Gender bias in gastroparesis: is nitric oxide the answer?

Accumulating evidence suggests that gender-related differences are prominent in gastric motility functions in both health and disease. Women are more susceptible to gastroparesis than men. Though the mechanism(s) involved are not fully understood, impairment of the nitrergic system is one of the main factors responsible for the disease. Uncoupling of neuronal nitric oxide synthase (nNOS) causes a decreased synthesis of NO leading to a reduction in smooth muscle relaxation. Tetrahydrobiopterin (BH(4)) (an essential cofactor for nNOS) is a key regulator of nNOS activity for stomach dysfunction and gastroparesis. In addition, BH(4) has been shown to be a potent antioxidant and anti-inflammatory agent. Well established by results from our laboratory, a diminished intracellular (BH(4):total biopterin) ratio in diabetic female rats significantly impairs nNOS activity and function. Recent research has been focused on BH(4) biosynthesis and gastroparesis because reduced BH(4) cofactor levels can alter the production of NO by nNOS. Researchers are now paying more attention to the possibility of using BH(4) as a therapeutic strategy in gastroparesis. The purpose of this review is to provide an overview of the regulation and function of nNOS by sex hormones and BH(4) and its potential role in the treatment of gastroparesis.

Increased cell migration and plasticity in Nrf2-deficient cancer cell lines.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression is deregulated in many cancers. Genetic and biochemical approaches coupled with functional assays in cultured cells were used to explore the consequences of Nrf2 repression. Nrf2 suppression by Keap1-directed ubiquitylation or the expression of independent short hairpin RNA (shRNA)/siRNA sequences enhanced cellular levels of reactive oxygen species, Smad-dependent tumor cell motility and growth in soft agar. Loss of Nrf2 was accompanied by concomitant Smad linker region/C-terminus phosphorylation, induction of the E-cadherin transcriptional repressor Slug and suppression of the cell-cell adhesion protein E-cadherin. Ectopic expression of the wildtype but not dominant-negative Nrf2 suppressed the activity of a synthetic transforming growth factor-beta1-responsive CAGA-directed luciferase reporter. shRNA knock-down of Nrf2 enhanced the activity of the synthetic CAGA reporter, as well as the expression of the endogenous Smad target gene plasminogen activator inhibitor-1. Finally, we found that Nrf2/Smad3/Smad4 formed an immunoprecipitable nuclear complex. Thus, loss of Nrf2 increased R-Smad phosphorylation and R-Smad signaling, supporting the hypothesis that loss of Nrf2 in an oncogenic context-dependent manner can enhance cellular plasticity and motility, in part by using transforming growth factor-beta/Smad signaling.

High biochemical selectivity of tadalafil, sildenafil and vardenafil for human phosphodiesterase 5A1 (PDE5) over PDE11A4 suggests the absence of PDE11A4 cross-reaction in patients.

The physiological role of phosphodiesterase (PDE)11 is unknown and its biochemical characteristics are poorly understood. We have expressed human His-tagged PDE11A4 and purified the enzyme to apparent homogeneity. PDE11A4 displays K(m) values of 0.97 microM for cGMP and 2.4 microM for cAMP, and maximal velocities were 4- to 10-fold higher for cAMP than for cGMP. Given the homology between PDE11 and PDE5, we have compared the biochemical potencies of tadalafil (Cialis, Lilly-ICOS), vardenafil (Levitra, Bayer-GSK), and sildenafil (Viagra, Pfizer Inc.) for PDE11A4 and PDE5A1. PDE5A1/PDE11A4 selectivities are 40-, 9300-, and 1000-fold for tadalafil, vardenafil, and sildenafil, respectively. This suggests that none of these three compounds is likely to crossreact with PDE11A4 in patients.

Inhibition of the 26S proteasome induces expression of GLCLC, the catalytic subunit for gamma-glutamylcysteine synthetase.

