Current Eye Research

ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20

Endothelin-Like Immunoreactivity in the Aqueous Humour and in Conditioned Medium from Cultured Ciliary Epithelial Cells Albrecht Lepple-Wienhues, Marion Becker, Frank Stahl, Susanne Berweck, Johannes Hensen, Walter Noske, Michael Eichhorn & Michael Wiederholt To cite this article: Albrecht Lepple-Wienhues, Marion Becker, Frank Stahl, Susanne Berweck, Johannes Hensen, Walter Noske, Michael Eichhorn & Michael Wiederholt (1992) Endothelin-Like Immunoreactivity in the Aqueous Humour and in Conditioned Medium from Cultured Ciliary Epithelial Cells, Current Eye Research, 11:11, 1041-1046, DOI: 10.3109/02713689209015075 To link to this article: http://dx.doi.org/10.3109/02713689209015075

Published online: 02 Jul 2009.

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Date: 29 June 2016, At: 12:07

Current Eye Research

Volume 11 number 11 1992, 1041- 1046

Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells Albrecht Lepple-Wienhues, Marion Becker, Frank Stahl, Susanne Berweck, Johannes Hensen', Walter Noskez, Michael Eichhorn3 and Michael Wiederholt

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Institut fiir Klinische Physiologie, 'Medizinische Klinik, 2Augenklinik, Klinikum Steglitz, Freie Universitat Berlin, W- 1000 Berlin 45 and 3Anatomisches Institut, Lehrstuhl 11, Universitat Erlangen-Niirnberg, W-8520 Erlangen, Germany ABSTRACT Endothelin-like immunoreactivity was detected in human (15.6 2 2.7 pg/ml) and bovine (11.1 2 0.98 pg/ml) aqueous humour of the eye. These concentrations are 2-3 times higher than the corresponding plasma levels. Cultured human nonpigmented ciliary epithelial cells released endothelin-like immunoreactivity with a maximum of 2.1 2 0.32 pg/(cmZ * 48 h). The release was stimulated by fetal calf serum, thrombin, carbachol and phorbol ester and blocked by cycloheximide. Immunocytochemistry showed cytoplasmic staining of cultured human nonpigmented ciliary epithelial cells for endothelin-1. Endothelin-1 was shown to induce contractions in isolated human ciliary muscle by isometric force measurements. Endothelin in the aqueous humour may play a role in the regulation of intraocular pressure.

MATERIAL AND METHODS Bovine eyes were obtained from a local abattoir, transferred to the laboratory on ice within 30 min after death and aqueous humour was sampled and frozen. Bovine plasma samples were obtained from the Institute for Veterinary Medicine. Human aqueous humour was obtained from patients undergoing cataract surgery by puncture of the anterior chamber carefully avoiding damage to the iris or other intraocular structures. The amount of aqueous humour obtained from each patient was 70-200 111. Therefore, the samples from 39 patients have been distributed arbitrarily

INTRODUCTION The regulation of intraocular pressure in the normal and glaucomatous eye is poorly understood. Previously we were

into 5 pools, and the immunoreactive endothelin-like immunoreactivity (ET-IR) was determined for each pool. The patients suffered from no other ocular disease except

able to demonstrate contractile properties of isolated

cataract. Following the anamnestic and clinical examination,

trabecular meshwork, probably influencing outflow resistance

no patient showed signs of heart failure, diabetes or renal

[l] . Furthermore, we have demonstrated endothelin (ET)-1-

failure. All specimen were kept on ice and frozen at -20°C

mediated contractions in bovine trabecular meshwork and

immediately. All human samples were obtained after

ciliary muscle [2]. ET has been characterized as a potent vasoconstrictive peptide acting on several types of smooth muscle, among them vascular, bronchial, uterine, intestinal and mesangial contractile cells [for review see 31. The aim of the present study was to investigate the possible role of this peptide in the regulation of intraocular

-

information and with the consent of the patients according to

the Declaration of Helsinki.

Cultures of SV-40 transformed human nonpigmented

ciliary epithelial cells ( W E ) , as well as primary cultures of

human ciliary muscle cells (HCM) and bovine corneal

pressure. The source of ET possibly acting on targets in the

endothelial cells (BCE) were maintained as described

aqueous humour inflow/outflow system was to be

previously [4].In short, using standard culture conditions the

investigated as well as the mechanisms of ET-release into

cells were incubated with Dulbeccos modified minimal

the anterior chamber of the eye. We tested the influence of

essential medium containing 100 IU/ml penicillin, 100 /ig/ml

several agents interfering with the intraocular pressure

streptomycin and 10 vol%fetal calf serum (Biochrom, Berlin,

regulation on ET-production. Isometric force measurements

FRG), gassed with 5%COz in air. The cells were trypsinized

were performed using human ciliary muscle strips to test the

and split in a 1:2 ratio. For the determination of the time

contractile action of ET-1 on this outflow regulating muscle.

course of ET-IR-release the medium was removed after

Received on April 15, 1992; accepted on October 20, 1992

0 Oxford University Press

1041

Current Eye Research ~~

seeding every 48 h and ET4R was determined in the

immunoglobulin (Dakopatts, Glostrup, Denmark), diluted in

Supernatants. To test the stimulating potency of several

TBS (1:200) containing 2%bovine serum albumin (Sigma,

agents all cells were incubated for the first 48 h after

%.Louis, USA). After washing in TBS for 3 times

seeding with medium containing 10%fetal calf serum, 24 h

streptavidin-biotin horseradish peroxidase (Dakopatts,

with serum-free medium, and then with the medium

Glostrup, Denmark) was applied for 60 min. The cells were

containing the respective agents for further 24h. This latter

then washed and developed with 0.05%diaminobenzidin

incubation was performed without serum except the

(Sigma) and dissolved in TBS containing 0.006% HzOZ.

specimen in which serum was tested as the stimulating

Finally, the cells were washed in distilled water and mounted

agent.. The medium was then removed and the ET-IR-content

in glycerin-gelatine. Control experiments were performed

was measured as described below. When used for release

using TBS instead of i.he primary antibody. nts of i

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measurements, HNPE was iin the 13th, HCM in the 9th, and BCE in the 3rd passage. Mean cell density was 3.5 0.12

* lo4 cells/cm2

* lo4 5

and no difference between the different

cell lines was found.

s

0

..

0

Sm.x Human donor eyes were obtained from the Florida Lions Eye Bank, Tampa, USA. They were shipped on ice, and experiments were perf'ormed within 48-72h after enucleation.

All samples were frozen and stored at -20°C until

Meridional strips of ciliary muscle were prepared as

extraction. After centrifugation the extraction was performed

described previously 111. Forces were recorded isometrically

using Seppak C18 cartridges (Water, Millipore, Milford,

during superfusion with bicarbonate/COz-buffered Ringer's

USA). A commercial RIA kit was used to determine ET-IR

solution at 37°C.

levels (ITS, Wychen, Netherlands). Each specimen was

Statistics

measured in duplicates. The procedure has been described previously [5]. The cross-reactivities have been announced

The results have been statistically tested using Student's t-test. The levels of significance are indicated in the text.

as follows: ET-1 loo%, ET-2 52%,ET-3 96%,Big-ET 7%,and ANP, angiotensin 11, ACTH and vasopressin

Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells.

Endothelin-like immunoreactivity was detected in human (15.6 +/- 2.7 pg/ml) and bovine (11.1 +/- 0.98 pg/ml) aqueous humour of the eye. These concentr...
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