Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

Transposition of the gene cluster CYC1-OSM1-RAD7 in yeast

A mutant of the yeast Saccharomyces cerevisiae contains an increased amount of iso-1-cytochrome c because two copies of a segment, denoted COR, were transposed to a new position on chromosome VII, while the original COR region was retained at the normal position on chromosome X; this COR segment encompasses the CYC1, OSM1 and RAD7 loci which determine, respectively, iso-1-cytochrome c, osmotic sensitivity and ultraviolet light sensitivity. The analysis of genomic DNA with cloned probes indicates that the length of the COR segment is approximately 12,000 base-pairs. We suggest that certain normal strains of yeast, which possibly may contain reiterated sequences, can produce extended transpositions similar to prokaryotes.     

Identification with a Monoclonal Antibody of a Phylogenetically Conserved Brain-Specific Determinant on a 130,000 Molecular Weight Glycoprotein of Human Brain

Human brain glycoproteins depleted of Thy-1 antigen were used to immunise Balb/c mice for monoclonal antibody production. The F3-87-8 antibody described in this paper interacts with a determinant present in large amounts on all human brain subregions studied (cerebral cortical grey matter, white matter, caudate, thalamus, dentate nucleus, putamen, cerebellar cortex) but absent from all other tissues examined (liver, heart, kidney, spleen, thymus, lymph node, erythrocyte, adrenal gland, and peripheral nerve). The determinant is conserved in mammalian evolution, as the brains of the rat and dog have amounts equal to that found in human brain. Balb/c mouse brain has approximately one-third as much antigen activity as these other mammalian brains, whereas brains of the frog and chicken have no detectable antigenic activity. Developmental studies showed that 16-week human foetal brain and neonatal dog brain had little or no antigen activity, indicating a dramatic increase in the amount of the determinant with brain maturation. Biochemical studies showed that the F3-87-8-bearing molecule was a major sialoglycoprotein of human brain with an apparent molecular weight of 130,000. It was shown by immunofluorescence to be particularly localised in what appeared to be fibre tracts in the thalamus and basal ganglia, and in the dentate nucleus, although all regions including grey matter were stained.     

Alternative Pathways of Glucose Utilization in Brain; Changes in the Pattern of Glucose Utilization in Brain Resulting from Treatment of Rats with 6-Aminonicotinamide

The effect of 6-aminonicotinamide (6AN) treatment on the activities of alternative pathways of glucose metabolism in 20-day-old rat brain was evaluated by measurements of yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, or 6 and uniformly labeled glucose, and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. At the highest dose of 6AN used (35 mg/kg body weight) there was a significant decrease in the 14CO2 yields via the pentose phosphate pathway, the glycolytic route, tricarboxylic acid (TCA) cycle, and via the glutamate-gamma-aminobutyric acid pathway. Giving a graded series of doses (20-35 mg 6AN/kg body weight) revealed a hierarchy of responses in which the pentose phosphate pathway, lactate, glyceride-glycerol, and fatty acid formation were most sensitive, followed, in sequence, by the pyruvate dehydrogenase reaction, the glutamate-gamma-aminobutyrate route and, finally, the TCA cycle. The nature of the blocks in the various pathways was examined by the use of metabolite profiles.     

Rat Brain 3-Hydroxy-3-Methylglutaryl-CoA Reductase: Subcellular Distribution and Cold Lability

Abstract: Data are provided indicating that the rat brain 3-hydroxy-3-methyl-glutaryl-CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half-life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3-hydroxy-3-methylglutaryl-CoA reductase in rat brain is discussed.     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

Use of [3H] Spiperone for Labelling Dopaminergic and Serotonergic Receptors in Bovine Caudate Nucleus

Abstract— [³H]Spiperone binding has been used to study neurotransmitter receptors in bovine caudate nucleus in displacement and saturation binding experiments. Displacement curves for several antagonists are biphasic and can be analysed into contributions from dopaminergic and serotonergic sites. Antagonist binding at each class of sites follows the simple mass action equations for binding at a homogeneous set of sites (slope factors close to unity). Agonist displacement curves also indicate complex behaviour, but agonist binding to the dopaminergic sites alone exhibits heterogeneous properties (slope factors less than unity). Saturation binding experiments have been conducted on each class of site, defining dopaminergic binding of [³H]spiperone as that binding displaced by 0.1 m m -dopamine and serotonergic binding as that displaced by 0.3 μ m -mianserin. In each case, a single class of binding sites was detected: the binding parameters derived in this way have been used to calculate the proportions of the two classes of binding site observed in displacement experiments. Good agreement was obtained between calculated and observed values.     

Endogenous opiates inhibit gastric acid secretion induced by central administration of Thyrotropin-Releasing Hormone (TRH)

Thyrotropin-releasing hormone (TRH) administered intraventricularly (ICV) to rats causes a dose-dependent increase in gastric acid secretion over a range of 0.01 μg to 10 μg in the pyloris ligated rat. The maximum increase in gastric acid secretion occurs in the first hour. This effect of TRH is not mediated by its metabolites, histidyl-proline diketopiperazine or pyroglutamyl-histidyl-proline (acid TRH). β-endorphin, D-alanine-methionine-enkephalin and the leucine-enkephalin precursor, dynorphin, all inhibit TRH-induced gastric acid secretion. Bombesin, which reduces basal gastric acid secretion had no effect on TRH-induced secretion.