Effects of insulin on the synthesis, intracellular degradation, and secretion of parathormone.

Bovine parathyroid organoids were maintained for up to 12 days of culture in the presence or absence of insulin. Insulin-treated organoids secreted more PTH and secretory protein-I (SP-I) than did untreated organoids at both 1.4 and 1.8 mM Ca, concentrations chosen to promote partially elevated and suppressed secretion rates, respectively. The insulin effect was dose dependent and reversible. To determine whether insulin might increase secretion by reducing degradation of cellular PTH, its effects on several parameters related to degradative processes were examined. Compared to control cultures maintained at either 1.4 or 1.8 mM Ca, insulin did not induce changes in the relative levels of intact hormone and COOH-terminal peptide fragments secreted into culture medium, nor did it decrease the total cellular levels of three lysosomal enzymes or mute the effects of 3-methyladenine (an agent that decreases formation of autophagosomes) to increase PTH secretion. Thus, insulin did not appear to increase PTH secretion by reducing the latter's cellular degradation. Synthesis of total proteins and of the secreted proteins SP-I and PTH was examined using short incubations of control and insulin-treated organoids with [3H] leucine. Incorporation of 3H into total acid-precipitable proteins was not elevated in insulin-treated organoids; that into PTH/pro-PTH and SP-I, however, was significantly greater in insulin-treated than in control organoids. The results suggested that the insulin-mediated increase in PTH and SP-I secretion is largely due to its regulation of PTH and SP-I biosynthesis.

Vasculature of the paraphysis cerebri of the frog.

The paraphysis cerebri is a glandular structure found in the third ventricle of lower vertebrates. It is well-developed in amphibians and reptiles. The function of the gland is not substantiated, but may play a role in calcium metabolism. To further elucidate its possible endocrine role, the paraphyseal vasculature was examined using casting techniques as well as transmission electron microscopy. Perfusion-fixed paraphyses of Rana pipiens and Rana catesbeiana were either: a) cast with Microfil (with subsequent dehydration, clearing, and macroscopic examination) or Batson's compound (followed by tissue digestion and examination by scanning electron microscopy); or b) processed for transmission electron microscopy. The paraphyseal capillary bed consists of a sinusoidal portal system which receives afferent blood from its associated choroid plexus. The choroid plexus of the third ventricle receives its blood supply via arterioles from the posterior telencephalic artery. These arterioles traverse in the periphery of the paraphysis to branch and supply the choroid plexus. Numerous venules exit from the choroid plexus and drain into sinusoids of the paraphysis. The sinusoid venules appear to empty into a midline venous structure which passes tangentially through the paraphysis. The sinusoids consist of fenestrated endothelium which is indicative of transport vessels. Nerve fibers were observed in the paraphysis, however, histofluorescence revealed no monoaminergic sympathetic innervation of the paraphyseal vasculature.

Aircraft automation: the problem of the pilot interface.

Aircraft operations, particularly in the IFR environment, are rapidly becoming very complex. Studies have shown that this complexity can frequently lead to accidents and incidents. Results of studies performed at NASA and elsewhere are presented to show that one of the major themes evident in both the accidents and incidents and in the research performed to solve the problems associated with them is that of human error. Examples of various incidents and blunders, recorded in several studies, illustrate and emphasize the hypothesis: "As systems become more and more automated and complex, the more they become prone to human error. The problem can be eliminated or reduced only if good human factor principles are incorporated in the implementation of the systems, to guarantee a good man/machine interface". Aircraft systems technology, however, (e.g.: electronics, avionics, automation) is evolving and developing at a very high rate. Examples of research are presented showing where this emerging technology has been employed to reduce the complexity and enhance the safety and utility of the aircraft operations.

Effects of calcium on synthesis and secretion of parathyroid hormone and secretory protein I.

