Nicotinamide nucleotide synthesis in regenerating rat liver

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.     

Cyclic nucleotide levels in activated human T lymphocytes

Phytohemagglutinin (PHA)-MR69 was used to activate human peripheral blood lymphocytes. The PHA concentration in the range of 1 to 4 micrograms/ml was optimal for lymphocyte stimulation. Cell activation occurred only in the presence of Ca ions and 5 min after it was followed by an increase in cGMP but not in cAMP. Immunomodulator, methylene bisphosphonic acid (10(-7) M and 4.10(-5) M), did not influence in culture. The cAMP and cGMP levels in PHA activated cells. Methylene bisphosphonic acid similar to 1-hydroxyenthylidene-1,1-bisphosphonic acid, aminomethylene bisphosphonic acid and phosphoneacetic acid on its addition to the culture (in the range from 10(-8) to 10(-4)M) 60 min before PHA or 24 or 48 hours after PHA administration produced no effect on the [3H]-incorporation into PHA-activated human blood lymphocytes.     

Neurotubule assembly at substoichiometric nucleotide levels using a GTP regenerating system

Tubulin derived from cold depolymerized bovine microtubules has been gel filtered to obtain a tubulin preparation with only 3% of the tubulin dimers containing exchangeable [³H]-guanine nucleotide. In the presence of acetyl-P and bacterial acetate kinase, this preparation polymerizes to form microtubules which are morphologically indistinguishable from microtubules formed in the presence of excess GTP. The extent of microtubule formation at substoichiometric nucleotide levels using the GTP regenerating system exceeds the extent of assembly obtained with excess GTP. It is concluded that the exchangeable guanine nucleotide site can be virtually unoccupied in intact neurotubules and this finding indicates that GDP can “catalyze” tubule assembly in the presence of a GTP regenerating system.     

Cyclic nucleotide levels during carbachol-induced smooth muscle contractions.

Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.     

Cyclic nucleotide levels in brains of control and hypertensive rats.

Adenosine 3′, 5′-monophosphate (cyclic AMP) and guanosine 3′,5- monophosphate (cyclic GMP) levels were measured in seven brain areas of spontaneously hypertensive rats (SHR) and two groups of control rats. In cerebral cortex, hypothalamus, pons-medulla oblongata and cerebellum cyclic AMP levels were higher in SHR than in Wistar-Kyoto controls. Cyclic GMP levels were higher in SHR than in Wistar-Kyoto rats in all brain areas except for the striatum and hippocampus where the levels were lower. There were also some differences in cyclic nucleotide levels between Wistar-Kyoto and Wistar-Charles River controls.     

Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

Cyclic nucleotide levels in the brain of inbred mice in emotional stress exposure

The content of cAMP and cGMP in different brain regions was studied in C57Bl/6 and BALB/c mice at rest and upon exposure to emotional stress induced by open field technique. Interstrain differences in baseline nucleotide content, differences in nucleotide distribution in the brain regions and changes in their concentration after stress have been revealed.     

Cyclic nucleotide phosphodiesterase in vertebrates

The cellular concentration of cyclic nucleotides is largely dependent upon the activity of the enzymatic system responsible for their degradation: cyclic nucleotide phosphodiesterase. This enzymatic system thus plays a crucial role in the regulation of the multiple functions which are modulated by cyclic nucleotides in the organism. Many methodological problems, as well as the complexity of the phosphodiesterase system have long maintained a confusion in this field. Recent progresses (purification to homogeneity of some enzymatic forms, discovery of regulatory mechanisms, particularly) have brought a considerable evolution in the knowledge of the system. It is now well established that cyclic nucleotide phosphodiesterase exists under several isoenzymatic forms, the properties and distribution of which largely differ from a tissue to another. Some of these forms are relatively well characterized, while the representativity of others is still discussed. The significance of this multiplicity of isoenzymes, and their interrelationships are presently under study. A very interesting aspect in the study of this enzymatic system is that it is submitted to several physiological regulatory processes. Recent studies on this point suggest that phosphodiesterase might play a major role in the response of the organism to several hormones. These fundamental studies of phosphodiesterase system find a most interesting application in the pharmacological field. Indeed, numerous synthetic compounds which inhibit the enzyme present a strong pharmacological interest.     

Cyclic nucleotide levels in rat embryo fibroblasts treated with tumor-promoting phorbol diester.

Intracellular concentrations of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and cyclic guanosine 3':5'-monophosphate were measured in rat embryo fibroblasts stimulated to divide by either the addition of 12-0-tetradecanoyl phorbol-13-acetate (TPA) or a serum-supplemented medium change. Cyclic nucleotide levels were altered within minutes and large oscillations occurred in a reciprocal fashion over the pre-replicative and the replicative phases. Patterns of oscillating levels depended on the growth state of the cultures. Intracellular content of cyclic nucleotide similarly changed in response to either mitogenic treatment with the exception of the early alterations in cyclic AMP. The medium-change stimulation resulted within minutes in a drop of the cyclic AMP levels at confluence and a rise in growing cells when TPA-induced stimulation proceeded without altering those levels. Treatment with 4-0-methyl-phorbol didecanoate, a TPA derivative that is inactive as a tumor promoter, did not affect the cyclic nucleotide levels.     

Cyclic nucleotide PDE-3

Type 3 cyclic nucleotide phosphodiesterase (PDE-3) isoforms exhibit a high affinity (“lowK _m”) for cAMP and are specifically inhibited by cGMP and a number of pharmacological agents, which increase myocardial contractility, inhibit platelet aggregation, and increase smooth muscle relaxation. The PDE-3 family consists of at least two isozymes, PDE-3A (cardiac type) and PDE-3B (adipocyte type), with distinct tissue-specific distributions. PDE-3A mRNA is highly expressed in the cardiovascular system, whereas PDE-3B mRNA is primarily expressed in adipocytes and hepatocytes. Toward understanding potential roles of PDE-3 in diabetes mellitus, we have established a specific and sensitive RNase protection assay (RPA) for quantitating PDE-3A and PDE-3B mRNA in rat diabetic models. In fatty Zucker diabetic (ZDF) rats, PDE-3A mRNA, but not PDE-3B mRNA, was expressed in heart, whereas liver and white and brown fat tissues predominantly expressed PDE-3B mRNA. Unexpectedly, PDE-3B mRNA expression was ≈2.5 times higher than PDE-3A mRNA in aorta from both ZDF and Sprague-Dawley (SD) rats. In contrast, expression levels of PDE-3A mRNA in heart were similar in both species. With this RPA, we were thus able to compare PDE-3A and-3B mRNA levels in different tissues as well as in different rat species.     

Cyclic nucleotide signalling in malaria parasites

Cyclic nucleotides are so-called intracellular second messenger molecules used by all cells to transform environmental signals into an appropriate response. Interest in the cyclic nucleotides cAMP and cGMP in malaria parasites followed early observations that both molecules might be involved in distinct differentiation events within the sexual phase of the life cycle that is required for transmission of parasites to the mosquito vector. Completed genome sequences combined with biochemical and genetic studies have confirmed the presence of the main enzymatic components of cyclic nucleotide signalling in the parasite. Dissection of their functions is underway and is giving initial insights into some of the cellular processes, which are regulated by these signalling pathways. Malaria parasites occupy terminally differentiated red blood cells for a significant proportion of their life cycle, but although there is some evidence of potential roles for the residual host cell signalling machinery in parasite development, details are few. A major gap in our knowledge is the nature of the cell surface receptors, which might trigger cyclic nucleotide signalling in the parasite.