Accelerated glycolysis and greater postischemic dysfunction in hypertrophied rat hearts are independent of coronary flow.

BACKGROUND: After ischemia, glycolysis and dysfunction are greater, while coupling of glucose oxidation to glycolysis is lower in hypertrophied hearts than in nonhypertrophied hearts. OBJECTIVE: To test the hypothesis that accelerated glycolysis, reduced coupling of glucose oxidation to glycolysis and increased postischemic dysfunction in hypertrophied hearts compared with nonhypertrophied hearts occur in the absence of differences in coronary flow. MATERIALS AND METHODS: Function, glycolysis and glucose oxidation were measured in isolated working control and hypertrophied rat hearts studied for 30 min before, and for 40 min after no flow global ischemia for 20 min under conditions in which coronary flow was comparable between the two groups. The hearts were perfused with 1.2 mmol/L palmitate, 5.5 mmol/L [5-3H/U-14C]-glucose, 0.5 mmol/L lactate, 100 mU/L insulin at a preload of 11.5 mmHg, and an afterload of 60 mmHg in control hearts or 80 mmHg in hypertrophied hearts. RESULTS: Despite comparable rates of coronary flow, functional recovery was lower in hypertrophied hearts than in control hearts. The rates of glycolysis were accelerated in hypertrophied hearts, while glucose oxidation did not significantly differ between the two groups. As a result, the coupling of glucose oxidation to glycolysis was lower in hypertrophied hearts than in control hearts. CONCLUSIONS: Increased postischemic dysfunction, accelerated glycolysis and reduced coupling of glucose oxidation to glycolysis in hypertrophied hearts compared with control hearts cannot be accounted for by differences in coronary flow. These data provide support for the concept that alterations in glucose metabolism contribute to the exaggerated postischemic dysfunction of hypertrophied hearts.

Hypertrophied rat hearts are less responsive to the metabolic and functional effects of insulin.

We determined the effect of insulin on the fate of glucose and contractile function in isolated working hypertrophied hearts from rats with an aortic constriction (n = 27) and control hearts from sham-operated rats (n = 27). Insulin increased glycolysis and glycogen in control and hypertrophied hearts. The change in glycogen was brought about by increased glycogen synthesis and decreased glycogenolysis in both groups. However, the magnitude of change in glycolysis, glycogen synthesis, and glycogenolysis caused by insulin was lower in hypertrophied hearts than in control hearts. Insulin also increased glucose oxidation and contractile function in control hearts but not in hypertrophied hearts. Protein content of glucose transporters, protein kinase B, and phosphatidylinositol 3-kinase was not different between the two groups. Thus hypertrophied hearts are less responsive to the metabolic and functional effects of insulin. The reduced responsiveness involves multiple aspects of glucose metabolism, including glycolysis, glucose oxidation, and glycogen metabolism. The absence of changes in content of key regulatory molecules indicates that other sites, pathways, or factors regulating glucose utilization are responsible for these findings.

Endotoxin-specific NF-kappaB activation in pulmonary epithelial cells harboring adenovirus E1A.

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-kappaB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-kappaB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c-jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-kappaB induction in these cells.

The effect of glucocorticoids on the expression of L-selectin on polymorphonuclear leukocyte.

When active bone marrow release is induced by inflammatory stimuli, it is associated with an increase in L-selectin expression on circulating polymorphonuclear leukocyte (PMN). This contrasts sharply with glucocorticoid-induced granulocytosis that is associated with decreased L-selectin expression on PMN. The present study was designed to determine if the reduced L-selectin expression observed after glucocorticoid treatment is the result of suppression of L-selectin synthesis in the bone marrow. New Zealand white rabbits treated with dexamethasone (2.0 mg/kg, a single dose intravenously) were shown to have decreased L-selectin expression on circulating PMN 12 to 24 hours after treatment (P <.01) with a return to baseline levels by 48 hours. When dexamethasone was administered 48 hours after the bone marrow PMN were pulse labeled with the thymidine analogue, 5'-bromo-2'-deoxyuridine (BrdU), L-selectin expression on BrdU-labeled PMN released from the bone marrow was decreased (P <.01). Dexamethasone decreased L-selectin expression on segmented PMN in the bone marrow (P <.05) but not on PMN already in the circulation. We conclude that glucocorticoids decrease L-selectin expression on circulating PMN by downregulating L-selectin expression in the maturation pool of bone marrow and speculate that this is an important glucocorticoid effect that influences the recruitment of PMN into inflammatory sites.

Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes.

BACKGROUND: Glucocorticoid-induced granulocytosis has been attributed to enhanced release of polymorphonuclear leukocytes (PMNs) from bone marrow, delayed apoptosis, and reduced egress of PMNs into tissues. This study was designed to determine the relative contributions of PMNs released from the bone marrow and those entering the circulation from the marginated pool to the granulocytosis produced by a single dose of dexamethasone (2.0 mg/kg) in rabbits. METHODS AND RESULTS: PMN transit through the mitotic and postmitotic pools of the bone marrow and rate of release of PMNs into the circulation were measured by use of the thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) to pulse-label PMNs in the bone marrow. The shift of PMNs from the marginated to the circulating pool was measured with BrdU-labeled PMNs transferred from donor rabbits to recipients before dexamethasone was delivered. The data show that dexamethasone increased bone marrow release of PMNs and shortened their transit time through the postmitotic pool (P<0.001) but not the mitotic pool of the bone marrow (P>0.05). Dexamethasone slowed the clearance of BrdU-labeled PMNs from the circulation (P<0.05) and lengthened their disappearance (half-life) from the circulation compared with control (half-life, 4.95 versus 9. 45 hours). At 6 hours after dexamethasone, bone marrow release contributed approximately 10%, mobilization from the marginated pool approximately 61%, and a lengthened half-life in the circulation approximately 29% to the glucocorticoid-induced granulocytosis. CONCLUSIONS: We conclude that a single dose of dexamethasone causes a granulocytosis primarily by a shift of PMNs from the marginated to the circulating pool, with a minor contribution from marrow release.

Identification of glucocorticoid- and adenovirus E1A-regulated genes in lung epithelial cells by differential display.

Adenovirus infection has been implicated in the pathogenesis of lung inflammatory diseases for which glucocorticoids provide effective antiinflammatory treatment. In this study, the differential display assay was used to identify messenger RNAs (mRNAs) differentially expressed in dexamethasone (1 microM for 24 h)-treated A549 lung epithelial cells compared to A549 cells transfected with the adenoviral E1A gene. Thirty-seven complimentary DNAs (cDNAs) (15 glucocorticoid-regulated, 22 adenovirus E1A-regulated) were isolated. DNA sequence analysis showed that 35 of these were unique, 2 were identical with each other, and 3 were common to the glucocorticoid- and E1A-regulated groups. Genes identified included those involved in transcription/translation, cytoskeletal/contractile element genes, metabolic enzyme genes, and genes associated with cell regulation/signal transduction. After further analysis of the isolated clones by Northern blotting, ribonuclease protection, and semiquantitative RT-PCR (reverse transcriptase-polymerase chain reaction), 10 of the 14 glucocorticoid-regulated and one of the three common to both the adenovirus E1A- and glucocorticoid-regulated cDNAs were confirmed for this control of their expression. We conclude that the strategy of identifying cDNAs regulated by both adenovirus E1A and glucocorticoids provides a promising approach for identifying genes that may be important in the pathogenesis of lung inflammation and therefore targets for glucocorticoid treatment.

L-selectin expression on polymorphonuclear leukocytes and monocytes in premature infants: reduced expression after dexamethasone treatment for bronchopulmonary dysplasia.

We examined the effect of dexamethasone on the expression of the adhesion molecule L-selectin on circulating polymorphonuclear leukocytes (PMLs) and monocytes from premature infants with bronchopulmonary dysplasia (BPD). Nineteen infants who received dexamethasone (Dex group) and 28 who did not receive dexamethasone (no Dex group) were studied. L-selectin expression, measured as mean fluorescence intensity, was lower on circulating PMLs (5.7 +/- 0.6 vs 10.6 +/- 0.7, p < 0.001) and monocytes (7.9 +/- 0.9 vs 12.5 +/- 0.9, p < 0.02) isolated from those who had received dexamethasone. Because L-selectin is important for the recruitment of PMLs to inflammatory foci in the lungs, we speculate that one of the mechanisms by which dexamethasone reduces inflammation in BPD is by impairing the ability of leukocytes to migrate into the BPD lesions.

Regression of cardiac hypertrophy normalizes glucose metabolism and left ventricular function during reperfusion.

