Threonine phosphorylations induced by RX-871024 and insulin secretagogues in betaTC6-F7 cells.

Treatment of the pancreatic beta-cell line betaTC6-F7 with an imidazoline compound, RX-871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (K(ATP)) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca(2+)](i)), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.

Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells.

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta.

Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-membered macrocycles containing a N-N'-bridged bisindolylmaleimide moiety is described. A panel of eight cloned human PKC isozymes (alpha, beta I, beta II, gamma, delta, epsilon, sigma, eta) was used to identify the series and optimize the structure and associated activity relationship. The dimethylamine analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene++ +-1,3(2H)-dione, inhibits the PKC beta I (IC50 = 4.7 nM) and PKC beta II (IC50 = 5.9 nM) isozymes and was 76- and 61-fold selective for inhibition of PKC beta I and PKC beta II in comparison to PKC alpha, respectively. The additional analogues described in the series are also selective inhibitors of PKC beta. LY333531 (1) exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol ester (4 beta-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the series with ED50 values ranging from 7.5 to 0.21 microM. A comparison of the PKC isozyme and related ATP dependent kinase inhibition profiles is provided for the series and compared to the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series is compared with that of the kinase inhibitor staurosporine.

Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor.

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

Beta-glucuronidase mediated pathway essential for retinal pigment epithelial degradation of glycosaminoglycans. Disease expression and in vitro disease correction using retroviral mediated cDNA transfer.

A beta-glucuronidase mediated pathway for the degradation of glycosaminoglycans is present in the retinal pigment epithelium. The pathway has been defined using ocular tissues and cultured cells from mutant animals having a recessively inherited deficiency of the lysosomal enzyme. In situ, storage products accumulate in secondary lysosomes of the retinal pigment epithelium, the cytoplasm fills with inclusions and the cells hypertrophy; severity of the disease increases with aging. Deficient activity of beta-glucuronidase is present in primary and second passage cultures. Radiolabel studies with 35SO4 show a significant retention of cell layer label by mutant retinal pigment epithelial cells during a 72-hr pulse or 24-hr chase period. The labels is in newly synthesized chondroitin sulfate and heparan sulfate, which are natural substrates for the deficient enzyme. There is no difference from normal in the total radioactivity and electrophoretic profile of the glycosaminoglycans that are synthesized and released into the media. A retroviral vector was used to transfer normal rat beta-glucuronidase cDNA into the mutant cells. The vector treatment results in restoration of enzyme activity and correction of the degradative defect; 35SO4 labeling shows that chondroitin sulfate and heparan sulfate levels return to normal. The vector treatment studies indicate that a single gene defect determines the abnormal beta-glucuronidase mediated pathway in the mutant retinal pigment epithelium.

Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.

Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder.

Retinal pigment epithelial glycosaminoglycan metabolism: intracellular versus extracellular pathways. In vitro studies in normal and diseased cells.

The synthesis and turnover of glycosaminoglycans (GAGs) in different fractions of cultured feline retinal pigment epithelium (RPE) were characterized. In one method of fractionation, trypsin was used to separate the extracellular components (referred to as trypsin-soluble glycocalyx) from the intracellular components. As a second method, the basal extracellular matrix (basal ECM) was separated from the rest of the GAGs (cell-associated GAGs) by extracting the cell layer with NH4OH. The incorporation of 35SO4 into cetylpyridinium chloride-precipitable GAGs in the cell-associated and the intracellular fractions increased throughout the labeling period, while in the trypsin-soluble glycocalyx and the basal ECM incorporation approached a maximum. While heparan sulfate was the predominant GAG in all compartments, most was located extracellularly. The majority of dermatan sulfate was localized in the intracellular fraction. GAGs in the trypsin-soluble glycocalyx exhibited a rapid rate of turnover, while GAGs in the intracellular compartment and basal ECM turned over much more slowly. Ascorbic acid increased the incorporation of 35SO4 into ECM chondroitin sulfate/dermatan sulfate, but not heparan sulfate, on a per cell basis. Cycloheximide reduced incorporation of 35SO4-GAGs into both the cell-associated compartment and the basal ECM. In contrast, monensin caused a reduction in basal ECM GAGs while increasing the GAGs in the cell-associated compartment. The intracellular accumulation of GAGs and resultant pathology in alpha-L-iduronidase (alpha-L-id)-deficient RPE indicated that this pathway for the intracellular degradation of GAGs is important in normal RPE function. However, the turnover of GAGs in the trypsin-soluble glycocalyx was not affected by deficient alpha-L-id activity or by the subsequent intracellular accumulation of GAGs. Therefore, normal lysosomal activity in the RPE is not a prerequisite for maintaining the rate of extracellular GAG turnover within normal limits.