Bovine parathyroid organoids were cultured for up to 3 wk in medium containing 1.4 or 1.8 mM calcium. Steady-state secretion of parathyroid hormone and secretory protein I was two- to fourfold greater at 1.4 mM. At the end of culture, organoids were incubated 3.5 h in 1 or 2 mM calcium to examine maximum and minimum acute secretory rates. Relative to organoids cultured at 1.8 mM calcium, culture at 1.4 mM induced a hypersecretory state, i.e., both the maximum and minimum acute secretory rates of organoids previously cultured at 1.4 mM calcium were up to threefold greater than those of organoids previously at 1.8 mM calcium. Proparathyroid hormone synthesis was up to 50% greater in organoids cultured at 1.4 mM calcium, whereas secretory protein I and total protein synthesis were unaltered. The results showed that parathyroid hypersecretion can be induced by chronic hypocalcemic conditions in vitro. We conclude that the secretory adaptation to chronic hypocalcemia in vitro involves alterations in both synthesis and degradation of parathyroid hormone.

Altering the expression of cell surface beta 1,4-galactosyltransferase modulates cell growth.

beta 1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is localized both in the Golgi complex and on the cell surface. Most studies of surface GalTase function have focused on its role in cellular interactions; however, surface GalTase has also been suggested to function during cellular proliferation. Consistent with this hypothesis, a variety of GalTase-specific perturbants inhibit cell growth in vitro and in vivo. However, all of these studies have been limited to the use of exogenous reagents to perturb GalTase function. Furthermore, all of these perturbants inhibit cell growth, irrespective of whether they stimulate or inhibit GalTase enzyme activity. Therefore, it remains unclear whether surface GalTase delivers a growth inhibitory or growth stimulatory signal. In this study, we took a more direct approach to defining surface GalTase function during growth by examining its expression during the cell cycle and by molecularly altering its expression in stably transfected cell lines. The expression of GalTase was shown to be cell cycle specific, with the cell surface and intracellular GalTase pools displaying independent expression patterns. Furthermore, multiple, independent, stably transfected cell lines with reduced levels of cytoskeletally associated surface GalTase grew faster than control cells, whereas cell lines that over-expressed surface GalTase grew slower than controls. These observations directly support the concept that surface GalTase delivers a growth inhibitory signal. Evidence is presented suggesting that surface GalTase interacts with the epidermal growth factor (EGF) receptor, as suggested by others. The activity of the EGF receptor was shown to be directly proportional to the growth rate of the various GalTase-transfected cell lines. Thus, the expression of surface GalTase directly affects cell proliferation rate and may do so by modulating the ability of the EGF receptor to transduce EGF-dependent signals.

Expressing murine beta 1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype.

Beta 1,4-Galactosyltransferase is traditionally viewed as a biosynthetic component of the Golgi complex, but a portion of galactosyltransferase is also expressed on the cell surface, where it has been suggested to function as a receptor for extracellular oligosaccharide ligands. Although results from a variety of studies are consistent with a cell adhesion function for galactosyltransferase, the most rigorous test of surface galactosyltransferase function is to produce a surface galactosyltransferase-dependent phenotype in cells that normally express negligible levels of surface galactosyltransferase. In agreement with previous reports, human HeLa cells were found to express low levels of galactosyltransferase on their surface and, therefore, were stably transfected with cDNAs encoding murine galactosyltransferase. Murine galactosyltransferase was expressed both within the presumed Golgi complex and on the cell surface, as assayed by enzyme activity and with antiserum raised against the bacterially expressed murine enzyme. HeLa cell transfectants adhered more strongly to their extracellular substrates than did control transfectants, as evidenced by a flatter morphology in culture and a more rapid spreading upon plating. In contrast, cell spreading was low and similar among all cell types when plated on extracellular substrates that did not contain binding sites for galactosyltransferase. Antibodies and Fab fragments against recombinant murine galactosyltransferase inhibited the increased cell spreading characteristic of galactosyltransferase transfectants, as did soluble recombinant galactosyltransferase and a variety of galactosyltransferase perturbants. Thus, expression of heterologous galactosyltransferase produces a surface galactosyltransferase-dependent phenotype, confirming its function as a cell adhesion molecule.

