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Neil D Goldberg Emeritus Chief of Gastroenterology, University of Maryland St. Joseph Medical Center, Towson, MD, USA Abstract: Iron deficiency anemia is the most common form of anemia worldwide, caused by poor iron intake, chronic blood loss, or impaired absorption. Patients with inflammatory bowel disease (IBD) are increasingly likely to have iron deficiency anemia, with an estimated prevalence of 36%–76%. Detection of iron deficiency is problematic as outward signs and symptoms are not always present. Iron deficiency can have a significant impact on a patient's quality of life, necessitating prompt management and treatment. Effective treatment includes identifying and treating the underlying cause and initiating iron replacement therapy with either oral or intravenous iron. Numerous formulations for oral iron are available, with ferrous fumarate, sulfate, and gluconate being the most commonly prescribed. Available intravenous formulations include iron dextran, iron sucrose, ferric gluconate, and ferumoxytol. Low-molecular weight iron dextran and iron sucrose have been shown to be safe, efficacious, and effective in a host of gastrointestinal disorders. Ferumoxytol is the newest US Food and Drug Administration-approved intravenous iron therapy, indicated for iron deficiency anemia in adults with chronic kidney disease. Ferumoxytol is also being investigated in Phase 3 studies for the treatment of iron deficiency anemia in patients without chronic kidney disease, including subgroups with IBD. A review of the efficacy and safety of iron replacement in IBD, therapeutic considerations, and recommendations for the practicing gastroenterologist are presented. Keywords: anemia, inflammatory bowel disease, intravenous iron, iron deficiency, oral iron, therapy

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury., (Copyright © 2016 the American Physiological Society.)

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

Background: Mycobacterium avium subspecies paratuberculosis (MAP) has been suspected of involvement in Crohn's disease (CD). We investigated this potential association by testing whole blood from CD patients and healthy controls for the presence of MAP by culture and molecular methods. In addition, each blood sample was analyzed for polymorphisms in the NOD2/CARD15 gene previously associated with CD., Methods: Four 4-mL K(2)-EDTA tubes of whole blood were drawn from each subject (n = 260, 130 CD patients and 130 healthy controls). Two tubes of blood were cultured for MAP by the following methods: Mycobacterial Growth Indicator Tube, Herrold's Egg Yolk Agar, BACTEC 460, and Hungate. The remaining 2 tubes of blood were tested for MAP DNA and polymorphisms in the NOD2/CARD15 gene by polymerase chain reaction (PCR)., Results: One healthy control patient was positive for MAP via PCR; however, no viable MAP was cultured from this individual. All blood cultures were negative for MAP. One CD patient's blood was culture-positive for M. tuberculosis complex. CD patients exhibited a higher rate of polymorphism in the NOD2/CARD15 gene than healthy control patients., Conclusions: In this study MAP was not recovered from the blood of CD patients or healthy controls. However, CD patients showed higher mutation rates in the NOD2/CARD15 gene, compared with healthy controls, supporting the findings of other investigators. No correlation between these polymorphisms and MAP bacteremia in CD patients could be identified in this study.

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by 18O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by 18O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K+ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2)ADP.

Adenine nucleotide metabolism was characterized in intact insulin secreting HIT-T15 cells during the transition from non-stimulated (i. e. 0.2 mM glucose) to the glucose-stimulated secretory state. Metabolic dynamics were monitored by assessing rates of appearance of 18O-labeled phosphoryls of endogenous nucleotides in cells incubated in medium enriched in [18O]water. Most prominent of the metabolic alterations associated with stimulated insulin secretion was the suppression in the rate of adenylate kinase (AK)-catalyzed phosphorylation of AMP by ATP. This was manifest as a graded decrease of up to 50% in the rate of appearance of beta-18O-labeled species of ADP and ATP and corresponded to the magnitude of the secretory response elicited over a range of stimulatory glucose concentrations. The only nucleotide exhibiting a significant concentration change associated with suppression of AK activity was AMP, which decreased by about 50%, irrespective of the glucose concentration. Leucine-stimulated secretion also decreased the rate of AK-catalyzed phosphotransfer. This secretory stimulus-related suppression of AK-catalyzed phosphotransfer occurs within 45 s of glucose addition, precedes insulin secretion, depends on the internalization and metabolism of glucose, and is independent of membrane depolarization and the influx of extracellular calcium. The secretory stimulus-induced decrease in AK-catalyzed phosphotransfer, therefore occurs prior to or at the time of KATP+ channel closure but it is not associated with or a consequence of events occurring subsequent to KATP+ channel closure. These results indicate that AK-catalyzed phosphotransfer may be a determinant of ATP to ADP conversion rates in the KATP+ channel microenvironment; secretory stimuli-linked decreased rates of AK-catalyzed ADP generation from ATP (and AMP) would translate into an increased probability of ATP-liganded and, therefore, closed state of the channel.

