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D L Barber

Publications and source records attributed to D L Barber.

At least 55 records · Page 3Linked to original sources
PubMed1992

Mutant constructs of the beta-adrenergic receptor that are uncoupled from adenylyl cyclase retain functional activation of Na-H exchange.

beta-Adrenergic receptor (beta AR) agonists modulate a number of intracellular effectors; for example, they stimulate adenylyl cyclase and Ca2+ channels, inhibit Na+ channels and Mg2+ efflux, and activate Na-H exchange. Regulation of adenylyl cyclase, Ca2+, Na+, and Mg2+ by the beta AR is mediated through receptor coupling to the GTP-binding protein Gs. We have previously determined, however, that beta AR stimulation of Na-H exchange occurs independently of receptor coupling to Gs. In the present study, we analyzed mutant beta ARs containing deletions of amino acid residues within the third cytoplasmic domain, to determine whether there is a structural basis for the ability of the beta AR to couple divergently to the Gs-dependent stimulation of adenylyl cyclase and the Gs-independent activation of Na-H exchange. Receptor constructs with deletions of residues 222-229 and 258-270, which were previously shown to be defective in coupling to Gs and adenylyl cyclase, retained an isoproterenol-induced activation of Na-H exchange that was similar in time course and magnitude to that observed with the wild-type beta AR. These results confirm our previous findings that the beta AR activates Na-H exchange independently of Gs, and they further suggest that distinct molecular determinants of the receptor divergently stimulate adenylyl cyclase and Na-H exchange.

Adenylyl Cyclases↗
PubMed1991

The receptor for yolk lipoprotein deposition in the chicken oocyte.

The final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically synthesized yolk precursor proteins from the plasma. The two major yolk-forming components, very low density lipoprotein (VLDL) and vitellogenin (VTG), have been shown to interact with a 95-kDa protein present in detergent extracts of ovarian membranes; this protein is absent in hens of a mutant nonlaying chicken strain (Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398). Here, we have purified the 95-kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor-mediated endocytosis by ultrastructural immunolocalization, structural, and functional studies. The receptor was visualized exclusively in the oocyte proper and was absent from somatic cells, in agreement with the previously reported expression of two different lipoprotein receptors in somatic cells and oocytes, respectively, of laying hens (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Amino acid sequences of tryptic fragments of the oocyte receptor were obtained, and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains. In particular, the chicken receptor's internalization sequence, Phe-Asp-Asn-Pro-Val-Tyr, is identical with that in low density lipoprotein receptors from mammals as well as Xenopus laevis. The ligand-binding properties, specificity, and kinetic parameters of the oocyte receptor were characterized in filtration assays employing pure ligands and receptor. In conjunction with ligand-blotting experiments following limited protease digestion of the receptor, the binding assay data suggest that VTG recognizes a substructure of the VLDL-binding site. These studies establish that a cell-specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays a key role in control of oocyte growth.

Amino Acid Sequence↗
PubMed1991

The laying hen expresses two different low density lipoprotein receptor-related proteins.

