The haematopoietic stem cell and its clonal progeny: mechanisms regulating the hierarchy of primitive haematopoietic cells. Transplantation and marking studies have provided an accurate description of various aspects of developmental and proliferative behaviour of totipotent haematopoietic stem cells. In particular, the remarkable ability of different clones to contribute to all lineages in a continuous, stable and long term manner is a hallmark of stem cell behaviour. Taken together, in vivo reconstitution experiments also provide the basis for a model of stem cell regulation that incorporates stochastic and non-stochastic components. More recent approaches provide optimism that many descriptive aspects of stem cell biology will soon be supported by elucidation of molecular mechanisms.


The bcr-abl gene in chronic myelogenous leukaemia. The observation made over 30 years ago that the Philadelphia chromosome is present in nearly all patients with CML led to the identification of a novel fusion gene bcr-abl. In the past few years, the biochemical and biological properties of bcr-abl have been extensively explored. Bcr sequences appear to activate c-abl for transformation by binding to the SH2 domain of c-abl in an intramolecular interaction, presumably interfering with the adjacent SH3 regulatory domain. Upon introduction into bone marrow cells, bcr-abl can cause acute or chronic leukaemias in mice and can stimulate the growth of many cell types, including multipotent stem cells, in vitro. Although their growth is stimulated, these cells are not fully malignant blastic leukaemias. The molecular events that occur during the progression to blast crisis of CML remain largely undefined, but existing animal models and in vitro culture systems will be useful for identifying or testing candidate genes. The study of tyrosine kinase oncogenes in general will probably lead to the identification of relevant bcr-abl substrates. The elucidation of these molecules as well as more downstream events in the bcr-abl signalling pathway offers the hope for novel therapeutic interventions to control Philadelphia chromosome leukaemias.


Avian erythroblastosis: a model system to study oncogene co-operation in leukemia. Avian erythroblastosis virus, AEV-ES4, provides an ideal in vitro system to study oncogene co-operation in the development of erythroid leukaemias in chickens. Two oncogenes that use distinct signal transduction pathways have been identified; these act to produce a highly malignant phenotype and are an oncogenic version of a plasma membrane growth factor receptor that regulates haematopoietic progenitor self renewal and a mutated version of a transcription factor that suppresses specific gene expression. These two diverse types of oncogenes act together to generate a more malignant phenotype than would be expected merely from a summation of the individual oncogene effects. The aspects of normal growth control and the regulation of differentiation, which are altered in this system to generate the leukaemic phenotype, include changes in the balance between proliferation versus maturation, altered expression of differentiation genes and changes in growth factor responsiveness. These are exactly those features that are altered in human leukaemias. Thus, the avian leukaemia virus model has taught us important lessons that will help us to understand the basic molecular mechanisms that give rise to human leukaemia.


Role of rel family genes in normal and malignant lymphoid cell growth. The rel family of genes encodes transcription factors, such as the v-rel oncoprotein and cellular transcription complexes (eg NF-kappa B), consisting of c-rel and related polypeptides. The expression of these genes is correlated most closely with lymphoid cell differentiation and growth stimulation in a variety of cell types. Similarly, rel family proteins control a number of genes involved in lymphoid cell growth and differentiation. Thus, it is not surprising that mutations in rel genes have been implicated directly and circumstantially in a number of lymphoid malignancies, as have a number of other cellular and viral transcription factors (see also Cleary M, Hayman M and Beug H, this issue and reviewed in Rabbitts, 1991). In addition, rel proteins are likely to be involved in T cell diseases caused by the human retroviruses HTLV-I and HIV-1. Therefore, rel proteins could serve as targets for anti-viral or anti-cancer therapies.


Transcription factors in human leukaemias. At the present time, two general mechanisms account for deregulation and subsequent oncogenic conversion of transcriptional proteins in human leukaemias. One involves quantitative alterations in expression, suggesting that activity of the involved factors is primarily controlled by their accessibility within the cell. Neoplastic transformation may result from excessive expression or, conversely, complete loss of functional products (eg tumour suppressor proteins not described here). The second mechanism involves mutation by protein fusion (or truncation) and illustrates the modular composition of transcriptional proteins. The loss or inappropriate combination of specific modules creates chimaeric proteins with presumably altered transcriptional properties that may contribute to the neoplastic phenotype. Both mechanisms underscore the importance of cognate interactions, particularly heterodimerization between various transcriptional proteins with other members of the transcription complex. Future efforts will continue to focus on the interactions of oncogenic transcription factors with other cellular proteins and their biologically relevant target genes.


