Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva.

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.

Modulation of chloride secretion across the pigmented rabbit conjunctiva.

The purpose of the present study was to investigate whether active Cl- secretion in the pigmented rabbit conjunctiva was subject to cAMP, Ca2+ and protein kinase C (PKC) modulation. The excised pigmented rabbit conjunctivas were mounted in the modified Ussing-type chambers for measurement of unidirectional 36Cl fluxes under the open-circuit condition and of the short-circuit current (Isc), potential difference, and transconjunctival electrical resistance. The results indicate that Cl- secretion across the conjunctiva was abolished by mucosal application of 1 mM N-phenylanthranilic acid and was reduced by 40% by serosal application of 10 microM bumetanide. Net Cl- flux was stimulated by 133% by 1 mM 8-Br cAMP, 107% by 10 microM A23187, and 87% by 1 microM phorbol 12-myristate-13-acetate (PMA), suggesting that cAMP, Ca2+, and PKC all modulated active Cl- secretion, respectively. There existed a linear correlation between measured changes in net Cl- flux and observed changes in Isc (r2=0.99). The serial treatment of the conjunctiva with (a) 1 mM 8-Br cAMP and 10 microM A23187 and (b) 10 microM A23187 and 1 microM PMA resulted in sequence-independent, additive stimulation of Isc. In the case of 1 mM 8-Br cAMP and 1 microM PMA, additive stimulation of Isc was observed only when 1 mM 8-Br cAMP was added prior to 1 microM PMA. These results suggest that a given pharmacological agent may affect more than one channel type and that there might be a possible connection among the channels at the signal transduction level. In summary, Cl- appears to enter the pigmented rabbit conjunctiva from the serosal fluid via Na+-(K+)-2Cl- cotransport process and exit to the mucosal fluid via channels, resulting in active Cl- secretion. Active Cl- secretion in the pigmented rabbit conjunctiva appears to be modulated by cAMP, Ca2+, and PKC.

Cyclic AMP modulation of active ion transport in the pigmented rabbit conjunctiva.

This study was conducted to determine whether active ion transport in the pigmented rabbit conjunctiva was subjected to cyclic AMP modulation. 8-BrcAMP and compounds that may affect intracellular cAMP levels were tested for their effects on the conjunctival short-circuit current (Isc) following mucosal application. Conjunctival Isc was increased by 1 mM 8-BrcAMP, 0.001-20 microM forskolin, 0.01-2 mM theophylline, 2 microM 1-epinephrine, and 20 microM terbutaline in a Cl(-)-containing but not in a Cl(-)-free medium. The increase in Isc induced by 1 mM 8-BrcAMP was reversed by 1 mM N-phenylanthranilic acid, suggesting enhancement of Cl- secretion by cAMP. Similarly, the increase in Isc induced by 20 microM terbutaline was blunted by 1 microM timolol, suggesting a role for beta 2 adrenergic modulation of Cl- secretion. Collectively, these results are consistent with the notion of cAMP modulation of conjunctival active ion transport.

Possible existence of Na(+)-coupled amino acid transport in the pigmented rabbit conjunctiva.

The objective of the present study was to determine whether Na(+)-amino acid cotransport contributed to the short-circuit current (ISC) in the isolated pigmented rabbit conjunctiva. Glycine, L-arginine, D-arginine, and L-glutamic acid were the amino acids tested, and bioelectric measurements were made in the Using chamber. The ISC was increased from 4% (L-glutamic acid) to 44% (L-arginine). The EC50 was 0.35 mM for glycine, 0.06 mM for L-arginine, 0.16 mM for D-arginine, and 1 mM for L-glutamic acid. No elevation in ISC was seen in the absence of Na+ in the mucosal bathing fluid. The above findings are consistent with the possible existence of a Na(+)-amino acid cotransport process on the apical side of the pigmented rabbit conjunctiva.

Plasma-induced graft copolymerization of HEMA onto silicone rubber and TPX film improving rabbit corneal epithelial cell attachment and growth.

A poly(2-hydroxyethyl methacrylate) (pHEMA)-grafted polymer film was prepared by plasma-induced graft copolymerization onto an elastic material, silicone rubber, and a plastic material, poly(4-methyl-1-pentene) (TPX). The control, Ar plasma-treated and pHEMA-grafted silicone rubber and TPX surfaces were characterized by ESCA, FTIR-ATR, SEM and contact angle techniques. ESCA verified the respective chemical shift of control and Ar plasma-treated films. The presence of the grafted pHEMA was also verified by ESCA. The introduction of pHEMA onto a hydrophobic support provided an adequate surface for rabbit corneal epithelium cell attachment and growth. Cell attachment and growth onto these surfaces were examined by light microscopy. Cell attachment onto the control and Ar plasma-treated surfaces was negligible, while improved attachment and growth of rabbit corneal epithelium cells was demonstrated on the pHEMA-grafted polymeric surface. At 72 h, the pHEMA-grafted silicone rubber surface attached and grew more cells as compared with those on a pHEMA-grafted TPX surface. The pHEMA-grafted silicone rubber surface demonstrated a confluent cell layer after 72 h.

ppHEMA-modified silicone rubber film towards improving rabbit corneal epithelial cell attachment and growth.

A plasma polymerized HEMA (ppHEMA) film was prepared by plasma deposition polymerization onto an elastic material, silicone rubber. The surfaces of control, argon plasma-treated, and ppHEMA-modified silicone rubber were characterized by ESCA, FTIR-ATR and SEM techniques. ESCA verified the respective chemical shift of control and ppHEMA-modified films. The presence of the ppHEMA was also verified by ESCA. The introduction of ppHEMA onto a hydrophobic support provided an adequate surface for rabbit corneal epithelial cell attachment and growth. Cell attachment and growth onto these surfaces were examined by light microscopy. Cell attachment onto the control and the argon plasma-treated surface was negligible, while improved attachment and growth of rabbit corneal epithelium cells was demonstrated on the ppHEMA-modified polymeric surface. The ppHEMA-modified silicone rubber surface demonstrated a confluent cell layer after 72 h.