Early membrane damage during coronary reperfusion in dogs. Detection by radiolabeled anticardiac myosin (Fab')2.

There is currently great interest in acute coronary reperfusion as a therapeutic modality for severe myocardial ischemia. While some studies have demonstrated a reduction in the overall extent of necrosis by early reperfusion, other studies have identified potentially deleterious effects produced by reflow. Because membrane disruption may be an important mechanism of irreversible cell injury, we measured changes in cell membrane integrity early during reperfusion using radiolabeled anticardiac myosin (Fab')2 antibody fragments in dogs. Our method involved brief periods of exposure to the (Fab')2 so that the levels of (Fab')2 binding indicated the degree of membrane disruption at discrete times during the progression of cell injury. In the first protocol (Fab')2 fragments labeled with either 125I and 131I were injected into the left circumflex coronary artery at the onset of reflow and at 45 min of reflow after a 1-h circumflex artery occlusion. Coronary sinus flow was diverted for 5 min following each injection to prevent recirculation. The (Fab')2 binding ratio (ischemic/control) increased during the first 45 min of reflow in each of eight experiments (mean increase 170%, P less than 0.01). No significant increase in (Fab')2 binding was observed in five additional experiments in which nonspecific (Fab')2 was injected. This indicates that the increase in binding seen with antimyosin-specific (Fab')2 was due to changes in specific binding rather than to alterations in (Fab')2 delivery produced by changes in blood flow distribution. The increase in membrane damage during reflow was confirmed by a second protocol in which each animal received only a single left atrial injection of (Fab')2 followed by rapid excision of the heart. The (Fab')2 binding ratio was 1.7 +/- 0.3 (SEM) in the group that received (Fab')2 at the onset of reflow and 3.7 +/- 0.6 (SEM) (P less than 0.05) in the group that received (Fab')2 after 45 min of reflow. In a third set of experiments in which hyperosmotic mannitol was infused during reflow the mean increase in (Fab')2 binding using the first protocol was only 80 +/- 40 vs. 170 +/- 30% without mannitol (P less than 0.05). Thus, membrane damage develops early during coronary reperfusion following 1 h of circumflex coronary artery occlusion, and part of this membrane damage can be prevented by altering the conditions of reflow. A method involving brief exposure of the myocardium to antimyosin (Fab')2 is promising for detecting changes in membrane integrity during evolving ischemic injury.

Effect of recainam on the energy required for ventricular defibrillation in dogs as assessed with implanted electrodes.

The effect of recainam (WY-42,362), a new class IC antiarrhythmic drug, on ventricular defibrillation was evaluated with the use of implanted superior vena cava and left ventricular patch electrodes in 17 normal dogs anesthetized with sodium pentobarbital. The energy required for a 50% probability of successful defibrillation (E50) was used as the index of ventricular defibrillation threshold. The dogs were classified into three groups: a saline group (n = 6), a low dose recainam group (n = 6) and a high dose recainam group (n = 5). The low dose infusion involved an intravenous loading dose of 3.75 mg/kg body weight over 20 min followed by a maintenance infusion of 0.0375 mg/kg per min. The high dose infusion was double those rates. The low dose recainam infusion produced a plasma recainam concentration of 3.1 +/- 0.3 micrograms/ml and significantly increased QRS duration by 11.3 +/- 3% during sinus rhythm. The high dose recainam infusion produced a plasma concentration of 7.7 +/- 0.9 microgram/ml and significantly increased QRS duration by 27 +/- 7% in sinus rhythm. Recainam did not change ventricular effective refractory period or sinus cycle length. The mean change in E50 between control and infusion periods was 1 +/- 5% in the saline group (8.5 +/- 1.4 versus 8.6 +/- 1.6 joules); 42 +/- 11% in the low dose recainam group (8.1 +/- 1.0 versus 11.3 +/- 1.3 joules) and 92 +/- 17% in the high dose recainam group (11.2 +/- 2.1 versus 20.5 +/- 2.5 joules).(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of reentry and acceleration due to double-wave reentry: two mechanisms for failure to terminate tachycardias by rapid pacing.

