2-(Anilinomethyl)imidazolines as alpha(1)-adrenoceptor agonists: the identification of alpha(1A) subtype selective 2'-carboxylic acid esters and amides.

2-(Anilinomethyl)imidazolines with 2'-esters or 2'-amides are potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The size and shape of the ortho substituent can have significant effects on the potency, efficacy, and subtype selectivity of these 2-(anilinomethyl)imidazolines. alpha(1A)-subtype selective agonists have been identified.

Time at confluence for human RPE cells: effects on the adherens junction and in vitro wound closure.

PURPOSE: To determine how time at confluence affects the properties of cultured human retinal pigment epithelium (RPE) cells, with emphasis on the adherens junction. METHODS: Cultures were maintained at confluence without passage for intervals to several months. Adherens junction proteins (N-cadherin, E-cadherin, alpha-catenin, beta-catenin, plakoglobin, and actin) and the proliferation marker Ki-67 were localized in the cultures by fluorescence microscopy, and in vitro wound healing was compared. Adherens junctions were analyzed for protein solubility in detergent buffers and sensitivity to disruption by treatment with anti-cadherin antibodies and low calcium conditions. RESULTS: Compared with cultures in early-confluence (2-3 days), postconfluent cultures (weeks) had more mature adherens junctions characterized by a circumferential (rather than linear) actin organization, and a zonular (rather than punctate) distribution of more detergent resistant cadherin and catenins. Postconfluent cultures also had fewer Ki-67-positive cells and a higher cell packing density. Early-confluence cells migrated into in vitro wounds as dissociated single cells, whereas postconfluent cells moved as contiguous sheets, retaining an intact junction during wound-induced cell migration and proliferation. Mature junctions were not disrupted by treatment of living cells with N-cadherin antibodies, which bound to and remained detectable at junctions for several days. Calcium withdrawal displaced N-cadherin from mature junctions and rendered it more soluble, but the dominant circumferential pattern of actin was stable. Restoration of medium calcium resulted in a rapid (hours) recovery of a nearly complete zonular pattern of insoluble N-cadherin. CONCLUSIONS: Over long postconfluent periods, cultured RPE cells became more growth quiescent, and intercellular cadherin adhesions became more stable, exhibiting increased resistance to calcium removal and greater retention of junctional integrity during in vitro wound closure. Consideration should be given to whether the behavior of RPE cells in postconfluent cultures, where intercellular adhesions are more mature, more closely simulates RPE cells in situ than cells in early-confluence cultures, which are more commonly used for analysis.

High levels of E-/P-cadherin: correlation with decreased apical polarity of Na/K ATPase in bovine RPE cells in situ.

PURPOSE: The adherens junction protein E-cadherin induces a basolateral polarity of Na/K ATPase in most epithelial cells that express it, whereas in retinal pigment epithelium (RPE) cells, Na/K ATPase is largely apical. The purpose of this study was to determine whether the distribution of Na/K ATPase differs in RPE cells in situ, that differ in levels of junctional E-cadherin. METHODS: Bovine RPE cells in situ were immunostained with an E-cadherin antibody (which has some cross-reactivity with the closely related epithelial cadherin P-cadherin), and RPE cells with different levels of junctional stain were identified. RPE cells with low and high E-/P-cadherin were costained in various combinations with Na/K ATPase and interacting proteins of the membrane cytoskeleton (ankyrin, fodrin, and actin) and analyzed by confocal imaging. RESULTS: Individual RPE cells within the same monolayer differed in amount of Na/K ATPase, with a lower frequency of high expressing cells in the area centralis. High expressing Na/K ATPase cells were found among cells with both low and high E-/P-cadherin levels. In cells with low E/P-cadherin, Na/K ATPase localized to apical microvilli, whereas in high E-/P-cadherin cells, Na/K ATPase was on basolateral surfaces in addition to microvilli. Actin staining showed that microvillar domains were smaller and that lateral membrane domains were taller in high E-/P-cadherin cells. In high but not low E-/P-cadherin cells, ankyrin and fodrin levels varied among cells, with a subset of cells showing distinctly higher expression. Both ankyrin and fodrin had complex subcellular distribution patterns, although they tended to be enriched basal to rather than apical to the adherens junction. Cells with high Na/K ATPase did not necessarily have commensurately higher levels of ankyrin or fodrin. Where both Na/K ATPase and ankyrin were high, they codistributed weakly in apical microvilli but more prominently on the basal cell surface. CONCLUSIONS: Within the same RPE monolayer, the polarity of Na/K ATPase differs among cells, with a more basal polarity found in cells with high levels of junctional E-/P-cadherin. The increased basal Na/K ATPase was due to a combination of a smaller microvillar domain, a taller lateral domain, and more basolateral staining for Na/K ATPase, perhaps because of an enrichment of a basal ankyrinfodrin membrane cytoskeleton with which Na/K ATPase is known to associate.

