Quercetin induced tissue-type plasminogen activator expression is mediated through Sp1 and p38 mitogen-activated protein kinase in human endothelial cells.

BACKGROUND: Wine polyphenol quercetin upregulates tissue-type plasminogen activator (t-PA) transcription in cultured human umbilical cord vein endothelial cells (HUVECs). However, the regulatory elements and signaling pathways involved in this regulation are unknown. OBJECTIVES: We aimed to localize quercetin-responsive t-PA promoter elements, identify the proteins that bind these elements, and decipher signaling pathways involved in the regulation of t-PA. METHODS: To localize quercetin-responsive elements, HUVECs were transiently transfected with various t-PA promoter-reporter constructs. Element functionality was evaluated by mutational analysis. Nuclear protein-t-PA element interactions were evaluated by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) analysis. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the signaling pathways involved in t-PA regulation. MAPK inhibition effects were evaluated by real-time PCR, immunoblotting analysis, and transfections. Coimmunoprecipitation was used to evaluate MAPK and transcription factor interaction. RESULTS: Deletion of the t-PA promoter region - 288 to - 250 resulted in loss of quercetin responsiveness. This region contains putative Sp1-binding elements, which we termed Sp1a and Sp1b. Sp1b mutation abolished the quercetin-inducible response, whereas Sp1a mutation had no effect. EMSA and ChIP analysis demonstrated quercetin-enhanced Sp1 binding to Sp1b. Inhibition of p38 MAPK abrogated basal and quercetin-induced t-PA expression and promoter activity, as well as quercetin-induced Sp1 binding to Sp1b. Quercetin enhanced p38 MAPK and Sp1 physical association, which was similarly diminished by p38 MAPK inhibition. CONCLUSIONS: We showed, for the first time, the presence of a functional Sp1-binding element in the t-PA promoter controlling quercetin induction via the p38 MAPK pathway. Understanding these mechanisms may provide new insights into polyphenol cardioprotective effects.

Activation of leptin expression by an inhibitor of carnitine palmitoyltransferase-1.

Leptin may regulate peripheral fatty acid oxidation and invoke a feedback mechanism that affects leptin expression in adipocytes. The objective of this study, therefore, was to determine whether inhibiting systemic fatty acid oxidation at the level of carnitine palmitoyltransferase-1 (CPT1) affects leptin expression. To accomplish this objective, fed or overnight fasted rats were treated with 2-tetradecylglycidic acid (TDGA), a specific, irreversible CPT1 inhibitor, and acute changes in rat epididymal leptin expression and serum leptin content were measured using Northern, RT-PCR, and radioimmunoassay analyses. Overnight fasting decreased both epididymal leptin mRNA content and serum leptin. Treating overnight fasted rats with TDGA increased both their epididymal leptin mRNA and their serum leptin significantly in a time- and concentration-dependent manner. TDGA affected neither epididymal leptin mRNA nor serum leptin in fed rats where systemic fatty acid oxidation is low. These results support the conclusion that CPT1-linked fatty acid oxidation is a key modulator of leptin expression in fasting rats.

1H-MRS detected lipolysis in diabetic rat hearts requires neutral lipase.

PURPOSE: Triacylglycerol (TAG) lipolysis increases in diabetic hearts. However, it is not known which pathway for lipolysis catalyzes this process. Thus, using 1H-magnetic resonance spectroscopy (MRS), we determined whether TAG lipolysis in diabetic rat hearts requires acid lipase or neutral lipase activity. METHODS: Rats were given IP injections of 110 mg streptozotocin (STZ)/kg. Forty-eight to 72 h after this treatment, all rats exhibited ketotic diabetes. The hearts of these ketotic rats were isolated, perfused isovolumically, and analyzed using 1H-MRS. RESULTS: The content of methylene protons (CH2)n--and otherfatty acid protons, measured using 1H-MRS, increased in hearts isolatedfrom STZ-treated compared to untreated rats. This increase in heart--(CH2)n--was directly related to the chemical content of heart TAGs. If isolated diabetic hearts were perfused with either glucose or glucose plus the acid lipase inhibitor methylamine, then heart content of TAG, measured as (CH2)n, decreased at rates of approximately 130 nmol TAG/gdw/min throughout a 55-min perfusion. If diabetic hearts were pretreated with the neutral lipase inhibitor diethyl-p-nitro-phenylphosphate (DNPP) and perfused with glucose, then heart TAG content, measured as (CH2)n, did not change during perfusion. CONCLUSIONS: 1H-MRS can detect the TAG and the net lipolysis of TAG in diabetic rat hearts. Net TAG lipolysis in diabetic rat hearts requires neutral lipase.

Identification of a 251-bp fragment of the PAI-1 gene promoter that mediates the ethanol-induced suppression of PAI-1 expression.

BACKGROUND: Moderate alcohol consumption reduces the risk for coronary heart disease. This cardioprotection may be due to ethanol enhancement of fibrinolysis. Fibrinolysis involves the interaction of plasminogen activators (PAs) and the plasminogen activator inhibitor type-1 (PAI-1). Factor(s) that decrease endothelial cell (EC) PAI-1 expression increase fibrinolysis and may decrease the risk for cardiovascular disease. METHODS: Five promoter deletion fragments were generated from a 1.1-kb PAI-1 promoter fragment and ligated to a luciferase reporter gene. Cultured human umbilical vein endothelial cells (HUVECs) were transiently transfected with these PAI-1 deletion constructs. A 251-base pair (bp) fragment of the PAI-1 promoter, positions -800 to -549, was cloned upstream of a heterologous promoter/enhancer. ECs luciferase activity was measured in the absence/presence of 20 mM ethanol. Electrophoresis mobility shift assays were performed with nuclear extracts from untreated and ethanol-treated ECs using this 251-bp fragment. RESULTS: Deletion analysis showed a region between position -800 and -549 mediated ethanol repression of luciferase activity. This 251-bp promoter fragment also repressed the activity of a heterologous promoter/enhancer in the presence of ethanol. Using the labeled 251-bp fragment, nuclear extracts from ethanol-treated ECs contained two inducible bands and one enhanced band. Non-ethanol treated nuclear extracts also contained a band that was not observed in ethanol-treated samples. Competition using 100-fold molar excess of unlabeled probe abolished these four bands. CONCLUSIONS: Repression of PAI-I gene transcription in cultured HUVECs exposed to ethanol may involve the interaction of several transcription factors with binding sites localized between positions -800 and -549 of the PAI-1 gene promoter.

Stereoselective synthesis of a conformationally defined cyclohexyl carnitine analogue that binds CPT-1 with high affinity.

Carnitine (1, 3-hydroxy-4-trimethylammoniobutyrate) is important in mammalian tissue as a carrier of acyl groups. In order to explore the binding requirements of the carnitine acyltransferases for carnitine, we designed conformationally defined cyclohexyl carnitine analogues. These diastereomers contain the required gauche conformation between the trimethylammonium and hydroxy groups but vary the conformation between the hydroxy and carboxylic acid groups. Here we describe the synthesis and biological activity of the all-trans diastereomer (2), which was prepared by the ring opening of trans-methyl 2,3-epoxycylohexanecarboxylate with NaN3. Racemic 2 was a competitive inhibitor of neonatal rat cardiac myocyte CPT-1 (K(i) 0.5 mM for racemic 2; K(m) 0.2 mM for L-carnitine) and a noncompetitive inhibitor of neonatal rat cardiac myocyte CPT-2 (K(i) 0.67 mM). These results suggest that 2 represents the bound conformation of carnitine for CPT-1.

2-Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase-1, induces myocardial hypertrophy via the AT1 receptor.

Activation of the antiogensin II, type 1 (AT1) receptor mediates the myocardial response to numerous hypertrophic stimuli. This study tested the hypothesis that 2-tetradecylglycidic acid (TDGA), an oxirane carboxylate inhibitor of mitochondrial carnitine plamitoyltransferase-1, induces myocardial hypertrophy via the AT1 receptor system. Male Sprague-Dawley rats treated with 10 mg TDGA/kg/day for 7 days had a heart wet weight:body weight ratio of 3. 58+/-0.16 mg/g compared with a ratio of 2.79+/-0.07 for rats treated with vehicle (P<0.05). The plasma level of antiogensin II was 117. 75+/-17.39 pg/ml in rats treated with 10 mg TDGA/kg/day compared with 54.0+/-11.38 pg/ml for rats treated with vehicle (P<0.05). The plasma level of angiotensin I in these two groups of rats was not different statistically. Rats treated with TDGA and given drinking water containing 1 mg losartan/ml had a heart wet weight:body weight ratio of 2.84+/-0.05 mg/g. This value was not statistically different from the value measured in rats given drinking water containing 1 mg losartan/ml and treated with vehicle alone. No significant difference in the heart wet weight:dry weight ratio occurred among these groups of rats. Finally, treating rats with TDGA or giving rats drinking water that contained 1 mg losartan/ml altered neither their heart rate nor their mean arterial blood pressure when compared with untreated rats. This data, therefore, suggests that oxirane carboxylates induce myocardial hypertrophy by activating the AT1 receptor independent of changes in systemic hemodynamics.

MDL-28170, a membrane-permeant calpain inhibitor, attenuates stunning and PKC epsilon proteolysis in reperfused ferret hearts.

OBJECTIVES: This paper tests the hypothesis that calpains are activated in the ischemic (I)/reperfused (R) heart and contribute to myocardial stunning. METHODS: Isolated ferret hearts were Langendorff perfused isovolumically, and subjected to 20 min of global I followed by 30 min of R in the presence or absence of 0.2 microM MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa(2+)-force curves obtained. Samples of left ventricle were extracted subjected to SDS-PAGE, and Western analyzed for PKC epsilon and PKM epsilon. RESULTS: Perfused ferret hearts exhibit a 43% decline in left ventricular developed pressure during R. Pre-treatment of hearts with MDL-28170 prior to I significantly improves function during R. Trabecular myofilaments from normal hearts have a KD for Ca2+ of 6.27 +/- 0.06; I/R decreased the KD to 6.09 +/- 0.04; trabeculae from I/R hearts pre-treated with MDL-28170 have a KD of 6.28 +/- 0.04. Western analysis shows ferret hearts to contain a single approximately equal to 96 kDa species of PKC epsilon. I/R hearts contain the native PKC epsilon and a approximately equal to 25 kDa smaller species of PKC epsilon which corresponds to PKM epsilon, the calpain proteolyzed form of PKC epsilon. Pre-treatment of I/R hearts with MDL-28170 markedly diminishes PKM epsilon in reperfused hearts. CONCLUSIONS: Mechanical stunning during R is sensitive to MDL-28170. Depressed mechanical function is reflected in a hyposensitization of trabecular myofilaments to Ca2+. Western analysis shows that PKM epsilon is present in R hearts.

1H NMR measurement of triacylglycerol accumulation in the post-ischemic canine heart after transient increase of plasma lipids.

