Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.     

Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Summary Bovine chromaffin cells maintained in culture for eight days were loaded with [³H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K⁺. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus.The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K⁺ produced 91±2% (n = 5) depletion in the [³H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells. Under the same conditions of stimulation, the distribution of the clathrin fluorescence was unaltered suggesting either that K⁺induced stimulation of the chromaffin cells does not change the cellular distribution of clathrin, or that the changes in the distribution of clathrin are of such low magnitude that they escape detection by fluorescence microscopy.     

The molecular shape of dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla

The structural features of the soluble dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla were studied using negative staining and platinum shadowing electron microscopic methods. The enzyme was shown to be highly asymmetric as suggested in earlier hydrodynamic studies. The tetramer of the enzyme appeared as four subunits arranged in the shape of a planar rose with an estimated width of 15 nm. A minimum thickness of 3.0 nm for the enzyme monomer was calculated from the shadow length of unidirectionally shadowed molecules. A model composed of four oblate ellipsoid monomers in a tetrameric rose arrangement is proposed for the shape of the dopamine beta-hydroxylase molecule. Two monomers associate edge to edge to form an in-plane dimer and two dimers associate side-by-side with their respective long axes at a slight angle to form a tetramer. Theoretical calculations based on the model are consistent with previous hydrodynamic studies.     

Ascorbic acid specifically enhances dopamine beta-monooxygenase activity in resting and stimulated chromaffin cells

Ascorbic acid enhancement of norepinephrine formation from tyrosine in cultured bovine chromaffin cells was characterized in detail as a model system for determining ascorbate requirements. In resting cells, ascorbic acid increased dopamine beta-monooxygenase activity without changing tyrosine 3-monooxygenase activity. [14C]Norepinephrine specific activity was increased by ascorbic acid, while [14C]dopamine specific activity was unchanged. Dopamine content, dopamine biosynthesis, tyrosine content, and tyrosine uptake were also unaffected by ascorbic acid. Furthermore, increased norepinephrine formation could not be attributed to changes in norepinephrine catabolism. Enhancement of dopamine beta-monooxygenase activity was specific for ascorbic acid, since other reducing agents with higher redox potentials were unable to increase norepinephrine formation. The specific effect of ascorbic acid on enhancement of norepinephrine formation was also observed in chromaffin cells stimulated to secrete with carbachol, acetylcholine, veratridine, and potassium chloride. In stimulated cells with and without ascorbate, there were no differences in dopamine content, tyrosine uptake, dopamine specific activity, and norepinephrine catabolism. These data indicate that, under a wide variety of conditions, only one catecholamine biosynthetic enzyme activity, dopamine beta-monooxygenase, is specifically stimulated by ascorbic acid alone in cultured chromaffin cells. This model system exemplifies a new approach for determining ascorbic acid requirements in cells and animals.     

Immunoelectron microscopic localization of dopamine beta-hydroxylase and chromogranin A in adrenomedullary chromaffin cells

Dopamine beta-hydroxylase (DBH) was purified from bovine adrenal medullae. Rabbit IgG raised against DBH inhibited its activity by 80%. In an immunoblot analysis, the IgG specifically recognized two subunits of DBH the 72 and 75 KD components. Chromogranin A (CGA) also was purified from bovine adrenal medullae, and rabbit IgG against CGA recognized this chromogranin A in the immunoblot analysis. The intracellular distribution of DBH and CGA in bovine chromaffin cells was determined quantitatively by immunoelectron microscopy using post-embedding protein A-gold technique. DBH and CGA were localized exclusively on chromaffin granules. The binding of gold particles to these granules was saturable. The maximum number of gold particles bound to the granules roughly corresponded to the number of DBH or CGA molecules in the granules estimated biochemically. DBH was observed evenly in the periphery and in the dense matrix of the chromaffin granules.     

Insulin-like growth factor I enhances gonadotropin-releasing hormone-stimulated luteinizing hormone release from bovine anterior pituitary cells

The role of insulin-like growth factor I (IGF-I) in the release of luteinizing hormone (LH) is unclear in ruminants. In the present study, the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) were examined in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17beta (E(2)) and IGF-I was characterized. GnRH(100nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36h after addition of IGF-I (250ng/ml), with a maximum at 12h (48.4ng/ml media versus 35.4ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500ng/ml increased the release by 18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2ng/ml media). E(2) (10nM), IGF-I (250ng/ml) and combined treatment of E(2) plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E(2) alone was 37.3% greater than with IGF-I alone (39.0ng/ml media versus 28.4ng/ml media) (P<0.05). When E(2) and IGF-I were added together (45.6ng/ml media), the release of LH was significantly greater than with either E(2) alone or IGF-I alone (P<0.05). E(2) (10nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250ng/ml) failed to increase GnRH binding. These results show that IGF-I enhances GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interacts with E(2) to increase the response to GnRH.     

