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M D Hollenberg

Publications and source records attributed to M D Hollenberg.

At least 127 records · Page 7Linked to original sources
PubMed1987

Mechanisms of receptor-mediated transmembrane signalling.

Although this paper has dealt with general mechanisms whereby a hormonal signal is transmitted across the cell membrane, advances in work with a number of receptors should permit a precision of description of these mechanisms that would have delighted both Langley and Ehrlich. For instance, the detailed sequences now known for the separate subunits of the nicotinic cholinergic receptor and the cellular manipulations made possible by the cloning of the separate subunit genes will make it possible to determine the precise receptor sequence involved either in acetylcholine binding or in ion channel function. The complete sequences and biochemical properties now known for the insulin and EGF-URO receptors to be dealt with in part by a subsequent article (van Obberghen and Gammeltoft, this series) should lay the groundwork for elucidating the transmembrane signalling mechanisms used by the kinase family of growth factor receptors. Continuing work on the structure of the beta-adrenergic receptor, and on the interaction of such receptors with guanine nucleotide regulatory complexes and on the detailed properties of the family of so-called G-proteins and their associated regulatory subunits should unravel the details for a variety of transmembrane signalling reactions. Thus, at least for three basic transmembrane signalling mechanisms: ligand modulated ion transport; ligand-modulated receptor enzyme activity (e.g. tyrosine kinase); and ligand-modulated liberation of cryptic mediators (like the alpha- and beta-subunits of the guanine nucleotide regulatory complexes) one can look forward with excitement to the elucidation in the not-too-distant future of a number of specific biochemical reaction pathways that lead to cell activation.

Animals↗
PubMed1987

Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin.

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.

Amino Acid Sequence↗
PubMed1987

Effects of oxytocin and vasopressin on the preadipocyte 3T3-F442A cell line.

The 3T3-F442A mouse fibroblast cell line, triggered by factors present in fetal calf serum (FCS), converts either spontaneously or, in the simultaneous presence of FCS and insulin, at an accelerated rate into cells exhibiting the adipocyte phenotype. The effects of the neurohypophysial hormones in differentiated cells on glucose metabolism (glucose oxidation and lipogenesis) were compared with the stimulatory actions of insulin, which had its most pronounced effects in cells differentiated spontaneously with FCS in the absence of insulin. The differentiated 3T3-F442A cells were sensitive to physiological levels of insulin and exhibited manyfold increases in glucose metabolism in response to it. This result demonstrated that these cultured cells respond to insulin, in a manner analogous to freshly isolated adipocytes. In contrast to its insulin-like effects in isolated epididymal adipocytes, oxytocin was not reproducibly able to stimulate glucose metabolism in differentiated 3T3-F442A cells. Vasopressin was similarly inactive. In contrast, both oxytocin and vasopressin blocked adipocyte conversion triggered by FCS, either in the presence or absence of insulin; vasopressin was more potent than oxytocin, indicating that a vasopressin receptor was responsible for the observed inhibition of differentiation. Our work suggests that vasopressin could potentially play a role in the regulation of the adipocyte differentiation process.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Basal, oxytocin-, and insulin-stimulated glucose oxidation in human endometrium.

Samples of endometrium from regularly cycling women (28 +/- 2 days cycle) were assayed for [U-14C]glucose oxidation activity in the presence or absence of 100 nM oxytocin or 1.7 nM insulin. The basal rate of glucose oxidation in the tissues obtained from women in early and midfollicular phase and late luteal phase was approximately 125 pmol/(h X mg tissue). Late follicular and midluteal phases had higher basal rates, up to 400 pmol/(h X mg tissue). Oxytocin increased glucose oxidation by 50-100 pmol X h-1 X mg-1 in early and midfollicular phase and in early luteal phase endometrial fragments. Insulin did not stimulate glucose oxidation in these tissues. In samples of late luteal phase, glucose oxidation was stimulated by both oxytocin and insulin. High and low basal glucose oxidation activity in the endometrium corresponded, respectively, to reported periods of high and low plasma estradiol in normal menstruating women. In contrast, oxytocin stimulated glucose oxidation in endometria from women with anticipated low plasma estradiol.

Endometrium↗
PubMed1987

Epidermal growth factor-urogastrone causes vasodilatation in the anesthetized dog.

