Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

Background-Dilated cardiomyopathy is characterized by an imbalance between left ventricular performance and myocardial energy consumption. Experimental models suggest that oxidative stress resulting from increased xanthine oxidase (XO) activity contributes to this imbalance. Accordingly, we hypothesized that XO inhibition with intracoronary allopurinol improves left ventricular efficiency in patients with idiopathic dilated cardiomyopathy. Methods and Results-Patients (nϭ9; ejection fraction, 29Ϯ3%) were instrumented to assess myocardial oxygen consumption (MV O 2 ), peak rate of rise of left ventricular pressure (dP/dt max ), stroke work (SW), and efficiency (dP/dt max /MV O 2 and SW/MV O 2 ) at baseline and after sequential infusions of intracoronary allopurinol (0.5, 1.0, and 1.5 mg/min, each for 15 minutes). Allopurinol caused a significant decrease in MV O 2 (peak effect, Ϫ16Ϯ5%; PϽ0.01; nϭ9) with no parallel decrease in dP/dt max or SW and no change in ventricular load. The net result was a substantial improvement in myocardial efficiency (peak effects: dP/dt max /MV O 2 , 22Ϯ9%, nϭ9; SW/MV O 2 , 40Ϯ17%, nϭ6; both PϽ0.05). These effects were apparent despite concomitant treatment with standard heart failure therapy, including ACE inhibitors and ␤-blockers. XO and its parent enzyme xanthine dehydrogenase were more abundant in failing explanted human myocardium on immunoblot. Conclusions-These findings indicate that XO activity may contribute to abnormal energy metabolism in human cardiomyopathy. By reversing the energetic inefficiency of the failing heart, pharmacological XO inhibition represents a potential novel therapeutic strategy for the treatment of human heart failure.

PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca 2؉ -transporting ATPase in the Golgi, a hitherto unusual location for a Ca 2؉ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45 Ca 2؉ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca 2؉ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 3 Glu and Asp-371 3 Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H ؉ -ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca 2؉ ) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca 2؉ -ATPases. PMR1 therefore represents a new and distinct P-type Ca 2؉ -ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca 2؉ -ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.In eukaryotic cells, the vast bulk of cellular calcium is sequestered within intracellular calcium stores, which maintain cytoplasmic calcium ion concentrations at submicromolar levels and release calcium in response to physiological signals. A major intracellular calcium pool is the endoplasmic reticulum, which is well known for its prominent role in inositol 1,4,5-trisphosphate-and caffeine-mediated calcium release (2). Filling of this store is accomplished by a thapsigargin-sensitive Ca 2ϩ -ATPase, a member of the ubiquitous family of P-type ion pumps, and best characterized by the isoform in skeletal muscle sarcoplasmic reticulum (SERCA1 1 ; Refs. 3 and 4). There are data supporting the existence of another ionomycin-sensitive pool of intracellular calcium that is unresponsive to both inositol 1,4,5-trisphosphate and caffeine, is non-mitochondrial, and appears to be loaded by a thapsigargin-insensitive Ca 2ϩ

Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca 2؉ and Mn 2؉ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca 2؉ pumps and screened for loss of Ca 2؉ and Mn 2؉ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) and Mn 2؉ toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn 2؉ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45 Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45 Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn 774 and Asp 778 in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca 2؉ and Mn 2؉ ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.