Fatty acid binding protein 4 is expressed in distinct endothelial and non-endothelial cell populations in glioblastoma.

AIMS: Glioblastoma (GBM) is the most common and aggressive primary brain tumour in adults. Angiogenesis and vasculogenesis play key roles in progression of GBMs. Fatty acid binding protein 4 (FABP4) is an intracellular chaperone for free fatty acids. FABP4 is detected in microvascular endothelial cells (ECs) in several normal tissues and promotes proliferation of ECs. The goal of this study was to characterize the tissue distribution pattern of FABP4 in GBMs. METHODS: Immunohistochemistry for FABP4 was performed on paraffin-embedded tumour sections and the intensity and distribution of FABP4 immunoreactivity were analysed. Double immunofluorescence was employed for detailed characterization of FABP4-positive cells. RESULTS: FABP4 immunoreactivity was absent in normal brain tissue sections. FABP4-positive cells were detected in 33%, 43%, 64% and 89% of Grade I, Grade II, Grade III and Grade IV glial tumours, respectively. Thus, the percentage of FABP4-positive cells in GBMs was significantly higher than lower-grade gliomas. In general, FABP4-expressing cells were distributed in a non-homogenous pattern, as 'hot spots' in glial tumours. FABP4 expression was detected in a subset of vascular ECs as well as some non-ECs. CONCLUSION: FABP4 is expressed in a significantly higher percentage of GBMs in comparison to both normal brain tissues and lower-grade glial tumours. FABP4 is expressed in some tumour ECs as well as non-ECs in glial tumours. As FABP4 promotes proliferation of ECs, detection of FABP4 in GBM-ECs, but not normal brain ECs suggests that FABP4 may play a role in the robust angiogenesis associated with GBMs.

Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease.

In Alzheimer's disease (AD), affected neurons accumulate beta amyloid protein, components of which can induce mouse microglia to express the high-output isoform of nitric oxide synthase (NOS2) in vitro. Products of NOS2 can be neurotoxic. In mice, NOS2 is normally suppressed by transforming growth factor beta 1 (TGF-beta 1). Expression of TGF-beta 1 is decreased in brains from AD patients, a situation that might be permissive for accumulation of NOS2. Accordingly, we investigated the expression of NOS2 in patients with AD, using three monospecific antibodies: a previously described polyclonal and two new monoclonal antibodies. Neurofibrillary tangle-bearing neurons and neuropil threads contained NOS2 in brains from each of 11 AD patients ranging in age from 47 to 81 years. NOS2 was undetectable in brains from 6 control subjects aged 23-72 years, but was expressed in small amounts in 3 control subjects aged 77-87 years. Thus, human neurons can express NOS2 in vivo. The high-output pathway of NO production may contribute to pathogenesis in AD.

Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment.

The endothelium is morphologically and functionally adapted to meet the unique demands of the underlying tissue. At the present time, little is known about the molecular basis of endothelial cell diversity. As one approach to this problem, we have chosen to study the mechanisms that govern differential expression of the endothelial cell-restricted von Willebrand factor (vWF) gene. Transgenic mice were generated with a fragment of the vWF gene containing 2,182 bp of 5' flanking sequence, the first exon and first intron coupled to the LacZ reporter gene. In multiple independent lines of mice, beta-galactosidase expression was detected within endothelial cells in the brain, heart, and skeletal muscle. In isogeneic transplantation models, LacZ expression in host-derived auricular blood vessels was specifically induced by the microenvironment of the heart. In in vitro coculture assays, expression of both the transgene and the endogenous vWF gene in cardiac microvascular endothelial cells (CMEC) was upregulated in the presence of cardiac myocytes. In contrast, endothelial cell levels of thrombomodulin protein and mRNA were unchanged by the addition of ventricular myocytes. Moreover, CMEC expression of vWF was not influenced by the addition of 3T3 fibroblasts or mouse hepatocytes. Taken together, the results suggest that the vWF gene is regulated by vascular bed-specific pathways in response to signals derived from the local microenvironment.

