Endogenous Cyclic Nucleotides Research Articles

A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the determination of endogenous cyclic nucleotides in rat brain

A robust high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3′,5′-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3′,5′-cyclic guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure endogenous 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC–MS/MS. The calibration curves for both analytes ranged from 5 to 2000ng/g and showed excellent linearity (r2>0.996). Relative surrogate analyte-to-analyte LC–MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies.

Cyclic nucleotide phosphodiesterase activity in stem cells of human periodontal ligament (PDL-MSCs) before and after osteogenic induction

The aim of this work was to evaluate both the level of endogenous cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cAMP) and phosphodiesterase activity in mesenchymal stem cells (MSCs) before and during the osteogenic induction. Samples were organized into control (nondifferentiated) and test groups which were analyzed at 3 different time points: 1, 2, and 4 weeks. Periodontal ligament MSCs were isolated and then expanded in an MSCM medium while cyclic nucleotide levels and phosphodiesterase activity were assessed. cAMP and cGMP levels were markedly higher in the first week than in the following stages. Similarly, PDE activity increased during the first week and reached the peak in the second week. This work validates that cAMP, cGMP, and PDE activities are important factors in the first phase of the osteogenic induction of a human stem cell.

The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.

Timing is everything: the effects of putative dopamine antagonists on metamorphosis vary with larval age and experimental duration in the prosobranch gastropod Crepidula fornicata.

The signal transduction pathway through which excess potassium ion stimulates the larvae of many marine invertebrates to metamorphose is incompletely understood. Recent evidence suggests that dopamine plays important roles in the metamorphic pathway of Crepidula fornicata. Therefore, we asked whether blocking dopamine receptors might prevent excess potassium ion from stimulating metamorphosis in this species. Surprisingly, the effects of the three putative dopamine antagonists tested (all at 10 microM) varied with exposure duration and the age of competent larvae. Chlorpromazine, a nonspecific dopamine antagonist known to have a number of other pharmacological effects, blocked the inductive action of excess potassium ion during the initial 5-8-h exposure periods in most assays, particularly for younger or smaller competent larvae. However, chlorpromazine in the absence of excess potassium ion also stimulated metamorphosis, particularly over the next 18 h, and worked faster on older competent larvae than on younger competent larvae. The specific D(1) antagonist R(+)-Sch-23309 had similar effects, blocking potassium-stimulated metamorphosis in short-term exposures and stimulating metamorphosis in longer exposures, particularly for older competent larvae. Although the specific D(2) antagonist spiperone (SPIP) blocked the inductive effects of excess potassium ion in only 1 of 6 assays during the first 6 h of exposure, it blocked metamorphosis in 2 of the assays during 24-h exposures. Our results indicate that dopamine receptors are involved in the pathway through which excess potassium ion stimulates metamorphosis in C. fornicata. In addition, the largely latent inductive effects of chlorpromazine, an inhibitor of nitric oxide synthase, suggest that endogenous nitric oxide may play a natural role in inhibiting metamorphosis in this species. Overall, our results would then suggest that exposing larvae of C. fornicata to excess K(+) leads to a shutdown of nitric oxide synthesis via a dopaminergic pathway, a pathway that can be blocked by some dopamine antagonists. Alternatively, chlorpromazine might eventually be stimulating metamorphosis by elevating endogenous cyclic nucleotide (e.g., cAMP) concentrations, again acting downstream from the steps acted on directly by excess K(+).

Tansley Review No. 106: Cyclic nucleotides in higher plants: the enduring paradox.

