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The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3–5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut–liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

The progress of science is tied to the standardization of measurements, instruments, and data. This is especially true in the Big Data age, where analyzing large data volumes critically hinges on the data being standardized. Accordingly, the lack of community‐sanctioned data standards in paleoclimatology has largely precluded the benefits of Big Data advances in the field. Building upon recent efforts to standardize the format and terminology of paleoclimate data, this article describes the Paleoclimate Community reporTing Standard (PaCTS), a crowdsourced reporting standard for such data. PaCTS captures which information should be included when reporting paleoclimate data, with the goal of maximizing the reuse value of paleoclimate data sets, particularly for synthesis work and comparison to climate model simulations. Initiated by the LinkedEarth project, the process to elicit a reporting standard involved an international workshop in 2016, various forms of digital community engagement over the next few years, and grassroots working groups. Participants in this process identified important properties across paleoclimate archives, in addition to the reporting of uncertainties and chronologies; they also identified archive‐specific properties and distinguished reporting standards for new versus legacy data sets. This work shows that at least 135 respondents overwhelmingly support a drastic increase in the amount of metadata accompanying paleoclimate data sets. Since such goals are at odds with present practices, we discuss a transparent path toward implementing or revising these recommendations in the near future, using both bottom‐up and top‐down approaches. Standardizing the way data are described and shared is key to accelerating the progress of science. Building on recent advances in paleoceanography and paleoclimatology, we present the first community‐led reporting standard for such datasets. The Paleoclimate Community reporTing Standard (PaCTS) provides guidelines as to which information should be included when reporting data from various paleoclimate archives, as well as themes common to many fields, like uncertainty and other site‐specific information. The ultimate goal of this effort is to (1) make these datasets more re‐usable over the long term, and (2) provide a roadmap for implementing and revising the standard, as the field of paleoclimatology and its practitioners both evolve. The requirements are driven by the differing needs of data producers and the data consumers, who often have different goals in mind. Thus, agreeing on and writing up these requirements involves building consensus among the community to decide on their present and future goals. First version of a crowdsourced reporting standard for paleoclimate dataThe standards arose through collective discussions, both in person and online, and via an innovative social platformThe standard helps meet the interoperability and reuse criteria of FAIR (Findable, Accessible, Interoperable, and Reusable)

Abbreviations: PDGF = platelet-derived growth factor; PDGFR = platelet-derived growth factor receptor Platelet-derived growth factors (PDGFs) mediate tissue repair and wound healing. PDGFs are synthesized and secreted by most inflammatory [...]

Dupilumab, a fully human anti-interleukin-4 receptor α monoclonal antibody, inhibits interleukin-4 and interleukin-13 signalling, key drivers of type-2-mediated inflammation. Adults with uncontrolled persistent asthma who are receiving medium-to-high-dose inhaled corticosteroids plus a long-acting β2agonist require additional treatment options as add-on therapy. We aimed to assess the efficacy and safety of dupilumab as add-on therapy in patients with uncontrolled persistent asthma on medium-to-high-dose inhaled corticosteroids plus a long-acting β2agonist, irrespective of baseline eosinophil count.

Data from early-stage studies suggested that interleukin (IL)-4 and IL-13 are requisite drivers of atopic dermatitis, evidenced by marked improvement after treatment with dupilumab, a fully-human monoclonal antibody that blocks both pathways. We aimed to assess the efficacy and safety of several dose regimens of dupilumab in adults with moderate-to-severe atopic dermatitis inadequately controlled by topical treatments.

Abbreviations: ACTH = corticotropin; NA = nocturnal asthma; NL = control subject; NNA = nonnocturnal asthma Patients with nocturnal asthma (NA) have increased airway inflammation at night, a phenomenon not [...]

Abbreviations: DX = dexamethasone; IL = interleukin Airway remodeling in asthma patients has received a great deal of attention as a process whereby significant structural abnormalities of the bronchial wall [...]

The increasing prevalence of both asthma and obesity is associated with substantial morbidity and healthcare utilization. Herein, we review recent data suggesting that the obese asthmatic may represent a distinct clinical phenotype.