OBJECTIVES: We sought to demonstrate mechanisms by which rapid pacing can cause conduction block without terminating reentry. BACKGROUND: Rapid pacing can fail to terminate or can accelerate tachycardias in patients. Mechanisms for these responses are poorly understood. METHODS: We studied reentry in the canine atrial tricuspid ring and a left ventricular ring in vitro in 12 preparations. Activations were recorded from 10 sites around the ring, and monophasic action potentials were recorded from critical sites of block. Rapid pacing at cycle lengths that intermittently caused conduction block was performed at multiple sites. RESULTS: Action potential alternans contributed to block of an orthodromic impulse during rapid pacing. When pacing continued for two stimuli after orthodromic block, a second episode of block could reverse the direction of tachycardia. Continued pacing at this site was likely to produce block of an antidromic impulse, which may initiate double-wave reentry. Double-wave reentry could be sustained or nonsustained. Its cycle length was 56% to 77% of the single-wave cycle length. The ratio of double-wave cycle length to single-wave cycle length was inversely correlated with the relative excitable gap (p < 0.01). Double-wave reentry can be a mechanism for persistent cycle length alternation during tachycardia. CONCLUSIONS: Successful termination of reentry by rapid pacing required block of an othrodromic impulse and stopping pacing within one stimulus after orthodromic block. Reversal of reentry makes the circuit resistant to termination from this site of pacing. Antidromic block can cause acceleration due to double-wave reentry when there is a substantial excitable gap.

Analysis of the resetting phenomenon in sustained uniform ventricular tachycardia: incidence and relation to termination.

UNLABELLED: Although the phenomenon of resetting has been studied in several experimental and clinical rhythms, it has not been systematically analyzed in ventricular tachycardia. To define the incidence and determinants of resetting as well as its relation to ventricular tachycardia termination, the response to programmed stimulation was prospectively studied during 78 electrically induced episodes of sustained, uniform ventricular tachycardia (mean cycle length 365 +/- 59 ms) in 53 patients. Single and double ventricular extrastimuli were introduced during 78 and 39 episodes of ventricular tachycardia, respectively. Rapid ventricular pacing was performed during 27 episodes. Resetting occurred in response to single ventricular extrastimuli in 43 (55%) of 78 ventricular tachycardias, to double extrastimuli in 31 (79%) of 39 ventricular tachycardias and to rapid pacing in 23 (85%) of 27 ventricular tachycardias. No ventricular tachycardia characteristic distinguished those tachycardias that were reset from those not reset. Termination of ventricular tachycardia occurred in 7 (9%) of 78 episodes with single ventricular extrastimuli, 14 (36%) of 39 episodes with double ventricular extrastimuli and 13 (48%) of 27 episodes with rapid pacing. Termination was less frequent than resetting with both single (9 versus 55%) and double (36 versus 79%) extrastimuli, as well as rapid pacing (48 versus 85%). Resetting preceded termination in 7 of 7 ventricular tachycardias terminated with single ventricular extrastimuli, 12 of 14 terminated with double ventricular extrastimuli and 9 of 13 terminated by rapid pacing. Ventricular tachycardias that were terminated could not be differentiated from those that were reset without termination. IN CONCLUSION: Resetting with programmed extrastimuli is common in hemodynamically stable sustained ventricular tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)

Indirect immunofluorescent localization of prolactin to the cytoplasm of decidua and trophoblast cells in human placental membranes at term.

An indirect immunofluorescent technique was used to determine the localization of cytoplasmic human PRL (hPRL) in fresh and incubated human placental membranes at term. In both fresh and 8-h incubated samples of amnion, amniochorion decidua, or chorion decidua obtained from three placentas, we found specific reproducible localization of hPRL to the cytoplasm of decidua and trophoblast cells. The decidua cells appeared to be the most intensely fluorescent. No specific hPRL immunofluorescence was noted in the amniotic epithelium of fresh or incubated samples of amnion and amniochorion decidua. These data suggest that the trophoblast decidua cell layer is the site of PRL localization and possibly synthesis in placental membranes at term and may be the origin of amniotic fluid PRL in humans.

Procainamide-induced slowing of ventricular tachycardia with insights from analysis of resetting response patterns.

To investigate the mechanism of slowing of the rate of ventricular tachycardias (VTs) by procainamide, resetting response patterns were characterized in 24 VTs in 22 patients. All patients had coronary artery disease and inducible sustained VT during procainamide therapy. Only tachycardias with the same surface QRS morphology before and after procainamide were studied: all were slowed by procainamide. The mean cycle length was 292 +/- 61 ms before and 374 +/- 61 ms after procainamide (p less than 0.05). The mean effective refractory period, measured at the right ventricle, was 241 +/- 21 ms before and 261 +/- 24 ms after procainamide (p less than 0.05). During procainamide therapy, single and double extrastimuli were delivered during VT and resetting response patterns identified. Patterns were characterized as flat, increasing or flat plus increasing. Resetting was seen in 17 (71%) of these VTs and resetting response patterns were identified in 16 (94%) of these. The resetting response pattern was flat in 7, flat plus increasing in 5 and increasing in 4. The finding of some flat portion at the end of resetting response patterns in 12 VTs after procainamide indicates that the reentrant impulse conducts through fully recovered tissue within the circuit. It suggests that procainamide slowed these VTs by slowing conduction velocity in fully recovered tissue due to sodium channel blockade and not by prolongation of action potentials and refractory periods.

Caloric restriction as a mechanism mediating resistance to environmental disease.

It has been observed that susceptibility to many degenerative diseases increases concurrently with industrialization and rising living standards. Although epidemiologic studies suggest that specific environmental and dietary factors may be important, caloric intake alone (as reflected in body size) may account for much of the differential risk observed among diverse human populations. It has been suggested from animal studies that caloric intake may be the primary effector for many hormonal, metabolic, physiologic, and behavioral responses that coordinate reproductive strategy to apparent availability of food. When caloric intake is excessive, particularly at critical developmental stages, physiologic priorities are set for body growth and fecundity rather than for endurance and longevity. The converse occurs during periods of famine, thus increasing the probability that sufficient individuals survive to restore the population when conditions improve. Calorically restricted rodents have significantly longer reproductive and total life spans than their ad libitum-fed controls and exhibit a spectrum of biochemical and physiologic alterations that characterize their adaptation to reduced intake. These include reduced stature, hypercorticism in the absence of elevated adrenocorticotropic hormone levels, increased metabolic efficiency, decreased mitogenic response coupled with increased rates of apoptosis, reduced inflammatory response, induction of stress proteins and DNA repair enzymes, altered drug-metabolizing enzyme expression, and modified cell-mediated immune function. The overall profile of these changes is one of improved defense against environmental stress. This has been suggested as the mechanistic basis for the protective effects of low body weight on radiation and chemically induced cancers in experimental animals. It may also explain the significantly higher thresholds of acute toxicity observed when calorically restricted rodents are exposed to certain test compounds.

Cardiac cryolesions: factors affecting their size and a means of monitoring their formation.

Twenty-seven endocardial cryolesions were created in mongrel dogs and analyzed to determine the effects on cryolesion size of both the initial myocardial temperature (37 degrees C versus 12 degrees C) and the pressure within the nitrous oxide delivery line (tank pressure of more than 700 pounds per square inch [psi] versus tank pressure of less than 700 psi). In addition, local myocardial temperatures were monitored to determine their utility in the intraoperative determination of the extent of cryothermic cell death. Cryolesion volume was significantly affected by both the initial myocardial temperature (p less than 0.001) and the line pressure (p = 0.014). In a 37 degrees C myocardium, the mean lesion volume ranged from 0.501 +/- 0.183 cc at line pressures lower than 700 psi to 0.839 +/- 0.258 cc at line pressures greater than 700 psi. In a 12 degrees C myocardium, the mean volume was 1.151 +/- 0.436 cc at line pressures lower than 700 psi and 1.361 +/- 0.288 cc at line pressures higher than 700 psi. A myocardial temperature of 0 degrees C occurs at the edge of the area of cell death. When analyzing the range from -5 degrees to +5 degrees C, the probability of a point at or lower than 0 degrees C falling inside the cryolesion is 84.2%. Monitoring intramyocardial temperature will predict the border of a cryolesion.

Gel chromatographic properities of human prolactin released from decidua tissue in vitro.