For three decades, hypotheses relating to the occurrence and function of cyclic nucleotides in higher plants have been highly controversial. Although cyclic nucleotides had been shown to have key regulatory roles in animals and bacteria, investigations with higher plants in the 1970s and early 1980s were criticized on the basis of (i) a lack of specificity of effects apparently elicited by cyclic nucleotides, (ii) the equivocal identification of putative endogenous cyclic nucleotides and (iii) ambiguity in the identification of enzymes connected with cyclic nucleotide. More recent evidence based on more rigorous identification procedures has demonstrated conclusively the presence of cyclic nucleotides, nucleotidyl cyclases and cyclic nucleotide phosphodiesterases in higher plants, and has identified plant processes subject to regulation by cyclic nucleotides. Here we review the history of the debate, the recent evidence establishing the presence of these compounds and their role; future research objectives are discussed. contents Summary I. background 427 II. the history of the debate on camp in plants 431 III. evidence for the natural occurrence of camp in plants 432 IV. adenylyl cyclase 435 V. phosphodiesterases 437 VI. camp-dependent protein kinases, creb, camp-binding proteins and cyclicnucleotide-gated channels 439 VII. putative functions of camp 442 VIII. camp and other cyclic nucleotides 445 IX. future directions 447 References 449.

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3–5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.

Isolation from the microsomal fraction of rat liver of a subfraction highly enriched in uncoated endocytic vesicles with high H +-ATPase activity and a 50 kDa phosphoprotein

A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H +-ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 g ml , was shown morphologically to be a mixture of small, smooth-surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electro-neutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'-phosphate and 4-acetamide-4'-isothiocyanostilbene-2, 2'-disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 μM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N, N'-dicyclohexyl-carbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of M r 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide- and Ca 2+-independent protein kinase.

The reactivity of mononucleotides with cholecystokinin/gastrin antisera

Dibutyryl cyclic GMP has been reported to interact with antisera specific for C-terminal tetrapeptide amide common for cholecystokinin (CCK) and gastrin. Moreover, cyclic nucleotides elute by gel chromatography in the same position as the free CCK/gastrin tetrapeptide. Therefore, we have examined the reactivity of 25 mononucleotides with eight CCK and gastrin antisera. The results show that the nucleotides all bind poorly to the antisera (nucleotide concentration required 1 mM). Hence, endogenous cyclic nucleotides, which are present in biological extracts in pM to nM concentrations, do not interfere with immunochemical CCK or gastrin measurements. The antisera displayed highly individual patterns of reactivity without preferential binding of di- or monobutyryl cyclic nucleotides (AMP, GMP or IMP). Thus, the present results do not support the idea of structural resemblance between the C-terminus of CCK/gastrin peptides and butyryl derivatives of cyclic GMP. Enzymatic treatment of the antral tetrapeptide-like immunoreactivity showed that nucleotides do not contribute to this material, which appears exclusively peptidergic.

Characterization of a calmodulin-binding protein that is deficient in trifluoperazine-resistant variants of the macrophage-like cell line J774.

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein.

Thyrotropin controls cyclic nucleotide metabolism of thyroid follicular cells without affecting membrane potential or input resistance

The action of thyrotropin on the rat thyroid follicular cell has been investigated using continuous transmembrane potential and input resistance recording from individual cells in in vitro preparations. The membrane potential in this study was −68.0 mV±0.6 (mean±S.E.). Over a wide range of concentrations (1.5–50 mU/ml), thyrotropin failed to affect membrane potential or input resistance while 20 mU/ml thyrotropin was shown to elicit complex time-dependent changes in tissue levels of both cyclic AMP and cyclic GMP. The present results reveal that thyrotropin-receptor interaction does not affect plasma membrane permeability, but is characterized by complex changes in endogenous cyclic nucleotide metabolism.

Inotropic responses of the frog ventricle to dibutyryl cyclic AMP and 8-bromo cyclic GMP and related changes in endogenous cyclic nucleotide levels