Insulin-like growth factor I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells

Insulin-like growth factor I stimulates mitogenesis in smooth muscle cells, and upregulates elastin synthesis in embryonic aortic tissue. Increased smooth muscle elastin synthesis may play an important role in vascular remodeling in chronic pulmonary hypertension. Therefore, we studied the effect of IGF-I on elastin and total protein synthesis by pulmonary arterial smooth muscle cells in vitro. Tropoelastin synthesis was measured by enzyme immunoassay, and total protein synthesis was measured by [3H]-leucine incorporation. In addition, the steady-state levels of tropoelastin mRNA were determined by slot blot hybridization. Incubation of confluent cultures with various concentrations of IGF-I resulted in a dose-dependent stimulation of elastin synthesis, with a 2.4-fold increase over control levels at 1000 ng/ml of IGF. The increase in elastin synthesis was reflected by a stimulation of the steady-state levels of tropoelastin mRNA. We conclude that IGF-I has potent elastogenic effects on vascular smooth muscle cells, and speculate that it may contribute to vascular wall remodeling in chronic hypertension.     

Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)₂ vitamin D₃ significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 M), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.     

Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are attractive candidates for cell based therapies. However, the mechanisms responsible for stem cell migration and homing after transplantation remain unknown. It has been shown that insulin-like growth factor-1 (IGF-1) induces proliferation and migration of some cell types, but its effects on stem cells have not been investigated. We isolated and cultured MSC from rat bone marrow, and found that IGF-1 increased the expression levels of the chemokine receptor CXCR4 (receptor for stromal cell-derived factor-1, SDF-1). Moreover, IGF-1 markedly increased the migratory response of MSC to SDF-1. The IGF-1-induced increase in MSC migration in response to SDF-1 was attenuated by PI3 kinase inhibitor (LY294002 and wortmannin) but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Our data indicate that IGF-1 increases MSC migratory responses via CXCR4 chemokine receptor signaling which is PI3/Akt dependent. These findings provide a new paradigm for biological effects of IGF-1 on MSC and have implications for the development of novel stem cell therapeutic strategies.     

Insulin-like growth factor I supports differentiation of cultured osteoblast-like cells

Rat calvaria cells grown in culture for one week had properties of osteoblasts: a high content in alkaline phosphatase and a marked cyclic AMP response to parathyroid hormone (PTH). In short-term experiments, insulin-like growth factor I (IGF I) stimulated the incorporation of [14C] glucose into glycogen. When IGF I was present in the medium during 6 days the cell number increased slightly and there was a substantial, disproportionate rise in alkaline phosphatase activity of the cultures. Thus, IGF I stimulates growth, and in addition, and in contrast to other growth factors, mainly enhances differentiation of osteoblasts.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of dopamine beta-hydroxylase in rat adrenal glands.

Rat adrenal gland levels of dopamine beta-hydroxylase are subject to dual control. Activation of the splanchnic nerves to the adrenal medulla by reserpine induces the synthesis of dopamine beta-hydroxylase without altering the rate of enzyme degradation. In contrast, hypophysectomy causes a decline in steady state dopamine beta-hydroxylase levels by first accelerating the rate of degradation, then by slowing the rate of enzyme synthesis as well. Adrenocorticotropic hormone administration partially reversed the effect of hypophysectomy on dopamin beta-hydroxylase degradation. These findings suggest that the trans-synaptic factors controlling dopamine beta-hydroxylase induction act by a different mechanism (enzyme synthesis) than the hormonal controls regulating steady state levels (enzyme degradation). Thus, active inhibition of enzyme degradation may be an important control in maintenance of steady state enzyme levels.     