Synthesis and secretion of glycosaminoglycans in cultured retinal pigment epithelium.

The synthesis and secretion of glycosaminoglycans (GAGs) in primary cultures of feline retinal pigment epithelium (RPE) was measured. After 14 days in culture, the cells were incubated for 3 days with media containing 20 microCi/ml 35SO4 and 10 microCi/ml [3H]-glucosamine. The GAGs were precipitated from the media and cell layer with cetylpyridinium chloride and ethanol, separated in 0.1 M Ba acetate by cellulose acetate electrophoresis, visualized by Alcian blue staining and fluorography, and quantitated by scanning densitometry and liquid scintillation counting. The predominant radioactively-labeled GAG secreted into the media by RPE cultures was chondroitin sulfate (CS), which accounted for 63% of the 35SO4, and 54% of the 3H incorporated. Radioactively-labeled heparan sulfate (HS), dermatan sulfate (DS), and hyaluronic acid (HA) were also identified in the media. Heparan sulfate accounted for 24% of the 35SO4 and 22% of the 3H, DS contained 13% of the 35SO4 and 7% of the 3H, and HA accounted for 17% of the 3H incorporated into the media GAGs. In contrast, fibroblast cultures secreted primarily HA, which accounted for 84% of the 3H in the media GAGs. The profile of GAGs retained by the cell layer was different from that of the secreted GAGs. The predominant radioactively-labeled GAG associated with the cell layer was HS. This GAG contained 54% of the 35SO4, and 52% of the 3H incorporated into cell layer GAGs. Dermatan sulfate contained 23% of the 35SO4, and 23% of the 3H, while CS accounted for 23% of the 35SO4, and 18% of the 3H incorporated into cell layer GAGs. The remaining 7% of the incorporated 3H was associated with HA. Total GAG profiles were determined for primary cultures of feline RPE by Alcian blue staining, and compared with those of freshly isolated cell samples. The GAG profiles were similar in cultured and freshly-isolated samples; however, the percentage of HS in the freshly-isolated samples was about twice as high as the percentage of HS in cultured samples, while the proportion of DS in cultured samples was higher than in freshly-isolated samples. This study demonstrates that the profile of radioactively-labeled GAGs secreted by primary cultures of feline RPE is distinct from that retained by the cell layer. In addition, the total GAG profile of cultured RPE is similar, but not identical, to that of freshly-isolated cells.

Synergistic activation of retinal capillary pericyte proliferation in culture by inositol triphosphate and diacylglycerol.

Inositol triphosphate (IP3) and diacylglycerol (DG) are second messengers which control ionic events implicated in cell proliferation in a variety of tissues. In order to determine if these two second messengers control the proliferation of bovine retinal capillary pericytes (BRCP) or feline retinal pigment epithelial cells (FRPE) in culture, both intact BRCP or FRPE or BRCP or FRPE made permeable by saponin were used to study the effects of IP3 and DG on [3H]thymidine incorporation into DNA. [3H]Thymidine incorporation by BRCP made permeable to saponin showed specific IP3 dose-dependence; the apparent Km was 0.3 microM of IP3. Similar effects of A23187, a Ca2+ ionophore, or synthetic DG (1-oleoyl-2 acetyl-glycerol) were also observed. The combination of synthetic DG (0-,2-,4-, 8 micrograms ml-1) and 1 microM A23187 produced greater stimulation of [3H]thymidine incorporation by intact BRCP than was seen with DG or A23187 alone. In contrast to BRCP. [3H]thymidine incorporation by FRPE was not stimulated by IP3, A23187 or synthetic DG. The synergistic activation of IP3 and DG provided direct evidence to support the view that BRCP proliferation in vitro were regulated by the levels of the two second messengers.

Glycosaminoglycan synthesis and secretion by bovine retinal capillary pericytes in culture.