The majority of short- and long-lived cellular proteins are degraded by the activities of the 26S proteasome, a large multi-catalytic protease. Its unique function places it as a central regulatory activity for many important physiological processes. Lactacystin is a very specific 26S proteasome inhibitor and represents an excellent tool for demonstrating that a pathway exhibits proteasome-dependent biochemical regulation. Exposure of HepG2 cells to lactacystin resulted in robust elevation of GLCLC mRNA levels, followed by an increase in GSH concentrations. GLCLC is the gene that encodes the catalytic subunit for gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the synthesis of glutathione (GSH). Inhibition of non-proteasome, protease activities did not induce GLCLC. Gel mobility shift assays and expression of CAT activity from heterologous reporter vectors identified Nrf2 mediation of the GLCLC antioxidant response element, ARE4, as the mechanism by which lactacystin induced GLCLC. These studies have identified 26S proteasome activity as a central regulatory pathway for glutathione synthesis.

Multi-faceted regulation of gamma-glutamylcysteine synthetase.

Glutathione is an important antioxidant that is involved in numerous cellular activities. gamma-Glutamylcysteine synthetase (gammaGCS) is a key regulatory enzyme in the synthesis of glutathione. It is a heterodimeric zinc metalloprotein that belongs to a unique class of proteins that gain activity due to formation of a reversible disulfide bond. The two subunits of gammaGCS exhibit differential and coordinate transcription regulation. In addition, the subunits are regulated at the posttranscriptional and posttranslational levels. These various levels of regulation allow numerous stimuli to induce or inhibit activity.

Determination of terazosin in human plasma, using high-performance liquid chromatography with fluorescence detection.

A selective, sensitive, rapid and reproducible high-performance liquid chromatographic method for the determination of terazosin in plasma is described. The structurally related compound prazosin was used as an internal standard. The method comprises extraction with methylene chloride followed by chromatography on a C18 reversed-phase column. The compounds were detected using spectrofluorimetry. The absolute recoveries were more than 90% with a minimal detection of 1 ng/ml and calibration curve was linear between 1 and 80 ng/ml.

PEST sequences in proteins involved in cyclic nucleotide signalling pathways.

There is growing evidence that PEST sequences act as proteolytic recognition signals within polypeptides. PEST sequences are rich in proline (P), glutamic acid (E), serine (S), and threonine (T) and can be identified by the PEST-FIND program. Both the catalytic and regulatory subunits of the cAMP-dependent protein kinase have been shown to have conditional PEST sequences which are exposed upon cAMP binding to the enzyme. cAMP binding leads to rapid dissociation of C- and R-subunits, and both subunits have increased sensitivity to proteolysis. It is not known whether other proteins that participate in the cyclic nucleotide signalling pathway have PEST regions in their amino acid sequences. Therefore, we have screened amino acid sequences of proteins that are directly involved in cyclic nucleotide cascade, including cGMP-dependent protein kinases, anchoring proteins for cAMP-dependent protein kinase, cyclic nucleotide-gated ion channels, and cyclic nucleotide phosphodiesterases, for PEST sequences using the PEST-FIND program. Many PEST sequences with high scores have been identified in these proteins. The occurrence of the PEST sequences is very high in proteins involved in cyclic nucleotide signalling pathways (approximately 80%). This value is much higher than the percentage (10%) of PEST sequences that can be found in the primary structures of the proteins listed in the data bank. This frequent occurrence of PEST sequences in proteins involved in cyclic nucleotide action and metabolism suggests an important role of proteolysis of these key proteins of signal transduction.

A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5).

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.

Destabilization of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-specific, heat shock inducers results in thermal denaturation at 37 degrees C.

A number of protein reactive compounds, including the thiol reagents diamide and arsenite, are known inducers of heat shock protein (HSP) synthesis and thermotolerance. These compounds are thought to damage cellular protein, which has been proposed to serve as the signal for induction. The specific mechanism of protein damage and its relation to thermal denaturation are unknown. The Ca2+-ATPase of sarcoplasmic reticulum, a membrane protein that contains 24 cys residues, was used to determine the effect of diamide, arsenite, N-ethylmaleimide (NEM), and the cys-specific probes Br-DMC and IAEDANS, which label one or two specific cys residues, respectively, on protein conformation and stability. The Ca2+-ATPase was chosen because diamide has been shown to affect the thermal properties of a class of membrane proteins of CHO cells (Freeman et al., 1995). The labeling of one or two thiols has no effect on activity or conformation, while more extensive reaction (but with less than approximately five to eight groups titrated) results in destabilization of the Ca2+-ATPase such that it denatures thermally at 37 degrees C. Higher levels of titration result in greater destabilization such that the protein is no longer stable at room temperature, with the production of a state similar to the thermally denatured state as assayed by activity, differential scanning calorimetry, ANS binding, and light scattering. The fractional denaturation induced by these thiol reagents, determined by the decrease in the heat absorbed during thermal denaturation, is directly proportional to inactivation of ATPase activity. Thus, inactivation of the Ca2+-ATPase by thiol reagents occurs because of denaturation not through oxidation of essential thiols. These results indicate that these thiol-specific heat shock inducers function by two mechanisms: (1) destabilization of proteins such that they thermally denature at 37 degrees C and (2) direct denaturation, apparently driven by thermal processes at room temperature, following more extensive reaction which results in extreme destabilization. We suggest that these are general mechanisms by which heat shock inducers damage proteins.