It is not yet known if the alterations in myocardial glucose metabolism and the exaggerated left ventricular dysfunction that occur during reperfusion in hypertrophied hearts are reversible. Thus, we studied isolated working hearts from aortic-banded (n = 29) and sham-operated control (n = 32) male Sprague-Dawley rats with or without enalapril maleate treatment (25.6 +/- 0.8 mg/kg per day, p.o.) to determine the effect of regression of cardiac hypertrophy on myocardial glucose metabolism and post-ischemic heart function. Hearts were perfused with buffer containing 1.2 mM palmitate, 11 mM [5-3H]/[U-14C]-glucose, 0.5 mM lactate and 100 microU/ml insulin. Glucose metabolism [rates of glycolysis (3H2O production) and rates of oxidation (14CO2 production) of exogenous glucose] and heart function (heart rate x peak systolic pressure) were measured during 30 min pre-ischemic perfusion and 60 min of reperfusion following 20 min of global, no-flow ischemia. Hearts from untreated aortic-banded rats were hypertrophied, being 27.6 +/- 1.8% larger than hearts from untreated control rats. Enalapril treatment caused regression of cardiac hypertrophy that normalized heart weight in aortic-banded rats. Rates of glycolysis of exogenous glucose in hearts from untreated aortic-banded rats were accelerated compared to rates in hearts from untreated control rats during pre-ischemic perfusion (4391 +/- 97 v 2652 +/- 69 nmol glucose/min per g dry wt, respectively, P < 0.05) and reperfusion (2402 +/- 58 v 1597 +/- 88 nmol glucose/min per g dry wt. respectively, P < 0.05). In contrast, rates of glycolysis of exogenous glucose in hearts from enalapril-treated aortic-banded rats were normalized before and after ischemia. Rates of glycolysis of exogenous glucose in hearts of control rats were not affected by enalapril treatment. Oxidation of exogenous glucose was not different among groups either before or after ischemia. Function of hearts from untreated aortic-banded rats at the end of reperfusion was significantly less than that of hearts from untreated control rats (23.9 +/- 2.6 v 32.2 +/- 0.7 mmHg x beats per min/1000, respectively, P < 0.05). As with myocardial glucose metabolism function of hearts from aortic-banded rats treated with enalapril was normalized during reperfusion. Thus, pharmacologically induced regression of pressure-overload cardiac hypertrophy normalizes glucose metabolism as well as left ventricular function during reperfusion.

Serum from patients with chronic renal insufficiency alters growth characteristics and ANP mRNA expression of adult rat cardiac myocytes.

Left ventricular hypertrophy is very prevalent among patients with renal insufficiency. Known hypertrophic factors, such as systemic hypertension, do not adequately account for the prevalence of left ventricular hypertrophy in these patients. Circulating growth factors may stimulate cardiomyocyte growth and contribute to the development of left ventricular hypertrophy. The effects of sera from patients with (n = 30) and without (n = 5) chronic renal insufficiency on the growth of cultured adult cardiomyocytes were compared. An adult rat cardiomyocyte primary culture system was established with a high purity of cardiomyocyte population as confirmed by immunocytochemical staining of cardiac contractile proteins. Myocytes responded with increased [3H]thymidine incorporation when treated with angiotensin II, epidermal growth factor, hydrocortisone and insulin, and with increased [3H]phenylalanine incorporation when treated with parathormone, isoproterenol, phenylephrine and insulin. Renal insufficiency serum stimulated [3H]thymidine incorporation was 1.5 times that of the control (P < 0.02) and also tended to increase incorporation of [3H]phenylalanine compared to the control (P = N.S.). Increased [3H]thymidine incorporation by renal insufficiency serum did not correlate with serum insulin, parathormone or glucose in the renal insufficiency patients. A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method was used to measure renal insufficiency serum-induced atrial natriuretic peptide mRNA expression in cultured cardiomyocytes. Atrial natriuretic peptide (ANP) mRNA was increased 1-3-fold in cardiomyocytes treated with renal insufficiency sera in comparison to control sera. These data suggest that circulating growth factor(s) may contribute to the development of cardiac hypertrophy in patients with renal insufficiency.

Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases.

