Increase In AMP Levels Research Articles

Gene expression and metabolite levels converge in the thermogenic spadix of skunk cabbage.

The inflorescence (spadix) of skunk cabbage (Symplocarpus renifolius) is strongly thermogenic and can regulate its temperature at around 23 °C even when the ambient temperature drops below freezing. To elucidate the mechanisms underlying developmentally controlled thermogenesis and thermoregulation in skunk cabbage, we conducted a comprehensive transcriptome and metabolome analysis across 3 developmental stages of spadix development. Our RNA-seq analysis revealed distinct groups of expressed genes, with selenium-binding protein 1/methanethiol oxidase (SBP1/MTO) exhibiting the highest levels in thermogenic florets. Notably, the expression of alternative oxidase (AOX) was consistently high from the prethermogenic stage through the thermogenic stage in the florets. Metabolome analysis showed that alterations in nucleotide levels correspond with the developmentally controlled and tissue-specific thermogenesis of skunk cabbage, evident by a substantial increase in AMP levels in thermogenic florets. Our study also reveals that hydrogen sulfide, a product of SBP1/MTO, inhibits cytochrome c oxidase (COX)-mediated mitochondrial respiration, while AOX-mediated respiration remains relatively unaffected. Specifically, at lower temperatures, the inhibitory effect of hydrogen sulfide on COX-mediated respiration increases, promoting a shift toward the dominance of AOX-mediated respiration. Finally, despite the differential regulation of genes and metabolites throughout spadix development, we observed a convergence of gene expression and metabolite accumulation patterns during thermogenesis. This synchrony may play a key role in developmentally regulated thermogenesis. Moreover, such convergence during the thermogenic stage in the spadix may provide a solid molecular basis for thermoregulation in skunk cabbage.

Metabolic and microbiota response to arginine supplementation and cyclic heat stress in broiler chickens.

Little attention has been paid to the biological role of arginine and its dietary supplementation in broilers under heat stress (HS) conditions. Therefore, the main aim of this study was to assess the response of broilers to arginine supplementation and cyclic HS, with a focus on liver, pectoral muscle, and blood metabolic profiles and the cecal microbiota. Day-old male Ross 308 broilers (n = 240) were placed in 2 rooms with 12 pens each for a 44-day trial. Pens were assigned to one of two groups (6 pens/group/room): the control group (CON) was given a basal diet in mash form and the treated group (ARG) was fed CON diet supplemented with crystalline L-arginine. The total arginine:lysine ratio of CON diet ranged between 1.02 and 1.07, while that of ARG diet was 1.20. One room was constantly kept at thermoneutral (TN) conditions, while the birds in the other room were kept at TN conditions until D34 and subjected to cyclic HS from D35 onwards (∼34°C; 9:00 A.M.-6:00 P.M.). Blood, liver, Pectoralis major muscle, and cecal content were taken from 2 birds per pen (12 birds/group/room) for metabolomics and microbiota analysis. Growth performance data were also collected on a pen basis. Arginine supplementation failed to reduce the adverse effects of HS on growth performance. Supplemented birds showed increased levels of arginine and creatine in plasma, liver, and P. major and methionine in liver, and reduced levels of glutamine in plasma, liver, and P. major. HS altered bioenergetic processes (increased levels of AMP and reduced levels of fumarate, succinate, and UDP), protein metabolism (increased protein breakdown to supply the liver with amino acids for energy production), and promoted the accumulation of antioxidant and protective molecules (histidine-containing dipeptides, beta-alanine, and choline), especially in P. major. Arginine supplementation may have partially counterbalanced the effects of HS on energy homeostasis by increasing creatine levels and attenuating the increase in AMP levels, particularly in P. major. It also significantly reduced cecal observed diversity, while HS increased alpha diversity indices and affected beta diversity. Results of taxonomic analysis at the phylum and family level are also provided.

