Incorporation Of 3H-adenine Research Articles

Growth rates and production of heterotrophic bacteria and phytoplankton in the North Pacific subtropical gyre

In field work conducted at 26°N, 155°W, in the North Pacific subtropical gyre, phytoplankton growth rates μ p estimated from 14C labeling of chlorophyll a (chl a) averaged approximately one doubling per day in the euphotic zone (0–150 m). Microbial (microalgal plus heterotrophic bacterial) growth rates μ m calculated from the incorporation of 3H-adenine into DNA were comparable to or exceeded phytoplankton growth rates at most depths in the euphotic zone. Photosynthetic rates averaged 727 mg C m −2 day −1 Phytoplankton carbon biomass, calculated from 14C labeling of chl a, averaged 7.2 mg m −3 in the euphotic zone. Vertical profiles of particulate DNA and ATP suggested that no more than 15% of particulate DNA was associated with actively growing cells. Heterotrophic bacterial carbon biomass was estimated from a two-year average at station ALOHA (22°45′N, 158°W) of flow cytometric counts of unpigmented, bacteria-size particles which bound DAPI on the assumption that 15% of the particles were actively growing cells and that heterotrophic bacterial cells contained 20 fg C cell −1 The heterotrophic bacterial carbon so calculated averaged 1.1 mg m −3 in the euphotic zone. Heterotrophic bacterial production was estimated to be 164 mg C m −2 day −1 or 23% of the calculated photosynthetic rate. Estimated heterotrophic bacterial growth rates averaged 0.97 day −1 in the euphotic zone and reached 4.7 day − at a depth of 20 m. Most heterotrophic bacterial production occurred in the upper 40 m of the euphotic zone, suggesting that direct excretion by phytoplankton, perhaps due to photorespiration or ultraviolet light effects, was a significant source of dissolved organic carbon for the bacteria.

Measurement of thyroid-stimulating immunoglobulins by incorporation of tritiated-adenine into intact FRTL-5 cells: a viable alternative to radioimmunoassay for the measurement of cAMP.

To evaluate the utility of 3H-adenine incorporation into intact rat thyroid epithelial cells (FRTL-5) as an alternative to radioimmunoassay for the measurement of cAMP following stimulation of these cells by serum thyroid-stimulating immunoglobulins from patients with Graves' disease. We determined the cAMP produced by FRTL-5 cells following incubation with serum from patients with a spectrum of autoimmune thyroid and other diseases using the 3H-adenine assay. We studied 27 patients with untreated Graves' disease, 10 with Graves' disease complicated by ophthalmopathy (all on antithyroid medication), 11 with Hashimoto's thyroiditis, five with multinodular goitre, one with thyroid carcinoma, 23 with type 1 diabetes mellitus, 19 with other autoimmune diseases and 10 controls. The 3H-cAMP produced in cells incubated with either bovine TSH (bTSH) or polyethylene glycol-precipitated serum immunoglobulins, was separated by sequential chromatography on Dowex and alumina columns, and counted. The thyroid-stimulating immunoglobulins index (3H-cAMP patient immunoglobulins/3H-cAMP control immunoglobulins) was calculated for each serum and considered positive if greater than 1.5 (+ve thyroid-stimulating immunoglobulins index, i.e. > 2 standard deviations above control). The thyroid-stimulating immunoglobulins index was correlated with measurement of thyrotrophin binding inhibitory immunoglobulins (TBII) by radioreceptor assay. The 3H-adenine assay has a sensitivity of 10(-11) M bTSH with maximal stimulation at 10(-9) M bTSH (30-fold). Twenty-five of 27 patients (92%) with untreated Graves' disease and four of 10 patients with Graves' disease complicated by ophthalmopathy had +ve thyroid-stimulating immunoglobulin indices. The thyroid-stimulating immunoglobulins index in patients with untreated Graves' disease correlated with their TBII assay result (r = 0.63, P < 0.001). In addition, the index was negative in patients with Hashimoto's thyroiditis, multinodular goitre, thyroid carcinoma, and type 1 diabetes mellitus. Of the patients with other autoimmune diseases only one (a patient with systemic lupus erythematosis) had a +ve thyroid-stimulating immunoglobulin index. Direct comparison of cAMP measurement by 3H-adenine incorporation and commercial radioimmunoassay showed an equal sensitivity to both bTSH and Graves' immunoglobulins. After cell preparation, results are obtained more quickly with the 3H-adenine assay than with a cAMP radioimmunoassay (5 hours compared to 2 days), and far more cheaply than by commercial radioimmunoassays. Measurement of thyroid-stimulating immunoglobulins using the incorporation of 3H-adenine into cAMP in FRTL-5 cells is sensitive, reproducible, rapid and specific. These features make this assay a viable alternative to RIA for the measurement of thyroid-stimulating immunoglobulins in patients with Graves's disease.

