The LargPAD Trial: Phase IIA evaluation of l-arginine infusion in patients with peripheral arterial disease.

OBJECTIVE: Endothelial function is improved by l-arginine (l-arg) supplementation in preclinical and clinical studies of mildly diseased vasculature; however, endothelial function and responsiveness to l-arg in severely diseased arteries is not known. Our objective was to evaluate the acute effects of catheter-directed l-arg delivery in patients with chronic lower extremity ischemia secondary to peripheral arterial disease. METHODS: The study enrolled 22 patients (45% male) with peripheral arterial disease (mean age, 62 years) requiring lower extremity angiography. Endothelium-dependent relaxation of patent but atherosclerotic superficial femoral arteries was measured using a combination of intravascular ultrasound (IVUS) imaging and a Doppler FloWire (Volcano Corporation, Rancho Cordova, Calif) during the infusion of incremental acetylcholine (10-6 to 10-4 molar concentration) doses. Patients received 50 mg (n = 3), 100 mg (n = 10), or 500 mg (n = 9) l-arg intra-arterially, followed by repeat endothelium-dependent relaxation measurement (limb volumetric flow). IVUS-derived virtual histology of the culprit vessel was also obtained. Endothelium-independent relaxation was measured using a nitroglycerin infusion. Levels of nitrogen oxides and arginine metabolites were measured by chemiluminescence and mass spectrometry, respectively. RESULTS: Patients tolerated limb l-arg infusion well. Serum arginine and ornithine levels increased by 43.6% ± 13.0% and 23.2% ± 10.3%, respectively (P < .005), and serum nitrogen oxides increased by 85% (P < .0001) after l-arg infusion. Average vessel area increased by 6.8% ± 1.3% with l-arg infusion (acetylcholine 10-4; P < .0001). Limb volumetric flow increased in all patients and was greater with l-arg supplementation by 130.9 ± 17.6, 136.9 ± 18.6, and 172.1 ± 24.8 mL/min, respectively, for each cohort. Maximal effects were seen with l-arg at 100 mg (32.8%). Arterial smooth muscle responsiveness to nitroglycerin was intact in all vessels (endothelium-independent relaxation, 137% ± 28% volume flow increase). IVUS-derived virtual histology indicated plaque volume was 14 ± 1.3 mm3/cm, and plaque stratification revealed a predominantly fibrous morphology (46.4%; necrotic core, 28.4%; calcium, 17.4%; fibrolipid, 6.6%). Plaque morphology did not correlate with l-arg responsiveness. CONCLUSIONS: Despite extensive atherosclerosis, endothelial function in diseased lower extremity human arteries can be enhanced by l-arg infusion secondary to increased nitric oxide bioactivity. Further studies of l-arg as a therapeutic modality in patients with endothelial dysfunction (ie, acute limb ischemia) are warranted.

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor/Ca2+ release channel (RyR1): sites and nature of oxidative modification.

In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO2-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO2 only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO2-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO2-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca(2+) channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in "hot spot" regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO2-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.

The pharmacology of aminoadamantane nitrates.

Memantine, an aminodamantane, has recently been approved to treat moderate-to-severe Alzheimer's disease in the US after over 20 years on the market in Europe for treatment of Parkinson's disease. The unique properties of Memantine allow for its selective inhibition of abnormally active NMDA receptor channels while preserving normal glutamate activity and healthy neuronal function. Recently, it has been shown that compounds such as nitroglycerin, used for years for ischemic coronary disease, can also regulate the NMDA receptor channel. Novel compounds have been synthesized in an attempt to combine these activities, in an attempt to synergistically improve upon the activities of both nitrates and aminoadamantanes. We have subjected these compounds to several laboratory tests to compare their ability to affect the function of the NMDA receptor and to dilate blood vessels. These tests provide an initial indication of which of the compounds may have enhanced activity relative to memantine. The results also provide guidance for the synthesis of additional compounds that are likely to have the properties that are being sought.