Human decidua tissue releases immunoactive prolactin into the medium upon incubation in vitro. The prolactin secreted is indistinguishable from pituitary prolactin in its binding and displacement characteristics, using two different antisera. By gel chromatographic criteria more than 90% of the prolactin is monomeric.

Variability of human hepatic UDP-glucuronosyltransferase activity.

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases using photoaffinity labeling, immunoblotting and enzymatic assays. There was wide inter-individual variation in photoincorporation of the photoaffinity analogs, [32P]5-azido-UDP-glucuronic acid and [32P]5-azido-UDP-glucose and enzymatic glucuronidation of substrates specific to the two subfamilies of UDP-glucuronosyltransferases. However, the largest differences were between subjects with liver disease. Glucuronidation activities toward one substrate from each of the UDP-glucuronosyltransferases subfamilies, 1A and 2B, for control and liver disease, respectively, were 1.7-4.5 vs 0.4-4.7 nmol/mg x min for hyodeoxycholic acid (2B substrate) and 9.2-27.9 vs 8.1-75 nmol/mg x min for pchloro-m-xylenol (1A substrate). Microsomes from a patient with chronic tyrosinemia (HL32) photoincorporated [32P]5-azido-UDP-glucuronic acid at a level 1.5 times higher than the other samples, was intensely photolabeled by [32P]5-azido-UDP-glucose and had significantly higher enzymatic activity toward p-chloro-m-xylenol. Immunoblot analysis using anti-UDP-glucuronosyltransferase antibodies demonstrated wide inter-individual variations in UDP-glucuronosyltransferase protein with increased UDP-glucuronosyltransferase protein in HL32 microsomes, corresponding to one of the bands photolabeled by both probes. Detailed investigation of substrate specificity, using substrates representative of both the 1A (bilirubin, 4-nitrophenol) and 2B (androsterone, testosterone) families was carried out with HL32, HL38 (age and sex matched control) and HL18 (older control). Strikingly increased (5-8-fold) glucuronidation activity was seen in comparison to HL18 only with the phenolic substrates. The results indicate that one or more phenol-specific UDP-glucuronosyltransferase 1A isoforms are expressed at above normal levels in this tyrosinemic subject.

Lessons from animal models of atrial arrhythmias.

This article presents selected lessons from experimental studies of atrial fibrillation and atrial flutter that pertain to the mechanisms and predisposing factors for flutter and fibrillation and approaches to treatment by antiarrhythmic drugs. Experimental studies also provide lessons for the effects of ablation and surgical lesions on prevention or facilitation of atrial fibrillation and flutter.

Effects of caloric restriction on rodent drug and carcinogen metabolizing enzymes: implications for mutagenesis and cancer.

Caloric restriction in rodents results in increased longevity and a decreased rate of spontaneous and chemically induced neoplasia. The low rates of spontaneous neoplasia and other pathologies have made calorically restricted rodents attractive for use in chronic bioassays. However, caloric restriction also alters hepatic drug metabolizing enzyme (DME) expression and so may also alter the biotransformation rates of test chemicals. These alterations in DME expression may be divided into two types: (1) those that are the direct result of caloric restriction itself and are detectable from shortly after the restriction is initiated; (2) those which are the result of pathological conditions that are delayed by caloric restriction. These latter alterations do not usually become apparent until late in the life of the organism. In rats, the largest direct effect of caloric restriction on liver DMEs is an apparent de-differentiation of sex-specific enzyme expression. This includes a 40-70% decrease in cytochrome P450 2C11 (CYP2C11) expression in males and a 20-30% reduction of corticosterone sulfotransferase activity in females. Changes in DME activities that occur late in life in calorically restricted rats include a stimulation of CYP2E1-dependent 4-nitrophenol hydroxylase activity and a delay in the disappearance of male-specific enzyme activities in senescent males. It is probable that altered DME expression is associated with altered metabolic activation of chemical carcinogens. For example the relative expression of hepatic CYP2C11 in ad libitum-fed or calorically restricted rats of different ages is closely correlated with the amount of genetic damage in 2-acetylaminofluorene- or aflatoxin B1-pretreated hepatocytes isolated from rats of the same age and caloric intake. This suggests that altered hepatic drug and carcinogen metabolism in calorically restricted rats can influence the carcinogenicity of test chemicals.