Localization of the neurally mediated arrhythmogenic properties of digitalis.

Available evidence suggests that the propensity of digitalis glycosides to produce cardiac arrhythmias is due in part to their neuroexictatory effects. We have performed experiments in cats which support the existence of a neurogenic component in the etiology of digitalis-induced ventricular arrhythmias. Our data further indicate that the locus of this neural effect lies within an area of the medulla 2 millimeters above to 2 millimeters below the obex. These findings, when considered with the effects of polar cardiac glycosides that do not cross the blood-brain barrier, suggest that the area postrema may be the site of neural activation.

Rivaroxaban and apixaban induce clotting factor Xa fibrinolytic activity.

Essentials Activated clotting factor X (FXa) acquires fibrinolytic cofactor function after cleavage by plasmin. FXa-mediated plasma fibrinolysis is enabled by active site modification blocking a second cleavage. FXa-directed oral anticoagulants (DOACs) alter FXa cleavage by plasmin. DOACs enhance FX-dependent fibrinolysis and plasmin generation by tissue plasminogen activator. BACKGROUND: When bound to an anionic phospholipid-containing membrane, activated clotting factor X (FXa) is sequentially cleaved by plasmin from the intact form, FXaα, to FXaß and then to Xa33/13. Tissue-type plasminogen activator (t-PA) produces plasmin and is the initiator of fibrinolysis. Both FXaß and Xa33/13 enhance t-PA-mediated plasminogen activation. Although stable in experiments using purified proteins, Xa33/13 rapidly loses t-PA cofactor function in plasma. Bypassing this inhibition, covalent modification of the FXaα active site prevents Xa33/13 formation by plasmin, and the persistent FXaß enhances plasma fibrinolysis. As the direct oral anticoagulants (DOACs) rivaroxaban and apixaban bind to the FXa active site, we hypothesized that they similarly modulate FXa fibrinolytic function. METHODS: DOAC effects on fibrinolysis and the t-PA cofactor function of FXa were studied in patient plasma, normal pooled plasma and purified protein experiments by the use of light scattering, chromogenic assays, and immunoblots. RESULTS: The plasma of patients taking rivaroxaban showed enhanced fibrinolysis correlating with FXaß. In normal pooled plasma, the addition of rivaroxaban or apixaban also shortened fibrinolysis times. This was related to the cleavage product, FXaß, which increased plasmin production by t-PA. It was confirmed that these results were not caused by DOACs affecting activated FXIII-mediated fibrin crosslinking, clot ultrastructure and thrombin-activatable fibrinolysis inhibitor activation in plasma. CONCLUSION: The current study suggests a previously unknown effect of DOACs on FXa in addition to their well-documented anticoagulant role. By enabling the t-PA cofactor function of FXaß in plasma, DOACs also enhance fibrinolysis. This effect may broaden their therapeutic indications.

Ouabain-specific antibodies: immunochemical properties and reversal of Na + , K + -activated adenosine triphosphatase inhibition.

Antibodies with high affinity and specificity for the cardiac glycoside ouabain were raised in rabbits. The antigen used was a conjugate of ouabain linked through its rhamnose moiety to terminal alpha-amino groups of poly D,L alanyl-human serum albumin. Ouabain-specific antibodies were present as early as 3 wk, and rose steadily in titer over the initial 20-33 wk of immunization. Levels as high as 6.5 mg specific immunoglobulin per ml antiserum were reached in one rabbit at the end of 45 wk. The average intrinsic association constants for ouabain were 1.3 x 10(9) M(-1) and 1.6 x 10(9) M(-1) in antisera studied in detail, and there was evidence of restricted heterogeneity of binding site affinities. A high degree of specificity was demonstrated. Significant cross-reactivity occurred only with other cardioactive steroid compounds such as acetyl strophanthidin, digoxin, and digitoxin, while endogenous steroids did not cross-react even when present in 1000-fold excess. A rapid and convenient radioimmunoassay procedure for plasma or urine ouabain concentrations was developed using these antibodies. Competition between ouabain-(3)H tracer and unlabeled ouabain for specific antibody binding sites allowed the measurement of ouabain concentrations as low as 0.1 ng/ml or less without need for extraction procedures. The high association constants observed in these studies permit antibody reversal of established myocardial effects of ouabain. Both blockade and reversal of ouabain inhibition of canine myocardial microsomal Na(+), K(+)-activated ATPase by antibody were documented, suggesting a possible mechanism for reversal of cellular effects.