Insulin-like growth factor I levels in healthy adults

Insulin-like growth factor I (IGF-I) levels mainly reflect secretion of growth hormone (GH) in the body. The aims of this study were to compare different IGF-I assay methods in healthy individuals, test the reliability of the methods and discuss the utility of IGF-I measurement in adults. The Nichols Institute Diagnostics radioimmunoassay was used to evaluate IGF-I in two random population samples of men and women (aged 25-64 years, n = 392) taken 10 years apart, in 1985 and 1995. This method for IGF-I testing was also compared with an immunoradiometric assay (IRMA) method in 387 men and women participating in the World Health Organization MONICA (MONItoring of trends and determinants for CArdiovascular diseases) Project, Goteborg, Sweden, in 1995. Serum IGF-I decreased with increasing age in both men and women. IGF-I was higher in young women compared with young men in both cohorts, while the opposite was found in the highest age group. Age-adjusted significant correlations were found between IGF-I and smoking, fibrinogen, coffee consumption, lipoprotein (a), osteocalcin and IGF-binding protein 3. The two cohorts showed similar mean IGF-I concentrations irrespective of method. The correlation between the Nichols and the IRMA methods was high: r = 0.93 (p < 0.0001). Based on this and previous studies, population-based IGF-I measurements are robust irrespective of which commercially available method of assay is used. IGF-I levels can be used in diagnosing acromegaly as well as providing target values. IGF-I assay can be used as a complement to stimulation testing in the diagnosis of GH deficiency, and as a tool for GH dose titration.     

Insulin-like growth factor I and impaired glucose tolerance

The effects of circulating insulin-like growth factor I (IGF-I) on glucose metabolism are well recognized. IGF-I is also important in maintaining beta-cell mass and regulating endogenous growth hormone (GH) levels. Low IGF-I levels could explain links between small birth size and the risk of developing type 2 diabetes mellitus in short, obese adults. In a recent prospective study, childhood insulin secretion was related to IGF-I levels and statural growth, whereas insulin sensitivity was related to early post-natal weight gain. Common genetic polymorphisms in the IGF1 gene have been linked to small birth size, post-natal growth and future diabetes risk, but these results have been inconsistent. Recent adult studies have demonstrated that lower baseline IGF-I levels predict the subsequent development of impaired glucose tolerance (IGT), type 2 diabetes and cardiovascular disease. Administration of low-dose GH therapy, at a dose that minimizes the lipolytic effects of GH and has the ability to increase IGF-I levels, enhances insulin sensitivity in young healthy adults and in GH-deficient adults and increases insulin secretion in individuals with IGT. Whether the administration of low-dose GH, recombinant IGF-I or combined IGF-I/IGF-binding protein 3 therapy prevents future development of IGT or type 2 diabetes in high-risk normoglycaemic and GH-deficient individuals merits further long-term studies.     

Insulin-like growth factor I levels in healthy children

Measurement of insulin-like growth factor I (IGF-I) levels is used during the assessment of a child for the presence of growth hormone (GH) deficiency and to monitor the efficacy of GH replacement therapy. In either case, robust normative data are required to allow IGF-I values to be expressed as standard deviation scores, enabling comparison between individuals and assessment of change over time. However, IGF-I levels in healthy children are affected by a number of parameters, including age, gender, pubertal status, height, nutrient intake, body composition, intercurrent illness and ethnicity, and the generation of such data requires the collection of samples from significant numbers of healthy children. As external quality assurance schemes for IGF-I and an international standard based on authentic recombinant IGF-I are not widely used, it is imperative for the clinician to understand the performance characteristics and limitations of the IGF-I assay used and to be aware of the source and quality of control data. It must also be recognized that IGF-I measurement is only one component of the diagnostic process and has its limitations, as tissue sensitivity to circulating serum IGF-I levels will differ between individuals.     

Hepatocyte growth factor enhances MMP activity in human endothelial cells

Scatter factor (SF) or hepatocyte growth factor (HGF) has been identified as an angiogenic factor. Angiogenesis requires not only tube formation but also invasion of pericytes and extracellular matrix (ECM) remodeling to promote new vessel stabilization. In the current study, the effect of SF/HGF on endothelial cell (EC) production of matrix metalloproteinases (MMPs) was explored. We showed that SF/HGF enhanced MT1-MMP synthesis and induced MMP-2 activation in two human EC lines: dermal microvessel EC and coronary arterial EC. Furthermore, SF/HGF accelerated EC invasion into matrix, an activity that could be inhibited by a MMP inhibitor. We also demonstrated that the MAP kinase cascade is critical in signal transduction pathway from SF/HGF stimulation to MT1-MMP up-regulation. The current study indicates that MMP activation is a novel effect of SF/HGF on ECs.     

Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine beta-hydroxylase

Dopamine beta-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose-N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine beta-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine beta-hydroxylase subunits of Mr congruent to 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl-N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine beta-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix.