Potassium-dependent sodium-calcium exchange through the eye of the fly.

In this review, we describe the characterization of a Drosophila sodium/calcium-potassium exchanger, Nckx30C. Sodium/calcium (-potassium) exchangers (NCX and NCKX) are required for the rapid removal of calcium in excitable cells. The deduced protein topology for NCKX30C is similar to that of mammalian NCKX, with 5 hydrophobic domains in the amino terminus separated from 6 at the carboxy-terminal end by a large intracellular loop. NCKX30C functions as a potassium-dependent sodium-calcium exchanger and is expressed in adult neurons and during ventral nerve cord development in the embryo. Nckx30C is expressed in a dorsal/ventral pattern in the eye-antennal disc, suggesting that large fluxes of calcium may be occurring during imaginal disc development in the larvae. NCKX30C may play a critical role in modulating calcium during development as well as in the removal of calcium and maintenance of calcium homeostasis in adults.

Two-step process for photoreceptor formation in Drosophila.

The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1-R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulators and sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1-R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.

Normal phototransduction in Drosophila photoreceptors lacking an InsP(3) receptor gene.

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. We investigated the role of the InsP(3) receptor (InsP(3)R) by generating mosaic eyes homozygous for a deficiency of the only known InsP(3)R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors underwent late onset retinal degeneration; however, whole-cell recordings from young flies demonstrated that phototransduction was unaffected, quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores all being normal. Using the specific TRP channel blocker La(3+) we demonstrated that both TRP and TRPL channel functions were unaffected. These results indicate that InsP(3)R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors.

Role of asparagine-linked oligosaccharides in rhodopsin maturation and association with its molecular chaperone, NinaA.

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn(20) is the sole site for glycosylation. The Rh1(N20I) protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1(N20I); therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1(N20I)/NinaA interaction. We further show that Rh1(N20I) interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.

Cloning and characterization of a potassium-dependent sodium/calcium exchanger in Drosophila.

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.

Pravastatin prevents clinical events in revascularized patients with average cholesterol concentrations. Cholesterol and Recurrent Events CARE Investigators.

OBJECTIVES: This analysis was carried out to determine if revascularized patients derive benefit from the 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor pravastatin. BACKGROUND: The HMG-CoA reductase inhibitors result in substantial reductions in serum cholesterol and stabilization of atherosclerotic plaques in patients with coronary artery disease. METHODS: Pravastatin was found to reduce clinical cardiovascular events in the Cholesterol and Recurrent Events (CARE) trial consisting of 4,159 patients with a documented myocardial infarction and an average cholesterol level (mean 209 mg/dl and all <240 mg/dl). A total of 2,245 patients underwent coronary revascularization before randomization including 1,154 patients with percutaneous transluminal coronary angioplasty (PTCA) alone, 876 patients with coronary artery bypass graft (CABG) alone, and 215 patients with both procedures. Clinical events in revascularized patients were compared between patients on placebo and on pravastatin. RESULTS: In the 2,245 patients who had undergone revascularization, the primary endpoint of coronary heart disease death or nonfatal myocardial infarction (MI) was reduced by 4.1% with pravastatin (relative risk [RR] reduction 36%, 95% confidence interval [CI] 17 to 51, p = 0.001). Fatal or nonfatal MI was reduced by 3.3% (RR reduction 39%, 95% CI 16 to 55, p = 0.002), postrandomization repeat revascularization was reduced by 2.6% (RR reduction 18%, 95% CI 1 to 33, p = 0.068) and stroke was reduced by 1.5% (RR reduction 39%, 95% CI 3 to 62, p = 0.037) with pravastatin. Pravastatin was beneficial in both the 1,154 PTCA patients and in the 1,091 CABG patients who had undergone revascularization before randomization. CONCLUSIONS: Pravastatin reduced clinical events in revascularized postinfarction patients with average cholesterol levels. This therapy was well tolerated and its use should be considered in most patients following coronary revascularization.

Left atrial diameter in nonvalvular atrial fibrillation: An echocardiographic study. Stroke Prevention in Atrial Fibrillation Investigators.

BACKGROUND: The left atrium (LA) is usually enlarged in patients with nonvalvular atrial fibrillation (AF), but factors associated with LA diameter are incompletely defined. METHODS AND RESULTS: This transthoracic echocardiographic cohort study includes 3465 participants with nonvalvular AF in 3 multicenter clinical trials. LA diameter determined by M-mode echocardiography was correlated with clinical and echocardiographic features by cross-sectional multivariate regression analyses. The mean LA diameter was 47 +/- 8 mm, on average 6 mm larger in those with AF at the time of echocardiography than in those with sinus rhythm (48 vs 42 mm, P <. 001). Patient age and body weight were independently predictive of LA diameter (P <.0001), but sex, body surface area, and body mass index were not. The estimated independent contribution of atrial rhythm to LA diameter was approximately 2.5 mm. Prolonged duration of AF, left ventricular dilatation and increased muscle mass, mitral regurgitation, annular calcification, and hypertension were additional independent predictors of LA diameter. CONCLUSIONS: Multiple factors appear to contribute to LA enlargement in patients with nonvalvular AF, including the presence and persistence of the dysrhythmia.

Restenosis is associated with decreased coronary artery nitric oxide synthase.

The purpose of the present study was to test the hypothesis that restenosis is associated with decreased constitutive nitric oxide synthase activity. Male miniswine with moderately elevated serum cholesterol levels underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries, followed 2 weeks later by repeat injury on the same coronary segments. After 4 weeks, the coronary arteries were either immediately frozen in liquid nitrogen or pressure-perfusion fixed and prepared for histologic examination. Constitutive nitric oxide synthase activity was quantified using a fibroblast reporter cell method, while constitutive nitric oxide synthase protein was compared between balloon-injured and non-balloon-injured arteries using Western blot analysis. Immunohistochemical studies were performed using a specific antibody against constitutive nitric oxide synthase protein. Following balloon injury, there was decreased constitutive nitric oxide synthase activity in balloon-injured coronary arteries, compared to distal non-balloon-injured segments from the same artery. Histological examination demonstrated an intact endothelium. Specific antibody staining revealed that there was less constitutive nitric oxide synthase protein reactivity by immunohistochemical analysis. Western analysis confirmed less constitutive nitric oxide synthase protein. The data are consistent with the hypothesis that restenosis is associated with decreased endothelial cell nitric oxide production. The data suggest this is secondary to a decreased amount of constitutive nitric oxide synthase enzyme in the endothelium. A deficiency in constitutive nitric oxide synthase enzyme may contribute to the impaired second messenger and paracrine functions of the endothelium observed during restenosis following balloon injury, including abnormal vasomotion, extracellular matrix formation, and platelet aggregation.

Extracellular matrix collagen synthesis and degradation following coronary balloon angioplasty.

Percutaneous transluminal coronary angioplasty is associated with intimal hyperplasia and extracellular matrix deposition of collagen, leading to restenosis in a significant number of cases. The purpose of the present study was to determine the effects of balloon angioplasty on extracellular matrix collagen content and collagenase activity in a porcine coronary artery restenosis model 6 weeks following balloon injury. We tested the hypothesis that in balloon-injured arteries the neointimal extracellular matrix was characterized by increased collagen content and decreased metalloproteinase activity relative to non-injured arteries. Male miniswine maintained on a high cholesterol diet underwent cardiac catheterization and double balloon injury to the right and left circumflex coronary arteries. The coronary arteries were either pressure-perfusion-fixed and prepared for histological examination, or dissected free of adventitia for further collagen and matrix metalloproteinase studies. Collagen synthesis in balloon-injured coronary arteries was compared to non-injured arteries using Northern blot analysis and histochemical stains. Comparative studies on differences between balloon-injured and non-balloon-injured arterial matrix metalloproteinase activity were done using zymography. Balloon angioplasty arterial injury resulted in a significant increase in type I collagen mRNA expression, with increased collagen deposition in the extracellular matrix. In contrast, matrix metalloproteinase activity was markedly decreased. The results suggest that the increased neointimal extracellular matrix observed late in the injury response may be due to not only increased collagen synthesis, but also reduced degradation. The failure to achieve a balance between the synthesis and degradation of extracellular matrix collagen could serve as an important mechanism responsible for restenosis.

The effects of intravascular stents on vasomotion in porcine coronary arteries.

Intravascular stents are being increasingly utilized in the treatment of atherosclerotic coronary artery disease, however little is known about the effects of stents on coronary vasomotion. The purpose of the present study was to compare the effects of a heparin treated tantalum stent and balloon injury on coronary artery vasorelaxation and vasoconstriction. Male miniswine underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries. After two weeks, one artery was either balloon-injured again, or underwent implantation of a stent. Four weeks later, the coronary arteries were prepared for in vitro isometric ring studies. Vasodilator responses to bradykinin and A23187 calcium ionophore were significantly impaired in balloon-injured vessels. The bradykinin and A23187 responses in stented vessels showed significantly less vasorelaxation, compared to both balloon-injured and normal vessels. There were no significant differences between any of the groups in their vasodilator response to nitroprusside. Vasoconstrictor responses to acetylcholine were significantly greater in balloon-injured vessels, compared to normal vessels. Stented vessels, however, showed markedly less vasoconstriction to acetylcholine compared to both balloon-injured and normal vessels. The maximal KCI vasoconstrictor responses in balloon injured vessels and normal controls were not significantly different. However, the maximal KCI responses in stented vessels showed significantly less constriction compared to both balloon injured and normal vessels. In conclusion, the data demonstrated that coronary arteries implanted with a heparin treated tantalum stent were capable of vasomotor activity. Both conventional balloon angioplasty and stents resulted in impaired endothelium-dependent vasorelaxation. Endothelium-independent vasorelaxation, however, was not impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Restenosis following placement of an intracoronary heparin treated tantulum stent in the hyperlipidemic miniature swine model.

Restenosis persists as an important factor limiting a favorable long term outcome following mechanical revascularization. The objective of the present study was to compare the effects of an intracoronary heparin treated tantalum prototype stent and balloon angioplasty on intimal hyperplasia, luminal diameter, and thrombosis in a porcine restenosis model. Male miniswine maintained on a high cholesterol diet and 325 mg aspirin per day underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries. Two weeks later one artery was either balloon injured again or implanted with a stent. No additional anticoagulation following stent placement was given, however aspirin was continued throughout the study. At four weeks, the coronary arteries were harvested and prepared for histologic examination and blinded quantitative morphometric analysis. The prototype stent was successfully deployed in 10 coronary arteries. Histological examination at explant revealed no evidence for thrombus or platelet aggregation. The angiographic luminal diameter of stented vessels was not significantly different from the diameter measured prior to implantation. In contrast, the angiographic diameter of balloon injured vessels was significantly decreased (4.4 +/- 0.4 mm2, balloon injured, vs. 5.8 +/- 3.3 mm2, control; p < 0.05). Stented arteries showed significantly more intimal hyperplasia, compared to balloon injured vessels (2.99 +/- 0.58 mm2 intimal area, stented arteries vs. 0.38 +/- 0.15 mm2 intimal area, control arteries; p < 0.05). In conclusion, heparin treated tantalum wire prototype intracoronary stents were successfully deployed in swine coronary arteries with no evidence for thrombus formation. Despite a significant intimal response, luminal diameter was preserved in stented vessels. The data suggest that a heparin treated tantalum wire prototype intracoronary stent may be an effective method of coronary revascularization that results in the preservation of luminal diameter without thrombotic occlusion.

Early angioplasty in patients with acute myocardial infarction complicated by hypotension.

Emergency percutaneous transluminal coronary angioplasty was performed in 62 patients with acute myocardial infarction complicated by hypotension. All patients were treated within 12 hours of the onset of chest pain. Angioplasty was completely successful (residual lesion less than or equal to 50%) in 48 patients, partially successful (patent vessel greater than 50% residual lesion) in four patients, and unsuccessful in 10 patients. Patients in whom angioplasty was successful had a hospital mortality rate of 19%; those in whom angioplasty was unsuccessful or only partially successful had hospital mortality rates of 60% and 50%, respectively, (p = 0.012). Patients with occlusion of the proximal left anterior descending vessel had the highest failure rate (42%) and the highest mortality rate (67%). Other univariate predictors of hospital mortality were older age and elevated end-diastolic pressure. Successful emergency angioplasty improves mortality in patients with acute infarction complicated by hypotension.

Emergency angioplasty in acute anterior myocardial infarction.

