Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

Dynamic imaging of cell, extracellular matrix, and tissue movements during avian vertebral axis patterning

Vertebrate axis patterning depends on cell and extracellular matrix (ECM) repositioning and proper cell-ECM interactions. However, there are few in vivo data addressing how large-scale tissue deformations are coordinated with the motion of local cell ensembles or the displacement of ECM constituents. Combining the methods of dynamic imaging and experimental biology allows both cell and ECM fate-mapping to be correlated with ongoing tissue deformations. These fate-mapping studies suggest that the axial ECM components "move" both as a composite meshwork and as autonomous particles, depending on the length scale being examined. Cells are also part of this composite, and subject to passive displacements resulting from tissue deformations. However, in contrast to the ECM, cells are self-propelled. The net result of cell and ECM displacements, along with proper ECM-cell adhesion, is the assembly of new tissue architecture. Data herein show that disruption of normal cell-ECM interactions during axis formation results in developmental abnormalities and a disorganization of the ECM. Our goal in characterizing the global displacement patterns of axial cells and ECM is to provide critical information regarding existing strain fields in the segmental plate and paraxial mesoderm. Deducing the mechanical influences on cell behavior is critical, if we are to understand vertebral axis patterning. Supplementary material for this article is available online at http://www.mrw.interscience.wiley.com/suppmat/1542-975X/suppmat/72/v72.266.html.     

Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. 1969

Xeroderma pigmentosum (XP) is a recessively transmitted disorder of man characterized by increased sensitivity to ultraviolet light. Homozygous, affected individuals, upon exposure to sunlight, sustain severe damage to the skin; this damage is characteristically followed by multiple basal and squamous cell carcinomas and not uncommonly by other malignant neoplasia. A tissue culture cell line was derived from the skin of a man with XP. Our measurements of ultraviolet-induced pyrimidine dimers in cellular DNA show that normal diploid human skin fibroblasts excise up to 70 per cent of the dimers 24 hours, but that fibroblasts derived from the individual with XP excise less than 20 per cent in 48 hours. Alkaline gradient sedimentation experiments show that during the 24 hours after irradiation of normal cells a large number of single-stranded breaks appear and then disappear. Such changes are not observed in XP cells. XP cells apparently fail to start, the excision process because they lack the required function of an ultraviolet-specific endonuclease. These findings, plus earlier ones of Cleaver on the lack of repair replication in XP cells, raise the possibility that unexcised pyrimidine dimers can be implicated in the oncogenicity of ultraviolet radiation.     

Glycation end-product cross-link breaker reduces collagen and improves cardiac function in aging diabetic heart

Aging and diabetes mellitus (DM) both affect the structure and function of the myocardium, resulting in increased collagen in the heart and reduced cardiac function. As part of this process, hyperglycemia is a stimulus for the production of advanced glycation end products (AGEs), which covalently modify proteins and impair cell function. The goals of this study were first to examine the combined effects of aging and DM on hemodynamics and collagen types in the myocardium in 12 dogs, 9-12 yr old, and second to examine the effects of the AGE cross-link breaker phenyl-4,5-dimethylthazolium chloride (ALT-711) on myocardial collagen protein content, aortic stiffness, and left ventricular (LV) function in the aged diabetic heart. The alloxan model of DM was utilized to study the effects of DM on the aging heart. DM induced in the aging heart decreased LV systolic function (LV ejection fraction fell by 25%), increased aortic stiffness, and increased collagen type I and type III protein content. ALT-711 restored LV ejection fraction, reduced aortic stiffness and LV mass with no reduction in blood glucose level (199 +/- 17 mg/dl), and reversed the upregulation of collagen type I and type III. Myocardial LV collagen solubility (%) increased significantly after treatment with ALT-711. These data suggest that an AGE cross-link breaker may have a therapeutic role in aged patients with DM.     

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.     

An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8(+) T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen's developmental cycle. In addition to their use as candidate Ags for stimulating CD8(+) T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8(+) T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8(+) T cells specific for an H-2D(b)-restricted epitope from CrpA are elicited at a significant level (approximately 4% of splenic CD8(+) T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.     

The diversity of globin-coupled sensors

The recently discovered globin-coupled sensors (GCSs) are heme-containing two-domain transducers distinct from the PAS domain superfamily. We have identified an additional 22 GCSs with varying multi-domain C-terminal transmitters through a search of the complete and incomplete microbial genome datasets. The GCS superfamily is composed of two major subfamilies: the aerotactic and gene regulators. We postulate the existence of protoglobin in Archaea as the predecessor to the chimeric GCS.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.