Nitrendipine potentiates Bay k 8644-induced contraction of isolated porcine coronary artery: evidence for functionally distinct dihydropyridine receptor subtypes

Bay k 8644 and nitrendipine, dihydropyridines classified as calcium channel agonist and antagonist, respectively, produced concentration-dependent biphasic responses (contraction and relaxation) in porcine coronary artery rings. Nitrendipine relaxed rings (IC50 = 60 nM) that were contracted with 100 nM Bay k 8644. Pretreatment of rings with 60 nM nitrendipine caused paradoxical potentiation of Bay k 8644-induced contraction. The data are consistent with a model that consists of two functionally-distinct dihydropyridine "receptors" with which Bay k 8644 and nitrendipine interact as partial agonists. We propose that these excitatory and inhibitory dihydropyridine receptor subtypes mediate contraction and relaxation, respectively, by dihydropyridines.     

Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme in Aplysia neurons

We have shown previously that the subcellular distribution of a major calmodulin-binding protein is altered under conditions causing increased synthesis of cAMP in Aplysia neurons (Saitoh, T., and J. H. Schwartz, 1983, Proc. Natl. Acad. Sci. USA, 80:6708-6712). We now provide evidence that this Mr 55,000 protein is a subunit of a Ca2+/calmodulin-dependent kinase: (a) both the Mr 55,000 calmodulin-binding protein and kinase activity are loosely attached to the membrane-cytoskeletal complex; (b) both kinase activity and the Mr 55,000 protein are translocated from the membrane-cytoskeleton complex to the cytoplasm under conditions that cause the change in the subcellular distribution of the Mr 55,000 calmodulin-binding protein; and (c) calmodulin-binding activity of the Mr 55,000 protein and the ability to carry out the Ca2+/calmodulin-dependent phosphorylation of synapsin I are purified in parallel. The subcellular localization of the Ca2+/calmodulin-dependent protein kinase appears to be under control of two second messengers: Ca2+ and cAMP. We find that the Mr 55,000 subunit is phosphorylated when the extracted membrane-cytoskeleton complex is incubated with Ca2+, calmodulin, and ATP, with the concomitant release of this phosphorylated peptide from the complex. Previously, we had found that, when translocation occurs in extracts in the presence of cAMP and ATP (but in the absence of Ca2+), there was no detectable phosphorylation of the Mr 55,000 subunit itself. The subcellular distribution of the subunit thus appears to be influenced by (a) cAMP-dependent phosphorylation, which, we infer, modifies some as yet unidentified structural component, causing the release of the enzyme; and (b) Ca2+/calmodulin-dependent phosphorylation of the Mr 55,000 subunit. These studies also suggest that phosphorylation has an important regulatory consequence: during the Ca2+/calmodulin-dependent translocation of the Mr 55,000 subunit, the kinase appears to be activated, becoming independent of added Ca2+/calmodulin.     

Synthesis and structure of proteoglycan core protein

Studies of the structure and synthesis of cartilage proteoglycan core protein have been carried out. Deglycosylation of completed, secreted proteoglycan by HF-pyridine treatment yielded an intact homogeneous core protein of approximately 210,000 daltons, with a blocked amino-terminus. Greater than 95% of chondroitin sulfate chains and 80% of N- and O-linked oligosaccharides were removed by the procedure, which made the product an excellent xylosyltransferase acceptor. Little alteration of core protein structure occurred during the HF-pyridine treatment as shown by complete immunoreactivity with antiserums prepared against hyaluronidase-digested proteoglycan. In other studies, the initially synthesized precursor for proteoglycan core protein was found to be approximately 376,000 daltons and localized to the rough membrane fractions. This precursor already contained N-linked oligosaccharides, and was also able to accept xylose, thereby initiating chondroitin sulfate chains. The precursor was translocated intact in an energy-dependent manner to smooth membrane-Golgi fractions where further processing of high mannose type of oligosaccharides and addition of glycosaminoglycan chains occurred. The subcellular distribution pattern of the chondroitin sulfate-synthesizing enzymes corroborated the proposed topological modifications of the proteoglycan core protein precursor.     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

The expression and chromatin structure of the chicken glyceraldehyde-3-phosphate dehydrogenase gene in mouse cells