Inflammation of the distal lung, which consists of the small airways (internal diameter <2mm) and alveolar tissue, is an important feature of the asthma clinical syndrome comprising airway inflammation, airway hyperresponsiveness and bronchodilator-responsive expiratory airflow limitation. Support for this assertion is derived from histologic studies which have demonstrated evidence of inflammation in this anatomic compartment, along with additional studies, which have elucidated the radiologic and physiologic correlates of distal lung inflammation. Delivering inhaled drugs to this area is challenging and is dependent on a number of drug-and delivery device-related factors, as well as on a patient’s inhaler technique and bronchial anatomy. Newer chlorofluorocarbon-free formulations of inhaled corticosteroids such as hydrofluoroalkane propelled metered-dose inhalers and dry powder inhalers appear to have certain advantages with regard to drug delivery that facilitate improved drug delivery to the distal lung. Mounting evidence indicates that recognition and treatment of distal lung inflammation may be key components of appropriate asthma pharmacotherapy.

Chronic obstructive pulmonary disease (COPD) is a progressive syndrome of expiratory airflow limitation caused by chronic inflammation of the airways and lung parenchyma. The airway inflammatory response in COPD is initiated by smoking in the overwhelming majority of cases, and chronic exposure to cigarette smoke initiates a series of events that cause damage to central airways, peripheral airways, and terminal airspaces, leading to physiologic and clinical abnormalities. The contrasting inflammatory phenotypes of asthma and COPD have important implications for clinical and physiologic manifestations of disease, as well as for therapy. The outpatient treatment of COPD differs from the approach used in asthma and can be divided into 3 subgroups: health care maintenance, drug therapy, and nondrug therapy. Smoking cessation, regular spirometry, and immunization are important components of health care maintenance. Drug therapy consists of optimal bronchodilator therapy supplemented, when necessary, with either inhaled corticosteroids or theophylline. Nondrug therapies include pulmonary rehabilitation, supplemental oxygen, and surgery.

Limitation of physical activity occupies a central role in the symptom complex of patients with chronic obstructive pulmonary disease (COPD), and improvement in exercise capacity is a key outcome of response to COPD therapy. Maximum exercise capacity testing facilitates assessment of physiologic mechanisms of exercise and allows quantitation of the degree of limitation. This manuscript utilizes published data from the National Emphysema Treatment Trial to investigate the minimal clinically important difference (MCID) in maximum exercise capacity in patients with severe emphysema. Distribution- and opinion-based methods were used to estimate MCID. Expert clinician opinion yielded a value of 10 Watts as the MCID for change in maximum exercise capacity. Baseline standard deviation and error data yielded a one-half standard deviation-based estimate of 10.5 Watts and a standard error-based estimate of 4.2 Watts. In subjects randomized to medical therapy, the mean ( ± SD) 24-month change in maximum exercise capacity following medical therapy was − 9.2 ± 1.2 Watts, whereas among those randomized to lung volume reduction surgery, mean 24-month change in maximum exercise capacity was 1.7 ± 17.7 Watts, with a mean difference between the groups of 10.9 Watts. The observed difference in maximum exercise capacity after 24 months between subjects randomized to medical versus surgical therapy conforms to both opinion- and distribution-based estimates of MCID. Further investigation is needed to develop and validate estimates of MCID for maximum exercise capacity and other key clinical outcomes in COPD.

To review the role of distal lung inflammation in asthma.