Reversal of advanced digitoxin toxicity and modification of pharmacokinetics by specific antibodies and Fab fragments.

The effects of Fab fragments of high-affinity specific antibodies have been studied in a canine experimental model of lethal digitoxin toxicity. Selected antiserum from sheep immunized and boosted with a digoxin-serum albumin conjugate contained antibodies that cross-reacted with digitoxin with an average intrinsic association constant of 1.4 x 10(10) M(-1) as determined by equilibrium dialysis. Rapid second-order association kinetics (k(f) = 3.7 x 10(6) M(-1) per s) and slow dissociation kinetics (k(r) = 1.9 x 10(-4) per s) were documented for the antibody-digitoxin complex. Eight dogs given 0.5 mg/kg digitoxin intravenously developed ventricular tachycardia after 23+/-4 (SEM) min. Control nonspecific Fab fragments were then given. All animals died an average of 101+/-36 min after digitoxin administration. Another eight dogs given the same digitoxin dose similarly developed ventricular tachycardia after 28+/-3 min. This group then received a molar equivalent dose of specific Fab fragments intravenously over 3 min, followed by a 30-min infusion of one-third of the initial dose. All dogs survived. Conducted sinus beats reappeared 18+/-4 min after initial Fab infusion, and stable normal sinus rhythm was present at 54+/-16 min. Plasma total digitoxin concentrations increased threefold during the hour after initial Fab infusion, while plasma free digitoxin concentration decreased to less than 0.1 ng/ml. Effects on digitoxin pharmacokinetics of these Fab fragments and the antibody population from which they were derived were further investigated in a primate species. Unlike common laboratory animals previously studied, the rhesus monkey was found to have a prolonged elimination half-life, estimated at 135 and 118 h by radioimmunoassay and [(3)H]digitoxin measurements, respectively, similar to man and thus providing a clinically relevant experimental model. Intravenous administration of 2 mol of specific Fab fragments per mole of digitoxin 6 h after 0.2 mg of digitoxin produced a rapid 4.3-fold increase in plasma total digitoxin concentration followed by a rapid fall (t((1/2)) 4 h) accompanied by a 14-fold enhancement of urinary digitoxin excretion over control values during the 6-h period after Fab was given. Analytical studies were consistent with increased excretion of native digitoxin rather than metabolites, and the glycoside was found in equilibrium dialysis studies to be excreted in the urine in Fab-bound form. Administration of 2 mol of specific antibody binding sites per mole of digitoxin as intact IgG caused a greater and more prolonged increase in plasma total digitoxin concentration, peaking 13-fold above control levels. In contrast to the effects of Fab, however, specific IgG reduced the rate of urinary digitoxin excretion substantially below control values. We conclude that Fab fragments of antibodies with high affinity for digitoxin are capable of rapid reversal of advanced, otherwise lethal digitoxin toxicity, and are capable of reducing the plasma half-life and accelerating urinary excretion of digitoxin.

Effects of thyroid hormone on sodium pump sites, sodium content, and contractile responses to cardiac glycosides in cultured chick ventricular cells.