Echocardiographic and ultrasonographic evaluation of cardiac and vascular hypertrophy in patients with essential hypertension. High-resolution ultrasonography is a noninvasive technique that allows to investigate the cardiovascular system, in particular the wall thickness and the lumen diameter of the arteries with accuracy and reproducibility. We measured the intima-media thickness of the common carotid artery (CCA) and of its bifurcation (BIF) in 40 patients with essential hypertension, 20 of them with left ventricular hypertrophy (LVH; age 42 +/- 10 years) and 20 without LVH (age 44 +/- 12 years); no other major cardiovascular risk factor was present in all the patients. Both carotid axes have been scanned from different views (anterior, lateral, posterior) on a transversal and longitudinal section using a high-resolution steerable linear array of 5.0 MHz. Carotid diameter and thickness were measured in the longitudinal section. CCA parameters were assessed 20 mm caudally to the flow divider. In patients with LVH, blood pressure (172 +/- 21/108 +/- 9 mm Hg) and left ventricular mass index (156 +/- 38 g/m2) were significantly (p < 0.01) higher than in patients without LVH (blood pressure: 158 +/- 11/99 +/- 12 mm Hg; left ventricular mass index: 98 +/- 10 g/m2), while there was no difference in serum glycemia, triglycerides, total and fractioned cholesterol levels. The intima-media thickness of both the CCA and BIF was significantly higher in the hypertensives with LVH (CCA: 0.85 +/- 0.02 vs. 0.65 +/- 0.02 mm; BIF: 0.93 +/- 0.04 vs. 0.70 +/- 0.03 mm, p < 0.01). There was a statistically significant correlation between the carotid wall thickness and the left ventricular mass index.(ABSTRACT TRUNCATED AT 250 WORDS)


Loss of nocturnal increase in plasma concentration of atrial natriuretic peptide in hypertensive chronic renal failure. Diurnal change of plasma atrial natriuretic peptide (ANP) concentration was investigated in 12 patients with hypertension due to chronic renal failure (CRF) and in 12 patients with essential hypertension (EH) of comparable degree. Blood pressure (BP) monitoring was performed at 15-min intervals, while peripheral blood samples were obtained at 4-hour intervals starting from 8.00 h. The mean 24-hour plasma levels (+/- SEM) of ANP were 24.3 +/- 1.8 pmol/l in EH and 23.4 +/- 1.2 pmol/l in CRF. In EH, plasma ANP concentration was highest at 4.00 h (33.5 +/- 0.8 pmol/l) and lowest at 16.00 h (15.5 +/- 0.6 pmol/l). In CRF, no significant circadian change was present (22.2 +/- 3.1 and 20.4 +/- 3.6 pmol/l, respectively), and the nocturnal fall in BP was lost. Our data demonstrate that in CRF the loss and possible reversal of the nocturnal decline in BP is associated with the disappearance of any significant circadian variation in the circulating concentrations of ANP. These findings suggest a role for ANP in the alteration of BP variability of CRF, possibly mediated by autonomic dysfunction, and are further evidence for the existence of a relation between the circadian rhythms of ANP and BP.


Gallopamil in chronic stable angina: antianginal effect and mechanism of action. A randomized, placebo-controlled, double-blind, cross-over trial. A double-blind, placebo-controlled, cross-over trial of oral gallopamil was performed in 10 patients with stable angina. Gallopamil significantly increased mean exercise time and 1-mm ST time. The rate-pressure product was increased at 1-mm ST time, but unmodified at the highest comparable work load and at peak exercise. The ST segment depression was significantly reduced both at the highest comparable work load and at peak exercise. Gallopamil proves safe and effective; the mechanism of its anti-ischemic effect seems to be due both to an increase in myocardial oxygen supply and to a reduction in myocardial oxygen demand.


Role of adrenal androgens in the development of arteriosclerosis as judged by pulse wave velocity and calcification of the aorta. To evaluate the role of adrenal androgens in the development of arteriosclerosis, serum dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), aortic calcification and pulse wave velocity (PWV) were measured in 69 males and 119 females without overt cardiovascular disease. The steroids decreased with age in both sexes, and the reduction was significantly steeper in younger (< or = 40 years) than in older (> 40 years) subjects only in females. When adjusted for age, the steroids were significantly lower in subjects with aortic calcification than in those without it, and the PWV was significantly slower in the latter. Adrenal androgens appear to retard the development and/or progression of arteriosclerosis.


Fibrinogen values in patients with and without restenosis following percutaneous transluminal coronary angiography. Fibrinogen has turned out to be an independent risk factor for coronary heart disease (CHD). It is not known whether or not this parameter could be a prognostic factor for restenosis following percutaneous transluminal coronary angioplasty (PTCA), which represents the main problem limiting the long-term efficacy of this procedure. Therefore, we studied fibrinogen concentrations in a series of 50 males (mean age: 55, range: 38-70 years) with CHD and successful PTCA. Follow-up coronary angiography was performed 12 months following PTCA. Twenty-two patients had restenosis, and 28 patients were without restenosis. Both groups did not differ significantly in medical history (smoking habits, hypertension, positive family history for cardiovascular diseases), in routine lipid profile (total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apolipoproteins A1 and B). Fibrinogen values were 405 +/- 128 mg/dl (range: 202-725) in patients with restenosis and 352 +/- 94 mg/dl (range: 187-568) in patients without restenosis (not significant). Elevated fibrinogen levels of more than 400 mg/dl were found in 8 patients in each group. Although fibrinogen is a proven marker for CHD in men, fibrinogen is not a risk factor for restenosis following PTCA.


