摘要:

The interaction between the alpha2- and beta2- adrenergic receptors of ciliary processes has Been studied by examining dose-response curves for adrenergic agonist stimulation of cyclic AMP production by intact, excised rabbit ciliary processes.Stimulation of cyclic AMP production by 1-isoproterenol is maximum from 0.1 to 1.0 uM; at higher concentrations stimulation decreases and approaches basal levels. Decreased cyclic AMP production at high concentrations of isoproterenol is blocked by the specific alpha2-adrenergic antagonist, yohimbine, but not by the alpha1-adrenergic antagonist, prazosin. Ciliary processes from animals after bilateral cervical ganglionectomy also show reduced cyclic AMP production at high concentrations of isoproterenol and this reduction is blocked by yohimbine, but not prazosin. This experiment suggests that the inhibition at high concentrations of isoproterenol is mediated by postsynaptic alpha2-adrenergic receptors. Cyclic AMP production is relatively insensitive to epinephrine and norepinephrine, but their responces are potentiated by yohimbine.Catecholamines and clonidine, a specific alpha2-adrenergic agonist, exhibit dose-dependent inhibition of forskolin-stimulated cyclic AMP production by ciliary processes. I50sfrom the dose-response curves are consistent with the characteristic binding affinities of these adrenergic agonists for alpha2-adrenergic receptors: clonidine = epinephrine>norepinephrine>isoproterenol. Inhibition of forskolin-stimulated cyclic AMP production by clonidine is blocked by yohimbine but not by prazosin.The dose-response curves for catecholamine stimulation of cyclic AMP production can be understood in terms of the relative affinities of these adrenergic agonists for beta2- and alpha2-adrenergic receptors linked to adenylate cyclase via N and Nirespectively. The interaction between these two adrenergic receptors, one which stimulates and one which inhibits stimulation of adenylate cyclase may provide an explanation for some of the paradoxical observations from studies of the effects of catecholamines on aqueous flow.

ISSN:0271-3683    

DOI:10.3109/02713688709025206

出版商:Taylor&Francis    

年代:1987

数据来源: Taylor

摘要:

Corneal epithelium and the trigeminal ganglion neurons which normally innervate the epithelium have been grown in adjacent chambers of a 35 mm tissue culture plate. Dissociated nerve cells from late embryonic rats were plated inside an 8 mm cloning cylinder attached to the center of the culture plate by silicone grease. In 7–10 days neurites extended out of this inner chamber by growing through the grease seal and along parallel scratches in the collagen coating of the tissue culture plate. Once this occurred, pure corneal epithelial explants were isolated from young adult rats and plated in the area surrounding the cloning cylinder, i. e. in the outer chamber. Cultures were monitored regularly with phase microscopy and, at various times, were fixed for ultrastructural examination. Within 24–48 hours of the epithelial plating, there were both individual neurites and bundles of neurites in contact with the epithelium. This interaction increased substantially over the next few days. Growth cones of the neurites could be seen to approach the microvilli-covered surface of the epithelium, travel over the surface and penetrate between the epithelial cells. This tissue culture model of the innervated ocular surface may prove valuable in the study of a variety of ocular conditions or diseases, as well as provide a means to study functional relationships and mechanisms of cellular interaction between neurons and their target cells.

ISSN:0271-3683    

DOI:10.3109/02713688709025207

出版商:Taylor&Francis    

年代:1987

数据来源: Taylor

摘要:

We have previously found that a breakdown of tight junctions in retinal pigment epithelial cells of Royal College of Surgeons' rats is associated with a redistribution of intramembrane particles and Na-K-ATPase activity. Changes in the lipid and sterol composition of membranes can alter their fluidity, permeability and enzyme activity, and may contribute to changes in cell barrier function in the dystrophic epithelium. We have now used filipin and digitonin, which bind to membrane sterols and produce membrane deformations recognizable by freeze-fracture and thin-section electron microscopy, to study the distribution of cholesterol and related 3-B-hydroxysterols in the dystrophic epithelium. The results of these studies show that in the normal pigment epithelium and prior to tight junction breakdown in the dystrophic epithelium, filipinand digitoninsterol complexes are rare in the membranes between tight junctions and adhering junctions, and in areas of attachment between the plasma membrane and basal lamina. Complexes are more numerous in the basal infoldings, and most densely packed in the lateral and apical microvillous membranes. During junction breakdown, complexes increase substantially in apical, basal, junctional, and nuclear membranes. Later, after the junctions disappear, complexes decrease. These results indicate that alterations in the expression of membrane sterols accompany the changes in structure and function of tight junctions in the dystrophic retinal pigment epithelium.