Ninety-three patients with acute anterior myocardial infarction were treated with emergency percutaneous transluminal coronary angioplasty (PTCA). All were found to have a high-grade obstruction in the left anterior descending (LAD) vessel or the bypass graft to this vessel; 64 patients had a total occlusion. A completely successful PTCA, defined as a residual lesion of less than or equal to 50%, was achieved in 73 (78%) patients. A partially successful PTCA, with a residual lesion of 51% to 99%, was achieved in 12 (13%) patients. PTCA was unsuccessful in eight (9%) patients. Hospital mortality was 14%. Three parameters viewed separately each predicted hospital mortality: presence of shock, a proximal location of the LAD vessel occlusion, and the residual stenosis after PTCA. Reocclusion was found in only 11% of patients but 34% had evidence of restenosis on restudy.

Efficacy of physical conditioning exercise in patients with rheumatoid arthritis and osteoarthritis.

A group of 120 patients with rheumatoid arthritis or osteoarthritis volunteered to be subjects for this study of aerobic versus nonaerobic exercise. Patients were stratified by diagnosis and randomized into an exercise program of aerobic walking, aerobic aquatics, or nonaerobic range of motion (controls). The retention rate for the 12-week program was 83%. Exercise tolerance, disease-related measures, and self-reported health status were assessed. The aquatics and walking exercise groups showed significant improvement over the control group in aerobic capacity, 50-foot walking time, depression, anxiety, and physical activity after the 12-week exercise program. There were no significant between-group group differences in the change scores for flexibility, number of clinically active joints, duration of morning stiffness, or grip strength. Our findings document the feasibility and efficacy of conditioning exercise for people who have rheumatoid arthritis or osteoarthritis.

Exercise tolerance and disease related measures in patients with rheumatoid arthritis and osteoarthritis.

One hundred and twenty patients with symptomatic rheumatoid arthritis (RA) or osteoarthritis (OA) in weight bearing joints (RA = 40; OA = 80) performed subjective maximal graded exercise tests on a motor driven treadmill. Disease related measures were also assessed. Findings from this sample indicated that people with arthritis were significantly impaired in exercise tolerance, flexibility and biomechanical efficiency. Significant differences between diagnoses appeared on a number of disease related measures; however, there was little correlation between disease related measures and exercise tolerance. Women demonstrated a greater aerobic impairment than men; and women with RA had a greater aerobic deficit than women with OA.

Limitations of echocardiography in the diagnosis of left atrial myxoma: a case report.

The classical echocardiographic features of left atrial myxoma are quite distinctive but do not comprise the full spectrum of abnormalities encountered. This case demonstrates atypical echocardiographic features of left atrial myxoma that initially obscured the correct diagnosis. Our findings underscore the potential limitations of echocardiography in the diagnosis of left atrial myxoma.

Observer variation in the echocardiographic diagnosis of mitral valve prolapse.

To assess inter- and intraobserver variation in the echocardiographic diagnosis of mitral valve prolapse, three independent observers analyzed M-mode echocardiograms (n = 80) and two-dimensional echocardiograms (n = 65) of patients with a mobile midsystolic click with or without a late or holosystolic murmur. In addition, a control group of 100 normal echocardiograms were interspersed among the echocardiograms of patients with mitral valve prolapse and were then interpreted. Each of the three observers analyzed all M-mode and two-dimensional echocardiograms initially and then 2 weeks later for the presence or absence of mitral valve prolapse. M-mode echocardiographic criteria for mitral valve prolapse consisted of late systolic posterior motion (greater than or equal to 3 mm) of one or both mitral leaflets or holosystolic hammocking (greater than or equal to 3 mm) of one or both mitral leaflets. Two-dimensional echocardiographic criteria for mitral valve prolapse consisted of: posterior systolic arching of one or both mitral leaflets in the parasternal long-axis view, and/or posterior systolic bowing of one or both mitral leaflets in the apical four-chamber view posterior to the plane of the mitral anulus, and/or excessive posterior coaptation of the mitral leaflets in either view flush with or posterior to the plane of the mitral anulus. There was insignificant observer variation both in the M-mode and two-dimensional echo groups, as determined using Cochran's Q test.(ABSTRACT TRUNCATED AT 250 WORDS)