The kinetics of creatine kinase (CK) and adenylate kinase (AK) activities were monitored in intact diaphragm muscle by 18O phosphoryl oxygen exchange to assess whether these two phosphotransferases provide an interrelated function integral to high energy phosphoryl metabolism. This possibility was examined by quantitating the net rates of CK- and AK-catalyzed phosphoryl transfer in comparison to the total cellular ATP metabolic rate when CK activity in the intact diaphragm muscle was progressively inhibited by 2,4-dinitrofluorobenzene. In noncontracting muscle from untreated rats, net rates of CK- and AK-catalyzed phosphotransfer were equivalent to 88 and 7%, respectively, of the total ATP metabolic rate. These results were compared with reported 31P NMR analyses of total creatine phosphate flux to estimate that each creatine phosphate molecule produced undergoes about 50 unidirectional CK-catalyzed phosphotransfers in transit to an ATP consumption site in the intact muscles. Graded inhibition by 2,4-dinitrofluorobenzene of intracellular CK activity by up to 98% resulted in a progressive shift in phosphotransferase catalysis from the CK to the AK system; the sum of the net rates of phosphoryl transfer by combining the increasing AK and decreasing CK activities continued to approximate the total cellular ATP metabolic rate. These results indicate that in diaphragm muscle CK and AK operate as interrelated cellular high energy phosphoryl transfer systems through which the majority of newly generated ATP is processed prior to its utilization.

We previously suggested that an importance of adenylate kinase (AdK) in skeletal muscle is to function as a high energy phosphoryl transfer system regulating ATP generation in correspondence with its consumption by specific cellular processes. The present experiments are intended to define the ATP-generating system coupled to and regulated by AdK-catalyzed phosphotransfer in skeletal muscle and also to examine the relationship between AdK- and creatine kinase (CK)-catalyzed phosphotransfer. Rates of phosphoryl transfer catalyzed by AdK were assessed in intact, isolated rat diaphragm by determining rates of AMP phosphorylation with endogenously generated [gamma-18O]ATP under conditions of altered anaerobic and aerobic ATP production. AdK-catalyzed phosphoryl transfer rates accelerated incrementally up to 12-fold in direct proportion to stimulated contractile frequency in parallel with equivalent increases in rates of ATP generation by lactate producing glycolysis. Stoichiometric equivalent increases of AdK-catalyzed phosphotransfer and anaerobic ATP production also occurred up to more than 20-fold when oxidative phosphorylation was impaired by either O2 deprivation or treatment with KCN or p-(trifluoromethoxy)-phenylhydrazone. These enhanced rates of AMP phosphorylation were balanced by virtually identically increased rates of AdK-catalyzed generation of AMP. This AMP was traced to arise from AdK-catalyzed phosphotransfer involving ADP generated by a muscle ATPase. Increased AdK-catalyzed phosphotransfer paired with the apparent compensatory increase in ATP generation by anaerobic glycolysis in oxygen-deprived muscle occurred coincident with diminished rates of CK-catalyzed phosphoryl transfer indicative of a pairing between oxidatively produced ATP and CK-catalyzed phosphotransfer. A metabolic model consistent with these results and conforming to the Mitchell general principle of vectorial ligand conduction is suggested.

