Hormonal regulation of expression of the angiotensinogen gene in cultured mouse hepatoma cells.

To investigate the hormonal regulation of expression of the angiotensinogen (ANG) gene in the liver, we constructed fusion genes with various lengths of the 5'-flanking region of the rat ANG gene linked to a bacterial chloramphenicol acetyl transferase (CAT) gene as reporter and introduced them into mouse hepatoma cells (Hepa 1-6). As a negative control, we introduced them into a nonhepatic cell line, a mouse testicular Sertoli (TM4) cell line. The level of expression of ANG-CAT fusion genes, pOCAT (ANG N-1498/+18), pOCAT (ANG N-688/+18), pOCAT (ANG N-110/+18), pOCAT (ANG N-53/+18) and (ANG-35/+18) were 3.7, 4, 1.1, 4, and 3-fold higher than promoterless pOCAT in Hepa 1-6 cells. No significant expression of any of these ANG-CAT fusion genes over the promoterless pOCAT was observed in Sertoli TM4 cells. The addition of dexamethasone (10(-10) to 10(-4) mol/L) stimulated the expression of the pOCAT (ANG N-1498/+18) fusion gene in Hepa 1-6 cells in a dose-dependent manner with a maximum stimulation at 10(-4) mol/L and a half-maximal stimulation at 10(-8) mol/L. A combination of dexamethasone (10(-6) mol/L) and 8-bromo-cyclic AMP (cAMP) (10(-3) mol/L) further enhanced the effect of the dexamethasone alone although cAMP alone had no effect. Testosterone (10(-6) mol/L), estradiol (10(-6) mol/L), progesterone (10(-6) mol/L), and thyroid hormone (L-T3, 10(-6) mol/L) did not have this effect in either the presence or absence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of expression of the angiotensinogen gene in cultured opossum kidney proximal tubular cells.

Angiotensinogen (ANG) messenger RNA is expressed in cultured opossum kidney (OK) proximal tubular cells. The aim of these studies was to investigate whether steroid hormones (dexamethasone, estradiol, testosterone, and progesterone) could stimulate the expression of renal ANG gene in vitro. Fusion genes consisting of various lengths of the 5'-flanking region of the rat ANG gene linked to a chloramphenicol acetyl transferase (CAT) reporter gene were constructed and introduced into cultured OK cells. The level of expression of fusion genes was determined by the level of cellular CAT enzymatic activity. The addition of dexamethasone (10(-12) to 10(-6) M) stimulates the expression of the pOCAT (ANG N-1498/+18) fusion gene in OK cells in a dose-dependent manner with a maximum stimulation at 10(-6) M and a half-maximal stimulation at 10(-9) M. Combination of dexamethasone (10(-6) M) and thyroid hormone, L-T3 (10(-6) M), further enhanced the effect of the dexamethasone alone. Testosterone (10(-6) M), estradiol (10(-6) M), and progesterone (10(-6) M) did not have this effect. Moreover, dexamethasone also stimulates the expression of the pOCAT (ANG N-688/+18) but not pOCAT (ANG N-110/+18), pOCAT (ANG N-53/+18) and pOCAT (ANG N-35/+18). These studies demonstrate that the glucocorticoid hormone is effective at stimulating the transcription of the ANG gene in OK cells, but stimulation is not observed from testosterone, estradiol, or progesterone. Moreover, glucocorticoid and L-T3 act synergistically to stimulate the transcription of the ANG gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone, L-T3, stimulates the expression of the angiotensinogen gene in cultured opossum kidney (OK) cells.

