The effect of beta-endorphin on biogenic amines, insulin, and glucagon levels in the hepatic portal circulation of normal and pancreatectomized dogs.

The effect of peak concentrations of beta-endorphin on hepatic portal and peripheral levels of plasma catecholamines, free serotonin, glucose, insulin, and glucagon was studied in trained, conscious, normal adult dogs fitted with an indwelling portal catheter. An injection of synthetic human beta-endorphin (20 micrograms/kg BW) into a cephalic vein produced a significant rise in the portal concentration of dopamine, norepinephrine, and epinephrine. The rise was accompanied by a reduction of portal free serotonin levels. The changes were not seen in the peripheral circulation. No appreciable changes in plasma insulin, glucagon, and glucose concentrations were noticed either in the hepatic portal or in the peripheral circulation. The response of the biogenic amines to beta-endorphin was abolished by pretreatment with Naltrexone (1 mg/kg BW). A dose of somatostatin antiserum given before beta-endorphin did not alter the biogenic amine response to the opioid peptide. When beta-endorphin was administered to pancreatectomized dogs devoid of exogenous and endogenous insulin supply, the biogenic amine response remained virtually the same as in normal intact dogs. It is concluded that in the dog a pulse of beta-endorphin causes profound alterations of splanchnic biogenic amine concentrations that are independent of the ambient levels of insulin, somatostatin, and pancreatic glucagon.

Identification of a GATA motif in the cardiac alpha-myosin heavy-chain-encoding gene and isolation of a human GATA-4 cDNA.

In an attempt to identify the cardiac-specific genes regulated by the transcription factor GATA-4, we have identified a putative GATA-binding site located within the 5' flanking sequence of the human cardiac alpha-myosin heavy-chain-encoding gene. The 23-bp sequence surrounding the core GATA-binding site is conserved across species. The core motif and flanking sequences of this GATA-binding site are almost identical to that of a well-established GATA-binding site located within the 3' enhancer of the human beta-globin gene. Using electrophoretic mobility shift analysis, two distinct nuclear factors were found to bind specifically to this element. We have isolated a full-length cDNA clone for human GATA-4 (hGATA-4) by screening a human heart cDNA library. The hGATA-4 cDNA sequence shows 85% identity with murine GATA-4 in the protein coding region. The deduced amino-acid sequence within the two zinc-finger DNA-binding domains of human GATA-4 is 100% identical with murine GATA-4. Northern blot analysis reveals that this 4.4-kb transcript has higher expression in adult heart than in fetal heart. Our results suggest that GATA-4 may regulate a set of cardiac-specific genes and play a crucial role in cardiogenesis.

Hypophysectomy does not alter the effects of somatostatin or growth hormone on canine splanchnic biogenic amine release.

We have demonstrated previously that growth hormone (GH) and somatostatin (somatotropin release inhibitory factor, SRIF) exert comparable effects on the release of splanchnic biogenic amines. The purpose of the present investigation was to study further the response of the two hormones and see whether the similarity persists in dogs completely deprived of endogenous GH. Experiments were conducted in seven hypophysectomized dogs fitted with an indwelling portal catheter. Two to 4 weeks after surgery the responsiveness of their catecholaminergic neurons was tested by an injection of human beta-endorphin (20 micrograms/kg); it caused a rise in portal catecholamine levels equivalent to that seen in intact dogs. Then the effect of a spike concentration of SRIF or GH on hepatic portal and peripheral levels of free serotonin and catecholamines was studied, all by radioenzymatic methods. The intravenous injection of ovine GH (100 micrograms/kg) or equimolar amounts of SRIF (7.5 micrograms/kg) produced in the hepatic portal circulation a transient but statistically significant rise of serotonin and a concomitant reduction in the concentration of dopamine, norepinephrine, and epinephrine. No changes were found in the peripheral circulation. The response patterns to SRIF or GH were virtually identical, which is in keeping with our other data, suggesting that the effect of GH on splanchnic biogenic amine secretion is SRIF-dependent and mediated by SRIF-containing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of hormones to arterial glycosaminoglycans and atherosclerosis.

Glycosaminoglycan fractions were measured in representative large and medium sized arteries of normal, hypophysectomized and hormone treated young beagles. Hyaluronate, heparan sulphate, dermatan sulphate and the isomeric chondroitin sulphates were determined in the aortic arch, thoracic and abdominal segments, in the external iliac, superior mesenteric, renal, common carotid and coronary arteries. The hormones used for replacement therapy of hypophysectomized animals were growth hormone, thyroxine, cortisone and the sex hormones testosterone, estrogen and progesterone. The sensitivity to an individual hormone was found to differ in various segments of the arterial tree; the thoracic and abdominal aorta were most responsive but renal and superior mesenteric arteries were relatively inert. The hypothesis is advanced that arteries with a GAG metabolism highly sensitive to hormones are more prone to develop atherosclerosis than arteries that have a limited sensitivity to alterations in endocrine balance.

