Mari K. Haddox

Increased cyclic GMP and decreased cyclic AMP levels in the hyperplastic, abnormally differentiated epidermis of psoriasis

Summary The genetic skin disease psoriasis has been examined as a model system that may provide an understanding of the control of normal epidermal specialization (differentiation) and the perturbed regulatory processes in proliferatioe diseases . The excessive glycogen accwnulation, increased proliferation and decreased tissue specialization characteristic of psoriasis involve cellular pro- cesses that have been shown to be regulated by cyclic AMP in other cells and tissues . It has also been suggested that cyclic GMP is a cellular ef- fector that may be involved in promoting cell pro- liferation and other events that oppose those be- lieved to be mediated by cyclic AMP . It was postulated, therefore, that the epidermis of the psoriasis lesion might exhibit an imbalance in the cellular concentrations of these two cyclic nucleo- tides . In this study the levels of cyclic AMP were measured in the involved epidermis (IE) and unin- volved epidermis (UE) from 25 psoriasis patients . The concentrations of cyclic AMP were found as reported previously using a different analytical procedure, to be significantly lower in IE based on protein and DNA . A comparison of the levels of cyclic .GMP in IE versus UE of 12 other psoriasis patients showed the levels of this cyclic nucleo- tide to be significantly increased in IE based on protein, DNA and wet weight . We suggest that this imbalance in the ratio of these two cyclic nucleo- tides may have pathophysiological relevance to the initiation and/or the maintenance of the psoriasis lesion .

ATP (Mg2+) induced inhibition of cyclic AMP reactivity with a skeletal muscle protein kinase

Abstract Exposure of a skeletal muscle protein kinase (peak I DEAE fraction) to μmolar ATP in the presence of Mg2+ before initiating the binding reaction or the cAMP activable phosphotransferase reaction with cAMP increases the requirement for this cyclic nucleotide in both events about 10 fold. The binding of ATP (Mg2+) to a protein in the kinase preparation was shown by Millipore and Sephadex filtration to parallel the inhibitory effect of the triphosphate. The value of Ks for ATP was in the 10−7 M range and 50% inhibition of cAMP (10−8 M) binding occurred at approximately 2 × 10−7 M ATP. ATP binding activity was associated with protein peaks exhibiting both cAMP binding and phosphotransferase activity after subjecting the kinase (holoenzyme) to sedimentation in sucrose density gradients or electrophoresis by electrofocusing.

Periodate-induced increase in cyclic GMP in mouse and guinea pig splenic cells in association with mitogenesis.

INTERACTION of the lectins phytohaemagglutinin (PHA) and concanavalin A (con A) with the lymphocyte plasma membrane initiates a sequence of metabolic events leading to cell proliferation. The numerous alterations in cellular components and processes that occur early after mitogenic stimulation include enhanced uptake of nucleotides2, amino acids3,4, sugars5, Ca2+ (ref. 6), and K+ (ref. 7), enhanced activity of several membrane enzymes8,9, accelerated turnover of phospholipids10,11, increased membrane fluidity12 and an increased accumulation of cellular cyclic 3′, 5′-guanosine monophosphate (cyclic GMP)13–15. Demonstrations that exogenous cyclic GMP can stimulate definable functions in lymphocytes16,17, including proliferation18, and that there is an association between phytomitogen action and cyclic GMP accumulation suggest that this nucleotide is a positive effector of proliferation. To investigate further how cyclic GMP may be involved in this sequence of events, we have studied the effects that the oxidant periodate (IO4) and the reducing agent cysteine have on the cyclic nucleotide levels of rodent splenic cells1. IO4 stimulates lymphocyte proliferation19 and because the oxidant produces definable chemical alterations in membrane components, which can be prevented by reducing agents20, studies with these agents may provide insights into the regulation of cyclic GMP metabolism in relation to mitogenic stimulation.

pH Induced increase in cyclic GMP reactivity with cyclic AMP-dependent protein kinases☆

Abstract Cyclic AMP-dependent protein kinases have been found which exhibit an enhanced capacity to bind cyclic GMP at acidic values of pH. The binding of cyclic GMP to a protein kinase from skeletal muscle, eluted as a single peak from DEAE cellulose columns, is inversely proportional to pH between the values of 7 to 4; the enzyme exhibits a 5 fold greater ability to bind cyclic [ 3 H]-GMP (10 −8 M) at pH 4.0 than 7.0. Protein kinases prepared from skeletal or uterine muscle, eluted as the first of two peaks from DEAE cellulose, exhibited similar pH dependent changes in specificity for cyclic GMP as determined by inhibition of cyclic [ 3 H]-AMP binding. Acidic pH did not appreciably enhance the binding of cyclic [ 3 H]-AMP to kinases prepared from aged skeletal muscle or kinase eluted as the second peak from DEAE cellulose.

Ascorbic acid modulation of splenic cell cyclic GMP metabolism.