Epidermal growth factor-urogastrone (EGF-URO) administered intraarterially was a potent dilator in dog femoral (FEM), superior (cephalic) mesenteric (SMA), celiac (CAC), coronary (COR), carotid (CAR), and renal (REN) vascular beds. The effects of EGF-URO, which exhibited tachyphylaxis, could not be attributed either to recirculating EGF-URO or to the secondary release of other agonists or products of the cyclooxygenase pathway. Two vascular beds (FEM, SMA) showed a high maximum responsiveness to EGF-URO (maximum effect [Emax] approximately equal to 70% increase in flow) whereas another group (CAC, COR, CAR, and REN) exhibited lower responsiveness (Emax approximately equal to 20%). The ED50 for this effect of EGF-URO was in the range of 0.4 micrograms (FEM, SMA, CAR, and COR) to 0.9 micrograms (CAC and REN). In isolated dog COR helical strips, EGF-URO did not exhibit either a direct relaxing or a contractile effect. However, preincubation of strips with EGF-URO caused up to a 66% inhibition of contraction in response to norepinephrine (1 microM), with an ED50 for EGF-URO of 1 nM. This action of EGF-URO also showed marked tachyphylaxis. Our data point to a potential role for EGF-URO (and possibly for the structurally related alpha-transforming growth factor) in the regulation of blood flow in vivo.

Animals↗
PubMed1987

Receptors for insulin and other growth factors: rationale for common and distinct mechanisms of cell activation.

The insulin-like biological activities of serum can now be attributed not only to insulin but also to other structurally related polypeptides (somatomedins or insulin-like growth factors, IGF's). Other polypeptides, like epidermal growth factor-urogastrone (EGF-URO) or platelet-derived growth factor (PDGF) have also been observed to cause "insulin-like" responses in their target cells. The similar biological activities of the polypeptides that are structurally related to insulin (IGF-I, IGF-II) can result either from an activation of their own distinctive receptors (the insulin or IGF-I receptors) or from receptor crossover whereby one polypeptide (e.g., IGF-I) can activate a second receptor system (e.g., the one for insulin). The similarity of cellular responses triggered by the insulin and IGF-I receptors can now be understood in terms of the remarkable similarities between these receptors with regard to their structural, immunologic, and enzymatic (protein tyrosine kinase) properties. Furthermore, the common insulin-like biological activities triggered by other growth factor receptors, like the ones for EGF-URO and PDGF, can now be rationalized in terms of the common protein tyrosine kinase activities and homologous amino acid sequences that these receptors exhibit, along with the receptors for insulin and IGF-I. Although some of the activities of insulin and these growth factors are similar, other actions of these agents are distinct.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibroblasts↗
PubMed1987

Distinct vascular actions of epidermal growth factor-urogastrone and transforming growth factor-alpha.

We compared the effects of epidermal growth factor-urogastrone (EGF-URO) with those of transforming growth factor-alpha (TGF-alpha) on regional blood flow in the anesthetized dog in vivo, and on isolated canine helical coronary artery strips in vitro. Like EGF-URO, TGF-alpha was a potent stimulator of femoral arterial blood flow in vivo; and, when added to the tissue bath in vitro before an agonist, TGF-alpha like EGF-URO was a potent inhibitor of the contractile responses of helical coronary arterial strips to various smooth muscle agonists: norepinephrine (NE), prostaglandin F2 alpha (PGF alpha) and potassium chloride (KCl). Nonetheless, there were marked differences between EGF-URO and TGF-alpha in terms of the two biological responses measured. In terms of blood flow, although the ED50 for the increase in blood flow was similar for EGF-URO and TGF-alpha (ED50 = 0.4 micrograms EGF-URO equivalents per dose), the maximum responsiveness to TGF-alpha (130% increase in flow) was substantially greater than the maximum response to EGF-URO (70% increase in flow). Furthermore, TGF-alpha did not cause tachyphylaxis to subsequent doses of TGF-alpha, whereas a prior dose of either EGF-URO or TGF-alpha desensitized markedly the blood flow preparations to EGF-URO. Different desensitization patterns for the actions of EGF-URO and TGF-alpha also were observed in the arterial strip preparations. The inhibitory action of EGF-URO for agonist (NE, PGF2 alpha or KCl)-mediated contraction was diminished with repeated exposure of the tissue to EGF-URO, whereas the inhibitory action of TGF-alpha persisted with repetitive tissue exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1986

Adenosine modulation of fat cell responsiveness to insulin and oxytocin.

We have investigated the effects of adenosine on the stimulation of glucose oxidation and lipogenesis by oxytocin and insulin in rat epididymal adipocytes. The addition of adenosine deaminase (1 U/ml) to the assay medium reduced the maximal oxytocin response (glucose oxidation and lipogenesis) to between 25 and 50% of the maximum response in control cells. The maximal response to insulin was not appreciably affected under these conditions. The addition of adenosine (10 or 30 microM) increased the cell sensitivity to oxytocin by elevating the maximum rate of oxytocin-stimulated glucose metabolism. Adenosine also increased the cell sensitivity to insulin by decreasing its ED50. A change in ED50, however, was observed only when control or adenosine-treated cells were compared to adenosine deaminase-treated cells; but not when control and adenosine-treated cells were compared. On its own, adenosine also caused an appreciable increase in both glucose oxidation and lipogenesis (ED50 approximately equal to 3 microM adenosine). The difference in the effect of adenosine on oxytocin action, compared with the effect on insulin action, points to differences in the mechanisms by which insulin and oxytocin stimulate glucose metabolism in adipocytes.