Characterization of Zn(2+)-binding nuclear proteins present in the myocardium.

Nonhistone nuclear proteins were isolated from 3-5 day old neonatal as well as 3 month-old adult myocardium. The nuclear proteins were separated and analyzed by two-dimensional polyacrylamide gel electrophoresis. Using a blot transfer technique equilibrated with 65Zn2+, at least four polypeptides exhibited Zn(2+)-binding activity over the spectrum of nonhistone nuclear proteins. A protein with a molecular weight of 68kDa pI7.8, which has been characterized for its involvement in nucleosome structure, consistently binds Zn2+ in both the neonatal and adult myocardium. This nuclear protein has now been further characterized by partial amino acid microsequencing. It was found that this novel polypeptide is distinct from the pore-complex lamina proteins. Three other polypeptides with M tau 90kDa, pI7.8, M tau 68kDa, pI6.5 and M tau 35kDa, pI7.5 exhibited increased Zn(2+)-binding activity in neonatal myocardium as compared to adult myocardium. Together with results from our previous studies, this study provides the first evidence implicating Zn(++)-binding nuclear proteins in the processes of growth and differentiation of myocardial development.

Arterial glycosaminoglycans in diabetic dogs.

The glycosaminoglycan (GAG) composition of a number of large and medium-sized arteries was studied in 6 alloxan-diabetic beagles and was compared with 6 normal, age-matched controls. Diabetic animals were maintained on diet and insulin for 100 days. The aortic arch, thoracic and abdominal segments, external iliac, superior mesenteric, renal, common carotid and coronary arteries were analyzed for hyaluronic acid (HA) and for heparan (HS), dermatan (DS), and chondroitin (CS) sulphates. All diabetic dogs displayed significant alterations. The HA content was reduced in iliac arteries, and together with HS, also in the thoracic aorta. HS or CS were increased in carotid, iliac and renal arteries, DS, a GAG constitutent with very high affinity for low density lipoproteins, was significantly increased in coronary arteries alone. 2 additional animals which are excluded from this series did not become diabetic after alloxanization and showed no change in arterial GAG content. Early changes in the chemistry of the arterial ground substance seem to provide a clue to the precocious development of atherosclerotic disease in diabetes.

The effect of glucagon on the glycosaminoglycan content of canine arteries.

Hypophysectomized young beagles were treated for 21 days with pharmacological doses of glucagon, administered intramuscularly in two daily injections of 0.5 mg each. Hyaluronic acid, heparan, dermatan and isomeric chondroitin sulphates were determined in aorta, carotid, iliac, renal, mesenteric and coronary arteries. In comparison with age-matched hypophysectomized controls, glucagon-treated dogs showed a reduction in the heparan sulphate concentration in their carotid arteries and thoracic aorta. These results indicate that glucagon has a very limited effect on arterial glycosaminoglycan content.

Effect of hormones on the glycosaminoglycan content of canine renal arteries.

The effect of hypophysectomy and replacement therapy with individual hormones such as somatotropin, cortisone, estradiol, progesterone and testosterone on the glycosaminoglycan (GAG) composition of renal arteries was studied in young beagles. The renals from normal animals showed a uniformly low total GAG content of about two thirds of the value found for aorta and coronary arteries. Neither hypophysectomy nor hormone replacement altered the GAG content, with the exception of estradiol which showed a marked effect by raising the hyaluronic acid content to nearly double the normal value. These results by being at variance with those obtained for aorta and coronary arteries provide further evidence of the differential hormone sensitivity that exists in the various segments of the canine arterial tree with respect to GAG metabolism.

Characterization and nucleotide sequence of the cardiac alpha-myosin heavy chain gene from Syrian hamster.

In small mammals, the cardiac alpha-myosin heavy chain (MyHC) gene is predominantly expressed in the adult ventricle, while the beta-MyHC gene is predominantly expressed in the fetal ventricle. In order to perform comparative studies of these genes, we sequenced the Syrian hamster alpha-MyHC gene by determining a continuous 32,415 base pair (bp) sequence. It contained 39 exons encoding a predicted polypeptide of 1939 amino acid residues (aa) with a molecular weight of 223,644 Da. Sequence comparison revealed that the coding sequence is highly identical to that of the human alpha-MyHC gene. A marked feature of the Syrian hamster alpha-MyHC gene was the abundance of reiterated sequences within introns. Eleven B1 and four B2 retroposons were identified, a variety of di-, tri-, and tetra-nucleotide repeats were present, and three longer direct repeats were found. Some of them appear to be associated with genetic recombination events.