This study tests the hypothesis that increased levels of plasma lipids can accelerate accumulation of myocardial triacylglycerols in post-ischemic but viable myocardium. Two groups of dogs underwent 90 min of left anterior descending coronary artery (LAD) occlusion followed by 240 min of reperfusion. The first group of saline-treated dogs (n = 7) had physiological levels of plasma lipids during reperfusion: a second group treated with Liposyn and heparin (n = 5) experienced increased plasma lipids during reperfusion. The transmural content of triacylglycerols was determined during ischemia and reperfusion using 1H NMR one-dimensional chemical shift imaging (1D CSI), and at the end of reperfusion using Oil Red-O staining and chemical assay. TTC staining was used to identify the extent of irreversibly injured myocardium. Subepicardial and plasma triacylglycerol content, measured both by 1D CSI and chemically, did not change during reperfusion in saline-treated dogs. Infusing dogs with Liposyn and heparin for 90 min during reperfusion transiently elevated their plasma triacylglycerols, which returned to normal levels following Liposyn wash-out. During Liposyn wash-out, myocardial triacylglycerols measured by 1D CSI preferentially increased in the subepicardium of area-at-risk myocardium (P < 0.05). Triacylglycerol content, measured chemically, also increased in area-at-risk compared to non-ischemic subepicardium (P < 0.001). Significant endocardial damage occurred in both groups, but elevated levels of plasma lipids did not increase the size of the area-at-risk. Therefore, elevated plasma lipids caused a preferential accumulation of triacylglycerols in area-at-risk myocardium during reperfusion without exacerbating irreversible ischemic injury. These results are consistent with either inhibited fatty acid oxidation or mis-matched fatty acid extraction and oxidation in area-at-risk myocardium.

Model systems for modulating the free energy of ATP hydrolysis in normoxically perfused rat hearts.

This study has two objectives; first, to develop perfusion conditions that decrease the free energy of ATP hydrolysis, Delta GATP, in isolated hearts; and, second, to modulate the Delta GATP in these perfused hear models. To accomplish the first goal, a series of inhibitors was employed to restrict acetyl-CoA oxidation. The second goal was accomplished by increasing work demand. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either; (i) no inhibitors (G group hearts); (ii) 0.3 mm bromo-octanoate (BrO), an inhibitor of beta-oxidation (GB group); (iii) 0.4 mm amino-oxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (GA group); (iv) BrO and AOA (GBA group hearts); or (v) BrO, AOA, and 4 mm butyrate, an alternate substrate (GBA-Bu). Pacing hearts at 300 beats per min (beats/min), at 450 beats/min, and at 450 beats/min in the presence of 80 microgram/l dobutamine allowed three increasing levels of work demand to be attained. The Delta GATP values of the five groups of hearts were calculated for each workstate using the concentrations of high energy phosphate metabolites measured by 31P NMR spectroscopy. At the highest levels of workload demand, the G, GB, and GBA-Bu group hearts had Delta GATP values >/=-53 kJ/mol ATP. At the highest levels of workload demand, the GA and GBA hearts had Delta GATP values 20 min. The G, GB, and GBA-Bu hearts attained RPPs of >/=54x10(3) mmHg/min at the highest levels of workload demand. The GA and GBA hearts attained RPPs of

1H NMR spectroscopic imaging of myocardial triglycerides in excised dog hearts subjected to 24 hours of coronary occlusion.

BACKGROUND: Myocardial ischemic insult causes depression of fatty-acid beta-oxidation and increased fatty-acid esterification with triglyceride (TG) accumulation. This accumulation has been demonstrated to occur in the territory with diminished blood flow surrounding an infarct, ie, the region at risk. To evaluate whether the extent of TG accumulation in the canine heart after 24 hours of ischemia could be detected, we applied myocardial 1H nuclear magnetic resonance (NMR) spectroscopic imaging (SI). METHODS AND RESULTS: Seven adult mongrel dogs underwent 24 hours of left anterior descending coronary artery occlusion. Postmortem, the hearts were excised and the size and location of the infarct were determined. With a Philips 1.5-T clinical NMR imaging/spectroscopic system, two-dimensional (2D) 1H NMR SI was performed. TG 1H NMR chemical shift images were reconstructed from the frequency domain spectra by numerical integration. A statistically significant (P < .05) increase in TG signal intensity was demonstrated in the region at risk compared with the nonischemic control region. There was an intermediate quantity of TG in the infarct region. Biochemical determination of tissue TG content (milligrams per gram wet weight) in the control, at-risk, and infarct regions confirmed the 1H NMR measurements. Histological evaluation with oil red O staining also demonstrated graded TG accumulation in myocytes. The highest TG levels were found in the at-risk region and the lowest levels in the control region. CONCLUSIONS: By use of 2D 1H NMR SI, the present study confirms and extends previous work that demonstrates preferential accumulation of TG in the reversibly injured myocardium after 24 hours of coronary occlusion. This study provides an important step toward the clinical application of TG imaging. When TG imaging is ultimately possible, resultant data would have diagnostic, prognostic, and therapeutic implications.