Actinomycin D inhibits the rapid increase in translatable calcitonin mRNA provoked by acute calcium stimulation.

Calcium, injected to rats, elicits a rapid increase in translatable calcitonin mRNA, by acting probably at the post-transcriptional level, as no change in calcitonin mRNA could be detected by hybridization assay. In this study we have measured calcitonin mRNA extracted from rats subjected or not to acute hypercalcemia and pretreated or not with actinomycin D. Calcitonin mRNA was quantified by its ability to direct the synthesis of calcitonin (CT) precursors in a cell free system and by hybridization to a 32P cDNA probe specific for CT mRNA. Actinomycin D, injected 5 hours before calcium administration, decreased the incorporation of 3H adenine in liver and thyroid, but did not inhibit the rise in plasma levels of calcium and CT (measured by radioimmunoassay). The antibiotic was able to inhibit the eightfold increase in translatable mRNA elicited by calcium administration in the control animals. Hybridizable CT mRNA levels were not modified by the treatments. Thus the increase in translatable CT mRNA after calcium stimulation is independent of CT secretion and is probably due to post-transcriptional modifications involving the expression of other gene(s).

A microbiological study of Guaymas Basin high temperature hydrothermal vents

Water samples and suspended particulate matter were collected from three high temperature (156–319°C) hydrothermal vents in the Guaymas Basin during July 1985, and were analysed for a variety of chemical constituents and for the presence of viable microorganisms. Our results indicate that black smoker fluids (> 150°C) are devoid of recognizable bacteria and contain negligible concentrations of ATP (<10 ng 1 -1) and low concentrations of particulate organic matter. In contrast, vent water samples collected in the hydrothermal plume at a distance of only 25 cm from the point of hot fluid discharge contain a diverse, metabolically active bacterial assemblage, high ATP concentrations (up to 372 ng 1 -1) and high concentrations of particulate organic carbon and nitrogen (up to 999 and 163 μg 1 -1, respectively). The hottest waters (> 150°C) displayed a low, but measurable level of metabolic activity (incorporation of 3H-adenine and 14C-glutamate) at temperatures ranging from 25 to 80°C, 1 atm with maximal activity at 45°C. The maximum rate of incorporation, however, was only ∼ 1% of the activity measured in the samples collected in the hydrothermal plume. The strong metabolic preference for mesophilic growth temperatures (45°C) argues against a high temperature origin. Thermophilic sulfur-respiring bacteria were isolated from a variety of source materials including black smoker vent waters (112–319°C). All positive enrichments grew at 80°C, but none survived at temperatures in excess of 93°C. We conclude that these bacterial cells did not originate from the high temperature hydrothermal vent waters. Two different sample devices (reffered to as vent cap and smoker poker) were designed and employed during in situ collection-incubation experiments. Neither the vent cap attachment-colonization experiment nor the smoker poker collection device revealed the existence of bacterial cells in the black smoker fluids, despite evidence for a significant bacterial population in the hydrothermal plume. Our field and laboratory results indicate that thermophilic and mesophilic bacteria observed in hydrothermal vent plumes are not derived from the hot hydrothermal fluids but must originate in peripheral habitats.

Accumulation of adenosine 3',5'-monophosphate in slices of rat cerebral cortex induced by alpha-adrenergic agonists. II. Studies on mechanisms underlying the interaction with adenosine.

