Profound underestimation of glucose uptake by [18F]2-deoxy-2-fluoroglucose in reperfused rat heart muscle

BACKGROUND: [18F]2-deoxy-2-fluoroglucose (FDG) is widely used as a tracer for glucose uptake in ischemic heart muscle. We tested the effects of low-flow ischemia and reperfusion on the ratio of tracer/tracee (lumped constant, LC). METHODS AND RESULTS: Isolated working rat hearts were perfused with Krebs-Henseleit buffer containing only glucose 5 mmol/L (group 1) or glucose 5 mmol/L plus oleate 0.4 mmol/L (group 2, fed; group 3, fasted). Dynamic glucose uptake was measured simultaneously with [2-3H]glucose and with FDG. After 20 minutes, coronary flow was reduced by 75% for 30 minutes before it was returned to control conditions for the final 20 minutes. Hexokinase activity in the cytosolic and mitochondrial fractions and tissue metabolites were determined. Rates of glucose uptake were highest when glucose was the only substrate. Glucose uptake, FDG uptake, and the LC increased during ischemia only in group 3. There was no change of these parameters during ischemia in groups 1 and 2. FDG uptake decreased significantly with reperfusion in groups 2 and 3, and there was a striking fall in the LC (from >1.0 to <0.2, P<.001). The fall in the LC was associated with a significant increase in intracellular free glucose. Neither ischemia nor reperfusion affected the kinetic properties of hexokinase. CONCLUSIONS: FDG profoundly underestimates glucose uptake during reperfusion in the presence of fatty acids. In the fasted state, however, FDG overestimates glucose uptake during ischemia. The results indicate limitations in the use of FDG to quantify myocardial glucose uptake in human heart.     

Separation of early steps in endocytic membrane transport

We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane-bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC) biotin at 4 degrees C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol-glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore-forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.     

Early prostaglandin release from the ischemic myocardium in the dog

Cellular consequences of myocardial ischemia were studied in anesthetized dogs. Confirmation of myocardial ischemia was provided by electrocardiographic and biochemical indexes. Prostaglandin F2alpha release into coronary venous blood was significantly elevated during myocardial ischemia, whereas indomethacin treatment prevented this increase in coronary venous prostaglandin F2alpha concentrations. No significant increase in prostaglandin E2 release was observed in response to myocardial ischemia, but indomethacin treatment significantly reduced coronary venous prostaglandin E2 concentrations below those of control values. Within one hour after occlusion of the coronary artery, the S-T segment was significantly altered, and coronary venous prostaglandin F2alpha had increased significantly above the control concentration. These changes persisted during four hours of myocardial ischemia. Plasma creatine phosphokinase activity increased significantly after two hours of myocardial ischemia and remained elevated for the subsequent two hours of ischemia. After four hours of myocardial ischemia, myocardial creatine phosphokinase activity of ischemic myocardium was significantly reduced, and labilization of myocardial treatment prevented increases in prostaglandin release but did not influence other biochemical changes or the electrocardiographic response to ischemia. Thus, prostaglandin release by ischemic myocardial tissue is an early response to the ischemic stimulus.     

Effect of protein kinase C inhibitors on cardioprotection by ischemic preconditioning depends on the number of preconditioning episodes