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.

The first characterization of the kinetics and subcellular compartmentation of adenylate kinase activity in intact muscle has been accomplished using rat diaphragm equilibrated with [18O]water. Rates of adenylate kinase-catalyzed phosphoryl transfer were measured by appearance of 18O-labeled beta-phosphoryls in ADP and ATP resulting from the transfer to AMP of newly synthesized 18O-labeled gamma-ATP. Unique features of adenylate kinase catalysis were uncovered in the intact cell not predictable from cell free analysis. This enzyme activity, which in non-contracting muscle is limited to 1/1000 of the estimated Vmax (cell free) apparently because of restricted ADP availability, is localized in subcellular compartments that increase in size and/or number with contractile frequency. Contraction also causes frequency-dependent increments in adenylate kinase velocity (22-fold at 4 Hz) as does oxygen deprivation (35-fold). These enhanced rates of adenylate kinase activity, equivalent to processing all the cellular ATP and ADP in approximately 1 min, occur when levels of ATP, ADP, and AMP are maintained very near their basal steady state. These characteristics of the dynamics of adenylate kinase catalysis in the intact cell demonstrate that rapid rates of AMP production from ADP are balanced by equally rapid rates of AMP phosphorylation with no net synthesis or accumulation of any adenine nucleotide. This rapid processing of nucleotide phosphoryls conforms to a proposed scheme whereby the adenylate kinase system provides the unique function of transferring, as beta-ADP, high energy phosphoryls generated by glycolytic metabolism to ATP-utilizing components in muscle.

Purified prostaglandin endoperoxides (PGG2 and PGH2) and hydroperoxides (15-OOH-PGE2) as well as fatty acid hydroperoxides (12-OOH-20:4, 15-00H-20:4, and 13-OOH-18:2) were examined as effectors of soluble splenic cell guanylate cyclase activity. The procedures described (in the miniprint supplement) for the preparation, purification, and characterization of these components circumvented the use of diethyl ether which obscured effects of lipid effectors because of contaminants presumed to be ether peroxides which were stimulatory to the cyclase. Addition of prostaglandin endoperoxides or fatty acid hydroperoxides to the reaction mixture led to a time-dependent activation of guanylate cyclase activity; 2.5- to 5-fold stimulation was seen during the first 6 min. The degree of stimulation and rate of activation were dependent on the concentration of the fatty acid effector; when initial velocities (6 min) were assessed half-maximal stimulation was achieved in the range of 2 to 3 micrometer. However, by extending the incubation time to 90 min similar maximal increases in specific activity could be achieved with 3 or 10 micrometer PGG2 or PGH2. Activation of guanylate cyclase upon addition of prostaglandin endoperoxides or fatty acid hydroperoxides was prevented or reversed by the thiol reductants dithiothreitol (3 to 5 mM) or glutathione (10 to 15 mM). Na2S2O4, not known as an effective reducing agent of disulfides, prevented but was relatively ineffective in reversing activation after it had been induced by PGG2. Pretreatment of the enzyme preparation with increasing concentrations of N-ethylmaleimide in the range of 0.01 to 1.0 mM prevented activation by PGG2 without affecting basal guanylate cyclase activity. These observations indicate that fatty acid hydroperoxides and prostaglandin endoperoxides promote activation of the cyclase by oxidation of enzyme-related thiol functions. In contrast PGE2, PGF2a, hydroxy fatty acids (13-OH-18:2, 12-OH-20:4) as well as saturated (18:0) monoenoic (18:1), dienoic (18:2), and tetraenoic (20:4) fatty acids were ineffective in promoting cyclase activation in the range of 1 to 10 micrometer. Studies to identify the species of the rapidly metabolized prostaglandin endoperoxides that serve as effectors of the cyclase indicated that PGG2 but not 15-OOH-PGE2 (the major buffer-rearrangement product of PGG2) is most likely an activator. In the case of PGH2, a rapidly generated (30 s) metabolite of PGH2 was found which contained a hydroperoxy or endoperoxy functional group and was equally as effective as PGH2 as an apparent activator of the enzyme. The combined effects of PGG2 and dehydroascorbic acid, another class of activator, exhibited additivity with respect to the rate at which the time-dependent activation was induced. These results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryl groups that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.