Incubation of slices of rat cerebral cortex with the calcium ionophore A23187 produced small increases in the accumulation of adenosine 3',5'-monophosphate (cyclic AMP). While low concentrations of Ca2+ ions (e.g., 200 microM) were sometimes necessary, the presence of adenosine (e.g., 50 microM) was essential; no effect of ionophore was observed when isoproterenol or isobutylmethylxanthine was substituted for adenosine. These results are consistent with the previously advanced hypothesis that stimulation of alpha-adrenergic receptors in this issue may cause calcium mobilization and thereby produce a calmodulin-mediated stimulation of adenylate cyclase. However, there is no apparent explanation for the requirement for adenosine. In addition, the possibility that additional mechanisms may be operating was suggested by experiments in which the incorporation of 3H-adenine into cyclic AMP was examined under steady-state conditions. While brief exposure to 3H-adenine after maximal adenosine- or isoproterenol-induced accumulations had been achieved led to small increases in the specific activity of cyclic AMP, the combination of norepinephrine and adenosine (plus propranolol) produced substantial decreases in the specific activity of cyclic AMP. Since the rate of incorporation of radioactivity did not keep pace with the expansion of the cyclic AMP pool, it is possible that norepinephrine also caused some reduction in the rate of cyclic AMP degradation under these conditions. Other interpretations of these results are discussed.

Characterization of microbial activity in the surface layers of a coastal subtropical sediment

Microbial biomass, metabolic activity, and nucleic acid synthesis were measured throughout a vertlcal profile of a coastal sub-tropical marine sediment. Heterotrophic activity, as measured by the uptake and respiration of 14C-glutamic acid, varied between individual sediment cores but generally was constant with depth for a given sample. Blomass (as determined by ATP concentrations) and DNA and RNA synthesis rates (as measured by the incorporation of 3H-adenine) were consistently higher at the sediment-water interface and decreased with depth. Consequently, the biomass(ATP)-specific rate of heterotrophic uptake increased sharply wlth depth while the biomassspecific rates of RNA and DNA synthesis remained constant These data indicate the presence of a large microbial biomass associated with the sediment-water interface and a smaller but equally competent population deeper within the sediment. Furthermore, cell d iv is~on (DNA synthesis) does not appear to be directly correlated with heterotrophic activity; however, data from both methods yield important and independent information concerning the metabolism of the mlcrob~al population as a whole.

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles.

The effects of intravesicular NAD on Na+-dependent 32Pi uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-32P]-NAD or [adenine-2,8-3H]-NAD by SDS-polyacrylamide gel electrophoresis. It was found that the Na+-dependent Pi uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na+-dependent Pi uptake was not inhibited. 32P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of 3H-adenine could be detected. The results provide evidence that NAD does not inhibit Pi transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.

The mode of genetic transmission of gouty family with increased phosphoribosylpyrophosphate synthetase activity.

The mode of genetic transmission of gout and increased activity of phosphoribosylphrophosphate synthetase (PRPPS) was studied in one family. Among 15 members of Family F, two male members had gout and had PRPPS activity of erythrocyte lysates three times higher than normal subjects. Five female members had activity 2.5 times higher than normal. The difference between the activities of male and female affected members was statistically significant (P less than 0.05). To examine the genetic trait of this abnormal PRPPS, the incorporation of 3H-adenine into erythrocytes or lymphocytes was studied using autoradiography. The number of grains which show the uptake of labeled adenine into cells revealed a normal distribution pattern in two normal persons and in two male patients, and a mixed pattern of the two cell populations in two female affected members. These results suggested mosaicism in female members and X-linked dominant transmission of this trait. Thermal inactivation of PRPPS of an affected female was intermediate between that from a normal subject and that from the affected males. This result showed the heterogeneity of the PRPPS from the hemolysate of an affected female. The genotype of PRPPS on the X-chromosome was assumed and the lod score between PRPPS and Xg was also estimated. From these findings and electrophoretical study, it was suggested that the abnormal enzyme was a mutant enzyme transmitted in an X-linked dominant trait, and that the mutation occurred on the structural gene of the PRPPS.

