Retinol-induced modification of the extracellular matrix of endothelial cells: Its role in growth control

Summary The growth of the endothelial cell (EC) is tightly regulated throughout the body. Many factors have been implicated in modulating EC growth including diffusible compounds, cell-to-cell interactions, and the extracellular matrix (ECM). Retinol, or vitamin A alcohol, has recently been shown to inhibit the growth of bovine capillary ECs, in vitro. Retinoids are known to modify ECM in other cell systems, and pure ECM components have been shown to effect EC growth rates. We, therefore, examined the role of the matrix in the retinol-induced inhibition of ECs. Cell-free matrices from control and vitamin A-treated ECs were prepared by removing cells with EGTA treatment after 7 d of culture. Matrix proteins were analyzed by solubilizing the matrices in 5M quanidine-HCl and performing Western blot analysis using specific antibodies to matrix proteins. In isolating the ECM, we observed that retinol-treated cultures of ECs were resistant to EGTA removal; retinol-treated ECs required twice the exposure time to EGTA to detach from their matrix than did controls cells. Western blot analysis of matrix proteins derived from control and retinol-treated EC cultures demonstrated a 1.6-fold increase in lamininβ chains and a 2.5-fold increase in fibronectin in the ECM of retinol-treated EC compared to control cell matrix. Functional properties of these matrices were assessed by plating control and Day 6 retinol-treated ECs onto the matrices and measuring attachment and growth by determining cell numbers at 24, 72, and 144 h. These studies revealed that control cells attached in greatest numbers to a control matrix whereas retinol-treated ECs preferentially attached to a matrix derived from retinol-treated cells. Furthermore, control ECs which grew rapidly on a control matrix were growth inhibited on a retinol-derived matrix. These data indicate that vitamin A treatment of ECs effects both their phenotype and influences the composition and the functional properties of their underlying ECM. These studies also demonstrate that alterations of the matrix are at least in part responsible for the growth inhibition of EC by retinol.     

Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system.

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.     

Differential role of insulin receptor autophosphorylation sites 1162 and 1163 in the long-term insulin stimulation of glucose transport, glycogenesis, and protein synthesis.

The long-term regulatory effect of insulin on glucose transport activity and glucose transporter expression was examined in Chinese hamster ovary (CHO) transfectants that overexpress either human insulin receptors of the wild type (CHO-R cells) or human insulin receptors mutated at two major autophosphorylation sites, Tyr1162 and Tyr1163 (CHO-Y2 cells). Previous studies showed that, when acutely stimulated by insulin, CHO-Y2 cells exhibit decreased receptor kinase activity along with decreased signaling of several pathways, including that for glucose transport, as compared with CHO-R cells. We now report the following. (i) When treated for 24 h with insulin (10(-10) to 10(-6) M), CHO-R and CHO-Y2 cells displayed closely similar concentration-dependent increases in 2-deoxyglucose uptake. In both transfectants, the maximal insulin-induced increase (approximately 3.5-fold) in uptake was cycloheximide-sensitive and was paralleled by equivalent increases in the levels of GLUT-1 immunoreactive protein and mRNA. (ii) By contrast, under similar conditions, CHO-Y2 cells exhibited a marked decrease in their response to insulin for [U-14C]glucose incorporation into glycogen (decreased sensitivity and maximal responsiveness) and for [U-14C]leucine incorporation into protein (decreased sensitivity) as compared with CHO-R cells. (iii) After a 24-h treatment with 10(-7) M insulin, CHO-R (but not CHO-Y2) cells showed a decreased ability to respond to a subsequent acute insulin stimulation of either receptor exogenous kinase activity or 2-deoxyglucose uptake as compared with respective untreated controls. These results indicate that (i) insulin receptors mutated at Tyr1162 and Tyr1163 retain normal signaling of the long-term stimulatory effect of insulin on glucose transport activity and GLUT-1 expression, but not on glycogenesis and overall protein synthesis; (ii) these three insulin signaling pathways may be triggered by distinct domains of the insulin receptor beta-subunit; and (iii) wild-type (but not twin-tyrosine mutant) receptors undergo negative regulation by chronic insulin treatment for subsequent signaling of acute biological actions of insulin.     

Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism.

To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.     

Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation.

Mutations in an intracellular region of the alpha 1B-adrenergic receptor constitutively activate the receptor, resulting in G protein coupling in the absence of agonist, as evidenced by elevated levels of polyphosphoinositide hydrolysis. Remarkably, all 19 possible amino acid substitutions at a single site in this region (alanine 293) confer constitutive activity. This set of mutated receptors exhibits a graded range of elevated biological activities, apparently representing a spectrum of receptor conformations which mimic the "active" state of the wild type receptor. In addition to their constitutive activities, these mutated receptors all demonstrate a higher affinity for agonists, another primary characteristic of the "active" conformation of G protein-coupled receptors. The fact that all possible mutations at this particular site result in increased activity suggests that this region may function to constrain the G protein coupling of the receptor, a constraint which is normally relieved by agonist occupancy.     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K⁺ conductance. Whole-cell currents showed an N-shaped current-voltage (I–13;V) relation with inward current activated at potentials negative to E_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ and showed a time-dependent decline at negative potentials, which was reduced in Na⁺-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K⁺ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K⁺ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K⁺ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

Alternate splicing pathways of the immunoglobulin heavy chain transcript of a teleost fish,Ictalurus punctatus

Previously we sequenced a partial cDNA clone encoding the 3' region of the message for the membrane receptor form of the heavy (mu) chain of the channel catfish which indicated that the first transmembrane (TM1) exon is spliced directly to the C mu 3 exon and not into a cryptic site within the CH4 exon, as occurs in other vertebrates. Studies utilizing polymerase chain reaction analysis of mRNA and further analysis of cDNA clones now confirm that the only detectable splicing pattern used in micron production by the channel catfish utilizes this C mu 3----TM1 pathway of pre-mRNA splicing.     

PHENOTYPIC PLASTICITY IN THE SAILFIN MOLLY,POECILIA LATIPINNA (PISCES: POECILIIDAE). II. LABORATORY EXPERIMENT

Field studies indicate that the influence of environmental factors on growth rate and size and age at maturity in sailfin mollies (Poecilia latipinna) is inconsistent over time and suggest that the marked interdemic variation in male body size in this species is the result of genetic variation. However, the role of specific environmental factors in generating phenotypic variation must be studied under controlled conditions unattainable in nature. We raised newborn sailfin mollies from four populations in laboratory aquaria under all possible combinations of two temperatures, three salinities, and two food levels to examine explicitly the influence of these environmental factors. Males were much less susceptible than females to temperature variation and were generally less plastic than females in terms of all three traits. Members of both sexes matured at larger sizes and at later ages in less saline and in cooler environments. Food levels were not sufficiently different to affect the traits we studied. The effects of temperature and salinity were not synergistic. Males from different populations exhibited different average ages and sizes at maturity, but females did not. The magnitudes of the effects we found were not substantial enough to account for the consistent interdemic differences in male and female body size that have been observed previously. Our results also indicate that no single environmental factor is solely responsible for the environmental effects observed in field experiments on growth and development. These studies, together with other work, indicate that the strongest sources of interdemic variation are genetic differences in males and differences in postmaturation growth and survivorship in females.     

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.