Hemodynamic effects of ibopamine on the pulmonary circulation and right ventricular performance in patients with chronic obstructive pulmonary disease. We have studied the effects of ibopamine, an oral dopamine derivative, on pulmonary hemodynamics and blood gas values when given as a single oral dose (100 mg) to patients with severe hypoxic chronic obstructive pulmonary disease. A small but significant increase in mean pulmonary arterial pressure from 29 +/- 8 to 32 +/- 7 mm Hg (p < 0.01) was noted 30 min after ibopamine was given associated with a small increase in cardiac index. No other consistent hemodynamic changes were observed, and no alteration in blood gas values or oxygen saturation occurred. These results do not indicate a role for ibopamine in the treatment of patients with hypoxic chronic obstructive pulmonary disease and pulmonary hypertension, but, in addition, do not indicate any major deleterious effects of ibopamine on pulmonary hemodynamics or blood gas values in such patients.


Hemodynamic effects and pharmacokinetics of long-term therapy with ibopamine in patients with chronic heart failure. To evaluate the attenuation of the effectiveness of long-term ibopamine therapy, ibopamine was administered in single doses of 100 and 200 mg to 10 patients with chronic heart disease. The hemodynamic studies using Swan-Ganz catheter and pharmacokinetic studies were carried out. Ibopamine was found to increase cardiac output and stroke index and to decrease systemic vascular resistance in this acute study. Six patients underwent long-term therapy with the drug and were evaluated for the development of tolerance. Three out of 5 patients experienced an improvement in NYHA functional class after 12-23 weeks of treatment. There was no attenuation in the effects of ibopamine on hemodynamics, pharmacokinetic parameters remained almost unchanged, and tolerance was not observed. These results suggest that ibopamine is useful as an orally administered anti-heart failure drug.


Difference in the residual left ventricular pump function between anterior and inferior myocardial infarctions. To evaluate the left ventricular regional ejection fraction (EF) of noninfarcted area in relation to the left ventricular end-diastolic volume (EDV) in patients with recent myocardial infarction (MI), 75 patients with Q-wave MI (anterior: 51 patients; inferior; 24 patients) were studied. The regional EF of noninfarcted area was obtained by radionuclide angiocardiography 4 weeks after the onset of MI and was used to estimate the left ventricular regional function of the noninfarcted area. Peak creatine kinase and QRS scores were not significantly different between anterior and inferior MI in each left ventricular EDV (EDV < or = 100, 101-139 and > or = 140 ml). Global EF and regional EF of noninfarcted area in anterior MI with left ventricular EDV > or = 140 ml was significantly lower than in those with EDV < or = 139 ml (p < 0.01), whereas there were no significant differences in global EF and regional EF of noninfarcted area in the three groups of left ventricular EDV in inferior MI. Thus, the effect of left ventricular EDV on regional EF of noninfarcted area and on the total cardiac performance was more important in anterior than in inferior MI, because a similar degree of left ventricular dilatation resulted in more severe derangements after anterior MI.


Risk stratification after acute myocardial infarction by means of echocardiographic wall motion scoring and Killip classification. In order to perform risk stratification, 195 consecutive, unselected patients with acute myocardial infarction (AMI) underwent independent echocardiographic and clinical evaluation of their left ventricular function by means of the wall motion index (WMI) and Killip classification 5 days after AMI. The patients were prospectively allocated to a low, medium or high risk class depending on WMI alone, and the 1-year mortality in these classes was 2, 34 and 37%, respectively (p < 0.0001). The 1-year mortality of the patients in Killip class I, II, or III and IV was 6, 26 and 48%, respectively (p < 0.00001). The number of patients allocated to the low risk group by means of WMI was 87, and the number of patients in Killip class I was 86. Since these groups were not identical, a total of 103 patients, i.e. 53% of the study population, could be identified as low risk patients regarding 1-year mortality 5 days after AMI, when WMI and Killip classification were used in combination. We conclude that the combination of echocardiographic and clinical evaluation of left ventricular function after AMI provides a strong and yet very simple procedure to identify low risk patients, which could be easily implemented in the routine work of coronary care units.


Electrocardiographic Q-waves inconstancy during thrombolysis in acute anterior wall myocardial infarction. It is the purpose of this paper to describe the electrocardiographic inconstancy of Q-waves during administration of thrombolytic therapy. This was documented in four patients given streptokinase early in the course of anterior wall myocardial infarction. Understanding the pathogenesis of sequential dynamic variations of Q-waves in this setting may offer important insights into coronary physiology and management of acute coronary events. We discuss the possible explanations for such changes with respect to tissue viability, dynamic vascular changes and electrophysiological properties of the reperfused infarcted myocardium.