RNA synthesis as a measure of microbial growth in aquatic environments. II. Field applications

Rates of stable ribonucleic acid (RNA) synthesis have been calculated from time-series measurements of the uptake of 3H-adenine and labeling of the intracellular adenosine triphosphate (ATP) and stable RNA pools for microbial communities from a variety of freshwater and marine ecosystems. Total rates of microbial RNA synthesis ranged from 6.6 pmol to 3.8x105 pmol of adenine incorporated into RNA l−1 h−1. Most of the variation was attributable to differences in microbial biomass; if total rate data are normalized to living microbial carbon (based on ATP measurements), the variation in specific rates of RNA synthesis is decreased by a factor of 100 to 1 000 for microbial communities from diverse environments. Light/dark experiments indicated that the uptake and incorporation of 3H-adenine are uncoupled from photophosphorylation. The measurement of rates of stable RNA synthesis of microbial assemblages in nature can yield useful information concerning in situ growth rates and the response of communities to changes in environmental conditions such as nutrient additions.

Prostaglandins, platelets, and schizophrenia.

Prostaglandin (PG) E1 enhances formation of 3H-adenosine-3',5'-cyclic monophasphate (3H-cAMP) in platelets pulse-labeled with 3H-adenine. This response was assessed as an index of receptor sensitivity and of PG function. Prostagladin E1-stimulated 3H-cAMP accumulation in paltelets from schizophrenics was significantly reduced compared with control subjects. Platelet incorporation of 3H-adenine and basal 3H-cAMP accumulation. We discuss the results in terms of the possible role of PGs in the etiopathology of schizophrenia and derivative implications for treatment.

Regulation of yeast mitochondrial DNA synthesis

A single recessive nuclear gene mutation has been isolated from strain 123.1 C of Saccharomyces cerevisiae which is conditionally deficient in mitochondrial DNA metabolism and has been termed tpi. Growth of this mutant strain in media containing galactose at 36°C causes a reduction of mitochondrial DNA synthesis as analyzed by incorporation of radioactive adenine into the mitochondrial DNA. These cells continue to grow and divide producing petite cells which are neutral and have been found to lack mitochondrial DNA as measured by radioactive incorporation of 3H-adenine into the mitochondrial DNA in the presence of cycloheximide at the permissive temperature. The rate of mitochondrial DNA synthesis of the mutant strain grown at the restrictive temperature in dextrose or glycerol containing media was found to be greatly reduced following two hours of exposure to the restrictive temperature. In addition, the action of this mutant gene has been found to be independent of the respiratory capacity of the mutant strain.

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing 3H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl- trans -zeatin, trans -zeatin, N 6-(Δ 2-isopentenyl) adenosine and N 6-(Δ 2-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N 6-(Δ 2-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of cis -zeatin and ribosyl- cis -zeatin.

Regulation of yeast mitochondrial DNA synthesis

A single nuclear gene mutation has been isolated from strain 123.1C of Saccharomyces cerevisiae which is conditionally deficient in mitochondrial DNA metabolism. Growth of the haploid in media containing dextrose, a repressing carbon source, at 36 degrees C causes the rapid cessation of mitochondrial DNA synthesis as analyzed by radioactive 3H-adenine incorporation into mitochondrial DNA. These cells continue to grow and divide giving rise to neutral petites which are devoid of mitochondrial DNA as measured by radioactive incorporation of 3H-adenine at the permissive temperature. Growth of the haploid cells in media containing glycerol, a non-repressing carbon source, at 36 degrees C does not prevent mitochondrial DNA synthesis, however, the population of cells becomes partially petite. When such petites are analyzed, they are found to be suppressive and to contain mitochondrial DNA as measured in the manner described above. The action of this mutated gene appears to involve the sunthetic aspects of mitochondrial DNA metabolism, as haploid cells prelabeled in dextrose media with 3H-adenine show no loss or degradation of mitochondrial DNA at the restrictive temperature of 36 degrees C.

Evidence for a humoral factor in unilaterally nephrectomized dogs stimulating renal growth in isolated canine kidneys.