Soluble cyclic GMP-phosphodiesterase was measured in normal and psoriatic epidermis. The specific activity of the enzyme was increased almost four-fold in involved compared with normal epidermis, and two- to three-fold in involved compared with uninvolved epidermis. The enzyme activity from all three sources was inhibited by 40-50% by ethylene glycol tetraacetic acid (EGTA). These results indicate that in addition to the reported enhanced capacity of psoriatic epidermis to generate cGMP, it has an increased ability to hydrolyse this nucleotide, although to a lesser degree than the augmentation found in soluble guanylate activity from psoriatic epidermis. These observations are compatible with the elevated steady-state levels of this nucleotide observed in the involved epidermis of psoriasis.

The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.

Light, in the presence of ATP, has been reported to stimulate cGMP binding to a 58 kDa protein in ROS (rod outer segments, Fesenko and Krapivinsky, 1986b, Photobiochem. Photobiophys. 13 345-58). This apparent light-related redistribution of ROS cGMP has been suggested to eliminate any requirement for phosphodiesterase-promoted hydrolysis of cGMP in the mechanism subserving phototransduction. Using conditions identical to those previously reported, this effect of light and ATP was examined further by characterizing the metabolic products that arise and the nucleotides that become liganded. The increased binding of radiolabeled guanine nucleotide upon illumination of ROS in the presence of ATP was confirmed, but the species of guanine nucleotide that were stimulated to bind under these conditions were identified as [32P]GDP and [32P]GTP rather than [32P]cGMP. The precautions to prevent enzymic hydrolysis of cGMP, which included conducting the reactions at 0 degrees and the addition of 3-isobutyl-l-methylxanthine (250 microM) to the reaction mixture did not prevent about a 20-fold increase in the rate of phosphodiesterase-catalyzed hydrolysis of radiolabeled cGMP by light when ATP was also present. This stimulation of phosphodiesterase activity is undoubtedly related to transphosphorylation by exogenous ATP of endogenous GMP and GDP involving catalytic actions of guanylate kinase and nucleoside diphosphate kinase in isolated ROS. These enzymes can also serve to generate [32P]GDP and [32P]GTP, which subsequently bind to ROS components. Such a mechanism involving ATP as phosphoryl donor was supported by observing that an analog of ATP (beta,gamma-methyleneadenosine 5'-triphosphate), which cannot serve as a phosphoryl donor, did not increase radiolabeled guanine nucleotide binding. Although several ROS proteins can form filter-retainable complexes with GDP and GTP, the properties of the 58 kDa protein found to be photoaffinity labeled with radioactive guanine nucleotide are most characteristic of those attributable to tubulin. The previous report that illumination in the presence of ATP stimulates the binding of cGMP to ROS components finds no support from the data obtained in the present studies.

The relative changes in vascular tissue levels of cyclic 3':5'-guanosine monophosphate (cGMP) and cyclic 3':5'-adenosine monophosphate (cAMP) were determined in bovine and canine vein strips that were quickfrozen during changes in contractility elicited by prostaglandins (PG) or isoproterenol. Prostaglandin F(2alpha)-induced venoconstriction was associated with increased vascular tissue levels of cGMP and increased ratios of cGMP to cAMP concentrations, but no consistent qualitative change in cAMP levels. In contrast, vein cAMP concentrations were increased during administration of the vasodilators PGE(2) and isoproterenol. The results suggest possible opposing roles for the two cyclic nucleotides in vasomotion.