Acylcarnitine accumulation does not correlate with reperfusion recovery in palmitate-perfused rat hearts.

Carnitine palmitoyltransferase-I (CPT-I) inhibitors improve postischemic myocardial function either by decreasing muscle long-chain acylcarnitines (LCAC) during ischemia or by increasing oxidation of alternate substrates such as glucose during reperfusion. These possibilities were evaluated using oxfenicine, a CPT-I inhibitor, and alternate substrates that bypass carnitine-dependent metabolism. Isolated rat hearts subjected to 20 min of ischemia followed by 40 min of reperfusion with 1.8 mM palmitate as exogenous substrate recovered little function during reperfusion. Hearts made ischemic and reperfused with palmitate and 2.4 mM hexanoate as exogenous substrates had significantly improved reperfusion function compared to palmitate-perfused hearts. Addition of 2 mM oxfenicine to palmitate-hexanoate-perfused hearts gave an additional small improvement in reperfusion function. At the end of ischemia, the LCAC content of hearts perfused with palmitate or hexanoate and palmitate was identical. Palmitate-, hexanoate, and oxfenicine-perfused hearts had significantly decreased LCAC content at the end of ischemia compared with hexanoate-palmitate-perfused hearts. Therefore, depressed reperfusion function in long-chain fatty acid-perfused hearts can be ameliorated by alternate substrates, including medium-chain fatty acids. LCAC accumulation during ischemia apparently plays only a minor role in the postischemic dysfunction of long-chain fatty acid-perfused hearts.

3-Amino-5,5-dimethylhexanoic acid. Synthesis, resolution, and effects on carnitine acyltransferases.

The selective inhibition of individual carnitine acyltransferases may be useful in the therapy of diabetes and heart disease. Aminocarnitine (3) is a weak competitive inhibitor (K(i) = 4.0 mM) for carnitine acetyltransferase (CAT), although the N-acetyl derivative 4 is about 165 times more potent (K(i) = 0.024 mM) than 3. Compound 3 is also a potent competitive inhibitor for carnitine palmitoyltransferases 1 and 2 (CPT-1 and CPT-2) (IC50 for CPT-2 = 805 nM). We synthesized 3-amino-5,5-dimethylhexanoic acid (7) and its N-acetyl derivative (8) as isosteric analogs of 3 and 4 that lack the quaternary ammonium positive charge. Like 3 and 4, compounds 7 and 8 were competitive inhibitors of CAT with significantly different potencies, but in this case, 8 (K(i) = 25 mM) was 10 times less potent than 7 (K(i) = 2.5 mM). R-(-)-7 and S-(+)-7 were stereoselective inhibitors of CAT (K(i) = 1.9 and 9.2 mM, respectively). Racemic 7 was a weak competitive inhibitor of CPT-2 (K(i) = 20 mM) and had no effect on CPT-1. These results are consistent with differences among the carnitine-binding sites on carnitine acyl-transferases that may be useful in selective inhibitor design. Furthermore, the data suggest that the quaternary ammonium positive charge of carnitine may be important for the proper orientation of carnitine and its analogs in the binding site.

Carnitine acyltransferase enzymic catalysis requires a positive charge on the carnitine cofactor.

