Philippa Claude

Morphological factors influencing transepithelial permeability: A model for the resistance of theZonula Occludens

SummaryEpithelial cells are joined at their apical surfaces byzonulae occludentes. Claude and Goodenough (1973) demonstrated a correlation between the structure of thezonula occludens as seen in freeze-fracture preparations and the passive electrical permeability of several simple epithelia. In epithelia with high transepithelial resistance, thezonula occludens consisted of many strands. In epithelia with low transepithelial resistance thezonula occludens was much reduced, sometimes consisting of only one strand.Evidence is reviewed here that indicates that in a number of simple epithelia the structure of thezonula occludens is largely responsible for the magnitude of transepithelial conductance. An equation is derived relating transepithelial junctional resistance to the number of junctional strands:R=Rminp−n whereR is the transepithelial resistance of thezonula occludens,Rmin is the minimum resistance of the junction (as when there areno strands in the zonula occludens),p is the probability a given strand is “open” andn is the number of strands in the junction. Using published experimental values ofR andn for different epithelia, the calculated value ofp was found to be as high as 0.4, which suggests that the strands in thezonula occludens are remarkably labile.Other morphological parameters relevant to transepithelial permeability are also considered, such as the width and depth of the intercellular spaces, and the size of the epithelial cells themselves.

Acidic fibroblast growth factor atimulates adrenal chromaffin cells to proliferate and to extend neurites, but is not a long term survival factor

Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that is a mitogen for endothelial cells and glial cells, as well as a differentiation factor for PC12 cells and certain neurons. We show here that aFGF is as potent as nerve growth factor (NGF) in stimulating both neuritic outgrowth and proliferation in adrenal chromaffin cells from young rats, but it fails to support long-term survival. Heparin strongly potentiates aFGF-dependent neuritic outgrowth but not aFGF-dependent proliferation. As is the case with NGF, phorbol myristate acetate depresses aFGF-induced cell division and increases the outgrowth of neurites. On the other hand, dexamethasone antagonizes neuritic outgrowth elicited by both NGF and aFGF but inhibits only proliferation induced by NGF. The effects of basic FGF (bFGF) are similar but not identical to those of aFGF. Thus the regulatory pathways controlled by aFGF, bFGF, and NGF are partially distinct.

Prolactin synthesis in primary cultures of pituitary cells: regulation by estradiol.

Pituitary cells cultured with estradiol respond by a specific increase in prolactin synthesis. Extensive inhibition of DNA synthesis (61-78%) with hydroxyurea or cytosine arabinoside resulted in only 28-33% decrease in estrogen-induced prolactin synthesis. To assess the role of prolactin cell proliferation in the estrogen-induced response, mammotrophs were identified by immunocytochemistry. Cultures treated with estradiol for 1, 2 or 5 days contained 101 +/- 1, 113 +/- 2 and 132 +/- 1% of the number of mammotrophs in controls. Estradiol treatment for corresponding periods resulted in prolactin synthesis representing 94 +/- 5, 144 +/- 11 and 270 +/- 22% of controls and prolactin mRNA levels representing 115 +/- 7, 160 +/- 7 and 322 +/- 22% of controls. Thus estrogen caused a considerable increase in prolactin synthesis which paralleled the increase in prolactin mRNA levels and a much smaller relative increase in the number of mammotrophs. We conclude that regulation of prolactin synthesis by estrogen is mediated predominantly but not exclusively through stimulation of gene expression in existing pituitary cells.

Regulation of pituitary growth and prolactin gene expression by estrogen.

We presented evidence that primary cultures of rat pituitary cells respond to estradiol by increased incorporation of precursors into prolactin. The response is specific for estrogenic hormones and is maximal at physiological concentrations of estradiol. The time course and magnitude of the response in cultured cells is in agreement with that observed under in vivo conditions, suggesting that estrogen exerts its effect mainly through a direct action on the pituitary gland. The data presented indicate that estrogen stimulates prolactin synthesis predominantly through increased prolactin mRNA accumulation, and to a lesser extent, through mammotroph cell proliferation. Chronic treatment with DES caused sustained proliferation of pituitary cells leading to prolactin producing pituitary tumors in the Fischer 344 rat, but not in the Holtzman rat. The genetic basis for these differences are currently under investigation.

Nerve growth factor and glucocorticoids regulate phenotypic expression in cultured chromaffin cells from adult rhesus monkeys.

Adrenal chromaffin cells and sympathetic neurons are both derivatives of the neural crest. Despite their morphological and functional differences, chromaffin cells retain some developmental plasticity and if treated with Nerve Growth Factor (NGF), can express certain characteristics of sympathetic neurons. However, there is some age and species variability in the response of chromaffin cells to NGF: in general chromaffin cells from adult animals are not considered to be dependent on NGF for survival, and chromaffin cells from adults of several species fail to respond to NGF in vitro by growing neurites. This is in contrast to the dramatic effects of NGF on chromaffin cells from perinatal rats. We have examined the requirements of chromaffin cells from adult rhesus monkeys to survive, to proliferate, and to express a neuronal morphology in vitro. NGF greatly enhances the proportion of rhesus chromaffin cells that form neurites and the length of the neurites that are formed, but the conversion to a neuronal phenotype is more limited than in chromaffin cells cultured from young rats. NGF also enhances rhesus chromaffin cell survival, but fails to stimulate their proliferation, in contrast to its effect on perinatal rat cells [18]. Glucocorticoid hormones (GCs) specifically antagonize the effects of NGF on neuritic outgrowth while promoting chromaffin cell survival. Thus adrenal chromaffin cells from rhesus monkeys retain a degree of developmental plasticity even in the adult animal.