The tachykinin neuropeptides substance P and neurokinin (NK) A have been postulated to participate in the inflammatory reaction in airways of smokers and asthmatics. We have examined the hypothesis that the expression of one or more of the three cloned tachykinin receptors (NK1, NK2, and NK3) is increased in inflammatory airway disorders, which could result in augmentation of the effect of released tachykinin neuropeptides. NK1 receptor and NK2 receptor but not NK3-receptor mRNA were detected by ribonuclease protection assay in RNA from both cartilaginous and membranous bronchi and subpleural lung. In lung samples containing membranous airways, NK2-receptor mRNA expression was increased fourfold in asthmatics compared with nonsmoking controls, whereas NK1-receptor mRNA levels were similar in the two groups. NK1- and NK2-receptor mRNA expression was increased twofold in smokers without airflow obstruction compared with nonsmokers, whereas NK1-receptor mRNA expression was significantly lower in patients with chronic obstructive pulmonary disease compared with smoking controls. In situ hybridization indicated NK1-receptor mRNA was expressed in submucosal glands and airway epithelial cells, whereas NK2-receptor and NK3-receptor mRNA were not detected. These observations have implications for the pathophysiology and treatment of both asthma and tobacco smoke-induced airway inflammation.

Oncogenic osteomalacia: evidence for a humoral phosphaturic factor.

Oncogenic osteomalacia is a syndrome characterized by phosphaturia, hypophosphatemia, reduced vitamin D levels, and osteomalacia. The cause is not known, but all patients have had a tumor; usually of mesenchymal origin. Removal of the tumor reverses the metabolic abnormalities. We report a patient with osteomalacia, severe hypophosphatemia, elevated alkaline phosphatase, low 1,25-dihydroxyvitamin D3, and phosphaturia. A tumor was identified in the infratemporal fossa. The tumor was removed, and all of the biochemical abnormalities resolved over the subsequent 8 months. The bone density returned to normal values. The tumor had the appearance of a paraganglioma and was used to establish a cell culture line called JH-55. Electron microscopy of the original tumor and the JH-55 cells demonstrated the presence of neurosecretory granules. A bioassay using opossum kidney cells was used to evaluate phosphate transport. Conditioned medium from the JH-55 cells inhibited phosphate reabsorption by the kidney tubular cells. Maximal inhibition required a 24-h incubation period and was not altered by the presence of an inhibitor of protein synthesis (10 micrograms/mL cycloheximide). Immunoassays revealed no detectable PTH-related peptide or intact PTH in the JH-55 medium. The cause of this paraneoplastic syndrome is not known, but all of the evidence is consistent with the action of a hormone that produces phosphaturia. This putative factor is distinct from other hormones that cause phosphaturia.

Expression of beta 2-adrenergic receptor mRNA in peripheral lung in asthma and chronic obstructive pulmonary disease.

Previous studies have indicated an increased number of beta 2-adrenergic receptors (beta 2AR) on bronchial smooth muscle in fatal asthma. This study evaluates the utility of autopsy lung for studies of gene expression and examines the hypothesis that increased expression of beta 2 AR mRNA in peripheral lung underlies the increased receptor number reported in central airways in fatal asthma. beta 2AR mRNA levels have been quantitated using the ribonuclease protection assay on RNA from peripheral lung obtained both at autopsy and thoracotomy from subjects with normal lungs as well as subjects with asthma or chronic obstructive pulmonary disease (COPD). Glucocorticosteroid and serum induction of beta 2AR mRNA in human epidermoid carcinoma A431 cells, which display a high abundance of beta 2AR receptors, was also examined to provide aliquots of RNA containing relatively high levels of beta 2AR mRNA for use as positive controls and internal standards. In A431 cells maintained after confluence in serum-free media for 72 h, maximal beta 2AR mRNA levels in response to 10% fetal bovine serum were 85% of maximal levels following serum plus 10 microM dexamethasone. Both autopsy and resected lung yielded undegraded RNA with a similar relative abundance of beta 2AR mRNA. Although geometric mean beta 2AR mRNA levels were similar in all three patient groups, relatively high levels were observed in resected lung in a subpopulation of subjects with mild or moderate asthma but not in autopsy lung from subjects with severe asthma. High levels of beta 2AR mRNA, presumably reflecting lung growth or asthma, were demonstrated in peripheral lung of a 4-yr-old child with asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol ester, forskolin, and serum induction of a human colon nuclear hormone receptor gene related to the NUR 77/NGFI-B genes.