3-Hydroxy-5,5-dimethylhexanoic acid (HDH) is an analogue of carnitine which differs only in the substitution of a quaternary carbon atom for the quaternary ammonium nitrogen. Thus HDH is isosteric with carnitine but lacks the quaternary ammonium positive charge. Racemic HDH, each of its enantiomers, and the O-acetyl derivative (Ac-HDH) were evaluated as alternate substrates and inhibitors for several carnitine acyltransferases. HDH and Ac-HDH are not substrates for carnitine acetyltransferase (CAT) at concentrations up to 10 mM, suggesting that the positive quaternary ammonium charge on carnitine is essential for CAT catalysis. However, HDH competitively inhibits CAT (Ki = 8.3 mM), carnitine palmitoyltransferase-I (CPT-I) (Ki = 3.6 mM), and CPT-II (Ki = 2.8 mM). Ac-HDH is also a competitive inhibitor of CAT when assayed in the reverse direction (Ki = 4.1 mM). Similarly, R-(+)-HDH and S-(-)-HDH are not substrates for CAT, but they are stereoselective competitive inhibitors (Ki = 20.3 and 7.5 mM for the R and S enantiomers, respectively). Stereoselective inhibition by HDH is even more dramatic with CPT-I, since S-(-)-HDH inhibits CPT-I (Ki = 1.4 mM) but R-(+)-HDH has no effect in concentrations up to 5 mM. As with CAT, HDH is a stereoselective inhibitor of CPT-II, and the Ki values for S-(-)- and R-(+)-HDH are 2.2 and 6.7 mM, respectively. Since the observed Ki values are significantly larger than the Km for carnitine, the positive charge on carnitine must also be important, but not essential, for binding to the carnitine site on carnitine acyltransferases.

1H-NMR spectroscopy can accurately quantitate the lipolysis and oxidation of cardiac triacylglycerols.

Triacylglycerol metabolism in isolated, perfused hearts from rats fed a diet containing 20% rapeseed oil (RSO) was studied using 1H-NMR spectroscopy. RSO-induced elevation in cardiac triacylglycerols is associated with an increase in the peak area of fatty acid 1H-NMR resonances. The ratio of methyl, gamma-methylene or methylene protons adjacent to a carbon-carbon double bond to the number of methylene protons in these hearts measured by 1H-NMR spectroscopy gives values similar to those derived from previously reported chemical analyses. In addition, the triacylglycerol content of these hearts determined by chemical analysis directly correlates with their content of 1H-NMR visible fatty acid resonances. This quantitative relationship allows the real-time measurement of the rates of cardiac triacylglycerol lipolysis using 1H-NMR spectroscopy. Rates of triacylglycerol lipolysis measured using 1H-NMR spectroscopy are similar to those previously measured by chemical methods. Triacylglycerol lipolysis measured using 1H-NMR spectroscopy occurs at a significantly faster rate in hearts perfused in the presence or absence of glucose when compared to hearts perfused with glucose and acetate or medium-chain fatty acids. Finally, the rate of triacylglycerol lipolysis in glucose perfused hearts is linearly related to work output. These results demonstrate that 1H-NMR spectroscopy can accurately quantitate triacylglycerol content and metabolism in the rapeseed oil-fed rat model. 1H-NMR spectroscopic or imaging techniques may be useful in the real-time evaluation of cardiac triacylglycerol content and metabolism.

Morphometric analysis demonstrates that metabolically active cardiac triglycerides are 1H NMR visible.

The relationship between myocardial triglyceride content and 1H NMR visible fatty acid resonance intensity was investigated. Hearts from rats fed a 20% rapeseed oil diet contained markedly increased levels of triglycerides as judged by thin layer chromatographic analysis. This elevation in cardiac triglycerides was associated with sharp increases in the cell volume occupied by lipid droplets and in 1H NMR visible fatty acid resonances. Spin-lattice and spin-spin relaxation times of the 1H NMR visible fatty acid resonances from myocardium of rapeseed oil-fed rats were similar in value to those measured for neat triolein. Additionally, the fatty acids constituting these enhanced 1H NMR visible resonances were metabolically active. Perfusion of triglyceride enriched hearts in the presence or absence of glucose caused a time-dependent decrease in the intensity of their 1H NMR visible fatty acid resonances. In contrast, perfusion with glucose+acetate essentially prevented this time-dependent decrease in 1H NMR visible fatty acid resonances. Morphometric analysis of these hearts demonstrated that the decrease in 1H NMR resonance intensity correlated with changes in the cell volume of triglyceride-enriched lipid droplets. These results demonstrate that metabolically active stores of cardiac fatty acids, presumably triglycerides, are 1H NMR visible. Further, they indicate the possible utility of 1H NMR spectroscopy in the future study of myocardial triglyceride metabolism.