An AMP-activated protein kinase complex with two distinctive alpha subunits is involved in nutritional stress responses in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions, such as carbon source, pH, temperature and osmolarity, in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses such as osmotic stress, hypoxia, ischaemia and exercise that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study we report the characterization of AMPK complexes for the first time in T. cruzi and propose the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We show that there is phosphotransferase activity specific for SAMS peptide in epimastigotes extracts, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression.

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle.

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid β-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD+ levels following changes in NAD+ -dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD+ ) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.

AICAR-Dependent Activation of AMPK Kinase Is Not Accompanied by G1/S Block in Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) have the capacity for self-renewal and pluripotency. Due to high proliferative activity, ESCs use a specific pathway of the formation of ATP molecules, which can lead to the development of the adaptive metabolic response under the conditions of energy deficiency (which is different from the response of differentiated cells). It is known that metabolic signals are integrated with the cell cycle progression; however, the signaling pathways that connect the availability of nutrients with the regulation of cell cycle in ESCs are insufficiently studied. We have studied the effect of the AICAR agent, which imitates an increase in AMP level and induces the activation of the metabolic sensor AMPK, on proliferation, cell cycle distribution, and pluripotency of mouse ESCs (mESCs). It has been demonstrated that cells treated with AICAR do not stop at the control G1/S point of the cell cycle, since they do not accumulate P21/WAF1 (G1/S checkpoint regulator), despite P53 activation. On the contrary, AICAR increases the rate of mESC proliferation, which correlates with increased expression of pluripotency marker genes (OCT3/4, NANOG, SOX2, KLF4, ESRRB, PRDM14). In addition, an increase in the transcription of the HIFlα gene (a key regulator of the cell proliferation and viability, as well as glucose metabolism under stress) was detected. An increase in the expression of glycolytic enzyme genes (LDHA, ALDOA, PCK2, GLUT4) under the effect of AICAR indicates a change in mESC metabolism towards increased glycolysis. Thus, AICAR-dependent AMPK activation as one of possible mechanisms of the mESC adaptive response to the emergence of energetic imbalance is not accompanied by a cell cycle arrest at the G1/S checkpoint, but involves the processes of increasing glycolytic activity.

The metabolism of 9-chloro-β-lapachone and its effects in isolated hepatocytes. The involvement of NAD(P)H:quinone oxidoreductase 1 (NQO1)

A β-lapachone analogue (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione) (9-chloro β-lapachone), named CGQ, with antitumoral, antiviral and antitrypanocidal activities was assayed for cytotoxic effects on isolated rat hepatocytes. The incubation of hepatocytes with this o-naphthoquinone showed (a) decreased adenylate energy charge, as a result of a decrease in ATP, and an increase in AMP levels; (b) increased NADP+ content, with a concomitant decrease of NADPH, NADH and NAD+ content; (c) decreased GSH content, accompanied by an increase in GSSG formation; (d) stimulated oxygen uptake as well as increased superoxide anion production and hydrogen peroxide formation; (e) inhibited lipid peroxidation; (f) hepatocyte viability was not reduced unless the NQO1 inhibitor dicoumarol was present. We hypothesize that the cytotoxicity of CGQ in dicoumarol-treated hepatocytes was the result of inhibition of the NQO1 detoxification pathway, thus allowing more quinone to be metabolized towards the one-electron pathway to form reactive semiquinones and/or reactive oxygen species. The results obtained indicate a protective role of NQO1 in preventing CGQ cytotoxicity in isolated rat hepatocytes.

Why does the brain (not) have glycogen?

In the present paper we formulate the hypothesis that brain glycogen is a critical determinant in the modulation of carbohydrate supply at the cellular level. Specifically, we propose that mobilization of astrocytic glycogen after an increase in AMP levels during enhanced neuronal activity controls the concentration of glucose phosphates in astrocytes. This would result in modulation of glucose phosphorylation by hexokinase and upstream cell glucose uptake. This mechanism would favor glucose channeling to activated neurons, supplementing the already rich neuron-astrocyte metabolic and functional partnership with important implications for the energy compounds used to sustain neuronal activity. The hypothesis is based on recent modeling evidence suggesting that rapid glycogen breakdown can profoundly alter the short-term kinetics of glucose delivery to neurons and astrocytes. It is also based on review of the literature relevant to glycogen metabolism during physiological brain activity, with an emphasis on the metabolic pathways identifying both the origin and the fate of this glucose reserve.