Two closely related members (mouse NUR 77 and rat NGFI-B) of the serum-inducible "early intermediate" gene family are nuclear hormone receptors containing zinc fingers of the cys2-cys2 type. This paper describes the complementary DNA cloning of the human equivalent of the NUR 77/NGFI-B genes isolated from LS-180 colon adenocarcinoma cells and named the ST-59 gene. ST-59 RNA expression was shown to be rapidly and transiently induced by fetal calf serum. To a lesser extent, epidermal growth factor could induce ST-59 RNA expression, but nerve growth factor, insulin-like growth factor, and fibroblast growth factor were ineffective. ST-59 receptor induction by serum was greatly amplified by cycloheximide and could be detected in actively growing LS-180 cells. The serum induction of RNA expression in these cells could be augmented by treatment with phorbol esters (10(-5) M), forskolin (10(-5) M), and 8-bromo cyclic AMP (4 x 10(-3) M). These results suggest that at least two signal pathways (protein kinase C and protein kinase A) participate in the ST-59 gene mRNA induction.

Experimental metastatic ability of H-ras-transformed NIH3T3 cells.

We have used a quantitative "experimental" metastasis assay in the embryonic chick, an immunodeficient host, to examine in vivo growth properties of ras oncogene-transformed NIH3T3 cells. We found that two independently derived populations of NIH3T3 cells that had been morphologically transformed with the T24 human H-ras oncogene were able to grow in vivo following i.v. injection. Nontransformed control NIH3T3 cells with normal morphology did not grow in this assay. Spontaneously arising morphological transformants from control NIH3T3 cell populations were also tested and did not grow in this assay. We conclude that the H-ras gene can confer experimental metastatic ability on nonmetastatic NIH3T3 cells, that the ras gene alters the cells in some way beyond in vitro morphological transformation, and thus that the in vitro transformation assay detects only part of the malignant phenotype of these cells.

Exposure of JB-6 mouse epidermal cells to 12-O-tetradecanoyl-phorbol-13-acetate is not accompanied by a significant change in total DNA-cytosine methylation.

The extent of methylation of the cytosine bases in DNA is believed to be a major factor influencing gene expression in eukaryotic cells. We have asked whether the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the amount of 5-methylcytosine in DNA. The amount and relative distribution of 5-methylcytosine in the DNA of two subclones of the JB-6 mouse epidermal cell line were determined respectively by high performance liquid chromatography and digestion with the restriction enzymes MspI and HpaII. Exposure to TPA for up to several cell generations had no detectable effect on the degree of DNA methylation (3.9% of the total cytosine) in the two JB-6 lines or Friend erythroleukemia cells. Reduced methylation was readily detected in DNA extracted from cells exposed to 5-azacytidine. The data suggest that tumor promotion (at least that induced by TPA) is likely not the consequence of a generalized elevation or reduction in the amount of 5-methyl-cytosine in the DNA.

delta9-Tetrahydrocannabinol and 17beta-estradiol bind to different macromolecules in estrogen target tissues.

delta9-Tetrahydrocannabinol (delta9-THC), added to the limit of its solubility, did not compete with tritiated 17beta-estradiol for binding to estrogen receptor sites in mouse mammary or uterine cytosols. On sucrose density gradients of low-ionic strength, mammary cytosol labeled with [3H]estradiol exhibited a binding peak near the "8S" region typical of estrogen receptor whereas in cytosol labeled with delta9-[3H]THC binding was limited to the nonspecific 4- to 5S region. Differences in sedimentation properties and reciprocal competition studies strongly refute previous claims that delta-9-THC binds to estrogen receptor and that by so doing it directly acts as an estrogen.

Estrogen binding in mammary tissue of C3H mice with or without the mouse mammary tumor virus.

Substrains of C3H mice, with and without the milk-transmitted form of the mouse mammary tumor virus (MMTV)1, were compared to determine if the presence of MMTV altered estrogen action. Uterine weight response to oral diethylstilbestrol was the same for both substrains, (MMTV+ and MMTV-). The concentration of high-affinity estrogen binding sites ("receptors") in lactating mammary tissue did not differ significantly for MMTV+ versus MMTV- mice; the affinity of the estradiol-receptor interaction appeared higher, however, in cytosol from MMTV- mice. Sucrose density gradient analysis revealed no significant differences in sedimentation properties of cytosol receptor from the two substrains.