Visualization of altered myocardial lipids by 1H NMR chemical-shift imaging following ischemic insult.

Acute myocardial infarction is associated with an accumulation of lipids. Spectroscopic and chemical-shift imaging strategies which can depict the spatial distribution of these chemical species are evolving. The present study was undertaken to test whether the Dixon method could detect spatially lipids known to accumulate in myocardium after an ischemic insult. Seven dogs underwent a 24-h coronary artery occlusion (LAD = 4, Cx = 3). Post mortem, hearts were removed and imaged ex vivo. Myocardial samples were also evaluated by high-resolution 1H NMR spectroscopy. Lipid images revealed regions of increased signal intensity, in the regions corresponding to the myocardial infarction, particularly in the periphery of the infarction. An increase in mobile lipids was observed by 1H NMR spectroscopy of myocardial samples with moderately reduced blood flow and corresponding to regions with increased signal intensity on the lipid image. This study shows that chemical-shift imaging may be useful for detecting alterations in myocardial lipid levels following an ischemic insult.

Mitochondrial adenosine triphosphatase in the oxyphil cells of a renal oncocytoma.

Oxyphil cells are characterized by cytoplasm packed with large numbers of mitochondria. Study of these unusual cells may provide information about the regulation of mitochondrial biogenesis. Although it has been suggested that this is a compensatory proliferation due to a mitochondrial dysfunction, no such dysfunction has been well documented. In this study we considered the possibility of dysfunction in the mitochondrial enzyme F1/Fo-adenosine triphosphatase(ATPase) as a stimulating factor involved in the mitochondrial proliferation of oxyphil cells. Mitochondria isolated from frozen tissue of a renal oncocytoma showing structural integrity and purity by electron microscopy were studied. Submitochondrial particles formed by sonic disruption showed the presence of the F1 component of mitochondrial ATPase with electron microscopy which was functionally active. The oligomycinsensitive ATPase activity from the renal oncocytoma was 0.133 mumol/min.mg submitochondrial particle protein which was higher than the readings obtained from normal kidney tissue (0.091 mumol/min.mg SMP protein) obtained from hamsters. Normal human renal tissue obtained at autopsy contained only nonfunctional mitochondria and therefore could not be used as control tissue. Mitochondrial ATPase dysfunction does not appear to be the inciting factor in the proliferation of mitochondria seen in oxyphil cell metaplasia and future studies should consider other possibilities. Preliminary functional studies of this nature can be performed with properly prepared frozen surgical tissue.

Myocardial connective tissue alterations.

There is a complex network of collagen throughout the heart. It is composed of a hierarchy of fibrils and fibers ranging from 10 nm to 2-3 microns in diameter. This network can be broken down by ischemia, adriamycin administration, or disulfide administration in laboratory animals. Following loss due to coronary artery ligation, the ischemic area begins bulging within 3 h. General loss of portions of the collagen matrix is induced by intravenous oxidizing glutathione, and results in marked diffuse ventricular dilatation. Generalized collagen loss in the ventricles, as induced by disulfide administration or adriamycin infusion, persists for 6 months at which time evidence of some replacement is visible, and evidence of diffuse fibrosis is present. In humans, cardiac dilatation occurs in a variety of disease states without overstretch of sarcomeres. This presumes rearrangement of the muscle bundles, which can only occur with marked alterations of the collagen matrix. Ventricular dilatation, associated with viral myocarditis or puerperal cardiomyopathy, may persist for months, suggesting the collagen loss, as with the experimental animals, takes many months to repair. The cardiac dilatation may ameliorate, or, in some patients, deteriorate into heart failure. The animal experiments with loss of the collagen matrix, ventricular dilatation, and failure to replace the matrix for many months provide an explanation for persistent cardiac dilatation in various human diseases.