Effects of ibuprofen and indomethacin on the regional circulation in newborn piglets.

We investigated the effects of clinically comparable doses of ibuprofen and indomethacin on renal, gastrointestinal and cerebral perfusion in newborn piglets, and hypothesized that ibuprofen would have less effect on regional circulation. Animals were randomly assigned to receive ibuprofen (20 mg/kg, n = 8), indomethacin (0.3 mg/kg, n = 7) or vehicle (n = 6). Fluorescent microspheres were injected prior to and at 20, 40, 60, 90 and 120 min after drug administration. Regional blood flow was measured and vascular resistances were calculated. Cardiovascular and respiratory variables were not significantly affected by either study drug or vehicle. Ibuprofen increased renal cortical and medullary resistance by 44 and 52% (p < 0.05). However, ibuprofen had no significant effects on gastrointestinal or cerebral resistance. Indomethacin raised renal cortical and medullary resistance by 66 and 71% at 60 min postinjection, respectively (p < 0.05). Indomethacin increased duodenojejunal, ileal and colon resistance by 97, 102 and 75% at 60 min, respectively (p < 0.05). Indomethacin increased cerebral cortical and cerebellar resistance by 92 and 86% at 90 min (p < 0. 05). While indomethacin and, to a lesser extent, ibuprofen both increase renal vascular resistance, indomethacin vasoconstricts the gastrointestinal and cerebral circulations which are unaffected by ibuprofen.

Parathyroid hormone-related protein in tracheal aspirates of newborn infants.

PTH-related protein (PTHrP) is found with its receptor in a variety of normal mammalian embryonic tissues where it apparently regulates cellular growth and differentiation. PTHrP stimulates phosphatidylcholine synthesis in rat fetal lung explants, suggesting a role in fetal type II alveolar maturation and surfactant production. We investigated PTHrP levels in tracheal aspirates of newborn infants. We collected tracheal aspirates from 40 intubated newborn infants within the first 24 h of life. PTHrP levels were measured by a RIA using rabbit antisera to PTHrP peptide 38-64. We found significantly lower PTHrP levels in tracheal aspirates from infants born at less than 35 wk of gestation (p = 0.02) and with a birth weight less than 2 kg (p = 0.04). We also found significantly lower PTHrP levels in male preterm (<35 wk of gestation) infants compared with female infants (p = 0.01), and in preterm infants who required multiple doses of surfactant (p = 0.005). Preterm infants exposed to antenatal steroids had significantly higher levels of PTHrP in tracheal aspirates (p = 0.02). PTHrP is associated with various indices of lung maturation and may prove to be a mediator of differentiation and growth.

Reduction of a disulfide bond of thrombospondin in the supernatant solution of activated platelets.

Incubation of the material secreted by activated platelets leads to the formation of disulfide-linked dimers and multimers of one of the proteins, thrombospondin. To determine whether these complexes formed as a result of thiol-disulfide exchange (no change in the number of thiols) or of oxidation of thiols (a decrease in the number of thiols), the number of thiols in TSP was measured during formation of multimers. The number of thiols increased from about 3/mol to 4.8/mol. The half-time for the disappearance of monomers of thrombospondin was fourfold greater than the half-time for appearance of new thiols. The appearance of new thiols, as well as the formation of multimers, was inhibited by Ca2+. The appearance of new thiols was reversible; addition of Ca2+ reversed the process, and at pH 8, but not at pH 6 or 7, the appearance of new thiols spontaneously reversed. No new thiols formed during incubation of partially purified thrombospondin or after the supernatant solution had been treated with activated thiol-Sepharose to remove reactive thiol compounds. It is concluded that thrombospondin has a disulfide bond that is unstable in the absence of Ca2+. It can be attacked by a thiol of another molecule of thrombospondin to form disulfide-linked multimers, by a thiol of the same molecule of thrombospondin to generate isomerization of disulfide bonds or, as observed in this study, by another secreted thiol compound to give a mixed disulfide and a new thiol.

Free thiols of platelet thrombospondin. Evidence for disulfide isomerization.

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.