Photoreceptor metabolism of cGMP and its regulation were characterized in isolated toad retinas by determining the intensity and time dependence of light-induced changes in the following metabolic parameters: cGMP hydrolytic flux determined by the rate of 18O incorporation from 18O-water into retinal guanine nucleotide alpha-phosphoryls; changes in the total (protein-bound and unbound) concentrations of the guanine nucleotide metabolic intermediates; and changes in the concentration of metabolic (unbound) GDP calculated from the fraction of the alpha-GDP that undergoes labeling with 18O. The latter is interpreted to reflect the state of the equilibrium between GDP- and GTP-complexed forms of G-protein. With narrow band 500 nm light that preferentially stimulates red rod photoreceptors, a range of intensities covering approximately 5 log units produced increases of over 10-fold in cGMP metabolic flux. However, the characteristics of the cGMP metabolic response over the first 2.5 log units of intensity are readily distinguishable from those at higher intensities which exhibit progressive attenuation by an intensity- and time-dependent process. Over the range of low intensities (0.6-3 log photons.micron-2.s-1) the metabolic response is characterized by 1) increases in cGMP hydrolytic flux of up to 8-fold as a logarithmic function of intensity of photic stimulation that are sustained for at least 200 s; 2) small increases or no change in the concentration of total cGMP; 3) large increases of up to 10-fold in the concentration of metabolically active GDP as a linear function of intensity with no significant change in the tissue concentrations of total GDP or GTP; and 4) amplification of the photosignal by the metabolism of approximately 10,000 molecules of cGMP per photoisomerization with the major site of amplification at the level of the interaction of bleached rhodopsin with G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous uterine cyclic GMP and cyclic AMP concentrations were monitored at each stage of the estrus cycle; cyclic GMP concentrations were found to be highest and cyclic AMP concentrations lowest during proestrus when plasma estrogen levels have been shown to be maximal. After administration of estradiol-17beta or diethylstilbestrol, the concentrations of uterine cyclic GMP in ovariectomized rats underwent a progressive and significant elevation and the levels of cyclic AMP declined significantly below control values when near maximal accumulation of tissue cyclic GMP was achieved. The increase in uterine cyclic GMP concentrations produced by estradiol benzoate administration was prevented when progesterone and the estrogen were co-administered. These results raise the possibility that an enhanced cellular accumulation of cyclic GMP may be involved in the expression of some of the actions of estrogen in uterine tissue.

Uterine guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels are elevated significantly from 2 to 12 h after a single injection of estradiol-17 beta or diethylstilbestrol to mature, ovariectomized, or immature rats. The accumulation of cyclic GMP is greater in endometrial- than myometrial-enriched uterine tissue. The estrogen-induced increase in cyclic GMP can be prevented by administration of the protein synthesis inhibitors, cycloheximide and puromycin, or by relatively large doses of the RNA synthesis inhibitor, actinomycin D, but not by the muscarinic antagonist, atropine. The requirement for a protein with a relatively rapid rate of turnover is suggested by the demonstration that cycloheximide, when administered after estrogen, can within a 3-h period restore the estrogen-elevated levels of cyclic GMP to those of the non-estrogen-treated tissue.

Arachidonic acid- or collagen-induced aggregation was accompanied by a progressive elevation in the level of cyclic GMP in washed human platelets with no significant alteration in the concentration of cyclic AMP. The extent of the increase in cyclic GMP was proportional to the concentration of arachidonic acid added. Enhanced accumulation of cyclic GMP produced by arachidonic or collagen was prevented by prior exposure of platelets to aspirin or indomethacin. Prostaglandin endoperoxide G2 caused platelet aggregation and an increase in cyclic GMP concentration; neither event was blocked by prostaglandin synthesis inhibitors. These results indicate that the generation of prostaglandin endoperoxides is a step in the sequence of events in platelet aggregation leading to the enhanced accumulation of cyclic GMP.