Isolated frog ventricles were superfused with solutions containing either N 6, O 2-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl 3',5'-cyclic AMP) or 8-bromo guanosine 3',5'-cyclic monophosphate (8-br 3',5'-cyclic GMP) and changes in isometric twitch tension and in the levels of endogenous 3',5'-cyclic nucleotides were measured. The results show that dibutyryl 3',5'-cyclic AMP potentiates the twitch, whereas 8-br 3',5'-cyclic GMP depresses it. Both effects are dose-related. The magnitude of the decrease in contractile force produced by 8-br 3',5'-cyclic GMP, and of the increase caused by dibutyryl 3',5'-cyclic AMP, are each paralleled by quantitatively-equivalent changes in the ratio 3',5'-cyclic AMP: 3',5'-cyclic GMP. The effect of 8-br 3',5'-cyclic GMP on the twitch is associated with a marked reduction in endogenous 3',5'-cyclic AMP levels, which is linearly related to the increment in intracellular 3',5'-cyclic GMP (slope of regression line ± S.E.: −10.4 ± 0.6 pmoles 3',5'-cyclic AMP·pmole −1 increase in 3',5'-cyclic GMP). The entry of dibutyryl 3',5'-cyclic AMP into the fibres is accompanied by a relatively small change in intracellular 3',5'-cyclic GMP, in this case, an increase (slope of regression line ± S.E.: +0.018 ± 0.002 pmoles 3',5'-cyclic GMP·pmole −1 increase in 3',5'-cyclic AMP). These results suggest that both 3',5'-cyclic nucleotides are normally involved in regulating contractility, 3',5'-cyclic AMP potentiating the twitch and 3',5'-cyclic GMP exerting a counter-action; and that 3',5'-cyclic GMP may constitute part of a feedback mechanism which serves to regulate 3',5'-cyclic AMP levels.

Hormonal activation of protein kinase and lipid mobilization in the locust fat body in vitro

Cyclic nucleotide-activated protein kinase in locust fat body is elevated by extracts containing adipokinetic hormone (AKH) from locust corpora cardiaca or by synthetic redpigment-concentrating hormone (RPCH) which also exhibits lipid-mobilizing activity. Maximal activation is first attained in vitro 90 sec after RPCH is added to the incubation medium and 3.5 min after addition of AKH extract. This activation reflects the increase in endogenous cyclic nucleotides. Incubation of fat body in the presence of exogenous cyclic nucleotides activates lipase, and RPCH stimulation ultimately elevates the level of transported diacylglycerol 2.3-fold. RPCH stimulates fat body lipase in vivo.

Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are complex, but the accompanying change in isometric twitch tension is paralleled closely by corresponding changes in the ratio 3',5'cyclic AMP:3',5'-cyclic GMP. 5. It is concluded that ATP exerts a dual effect on the ventricle and that the contractile response is regulated by changes in the metabolism of 3',5'-cyclic nucleotides. The effects of indomethacin indicate a possible involvement of prostaglandins in mediating the ATP response. It is suggested that the initial effect of ATP on the ventricle is to increase the permeability of the fibres to Ca2+. 6. The relationship between 3',5' cyclic nucleotide levels and ventricular contractility is discussed. It is postulated that the antagonistic effects of 3',5'-cyclic AMP and 3',5'-cyclic GMP are expressed at the level of certain phosphoproteins which regulate both the availability of Ca2+ and the sensitivity of the contractile proteins to Ca2+.

Release of prostaglandins from the isolated frog ventricle and associated changes in endogenous cyclic nucleotide levels.

1. A study has been made of the decline in contractility and some associated metabolic changes which occur in the isolated frog ventricle during the development of hypodynamic depression. 2. The release of two identified prostaglandins (PG), E1 and E2, together with several as yet unknown prostaglandin-related substances (PRS), accompanies the development of hypodynamic depression. There is a close correlation between the extent to which the isometric twitch is depressed and the quantity of prostaglandin released into the superfusate. 3. Fractionation of extracts of 'used' superfusates, using preparative-scale thin-layer chromatography, revealed the presence of six major components, four of which (PGE1 and PGE2 and two unidentified components) were found to be cardioactive and potentiated contraction when tested subsequently on hypodynamic preparations. 4. Two agents which influence prostaglandin biosynthesis, arachidonic acid and indomethacin, are found to affect both the rate at which the hypodynamic state develops and the extent to which the 'steady-state' twitch tension is depressed, in a dose-dependent manner. Indomethacin, a PG-synthetase inhibitor, accelerates the decay and depresses the final 'steady-state' tension attained, whereas arachidonic acid, the principal precursor for prostaglandin biosynthesis, has the converse effects. 5. Measurements of endogenous 3'5'-cyclic nucleotide levels reveal a time-dependent decrease in intracellular adenosine 3'5'-cyclic monophosphate (3'5'-cyclic AMP) and a concomitant increase in guanosine 3'5' cyclic monophosphate (3'5'-cyclic GMP). The decline in isometric twitch tension is paralleled almost exactly by an equivalent reduction in the ratio 3'5'-cyclic AMP: 3'5'-cyclic GMP. 6. Superfusion of isolated ventricles with Ringer solution containing exogenous, lipid-soluble derivatives of 3'5'-cyclic AMP and 3'5'-cyclic GMP affects both the rate of decline of the isometric twitch and the steady-state tension ultimately reached: thus, 8-bromo-3'5'-cyclic GMP accelerates the decline in contractility and depresses the steady-state level, whereas dibutyryl 3'5'-cyclic AMP delays the development of hypodynamic depression, and elevates the final twitch tension. The effects of both 3'5' cyclic nucleotide derivatives are dose-dependent. 7. The possible involvement of prostaglandins and 3'5'-cyclic nucleotides as causal agents in the mechanism of hypodynamic depression is discussed. The biochemical basis for the implied antangonistic effects of 3'5'-cyclic AMP and 3'5'-cyclic GMP in regulating ventricular contractility is considered in the following paper (Flitney & Singh, 1980).