We have identified, by a combination of ligand, 45Ca2+, and immunoblotting, two large membrane proteins akin to the mammalian so-called low density lipoprotein (LDL) receptor-related protein (LRP) in chicken tissues. LRP has thus far been demonstrated only in mammalian species where it is thought to act as a receptor for proteinase-alpha 2-macroglobulin complexes and/or chylomicron remnants, lipoproteins not produced in birds. One of the chicken LRPs was demonstrated in liver, and has the same apparent Mr and hallmark biochemical properties as rat liver LRP. The other chicken LRP is smaller (approximately 380 kDa) and is expressed in ovarian follicles, but is undetectable in liver. Immunological analysis demonstrated a lack of cross-reactivity between the two LRPs, as well as between them and the previously identified chicken oocyte-specific 95-kDa receptor for the yolk precursors, very low density lipoprotein, and vitellogenin (Stifani, S., Barber, D. L., Nimpf, J., and Schneider, W. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 87, 1955-1959). As shown by ligand blotting, both chicken LRPs have the ability to interact with vitellogenin, a property they share not only with rat LRP, but also with mammalian LDL receptors. To obtain independent confirmation of the ligand blotting results, the smaller (follicular) LRP was purified and high-affinity binding of vitellogenin to it was demonstrated by a solid-phase filtration binding assay. Amino acid sequences of tryptic fragments of the smaller LRP were obtained, and its homology with human LRP demonstrated through unambiguous alignment of three fragments. Both chicken LRPs, the chicken oocyte 95-kDa receptor, as well as rat LRP, could be shown by ligand blotting to interact specifically with chicken serum alpha 2-macroglobulin. In addition, human apolipoprotein E, a ligand implicated in receptor-mediated metabolism of chylomicron remnants, also binds to the smaller chicken LRP, further emphasizing the similarities between LDL receptors and related proteins from a variety of species. In analogy to the known dichotomy of chicken LDL receptors, which is characterized by the production of the 95-kDa oocyte-specific receptor on one hand and a 130-kDa LDL receptor that is exclusively expressed in somatic cells (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139), it appears that the smaller and larger chicken LRPs also may be restricted to the oocyte and somatic cells, respectively.

Amino Acid Sequence↗
PubMed1991

Fatty acids stereospecifically stimulate neurotensin release and increase [Ca2+]i in enteric endocrine cells.

In primary cultures of canine enteric endocrine cells, fatty acids directly stimulated the release of neurotensin-like immunoreactivity (NTLI). This stimulatory effect was cell specific, selective for long-chain unsaturated fatty acids, and stereospecific. Saturated fatty acids of comparable chain length and trans isomers of long-chain unsaturated fatty acids had no effect on basal NTLI secretion. NTLI release in response to oleic acid (cis-11) was dose dependent with an apparent EC50 of 37 +/- 0.18 microM. Cyclooxygenase inhibitors had no effect on fatty acid-stimulated NTLI release, indicating the response was not mediated by the production of active arachidonic acid metabolites. Somatostatin (100 nM) inhibited maximal oleic acid-stimulated NTLI release by 92%. Long-chain unsaturated fatty acids also selectively and stereospecifically stimulated an increase in the mobilization of [Ca2+]i to 313.5 +/- 28.6% of resting [Ca2+]i. Staurosporine, an inhibitor of protein kinase C, dose dependently inhibited oleic acid-stimulated NTLI release with an IC50 value of 22 +/- 0.4 nM. Long-chain unsaturated fatty acids had no effect on basal NTLI secretion from rat pheochromocytoma cells and medullary thyroid carcinoma cells, two clonal lines that express NTLI. The cell-specific, selective stereospecific, and inhibitable action of fatty acids on NTLI secretion suggests that the effect of fatty acids on enteric endocrine cells is indicative of a receptor-mediated mechanism.

Adrenal Gland Neoplasms↗
PubMed1990

Evolution of lipoprotein receptors. The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand apolipoprotein E.

The laying hen expresses two different lipoprotein transport receptors in cell-specific fashion. On the one hand, a 95-kDa oocyte membrane protein mediates the uptake of the major yolk precursors, very low density lipoprotein, and vitellogenin; on the other hand, somatic cells synthesize a 130-kDa receptor that is involved in the regulation of cellular cholesterol homeostasis (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Here we show that the oocyte-specific receptor binds, in addition to the yolk precursor proteins, an apolipoprotein of mammalian origin, apolipoprotein E. Ligand blotting, a solid-phase binding assay, and antireceptor antibodies were employed to demonstrate that binding of vitellogenin, very low density lipoprotein (via apolipoprotein B), and apolipoprotein E occurs to closely related, if not identical, sites on the 95-kDa oocyte receptor. The binding properties of lipovitellin, which harbors the receptor recognition site of vitellogenin, are analogous to those of apolipoprotein E: both require association with lipid for expression of functional receptor binding. The ligand specificity of the avian oocyte lipoprotein receptor supports the hypothesis that vitellogenin, which has evolved in oviparous species, represents a counterpart to mammalian apolipoprotein E.