The effects of histamine on lipolysis and associated changes in adenosine 3',5'-monophosphate (cyclic AMP) levels were examined in the isolated canine fat cell. Histamine, like norepinephrine, caused a dose-dependent increase in free fatty acid (FFA) and glycerol levels. The lipolytic response to histamine was preceded by a rise in the levels of cyclic AMP and was greatly potentiated by the addition of theophylline. In isolated canine fat cells, histamine (2 muM) caused a 7-fold increase in FFA levels. This effect was inhibited more than 50% in the presence of insulin (0.4 mmu/ml) or prostaglandin E1 (2.8 muM). In similar experiments, cyclic AMP Levels were increased 11-fold by histamine (2 muM) in the presence of 1 mM theophylline. Burimamide (0.1 mM), a histamine H2-receptor antagonist, reduced the effect of histamine (2 muM) on FFA levels as well as the effect on cyclic AMP levels greater than 95% but did not inhibit the lipolytic response to norepinephrine (2 muM). Propranolol (0.01 mM), a beta adrenergic antagonist, reduced the lipolytic response to norepinephrine by 97% but did not inhibit the effects of histamine on FFA or cyclic AMP levels. Tripelennamine and 1,5-diphenyl-3-dimethylaminopyrrolidine, histamine H1-receptor antagonists, inhibited neither the lipolytic response to histamine nor the effect on cyclic AMP levels. It was concluded that histamine induces lipolysis in canine fat cells by a mechanism involving cyclic AMP and the histamine H2-receptor.

Soluble guanylate cyclase activity was measured in normal and psoriatic human epidermis. The specific activity of guanylate cyclase was determined to be increased 10-fold and 3-fold in involved and uninvolved epidermis of psoriatics, respectively, compared with normal epidermis. Arachidonic acid or HETE (5 to 50 microM) stimulated guanylate cyclase activity from involved epidermis 2 to 3-fold and from uninvolved epidermis up to 2-fold, but these fatty acids had no effect on the activity of this cyclase from normal epidermis. These results indicate that the biosynthetic capacity to generate cGMP is markedly increased in involved epidermis from psoriatics because of a markedly increased specific activity of guanylate cyclase and an alteration in a property of this enzyme activity which renders it stimulable by fatty acids reported to accumulate in this lesion.

A labile prostaglandin was isolated as one of the products generated from [1-14C] eicosatetraenoic acid incubated with sheep vesicular gland microsomes. The eicosatetraenoic acid metabolite amounted to ca. 16% of the total radiolabeled products. Formation of this new prostaglandin was prevented when heat-denatured microsomes were employed or when incubation mixtures were supplemented with indomethacin or phenol. However, incubation of prostaglandin G2 (PGG2) with hematin in the presence or absence of catalytically active or heat-inactivated microsomes led to production of approximately the same quantity of the new prostaglandin. These results indicated that the new prostaglandin can be formed nonenzymically. The new prostaglandin was conclusively identified by gas liquid chromatography-mass spectrometry analysis as 15-keto-9,11-peroxidoprosta-5,13-dienoic acid (15-keto-PGG2) after chemical conversion to known prostaglandins. The effects of 15-keto-PGG2 and PGG2 were similar on canine lateral saphenous vein; both promoted contraction followed by prolonged relaxation, but 15-keto-PGG2 appeared to be 1/50 as potent as PGG2.

Soluble guanylate cyclase activity was measured in normal and psoriatic human epidermis. The specific activity of guanylate cyclase was determined to be increased 10-fold and 3-fold in involved and uninvolved epidermis of psoriatics, respectively, compared to normal epidermis. Arachidonic acid (5 to 100 micrometers) or 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) (5 to 50 micrometers) stimulated guanylate cyclase activity from involved epidermis 2- to 3-fold and from uninvolved epidermis up to 2-fold, but these fatty acids had no effect on the activity of this cyclase from normal epidermis. These results indicate that there is an increase in the cGMP biosynthetic capacity of involved epidermis from psoriatics that derives from a markedly increased specific activity of guanylate cyclase and an alteration in a property of this enzyme activity which renders it responsive to fatty acids reported to accumulate in this lesion. These observations are consistent with the report that an elevated steady-state level of cGMP is one of the consequences of the strikingly altered metabolism of cGMP in psoriatic epidermis.