Sensitivity of cardiac muscle to digitalis glycosides depends on the thyroid state. The mechanism of this interaction was investigated at the cellular level using spontaneously beating monolayers of cultured chick embryo ventricular cells. Cells were grown for 48 h in serum-free medium containing concentrations of triiodothyronine (T3) from zero to 10(-7) M, and the total number of sodium pump sites, sodium content, and contractile amplitude in the presence and absence of various concentrations of ouabain were determined. T3 caused a concentration-dependent increase in the number of specific ouabain binding sites; the maximal increase to 160% of control was observed in response to 10(-8) M T3. T3 lowered steady-state cellular sodium content in a concentration-dependent manner, also. Ouabain (1 microM) exposure elevated cellular sodium content in all cells, but the increase was greatest in cells grown in T3-free medium and least in cells grown in 10(-8) M T3. The positive inotropic and toxic effects of ouabain in cells grown in 10(-8) M T3 were diminished at any given ouabain concentration, and thus, the dose-response curve was shifted to the right. These results indicate that T3 causes induction of additional sodium pump sites that are functional. The increased tolerance of hyperthyroid cells and reduced tolerance of hypothyroid cells to cardiac glycosides can be explained by these changes in the number of sodium pump sites and cellular sodium content, and consequently, calcium influx via sodium-calcium exchange.

Reversal of ouabain and acetyl strophanthidin effects in normal and failing cardiac muscle by specific antibody.

Isolated cat right ventricular papillary muscles were used to study the effects of antibodies with high affinity for ouabain and acetyl strophanthidin on myocardium exposed to these cardioactive steroids. Antibodies with average intrinsic affinity constants for ouabain and acetyl strophanthidin of the order of 10(8) M(-1) were raised in rabbits challenged by repeated injection of a conjugate of ouabain covalently linked to a poly D,L-alanyl derivative of human serum albumin. Effects were assessed in terms of time-course and extent of inotropy reversal, influence of experimentally induced ventricular failure, digitalis-antibody concentration relations, influence of digitalis-antibody complex on response to additionally added digitalis, and relation of antibody effects on digitalis-induced automaticity and contracture to reversal of inotropy. Specific antibody (but not control antibody) in 1.1-1.5-fold molar excess over cardioactive steroid concentrations blocked positive inotropic effects of ouabain and acetyl strophanthidin, and gradually reversed established contractile effects of these agents with a mean time for half-reversal of ouabain-induced inotropy of 124+/-6 (SEM) min and 37+/-3 min for half-reversal of acetyl strophanthidin-induced inotropy. Papillary muscles from cats with right ventricular failure induced by chronic pulmonary artery constriction responded similarly. Both normal and failing muscles returned to but not below levels of contractility existing before cardioactive steroid exposure, and time for half-reversal of inotropy by antibody was significantly shorter than time for half-reversal after removal of ouabain or acetyl strophanthidin by muscle bath washout alone. Presence of ouabain- or acetyl strophanthidin-antibody complex did not alter the myocardial contractile response to subsequently added cardioactive steroids. Spontaneous automaticity occurring as a toxic response to ouabain or acetyl strophanthidin in eight muscles was rapidly reversed by specific antibody at a time when positive inotropic effects were still fully manifest. Early contracture was also reversed by specific antibody. These studies provide further support for the concept that cardiac glycoside-specific antibodies are capable of reversing established cellular effects of cardioactive steroids.

Digoxin intoxication: the relationship of clinical presentation to serum digoxin concentration.

A radioimmunoassay for serum digoxin concentration has been used to study the interrelationships of circulating levels of the drug and various factors in the clinical setting in 48 hospitalized patients with cardiac rhythm disturbances due to digoxin intoxication. 131 patients on maintenance doses of digoxin without toxicity and 48 patients with equivocal evidence of digoxin excess were also studied and compared with the toxic group. Patients with cardiac rhythm disturbances due to digoxin intoxication tended to be older and to have diminished renal function compared with the nontoxic group; body weight, serum potassium concentration, underlying cardiac rhythm, and nature of cardiac disease were not significantly different for the groups as a whole. Despite comparable mean daily digoxin dosages, digoxin intoxicated patients had a mean serum digoxin concentration of 3.7 +/-1.0 (SD) ng/ml, while nontoxic patients had a mean level of 1.4 +/-0.7 ng/ml (P < 0.001), 90% of patients without evidence of toxicity had serum digoxin concentrations of 2.0 ng/ml or less, while 87% of the toxic group had levels above 2.0; the range of overlap between the two groups extended from 1.6 to 3.0 ng/ml. Patients with atrioventricular block as their principal toxic manifestation had a significantly lower mean serum digoxin concentration than those in whom ectopic impulse formation was the chief rhythm disturbance. Patients with equivocal evidence of digoxin excess had received comparable daily maintenance doses of digoxin but had a mean serum concentration of 1.9 +/-0.8 ng/ml, intermediate between those of the nontoxic (P < 0.005) and toxic (P < 0.001) groups. Renal function as judged by mean blood urea nitrogen concentration was also intermediate. The data indicate that knowledge of the serum digoxin concentration, weighed in the clinical context, is useful in the management of patients receiving this drug.

