Saturation of anti cyclic nucleotides antibodies by endogenous cyclic nucleotides.

Rabbits immunized against cyclic AMP or cyclic GMP produce antibodies which are fully saturated by their respective endogenous cyclic nucleotides. This was proved a) in comparing radioimmunological measurements of cyclic nucleotides in antiserum and the binding site concentration determined by equilibrium dialysis, b) in showing the ineffectiveness of serum phosphodiesterase to hydrolyze the cyclic AMP present in the anti-cyclic AMP antiserum. Immunological and radioimmunological implications of this phenomenon are discussed.     

Regulation and function of cyclic nucleotides.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.     

Phosphodiesterase of cyclic nucleotides

Problems are considered on form multiplicity, purification and molecular weight of cyclic nucleotides phosphodiesterase. A supposition is made that the molecular weight of the catalytic subunit of the enzyme for most studied objects is about 60000. The catalytic subunit may form di- and trimers and be associated with regulatory proteins of different type. The problem of phosphodiesterase regulation is analyzed on the basis of potentialities of the equilibrium shift between the protein subunits of the enzyme; the role of cyclic nucleotides as well as of triphosphonucleotides are shown to influence the regulation of the enzymic activity. In some cases the mechanism of changes in the activity of phosphodiesterase bound with the receptor is shown to be similar to that for adenylate cyclase. In particular, the role of GTP and one of the protein subunits of phosphodiesterase in this process is stated.     

Effect of serotonin on cyclic nucleotides of human platelets.

Serotonin produced a 6 to 10 fold increase of cyclic GMP over baseline levels of this nucleotide in platelets. Maximum stimulation was reached within 30 sec to 1 min after addition of serotonin and was dependent upon its concentration in the medium. Inhibition of serotonin uptake by methysergide, dihydroergotamine and chloroimipramine did not influence the serotonin-induced stimulation of cyclic GMP but glutaraldehyde and formaldehyde blocked it completely. Cyclic AMP levels in platelets were not affected by serotonin. The serotonin-induced stimulation of cyclic GMP is independent of the uptake of this biogenic amine by platelets and is not due to platelet aggregation.     

Role of cyclic nucleotides in modulating ovarian hCG action.

The role of cyclic nucleotides in mediating hormonally responsive adenylate cyclase and cAMP-dependent protein kinase was examined in vivo and in vitro when pseudopregnant rats were injected with hCG. Intracellular ovarian levels of cAMP increased, as expected, but no change in cGMP concentrations was observed. However, both cGMP and cAMP activated ovarian CDPK holoenzyme in vitro but cGMP had a lower affinity. The subunits of hCG were without effect. Even though cGMP and cAMP dissociate partially purified ovarian CDPK holoenzyme in vitro, the receptor sites of the regulatory subunit of CDPK would appear to be relatively specific for cAMP. Moreover, cGMP probably does not mediate hCG action in vivo.     

Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.     

The role of cyclic nucleotides and prostaglandins in heart function.

A short review of the role of cyclic nucleotides and prostaglandins (PGs) in normal and pathological functions of the heart is given. Possible interrelationships of these two regulatory systems have been studied by using spontaneously beating rat atria preparations. Addition of noradrenaline (NA) to the incubate (1 . 10(-6) M) caused an increase in amplitude and frequency which was preceded and parallelled by an elevation of the tissue cAMP level. A transient increase in cGMP and PGE values was also seen. Propranolol (5 . 10(-6) M) abolished the increase in amplitude and frequency as well as in cAMP and PGE concentrations. Indomethacin (1 . 10(-5) M) inhibited the formation of PGE. The increase in cGMP was blocked by phenoxybenzamine. Interchange between beta- and alpha-receptors according as the temperature is lowered has been described earlier. Hypothermia (20 degrees C) had a positive inotropic effect on the atria and increased the tissue cAMP concentration. Loading of the atria caused an increase in cAMP without any effects on cGMP or PGs. Slight hypoxia did not change the cAMP or PG levels, but elevated the cGMP values. Arrhythmias induced by hypo- or hyperpotassemia did not modify the biochemical parameters measured. PGF2alpha (1. 10(-5) M) normalized the atrial rhythm and increased the amplitude without changing cyclic nucleotide or PG levels. PGE1 (1 . 10(-4) M) increased the amplitude of normorhythmic atria and the tissue concentration of cAMP. PGE2 was the only PG tested which stimulated the heart adenylate cyclase in vitro. There seems to be close but complicated relationships between cyclic nucleotides and PGs in the heart.     

Antibody-dependent lymphocyte mediated cytotoxicity mechanism and modulation by cyclic nucleotides.

