Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.

Aspirin prodrugs and related nitric oxide releasing compounds hold significant therapeutic promise, but they are hard to design because aspirin esterification renders its acetate group very susceptible to plasma esterase mediated hydrolysis. Isosorbide-2-aspirinate-5-salicylate is a true aspirin prodrug in human blood because it can be effectively hydrolyzed to aspirin upon interaction with plasma BuChE. We show that the identity of the remote 5-ester dictates whether aspirin is among the products of plasma-mediated hydrolysis. By observing the requirements for aspirin release from an initial panel of isosorbide-based esters, we were able to introduce nitroxymethyl groups at the 5-position while maintaining ability to release aspirin. Several of these compounds are potent inhibitors of platelet aggregation. The design of these compounds will allow better exploration of cross-talk between COX inhibition and nitric oxide release and potentially lead to the development of selective COX-1 acetylating drugs without gastric toxicity.

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

We describe a new type of barbiturate-based matrix metalloproteinase (MMP) inhibitor incorporating a nitric oxide (NO) donor/mimetic group (series 1). The compounds were designed to inhibit MMP at enzyme level and to attenuate MMP-9 secretion arising from inflammatory signaling. To detect effects related to the nitrate, we prepared and studied an analogous series of barbiturate C5-alkyl alcohols that were unable to release NO (series 2). Both series inhibited recombinant human MMP-2/9 activity with nanomolar potency. Series 1 consistently inhibited the secretion of MMP-9 from TNFα/IL1β stimulated Caco-2 cells at 10 μM, which could be attributed to NO related effects because the non-nitrate panel did not affect enzyme levels. Several compounds from series 1 (10 μM) inhibited tumor cell invasion but none from the non-nitrate panel did. The work shows that MMP-inhibitory barbiturates are suitable scaffolds for hybrid design, targeting additional facets of MMP pathophysiology, with potential to improve risk-benefit ratios.