PRKA/AMPK: Integrating Energy Status with Fertility in Pituitary Gonadotrophs

Polycystic ovarian syndrome (PCOS) is a common reproductive disorder, characterized by irregular menses, hyperandrogenism, and polycystic ovaries. Patients with PCOS also commonly suffer from infertility, obesity, and insulin resistance [1]. The biochemical changes that are commonly associated with PCOS are elevated luteinizing hormone (LH) to follicle-stimulating hormone (FSH) ratios, elevated bioavailable and total testosterone levels, and elevated insulin levels associated with insulin resistance. Historically considered to be a hyperandrogenic disease, PCOS has been treated by focusing on weight reduction and alleviating the symptoms associated with excess androgens when pregnancy was not desired or on the induction of ovulation with the antiestrogen clomiphene citrate when pregnancy was desired. However, effective treatment of PCOS symptoms with insulin-sensitizing agents for the treatment of type II diabetes has resulted in an increasing emphasis on insulin resistance in the pathogenesis of this disease [2]. Currently, the biguanide insulin-sensitizing agent metformin is increasingly used both to alleviate the symptoms of PCOS and to treat the associated infertility. The successful use of this therapy has resulted in a closer examination of the mechanism of action by metformin at targets throughout the hypothalamicpituitary-gonadal axis. In the treatment of type II diabetes, metformin decreases hepatic glucose production and increases glucose consumption in peripheral tissues, resulting in decreased blood glucose levels, decreased insulin release, and decreased fat accumulation [3]. At the cellular level, metformin inhibits the mitochondrial electron-transport chain at complex I to decrease ATP production; the resulting increase in AMP levels activates the AMP-dependent kinase (AMPK; official symbol PRKA). PRKA serves as a regulator of cellular and systemic energy homeostasis [3]. PRKA is a heterotrimeric protein consisting of alpha, beta, and gamma subunits. It can be activated by any cellular stress that increases AMP levels by means of the allosteric binding of AMP to sites in the gamma subunit [4] as well as through phosphorylation of Thr172 in the alpha subunit by serine/threonine kinase 11 (STK11/LKB1), calcium/calmodulin-dependent protein kinase kinase (CAMKK), and most recently, the transforming growth factor-b-activated kinase (TAK1) [4–6]. A number of hormones that regulate energy homeostasis (leptin, ghrelin, adiponectin, and resistin) have also been shown to exhibit tissue-specific stimulation or inhibition of PRKA kinase [4, 7]. Treatment with metformin may improve reproductive health through a combination of effects on multiple organ systems, resulting in increased insulin sensitivity and normalization of insulin, insulin-like growth factor 1, and androgen levels. The study by Tosca et al. [8] provides a novel point of integration between mediators of energy homeostasis and regulation of reproductive function at the level of the pituitary gland. Using both pharmacologic and molecular tools, the authors have presented compelling data to link this energysensing pathway to both activin-mediated and gonadotropinreleasing hormone (GNRH)-mediated regulation of gonadotropin expression [8]. Because PCOS is associated with elevated LH levels, the authors first asked if metformin treatment could alter the secretion of LH and FSH. Using primary rat pituitary cultures, these investigators determined that metformin had no effect on its own but was able to inhibit LH release in response to GNRH. FSH release was inhibited by metformin in response to both GNRH and activin. Because metformin activates PRKA, the authors also examined the expression of the various PRKA subunits in rat pituitaries and primary cultures of rat pituitary cells. They assessed expression of the six PRKA subunits by RT-PCR and Western blot analysis. They used immunofluorescent analysis to assess the expression of the alpha 1 subunit of PRKA and colocalize its expression with protein hormone expression from each of the five cell types in the anterior pituitary gland. This subunit was strongly expressed in gonadotrophs and thyrotrophs but less so in somatotrophs and lactotrophs, and it was undetectable in corticotrophs. An earlier study indicated that corticotrophs primarily express the alpha 2 subunit of the kinase. For a negative control, the authors showed that no staining occurred in Prkaa1 knockout mouse pituitaries. The authors next examined the effect of metformin on PRKAA-activating phosphorylation and a downstream target of PRKA, acetyl-coenzyme A carboxylase. They demonstrate doseand time-dependent changes in phosphorylation of these targets. Using compound C, a specific inhibitor of PRKA activity, they verified the role of PRKA in the inhibition of LH and FSH release by metformin. Similarly, a dominant negative PRKA construct also blocked the ability of metformin to inhibit Fshb mRNA transcription in response to activin A. The role of SMAD2, AKT, mitogen-activated protein kinase (MAPK) 14 (p38), and MAPK3/1 (Erk1/2) in activin A and GNRH signaling was examined as well, and a time course of response for those activators was established. Having validated this model, the authors determined that metformin treatment is Correspondence: FAX: 970 297 1275; e-mail: dawn.duval@colostate.edu