OBJECTIVES: This study examined the possibility that the role of PKC in [corrected] PC, and thus the response to PFC inhibitors, may differ depending on how many ischemic episodes are employed to precondition the heart. METHODS: In the first series of experiments, myocardial infarct was induced by 30 min of coronary occlusion and 3 h of reperfusion in the rabbit. Infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Prior to the 30-min ischemia, rabbits were subjected to no PC, single PC (i.e., PC with an episode of 5 min ischemia/5 min reperfusion), and repetitive PC (2 cycles of 5 min ischemia/5 min reperfusion) with or without one of three treatments: polymyxin B (PolyB), staurosporine (Stauro), and 8-sulfophenylthephylline (SPT). In the second series of experiments, the rabbits received 5 min of coronary occlusion after repetitive PC with or without PolyB or Stauro treatment. Then, myocardial tissue in the ischemic region was sampled for assay of PKC activity. Untreated rabbits served as controls. RESULTS: Single and repetitive PC limited %IS/AR to the same extent (%IS/AR = 9.8 +/- 1.9 and 10.4 +/- 2.3, both p < 0.05, vs. the control value of 44.5 +/- 3.4), and single PC was blocked by PolyB (%IS/AR = 43.9 +/- 2.7) and Stauro (%IS/AR = 31.5 +/- 3.2). Although the protocol of PolyB injection maintained the plasma PolyB level during sustained ischemia well above its Ki for PKC, this agent and also Stauro failed to abolish the protection by repetitive PC (%IS/AR = 21.6 +/- 3.0 and 11.4 +/- 4.3, respectively). SPT, an adenosine receptor antagonist, not only blocked single PC (%IS/AR = 44.4 +/- 4.4) but also attenuated protection by repetitive PC (%IS/AR = 28.3 +/- 3.6). Infarct sizes in non-preconditioned hearts were not modified by PolyB, Stauro, or SPT. The ratio of membrane fraction PKC activity to cytosolic fraction PKC activity was elevated by repetitive PC plus 5 min ischemia, and this change in PKC was inhibited in hearts given PolyB and Stauro. CONCLUSIONS: In contrast to single PC, repetitive PC protects the heart against infarction even when PolyB and Stauro are administered to inhibit PKC during ischemic insult. This difference may be attributable to a PKC-independent mechanism, in which the adenosine receptor may be partly involved.     

Post-ischemic stimulation of 2-deoxyglucose uptake in rat myocardium: role of translocation of Glut-4

Myocardial ischemia elicits translocation of the insulin-sensitive glucose transporter GLUT-4 from intracellular membrane stores to the sarcolemma. Because glucose metabolism is of crucial importance for post-ischemic recovery of the heart, myocardial uptake of [3H]-labeled 2-deoxyglucose and subcellular localization of GLUT-4 were determined during reperfusion in isolated rat hearts perfused with medium containing 0.4 mm palmitate and 8 mm glucose. Hearts were subjected to 20 min of no-flow ischemia, followed by reperfusion for up to 60 min. Subcellular localization of GLUT-4 was determined by cell fractionation followed by immunoblotting. After 15 and 60 min of reperfusion uptake of 2-deoxyglucose was significantly higher (91+/-9 and 96+/-8 nmol/min/g wet weight, respectively) as compared to control values (65+/-1 nmol/min/g wet weight). Ischemia elicited translocation of GLUT-4 to the sarcolemma, which persisted after 15 min of reperfusion. However, after 60 min of reperfusion the subcellular distribution of GLUT-4 was similar to control hearts. In conclusion, reversal of ischemia-induced translocation of GLUT-4 to the sarcolemma is rather slow, possibly facilitating glucose uptake early during reperfusion. However, myocardial uptake and phosphorylation of 2-deoxyglucose remains enhanced late during reperfusion, when pre-ischemic distribution of GLUT-4 is almost completely restored, indicating that additional mechanisms are likely to be involved in post-ischemic stimulation of glucose uptake.     

Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.     

Calcium uptake and binding by membrane fractions of human placenta: ATP-dependent calcium accumulation

An ATP-dependent calcium (Ca2+) sequestration activity was demonstrated in membrane vesicles prepared from the human term placenta. Microsomal and brush border membrane fractions accumulated Ca2+ within a vesicular space by a saturable process requiring Mg2+ and ATP. The "uptake" activity was enriched six-fold in a microsomal membrane fraction and was only 1.5-fold enriched in purified brush border membranes compared to the activity present in the filtered homogenate. Mitochondrial inhibitors such as azide and oligomycin did not inhibit Ca2+ uptake in these preparations. The process was temperature dependent and displayed Michaelis-Menten-like kinetics with respect to free Ca2+ concentrations. At 30 degrees C, the Vmax was 1.05 nmole/mg/min; Km = 74 nM for free Ca2+ in the microsomal fraction. Oxalate and phosphate enhanced uptake in both fractions. Ca2+ uptake activity was not associated with Ca2+-stimulated ATPase, alkaline phosphatase, or other brush border markers during cell fractionation. The characteristics of the Ca2+ uptake process contrasted sharply with those of Ca2+-stimulated ATPase, and a Ca2+-stimulated, Mg2+-dependent ATPase activity could not be identified in these membrane vesicle preparations.     