Effect of thyroid hormone on slow calcium channel function in cultured chick ventricular cells.

The hyperthyroid state is associated with increased myocardial contractility. To clarify responsible mechanisms, we examined the effects of thyroid hormone on slow Ca channels, beta-adrenergic receptors, transsarcolemmal 45Ca flux and cytosolic free calcium in cultured chick ventricular cells. Compared with cells grown without triiodothyronine (T3), cells grown in 10 nM T3 possessed 67% (P less than 0.05) more dihydropyridine 3H-PN200-110 binding sites, 24% (P less than 0.05) more beta-adrenergic antagonist 3H-CGP12177 binding sites, a 57% (P less than 0.05) greater nifedipine-sensitive initial 45Ca uptake rate, and a 31% (P less than 0.05) greater nifedipine-sensitive 45Ca uptake rate in response to BAY k 8644. Time-averaged mean intracellular free Ca concentration ([Ca]i) measured with fura-2, total protein content, and dissociation constant values for 3H-PN200-110 or 3H-CGP12177 binding was not significantly different in the two groups of cells. BAY k 8644 (1 microM) increased mean [Ca]i 2.85- or 2.16-fold in cells grown with or without 10 nM T3, respectively. l-Isoproterenol (1 microM) increased [Ca]i 1.53- or 1.28-fold in cells grown with or without 10 nM T3, respectively. We conclude that thyroid hormone augments transsarcolemmal Ca influx, at least in part via slow Ca channels associated with increased numbers of these channels. T3-treated cells appear to be more responsive to the effects of BAY k 8644 or isoproterenol on [Ca]i.

Differing sensitivities of Purkinje fibers and myocardium to inhibition of monovalent cation transport by digitalis.

The extent of inhibition of monovalent cation active transport in Purkinje fibers and myocardium in response to toxic and inotropic doses of digitalis were studied in the dog to elucidate the factors underlying the different relative sensitivities of these tissues to the toxic arrhythmogenic effects of digitalis. Monovalent cation transport inhibition was assessed by measuring uptake of the K(+) analog Rb(+) in samples of myocardium and Purkinje fibers after in vitro ouabain exposure and after acute or chronic administration of digoxin in vivo. The active uptake of Rb+ was determined as the difference between total uptake and uptake in the presence of 1.0 mM ouabain. Mean active uptake of Rb(+) by Purkinje fibers from control hearts was 1.62+/-0.11 (SEM) nmol/mg wet wt per 15 min, significantly greater than the value of 0.49+/-0.05 for myocardium (P < 0.001). Concentration-effect curves for inhibition of monovalent cation active transport by in vitro exposure to graded concentrations of ouabain showed that the concentration for half-maximal inhibition of monovalent cation transport, IC(50), for Purkinje fiber transport was 0.4 muM, significantly less than the value of 1.4 muM for myocardial samples. Dogs were given toxic doses of digoxin (0.3 mg/kg i.v.). At onset of sustained ventricular tachycardia, they were killed and monovalent cation transport measured in myocardial and Purkinje fiber samples. Active Rb(+) uptake was inhibited by 44% in myocardial samples, whereas a significantly greater inhibition of 76% was noted in Purkinje fibers (P < 0.01). Similar data were obtained for both transmural myocardial biopsy samples and subendocardial trabecular samples obtained from regions adjacent to Purkinje fibers. Another group of dogs received a nontoxic dose of 0.02 mg/kg i.v. daily for 6 d. Myocardium showed a 17% reduction in Rb(+) active uptake compared to control animals receiving no drug, whereas Purkinje fiber transport was reduced in these dogs to a significantly greater extent averaging 44% (P < 0.001). Thus, both toxic and inotropic (non-toxic) doses of digoxin inhibited monovalent cation transport in Purkinje fibers to a greater extent than in myocardium. This difference in apparent sensitivity of monovalent cation transport to digoxin may contribute to the previously reported tendency of digitalis toxic arrhythmias to arise in Purkinje fibers.