Involvement of cyclic GMP in the release of stereotyped behaviour patterns in moths by a peptide hormone.

A peptide hormone, the eclosion hormone, triggers two behavioural patterns--the pre-eclosion and eclosion patterns--when injected into pharate silkmoths. Injection of cyclic nucleotides caused the same behavioural responses with cGMP being 10 to 100 times more potent than cAMP. Exogenous cGMP also acted directly on the isolated nervous system to evoke the characteristic motor programmes. Protection of endogenous cyclic nucleotides by pretreatment of moths with a phosphodiesterase inhibitor, theophylline, markedly enhanced the sensitivity of the moths to the hormone. Injection of partially purified hormone preparations was followed by an increase in nervous system cGMP but not cAMP. The increase preceded the behavioural effectiveness of each dose was correlated with its ability to cause a cGMP increase. It was concluded that the behavioural effects of the eclosion hormone are mediated through an increase in cGMP in the nervous system.

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.

Biochemical characteristics of phagocytosis in the P388 D 1 cell

Adherence and phagocytosis of 51chromium labeled sheep red blood cells ([ 51Cr]-sRBC) by P388 D 1 cells in tissue culture were studied under various conditions and were found to possess certain requirements including opsonization, temperature, microfilaments and cyclic nucleotide levels. Exogenous administration of 10 −2 M N 6, O 2-dibutyryl adenosine 3′–5′ cyclic monophosphoric acid (db-cAMP) or adenosine 3′–5′ cyclic monophosphoric acid (cAMP) inhibited phagocytosis of opsonized [ 51Cr]-sRBC by 36 and 42%, respectively. Aminophylline potentiated the inhibitory response to both cAMP and db-cAMP. The measurement of endogenous cyclic nucleotide levels during phagocytosis of opsonized sRBC showed a rise in guanosine 3′–5′ cyclic monophosphate (cGMP) during the first 5 min with a gradual decline to control levels at 45 min and a rise in cAMP levels reaching a peak at 30 min which remained above control values for the duration of the experiment. As the rate of phagocytosis decreased the ratio of cAMP cGMP increased. These observations emphasize the importance of metabolic functions and cyclic nucleotides during phagocytosis by the P388 D 1 cells and strengthen the usefulness of the P388 D 1 cells as a model in evaluating various macrophage activities.

Transport of methotrexate and other folate compounds: Components, mechanism and regulation by cyclic nucleotides

Transport of Methotrexate and other folate compounds into cells of L1210 mouse leukemia or Lactobacillus casei occurs via “active,” carrier-mediated processes. In the bacterial system, transport is linked to glycolysis and is unaffected by cyclic nucleotides. A folate-binding protein, believed to be the carrier in the transport process, has been solubilized by treatment of L. casei membranes with Triton X-100 in the presence of [ 3H]folate and purified to homogeneity. The protein (MW 25,000) binds an equimolar amount of folate. Owing to a preponderance of hydrophobic amino acids, the binding protein is insoluble in aqueous media unless encased in a Triton micelle. Similar experiments suggest that L1210 cells may contain a Triton-extractable membrane protein that binds 5-methyl tetrahydrofolate and Methotrexate. Transport of Methotrexate into L1210 cells is depressed by exogenous cyclic nucleotides or compounds that elevate the level of endogenous cyclic nucleotides, such as phosphodiesterase inhibitors, adenyl cyclase stimulators, glucose, ATP, ADP and GTP. Implications of these observations for cancer chemotherapy are discussed.