Amodal completion in the absence of image tangent discontinuities

The localization of the low-affinity adenosine binding protein adenotin-1 with respect to distribution in rat organs and subcellular compartments was investigated. Adenotin-1 was characterized by 5'-N-ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binding and Western blotting. Cytosolic as well as membrane fractions of all tissues contained adenotin-1. Highest levels of membrane-bound adenotin-1 were found in the liver (liver > kidney approximately spleen approximately lung > forebrain approximately cerebellum > fat heart - striated muscle), whereas highest levels of cytosolic adenotin-1 were detected in spleen, liver, lung and fat. Subcellular fractions from rat liver were prepared by differential and density gradient centrifugation. Like the homologous proteins endoplasmin or gp96, adenotin-1 is enriched in the endoplasmic reticulum. Cytosolic and membrane-bound adenotin-1 species are pharmacologically distinct, because in the liver particulate fraction adenotin-1 showed a more rapid binding kinetics, a twofold lower affinity for [3H]NECA (KD 227 nM vs. 105 nM) and a sevenfold higher affinity for 2-chloroadenosine than the cytosolic protein (Ki 1.48 microM vs. 9.25 microM). In rat liver cytosol, two different binding sites were found, which differed in [3H]NECA binding kinetics and displayed a hundredfold difference in their affinity for 2-chloro-5'-N-methylcarboxamidoadenosine (Ki 45.8 nM vs. 4.76 microM). The presence of adenotin-1 in subcellular fractions, as determined by radioligand binding, was confirmed by Western blotting. Adenotin-1 was detected as a 98-kDa band in all rat liver subcellular fractions, which agrees with the molecular mass determined for the purified protein. In the cytosol, a 65-kDa hand was labeled more intensely than the 98-kDa band. This additional band probably represents the pharmacologically distinct species of adenotin-1 found in the cytosol.     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Myocardial distribution and biotransformation in vitro and in vivo of nicorandil in rats, with special reference to mitochondria

This study reports subcellular localization of nicorandil in the myocardium and metabolism in mitochondria after oral dosing of 3 mg/kg nicorandil to rats. In the in vitro experiments, nicorandil, which was incubated with tissue homogenates (liver, kidney, heart, and small intestine), was metabolized to its denitrated compound, SG-86, and unknown substances. In the absence of a NADPH-generating system in the heart, the metabolic activity existed only in the mitochondrial fraction, but not in cytosolic and microsomal fractions. In the presence of the system, the activity in the mitochondrial fraction became much higher. To examine subcellular distribution of nicorandil in the myocardium, [14C]nicorandil was orally given to rats. Fifteen minutes after oral dosing of 3 mg/kg [14C]nicorandil, of which myocardial concentration reached a peak, nicorandil and SG-86 were found in mitochondrial fractions as well as in cytosolic and microsomal ones of the heart. Electron-microscopic autoradiograms, 15 min after oral dosing of 3 mg/kg [3H]nicorandil to rats, also showed the existence of the silver grains (showing radioactivity) in mitochondria of the heart. We conclude that nicorandil given orally is distributed in mitochondria of the heart, being partly transformed into SG-86, and that the myocardial mitochondria may be a potential site of action of nicorandil, an opener of KATP channels, which have been demonstrated to be present in this subcellular particle.     

Persistent myocardial ischemia increases GLUT1 glucose transporter expression in both ischemic and non-ischemic heart regions

Persistently ischemic myocardium exhibits increased glucose uptake which may contribute to the preservation of myocardial function and viability. Little is known about the specific molecular events which are responsible for this increase in uptake. Therefore, we investigated whether myocardial ischemia induces the gene expression of the major cardiac facilitative glucose transporters, GLUT4 and GLUT1. We determined the expression of myocardial glucose transporter mRNAs and polypeptides after 6 h of regional ischemia in a dog model by semi-quantitative Northern blotting and immunoblotting. GLUT1 but not GLUT4 expression was significantly increased in both ischemic and non-ischemic regions from the experimental hearts when compared to surgical control and normal hearts. GLUT1 mRNA expression was increased 3.4-fold and GLUT1 polypeptide expression was increased 1.7-fold in ischemic hearts when compared to normal or surgical-control hearts. There were no significant regional differences in GLUT1 expression in either normal or ischemic hearts. However, there was a tendency for GLUT1 mRNA expression to be highest in the non-ischemic regions from the 6-h ischemia hearts. These findings suggest that myocardial ischemia induces a factor or factors which stimulate GLUT1 expression in non-ischemic as well as ischemic myocardial regions. Increased GLUT1 expression may play a role in augmenting glucose uptake during ischemia.     