Modulation of endocrine pathways by 4,4'-DDE in the deer mouse Peromyscus maniculatus.

4,4'-DDT and 4,4'-DDE are widespread environmental contaminants that cause eggshell thinning in birds, altered sex ratios in the American alligator, and changes in the anal-genital distance in rodents. These contaminants are known to cause some of their toxicity by altering steroid receptor-mediated mechanisms. However, chemical-specific alterations in the expression of hormone-metabolizing enzymes may also be a mechanism for endocrine disruption, by altering the half-life of hormones in critical tissues. Previously, we showed that 4,4'-DDE causes a dose-dependent increase in ethoxyresorufin-O-deethylase (EROD) activity, but not pentoxyresorufin-O-dealkylase (PROD) activity, in the deer mouse. In this study, we demonstrated that 4,4'-DDE elicited a corresponding increase in CYP1A protein expression but not CYP2B using Western blotting and immunoprecipitation. 4,4'-DDE-mediated changes in phase II conjugating enzymes; UDP-glucuronosyltransferase (UGT) and phenolsulfotransferase (ST), were also investigated for the first time. Prepubescent female deer mice were dosed with 4,4'-DDE by gavage on days 1 and 2, then euthanized on day 4. As anticipated, dose-dependent increases in hepatic EROD and MROD activities, but not PROD or BROD, were observed. UGT activity was monitored by incubating liver microsomes and 14C-UDP-GA with potential substrates and measuring incorporation of radioactivity into TLC-resolved glucuronides. Dose-dependent increases in conjugation were observed with p-nitrophenol (a general UGT substrate) but not testosterone. Interestingly, a biphasic dose-response curve was observed for ST activity, with a peak at the 3 mg/kg dose. Dose-dependent increases in CYP1A1 and UGT-specific immunoreactive proteins were observed, suggesting de novo synthesis as a consequence of 4,4'-DDE exposure. We also measured Phase I and II enzymes in deer mouse platelets. Preliminary results indicate that the 4,4'-DDE-induced changes in liver Phase I and II enzyme activity were similar, but not identical, to those found in platelets. These results indicate that environmentally-relevant levels of 4,4'-DDE modulate the activity and expression of CYP1A1 and phase II enzymes in the deer mouse and that certain changes may be measured non-lethally.

Metabolic differences and their impact on human disease Sulfotransferase and colorectal cancer.

Gene-environment interaction is an important aspect of human cancer risk. Genetic polymorphisms in acetylation and N-oxidation have previously been described regarding their impact on the heterocyclic amine-induced risk for colon cancer. Here, we report that another enzyme involved in the metabolism of food-borne carcinogens, sulfotransferase (ST1A3 measured by 2-naphthol activity), may function as a potential protective factor for colon cancer in humans. Initially characterized in human liver and colon (Chou et al., 1995), TS-PST activity can also be measured in platelets. A simple microtiter-based colorimetric technique was developed for use in this case-control study. African-Americans had a higher mean ST activity than Caucasians (2.32±0.24 versus 1.77±0.09 nmols/min per mg cytosolic protein, P=0.036). Furthermore, the slow ST phenotype (ST≤1.53) was more frequently associated with colon cancer than controls (57 versus 40%, P=0.026). These data suggest that the ST1A3 isoform may play a role in the differential risk for colorectal cancer.

Understanding reentry to out-of-home care for reunified infants.

Although many children placed in out-of-home care are reunified with their families of origin, a significant portion reenter care, reflecting continued family problems and weaknesses within the child welfare system. For infants, the stability of reunification is particularly crucial, given their developmental stage. This study reviewed the case records of 88 randomly selected infants who had been reunified with their families. Thirty-two percent of those infants reentered care within four to six years of their reunification. The identification of factors predictive of reentry into care has both policy and practice implications.

Analysis of molecular markers for metamorphic competency and their response to starvation or feeding in the mosquito, Aedes aegypti (Diptera: Culicidae).