The synthesis and secretion of glycosaminoglycans (GAGs) was characterized in subcultures of bovine retinal capillary pericytes. The GAGs were metabolically labeled with [3H]glucosamine and 35SO4 for 3 days, and then precipitated from the cell layer or media by cetylpyridinium chloride and ethanol, separated by cellulose acetate electrophoresis and further identified by their susceptibility to degradative procedures. The predominant radioactively labeled GAG associated with the pericyte-cell layer was heparan sulfate (HS). Radioactively labeled chondroitin sulfate (CS) and hyaluronic acid (HA) were also present in the pericyte-cell layer. No radioactively labeled dermatan sulfate (DS) was detected. The profile of radioactively labeled GAGs secreted by pericytes into the media differed considerably from that associated with the cell layer. Equal amounts of radioactivity were incorporated into HS and CS. Small quantities of radioactively labeled HA were also present in the media. Although no radioactively labeled DS was detected in the pericyte-cell layer, it was present in the media. The total pericyte-cell layer GAG profile was determined by scanning densitometry of the three bands resolved after cellulose acetate electrophoresis and Alcian Blue staining. The slowest band was identified as HS, and accounted for 17% of the total GAGs. The middle band was identified as DS, and accounted for 34% of the total GAGs. The fastest band was tentatively identified as either DS or chondroitinase AC-resistant CS, and constituted 49% of the total GAGs. The GAGs associated with the fibroblast-cell layer and secreted into the media by fibroblasts also were characterized and compared with those produced by pericytes. The major differences were in the secretion of large amounts of HA into the media by fibroblasts, and the presence of radioactively labeled DS in the cell layer of fibroblasts.

Ascorbate transport in cultured cat retinal pigment epithelial cells.

Transport of ascorbate by primary cultures of cat retinal pigment epithelial cells (RPE) was studied. Confluent primary cultures were incubated with 10-500 microM L-[carboxyl-14C] ascorbic acid in balanced salt solution (BSS) at 37 degrees C for 1 to 40 min. The uptake of radioactive ascorbate followed saturation kinetics with a Km of 42 microM and Vmax of 117 pmol min-1 microgram-1 DNA. Cells incubated with 10 microM radioactive ascorbate for 40 min showed a ratio of intracellular to extracellular radioactive ascorbate of greater than 40. The transport of ascorbate was sodium- and energy-dependent. Replacement of 150 mM NaCl in BSS with 150 mM LiCl reduced ascorbate uptake significantly. Ouabain, 2,4-dinitrophenol, alpha-D-glucose, 3-O-methyl-D-glucose, and the ascorbate analogues, D-isoascorbate and dehydroascorbate, each inhibited ascorbate uptake into RPE cells. The efflux of radioactivity into the incubation media was slow when cells were preloaded with either 50- or 500 microM radioactive ascorbate, but increased when cells preloaded with 50 microM ascorbate were incubated in the presence of excess non-radioactive ascorbate. These studies demonstrated that a sodium-dependent carrier system is involved in transport of ascorbate in primary cultures of cat RPE.

Arylsulfatase B activity in cultured retinal pigment epithelium: regional studies in feline mucopolysaccharidosis VI.

Feline mucopolysaccharidosis VI (MPS VI) is a recessively inherited lysosomal storage disease resulting from a deficiency of arylsulfatase B (ASB). Previous histopathologic findings have indicated that the disease is expressed morphologically in non-pigmented retinal pigment epithelial cells (RPE) in the posterior pole and superior equatorial regions by the accumulation of vacuolated inclusions and eventual cellular hypertrophy, while pigmented regions in the periphery are minimally affected. To determine if the regional and age-dependent variations in disease severity result from differences in residual enzyme activity, primary cultures of feline MPS VI-affected RPE were initiated from defined regions of the eye and maintained in vitro for 14 days. Cultures initiated from nonpigmented areas of affected adult eyes (posterior pole, superior equatorial) were more diseased than those from pigmented (inferior-equatorial, peripheral) areas. In the nonpigmented cultures, the disease was expressed by the accumulation of single membrane-bound inclusions and cellular hypertrophy. These inclusions were indistinguishable in their morphologic appearance and distribution from those found in situ. In contrast, the cultures initiated from pigmented areas remained normal or minimally affected. The same spatial disease distribution was present in young affected eyes, but the expression of the disease was much less severe. It is apparent that temporal, spatial, and pigmentation factors were correlated with disease expression in vitro as well as in situ. Arylsulfatase B activity was measured biochemically, and found to be deficient in all regions of young and adult eyes. It was notable that there was no correlation between the level of residual enzyme activity, and the pigmentation or spatial position from which the cells were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Translation of type IV procollagen messenger RNA from cultured cat retinal pigment epithelial cells.

The activity of type IV procollagen mRNA was detected in the total cytoplasmic RNA prepared from normal feline retinal pigment epithelial cells in culture. The translation products contained two distinct bands in the pro alpha chain region on SDS-PAGE, which were sensitive to collagenase digestion. Corresponding bands were identified in the immunoprecipitate of the translation products after reaction with an anti-type IV collagen antiserum and separation by protein A-Sepharose column chromatography.