Totally isolated canine kidneys were perfused at 37 degrees C. for 240 minutes with whole blood from dogs that had undergone a sham operation or a unilateral nephrectomy 24 hours previously. Renal blood flow, glomerular filtration rate, serum sodium, and serum osmolality were not significantly different in the experimental and control preparations. Nucleic acid synthesis as measured by 3H-adenine incorporation was 75 percent higher (p less than 0.01) in the kidneys perfused with blood from the neprectomized dogs. This result supports the theory that a blood-borne factor stimulates nucleic acid synthesis and initiates compensatory renal growth.

Macromolecule synthesis in a mutant of Saccharomyces cerevisiae inhibited by S-adenosyimethionine.

Saccharomyces cerevisiae strain 83384-B3 carries the sai-1 mutation which confers sensitivity to S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). It was shown that the mutant is impermeable to precursors of ribonucleic acid (RNA) and protein during inhibition by SAM (0.2 mM). Inhibition of uptake of adenine and uracil was nearly complete 3 h after growth in the presence of SAM and the uptake of leucine was at least 10-fold lower. The incorporation of 3H-adenine into ribosomal RNA, transfer RNA and heterodisperse RNA, believed to be messenger, was reduced 10-fold when measured after 1 h inhibition. The inhibition of growth was completely reversed by methionine (2.0 mM) in cells previously exposed to SAM for 90 min. The polysome content in cells inhibited by SAM was 25% less than the control after 4 h inhibition. Ribosome synthesis increased only about 40% in the presence of SAM and about 5-fold in the control over an 8 h period. All classes of RNA were synthesized during inhibition.

Molecular and genetic events accompanying petite induction and recovery of respiratory competence induced by ethidium bromide.

The treatment of yeast cells with high levels of ethidium bromide causes a rapid induction of respiratory deficient mutants followed by a period of recovery to respiratory competence in 60 to 70% of the cells. Prolonged exposure then results in a final irreversible phase of petite formation. Sucrose gradient sedimentation analysis of 3H-adenine labelled mtDNA indicates that limited fragmentation (to about 16-18S) occurs during the initial phase of petite induction followed by a reassembly of the fragments during the period corresponding to the recovery of respiratory competence. The reassembly is associated with an ethidium bromide insensitive incorporation of 3H-adenine into mtDNA at a level consistent with repair synthesis. Genetic analyses, based on the transmission of five markers carried on the mtDNA of "repaired rho+" clones, suggests that reassembly occurs with a high degree of fidelity, though in two of a total of twenty five clones differences in marker transmission frequency were observed which could possibly reflect an altered gene order. In addition, a description is given of the marked changes in the suppressive nature of the treated cells and the temporary reduction in the capacity for marker transmission seen to accompany the transitory fragmentation of the mtDNA. The final phase of petite induction is an energy dependent degradation of the mtDNA to produce a rho degrees culture.

Loss of mitochondrial DNA in respiration-deficient mutant of yeast induced by 4-nitroquinoline 1-oxide.

The loss of mitochondrial DNA was found in a cytoplasmic respiration-deficient mutant of yeast, strain N-1, induced by treatment of a normal yeast with 4-nitroquinoline 1-oxide. In cesium chloride density gradient centrifugation, mitochondrial DNA from the normal yeast showed a density of 1.684 g/ml, but mitochondrial fraction of respiration-deficient mutant strain N-1 had no detectable DNA at a densty of 1.684 g/ml or near it. Though an incorporation of 3H-adenine into mitochondrial DNA was amplified by the presence of cyclohemimide, the mutant had no detectable radioactivity in its mitochondrial fractions. Accordingly, it was concluded that respiration-deficient mutant strain N-1 has lost its mitochondrial DNA which was indispensable for the respiratory activity of yeast cells.

Replication of the nuclear genome in yeast does not require concomitant protein synthesis

Incorporation of 3H-adenine into nuclear DNA in a short pulse in mid-S in a synchronised culture of Saccharomyces cerevisiae was unaffected by the presence of 100 μg/ml cycloheximide. However, colorimetric DNA analyses showed that entry into S was completely blocked by adding the drug at times earlier than about 10 min before initiation of replication. Cell autoradiography of cultures labelled in various regimes showed that at this time there is a cycloheximide-transition point at which the cell acquires the capacity to both initiate and complete a whole round of replication in the presence of 100 μg/ml of cycloheximide. Thus, all the proteins required for passage through one S period are made in advance of initiation.