Animals↗
PubMed1990

Multiple receptors coupled to adenylate cyclase regulate Na-H exchange independent of cAMP.

We have previously determined that beta-adrenergic and somatostatin receptors stimulate and inhibit, respectively, Na-H exchange independent of changes in cAMP accumulation (Barber, D.L., McGuire, M.E., and Ganz, M.B. (1989) J. Biol. Chem. 264, 21038-21042). The present study extends our work on the beta-adrenergic receptor (beta AR) by investigating receptor activation of Na-H exchange in multiple cell types that either endogenously express the beta AR or that have been transfected with cDNA of the hamster lung beta 2AR or the turkey erythrocyte beta AR. Exchanger activity was determined by monitoring intracellular pH in cell populations loaded with the pH-sensitive dye BCECF (2,7-biscarboxyethyl-5(6)-carboxyfluorescein). In addition to the action of the beta AR, activation of prostaglandin E1 and parathyroid hormone receptors induced an intracellular alkalinization by stimulating a Na(+)-dependent amiloride-sensitive Na-H exchange. In contrast, activation of D2-dopaminergic receptors induced an intracellular acidification by inhibiting Na-H exchange. beta-Adrenergic, prostaglandin E1, and parathyroid hormone receptors activated Na-H exchange independent of changes in intracellular cAMP accumulation and independent of a cholera toxin-sensitive stimulatory GTP regulatory protein. D2-dopaminergic receptors inhibited exchanger activity independent of a pertussis toxin-sensitive inhibitory GTP regulatory protein. We suggest that these receptors are functionally coupled to adenylate cyclase and Na-H exchange through divergent signaling mechanisms.

Adenylyl Cyclases↗
PubMed1990

A single chicken oocyte plasma membrane protein mediates uptake of very low density lipoprotein and vitellogenin.

Specific cell-surface receptors mediate the uptake of plasma proteins into growing oocytes of oviparous species, thereby forming yolk. Quantitatively the most important yolk precursors are the lipoproteins, very low density lipoprotein, and vitellogenin. We show that a single major chicken oocyte plasma membrane protein with an apparent molecular mass of 95 kDa as determined by SDS/PAGE under nonreducing conditions is the receptor for both of these ligands. Binding activities for the two ligands copurified on ligand affinity matrices and were inhibited by the same antibody preparations, and the ligands competed with each other for binding to the 95-kDa protein. In addition to these biochemical and immunological lines of evidence for the identity of the vitellogenin receptor with the very low density lipoprotein receptor, genetic proof was obtained. We have previously shown that the mutant nonlaying "restricted-ovulator" hen carries a defect in the gene responsible for functional expression of the oocyte 95-kDa protein. Here we demonstrate that this single gene defect in the restricted-ovulator hen has detrimental consequences for the binding not only of very low density lipoprotein but also of vitellogenin to the 95-kDa receptor normally present in oocytes. The intriguing bifunctionality of this chicken oocyte membrane protein possibly relates to its crucial role in receptor-mediated control of oocyte growth.

Animals↗
PubMed1989

Beta-adrenergic and somatostatin receptors regulate Na-H exchange independent of cAMP.