Extracellular matrix production by cat retinal pigment epithelium in vitro: characterization of type IV collagen synthesis.

Feline retinal pigment epithelial cells (RPE) produced an extracellular matrix (ECM) in vitro which was located between the basal surface of the RPE and the culture plate. This ECM had three morphological components: bundle, granular and fibrillar. After 14 days in culture the basal extracellular space contained small amounts of bundle material; granular and fibrillar material were infrequently observed at this time. The amount of ECM material increased with increasing time in culture. The accumulation of the granular component extracellularly was greatest between 60 and 108 days. Fibrillar material, although occasionally observed in the ECM, appeared to be an infrequent component. By 145 days, the ECM filled the extracellular space between the RPE and the culture plate. The time-dependent increase of the ECM indicated continued synthesis and secretion of ECM into the basal extracellular space by the RPE. Confluent RPE cultures, or choroidal/scleral fibroblasts, were incubated for 24 hr with [14C]-proline. Newly synthesized collagen, either in the culture medium or the cell layer, was co-precipitated with added carrier collagen by (NH4)2 SO4. The samples, with or without reduction and alkylation, were digested with pepsin and fractioned by selective salt precipitation and carboxymethyl(CM)-cellulose chromatography. The resulting fractions were further analyzed, or purified for thin layer chromatography (TLC) amino acid analysis, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Cultured RPE cells, but not choroidal/scleral fibroblasts, produced labelled peptides which were characterized as alpha 1 (IV), and alpha 2 (IV) collagen chains by CM-cellulose chromatography, SDS-PAGE, proline: hydroxyproline ratios and sensitivity to bacterial collagenase. In contrast, choroidal/scleral fibroblasts produced labelled alpha 1 (I), beta 12 (I) and alpha 2 (I) collagen chains. The synthesis of type IV collagen by RPE cells may reflect the production of ECM observed by electron microscopy in cultured feline RPE cells.

Tissue culture of cat retinal pigment epithelium.

To culture retinal pigment epithelial (RPE) cells from normal cats, the cells were enzymatically dissociated from the eyecup and grown in either Ham's F-10 Nutrient Mixture or Eagle's Minimum Essential Media supplemented with 20% fetal calf serum. Cultures reached confluency between 6 and 10 days and contained monolayers of polygonally shaped cells. Light and electron microscopy demonstrated that most of the normal morphological characteristics of cat RPE cells in vivo were maintained in vitro; these included apical microvilli, apicolateral junctional specializations, basal infoldings and intracellular organelles. Pigment granules appeared to be diluted by cell division. No evidence of a basal membrane formation was seen; however, a fine granular or fibrillar extracellular matrix was observed in some cultures and was located between the culture plate surface and the basal surface of the RPE. Primary cultures were viable for up to 145 days. The activities of two lysosomal hydrolases (arylsulfatase A and arylsulfatase B) involved in the metabolism of sulfatide and dermatan sulfate were measured in confluent cultures. Mean arylsulfatase A activity was 1297 nmol nitrocatechol/mg protein/hr and arylsulfatase B activity was 553 nmol nitrocatechol/mg protein/hr. These activities were approximately 5 to 10-fold higher than present in cat peripheral leukocytes and skin fibroblasts in vitro. This in vitro system will facilitate studies on normal function and in conditions where the RPE has been compromised by inherited diseases (i.e. gyrate atrophy, mucopolysaccharidosis I and VI).

Transport of 3-O-methylglucose in isolated rat retinal pigment epithelial cells.

The uptake of 3-O-methylglucose in isolated rat retinal pigment epithelial cells exhibits complex kinetics as revealed by a plot of the Lineweaver-Burke transformation of the Michaelis-Menten equation. Conventional analysis of the linear portion of the double-reciprocal plot yields a Km of 7 . 69 mM and a Vmax of 1 . 95 nM/mg protein/min. If data are analyzed to take into account the existence of two carrier-mediated processes, a high affinity system (Km = 1 . 94 mM, Vmax = 0 . 012 nM/mg protein/min) and a low affinity system (Km = 640 mM, Vmax = 12 . 20 nM/mg protein/min) for 3-O-methylglucose uptake results. Three-O-methylglucose accumulation over a 10-min period was insensitive to phloridzin (0 . 1 mM), ouabain (1 mM) and dinitrophenol (1 mM), but was inhibited by mercuric chloride (1 mM). Phloretin (0 . 1 mM) increased the accumulation of both glucose and 3-O-methylglucose, which may indicate the existence of carrier-mediated efflux system. These findings demonstrate that glucose transport in rat retinal pigment epithelial cells is a complex process involving carrier mediation.