Dietary fat influences electric membrane properties of neurons in cell culture

1. SJL/J mice were maintained on semipurified diets which differed in the ratio of polyunsaturated/saturated fatty acid content (P/S). Exposure was from conception and was maintained for periods ranging from 6 to 34 weeks. 2. Neural cell cultures were prepared from dorsal root ganglia (DRG). After 6 and 20 days of culture, neuronal electric membrane properties were determined quantitatively by intracellular recording. 3. A number of significant differences were observed for the two dietary conditions. DRG from mice on the low-P/s diet had an increase in the rate of fall of both phases of repolarization which, in conjunction with the reduced action potential overshoot, led to a reduced action potential duration. This shift to shorter-duration action potentials was accompanied by a shift to more monophasic falling phases. The low-P/S neurons also exhibited a decreased afterhyperpolarization, decreased specific membrane resistance, and decreased membrane electrical time constant compared to high-P/S neurons. 4. It was concluded that the P/S ratio in the diet can have a significant effect on the electric properties of neurons. The high-P/S neurons tended to have action potentials with biphasic repolarizations and longer durations. In contrast, the low-P/S neurons tended to have action potentials with monophasic repolarizations and shorter durations. Moreover, the known ionic dependence of these two types of action potentials suggested that the low-P/S diet resulted in action potentials with a more exclusive Na dependence, while the high-P/S diet resulted in action potentials with both Na and Ca dependence.     

An immunocytochemical study of thyrotropin releasing hormone in the porcine, ovine and rodent pineal gland

A biotin-avidin immunoperoxidase method was used to localize a pineal TRH-like compound in histological sections. TRH-like immunoreactivity was observed in porcine, ovine and rodent pineal glands. The immunoreaction was located on positively stained cell bodies and cellular processes throughout the glands. The exact location of the TRH (in pinealocytes, glial cells, or both) as well as the physiologic significance of the immunoreactive material remain to be elucidated.     

Endothelial cells stimulate ANF secretion from atrial myocytes in co-culture

Using a novel in vitro co-culture system, we investigated the possible influence of vascular endothelial cells on the secretion of atrial natriuretic factor (ANF) from atrial myocytes. Co-culture of bovine aortic endothelial cells grown on Cytodex-3 microcarrier beads with primary monolayer cultures of neonatal rat myocytes induced a 2.1-fold increase in immunoreactive ANF (irANF) in the medium, compared with irANF in medium from atrial cultures alone. This increase did not appear to be the result of processing of prohormone to more immunoreactive species, and could be inhibited by 47% with 10 microM acetylcholine. The endothelium-derived vasoconstrictor peptide, endothelin, elicited a dose-dependent increase in ANF secretion from atrial cultures, but, contrary to vasopressin, was incapable of further stimulating release from atrial-endothelial co-cultures. These experiments suggest that endothelium stimulates the release of ANF from myocytes, possibly by the action of the peptide endothelin.     

Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.     

Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[a]pyrene.

Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[a]pyrene, 9r,10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D0's, the DNA-repair-deficient cell lines were found to be 4-5-fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells.     

Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate

The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.