Adenosine↗
PubMed1986

Mechanisms of receptor-mediated transmembrane signalling.

Although this paper has dealt with general mechanisms whereby a hormonal signal is transmitted across the cell membrane, advances in work with a number of receptors should permit a precision of description of these mechanisms that would have delighted both Langley and Ehrlich. For instance, the detailed sequences now known for the separate subunits of the nicotinic cholinergic receptor and the cellular manipulations made possible by the cloning of the separate subunit genes will make it possible to determine the precise receptor sequence involved either in acetylcholine binding or in ion channel function. The complete sequences and biochemical properties now known for the insulin and EGF-URO receptors to be dealt with in part by a subsequent article (van Obberghen and Gammeltoft, this series) should lay the groundwork for elucidating the transmembrane signalling mechanisms used by the kinase family of growth factor receptors. Continuing work on the structure of the beta-adrenergic receptor, and on the interaction of such receptors with guanine nucleotide regulatory complexes and on the detailed properties of the family of so-called G-proteins and their associated regulatory subunits should unravel the details for a variety of transmembrane signalling reactions. Thus, at least for three basic transmembrane signalling mechanisms: ligand modulated ion transport; ligand-modulated receptor enzyme activity (e.g. tyrosine kinase); and ligand-modulated liberation of cryptic mediators (like the alpha- and beta-subunits of the guanine nucleotide regulatory complexes) one can look forward with excitement to the elucidation in the not-too-distant future of a number of specific biochemical reaction pathways that lead to cell activation.

Animals↗
PubMed1986

Epidermal growth factor (urogastrone)-mediated phosphorylation of a 35-kDa substrate in human placental membranes: relationship to the beta subunit of the guanine nucleotide regulatory complex.

We have identified a component of about 35 kDa (pp35), present in human placental membrane preparations, that is a substrate for epidermal growth factor (urogastrone) [EGF(Uro)]-mediated phosphorylation. The EGF(Uro)-stimulated phosphorylation of pp35 was calcium-dependent and was markedly enhanced in membranes prepared in the presence (but not in the absence) of calcium. The phosphate incorporated into pp35 in the presence of EGF(Uro) was alkali-stable and was present as O4-phosphotyrosine. Under identical conditions, insulin did not stimulate pp35 phosphorylation. Either in its native or in its phosphorylated form, pp35 could be released from the membranes in the presence of calcium-chelating agents (EDTA/EGTA); and EGF(Uro)-stimulated phosphorylation was reconstituted by adding back EDTA/EGTA eluates to EDTA/EGTA-washed membranes in the presence of calcium. The properties of pp35 were similar if not identical to those of beta-35, a 35-kDa polypeptide similar to the beta subunit of the guanine nucleotide-binding oligomers that stimulate (Gs) or inhibit (Gi) the adenylate cyclase system. As with pp35, EGF(Uro)-stimulated phosphorylation of isolated rabbit liver beta-35 was observed in a reconstituted system using either EDTA/EGTA-washed placental membranes or solubilized EGF(Uro) receptor immobilized on concanavalin A-agarose. In contrast, the addition of beta subunits derived from rabbit liver Gi or bovine transducin did not result in phosphorylation of a 35-kDa substrate in the reconstituted system. Further, a 35-kDa protein released from placental membranes crossreacted with an anti-transducin antibody that can recognize the beta subunit isolated from a variety of sources. We conclude that the human placental pp35 substrate likely represents the placental equivalent of the beta-35 protein. Our data point to a possible link between those receptors involved in growth-factor action and the regulatory systems that utilize GTP-binding proteins as transducing elements.

Calcium↗
PubMed1986

Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium.

We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 x 10(5) +/- 0.3 x 10(5)) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Modulation by epidermal growth factor--urogastrone of contraction in isolated canine helical mesenteric arterial strips.