Metabolic and Developmental Adaptations of Growing Potato Tubers in Response to Specific Manipulations of the Adenylate Energy Status

Heterotrophic carbon metabolism has been demonstrated to be limited by oxygen availability in a variety of plant tissues, which in turn inevitably affects the adenylate status. To study the effect of altering adenylate energy metabolism, without changing the oxygen supply, we expressed a plastidially targeted ATP/ADP hydrolyzing phosphatase (apyrase) in tubers of growing potato (Solanum tuberosum) plants under the control of either inducible or constitutive promoters. Inducible apyrase expression in potato tubers, for a period of 24 h, resulted in a decrease in the ATP-content and the ATP-ADP ratio in the tubers. As revealed by metabolic profiling, this was accompanied by a decrease in the intermediates of sucrose to starch conversion and several plastidially synthesized amino acids, indicating a general depression of tuber metabolism. Constitutive tuber-specific apyrase expression did not lead to a reduction of ATP, but rather a decrease in ADP and an increase in AMP levels. Starch accumulation was strongly inhibited and shifted to the production of amylopectin instead of amylose in these tubers. Furthermore, the levels of almost all amino acids were decreased, although soluble sugars and hexose-Ps were highly abundant. Respiration was elevated in the constitutively expressing lines indicating a compensation for the dramatic increase in ATP hydrolysis. The increase in respiration did not affect the internal oxygen tensions in the tubers. However, the tubers developed a ginger-like phenotype having an elevated surface-volume ratio and a reduced mass per tuber. Decreased posttranslational redox activation of ADP-glucose pyrophosphorylase and a shift in the ratio of soluble starch synthase activity to granule-bound starch synthase activity were found to be partially responsible for the alterations in starch structure and abundance. The activity of alcohol dehydrogenase was decreased and pyruvate decarboxylase was induced, but this was neither reflected by an increase in fermentation products nor in the cellular redox state, indicating that fermentation was not yet induced in the transgenic lines. When taken together the combined results of these studies allow the identification of both short- and long-term adaptation of plant metabolism and development to direct changes in the adenylate status.