Stimulation of monovalent cation active transport by low concentrations of cardiac glycosides. Role of catecholamines.

The stimulatory effect of low concentrations of ouabain on the Na-K pump in isolated guinea pig left atria was studied in vitro by assessing active transport of the K(+) analog Rb(+). Active transport of Rb(+) was stimulated 20+/-8% (SEM, P < 0.05) above control values by 3 nM ouabain, but was inhibited by concentrations >10 nM. Preincubation with the beta-adrenergic antagonist propranolol (1 muM) completely blocked stimulation of active transport of Rb(+) by 3 nM ouabain. Norepinephrine, 10 nM, increased Rb(+) active transport 29+/-10% (P < 0.02) above control values. The beta-adrenergic agonist l-isoproterenol, 10 nM, increased active transport of Rb(+) by 33+/-10% (P < 0.01) above control levels. This stimulatory effect was abolished if tissues were first exposed to propranolol. Tyramine (0.1 muM), a stimulator of endogenous catecholamine release, increased active transport of Rb(+) 26+/-12% (P < 0.05) above control values. Rb(+) active transport was not significantly changed when left atrial tissues were incubated with alpha-adrenergic agonists or antagonists. Ouabain stimulation of Rb(+) active transport was prevented by in vivo depletion of myocardial endogenous catecholamines by either reserpine or 6-hydroxydopamine. These findings indicated that in myocardial tissue, Na-K pump stimulation by low concentrations of ouabain is mediated at least in part through beta-adrenergic effects of endogenous catecholamines.

Enkephalins increase cyclic adenosine monophosphate content, calcium uptake, and contractile state in cultured chick embryo heart cells.

Peripheral vascular effects of opioid peptides are well known, but direct myocardial effects have not been established. We studied the inotropic response of spontaneously beating cultured chick embryo ventricular cells to the enkephalin analogue [D-Ala2]-enkephalin. Amplitude of cell motion increased in a concentration-dependent manner with 0.53 microM [D-Ala2]-enkephalin producing half-maximal response. The mechanism of this positive inotropic effect was investigated by examining alterations in 45Ca influx, cyclic AMP accumulation and adenylate cyclase activity in response to [D-Ala2]-enkephalin. At maximally inotropic concentrations, the 45Ca influx rate increased 39%, adenylate cyclase was stimulated by 30%, and cyclic AMP content rose more than twofold. Thus, in contrast to neural tissue, receptors for enkephalin in cultured heart cells are coupled to adenylate cyclase in a stimulatory manner. Occupancy of these receptors produces an increase in cyclic AMP levels and exerts a positive inotropic effect via a verapamil-sensitive enhancement of Ca influx.

The central nervous system as a site of action for the coronary vasoconstrictor effect of digoxin.

Digitalis is known to have a vasoconstrictor effect in the coronary circulation. Recent studies have demonstrated that the coronary vasoconstrictor effects of acetylstrophanthidin and digoxin are neurally mediated via alpha adrenergic fibers. In the present study, experiments were done in 20 dogs anesthetized with chloralose and urethane to study the central nervous system as a possible site of action for this vasoconstrictor effect of digoxin. After the intravenous administration of 1.0 mg digoxin, cerebrospinal fluid concentrations of digoxin rose to a peak of 2.3+/-0.4 (SEM) ng/ml at 15 min, temporally corresponding to the peak in coronary vascular resistance change of +20.0+/-2.5% of control in the paced canine heart. Submicrogram digoxin injections into the lateral cerebral ventricle produced a significant increase in coronary vascular resistance, the latter injection producing a peak increase in coronary vascular resistance of 12.4+/-1.2% of control. Cross-perfusion experiments, where the isolated head of the operative dog was perfused from a donor dog receiving digoxin, thus keeping digoxin levels in the remainder of the operative dog very low, showed a similar degree of coronary vasoconstriction. Thus, the central nervous system appears to be an important site of action for the early coronary vasoconstrictor effect of digoxin.

