Alteration of sodium transport by the choroid plexus with amiloride

Cerebrospinal fluid (CSF) production results from active transport of Na+ from blood to CSF, which is followed by H2O and anions. Amiloride reduces Na+ movement in epithelial tissues. To ascertain if amiloride alters transport of Na+ in the choroid plexus, the drug was administered either i.p. to male Sprague-Dawley rats that were bilaterally nephrectomized to determine in vivo effects, or added to artificial CSF to incubate the choroid plexus in vitro. Choroid cell [Na+] was reduced after amiloride treatment both in vivo and in vitro. In addition, the rate of 22Na uptake into the CSF and choroid plexus (CP) was decreased after amiloride. Alterations in choroid cell [Na+] and 22Na penetration into CSF and CP occurred at relatively high doses of drug (1 mumol/ml, in vitro and 100 micrograms/g in vivo), but lower doses were less effective (0.1 mumol/ml in vitro and 10 micrograms/g in vivo). It is concluded that the effects of amiloride on Na+ distribution and transport in the CP are due to inhibition of basolateral Na+-H+ exchange.     

Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.     

Bicarbonate-Reversible and Irreversible Inhibition of Photosystem II by Monovalent Anions

We tested a number of inhibitory monovalent anions for their primary site of action on photosystem II(PSII) in chloroplasts. We find that the inhibitory effects of F⁻, HCO₂⁻, NO₂⁻, NO₃⁻, and CH₃CO₂⁻ are all reversed by addition of a high concentration of HCO₃⁻. This class of anions competitively inhibits H¹⁴CO₃⁻ binding to PSII. All of those anions tested reduced H¹⁴CO₃⁻ binding more in the light than in the dark. We conclude that the primary inhibitory site of action of a number of monovalent anions is at the HCO₃⁻ binding site(s) on the PSII complex. The carbonic anhydrase inhibitor gold cyanide, and also azide, inhibit PSII but at a site other than the HCO₃⁻ binding site. We suggest that the unique ability of HCO₃⁻ to reverse the effects of inhibitory anions reflects its singular ability to act as a proton donor/acceptor at the anion binding site. A similar role has been proposed for non-substrate-bound HCO₃⁻ on carbonic anhydrase by Yeagle et al. (1975 Proc Natl Acad Sci USA 72: 454-458).     

Changes in lung volume and deflation stability in hyaline membrane disease

Total lung capacity (TLC), inspiratory capacity, functional residual capacity, and deflation stability of prematurely delivered Macaca nemestrina primates were measured serially during development of, and recovery from, hyaline membrane disease (HMD) to relate changes in lung volumes to changes in deflation stability. Gestational age-matched primates that did not develop HMD served as controls. TLC, measured by N2 washout, fell at 2-12 h of age (P less than 0.0001) in animals with HMD and remained lower than controls for at least 48 h (P less than 0.005). However, deflation stability, defined as the fraction of TLC remaining upon deflation to 10 cm H2O, improved from 2 to 12 h of age (P less than 0.001). Postmortem studies confirm the measurements of TLC and deflation stability and provide evidence that interstitial thickening and obstruction of air spaces with debris may be partially responsible for the observed changes in TLC in primates that develop HMD. It has been assumed that TLC is reduced in HMD because of atelectasis from elevated alveolar surface tension, but the sequential measurements in these animals suggest that other mechanisms also contribute.     

Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.     

A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

The effect of ethanol on arachidonic acid metabolism in the murine peritoneal macrophage

Exposure to ethanol in man has been linked to an alteration of the immune surveillance system and reduced ability of the macrophage to undergo phagocytosis. Since ethanol has been suggested to alter membrane function and inhibit the production of calcium ionophore stimulated synthesis of prostaglandins and leukotrienes by the human neutrophil and transformed murine mast cell, the dose response effect of ethanol on the biosynthesis of icosanoids by the peritoneal macrophage during zymosan phagocytosis was studied. Peritoneal macrophages from two inbred strains of mice derived from a common stock (HS) and selected for sensitivity to ethanol (shoprt sleep [SS]/long sleep [LS]) were studies. Zymosan phagocytosis was found to lead to synthesis of LTC₄ (70 ng/10⁶ cells), 6-keto-PGF_1a (5 ng/10⁶ (3 ng/10⁶ cells). For the HS macrophage, ethanol caused a dose dependent inhibition of these lipid mediators as well as inhibition of phagocytosis and release of beta-hexosaminidase. However, a difference was observed in arachidonate metabolism stimulated by phagocytosis between the LS and SS mice below 100 mM ethanol. The SS mouse had a 50% inhibition of cyclooxygenase products at 86 mM ethanol with no inhibition of lipoxygenase metabolites. The LS mice had a trend suggesting increased lipoxygenase metabolites below 100 mM ethanol. At these levels of ethanol which can be found in man, these results suggest there may be differential production of lipid mediators under genetic control.     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.