02/2021 No. 1 HUMAN POSTERIOR CILIARY ARTERY PHARMACOLOGY / Yu er ol 49 Table 1. Summary of donor details Patient Age Delay time number (yr) (hr) History Medications HI 78 9 Not avilable Not available H2 77 6 Hypertension None H3 76 4 Hypertension diabetic (type 2) Daonil, aspirin, aldomet, propranolol H4 63 8 Carcinoma, lung Becotide, ventolin, oxycodone H5 73 6 Hypertension Diltiazem H6 43 2 Enucleation, detached retina, None calcined vitreous H7 78 8 Hypertension Becotide, prednisone, ventolin, spirondactone All tissue was postmortem except for H6, which was from a surgical enucleation. and medication information were (Table 1). The pa- the chamber was emptied by suction, and the feed line tients ranged in age from 43-78 yr. Before tissue was for one solution opened until the bath was full to the used, blood samples from donors were screened by level of the suction overflow line. State Health Laboratory Services for human immuno- deficiency virus, hepatitis B, and syphilis, and limbal Isolated Vessel Preparation swabs were cultured for bacterial growth against a The lateral posterior ciliary artery plus connective gentamicin-sensitive disc. The time between death or tissue was carefully dissected free from adherent or- operation before the tissue was placed into oxygen- + bital tissue. The vascular ring segments (1-2 mm in ated Na -Krebs solution at 4°C varied from 2-9 hr. length) were cut and mounted in the myograph sys- All ring segments were tested in the myograph bath on tem. One or two segments were obtained from each day 0 and a few on day 1. eye. The mounting of the ring segment was done under microscopic control (Zeiss stereomicroscope Myograph System DR, magnification X40; Zeiss, Oberkochen, Ger- The myograph was modified from another study.5 many). Two 50-/im tungsten wires, coated with gold It was custom-made and consisted of a hollow cylin- paint to render the surface smooth, were threaded dric stainless-steel block in which there was a concen- through opposite ends of the vessel lumen. We used tric cylindric depression (volume, 5 ml) which formed forceps for all vessel and remaining connective tissue the myograph bath. An isometric force transducer handling until the vessel was mounted completely on (Grass FTO3) and an XYZ microdrive system (Nari- the tungsten wires; after this, the connective tissue shige, MM 3, driven in the x direction by a hydraulic was dissected away. The tungsten wires were attached microdrive, MO-8; Narishige, Tokyo, Japan) were rigidly to the transducer and microdrive system, re- spectively, and the wires were kept parallel to each mounted on a base plate on either side of the organ + bath. These were used to measure the "isometric" other. The bath was filled with the Na -Krebs bathing tension developed by the muscle and to apply the pas- solution, and the vessel left to equilibrate for at least sive stretch, respectively. Bath fluid and bathing solu- 30 min under zero tension. The isometric tension out- tions were maintained at 37°C by circulating water put from the transducer was amplified (low level di- from a temperature bath (Techne TE-8D, Cambridge, rect current amplifier, 79122E; Grass, Quincy, MA), UK) through the stainless-steel base block and outer filtered (direct current to 3 Hz), and continuously re- jackets of the glass containers. Bath temperature corded on a six-channel chart recorder (R50; Rika- (LC87 temperature probe; T.P.S., Brisbane, Austra- denki, Tokyo, Japan). The overall compliance of the lia), pH (Activon 209 pH probe and meter; Granville, recording system was 5-10 jtm/mN; this was not corrected for in the data we present. Australia), and Po2 (using a D02M dissolved O2 meter; WPI, New Haven, CT) were monitored contin- uously and displayed digitally and on a chart re- Solutions and Agonists corder. They were maintained close to 37°C, 7.4, and Solutions: Vessels usually were bathed in normal 95% O2, respectively. The two organ bathing solutions Na+-Krebs solution containing: NaCl 119 mM, KC1 were delivered to the bath through a hydrostatic pres- 4.6 mM, CaCl 1.5 mM, MgCl 1.2 mM, NaHCO 15 sure head by opening a valve in the appropriate line. 2 2 3 mM, NaH2PO4 1.2 mM, and glucose 6 mM. For These solutions plus that in the bath were equilibrated 0.124 M K+-Krebs solution, the composition was KC1 with carbogen (95% O2, 5% CO2), by continuous slow 124 mM, CaCl 1.5 mM, MgCl 1.2 mM, NaHCO 15 bubbling. When a change of bath fluid was required, 2 2 3 mM, NaH2PO4 1.2 mM, and glucose 6 mM. Downloaded from iovs.arvojournals.org on 10/02/2021 50 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1992 Vol. 33 Agonists: Agonists used were (±)-norepinephrine HC1, (-)-epinephrine, 5-HT creatinine sulfate, dopa- mine HC1, (-)-phenylephrine HC1, isoproterenol HC1, tyramine HC1, histamine, xylazine HC1, and acetylcholine chloride (ACH). All chemicals were ob- tained from Sigma (St. Louis, MO) and were dis- solved in 0.9% NaCl. Stock solutions were stored at 0.5 8 1 -70°C, and fresh dilutions of 10" to 10" M were mN/mm made daily. All pharmacologic agents were pipetted directly into the muscle bath in a 50-/ul volume. The concentrations reported (10"'° to 10~4 M) are the cal- culated final concentrations in the bath solution.
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