Insulin dependence of the actions of growth hormone and somatostatin on splanchnic biogenic amines of the dog.

It is known from studies previously conducted in this laboratory that an iv injection of ovine growth hormone (GH, 100 micrograms/kg BW) or an equimolar amount of somatostatin (SRIF, 7.5 micrograms/kg BW), given to normal conscious dogs into a saphenous vein, leads to a significant increase in hepatic portal plasma serotonin and a simultaneous decrease in the concentrations of dopamine, norepinephrine and epinephrine. The changes take place within 12 minutes after the injection and are observed only in the portal circulation. The purpose of the present experiment was to investigate whether or not similar results could be obtained in diabetic animals. Mongrel dogs were rendered diabetic by surgical pancreatectomy and fitted with an indwelling hepatic portal catheter. Radioenzymatic methods were employed for quantitative measurements of plasma free serotonin and catecholamines. No response was noted when the same type of experiments as those conducted in normal dogs were now carried out in trained, fully conscious totally pancreatectomized dogs deprived of exogenous insulin supply. When the same animals were given an injection into a peripheral vein of 50 mU/kg BW regular crystalline insulin (a small dose that affected neither plasma glucose nor biogenic amine levels) 10 minutes prior to the administration of the other hormones, the usual response to both GH and SRIF was restored, i.e. the data were comparable to those of normal dogs. It is concluded that the GH/SRIF effect on gut biogenic amines is insulin dependent.

Identification of genes encoding zinc finger motifs in the cardiovascular system.

The Zn2+-finger DNA-binding domain has been identified in several developmental control proteins, transcription factors and gene products associated with diseases, as well as in several RNA-binding proteins. We applied library screening, expressed sequence tagging (EST sequencing), Zn2+-binding assays and Northern blot hybridization, in order to characterize novel cDNA clones of the human cardiovascular system which contain Zn2+-finger motifs. An embryonic (8-10 weeks gestation) heart lambda ZAP Express cDNA library was screened with an oligonucleotide probe deduced from a consensus amino acid sequence which is highly conserved for Zn2+-finger proteins, and approximately 350 positive clones were isolated from 1 x 10(4) plaque-forming units (pfu) initially plated. The isolated clones were classified as known and novel following single pass automated DNA sequencing. Analysis of Northern blot hybridization delineated the tissue specificity of these clones, as well as their association with cardiac growth and development. Existence of Zn2+-finger motifs in the novel clones was confirmed by Zn2+-binding assay. In this report, we present the characterization of eight novel clones, including the complete cDNA sequences of one of these clones (HHZ-123).

Localization of a novel zinc finger gene to human chromosome 7q22 region by fluorescence in situ hybridization.

As part of an endeavour to identify and characterize zinc finger genes of the cardiovascular system, a novel zinc finger gene, HHZ105, was isolated from a human fetal heart cDNA library. This gene was mapped by fluorescence in situ hybridization to human chromosome 7q22, a region associated with a number of genetic disorders and developmental deficiencies.

Localization of three novel zinc finger genes to the centromeric region of human chromosome 10 by fluorescence in situ hybridization.

An oligonucleotide probe for the consensus sequence of the linker region of zinc finger proteins was used to isolate cDNA clones from a human fetal heart cDNA library. Following DNA sequencing analysis and comparison, genes for the novel clones were mapped by fluorescence in situ hybridization. We report the chromosomal localization of three zinc finger-coding genes to the region of centromere on human chromosome 10p11.1q-11.2, indicating involvement in gene duplication and chromosome rearrangement during primate evolution.

Localization of a novel zinc finger gene to the human chromosome 7p11.2-p12 by fluorescence in situ hybridization.

A novel zinc finger gene (ZNF182) was isolated from a human fetal cardiac cDNA library, using a consensus C2H2 zinc finger oligonucleotide probe. This gene was assigned to human chromosome 7p11.1-p12 by fluorescence in situ hybridization (FISH). Additional FISH signals were identified on both the long and the short arms of chromosome 19, suggesting the presence of homologous genes at these loci.

Identification of a locus of zinc finger genes in human chromosome 19q13.1-q13.3 region by fluorescence in situ hybridization.

A group of zinc-binding cDNA clones from a human fetal heart library was isolated using an oligonucleotide probe to the consensus sequence of the linker region of zinc finger proteins. Genes for novel clones were mapped by fluorescence in situ hybridization. In the process, we identified a previously unrecognized locus for two zinc finger-coding genes in human chromosome 19q13.1-q13.3 (ZNF180,ZNF181), where genomic rearrangements were shown to be accompanied by various developmental abnormalities, DNA repair deficiencies, and cellular malignancies.