Progression of systolic dysfunction correlated with the ultrasonographically assessed myocardial tissue damage in patients with dilated cardiomyopathy. An echocardiographic follow-up study was performed in 59 patients with dilated cardiomyopathy to characterize the relationship between left ventricular systolic dysfunction and the myocardial tissue character assessed by echocardiographic myocardial gray level distributions. All the selected patients were divided into two groups; those with (group A) and those without (group B) progressive systolic dysfunction. In group A, the ejection fraction decreased from 47 +/- 5 to 23 +/- 4% (p < 0.05) during the follow-up period. The histogram of the myocardial gray level distribution became wider, less peaked and more right-sided in those patients. The relative echo intensity of the myocardium increased from 1.29 +/- 0.19 to 1.60 +/- 0.18 (p < 0.01). In group B, however, neither the shape of the gray level distribution nor ejection fraction changed during the same follow-up period. The relative echo intensity did also not change. Our present results suggested that some of the patients with dilated cardiomyopathy showed progressive systolic dysfunction with extensive myocardial tissue damage.


Effects of a 1-year exercise training program on myocardial ischemia in patients after myocardial infarction. To determine the effects of exercise training on exercise-induced ischemia in patients following myocardial infarction, the experience of 13 patients with exercise-induced ST depression, who were moderate-to-high intensity trained for 1 year, has evaluated. After training, the maximum ST depression was significantly less (1.9 +/- 0.8 vs. 1.1 +/- 0.8 mm; p < 0.01), despite an increased maximal rate-pressure product (RPP; heart rate x blood pressure/100; 241.3 +/- 44 vs. 262.0 +/- 58; p < 0.01). For the onset of 0.1 mV of ST depression, we found a significant increase in RPP from 204.1 +/- 34.7 to 234.1 +/- 49.4 (p < 0.01) and also in heart rate (117.1 +/- 15.1 vs. 125.1 +/- 21.7 b.p.m.; p < 0.05), blood pressure (167.6 +/- 18 vs. 180.3 +/- 18 mm Hg; p < 0.01) and workload (93.8 +/- 17.4 vs. 121.1 +/- 23.2 W; p < 0.01). The relationship between ST depression and RPP (RPP/STmax) was favorably modified after training. The ratio RPP/STmax improved significantly from 143.6 +/- 49.4 to 209.1 +/- 69.5 (p < 0.0001). These findings support the hypothesis that a 1-year moderate-to-high training program in some patients following myocardial infarction can elicit adaptations that may well be attributed, at least in part, to an improvement in coronary blood flow.


Menopause-related changes in left ventricular function in healthy women. Using technetium scans, this study was aimed at examining possible changes in left ventricular function related to the natural process of cessation of ovarian estrogen production. Fourteen healthy postmenopausal women, divided into two groups according to the time-lapse from menopause (A > 3 years; B < 5 years), underwent a technetium heart scan. The two groups did not differ in heart rate, blood pressure, double product, systemic vascular resistance and cardiac index. The mean end-systolic volume in group A was 14.9 and 25.7 ml/m2 in group B (p = 0.003). The mean pressure/volume ratio was significantly higher in group A than in group B (8.6 vs. 4.7 mm Hg/ml/m2, p = 0.02). Peak ejection rate and peak filling rate were also significantly greater in group A compared to group B (3.3 vs. 2.8 end-diastolic volumes/s, p = 0.02; 2.8 vs. 2.1 end-diastolic volumes/s, p = 0.001, respectively). Our findings suggest that women at an early phase of menopause have a higher degree of myocardial contractility than women of a similar age whose menopause is of longer duration.


Physical rehabilitation in coronary patients who have suffered from episodes of cardiac failure. The effects of bicycle training for 5 weeks were evaluated in 12 patients after myocardial infarction with left ventricular dysfunction (left ventricular ejection fraction < 40%) and at least one episode of cardiac failure in the past. The patients were divided into two groups of six according to the Weber classification: Group B (VO2/kg/min: 16-20) and Group C (VO2/kg/min: 10-15). Cardiopulmonary and hemodynamic parameters were evaluated during a maximal exercise test and a simultaneous catheterization of the right side of the heart before and after the training. An increase in the capacity for work was recorded in Group B (p < 0.02), while Group C remained unchanged. A statistically significant increase in minute ventilation (p < 0.05) and VO2/kg/min (p < 0.0006) was recorded in Group B. Group C showed an increase in the mean pulmonary arterial pressure (p < 0.03). All of the other parameters remained unchanged after training. We conclude that physical rehabilitation improves the tolerance to exercise in patients with a mildly depressed cardiac function (Group B) but not in patients with a very depressed cardiac function (Group C).