We have examined underlying causes for observations made in hepatocytes in which catalytic subunits of Na(+)-K(+)-ATPase are found both in bile canalicular (apical) and sinusoidal (basolateral) membrane domains, whereas functional activity is associated preferentially with sinusoidal membrane sites. In a series of parallel studies, we determined by both light and electron microscopy that Na(+)-K(+)-ATPase alpha-subunits were localized to both membrane domains of hepatocytes. With the use of purified liver plasma membrane subfractions, ouabain inhibition curves demonstrated similar inhibition constants (inhibition constant 10(-5) M), and immunoblots using alpha 1-, alpha 2-, and alpha 3-polyclonal and monoclonal antibodies demonstrated antigenic sites predominantly for alpha 1 in both membrane fractions. Also, Northern blot hybridization analysis revealed only the alpha 1-isoform in hepatocytes. In contrast to the bipolar distribution of the alpha 1-subunit, the beta-subunit was identified only at the sinusoidal surface using fluorescence labeling with a monoclonal antibody. The beta 1-isoform was demonstrated by Northern blot analysis and was present predominantly at the sinusoidal domain by immunoblotting with polyclonal antibodies. In addition to the bipolar distribution of alpha 1, immunoblotting of liver plasma membrane subfractions demonstrated a symmetrical distribution of fodrin, ankyrin, actin, and E-cadherin at both domains. These results suggest that functionally competent alpha/beta-complexes form at the sinusoidal domain, whereas only alpha 1-subunits are present at the apical pole.

The ferrochelatase-inhibitory activity, porphyrin-inducing activity, and cytochrome P-450- and heme-destructive effects of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were studied in chick embryo liver cells. The ferrochelatase-inhibitory activity of the 4-butyl, 4-pentyl, 4-hexyl, and 4-cyclopropylmethyl analogues of DDC was considered to be due to the formation of the corresponding N-alkylporphyrins. These N-alkylporphyrins were isolated from the livers of phenobarbital-pretreated rats following administration of the corresponding DDC analogues. The 4-isobutyl analogue did not have ferrochelatase-inhibitory activity despite its ability to cause formation of an N-isobutylporphyrin in rat liver. The 4-chloromethyl analogue of DDC inhibited ferrochelatase activity. The inability to isolate an N-alkylporphyrin from rat liver with this analogue may be due to its lability. The porphyrin-inducing activity of these analogues depended on their ferrochelatase-inhibitory potency and lipophilicity. The DDC analogues caused cytochrome P-450 and heme destruction. The relative ferrochelatase-inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site.

The ferrochelatase-reducing activity and cytochrome P-450- and heme-destructive effects of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were studied in chick embryo liver cells. A group of DDC analogues was found in which an inability to reduce ferrochelatase activity corresponded with an inability to cause cytochrome P-450 and heme destruction. In a second group of DDC analogues, the ability to reduce ferrochelatase activity corresponded with the ability to cause cytochrome P-450 and heme destruction. These observations support the idea that the protoporphyrin IX moiety of N-alkylprotoporphyrin IX originates from the heme moiety of cytochrome P-450. A third group of DDC analogues caused cytochrome P-450 and heme destruction despite an inability to reduce ferrochelatase activity. With this third group of DDC analogues, the heme moiety of cytochrome P-450 is likely degraded to products other than N-alkylporphyrins. The inability of several lipophilic DDC analogues [4-benzyl, 4-isopropyl, 4-cyclohexyl, 4-(3-cyclohexenyl)] to reduce hepatic ferrochelatase activity may explain their low porphyrinogenicity. The pattern of porphyrin accumulation produced in response to two DDC analogues that did not inhibit ferrochelatase was investigated using high performance liquid chromatography. Coproporphyrin was the major porphyrin to accumulate in response to the 4-isopropyl analogue and uro- and heptacarboxylic acid porphyrins in response to the 4-benzyl analogue. These patterns of porphyrin accumulation are consistent with the inability of these analogues to inhibit ferrochelatase.

A somatic cell mutant (CR1) of the Chinese hamster ovary cell line (CHO-K1) that is defective in the regulation of cholesterol biosynthesis can be grown under conditions in which plasma membranes from these cells display various cholesterol contents and acyl chain order parameters. The (Na+ + K+)-stimulated adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from these cells was shown to vary in activity by a factor of 10 as the order parameter was varied, and the activity exhibited an exponential dependence on this parameter. Under these conditions the number of Na+,K+-ATPase molecules was shown to remain constant by affinity labeling with [gamma-32P]ATP in the absence of Na+. Control experiments showed that alteration in cholesterol content without change in order parameter did not result in altered enzyme activity. It is concluded that, under our conditions, the rate of catalysis by the Na+,K+-ATPase is determined by the order parameter. These studies suggest a physical mechanism by which variation of membrane lipid composition or other factors that determine membrane lipid acyl chain order parameter can result in variation in membrane enzyme activity.