Effects of ions on thyrotrophin and prostaglandin E 1 stimulation of glucose oxidation and adenyl cyclase-cyclic AMP system in dog thyroid slices

Substitution of K + or choline for Na + in Krebs-Ringer bicarbonate buffer increased basal glucose oxidation and 3H-adenine incorporation into 3H-CAMP but had no effect on CAMP concentrations. In high K + buffer, TSH (3 mU/ml and 50 mU/ml) and prostaglandin E 1 (PGE 1) (0.1 μg/ml and 10 μg/ml) had little or no effect on glucose oxidation while their stimulation of 3H-CAMP formation and CAMP levels was equivalent to that obtained using regular Krebs-Ringer bicarbonate buffer. These results indicated that high K + inhibited the effects of CAMP and not its generation. The increased basal glucose oxidation in high K + buffer probably reflected increased membrane permeability since slices incubated with 14C-1-glucose and regular Krebs-Ringer bicarbonate buffer did not have augmented 14CO 2 production when they were later transferred to high K + buffer. The increased 3H-adenine incorporation into 3H-CAMP cannot be explained by increased membrane permeability, but appeared to reflect an effect on the intracellular conversion of 3H-adenine to 3H-ATP or 3H-ATP to 3H-CAMP. The effect of high K + probably reflected low Na + in the buffer since similar results were obtained when choline chloride was substituted for NaCl in Krebs-Ringer bicarbonate buffer. Exclusion of Ca ++ from Krebs-Ringer bicarbonate buffer decreased basal glucose oxidation, but had no effect on 3H-adenine conversion to 3H-CAMP or CAMP concentrations. Although effects of TSH and PGE 1 on glucose oxidation were diminished in buffer devoid of Ca ++, their stimulation of 3H-CAMP formation and CAMP levels was similar in the presence and absence of Ca ++. These results demonstrate that changes in the ionic composition of the buffer do not interfere with activation of the adenyl cyclase-CAMP system but modify some of the subsequent actions of the CAMP formed.

Effects of chlorpromazine, propranolol and phospholipase C on thyrotropin and prostaglandin stimulation of adenyl cyclase-cyclic AMP system in dog thyroid slices

The effect of chlorpromazine, propranolol and phospholipase C on the TSH and PGE 1, stimulation of the adenyl cyclase-cyclic AMP system and 14C-1-glucose oxidation was studied in dog thyroid slices. Chlorpromazine (2 × 10 −4 M) completely inhibited TSH (3 mU./ml.) stimulation of glucose oxidation, 3H-adenine incorporation into 3H-cyclic AMP and cyclic AMP concentrations. The inhibition could be partially overcome by increasing the amount of TSH to 50 mU./ml. Chlorpromazine also reduced the stimulation of 3H-cyclic AMP formation and cyclic AMP concentrations induced by prostaglandin E 1 (1 μg./ml.). Propranolol (10 −3 M) markedly diminished the TSH (5 mU./ml.) augmentation of adenyl cyclase activity, 3H-cyclic AMP formation and cyclic AMP levels while 10 −4 M propranolol had no effect. Neither concentration of propranolol nor chlorpromazine influenced basal levels of these parameters. Incubation of thyroid slices with phospholipase C was associated with some increase in basal glucose oxidation but marked reduction of the subsequent TSH (10 mU./ml.) stimulation of 14CO 2 production, cyclic AMP levels and 3H-cyclic AMP formation. These results are all compatible with the concept that the chlorpromazine, propranolol and phospholipase C inhibition of TSH stimulation of glucose oxidation reflect and effect on adenyl cyclase and cyclic AMP generation. Prostaglandin E 1 and F 1α (100 mg./ml.) increased 32P incorporation into phospholipids while an equivalent amount of prostaglandin B 1 had no effect and A 1 inhibited 32P incorporation. These results provide further evidence that stimulation of 32P incorporation into phospholipids may be independent of changes in cyclic AMP concentrations.