Endomyocardial biopsies in chronic chagasic cardiomyopathy. Immunohistochemical and ultrastructural findings. Mononuclear cellular infiltrates and extensive fibrosis, with or without apical ventricular aneurysms, are the usual morphological findings in chronic chagasic cardiomyopathy. These lesions are thought to be mediated by immune phenomena rather than by continuing parasitic invasion of the heart. In the present report, we correlated clinical, immunohistochemical and ultrastructural findings in 30 endomyocardial biopsies from patients with chronic chagasic cardiomyopathy. In 12 of these biopsies, immunocytochemical techniques were used to identify and count leukocytes (common leukocyte antigen, CLA), T lymphocytes (UCHL-1 antibody) and B lymphocytes (L-26 antibody). The biopsy specimens showed variable degrees of myocardial hypertrophy and mononuclear infiltrates. No tissue forms of trypanosomes were found. The endocardium averaged 24 +/- 12.6 microns (mean +/- SD) in thickness. The mean myocyte diameter was 20 +/- 7.33 microns. The hearts were severely fibrotic containing a mean of 24.1 +/- 12.8% of fibrous tissue (range 8.2-49%), mast cells were scarce. Mononuclear cell infiltrates were found in 25 of the 30 biopsies. In 12 biopsies, immunohistochemical studies showed that the majority of the lymphocytes were T lymphocytes and associated with necrotic or degenerating myocytes. 10 of the 12 biopsy samples showed 5 or more CLA-positive mononuclear cells/high power field. In these 10 patients, T and B lymphocytes represented 32 and 13% of the total mononuclear infiltrating cells, respectively. The remaining cells were monocytes and macrophages.


Physiological features of pericardial constriction in the absence of pericardial disease. To sustain a clinical diagnosis of constriction it is classically held that the fibrous pericardium must be thickened and adherent to the surface of the heart. A case is presented in which leukaemic infiltration of the fat overlying the myocardium resulted in the physiological features of constriction, although all layers of the pericardium itself were normal. Constriction is thus a physiological diagnosis; it may develop in the absence of the classical anatomical findings.


Variability in the transtricuspid gradient by continuous wave Doppler echocardiography. In order to examine the day-to-day variability in the tricuspid regurgitant velocity jet and to determine the degree of physiological changes under exercise and volume loading, repeated echo cardiographic Doppler measurements in 1 single subject were performed.


Effects of intravenous amiodarone on electrical dispersion in normal and ischaemic tissues and on arrhythmia inducibility: monophasic action potential studies. The aim was to study the effects of intravenous amiodarone on: (1) repolarisation and other monophasic action potential characteristics of normal and ischaemic tissues, and (2) vulnerability to pacing induced repetitive ventricular firing following acute left anterior descending coronary artery ligation. Epicardial monophasic action potentials were continuously and simultaneously recorded from ischaemic and non-ischaemic regions of the hearts of 24 pentobarbitone anaesthetised dogs. Monophasic action potential duration, rise time, and amplitude, as well as the degree of dispersion of repolarisation between normal and ischaemic areas and vulnerability to electrical induction of repetitive ventricular firing, were determined using hand held silver-silver chloride "contact pressure" electrodes, before and 15 min after coronary ligation; and 15 min after exposure to low (10 mg.kg-1) doses of amiodarone. Similar determinations were made in drug free controls. Under control conditions, monophasic action potentials from both the (potentially) ischaemic and non-ischaemic regions were comparable, dispersion of repolarisation was minimal, and repetitive ventricular firing could not be induced. Coronary ligation significantly decreased monophasic action potential duration and increased rise time in the ischaemic, but not in the non-ischaemic, regions. Dispersion of repolarisation increased markedly. Repetitive ventricular firing could be induced in all dogs. "Low dose" amiodarone caused a much greater prolongation of repolarisation duration at ischaemic than at non-ischaemic sites. Dispersion of repolarisation decreased virtually to control levels and repetitive firing could no longer be induced in six of seven dogs. "High dose" amiodarone increased ischaemic region repolarisation duration and rise time relative to the non-ischaemic region to an even greater extent than the "low" dose, with the result that dispersion of repolarisation increased rather than decreased and repetitive ventricular firing again became inducible. The data indicate that: (1) intravenous amiodarone prolongs repolarisation of ischaemic tissues to a greater extent than that of normal tissues; (2) "low" dose amiodarone resulted in an overall decrease in dispersion of repolarisation, with little or no change in dispersion of rise time; (3) the "high" dose increased dispersion of both repolarisation and rise time. These changes were paralleled by changes in vulnerability to induction of repetitive ventricular firing by programmed ventricular pacing, the "low" dose being associated with decreases, and the "high" dose with further increases, in vulnerability, respectively.