Activation of beta-adrenergic and somatostatin receptors increases and attenuates, respectively, cAMP. We have determined, however, that in enteric endocrine cells beta-adrenergic and somatostatin receptors also regulate Na-H exchange activity, independent of their effects on cAMP. In cells loaded with a pH-sensitive dye, epinephrine, acting at a beta 2-adrenergic receptor induced an alkalinization while somatostatin caused an acidification of intracellular pH (pHi). These pHi changes were dependent on extracellular Na+ and inhibited by amiloride. Forskolin, dibutyryl-cAMP and 8-bromo-cAMP, however, had no effect on pHi. Cholera toxin, while decreasing the EC50 for epinephrine-stimulated increases in cAMP, had no effect on epinephrine-induced alkalinization, suggesting receptor coupling to Na-H exchange was not mediated by a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). Additionally, epinephrine stimulated Na-H exchange in cyc- variants of S49 lymphoma cells, which lack a fundamental Gs. In the presence of pertussis toxin, somatostatin attenuation of cAMP was completely reversed; however, somatostatin inhibition of Na-H exchange was not affected. We suggest that beta-adrenergic and somatostatin receptors regulate Na-H exchange independent of changes in cAMP and possibly independent of GTP-binding proteins previously described as being coupled to these receptors.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Canine enteric submucosal cultures: transmitter release from neurotensin-immunoreactive neurons.

A culture system of dispersed submucosal neurons from canine ileum has been developed. The neuronal nature of over 80% of the cells in culture was confirmed by positive staining with a neurofilament antibody. In this culture system, neurotensin-immunoreactive neurons constituted greater than 50% of the total cell population. Neurotensin immunoreactivity in these cells was chromatographically characterized as a single molecular form coeluting with synthetic neurotensin (1-13). We have assessed the release of immunoreactive neurotensin by stimulatory and inhibitory transmitters, and by post-receptor activators of cell function. Forskolin (10 microM), the calcium ionophore A23187 (100 nM), and the active phorbol ester beta-12 myristrate 13-acetate (10 nM), each significantly increased neurotensin release compared with basal peptide secretion. The concomitant application of ionophore and phorbol ester resulted in a marked increase in neurotensin release and this stimulatory response was inhibited over 70% by somatostatin (100 nM). Substance P (0.1-100 nM) caused a dose-dependent increase in neurotensin release. Somatostatin (100 nM) reduced maximal stimulation with 100 nM substance P by 79%. Our results suggest that this submucosal culture system represents an entirely new model for characterizing transmitter release from enteric neurons.

Animals↗
PubMed1987

Characterization of the chicken oocyte receptor for low and very low density lipoproteins.

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.

Animals↗
PubMed1987

Neurotensin-containing neurons in the canine enteric innervation.

The intrinsic innervation of the gastrointestinal tract has been demonstrated to contain numerous peptidergic neurons. Neurotensin, originally isolated from bovine hypothalamus, has been localized in intestinal epithelial endocrine cells but not convincingly in the enteric innervation. The present study demonstrates the presence of neurotensin-immunoreactive neurons and nerve fibers in the canine submucous and myenteric ganglia. The peptide was characterized as neurotensin 1-13 by high pressure liquid chromatography and there was a mean concentration of 18.4 +/- 3.9 pmol (+/- S.E.M., n = 3) per g wet weight of submucosal extract. These neurons were a separate population from the vasoactive intestinal peptide- and somatostatin-immunoreactive cell bodies. These results demonstrate that neurotensin is present in significant amounts in the canine submucous plexus.

Animals↗
PubMed1987

Morphologic and physiologic studies of canine ileal enteroglucagon-containing cells in short-term culture.

Enteroglucagon-containing cells have been maintained in short-term culture, and the morphologic characteristics of these cells and their response to selected agents have been determined. After 48 h in culture the ultrastructural appearance of the enteroglucagon-immunoreactive cells showed evidence of polarization with re-formation of apical microvilli and the secretory granules concentrated at the opposite pole of the cell. The size of the intracellular secretory granules was 370 +/- 15 nm. The release of enteroglucagonlike immunoreactivity was stimulated in a dose-dependent manner by the adrenergic agonists epinephrine and isoproterenol. The response to epinephrine was competitively inhibited by propranolol, producing a rightward shift of the dose-responsive curve. The alpha-adrenergic agonists methoxamine and clonidine did not stimulate enteroglucagon release above basal. The adenyl cyclase activator forskolin also stimulated release of the peptide in a dose-dependent manner. Carbachol and somatostatin produced a dose-dependent inhibition of epinephrine-stimulated release, indicating direct inhibitory modulation of enteroglucagonlike immunoreactive cells. Somatostatin also inhibited forskolin-stimulated release. These data indicate that canine ileal enteroglucagon cells in short-term culture respond to a number of specific stimuli.