Expression of E-cadherin by human retinal pigment epithelium: delayed expression in vitro.

PURPOSE: To determine whether retinal pigment epithelial (RPE) cells, which reportedly express N-cadherin as their major cadherin cell adhesion protein, also express the more common epithelial cadherin, E-cadherin. METHODS: Cadherins expressed by human RPE cells in situ were examined by western blot analysis of extracts prepared from the RPE of human adult eyes. Cadherins expressed in vitro were examined by analysis of confluent and postconfluent human RPE cultures, using the methods of reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Protein distribution was examined by conventional fluorescence microscopy, confocal imaging, or both. Proteins whose expression, distribution, or both correlated with E-cadherin expression in other epithelial cells were examined by similar methods in cultured RPE cells. RESULTS: In addition to N-cadherin, E-cadherin (and P-cadherin) was found in adult human RPE in situ. In cultured human RPE cells, N-cadherin was ubiquitous, but E-cadherin was limited to patches of cells and was not expressed until several weeks after confluence, a time when several phenotypic variants become prominent. E-cadherin was absent from RPE cells of fusiform shape but was found in only a subset of epithelioid RPE cells. Unlike epithelial cell lines expressing E-cadherin, cultured RPE cells with E-cadherin did not show diminished coexpression of N-cadherin, increased expression of desmosomal proteins, or a preferential expression of the alphaE- (rather than alpha-N) isoform of the cadherin linker protein alpha-catenin. Na/K ATPase distributed to both apical and basolateral membranes in RPE cells with junctional E-cadherin and not preferentially to the basolateral domain as in most epithelial cells with E-cadherin. CONCLUSIONS: RPE cells express E-cadherin, a cadherin found in most other epithelial cells, but which was believed to be absent from RPE. In RPE in vitro, E-cadherin expression is a late developmental event, occurring in late confluence in cells that already express N-cadherin. E-cadherin is an established epithelial morphoregulatory protein, but it does not induce the same properties in RPE cells as in other epithelial cells, suggesting tissue-specific differences in the potential of E-cadherin to determine an epithelial phenotype.

Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells.

For most epithelial cells, the adherens junction protein E-cadherin is an epithelial morphogen, inducing the development of an epithelial phenotype in vitro after cell contact at confluency. Here retinal pigment epithelial cells (RPE), which lack E-cadherin but express a cadherin that is also found in many non-epithelial cells (N-cadherin), were examined for the ability to produce an epithelial phenotype in vitro. Subpopulations of grossly epithelioid or fusiform cells were selected for analysis from RPE cultures derived from adult human donors. After confluency, epithelioid RPE cells were observed to undergo time-dependent changes that were similar to those previously found in epithelial cells expressing E-cadherin: the cadherin gradually developed a zonular distribution of detergent-resistant protein that co-localized with forming circumferential actin bundles; Na/K ATPase accumulated at cell contact sites, then polarized to its tissue-specific domain (the apical membrane for RPE); the cells formed elevated domes on the impermeant culture substrate. In contrast to cells expressing E-cadherin, these events in RPE required weeks rater than days at confluency. Additional proteins were examined in epithelioid RPE cells revealing that cytokeratins reorganized after confluency producing a zonular array, and several other adhesion proteins (alpha5beta1 integrin, ICAM-1, PECAM-1, NCAM) became enriched at cell-cell contact sites, each developing a distinct pattern at a distinct postconfluency interval. In contrast to epithelioid RPE, in fusiform RPE the adhesion molecules did not develop discrete distribution patterns after confluency, although the same complement of adhesion proteins was expressed. In cells expressing E-cadherin, the absence of epithelial properties is often due to underexpression of the cadherin or of the catenins, adherens junction proteins that link the cadherin to actin. Fusiform RPE, however, were not deficient in these proteins, expressing amounts of N-cadherin, alpha-catenin, beta-catenin, plakoglobin, p120, alpha-actinin and vinculin that were equivalent to epithelioid cells. It appears, therefore, that a subset of epithelial cells that express N-cadherin can produce a highly-developed epithelial phenotype in vitro through a slow morphogenetic process. However, the expression alone of adhesion molecules, including those with a morphoregulatory function in other cells, is insufficient to produce an epithelial phenotype in all cells derived from the pigment epithelium.

Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ.

We have previously developed methods based upon differential cell adhesion to select for cells of two different phenotypes, epithelioid and fusiform, from cultures of human RPE. Here we considered whether the differences in cell shape correlated with differences in protein tyrosine phosphorylation since it is known that elevated phosphorylation perturbs the stability of the adherens junction, a major determinant of epithelial phenotype. In cultures of both phenotypes we found low tyrosine phosphorylation levels in postconfluent cultures, and the same complement of tyrosine phosphoproteins after treatment with a phosphatase inhibitor. However, in contrast to epithelioid cells, fusiform RPE failed to localize the phosphoproteins to junctional sites on the cell periphery. We also re-evaluated primary and passaged RPE cultures for additional cell shape variants. Several discrete phenotypes were identified within the same cultures. They required several weeks at confluency to develop in primary as well as in passaged cultures, but after developing they remained stable for months. Since explanted RPE cells manifest several shape variants in an identical culture environment we examined bovine RPE cells in situ to determine whether the cells were heterogenous with regards to some cell surface and cytoskeletal proteins that might contribute to cell shape. Circumferential actin microfilament bundles and the occluding junction protein ZO-1 had fairly uniform distributions among cells in the monolayer, but the intermediate filament protein vimentin and the pericellular expression of phosphotyrosine varied among individual cells. Therefore, despite its grossly homogeneous appearance, the RPE monolayer in situ might be considered a mosaic of structurally heterogeneous cells which can give rise to phenotypically-distinct subpopulations when propagated in vitro.

The relative efficiency of beta adrenoceptor coupling to myocardial inotropy and diastolic relaxation: organ-selective treatment for diastolic dysfunction.

The relative effects of drugs which elevate cytosolic cyclic AMP on inotropy and diastolic relaxation (lusitropy) of guinea pig atria were quantified in vitro. There was a temporal difference between these responses in that inotropy reached peak response considerably faster than lusitropy. Also, although the relaxation response was sustained to an elevated steady state, the inotropic responses to beta adrenoceptor agonists were transient and returned to base line over 90 min. However, the inotropic responses to forskolin and dibutyryl cyclic AMP (cAMP) were sustained. For all of the drugs tested, the lusitropic response was at least 4 times more sensitive than the inotropic response (i.e., the concentration response curve for relaxation was shifted to the left of the curve for inotropy). In the case of beta adrenoceptor agonists, these differences were greater, presumably because of the fading inotropic response over 90 min. It was found that although high efficacy beta adrenoceptor agonists such as isoproterenol (and the direct activator of adenylate cyclase forskolin) produced both inotropy and lusitropy, lower efficacy agonists produced predominant lusitropy. The low efficacy agonist prenalterol produced insignificant inotropy but 60% maximal lusitropy. These data were modeled mathematically by a "differential coupling model" which assumed that a uniform cytosolic level of elevated cAMP activated two biochemical processes of differing sensitivity. Thus, the lusitropic response (phosphorylation of phospholamban) was coupled more efficiently to the cAMP response than the inotropic response (phosphorylation of calcium channels). A second model ("differential messenger concentration model") which calculated the effects of a compartmentalization of cAMP concentration within the cardiac cell by restricted diffusion and/or selective degradation by phosphodiesterases also was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of pentobarbital-induced narcosis by thyrotropin-releasing hormone (TRH) in dogs.

A growing body of evidence suggests that thyrotropin-releasing hormone (TRH), and endogenous brain tripeptide, produces behavioral excitation in a variety of mammalian species. This study evaluated the cardiopulmonary and antidepressant response to a single intravenous (i.v.) bolus of TRH in sodium pentobarbital (33 mg.kg-1) anesthetized dogs. TRH (0.5 and 1 mg) produced a significant dose-dependent decrease in sleeping time (33% and 51%, respectively) when compared to i.v. vehicle (1 ml of 0.9% NaCl)-treated animals. Of interest was the finding that the high (1 mg), but not the low (0.5 mg) dose of TRH significantly (P less than 0.01) increased mean arterial pressure and heat rate. In addition, i.v. TRH (1 mg) significantly (P less than 0.01) decreased tidal volume. A trend toward increased respiratory frequency in TRH-treated dogs was noted, this difference, however, did not reach statistical significance. In conclusion, the results of this study support the view that TRH, and endogenous brain hormone, may have an important clinical application in cases where stimulation of the central nervous system is required.