Abstract Chronic ascorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80%); ascorbate (1 mM) addition to these cells in vitro restored the cellular concentration to control levels. Splenic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to exogenous ascorbate whereas thiol reducing agents diminished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of ascorbate while agents known to interfere with or promote H 2 O 2 production had no effect. Guanylate cyclase activity in cell lysates increased after treatment of intact cells with ascorbate; dithiothreitol reversed this effect. Ascorbate also enhanced guanylate cyclase activity in cell lysates. The results suggest that oxidizing equivalents in the form of the monoanionic free radical of ascorbate alter cyclic GMP metabolism in these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component.

Oxidative activation of guinea pig splenic cell guanylate cyclase activity by dehydroascorbate, ascorbate, fatty acid hydroperoxides and prostaglandin endoperoxides

The modulation of cyclic GMP metabolism by oxidative-reductive related processes was studied in intact guinea pig splenic cells and with respect to the activities of soluble and particulate guanylate cyclase. In intact cells the oxidant, dehydroascorbic acid (DHA), produced time and concentration dependent increases in cyclic GMP steady state levels. The thiol reductants, cysteine or DTT, decreased cellular cGMP levels 50 to 70%. Exposure of cells to sulfhydryl reactive reagents such as N-ethylmaleimide (NEM), p-hydroxymercuribenzoate, or 5,5′ dithiobis- [2-nitrobenzoic acid] or to the transition metal chelator, 4,7 -diphenyl-1,10 phenanthroline also caused striking lowering of cGMP levels. DHA-induced elevation of cGMP in intact cells persisted after removal of the oxidant and could be reversed upon reduction by DTT. Depression of cGMP steady state levels was induced by DTT which was also shown to persist after removal of the reductant and to be reversed by exposure of the cells to the oxidant, DHA. The effects produced by DHA, DTT, and NEM on soluble and particulate fractions of guanylate cyclase from these cells corresponded to the alterations each produced on cGMP steady state levels in the intact cells. DHA (0.1 to 10 mm) activated while DTT (0.1 to 5 mm) or NEM (0.5 to 5 mm) inhibited guanylate cyclase activity. DHA activation of the soluble enzyme was rapid and additive with the slower activation that occurred spontaneously in air; DTT could prevent or reverse DHA as well as spontaneous air activation. Activation or inhibition by DHA or DTT, respectively, of the particulate enzyme persisted after removal of the effectors but the stable effect produced by the oxidant could be reversed by the reductant and vice versa. Low concentrations of NEM (0.05–0.1 mm) which inhibited basal guanylate cyclase activity minimally (20–25%) completely abolished sensitivity to DHA-activation. Cells exposed to DHA, resuspended in DHA-free media and lysed, exhibited 3 to 4 fold enhanced activity of guanylate cyclase which was restored to the lower basal level of activity upon addition of DTT during the course of the cyclase reaction. Ascorbic acid has similar effects on cGMP metabolism in intact cells including the effect to produce a stable enhancement of guanylate cyclase activity which persists after cell disruption and is suppressed by DTT. However, unlike DHA, ascorbate has no effect on guanylate cyclase activity after cell disruption. Fatty acid hydroperoxides 12-OOH-20:4 (HPETE), 15-OOH-20:4 13-OOH-18:2, and PGE2-OOH and the prostaglandin endoperoxides PGG2 and PGH2 were also shown to be effective activators of soluble (but not particulate) splenic cell guanylate cyclase activity. The hydroxy analogues of these fatty acids and the prostaglandins PGE2 and PGF2 α as well as arachidonic, linoleic, oleic and stearic acids had little or no effect. The stimulatory effects of the lipid hydroperoxides and endoperoxides were demonstrable in air or an argon atmosphere and were prevented and reversed by thiol reductants. Stimulation by both DHA and PGG2 exhibited additivity with respect to promoting the rate at which activation proceeded to achieve a state of maximal catalysis. These results indicate that the soluble guanylate cyclase from splenic cells exhibits regulatory sites for hydrophobic and hydrophilic oxidants which can activate the enzyme by promoting a sulfhydryl-disulfide interconversion at these sites. It is suggested that the action of certain hormones and other membrane active substances may be linked to guanylate cyclase through specific redox systems. The components comprising the link, which remain to be identified, may differ depending upon the cell-type or specific effector at the cell plasma membrane.

[11] The measurement of cyclic GMP with Escherichia coli elongation factor tu

Publisher Summary Elongation factor Tu (EF-Tu) is a soluble factor from E. coli that promotes the binding of aminoacyl-tRNA to ribosomes in the process of peptide chain elongation. EF-Tu is easily obtainable in a reasonably good state of purity, is relatively stable, exhibits a high degree of specificity for GDP (and GTP) and a dissociation constant for GDP in the range of 10 -9 M (10 -7 M for GTP). Highly purified EF-Tu can be prepared from Escherichia coli (B cells obtainable from Grain Processing Corporation, Muscatine, Iowa) by the method of Miller and Weissbach or Arai. The procedure involves preparation of a high speed supernatant fraction from broken cells followed by partial purification of an EF-Tu-GDP complex by ammonium sulfate fractionation, chromatography on DEAE Sephadex A-50, and Sephadex G-100. The specificity of the assay derives from three steps in the procedure: (a) the chromatographic purification of tissue extracts, (b) the selective conversion of GMP catalyzed by ATP-GMP phosphotransferase, and (c) the selective binding by EF-Tu of GDP (and GTP).