Expression of glutathione and gamma-glutamylcysteine synthetase mRNA is Jun dependent.

The gene GLCLC encodes the catalytic subunit of gamma-glutamylcysteine synthetase (glutamate-cysteine ligase E.C. 6.3.2.2), the rate limiting enzyme for glutathione synthesis. When HepG2 cells were exposed to the serine/threonine phosphatase inhibitor okadaic acid (OA), increased expression of GLCLC was observed, as was the development of resistance to xenobiotic induced GSH depletion. Okadaic acid is known to activate both NF-kappaB and AP-1 activity. Inhibition of NF-kappaB activity by overexpression of an IkappaB alpha transdominant inhibitor or exposure to the protease inhibitor TLCK did not inhibit the OA mediated increase in GLCLC transcripts. Fibroblasts derived from a mouse containing a c-Jun null mutation exhibited diminished AP-1 binding activity, reduced levels of GLCLC message, and a correspondingly low GSH concentration compared to wild type cells. When the null cells, which express Jun B and Jun D, were exposed to OA, AP-1 binding activity increased, as did expression of GLCLC message. These results indicate that AP-1 transcription factors participate in the regulation of glutathione metabolism.

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca2(+)-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response.

Alteration of transcriptional and post-transcriptional expression of gamma-glutamylcysteine synthetase by diethyl maleate.

Gamma-glutamylcysteine synthetase (gamma-GCS), also known as glutamate-cysteine ligase (EC 6.3.2.2), is the rate-limiting enzyme in the synthesis of glutathione (GSH). The gene GLCLC encodes the catalytic subunit while GLCLR encodes the regulatory subunit. Although it has been shown that GLCLC can respond to a variety of stresses by increased transcription, it is not known whether a similar response occurs for GLCLR. Nor is it known whether post-transcriptional regulation of either gene product is altered during stress. The present investigation was undertaken to explore transcriptional and post-transcriptional regulation of GLCLC and GLCLR gene products when HepG2 cells were challenged with the radiation sensitizer diethyl maleate (DEM). Expression of steady-state GLCLC and GLCLR mRNA was enhanced 5-20-fold after DEM challenge. Nuclear run-off assays were performed on unstressed and stressed cells to determine whether the increased expression of GLCLC and GLCLR mRNA was due to altered transcriptional activity of these genes. The DEM treatment increased the transcription rates of both genes 2-5-fold. In unstressed HepG2 cells, the half-life of GLCLC mRNA transcripts was approximately 4 h. In contrast, the half-life of GLCLR transcripts was approximately 8 h. In cells treated with DEM, the half-lives of all transcripts were increased, indicating that message stabilization contributed to the increased expression of gene products. Finally, a PEST algorithm has identified a PEST (proline, glutamate, serine, threonine) motif within the catalytic subunit of gamma-GCS, suggesting that this subunit might exhibit conditional proteolytic regulation. These results imply that regulation of the products of the GLCLC and GLCLR genes may be altered at multiple levels during exposure to stress.

Identifying protein kinases in crude extracts that phosphorylate cyclic GMP-binding cyclic-GMP specific phosphodiesterase.

The main protein kinase that phosphorylates cyclic GMP-binding cyclic GMP-specific phosphodiesterase (cG-BPDE) in crude extracts of bovine lung is cyclic GMP-dependent protein kinase. This can be shown by the use of either exogenous or endogenous cG-BPDE as substrate for endogenous cyclic GMP-dependent protein kinase. The characteristics of this phosphorylation suggest a physiological significance.

Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type I alpha, compared with type I beta, cGMP-dependent protein kinase.

Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.