Ischemia-induced alterations in myocardial (Na+ + K+)-ATPase and cardiac glycoside binding.

The effects of ischemia on the canine myocardial (Na+ + K+)-ATPase complex were examined in terms of alterations in cardiac glycoside binding and enzymatic activity. Ability of the myocardial cell to bind tritiated ouabain in vivo was assessed after 1, 2, and 6 h of coronary occlusion followed by 45 min of reperfusion, and correlated with measurements of in vitro (Na+ + K+)-ATPase activity and in vitro [3H]ouabain binding after similar periods of ischemia. Regional blood flow alterations during occlusion and reperfusion were simultaneously determined utilizing 15 mum radioactive microspheres to determine the degree to which altered binding of ouabain might be flow related. Anterior wall infarction was produced in 34 dogs by snaring of confluent branches of the left coronary system. Epicardial electrograms delineated ischemic and border zone areas. Coronary reperfusion after 2 and 6 h of occlusion was associated with impaired reflow of blood and markedly impaired uptake of [3H]ouabain in ischemic myocardium. In both groups, in vivo [3H]ouabain binding by ischemic tissue was reduced out of proportion to the reduction in flow. Despite near-complete restoration of flow in seven dogs occluded for 1 h and reperfused, [3H]ouabain remained significantly reduced to 58 +/- 9% of nonischemic uptake in subendocardial layers of the central zone of ischemia. Thus, when coronary flow was restored to areas of myocardium rendered acutely ischemia for 1 or more hours, ischemic zones demonstrated progressively diminished ability to bind ouabain. To determine whether ischemia-induced alteration in myocardial (Na+ + K+)-ATPase might underlie these changes, (Na+ + K+)-ATPase activity and [3H]ouabain binding were measured in microsomal fractions from ischemic myocardium after 1, 2, and 6 h of coronary occlusion. In animals occluded for 6 h, (Na+ + K+)-ATPase activity was significantly reduced by 40% in epicardial and by 35% in endocardial layers compared with nonischemic myocardium. Comparable reductions in in vitro [3H]ouabain binding were also demonstrated. Reperfusion for 45 min after occlusion for 6 h resulted in no significant restoration of enzyme activity when compared to the nonreperfused animals. In six animals occluded for 2 h, a time at which myocardial creatine phosphokinase activity remains unchanged, (Na+ + K+)-ATPase activity was reduced by 25% compared with nonischemic enzyme activity. In five dogs occluded for 1 h, (Na+ + K+)-ATPase activity in ischemic myocardium was unchanged from control levels. We conclude that reduced regional myocardial blood flow, local alterations in cellular milieu, and altered glycoside-binding properties of (Na+ + K+)-ATPase all participate in the reduction of cardiac glycoside binding observed after reperfusion of ischemic myocardium. In addition, after 2 or more hours of severe ischemia, myocardial (Na+ + K+)-ATPase catalytic activity is significantly reduced despite incubation in the presence of optimal substrate concentrations.

Thyroid-induced alterations in myocardial sodium-potassium-activated adenosine triphosphatase, monovalent cation active transport, and cardiac glycoside binding.