The ultrastructure of chicken gizzard vinculin as visualized by high-resolution electron microscopy

OBJECTIVES: A myocardial calcium-independent PLA2 has been described that is activated during myocardial ischemia and this enzyme may modulate ATP-sensitive potassium channels (KATP). The aim of this study was to determine the effect of an inhibitor of this enzyme, a bromoenol lactone, in isolated globally ischemic rat hearts. METHODS: Isolated rat hearts were treated for 10 min with 0.3-6 microM bromoenol lactone and then subjected to 25 min ischemia and 30 min reperfusion. RESULTS: The bromoenol lactone significantly increased coronary flow in nonischemic myocardium, and slightly reduced cardiac function at 6 microM. During global ischemia, time to contracture was significantly increased from vehicle group values in the presence of the bromoenol lactone (EC50 = 1.2 microM). During reperfusion, a concentration-dependent increase in function and a reduction in LDH release were observed for the PLA2 inhibitor. The concentrations of the PLA2 inhibitor which were significantly cardioprotective, inhibited this enzyme in membrane fractions of rat myocardium (IC50 = 0.87 microM). The KATP blocker sodium 5-hydroxydecanoate (5-HD) inhibited the increase in time to contracture observed for the bromoenol lactone. During reperfusion, 5-HD abolished the protective effects of the bromoenol lactone on cardiac function and LDH release. Glyburide had similar effects on the cardioprotective activity of the bromoenol lactone, although it only partially abolished the LDH reducing effect of this agent. CONCLUSIONS: The bromoenol lactone protects ischemic myocardium at concentrations which also inhibit calcium-independent PLA2. This cardioprotection can be attenuated by blockers of KATP, suggesting a potential mechanism for modulation of myocardial KATP.     

TrfA dimers play a role in copy-number control of RK2 replication

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage. The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42 degrees C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min. Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 +/- 14 (control group) to 17 +/- 5 units/g wet wt per 45 min (heat-pretreated group) (p < or = 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 +/- 3.3. vs. 17.8 +/- 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.     

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above. It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Curare: a preventive of traumatic complications in convulsive shock therapy (including a preliminary report on a synthetic curare-like drug). 1940

Yellow fluorescent lipofuscin deposited in rat kidney was extracted in an aqueous solution and characterized after separation. Centrifugal fractionation of the extract revealed that most of the yellow fluorescence was detected in the 105,000 x g-supernatant, and little in nuclei, cell debris, mitochondria, lysosomes, microsomes and plasma membrane. The yellow fluorescence in the supernatant was fractionated by gel filtration through Sephadex columns into 5 yellow fluorescent fractions A, B (B1, B2 and B3) and C showing the same fluorescence spectra with excitation maximum/emission maximum at 400/620 nm. The components in fraction A were converted into the smaller molecular-weight components in fraction B on treatment with 4 M urea or protease, suggesting that they were proteinaceous. The smallest molecular-weight fluorescent components in fraction C were adherent to solid cellulose materials. The fluorescent components in all the fractions were soluble in water and insoluble in chloroform-methanol, indicating that they were not lipidic materials. The fluorophores in these fractions were kept stable on borohydride treatment, but readily converted into non-fluorescent components on heavy-metal ion treatment. The characteristics of the yellow fluorescence in these fractions were quite different from those of bluish lipofuscin-like fluorophores that may be generated in tissues during lipid peroxidation.     