Chicken glyceraldehyde-3-phosphate dehydrogenase gene (GAPD) and thymidine kinase gene (TK) were co-transfected into mouse LMTK- cells by the calcium phosphate precipitation technique. Four of the eight hypoxanthine/aminopterin/thymidine-containing medium-resistant, TK+ transfectants were shown to produce different amounts of chicken glyceraldehyde-3-phosphate dehydrogenase by zymogram analysis. Subcloning and further analysis revealed that the chicken GAPD was stably inherited and that its enzyme subunits randomly combined with mouse subunits in heterotetramers. Although the contribution of chicken enzyme varied from approximately 30 to approximately 90% of the total glyceraldehyde-3-phosphate dehydrogenase activity with a proportional increase in total activity in the different subclones, it did not appear to affect the expression of mouse endogenous glycolytic enzymes since there was no distinct change in the levels of either mouse glyceraldehyde-3-phosphate dehydrogenase mRNA nor mouse phosphoglycerate kinase enzyme activity. The levels of chicken GAPD copy number, mRNA, and enzyme apparently were generally correlated in the different subclones, suggesting that the chicken GAPD in the mouse cells were expressed constitutively. In situ hybridization revealed that the transfected genes were integrated into mouse chromosomes in one cluster, and the locations of these clusters were different in different clones. Chromatin structure analyses of the chicken GAPD in four different transfectants revealed three DNase I-hypersensitive sites located around 0.2, 2.0, and 3.4 kilobases upstream from the 5' side of the gene. These sites are also present in the same locations in chicken lymphoblastoid cells (Kuo, M. T., Iyer, B., and Schwartz, R. J. (1982) Nucleic Acids Res 10, 4565-4579), indicating the dominant transmission of DNase I-hypersensitive cleavage sites in the transfected gene.     

Extracellular matrix microfibrils are composed of core proteins coated with fibronectin

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.     

Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment

Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.     

A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR/8/34 influenza virus hemagglutinin

Approximately 50% of the primary antibody response of BALB/c mice to the A/PR/8/34 influenza virus hemagglutinin is directed to the Cb site, one of the four major antigenic regions of the molecule. To determine the structural basis of the anti-Cb site response, we have examined the paratypic and genetic diversity exhibited by a panel of 24 primary and 4 secondary response mAb specific for this antigenic region. Reactivity pattern analysis demonstrated 20 distinct fine specificities among these antibodies, and V region gene sequence analysis showed that they are encoded by 17 different VH gene segments from 6 VH gene families and 14 different VK gene segments from 6 VK gene groups. Despite this overall diversity, many of the antibodies can be placed in a limited number of sets based on the shared expression of VH and/or VK genes. One set contains antibodies encoded by a single gene of the VK4/5 group in combination with one of two closely related genes from the J558 VH family. This set accounts for half of the Cb site-specific primary response hybridomas, indicating that the representation of the various anti-Cb site B cell specificities during the primary response to A/PR/8/34 influenza virus is not uniform. The preferential participation of B cells expressing this VH/VK combination is largely responsible for the dominance of anti-Cb site antibodies in the primary anti-hemagglutinin response.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

Individual VH genes detected with oligonucleotide probes from the complementarity-determining regions

The germ-line and expressed Ig repertoire was examined with three oligonucleotide probes from the CDR regions of VH18/2, a VH gene from the largest human VH gene family, VHIII. Each oligonucleotide probe detected small numbers of germ-line bands (1-5) under conditions in which single base differences can be detected; more than half of these bands were polymorphic. The combined results from pairs of oligonucleotides from CDR1 and CDR2 identified a single band on Southern blots, as did a probe from the 5' end of CDR2. This band contains the 18/2 germ-line gene. The nucleotide sequence of expressed VH genes that hybridized to both CDR probes or to the 5' CDR2 probe were greater than or equal to 97% homologous to 18/2 in both the framework and CDR regions. This group of closely related VH genes, the 18/2 CDR family, appears to be overexpressed. The role of polymorphisms and differential expression of individual V genes in multigenic autoimmune diseases, as well as the organization and expression of individual V genes, can be examined with pairs of oligonucleotides from CDR1 and the 3' end of CDR2, or with probes from the 5' end of CDR2.