We have examined the effect of epidermal growth factor--urogastrone (EGF-URO) on the response of isolated canine helical mesenteric arterial strips contracted by norepinephrine (NE), KCl, and transmural electrical stimulation (TES). Although EGF-URO alone did not affect resting arterial tone, contraction caused by all three modes of stimulation (NE, KCl, and TES) was inhibited up to 50% in the presence of EGF-URO. The action of EGF-URO did not depend on the presence of intact endothelial cells. The most pronounced effect of EGF-URO was observed on KCl-mediated contraction. The inhibitory effect of EGF-URO was maximal at about 15 min after addition of the polypeptide to the organ bath and persisted (e.g., electrical stimulation) for up to 1 h. A half-maximal inhibitory effect of EGF-URO was observed at a concentration of about 1 nM. Washing the tissue free of EGF-URO reversed its inhibitory action. Although in the presence of indomethacin (3 microM) EGF-URO caused a small, variable elevation in resting tension, the presence of indomethacin did not affect the ability of EGF--URO to inhibit contraction mediated by KCl. Under conditions wherein contraction in response to maximally effective concentrations of either NE or KCl was made dependent on the addition of calcium, EGF-URO was able to inhibit the response in the presence of KCl but not in the presence of NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone.

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.

Brain↗
PubMed1985

Receptors for insulin and epidermal growth factor: interaction with organomercurial agarose.

The receptor for both insulin and epidermal growth factor (EGF) from human placental membranes, after crosslink labeling with 125I-labeled insulin and EGF, can be absorbed to an organomercurial-agarose derivative (Affi-Gel 501) and can be recovered from the gel by elution with dithiothreitol (DTT). Pretreatment of crosslink-labeled membranes with N-ethylmaleimide (NEM) blocks the ability of the receptor to react with the organomercurial column. NEM also abolishes the protein kinase activity of both receptors. Under appropriate conditions, insulin can promote the reaction of the insulin receptor with the organomercurial-agarose derivative. For both the insulin and EGF receptors, our results provide an avenue for the isolation of the sulfhydryl-containing receptor domains that may play a role in the control of receptor function.

Chromatography, Affinity↗
PubMed1985

Oxytocin and glucose oxidation in the rat uterus.

The effects of oxytocin on the biochemical pathways of glucose oxidation were investigated in the rat uterus. In the presence of oxytocin, glucose oxidation in uterine segments obtained from Sprague-Dawley rats at diestrus increased 1.5-2.0-fold above the basal rate. A half-maximal response was observed at about 3 nM oxytocin; the maximum response was equal to or greater than the response to 1.7 nM insulin. In stripped myometrial segments (denuded of the endometrial component), oxytocin stimulated glucose oxidation at estrus only; whereas in intact uterine segments, the stimulation of oxidation was observed at both estrus and diestrus. In contrast, stimulation of oxidation by carbachol in stripped myometrial segments was independent of the estrous state of the tissue. The ratio of [1-14C]glucose to [6-14C]glucose oxidation was measured to estimate the relative involvement of the pentose phosphate and the tricarboxylic acid pathways of metabolism. In myometrial tissue, stimulation of glucose oxidation by oxytocin appeared to proceed through the tricarboxylic acid cycle. In intact uterine segments, at diestrus, glucose oxidation involved largely the pentose phosphate pathway (suggesting increased glucose metabolism in endometrial tissue), whereas at estrus, in the intact tissue segments, oxytocin increased glucose oxidation largely via the tricarboxylic acid cycle, and appeared to do so predominantly in the myometrial tissue. Carbachol-stimulated glucose oxidation appeared to proceed mainly via the tricarboxylic cycle in the myometrial tissue, irrespective of the stage of the estrous cycle. In the uterus of the Brattleboro rat (either intact uterine segments or stripped myometrial strips), oxytocin stimulated glucose oxidation only at estrus, predominantly through the tricarboxylic acid cycle. These findings suggest that oxytocin, in addition to its known effect on the contractility of uterine and myoepithelial smooth muscle, may regulate glucose metabolism in both the myometrial and endometrial components of uterine tissue.

Animals↗
PubMed1985

Effect of epidermal growth factor/urogastrone on glycosaminoglycan synthesis and accumulation in vitro in the developing mouse palate.

Epidermal growth factor/urogastrone (EGF-URO) has previously been implicated in murine secondary-palate formation. We report here that, in correlation with its effects on palate fusion, EGF-URO in physiological amounts (1.7 nmol/l) markedly affects glycosaminoglycan (GAG) production in organ cultures of mouse palate tissue; the effects of EGF-URO are dependent on the developmental stage of the palate. GAG production, particularly that of hyaluronic acid (HA), is stimulated two- to eight-fold by EGF-URO in cultures of palate tissue obtained between days 11-12 and 13-15 of development; by the time of birth, EGF-URO no longer stimulates GAG production in such cultures. EGF-URO increases the amount and alters the distribution of HA within the palate. The results suggest a role for EGF-URO and for HA in the process of normal palatal development.

Animals↗