Angiotensinogen (ANG) messenger RNA is expressed in opossum kidney (OK) proximal tubular cells. To examine whether thyroid hormone, L-T3, could stimulate the expression of the ANG gene in OK proximal tubular cells, fusion genes, consisting of various lengths of the 5'-flanking region of the rat angiotensinogen gene linked to a human growth hormone reporter gene, were constructed and introduced into OK cells. As a negative control, they were introduced into a nonkidney cell line, a human choriocarcinoma cell line (JEG-3). The level of the expression of fusion genes in these cells were determined by the level of immunoreactive human growth hormone secreted into the culture medium. The expression of ANG-growth hormone (ANG-GH) fusion genes pOGH (ANG N-1498/+18), pOGH (ANG N-688/+18), pOGH (ANG N-110/+18), pOGH (ANG N-53/+18), and pOGH (ANG N-35/+18) was 226-, 4.5-, 1.0-, 12-, and 2.5-fold higher than promoterless pOGH in the expression of growth hormone activity in OK cells. No significant expression of any of these ANG-GH fusion genes over the promoterless pOGH was observed in JEG-3 cells. The addition of L-T3 stimulates the expression of pOGH (ANG N-1498/+18) in a dose-dependent manner with a maximal and half-maximal effect at 10(-7) M and at 10(-8) to 10(-9) M, respectively. Thyroid hormone (10(-7) M) also stimulates the expression of pOGH (ANG N-688/+18) but not pOGH (ANG N-110/+18), pOGH (ANG N-53/+18), or pOGH (ANG N-35/+18).(ABSTRACT TRUNCATED AT 250 WORDS)

Functional diversity of bioactive peptides in the nervous system itself: "how the brain may understand".

The interactions involving cells of the nervous system are a complex form of intercellular communication. Biosynthesis of peptide hormones or active neuropeptides is generally through a precursor which provides increased product choices as a function of the processing pathway. Proteolytic processing as well as other molecular modifications lead to a wide range of mature products which may vary in different tissues even though they are derived from the same precursor. Also the same neuropeptide may exhibit different bioactivities for different target cells. Finally, by means of collective packaging in secretory organelles, a cell may be able by synergism to further broaden its biologic effects. In these ways, what is seen as added complication in the CNS, may be from the point of view of the cell, a successful attempt to increase its survival ability to adapt and influence its bioenvironment.

Immunoreactivity of vasopressin and a novel pituitary protein '7B2' in Long-Evans and Brattleboro rat hypothalamus and hypophysis.

The unlabeled antibody (peroxidase-anti-peroxidase) method was used to simultaneously localize vasopressin and a novel pituitary protein designated '7B2' in rat hypothalamus and pituitary. Results showed the common localization of both substances within magnocellular neurons of supraoptic, supraoptic retrochiasmic and paraventricular nuclei. The distribution was also similar in the inner zone of the median eminence and in the posterior lobe of the pituitary gland. Only 7B2 antiserum labeled the external zone of the median eminence and the intermediate and anterior lobes of the pituitary. In the Brattleboro rat the anterior and intermediate lobes were strongly labeled with 7B2-IR and there was some 7B2-staining in the hypothalamus and the posterior lobe, but the intensity of the reaction was diminished.

Subcellular fractionation of pituitary neurointermediate lobes: revelation of various basic proteases.

Homogenates of rat neurointermediate lobes were purified by centrifugation on a Percoll gradient. Lysates of the Percoll gradient fractions were incubated with a synthetic octapeptide and pentapeptide substrate (N-acetyl Lys-Arg-Tyr-Asn-Leu-Thr-Ser-Val-amide and N-acetyl Lys-Arg-Tyr-Asn-Leu-amide), and enzymatic characteristics were compiled. Early assays on nonamidated forms revealed carboxypeptidase activity, whereas with the amide derivatives no carboxypeptidase activity could be detected. These amide substrates were therefore used in all subsequent incubations. High levels of a Leu/Thr and Lys/Arg cleavage were present in fractions almost throughout the Percoll gradient. Cleavages at Tyr/Asn, Thr/Ser, and Arg/Tyr were localized at different regions of the Percoll gradient. Surprisingly, none of the five enzymatic activities appear to be localized in the secretory granule fractions as defined by the presence of immunoreactive beta-endorphin in the gradient. All of the five proteolytic activities have a basic pH optimum (pH 8-9), and four of them seem to be thiol proteases, as categorized by inhibitor studies. The fifth one, namely the Try/Asn cleavage, is more likely to be due to a metalloendopeptidase, since it is activated by Zn2+ and Co2+.