Protozoa from cooling towers may serve as hosts for legionellae, but such protozoa have not been examined with respect to effects of cooling tower biocides. In this study, two ciliate species,Tetrahymena sp andColpoda sp, and two amoebae species,Vannella miroides andAcanthamoeba hatchetti, were isolated from a cooling tower and tested for survival in the presence of four cooling tower biocides. The protozoa were exposed for 24 h to a thiocarbamate compound, an isothiazolone compound, quaternary ammonium compounds (QAC), and tributyltin neodecanoate with quarternary ammonium compounds (TBT/QAC). After exposure, cells were examined for viability. The highest concentration of each biocide in which cells could survive was compared to the manufacturers' recommended maintenance dosage (MRMD) of the biocides.Tetrahymena andColpoda survived concentrations within the range of the MRMD of thiocarbamate and QAC.Vannella andAcanthamoeba survived concentrations within the MRMD of thiocarbamate, isothiazolone, and QAC.Colpoda cysts andAcanthamoebae cysts remained viable after exposure to concentrations much greater than the MRMD of thiocarbamate, isothiazolone, and QAC. None of the protozoa in any stage could survive the MRMD of TBT/QAC. These results show that protozoa indigenous to cooling towers may survive the recommended concentration of certain biocides, and this information may be important in devising procedures for eradicating hosts for legionellae.

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.

Cellular and molecular mechanisms regulating the activity of the sodium pump or Na,K-ATPase during proliferation of hepatocytes following 70% liver resection have not been defined. Na,K-ATPase may be regulated by synthesis of its alpha- and beta-subunits, by sorting to either the sinusoidal or apical plasma membrane domains, or by increasing membrane lipid fluidity. This study investigated the time course of changes during hepatic regeneration for Na, K-ATPase activity, lipid composition and fluidity, and protein content of liver plasma membrane subfractions. As early as 4 h after hepatic resection, Na,K-ATPase activity was increased selectively in the bile canalicular fraction. It reached a new steady state at 12 h and remained elevated for 2 days. Although hepatic regeneration was associated with a reduced cholesterol/phospholipid molar ratio and increased fluidity, measured with two different probes, these changes in lipid metabolism were in the sinusoidal membrane domain. The Na,K-ATPase beta1-subunit, but not the alpha1-subunit, was increased selectively at the bile canalicular surface as shown by immunoblotting of liver plasma membrane subfractions and the morphological demonstration at both the light and electron microscopic levels. Furthermore, cycloheximide blocked the rise in beta1-subunit mRNA levels. Since the time course for beta1-subunit accumulation was similar to that for activation of Na,K-ATPase activity, this change implicated the beta1-subunit in activating sodium pump activity.

We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.

Recent research on reasoning has resulted in a number of authors urging a convergence between ideas in the hitherto disparate fields of deduction and decision making. The deontic reasoning literature in particular refers increasingly to the decision-making constructs of subjective utility and subjective probability. Although the former construct has received some attention from experimenters, the latter has remained relatively unexplored. In this paper a set of experiments is reported in which a modified form of Wason's selection task using an enlarged array was used to investigate the role of a probabilistic factor in reasoning with conditional obligations. Results showed that this factor played a significant role in mediating this reasoning, when probabilistic information was added both to antecedent and to consequent items. Other results indicated that the effect occurred principally by suppressing selections of items with relatively low subjective relevance.