Energy Metabolic Changes in the Early Post-injury Period Following Traumatic Brain Injury in Rats

Impaired cerebral energy metabolism may be a major contributor to the secondary injury cascade that occurs following traumatic brain injury (TBI). To estimate the cortical energy metabolic state following mild and severe controlled cortical contusion (CCC) TBI in rats, ipsi-and contralateral cortical tissues were frozen in situ at 15 and 40 min post-injury and adenylate (ATP, ADP, AMP) levels were analyzed using high-performance liquid chromatography (HPLC) and the energy charge (EC) was calculated. At 15 min post-injury, mildly brain-injured animals showed a 43% decrease in cortical ATP levels and a 2.4-fold increase in AMP levels (P < 0.05), and there was a significant reduction of the ipsilateral cortical EC when compared to sham-injured animals (P < 0.05). At 40 min post-injury, the ipsilateral adenylate levels and EC had recovered to the values observed in the sham-injury group. In the severe CCC group, there was a 51% decrease in ipsilateral cortical ATP levels and a 5.3-fold increase in AMP levels with a significant reduction of cortical EC at 15 min post-injury (P < 0.05). At 40 min post-injury, a 2.6-fold ipsilateral increase in AMP levels and an 11% and 44% decrease in EC and ATP levels, respectively, remained (P < 0.05). A 37-38% reduction of the total adenylate pool was observed ipsilaterally in both CCC severity groups at the early time-point, and a 19% and 28% decrease remained in the mild and severe CCC groups, respectively, at 40 min post-injury. Significant contralateral ATP and EC changes were only observed in the severe CCC group at 40 min post-injury (P < 0.05). The energy-requiring secondary injury cascades that occur early post-injury do not challenge the brain tissue to the extent of ATP depletion and may provide a window of opportunity for therapeutic intervention.

High-energy phosphate compounds in spermatozoa of the sea urchins Arbacia lixula and Paracentrotus lividus

Changes in the levels of high-energy phosphate compounds following incubation in seawater were measured in spermatozoa of the sea urchins Arbacia lixula and Paracentrotus lividus, which belong to the orders Arbacioida and Echinoida, respectively. Following incubation in seawater, the levels of ATP in both A. lixula and P. lividus spermatozoa decreased rapidly. In contrast, a remarkable increase in AMP levels was observed, although ADP levels changed slightly. A. lixula and P. lividus spermatozoa contained high concentrations of creatine phosphate, but arginine phosphate was present at a trace level. The creatine phosphate levels in these spermatozoa decreased immediately following incubation in seawater. There were high activities of creatine kinase and adenylate kinase in A. lixula and P. lividus spermatozoa. However, arginine kinase activity could not be detected. These results suggest that creatine phosphate is able to substitute for ATP in both A. lixula and P. lividus spermatozoa. It is also possible that not only the creatine-phosphate shuttle but also adenylate kinase plays an important role for ATP supply to use their flagellar motility.

Effect of catecholamines on Na/H exchange in vascular smooth muscle cells.

Catecholamines were found to activate Na/H exchange in a concentration-dependent manner in primary cultures of vascular smooth muscle cells (VSMC). The potency order was found to be epinephrine greater than norepinephrine greater than isoproterenol. The major pathway for catecholamine effects appeared to be via interaction with an alpha 1 adrenergic receptor. In addition, it was found that alpha 1 receptor-mediated Na/H exchange in VSMC was increased by angiotensin II and inhibited by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Adrenergic receptors have been shown to be coupled to both adenylate cyclase and to inositol phosphate release (Leeb-Lundberg, L. M. F., S. Cotecchia, J. W. Lomasney, J. F. DeBernadis, R. J. Lefkowitz, and M. G. Caron, 1985, Proc. Natl. Acad. Sci. USA, 82:5651-5655.). It was found that catecholamines increased AMP levels in the potency order isoproterenol greater than norepinephrine greater than epinephrine and the receptor involved was a beta adrenergic receptor. Since these findings did not parallel the results obtained for catecholamine stimulation of Na/H exchange, an increase in AMP levels was probably not the mechanism by which major pathway for catecholamine-stimulated Na/H exchange in VSMC (via the alpha 1 receptor) was activated. When the effects of catecholamines were measured on inositol phosphate release, the potency order for catecholamine stimulation was epinephrine greater than norepinephrine greater than isoproterenol, and the receptor involved was an alpha 1 adrenergic receptor. In addition, angiotensin II increased and TPA inhibited catecholamine-stimulated inositol phosphate release. Since these findings paralleled the results obtained for catecholamine stimulation of Na/H exchange, inositol phosphate release may be the mechanism by which the major pathway for catecholamine-stimulated Na/H exchange in VSMC (via the alpha 1 receptor) was activated.