Somatostatin mediates the effect of growth hormone surges on splanchnic biogenic amines.

Experiments were conducted in trained, conscious dogs fitted with an indwelling portal catheter. Radioenzymatic methods were employed for the quantitative measurement of plasma-free serotonin and catecholamines. An injection of ovine growth hormone (GH, 100 micrograms/kg) or an equimolar amount of somatostatin (somatotropin release inhibitory factor, SRIF, 7.5 micrograms/kg) into a saphenous vein led, within the first 15 min, to a transient but significant increase in plasma serotonin and a decrease in the concentrations of dopamine, norepinephrine, and epinephrine. The changes were frequently in excess of 40% of baseline values, and were found only in the portal and not in the peripheral circulation. When the animals were pretreated with an antiserum specifically directed against SRIF, GH surges no longer caused alterations in the portal levels of biogenic amines. Thus, the effects of spike concentrations of GH on plasma serotonin and catecholamines are apparently mediated by SRIF, a novel and unexpected function for a hormone that is known as an inhibitor of GH secretion.

Growth hormone stimulates serotonin secretion into the hepatic portal circulation.

Hepatic portal plasma concentrations of free serotonin were found to be transiently but significantly elevated in normal dogs following a single injection of ovine growth hormone into a peripheral vein. The data are consistent with the concept that spike concentrations of growth hormone are capable of altering the splanchnic concentration of agents that are concerned with glucose homeostasis.

The effect of growth hormone on biogenic amines in the hepatic portal circulation of the dog.

The effects of a spike concentration of growth hormone (GH) on hepatic portal and peripheral levels of free serotonin and catecholamines were studied by improved radioenzymatic methods in trained, conscious, normal, adult dogs fitted with an indwelling portal catheter. An injection of ovine GH (6 or 100 micrograms/kg) into a cephalic vein produced in the hepatic portal circulation a transient, statistically significant rise of serotonin and a concomitant significant reduction in the concentration of dopamine, norepinephrine, and epinephrine. No change was found in the peripheral circulation, partly because the amines were conjugated to sulfates and glucuronides and these derivatives are not detectable by our assays. Thus, a pulse of GH not only stimulates the release of pancreatic hormones and glucose turnover, but also affects the portal profile of glucoregulatory bioamines. The present investigation lends further support to our view that the splanchnic area represents an endocrine system whose preferential target is the liver.

The effect of tryptophan on biogenic amines in the hepatic portal circulation of the dog.

Mongrel dogs were fitted with indwelling hepatic portal catheters. After recovery from surgery, experiments were conducted in fasting, unrestrained, fully conscious, normal dogs which were accustomed to handling and withdrawal of blood samples. L-Tryptophan, a specific serotonin precursor, was injected into a saphenous vein, 10 microM/kg body weight, dissolved in saline. Plasma serotonin, dopamine, norepinephrine, and epinephrine were determined by radioenzymatic assays in blood samples withdrawn at frequent intervals for 2 h, simultaneously from the indwelling catheter and from a catheter temporarily inserted into a saphenous vein other than the one used for the injection of tryptophan. The injection of the amino acid caused a significant elevation of the concentration of platelet-free serotonin within 60 min and this was accompanied by a reduction in the concentration of the catecholamines, dopamine, norepinephrine, and epinephrine. The changes occurred only in the portal circulation and were not detected in peripheral blood samples. The results of these experiments indicate the existence of a cause and effect related interdependence between the splanchnic serotonergic and adrenergic systems in that the tryptophan-stimulated increase in serotonergic activity resulted in a concomitant reduction in gut adrenergic activity.

Secretory pattern of canine growth hormone.

Our aim was to define the secretory pattern of growth hormone (GH) under basal conditions in fasted, conscious, male dogs accustomed to handling. Blood samples were withdrawn from a cephalic vein at 15-min intervals. In this way, any ultradian rhythms, if present, could be detected within the frequency range of 0.042-2 cycles/h. In addition, samples were drawn at either 1- or 2.5-min intervals for 2.5 or 5 h to determine whether frequency components greater than 2 cycles/h were present. GH was measured by radioimmunoassay and the raw data were submitted to time series analysis employing power spectral estimation by means of fast Fourier transformation techniques. Peak plasma levels were up to 12 times higher than the baseline concentration of approximately 1 ng/ml. Spectral analysis revealed an endogenous frequency of 0.22 cycles/h, i.e., a periodicity of 4.5 h/cycle. The results indicate that under basal conditions the secretory bursts of canine GH are limited to one peak every 4.5 h.