Left ventricular hypertrophy in a canine model of reversible pressure overload. The goal of therapy for left ventricular pressure overload should include regression of the associated left ventricular hypertrophy, but this process is incompletely understood. The aim of the study was to characterise the extent and time course of the progression and regression of pressure overload left ventricular hypertrophy in a canine hypertrophy model. Six puppies were studied longitudinally with haemodynamic and echocardiographic measurements for 10 months. The study animals underwent ascending aortic banding at nine weeks of age which produced an initial gradient of 30 mm Hg. Subsequent growth led to an increase in gradient and the development of left ventricular hypertrophy. Then thoracotomy was again performed to remove the band. One month later, balloon aortoplasty was performed to remove the residual gradient. The animals were then observed for six months. Growth increased the gradient to 105(SEM 10) mm Hg three months after banding. The left ventricular weight to body weight ratio (g.kg-1), an index of hypertrophy, was 7.2(0.5) after three months of pressure overload. Subsequently the band was surgically removed, reducing the gradient to an average of 58(10) mm Hg. Balloon dilatation of the residual aortic stricture reduced the gradient further to 6(5) mm Hg. Over the ensuing six months, echocardiographic determination of left ventricular mass showed the regression in left ventricular hypertrophy. After six months, left ventricular weight to body weight ratio in the previously banded animals was significantly reduced from 7.2(0.5) to 5.3(0.2) (p less than 0.05). The model produced over 100% left ventricular hypertrophy, most of which regressed following removal of the pressure overload.


Effects of chronic oral administration of nifedipine and diltiazem on occlusive thrombus of small coronary arteries in (NZW x BXSB)F1 male mice. (NZWxBXSB)F1 male mice were used as a model to study the effects of chronic administration of nifedipine and diltiazem on small coronary artery disease and mortality. 16 week old (NZWxBXSB)F1 male mice were given either nifedipine (n = 29) at 20 mg.kg-1.d-1 orally or diltiazem (n = 28) at 100 mg.kg-1.d-1 orally in two divided doses until they were 24 weeks old (ie, for 60 d). Age matched control mice were given an equivalent volume of vehicle (n = 30). Mice that died received immediate necropsy. After 60 d, all surviving mice were killed and hearts and kidneys were examined histologically. The survival rate at 24 weeks was significantly higher in mice given nifedipine than in the controls (71% and 47%, respectively, p less than 0.05). The number of mice with myocardial necrosis, scar formation, or both, the percentage of areas of the ventricular wall with myocardial necrosis and scar formation, and the number of mice with small intramyocardial arteries showing greater than or equal to 75% stenosis were all significantly lower in the mice given nifedipine than in the controls. In the mice given diltiazem, there were no such differences from the controls. There was no evidence of significant stenosis or thrombosis in the extramyocardial coronary arteries in any of the mice. Systolic blood pressure and the rate-pressure product at 24 weeks of age, as well as the histological score for glomerulonephritis changes, were significantly lower in mice given nifedipine or diltiazem than in the controls. Despite an equivalent improvement of systolic blood pressure, the double product, and renal disease in nifedipine treated and diltiazem treated mice, only nifedipine prevented small coronary artery disease and increased the survival of (NZWxBXSB)F1 male mice, suggesting that nifedipine prevents occlusive thrombi of small coronary arteries better than diltiazem.


Histochemical demonstration of endothelial superoxide and hydrogen peroxide generation in ischaemic and reoxygenated rat tissues. The aims were to test and evaluate two novel and independent histochemical methods for detecting the initial postischaemic burst of superoxide and hydrogen peroxide in buffer perfused rat tissues during reflow after 60 min warm ischaemia. The first is a high manganese/diaminobenzidine technique, in which superoxide oxidises Mn2+ to Mn3+, which in turn oxidises diaminobenzidine to form amber coloured polymers, observable by light microscopy. The second is a high iron/diaminobenzidine technique, in which hydrogen peroxide oxidises diethylenetriaminepenta-acetate chelated Fe2+ to form intermediate species, which in turn oxidise diaminobenzidine similarly to Mn3+. Various isolated organs of the rat were rendered ischaemic for 60 min, and reperfused with oxygen or air equilibrated buffers containing diaminobenzidine and either Mn2+ or Fe2+. Tissues were fixed by perfusion with Trump's solution and processed for light microscopy. Both manganese and iron methods consistently showed the appearance of reaction product on the luminal surfaces of arterial, capillary, and venular endothelial cells in lung, heart, and intestine of the rat during the first 2 to 3 min of reoxygenation after ischaemia. The histochemical reactions were nearly absent in non-manganese-treated and non-iron-treated controls. Superoxide dismutase strongly inhibited Mn2+/diaminobenzidine reaction product formation and catalase strongly inhibited Fe2+/diaminobenzidine reaction product formation, when tested in specially perfused lung preparations in which these specific antioxidant enzymes were concentrated. These histochemical techniques provide direct, visual evidence that a burst of reactive oxygen species is generated in postischaemic rat tissues. The Mn2+/diaminobenzidine and Fe2+/diaminobenzidine techniques permit investigation of the endothelium derived reactive oxygen by simple laboratory procedures available to almost any investigator at low marginal cost. The endothelial oxidants so revealed may be of pathophysiological significance in a variety of cardiovascular disorders.