The effect of epidermal growth factor on neonatal incisor differentiation in the mouse.

The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine ([3H]TDR) and tritiated proline ([3H]PRO). On days 0 (day of birth), 1, and 2, EGF was administered (3 micrograms/g body wt) sc to neonates. Mice were killed on Days 1, 4, 7, 10, and 13 after birth and were injected with either [3H]TDR or [3H]PRO 1 hr before death. [3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) fibroblasts. [3H]PRO was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [3H]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-microns-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-microns level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 microns, 45 microns,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in vivo 125I-EGF binding was analyzed autoradiographically.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal growth factor in the neonatal mouse salivary gland and kidney.

In the adult mouse, epidermal growth factor (EGF) is synthesized in granular convoluted tubule (intralobular) duct cells of the submandibular gland and in distal tubule cells of the kidney. The presence of EGF in developing tissues and maternal milk and the localization of EGF receptors in developing tissues suggest a role for EGF in developmental processes. The primary aims of the present study were to: (1) localize EGF and EGF-binding sites in the kidney and submandibular gland during neonatal development and (2) to determine the effect of exogenously administered EGF on cell proliferation in these two developing organs. In the present study, EGF was localized by immunocytochemistry in granular convoluted tubule cells of the submandibular gland initially on day 21 after birth and in distal tubule cells of the kidney on postnatal day 6. EGF binding in the kidney decreased after birth with some localization to the glomerulus. In submandibular glands of newborn and 10-day-old mice, EGF-binding sites were associated with both acinar and duct cells with peak binding at 10 days postnatally. Submandibular glands from 20-day-old mice demonstrated primarily ductal EGF-binding sites. Exogenously administered EGF induced a mitogenic response in acinar and interlobular duct cells of submandibular glands during the first week after birth. EGF treatment during this period had an inhibitory effect on 3H-thymidine incorporation into cellular compartments in the developing kidney. The identification of EGF-binding sites in the kidney and submandibular gland before the presence of EGF suggests that an EGF-like molecule such as transforming growth factor alpha (TGF-alpha) may be present as a potential ligand in these organs. In order to assess this possibility, developing kidneys and submandibular glands were stained with anti-TGF-alpha. These immunocytochemical studies localized TGF-alpha to the proximal tubule of the kidney and immature acinar cells of the newborn mouse. Our data strongly support an autocrine, juxtacrine or paracrine role for EGF and/or TGF-alpha in the regulation of cell proliferation and cytodifferentiation in the kidney and submandibular gland.

Atrial natriuretic peptide in heart and specific binding in organs from fetal and newborn rats.

To assess the possibility that atrial natriuretic peptide plays a role in salt and water balance during early mammalian development, we examined hearts from fetal and neonatal rates for the presence of this peptide and presumed target tissues for their ability to bind the hormone. Immunohistochemistry was used to localize and radioimmunoassay to quantify this peptide in heart. Immunoreactive atrial natriuretic peptide was visualized in the fetal heart on day 17.5 post-conception. It was distributed throughout the atrial appendages and free wall and, in ventricle, in the trabeculae carnae and chordae tendineae. The concentrations of immunoreactive atrial natriuretic peptide in atria of rats on day 19.5 post-conception were one-tenth of those in the adult. Levels of this peptide in fetal ventricle were low and virtually absent from the adult tissue. Specific binding of radiolabelled atrial natriuretic peptide measured by whole organ counting occurred in several organs from 19.5-day fetal and neonatal rats. A number of these tissues, including the kidney, ileum, adrenal, lung and liver, are targets for and/or bind the peptide in adult rats. Specific binding in these tissues was localized using autoradiography at anatomical sites similar to those in adult organs. Specific binding was also seen in fetal but not neonatal skin. In the kidney, binding was associated with immature as well as mature glomeruli. These findings support the proposition that atrial natriuretic peptide may function in the perinatal rat as it does in the adult and, in addition, may play a unique role during fetal life.