Animals↗
PubMed1987

Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor.

Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional coding domain, resembling neuromedin N, occurs immediately after an Arg-Arg basic amino acid pair located in the central region of the precursor. Additional amino acid homologies suggest that tandem duplications have contributed to the structure of the gene. RNA blot analysis, using the cloned cDNA probe, has revealed several mRNA species ranging in size from 500 to 980 nucleotides in the canine enteric mucosa. In contrast, a single RNA species of 1500 nucleotides was detected in bovine hypothalamus poly(A)+ RNA. The ability of the canine probe to cross-hybridize with bovine mRNA suggests that this probe can be used to isolate neurotensin/neuromedin N genes from other mammalian species.

Amino Acid Sequence↗
PubMed1987

Somatostatin and muscarinic inhibition of canine enteric endocrine cells: cellular mechanisms.

Using a recently developed canine primary enteric endocrine cell culture system, we have investigated the role of adenosine 3',5'-cyclic monophosphate (cAMP) in mediating the release of neurotensin and enteroglucagon. Epinephrine-stimulated peptide release was concomitant with an increase in cAMP accumulation. Carbachol and somatostatin (SRIF) markedly inhibited the epinephrine effect on both peptide release and cAMP content. The addition of 3-isobutyl-1-methylxanthine potentiated epinephrine-stimulated peptide release without altering the relative inhibition by carbachol and SRIF, suggesting that these agents did not inhibit endocrine cell function by increasing phosphodiesterase activity. To determine the role of cAMP production in mediating inhibition of peptide release, cells were incubated with the bacterial toxin, pertussis toxin (PT). In cultures pretreated with PT, carbachol inhibition of both peptide release and cAMP accumulation was completely reversed. In contrast, SRIF inhibition of cAMP content was completely reversed after PT treatment, but inhibition of peptide release was only partially reversed. Additionally, toxin treatment only partially reversed SRIF inhibition of forskolin- and calcium ionophore-stimulated peptide release. These data suggest that muscarinic cholinergic inhibition of neurotensin and enteroglucagon release is mediated entirely through the guanine nucleotide-binding protein (Ni) or a similar toxin-sensitive, GTP-binding protein. SRIF-inhibited peptide release is mediated partially through a toxin-sensitive substrate, as evidenced by PT reversal of reduced cAMP levels. SRIF may also inhibit neurotensin and enteroglucagon release by a cAMP-independent pathway that is not coupled to Ni or a similar PT-sensitive, GTP-binding protein.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Studies on the origin of the hydroxyl spin adduct of DMPO produced from the stimulation of neutrophils by phorbol-12-myristate-13-acetate.

The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), is commonly used for the detection of short-lived free radicals produced by neutrophils during their respiratory burst. The spin adducts of both the superoxide ion (O2-) and the hydroxyl radical (.OH) are detectable during this process. Since myeloperoxidase (MPO), which is also active during the respiratory burst, produces hypochlorous acid (HOCl) (HOCl) in the presence of chloride ions (Cl-) and hydrogen peroxide (H2O2), this species has been investigated as a possible source of the DMPO-OH adduct. At concentrations of hypochlorous acid between 0.1 and 0.7 mumol/ml the DMPO-OH spin adduct is detected using electron spin resonance (ESR) techniques. Two possible mechanisms for the formation of this adduct are proposed. These findings suggest that the product of MPO, namely hypochlorous acid, is a possible source of the hydroxyl spin adduct detected during the respiratory burst.

Animals↗