Decreased collagen mRNA and regression of cardiac fibrosis in the ventricular myocardium of the tight skin mouse following thyroid hormone treatment. The aim was to study the effect of thyroid hormone on collagen gene expression in the myocardium of the tight skin mouse (TSK), a genetic model of myocardial fibrosis. Heterozygous male (TSK/+) (n = 20) and normal (+/+) homozygous mice (n = 20), 1.5-2 months old of the C57 BL/6 strain were studied. Ventricular hypertrophy following thyroid hormone treatment (L-thyroxine, 10 micrograms.100 g-1 body weight daily intraperitoneally) was examined by measurement of the heart weight/body weight ratios and histological changes. Expression of fibrillar collagen types I and III in the ventricular myocardium was examined by measurement of the abundance of their respective mRNAs. Collagen synthesis was examined by measurement of hydroxyproline. Deposition of collagen types was evaluated by immunofluorescence staining. Expression of non-collagenous proteins, sarcomeric and cytoskeletal actin, was measured at the mRNA level. After 12 days of treatment ventricular hypertrophy was induced in the heart of the TSK mice. The results of northern hybridisation analyses showed that in the hearts of TSK mice 24 h after thyroxine treatment the abundance of mRNA for pro alpha 2 (I) collagen was decreased by 32% (p less than 0.05), pro alpha 1 (III) collagen by 47% (p less than 0.002), cytoskeletal actin by 50% (p less than 0.005), and sarcomeric actin mRNA by 34% (p less than 0.01) compared to the untreated TSK mice. The abundance of mRNA for pro alpha 2 (I) and pro alpha 1 (III) collagens in the thyroxine treated TSK mice were nearly comparable to that in normal homozygous mice. In TSK mice which were treated for 12 d, collagen content of the ventricular myocardium, as determined by hydroxyproline measurements, was decreased by 22.5% (p less than 0.01) compared to that in the heart of normal homozygous mice. Effects of thyroid hormone on ventricular gene expression in TSK mice result in a diminished collagen mRNA and collagen content and the disappearance of cardiac fibrosis. Thyroid hormone may selectively prevent the induction of cardiac fibrosis and play an important role in regression of cardiac fibrosis via endocrine pathways.


Exogenous nitric oxide inhibits in vivo platelet adhesion following balloon angioplasty. The aim was to investigate the effects of an exogenous source of nitric oxide on in vivo platelet adhesion at the site of endothelial denudation after balloon angioplasty. The study group consisted of 12 anaesthetised Large White pigs. Pigs were randomised to receive SIN-1 (3-morpholino-sydnonimine), an exogenous donor of nitric oxide, or placebo before and during balloon induced vessel wall injury. Platelet deposition was quantified using the injection of autologous 111indium labelled platelets. Platelet function was also monitored by the measurement of bleeding time and ex vivo whole blood aggregometry. Superficial vessel wall injury was confirmed histologically and platelet monolayer formation was demonstrated by scanning electron microscopy. Platelet deposition at the site of endothelial denudation was markedly reduced following SIN-1 administration compared to placebo: 1.266(SEM 0.063) v 1.732(0.060) log platelets x 10(5).cm-2, p = 0.001. SIN-1 raised platelet cyclic GMP concentration, from 4.47(2.48) to 6.14(2.44) pg.platelet-1 (p less than 0.01) and prolonged the bleeding time, from 135(5) to 202(6) s (p = 0.001), but had non-significant effects on ex vivo whole blood aggregometry. Exogenous nitric oxide, through the activation of platelet soluble guanylate cyclase, inhibits platelet adhesion in vivo following balloon angioplasty.


Fluorescence spectroscopy for identification of atherosclerotic tissue. Vessel perforation and limited steerability of the laser light are the major limitations of laser angioplasty. To improve steerability fluorescence spectroscopy has been proposed for identification of atherosclerotic plaques. The aim was to investigate this. Fluorescence spectroscopy with three different excitation wavelengths (325 nm, 380 nm, 450 nm) was tested in an emission range of 400 nm to 600 nm. Intensity ratios at 480/420 nm were determined in different types of blood vessels. Necropsy material from 40 patients (punch biopsies of 4 mm diameter from the coronary and carotid artery as well as from the ascending and descending aorta) was studied spectroscopically. Histological alterations of the vessel wall were assessed by a semiquantitative score (0 to 10 points): (a) normal tissue, 0 to 2 points (mean = 0.25; n = 38); (b) mild atherosclerotic lesions, 3 to 5 points (mean = 3.35; n = 39); (c) severe atherosclerotic lesions, greater than or equal to 6 points (mean = 6.75; n = 43). Best spectroscopic results were obtained with an excitation wavelength of 325 nm. In samples with severe atherosclerotic lesions the fluorescence spectra showed a significant reduction of the emitted wavelength intensities when compared to normal tissue. There was a clear separation of the fluorescence spectra between normal and mild as well as between normal and severe atherosclerotic lesions; normal tissue showed an increased intensity in the range from 420 nm to 540 nm, whereas atherosclerotic lesions had no or only a small peak at 480 nm. There was a significant correlation between the semiquantitative score (n = 120) and the fluorescence ratio at 480/420 nm (excitation wavelength 325 nm) with a correlation coefficient of 0.87. The spectroscopic results showed no differences between the samples taken from different types of vessels. Fluorescence spectroscopy allows a reliable identification of normal and atherosclerotic lesions. The close correlation between the emitted light intensity ratio at 480/420 nm and the histological alterations of the vessel wall suggests a relationship between vessel wall fluorescence and the atherosclerotic alterations of the wall.