Subcellular localization of enterokinase (enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3% in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (Mr, 2 - 10(6)) and two larger peaks of free enzyme (Mr, 3 - 10(5) and 9 -10). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules

The subcellular distribution and segmental variations in location of gp330, a scavenger receptor for filtered proteins in renal proximal tubules, was analyzed. Kidney tissue from rats (4 different strains), rabbits and humans were analyzed by light- and electron microscope immunocytochemistry, using cryosections or Lowicryl sections from cryosubstituted tissue. Gp330 was located mainly in apical coated pits, small and large endocytic vacuoles and in dense apical tubules in the proximal tubule cells. The labeling density was markedly higher in segments 1 and 2 as compared to segment 3 of the proximal tubule. In addition to the location in the early part of the endocytic pathway, gp330 was also present in lysosomes, especially in segments 1 and 2. The lysosomal labeling was not restricted to the membrane, but was also seen in the matrix. Localization of gp330 in lysosomes was confirmed on sections from purified lysosomal fractions from rat renal cortex. The brush border localization of gp330 in proximal tubules exhibited a characteristic segmental variation. In the initial part of segment 1, there was virtually no brush border labeling. In the remaining part of segment 1 and in segment 2, there was a distinct but sometimes patchy labeling of the brush border. In segment 3, groups of microvilli of approximately 10 as seen in sections were intensively labeled from bottom to tip and there were often more than one of these groups on a single cell, the remaining microvilli were unlabeled. No differences in the cellular and subcellular localization of gp330 were observed between species or rat strains. In conclusion, the present study demonstrates that in addition to its location in the early endocytic and recycling pathway, gp330 is also present in microvilli and the protein and degradation products thereof is present in lysosomes, consistent with its role as a protein scavenger receptor.     

Compartmentation of the type I regulatory subunit of cAMP-dependent protein kinase in cardiac ventricular muscle

The species-dependent compartmentation of type I cAMP-dependent protein kinase (PKA I) and its dissociated regulatory subunit (RI) was examined in the heart by biochemical and immunohistochemical means. PKA I and RI were resolved from type II cAMP-dependent protein kinase and its regulatory subunit by DEAE-Sephacel chromatography of the supernatant and Triton X-100 soluble particulate fractions of heart homogenates. The relative amounts of holoenzymes and subunits were determined by cAMP-binding, protein kinase, 8-N3-[32P]cAMP photoaffinity labeling, and Western blot assays. Rat, rabbit, and guinea pig hearts all contained PKA I to varying degrees, but only in the supernatant fractions. Significant amounts of dissociated RI were found in the supernatant fractions, and to a lesser extent the particulate fractions, of these species. In contrast, though no PKA I was detected in the supernatant or particulate fractions of pig and beef heart, half of the cAMP-binding activity in the particulate fraction was attributed to RI. The results suggest that RI may associate with membrane fractions when it is not associated with the PKA catalytic subunit. Immunohistochemical studies of tissue sections from pig, beef, and rat cardiac ventricle using antibodies directed against RI also revealed species-dependent localization of RI. Cardiac myocyte intercalated discs were stained in pig and beef sections with additional sarcolemmal staining in beef sections. Rat ventricle, which contained large amounts of supernatant PKA I, showed nuclear staining. The localization of RI to cardiac myocyte intercalated discs and sarcolemma in certain species suggests a role(s) for this subunit in mediating cAMP-regulated events in these regions.     

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.     

Effects of hyperthermic stress on myocardial function during experimental coronary ischemia

We evaluated hyperthermic influences on ischemic hearts by comparing two groups of intact working swine hearts (n = 20) made globally ischemic. Heart muscle temperature was selectively increased from 37.5 +/- 0.3 degrees C to 39.7 +/- 0.3 degrees C in one group (n = 11) by warming the coronary perfusate. Ischemia in normothermic hearts significantly (P less than 0.05) decreased mechanical function (as reflected by increases in left ventricular end-diastolic pressure [LVEDP]), myocardial oxygen consumption (MVO2), glucose uptake, glycolytic flux, free fatty acid (FFA) uptake and oxidation, and tissue stores of high energy phosphates. Hearing in ischemic hearts further depressed mechanical function at similar reductions in coronary flow and MVO2. Glucose uptake was terminally increased over normothermic values (329 vs. 221 mumol/hr per g) as was glycolytic metabolism, FFA uptake (26 vs. 17 mumol/hr per g), and FFA oxidation (21 vs. 11 mumol/hr per g). However, these changes were not translated into increased energy stores of tissue creatine phosphate and ATP. Thus, in ischemic hearts, hyperthermia neither prevented the development of mechanical deterioration nor improved oxidative phosphorylation despite increases in metabolic substrate utilization. These data suggest that in experimental global ischemia heat is an added energy drain in already burdened myocardium.