The nutritional condition of fourth instar larvae of the yellow fever mosquito, Aedes aegypti, governs female longevity and egg production, both are key determinants of pathogen transmission. As well, nutrition provisions larval growth and development and attains its greatest pace in the last larval instar in preparation for metamorphosis to an adult. These developmental processes are regulated by a complex endocrine interplay of juvenile hormone, neuropeptides, and ecdysteroids that is nutrition sensitive. We previously determined that feeding for only 24h post-ecdysis was sufficient for fourth instar Ae. aegypti larvae to reach critical weight and accumulate sufficient nutritional stores to commit to metamorphosis. To understand the genetic basis of metamorphic commitment in Ae. aegypti, we profiled the expression of 16 genes known to be involved in the endocrine and nutritional regulation of insect metamorphosis in two ways. The first set is a developmental profile from the beginning of the fourth instar to early pupae, and the second set is for fourth instars starved or fed for up to 36 h. By comparing the two sets, we found that seven of the genes (AaegCYP302, AaegJHE43357, AaegBrCZ4, AaegCPF1-2, AaegCPR-7, AaegPpl, and AaegSlif) were expressed during metamorphic commitment in fourth instars and in fed but not starved larvae. Based on these results, the seven genes alone or in combination may serve as molecular indicators of nutritional and metamorphic status of fourth instar Ae. aegypti larvae and possibly other mosquito species in field and laboratory studies to gauge sub-lethal effects of novel and traditional cultural or chemical controls.

The effect of chronic oral and acute intravenous amiodarone administration on ventricular defibrillation threshold using implanted electrodes in dogs.

The effect of acute intravenous administration and chronic oral loading of amiodarone on defibrillation threshold was evaluated in normal anesthetized dogs using implanted superior vena caval spring and left ventricular patch electrodes. The effect of oral loading with amiodarone was evaluated by comparing three groups of six dogs each that received either no drug, 200 mg/day for 9 days, or 400 mg/day for 9 days. Defibrillation threshold was evaluated by administering a fixed sequence of shocks with increasing energies until defibrillation was successful. Defibrillation was determined 13 times in each animal. The mean defibrillation threshold (plus or minus standard error of the mean) was 7.5 +/- 0.3 J in the control group, 15.4 +/- 0.6 J in the group receiving amiodarone 200 mg/day, and 17.9 +/- 0.8 J for the group receiving 400 mg/day. These values are significantly different using analysis of variance and Tukey's test. The acute effect of intravenous amiodarone, 5 mg/Kg was evaluated in five dogs using each dog as its own control. The mean defibrillation threshold during control period was 10.8 +/- 0.4 J, and during the first two hours after amiodarone administration was 10.8 +/- 0.4 J. There was no significant difference. Thus, in this study oral administration of a loading dose of amiodarone comparable to that used in patients produced a dose dependent increase in defibrillation threshold, whereas no change in defibrillation threshold was observed acutely after intravenous administration.

Spontaneous termination of reentry after one cycle or short nonsustained runs. Role of oscillations and excess dispersion of refractoriness.

This study describes factors that contribute to spontaneous termination of reentry lasting one to 10 cycles after induction by a single premature stimulus. Reentry was studied in vitro in rings of canine atrial tissue from around the tricuspid valve orifice. Activation was recorded from a circular array of 10 extracellular bipolar electrodes equally spaced around the ring. In some experiments, transmembrane or monophasic action potential recordings were made near critical sites. Termination of reentry within one cycle after induction was recorded 110 times in 11 of 35 experiments. Important factors contributing to termination were 1) an obligatory reversal of the activation sequence that resulted in a long coupling interval in the critical region beyond the site of unidirectional block after the premature stimulus and 2) much longer refractory periods limited to this critical region, which facilitated unidirectional block but contributed to termination when this region was first activated with a short coupling interval at the end of the first reentrant cycle. Termination of nonsustained reentry lasting longer than one cycle resulted from oscillations of conduction and refractoriness initiated by the abrupt shortening of cycle length after initiation of reentry. Oscillations of conduction resulted from interval-dependent conduction of reentrant impulses that encountered partially refractory tissue. For reentry to become sustained, the oscillations after induction of reentry must dampen. Thus, damped cycle length oscillations after induction may identify clinical tachycardias caused by reentry with a partially excitable gap.