Inhibition by cyclic AMP and dibutyryl cyclic AMP of transport of organic acids in kidney cortex

1. 1.|Cyclic adenosine 3′,5′-monophosphate and N 6-2′- O-dibutyryl cyclic adenosine 3′,5′-monophosphate decrease the initial entry rate and the steady-state uptake of p-aminohippurate and uric acid by rabbit kidney cortex slices. 2. 2.| N 6-2′- O-Dibutyryl adenosine 3′-5′-monophosphate inhibits the tubular transport of p-aminohippurate competitively. 3. 3.|Isoproterenol, known to increase cyclic nucleotide concentration of the cortical tubules by activation of adenyl cyclase, decreases p-aminohippurate transport. Antidiuretic hormone which is known to stimulate only medullary adenyl cyclase has no effect on p-amino-hippurate uptake by cortical slices. 4. 4.|Theophylline, which inhibits cyclic nucleotide phosphodiesterase and, therefore, enhances the cellular accumulation of endogenous cyclic nucleotide, depresses p-aminohippurate transport.

Plasma clearance rates and renal clearance of 3H-labeled cyclic AMP and 3H-labeled cyclic GMP in the dog.

Previously, in an attempt to understand the mechanisms involved in the regulation of plasma cyclic nucleotides, we measured concentrations of adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) in plasma from selected blood vessels of anesthetized dogs. The observation that the renal venous plasma concentrations of both cyclic nucleotides were less than arterial concentrations suggested that the kidney might be an important site for the elimination of these compounds from plasma and prompted further investigation of the renal handling of these compounds. Tracer doses of either [(3)H]cAMP or [(3)H]cGMP were administered to anesthetized dogs by constant intravenous infusion, and metabolic clearance rates were determined. Concentrations of endogenous cyclic nucleotide and of cyclic nucleotide radioactivity were measured in aortic and renal venous plasma as well as in urine. Renal venous plasma [(3)H]cGMP was 39% and [(3)H]cAMP was 65% of the concentration in arterial plasma. Endogenous cyclic nucleotide levels showed a similar relationship. The plasma clearance rates (PCR) were 271+/-27 ml/min (mean+/-SE) for cGMP and 261+/-17 for cAMP. The total kidney clearance (calculated as the renal plasma flow x renal cyclic nucleotide extraction ratio) accounted for 52+/-4% and 30+/-2% of the PCR for cGMP and cAMP, respectively. Only about two-thirds of the total kidney clearance of each cyclic nucleotide could be accounted for by urinary excretion, the remainder presumably being the result of renal metabolism. The urinary clearances of (3)H-labeled cGMP (40.9+/-4.2 ml/min) and endogenous cGMP (45.0+/-2.3 ml/min) were not significantly different from each other. Both were approximately 50% greater than the glomerular filtration rate, which was 27.1+/-2.0 ml/min, indicating that a significant amount of urinary cGMP is derived from plasma by tubular secretion. In contrast, the urinary clearances of (3)H-labeled cAMP (23.7+/-1.9 ml/min) and endogenous cAMP (27.2+/-2.6 ml/min) were nearly equal both to each other and to the glomerular filtration rate, which was 24.6+/-1.7 ml/min. Thus, in the dog, glomerular filtration of plasma cAMP appears to be responsible for most of the cAMP found in urine. Renla production of cAMP, which in humans contributes from a third to a half of the urinary cAMP, was quantitatively of minor importance in the dog.Thus, under the conditions of these experiments in dogs, renal elimination appears to be responsible for half of the PCR of cGMP and about a third of the PCR of cAMP. About a third of the renal elimination of both cyclic nucleotides appears to be due to metabolic degradation within the kidney, and the balance is due to excretion in the urine.