ISSN:0271-3683    

DOI:10.3109/02713688709025208

出版商:Taylor&Francis    

年代:1987

数据来源: Taylor

摘要:

Experiments were undertaken to determine if serotonin (5-HT) radioligand binding sites were present in a membrane fraction of iris + ciliary body from adult, albino rabbits. The total binding of 3H-5-HT,3H-spiroperidol (SPI) and3H-ketanserin (KET), all at 2 nM, was determined in the absence and in the presence of ketanserin, mianserin, methysergide or 5-methoxytryptamine (5-MT), all at 1μM. Except for ketanserin, which displaced 15% of3H-KET binding, none of the agents altered3H-SPI or3H-KET binding. Reductions of 12% and 14% in3H-5-HT binding were achieved by ketanserin and mianserin, respectively. In contrast, methysergide and 5-MT displaced3H-5-HT binding by 57% and 56%, respectively. 5-HT (1μM) also displaced3H-5-HT binding by approximately 60% and this was used to measure nonspecific binding. Specific binding of3H-5-HT was saturable and of high affinity with one population of binding sites being labelled. Kdand Bmaxvalues of 1.1 nM and 57.8 fmoles/mg protein were obtained. Seven 5-HT antagonists possessing various affinities for 5-HT1and 5-HT2binding sites were assessed for their ability to displace specific3H-5-HT binding. Ketanserin was the least potent (Ki>3μM). In contrast, the respective Kivalues for metergoline and methysergide were 13 nM and 20 nM. These observations indicate the presence of 5-HT1binding sites. The indole-containing agonists 5-HT, 5-MT and 5-methoxy-N, N-dimethyltryptamine were potent displacers of specific3H-5-HT binding, the observed Kivalues being 0.6 nM, 7.0 nM and 3.6 nM, respectively. In contrast, weaker affinities were displayed by piperazine substituted 5-HT agonists, e. g. a Kivalue of 757 nM for trifluoro-m-tolyl-piperazine. Findings for 5-HT agonists suggest that 5-HT1Areceptors are present in the rabbit iris + ciliary body. The high affinity (Kiof 2.8 nM) observed for 8-OH-DPAT in this study is in harmony with this concept.

ISSN:0271-3683    

DOI:10.3109/02713688709025209

出版商:Taylor&Francis    

年代:1987

数据来源: Taylor

摘要:

The EDTA-extractable protein (EEP) is a major extrinsic protein of lens membrane. The 35 kilodalton (kDa) polypeptide of the EEP cross-reacted to antibody prepared against calpactin I, a substrate for the src protein and an inhibitor of phospholipase A2. Calpactin I is also thought to play a structural role in linking cytoskeleton to membrane. The 35 kDa protein in bovine lens contained phosphotyrosine residues that can be detected by affinity purified antibody to this moiety. Although there is some microheterogeneity of EEP using two dimensional gel electrophoresis, at least one of the chick polypeptides, immunoreactive for calpactin I, can be phosphorylated in whole lens culture. These results suggest a regulatory function for the EEP in lens.

ISSN:0271-3683    

DOI:10.3109/02713688709025210

出版商:Taylor&Francis    

年代:1987

数据来源: Taylor

摘要:

Previous work (1,2,3) has indicated that the in vivo post-translational modification of the alpha crystallin primary gene product A is due to a specific phosphorylation process involving a serine residue located in a chymotryptic fragment with the sequence ARG-LEU-PRO-SER-ASN-VAL-ASP-GLN-SER-ALA-LEU which corresponds to the residues 119 to 129 of the polypeptide chain. To define which of the two serines is phosphorylated, thepresent experiments were carried out. The32P-labeled chymotryptic fragment was obtained from alpha crystallin isolated from the outer cortex of calf lenses incubated in the presence of [32P]-orthophosphate. By analyses of the products obtained after Edman degradation, utilizing electrophoresis in cellulose TLC plates and radioautography, it was possible to locate the phosphate in the serine residue at position 122 in the polypeptide chain. No phosphate could be detected in the serine residue at position 127.

ISSN:0271-3683    

DOI:10.3109/02713688709025211

出版商:Taylor&Francis    

年代:1987

数据来源: Taylor