The injury to antibody coated thymocytes by the “K cell” among nonsensitized rat splenocytes was modulated by altering the cellular level of cAMP and cGMP. Preincubation of the attacking cell population with 1 μg/ml cholera toxin caused an elevation of cAMP levels of 1–18 pM per 10⁷ cells and was associated with a proportionate reduction in cytotoxicity. Agents which are known to raise cAMP levels including the Prostaglandins (PG) E₁ (10^?7–10^?5 M), PGF_2α (10^?5–10^?3 M), PGA₁ (10^?9–10^?5M), and theophylline (10^?3 M) also produced marked suppression of antibody dependent lymphocyte mediated cytotoxicity. Direct elevation of cellular levels of cGMP by the analog 8-bromo cGMP (5 × 10^?6M) led to augmentation of cytotoxicity. Removal by EDTA and MgEDTA of calcium and magnesium ions from the culture media markedly inhibited cytotoxicity.     

Regulation of cyclic nucleotides in retinal photoreceptors

Summary The distribution of cyclases in retinal photoreceptors of dark- and light-adapted brook trout was studied by means of a cytochemical method (lead precipitation). It confirms earlier reports that retinal photoreceptors contain high levels of cyclic nucleotides, and that cAMP predominates in cones and cGMP in rods. There is an apparent difference in the level of the cyclases with the adaptive states. In addition, the catalytic unit of cyclase is interlamellar in cones. In rods, adenylate cyclase is intradiscal, while the location of guanylate cyclase varies with the adaptive state. The variation of cyclase with adaptation indicates that this enzyme has a role in the process of visual transduction.     

Prostaglandins, cyclic nucleotides and cell function

Based upon the ability of the E-prostaglandins to stimulate cyclic AMP formation in a dose-related manner and the correlation between this property and their affinity for a membraneous receptor, the action of these prostaglandins was proposed to be expressed largely via cyclic AMP. The failure of the F-prostaglandins to demonstrate significant activity in these two parameters led to the suggestion that they must act at another receptor via a different mediator. The recent isolation of a receptor unique to PGF_2α and the demonstrated ability of this prostaglandin to increase tissue cyclic GMP levels are consistent with this concept that the E-prostaglandins and PGF_2α play distinctly different roles in cell regulation.     

Influence of cyclic nucleotides on amphibian ectoderm

Summary Isolated amphibian (Triturus alpestris) gastrula ectoderm was treated with cyclic nucleotides for 24 h and cultured up to 12 days. Explants treated with$cyclic N⁶-Monobutyryl-adenosine-35-monophosphate, cyclic Dibutyryladenosine-35-monophosphate and cyclic Dibutyrylguanosine-35-monophosphate in a concentration of 10^–3 and 10^–5 M did not differentiate into mesoderm- or endoderm-derived tissues. The number of explants with small neural and neuroid structures did not exceed the percentage found in the control series. Inductions could also not be obtained when ectoderm was dissociated prior to the treatment with cyclic nucleotides, or when theophylline (which inhibits phosphodiesterase) was added to the culture medium. The results are discussed with regard to the possible mode of action of the vegetalizing factor.     

Effect of cyclic nucleotides on the cyanide-insensitive respiration of polymorphonuclear leucocytes.

The effects of N6,O2'-dibutyryladenosine 3',5'-monophosphate (Bu2-cyclic AMP) and N2,O2'-dibutyrylguanosine 3',5'-monophosphate (Bu2-cyclic GMP) on the cyanide-insensitive respiration of guinea pig peritoneal exudate polymorphonuclear leucocytes were studied. Bu2-cyclic AMP inhibited the respiration induced both by phagocytosis of E. coli and by the interaction with trypsin-digested rat liver microsomes. The addition of theophylline gave rise to an inhibitory pattern similar to that with Bu2-cyclic AMP against both the respirations induced. On the other hand, Bu2-cyclic GMP did not affect the respiration induced by phagocytosis whereas it inhibited the respiration induced by the addition of myristic acid was inhibited by Bu2-cyclic AMP, which was similar to that with E. coli. The respiration induced by methylene blue was inhibited neither by Bu2-cyclic AMP nor by Bu2-cyclic GMP. These observations suggest that the cyanide-insensitive respiration of polymorphonuclear leucocytes may be classified into at least three types from the inhibitory pattern of cyclic AMP and cyclic GMP.     

Effect of cyclic nucleotides in vitro assays of rat liver galactosyltransferase.

Experiments with rat liver microsomal galactosyltransferase has been developed to test the effect of cyclic nucleotides on the transfer activity. An overall stimulation is observed when cAMP or cGMP (concentration higher that 10(-6) M) are added to the incubation medium. However, more detailed experiments show that the cyclic nucleotides do not act as direct effectors of the enzyme but present the precursors degradation by the glycosylnucleotide pyrophosphatases.     

Stimulation of cyclic nucleotides of calcium efflux in barnacle muscle fibers.

Internal application of 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷M cGMP and cAMP caused an increase in ⁴⁵Ca efflux in barnacle muscle fibers. Stimulation by either nucleotide occurred in the absence of external calcium and could be prevented by external application of 10 mM procaine or by prior internal treatment of these fibers with EGTA. The results indicate that cyclic nucleotides increase calcium efflux by releasing calcium from internal stores.