Kinetic evidence for cytoplasmic calcium as an inhibitory messenger in parathyroid hormone release

A sudden change of extracellular Ca2+ from 0.5 to 3.0 mM resulted in a transient rise of the cytoplasmic Ca2+ concentration (Ca2+i) followed by a sustained increase in parathyroid cells loaded with the Ca2+-indicator fura-2. The initial transient could be eliminated by increasing the Ca2+ buffering capacity of the cytoplasm. Under such conditions the rise of Ca2+i exhibited kinetics reminiscent of those for 45Ca uptake and cell depolarization. Addition of 0.5 mM Mn2+ mimicked the effect of raising the extracellular Ca2+ concentration, since there was an initial Ca2+i transient followed by a slower entry of Mn2+ into the cells. This reaction pattern was different from that of pancreatic alpha 2-cells in which there was no substantial influx of Mn2+ before depolarization with arginine. When measuring the kinetics of parathyroid hormone (PTH) release it was apparent that Ca2+ inhibition of secretion followed Ca2+i and thus became substantially delayed after eliminating the initial transient. The results support the concept of a depolarizing Ca2+ permeability in the parathyroid cell membrane which can be activated by external Ca2+, and indicate that Ca2+i is an inhibitory messenger of importance for the physiological regulation of PTH release.     

Lipid phase transitions in membranes involving intrinsic periodic curvature

A conformation of the lipid bilayer of membranes is proposed, with periodic curvature corresponding to the minimal surface structure of cubic lipid phases. Evidence is given indicating that activities of lipid synthesis/modification enzymes embedded in the membrane are controlled by the lateral "packing-pressure", so that the lipid bilayer is close to a transition from the lamellar (L alpha) type of conformation to this periodically curved conformation. Such a phase transition mechanism is assumed to be involved in numerous cooperative membrane functions.     

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.     

Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase

Mixed micelles of bile salt and phospholipids inhibit the lipase-colipase-catalysed hydrolysis of triacylglycerols. Free fatty acids can reverse this inhibition and reactivate lipase-colipase. This reactivation is either due to the formation of a high-affinity complex between lipase and colipase induced by free fatty acids and/or to a change of the quality of the interface. Lauric acid, oleic acid and linoleic acid are the most potent reactivators, while short-chain free fatty acids have no effect and long-chain, saturated free fatty acids inhibit the lipase-colipase activity further. The physiological relevance of these results is evident as the glyceride emulsion reaching the duodenum already contains free fatty acids due to the activity of lingual lipase in the stomach.     

Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues

Two proteins of Mr = 58,000 and 59,000, respectively, were purified from 4 M guanidinium chloride extracts of articular cartilage by dissociative CsCl-density gradient centrifugation followed by gel chromatography on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. The two proteins differ in ionic properties and only the one with Mr = 59,000 bound to the ion exchanger. Although the two proteins showed dissimilar peptide patterns after proteolysis, their amino acid composition was similar, with very high contents of leucine and aspartic acid/asparagine. The two proteins showed no cross-reactivity in radioimmunoassays. By use of these assays, the proteins were demonstrated in extracts of most connective tissues, with high contents of about 0.1% of tissue wet weight determined in several types of cartilage. Among the non-cartilage connective tissues, tendon and sclera had the highest contents of the proteins, i.e. about 0.1% of the tissue wet weight. Bone extracts, on the other hand, contained insignificant amounts of the proteins. Only the Mr = 59,000 protein was detected in serum, its concentration being about 33 micrograms/l. Both proteins were shown to be localized in the extracellular matrix of cartilage, predominantly in the territorial matrix, by using indirect immunofluorescence.     

Sequential Hormonal Changes in the Postpartum Dairy Cow

Peripheral plasma levels of 15-keto-13,14-dihydro-PGF2α, progesterone, Cortisol, LH and prolactin were studied in 6 primiparous postpartum dairy cows. The cows were followed by hormone measurements and clinical examinations from parturition until pregnancy was established. Blood was collected 3 times per day. The cervix, uterus and ovaries were examined by rectal palpation at 6–10 days intervals. The cows were observed for signs of oestrus twice daily and were additionally teased with a bull to provoke standing heat. Four cows had a normal parturition and dropped their fetal membranes shortly afterwards. (NR group). The remaining 2 retained their fetal membranes for more than 24 h following parturition (RFM group). One out of 6 cows showed standing oestrus at the first ovulation, 4 animals were in oestrus at the second ovulation and all cows showed signs of oestrus at the third ovulation. Although the length of the first luteal phase varied from 9 to 22 days a corpus luteum was in all cases palpated. The secretion of progesterone during the first luteal phase was terminated by a PGF2α release. A significant difference in 15-keto-13,14-dihydro-PGF2α levels between the 2 groups was found on days 0–4 (2.39 vs 6.87 nmol/1 at Ρ < 0.06). Postpartum prostaglandin F2α release as reflected by the level of 15-keto-13,14-dihydro-PGF2α lasted shorter in the NR group than in the RFM group (15–17 vs 21 days). Significant positive correlations between 15-keto-13,14-dihydro-PGF2α and Cortisol as well as between prolactin and Cortisol during the first 24 days postpartum were noted only in cows having normal parturition. The most pronounced daily prolactin variations occurred during the second luteal phase (NR group), when a significant difference between the times 8.00, 12.00 and 15.00 was recorded (14.7, 31.5 and 19.7 μg/l, respectively). Moreover, a partial negative correlation between log value of prolactin and arithmetical value of LH was found in these cows only during the first luteal phase after parturition.     

Selectivity in the binding of hydroxylated benzo[a]pyrene derivatives to purified cytochrome P-450c

The interaction of rat liver microsomal cytochrome P-450c with potential benzo[a]pyrene (BP) metabolites has been compared with the binding of BP by optical and fluorescence spectroscopy. Fluorescence quenching of the phenolic derivatives of BP derives from 1:1 complex formation with P-450c, is a function of the position of the hydroxyl substituent, and correlates with the concomitant increase in high-spin cytochrome observed in parallel optical titrations. The proportion of high-spin cytochrome seen when P-450c was reconstituted in dilauroylphosphatidylcholine vesicles (60 micrograms/mL) ranged from about 7% for the 3- and 7-phenols to 75% for 11- and 12-phenols. BP and all 12 methyl-BP derivatives have comparable high affinities for P-450c (50-70% high spin). Kd determinations with purified P-450c indicated very strong binding of BP phenols that induce high-spin complexes (4-, 5-, 9-, 10-, 11-, and 12-phenols; Kd = 3-25 nM). Inhibition of n-octylamine binding by the 3- and 7-phenols indicated weak interactions (Kd = 80-90 nM), even though low-spin complexes were formed. Inhibition of BP metabolism catalyzed by P-450c with BP phenols correlated with their respective dissociation constants. These results suggest that phenolic substitution at certain positions on BP (1, 2, 3, 7, or 8) interferes with binding to the active site while substitutions at the other positions either enhance or have no effect on binding. BP dihydrodiols [including the (+)- and (-)-BP 7,8-dihydrodiols] were relatively ineffective in forming high-spin complexes (approximately 20%), and fluorescence quenching of dihydrodiols by P-450c also saturated at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel ganglioside on erythrocytes with blood group Cad specificity

The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.