A factor [the feedback regulator (FR)] formed by adipocytes after the stimulation of a cAMP raising hormone has been found to be a potent inhibitor of membrane-bound adenylate cyclase [EC 4.6.1.1.; ATP pyrophosphate-lyase (cyclizing)]. In a standard assay system using rat adipocyte plasma membrane as the source of adenylate cyclase, the FR inhibited adenylate cyclase by lowering the Vmax without affecting the apparent Km for ATP (0.3-0.6 mM). The apparent Ka for epinephrine (5-6 muM) was also not affected by FR. The inhibitory action of FR was partially countered by Mg2+ ions. An increase in phosphorylation of plasma membrane was observed when FR was present in the incubation system. The concentration required for a 50% inhibition was four times higher when adenosine 5-(beta,gamma-imino) triphosphate [AMP-P(NH)P] replaced ATP as the substrate for adenylate cyclase, implying that adenylate cyclase was inactivated by phosphorylation caused by FR. Increase in FR inhibition obtained by adding low concentrations of adenosine 5-(alpha,beta-methylene) triphosphate or ATP to AMP-(NH)P as the substrate supports this view. The inhibitory action was reversible. These results are consistent with the previously reported phenomena that (1) the undue to the formation of FR, and (2) the recovery of responsiveness of the stimulated cells by washing the cells with regular buffer medium is a result of the removal of FR. The hormone-initiated biphasic curve of cAMP levels in adipocytes is believed to be due to the negative feedback action of FR on adenylate cyclase. The mechanism of action of FR on inhibition of adenylate cyclase appears to be related to the phosphorylation of certain membrane components.

A sensitive enzymatic procedure has been developed for the determination of guanosine 3′:5′-cyclic monophosphate (cyclic GMP). It is based on the conversion of cyclic GMP to GMP by cyclic nucleotide phosphodiesterase and on the transfer of 32P from [γ-32P]ATP to GMP by the action of a specific ATP:GMP phosphotransferase (EC 2.7.4.8). The [32P]GDP is separated from the remaining [32P]ATP by enzymatic degradation of ATP by myosin and by precipitation of the 32Piformed. The reaction blank, which is mostly caused by the nucleotide content of the enzymes, is doubled by about 0.1 pmol of cyclic GMP. The procedure has advantages in speed and/or accuracy over other methods in current use.Cyclic nucleotide concentrations were studied in the ductus deferens of the rat; two agents were used, carbachol and norepinephrine, which cause contraction. Incubation with 0.1 mM carbachol caused a 3-fold increase in cyclic GMP content, which was maximal about 2 min after carbachol addition. Cyclic AMP concentrations were not significantly changed. Addition of 0.01 mM norepinephrine increased cyclic GMP content by about 25% within 1 min and by 40% within 3 min; cyclic AMP concentrations were only slightly increased. A 3-min incubation with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (0.1 mM) doubled the cyclic GMP content and increased cyclic AMP concentration by 50%.

Guanosine 3′:5′-cyclic monosphosphate (cyclic GMP) levels in the ductus deferens of the rat were increased 2- to 3-fold by acetylcholine (10-1000 μM) or by 125 mM KCl, while adenosine 3′:5′-cyclic monophosphate (cyclic AMP) levels were not changed. After incubation for 30 min in the absence of Ca++, cyclic GMP control levels were decreased by 85% and were not affected by acetylcholine or KCl. The readdition of Ca++(1.8 mM) for 3 min to Ca++-deprived tissue partially restored basal cyclic GMP levels and the effects of acetylcholine and KCl. The addition of Sr++(3.6 mM) or of Ba++(1.8 or 10 mM) also caused an increase in basal cyclic GMP in Ca++-deprived tissue. Cyclic AMP levels were not significantly changed under any of these conditions. The addition of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.1 mM), to ductus deferentes increased the amount of cyclic AMP about 50% and that of cyclic GMP about 2-fold. The later effect also depended on the presence of Ca++. 1-Methyl-3-isobutylxanthine (0.1 mM) increased cyclic GMP and cyclic AMP levels in slices of rat submaxillary glands. Methacholine increased cyclic GMP if added in the presence of methyl isobutylxanthine. Cyclic GMP control levels and the effect of methyl isobutylxanthine were unchanged by Ca++omission, but the effect of methacholine was abolished.These findings indicate that calcium ions are important for the control of cyclic GMP levels in these tissues.