Galactose transfer to endogenous acceptors within Golgi fractions of rat liver.

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.

Membrane fusion and glycosylation in the rat hepatic Golgi apparatus.

When purified Golgi fractions were incubated with UDP-[3H]galactose in the absence of Triton-X-100, radioactivity was incorporated into an endogenous lipid and several peptide acceptors. Electron microscope analysis of Golgi fractions incubated in the endogenous galactosyl transferase assay medium revealed extensive fusion of Golgi saccules. Systematic removal of constituents in the galactosyl transferase assay medium showed enhanced (minus beta-mercaptoethanol) or reduced (minus ATP, minus sodium cacodylate buffer or minus MnCl2) fusion of Golgi membranes compared to the complete medium, Stereologic analysis revealed a correlation between membrane fusion and galactosyl transferase activity (r = 0.99, P less than 0.001). Electron microscope radioautography was carried out after incubation of Golgi fractions with UDP-[3H]galactose. Silver grains were not observed over trans elements of Golgi but were revealed mainly over large fused saccules with the number of silver grains being proportionate to membrane fusion (r = 0.92, P less than 0.001). Bilayer destabilization at points of Golgi membrane fusion may act to translocate galactose across the Golgi membrane and thereby provide a fusion regulated substrate for terminal glycosylation.

Radioautographic visualization of in vivo insulin binding to the exocrine pancreas.

Two minutes after the iv injection of [125I]insulin, hormone was linked to specific binding sites distributed randomly along the basal and lateral membranes of plasmalemma of pancreatic exocrine cells. The interaction had all of the properties of bona fide binding to a true insulin receptor, and under the described experimental conditions, estimates of receptor densities indicated the exocrine cell plasmalemma to be one of the richest sources of insulin receptors in the male rat.

Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography.

A tissue-screening survey employing quantitative radioautography was carried out at 2 min after the intravascular injection of 125I-insulin into laboratory rats. The results revealed a substantial binding of insulin to cells forming the proximal convoluted tubule in kidney, hepatocytes of liver, acinar cells of the pancreas, parenchymal cells of the adrenal cortex and medulla, and epithelial cells of the gastrointestinal tract. Control experiments indicated that this binding was due to a specific interaction with the insulin receptor, except in the case of kidney where the binding was shown to be nonspecific. Although the major target for insulin action (liver) clearly demonstrated specific insulin binding, several other classical targets (adipocytes, skeletal, cardiac, and smooth muscle cells) showed no specific 125I-insulin binding and therefore indicated the limits of sensitivity of the in vivo radioautographic method. Nevertheless, the working hypothesis of a direct correlation of insulin receptor density with insulin action points to the hitherto unemphasized targets of pancreas, adrenal gland, and gastrointestinal tract as major sites of insulin action in the body.

A morphologic analysis of insulin binding sites in isolated rat liver nuclei.

Under conditions which demonstrated a high degree of specific 125I-insulin binding to purified plasmalemma, no specific binding could be found in freshly prepared nuclei. Analysis of light and electron microscope radioautographs of nuclei incubated with 125I-insulin with and without additional excess unlabeled insulin also failed to provide any evidence for 125I-insulin binding to rat liver nuclei in vitro. It is concluded that current methods are too insensitive to detect insulin receptors in nuclei (if indeed such receptors exist in this organelle to any appreciable extent.

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study.

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.

Polypeptide hormone binding sites in vivo: initial localization of 125I-labeled insulin to hepatocyte plasmalemma as visualized by electron microscope radioautography.

The rationale of the specific-binding assay was applied to the detection of the liver insulin receptor in vivo. Quantitative electron microscope radioautography indicated that, 3 min after an intraportal injection, 125I-insulin was exclusively located to the hepatocyte plasmalemma.

An enzymic analysis of a nuclear envelope fraction.

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104-125 (1972) is characterized by high levels of glucose-6-phosphatase and 5'-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes b5 and P-450 as well as NADPH- and NADH-cytochrome c reductase activities are present but at lower levels than found in microsomes. Cytochrome c oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5'-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside triphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.