The effects of thyroid hormone on guinea pig myocardial NaK-ATPase activity, transmembrane monovalent cation active transport, and cardiac glycoside binding were were examined. NaK-ATPase activities of left atrial and left ventricular homogenates of control and triiodothyronine (T3)-treated animals were determined, and compared to activities of skeletal muscle and liver. T3 administration was associated with a significant increase of 18% in left atrial and left ventricular NaK-ATPase specific activities. This increment was less than that noted in skeletal muscle (+42%) and liver (+30%). To determine if enhanced NaK-ATPase activity was accompanied by increased monovalent cation active transport, in vitro 86Rb+ uptake by left atrial strips and hemidiaphragms was measured. Transition from the euthyroid to the hyperthyroid state resulted in a 68% increase in active 86Rb+ uptake by left atrium, and a 62% increase in active uptake by diaphragm. Passive 86Rb+ uptake was not affected in either tissue. Ouabain binding by atrial and ventricular homogenates of T3-treated animals was increased by 19 and 17%, respectively, compared to controls, in close agreement with thyroid-induced increments in NaK-ATPase activiey. Taken together, these results are consistent with enhanced myocardial NaK-ATPase activity and monovalent cation activt transport due to an increase in the number of functional enzyme complexes.

Nitric oxide synthase (NOS3) and contractile responsiveness to adrenergic and cholinergic agonists in the heart. Regulation of NOS3 transcription in vitro and in vivo by cyclic adenosine monophosphate in rat cardiac myocytes.

Cardiac myocytes express the nitric oxide synthase isoform originally identified in constitutive nitric oxide synthase cells (NOS3), which mediates the attenuation by muscarinic cholinergic agonists of beta-adrenergic stimulation of L-type calcium current and contractility in these cells. However, calcium current and contractility in these cells. However, the reciprocal regulation of NOS3 activity in myocytes by agents that elevate cAMP has not been reported. In this study, we show that NOS3 and mRNA and protein levels in cardiac myocytes are reduced both in vitro after treatment with cAMP elevating drugs, and in vivo after 3 d of treatment with milrinone, a type III cAMP phosphodiesterase inhibitor. This effect on NOS3 activity by cAMP is cell type specific because treatment of cardiac microvascular endothelial cells in vitro or in vivo did not decrease NOS3 mRNA or protein in these cells. NOS3 downregulation in myocytes appeared to be at the level of transcription since there was no modification of NOS3 mRNA half-life by agents that increase intracellular cAMP. To determine the functional effects of NOS3 downregulation, we examined the contractile responsiveness of isolated electrically paced ventricular myocytes, isolated from animals that had been treated in vivo with milrinone, to the beta-adrenergic agonist isoproterenol and the muscarinic cholinergic agonist carbamylcholine. There was no difference in baseline contractile function in cells that had been pretreated with cAMP elevating agents compared to controls, but cells exposed to milrinone in vivo exhibited an accentuation in their contractile responsiveness to isoproterenol compared to controls and a loss of responsiveness to carbamylcholine. Downregulation of myocyte NOS3 by sustained elevation of cAMP may have important implications for the regulation of myocardial contractile state by the autonomic nervous system.

Identification of the viral genes responsible for growth of strains of reovirus in cultured mouse heart cells.

Viral growth in specific tissue is usually required in order to lead to pathology. Two reovirus isolates (type 1 Lang and type 3 Dearing) differ in their capacity to grow in cultured mouse heart cells. The mammalian reoviruses contain a genome of 10 double-stranded RNA gene segments. By the use of 37 reassortant viruses (consisting of viruses with different combinations of genes derived from the two parents), difference in capacity of different strains to grow in heart cells was mapped to three different genes, all of which encode viral core proteins: the M1 gene (P less than 0.000044); the L1 gene (P = 0.00094); and the L3 gene (P = 0.019). Using the same set of reassortant viruses, the L1 (P = 0.00015) and L3 (P = 0.0065) genes were involved in differences of the ability of viral strains to grow in mouse L cells (fibroblasts), but the M1 gene (P = 0.12) was not. These findings suggest that the M1 gene plays an important and specific role in determining the relative capacity of certain viral strains to grow in the heart. Thus, we have identified viral genes responsible for differing growth capacity in heart muscle cells in culture. These findings provide a novel system for studies of viral myocarditis at a molecular genetic level.