Helen C. Turner

Presence of CFTR in the conjunctival epithelium

Purpose. To determine the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) in the conjunctiva and examine the possibility of its regional expression in rabbit, rat and porcine conjunctival epithelia given distinct differences in morphological appearance between the bulbar and palpebral epithelia. Methods. Two specific anti-CFTR antibodies, against different epitopes in the R domain of the CFTR molecule were used in immunofluorescent labeling of frozen fixed sections isolated from the bulbar and palpebral regions of fresh rabbit, porcine and rat conjunctivae. CFTR expression was also determined in the rabbit conjunctival epithelium using RT-PCR methods. Results. CFTR immunoreactivity in the conjunctival epithelium exhibits polarized expression and is associated with the apical domain of conjunctival epithelial cells. An identical pattern of staining obtained in porcine cryosections using either of the anti-human CFTR antibodies confirmed the specificity of the positive apical staining. RT-PCR analysis produced bands at the predicted size for CFTR mRNA transcripts in both bulbar and palpebral portions of the rabbit conjunctival epithelium. Conclusions. Apical localization of CFTR in the conjunctival epithelium is consistent with the function of this protein as a chloride channel or as a regulator of channel activity. The identification of CFTR in both bulbar and palpebral portions of the conjunctiva provides evidence that the mechanisms for Cl secretion reside throughout the conjunctiva. This finding suggests that manipulations of the CFTR Cl channel could affect transepithelial Cl transport rates and water movement into the tear film.

Immunolocalization of Na-K-ATPase, Na-K-Cl and Na-glucose cotransporters in the conjunctival epithelium.

Purpose. To investigate possible regional expression of transport systems in the conjunctival epithelium given distinct differences in morphological appearance between the bulbar and palpebral epithelia as well as variations found in the proportions of Na absorptive versus Cl secretory activities in electrophysiological studies. Methods. Mouse monoclonal antibodies against the a1-subunit of Na-K-ATPase and Na-K-Cl cotransporter (NKCC1) and a rabbit polyclonal antibody against the Na-glucose cotransporter (SGLT1) were used in immunoblotting and immunofluorescent labeling of frozen fixed sections isolated from either the bulbar and palpebral regions of the conjunctiva. Results. Western blot analysis clearly demonstrated the presence of Na-K-ATPase, NKCC1 and SGLT1 proteins in both bulbar and palpebral conjunctiva. Indirect immunofluorescence studies on bulbar and palpebral conjunctival portions revealed intense staining by the anti-NKCC1 and anti-a1-Na-K-ATPase antibodies exclusively at the basolateral surfaces, whereas the anti-SGLT1 antibody was used with porcine conjunctiva to elicit strong and unambiguous staining along the apical plasma membrane. Conclusions. Proteins that mediate the transport activities of the Na-K-ATPase and Na-K-Cl cotransporter are uniformly distributed throughout the palpebral and bulbar regions of the rabbit conjunctival epithelium. Although the Na-glucose cotransporter could be detected in immunoblots of the rabbit, this cotransporter appears to be uniformly distributed as well, based upon immunohistochemical sections of the pig conjunctiva. Thus, it appears likely that mechanisms for Cl secretion and Na absorption reside in both bulbar and palpebral segments of the conjunctival epithelium.

Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae

Purpose. To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. Methods. Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I sc) across rabbit and porcine conjunctivae were contrasted. Results. RT-PCR assays indicated the expression of 5-HT 1B and 5-HT 1D receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 4 and 5-HT 7 mRNA’s in the porcine conjunctiva, and 5-HT 1D, 5-HT 1F and 5-HT 7 in the human conjunctiva. Since the 5-HT 4 and 5-HT 7 receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT 7 moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I sc across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I sc, which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. Conclusions. The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

Beta-adrenergic inhibition of rabbit lens anterior-surface K(+) conductance.

Purpose. To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. Methods. The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I sc) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human ß 2 -adrenoceptors. Reverse transcription – polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human ß 2 receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. Results. Asymmetrical I sc reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the ß 2 -adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. Conclusions. The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I sc responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.