Excitation-contraction coupling in skeletal muscle is known to be under absolute control of plasmalemma voltage, but the steps from transverse (T)-tubule depolarization to Ca2+ release from the sarcoplasmic reticulum have not been elucidated. The effect of changing T-tubule membrane potential on inositol 1,4,5-trisphosphate (InsP3) stimulation of Ca2+ release from the sarcoplasmic reticulum was studied to explore a possible role for InsP3 as a chemical signal in excitation-contraction coupling. InsP3 was microinjected into peeled rabbit skeletal muscle fibers at a pipette concentration of 0.5 microM; Ca2+ release from the sarcoplasmic reticulum was monitored as an isometric tension transient. The response to 0.5 microM InsP3 was significantly larger when T-tubules were in a depolarized state than when they were in a polarized state, and this difference in response was independent of the ionic composition of the bathing solutions or the method for depolarizing the T-tubules. Thus, T-tubule depolarization may sensitize the sarcoplasmic reticulum to a preexisting low concentration of InsP3 and greatly reduce the need for InsP3 production. Plasmalemma voltage control of the stimulatory effects of InsP3 may have relevance for mechanisms in excitable nonmuscle cells.

The characteristics of the positive inotropic effect of different preparations of cholera toxin on the isolated 4-day-old embryonic chick heart were investigated. Crude (CCT) as well as partially purified and purified (PCT)preparations of cholera toxin were shown to have positive inotropic activity. Contractility was increased within 1 minute and reached a maximum at approximately 40 minutes after exposure to toxin. Activities of all three toxin preparations were abolished by heating, their effects were prevented by addition of antitoxin, but nort propranolol, before exposure of the heart to toxin. The positive inotropic effect of PCT was not reversed by washing, but was reversed by subsequent addition of antitoxin. The effects of CCT were reversed by insulin. Effects of both CCT and PCT were potentiated by theophylline, and both toxin preparations elevated tissue levels of cyclic adenosine 3',5'-monophosphate. A filtrate of CCT (MW is less than 30,000) exhibited similar cardiostimulant activity but displayed certain differences from the other toxin preparations. The filtrate was heat stable, and its effects were blocked by pretreatment with propranolol but not by antitoxin. The filtrate of CCT, unlike the toxin-containing preparations, did not stimulate intestinal fluid secretion in the dog.

Primary hepatic actinomycosis is seldom seen, and almost all cases have been diagnosed by laparotomy. Treatment with high-dose parental penicillin for prolonged periods is recommended. In the past concomitant surgical drainage also has been recommended, but the nonoperative success reported here and by others suggests that surgical drainage is not essential to the treatment.

Intracellular and extracellular levels of 3':5'-cyclic GMP and 3':5'-cyclic AMP were studied in synchronized Novikoff rat hepatoma cells. Intracellular levels of cyclic GMP increased spontaneously from 2-fold (without colcemid) to 10-fold (with colcemid), in proportion to the number of cells in mitosis. As cells entered mitosis, cellular cyclic AMP declined simultaneously with the rise in cyclic GMP. These reciprocal changes in cyclic nucleotide levels were reversed as cells passed out of metaphase and through anaphase. Maximum cyclic AMP and minimum cyclic GMP concentrations occurred during G-1. Less marked reciprocal fluctuations in both cyclic nucleotides were also found in S-phase and early G-2, where the ratio of cyclic AMP to cyclic GMP concentrations first fell and then increased. These changes in cyclic nucleotide ratios were closely correlated with major cell-cycle transitions at the boundaries between G-1/S-phase, S-phase/G-2, G-2/prophase, and metaphase/anaphase. Most, but not all, of the extracellular cyclic nucleotides were extruded when cells traversed mitosis. Colcemid or vinblastine completely prevented the appearance of extracellular cyclic AMP but augmented the appearance of extracellular cyclic GMP in parallel with the accumulation of mitotic cells. These results reflected changes in intracellular cyclic nucleotides and indicated that increased intracellular turnover of cyclic GMP and cyclic AMP occurred before and after metaphase, respectively. Elevated cyclic GMP levels during mitosis and S-phase are consistent with potential modulatory roles for this cyclic nucleotide in proliferation.