Neurotrophins in the Developing and Adult Primate Adenohypophysis: A New Pituitary Hormone System?

Expression of neurotrophins and of the low-affinity neurotrophin receptor p75 was examined immunocytochemically in pituitary glands of twelve developing and adult macaques, ranging in age from fetal day 100 through age 5 years. Neurotrophins were identified by labeling with a rabbit polyclonal antiserum raised against purified mouse nerve growth factor, which recognizes brain-derived neurotropic factor and neurotrophin-3 as well. During pituitary morphogenesis, neurotrophins were present in epithelial cells distributed throughout all divisions of the anterior pituitary (pars distalis, pars intermedia, and pars tuberalis). Near term and in the adult, neurotrophin-immunoreactive cells were fewer in number and their distribution was limited to the pars distalis and pars tuberalis. A monoclonal antibody against the human neurotrophin receptor p75 heavily labeled mesenchymal boundary structures and blood vessels in the developing gland, and several populations of glial-like cells with a presumed paracrine function (folliculostellate cells in the pars distalis, and pituicytes and tanycytes in the neural lobe and infundibulum, respectively) as well as axons innervating the portal vasculature in postnatal specimens. These complementary patterns of neurotrophin and receptor expression suggest a possible inductive role for neurotrophins in pituitary morphogenesis and in the establishment of hypothalamic neural and hormonal control of pituitary function. In the adult anterior pituitary, examined using double-label immunocytochemistry for neurotrophins and conventional anterior-pituitary hormones, neurotrophins did not colocalize with human prolactin, human adrenocorticotropic hormone, recombinant human growth hormone, or the beta subunits of human luteinizing hormone, human follicle-stimulating hormone, or human thyrotropin. Neurotrophin-containing cells therefore appear to be a distinct population, suggesting novel paracrine or endocrine functions for this family of neuropeptides.

Chronic exposure to an activator of protein kinase C mimics early effects of NGF in chromaffin cells

Adrenal chromaffin cells respond to nerve growth factor (NGF) in vitro by expressing neuronal characteristics and, over a period of 2 to 4 weeks, transdifferentiating into postmitotic sympathetic neurons. Phorbol myristate acetate (PMA) is a potent activator of protein kinase C (PKC); chronic exposure to PMA mimics the initial actions of NGF by promoting the outgrowth of neurites and increasing the incorporation of [3H] thymidine in primary cultures of adrenal chromaffin cells from young rats. PMA and NGF affect the same populations of cells and even individual neurites. These effects are specific for active phorbol ester and do not result from the release of NGF or FGF in the cultures. As in the case of NGF, the effects are inhibited by glucocorticoids. The PKC inhibitor staurosporine inhibits the effects of PMA, as well as those of NGF, in a dose-dependent manner. These results suggest that a modulation in activity of PKC is important in the neuritogenic and proliferative effects of NGF, at least for an initial period of approximately 1 week.

Brown adipose tissue from fetal rhesus monkey (Macaca mulatta): morphological and biochemical aspects

Brown adipose tissue (BAT) from fetal rhesus monkeys microscopically resembled adult rodent BAT containing multiocular fat cells with numerous mitochondria. Mitochondrial carnitine palmitoyl transferase activity was lower than that in adult rodents and adenine nucleotide translocase activity was similar to that reported for rats. Rhesus monkey BAT mitochondria (BATM) possess an uncoupling protein that is characteristic of BAT as evidenced by the binding of [3H]GDP, the inhibition by GDP of the high Cl- permeability or rapid alpha-glycerol-3-phosphate oxidation. Electrophoretic analysis of BATM showed the presence of a 32,000 mol.wt protein which was enriched by procedures established for the isolation of BATM uncoupling protein.

Effects of NGF and glucocorticoid on NGF receptor immunolabeling of cultured rhesus adrenal chromaffin cells.

Nerve growth factor (NGF) promotes the outgrowth of neurites from cultured adrenal chromaffin cells from adult rhesus monkeys, but little is known about the distribution, at the cellular level, of the NGF receptors (NGFR) responsible for this response. We examined changes in immunostaining for NGFR in chromaffin cells cultured for 4 weeks in the presence or absence of NGF, with or without dexamethasone (DEX), which inhibits neuritic outgrowth from these cells. Purified cultures of adrenal chromaffin cells from adult rhesus monkeys were grown for up to 9 weeks in NGF, DEX, NGF plus DEX, or control medium. Cells were immunolabeled with three different monoclonal antibodies directed against different epitopes of the human NGFR. Although the distribution of immunolabeling was not uniform from cell to cell, the overall intensity of NGFR immunolabeling varied dramatically between different growth conditions. Of greatest interest, DEX-treated cells stained the most intensely at all time points, while the intensity of immunolabeling was much fainter in NGF-treated cells and decreased with time in culture. In contrast to the intensity of labeling, the proportion of chromaffin cells with immunoreactivity increased with time in all treatment groups. Thus, GCs do not appear to antagonize the effects of NGF merely by decreasing the total number of immunoreactive NGFR on the surface of these cells.