Adenylate and Nicotinamide Nucleotides in Developing Wheat Grains

Contents of ATP and ADP per grain increased during grain development up to 21-35 d after anthesis (DAA). A sharp increase in AMP level was observed at 42 DAA. Of the four nicotinamide nucleotides, NAD was most abundant. Energy charge values were more than 0.7 during active grain filling stages and less than 0.6 when rate of starch deposition was low.

Variations in the Adenylate Levels During Phosphate Depletion in Isolated Soybean Cells and Wheat Leaf Fragments

Summary The adenylate contents and energy charge values of isolated soybean leaf cells and wheat leaf fragments were determined after incubation with non metabolizable sugars (glucosamine or mannose) which are considered to sequestrate the intracellular inorganic phosphate. Phosphate sequestration resulted in slightly diminished energy charge values and in a decrease in total adenylate levels. No AMP accumulation was observed. The adenylate levels were maintained when the sugar was added together with an equimolecular amount of inorganic phosphate. The intracellular inorganic phosphate contents were only slightly decreased by glucosamine but a dramatic inhibition of the photosynthetic CO 2 fixation activity was observed. The results suggest a regulation of the intracellular adenylate levels by inorganic phosphate the function of which would be to prevent large variations in energy charge and sharp increases in AMP levels.

Adenylate and Nicotinamide Nucleotides in Developing Soybean Seeds During Seed-Fill

Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity.Of the oxidized (NAD, NADP) and reduced (NADH, NADPH) nicotinamide nucleotide forms, NAD was the most abundant. Levels as high as 17.5 micrograms per seed were observed during the mid-seed-filling stages. NADP was found almost exclusively in the reduced form with a NADP: NADPH ratio of less than 0.35, whereas the reverse was noted for NAD which was found mainly in the oxidized form with a NAD:NADH ratio in the range of 5 to 25. NADP was detected in low concentrations compared to the other adenylate and nicotinamide nucleotides. The nicotinamide redox charge defined as (NADH + NADPH)/(NAD + NADH) + (NADP + NADPH) was calculated to express the state of the energy balance between the oxidized and reduced nicotinamide nucleotide forms. The nicotinamide redox charge varied between 0.15 and 0.30 during seed development and was significantly lower than that found for the adenylate energy charge.

Cycles of agglutination-disagglutination induced by ristocetin in thrombasthenic platelets.

An oscillatory pattern of platelet agglutination-disagglutination in response to Ristocetin (R) at narrow concentration ranges was observed in citrated platelet rich plasma (PRP) of 10 patients with Glanzmann's thrombasthenia. The cyclic pattern decreased in intensity over time, was reproducible, and was not pH dependent. Formalin-fixed thrombasthenic platelets agglutinated with R but did not show a cyclic pattern. Incubation with 2.5 microM ADP inhibited R oscillation response, but small increases in R dose overcame this inhibition. The addition of ATP or creatine phosphate/creatine phosphokinase to thrombasthenic platelets inhibited by ADP restored the R oscillation response. In the platelets of a single patient, intracellular levels of ADP and ATP were shown to diminish during an oscillation response to R. There was an increase in AMP levels during the same period of time. The changes in these three intracellular nucleotides were gradual over time and did not vary with phases of the oscillation. Acetyl salicylic acid (ASA), at concentrations shown to block cyclooxygenase activity in control platelets, enabled thrombasthenic platelets to respond to R with full agglutination without oscillations. Lower concentrations of ASA in the PRP gave a return of the oscillation response. Our data suggest that the disagglutination phase of the R response of thrombasthenic platelets is not a function of the known glycoprotein membrane defect, but depends on materials originating in the platelet whose release is blocked by ASA.