Afterload dependent prolongation of left ventricular relaxation: importance of asynchrony. Acute increases in afterload and in left ventricular asynchrony both independently prolong left ventricular isovolumetric relaxation. The aim of the study was to investigate whether an increased left ventricular afterload augments left ventricular asynchrony, which in turn could mediate the afterload dependent prolongation of left ventricular isovolumetric relaxation. Seven chloralose anaesthetised open chest dogs were instrumented with a left ventricular pressure gauge and two pairs of ultrasonic wall thickness crystals in the antero-apical and postero-basal left ventricular wall. At a constant heart rate of 149(SEM 7) beats.min-1, left ventricular pressure was acutely increased by brief manual clamping of the descending (AOCD) and ascending AOCA) thoracic aorta. Left ventricular asynchrony was quantified by the phase difference of the first Fourier harmonic between postero-basal and antero-apical wall motion. Global left ventricular relaxation was measured as the time constant of isovolumetric pressure fall, tau. Regional myocardial relaxation was assessed as the mean rate to half end diastolic thinning. AOCD increased left ventricular peak systolic pressure from 141.9(6.9) mm Hg to a maximum of 182.0(5.1) mm Hg and tau from 34.3(2.4) ms to 48.0(5.0) ms (p less than 0.05). Simultaneously, phase difference increased markedly during AOCD, from 12.7(3.5) degrees to 24.4(2.2) degrees (p less than 0.05). At matched left ventricular peak systolic pressures, AOCA increased tau from 33.4(2.5) ms to only 42.5(4.3) ms (p less than 0.05 v control and AOCD). Concomitantly, the increase in phase difference was smaller and statistically non-significant, at 13.7(2.9) degrees v 17.1(2.5) degrees. During 13 out of the 14 aortic clampings (7 AOCD, 6 AOCA), tau correlated linearly with phase difference [mean r = 0.74(0.03)]. In contrast to their effects on global left ventricular relaxation and asynchrony, neither AOCD nor AOCA influenced the rate to half end diastolic thinning. (1) left ventricular asynchrony may increase during an acute augmentation of left ventricular afterload; (2) this increased left ventricular asynchrony possibly contributes to the afterload dependent prolongation of left ventricular isovolumetric relaxation rate.


Variable effects of human and canine polymorphonuclear leucocytes on vascular smooth muscle tone. Previous studies have shown variable effects of human and canine polymorphonuclear leucocytes (neutrophils) on vascular tone. The aim of this study was to identify whether these variations in neutrophil function are due to species differences. Canine and human arterial rings (with and without endothelium) were contracted with the thromboxane A2 analogue U46619, and then exposed to isolated neutrophils. Human neutrophils caused a significant relaxation of the human mammary arterial rings, and the relaxation was unaffected by the cyclo-oxygenase inhibitor indomethacin, enhanced by superoxide dismutase (SOD), and inhibited by oxyhaemoglobin. The relaxant effect of human neutrophils was also diminished upon pretreatment with NG-monomethyl-l-arginine (L-NMMA), indicating that the vasorelaxant material released by the neutrophils was nitric oxide (NO). Human neutrophils also relaxed canine femoral arterial rings, and the relaxant effect was potentiated by SOD and inhibited by pretreatment with oxyhaemoglobin or L-NMMA, confirming that the vasorelaxation was via release of NO. Canine neutrophils, on the other hand, caused an endothelium dependent contraction of autologous femoral arterial rings. This vasoconstriction was not affected by indomethacin, SOD, oxyhaemoglobin, or L-NMMA. However, treatment of canine neutrophils with the 5-lipoxygenase inhibitor piriprost attenuated (p < 0.02) their contractile effect on vascular rings, suggesting that neutrophil generated 5-lipoxygenase products were probably responsible for smooth muscle contraction. Presence of the leukotriene C4 and D4 receptor antagonist FPL 55,712 totally blocked the contractile effects of canine neutrophils, indicating that femoral arterial ring contraction was mediated by peptido-leukotrienes. The endothelium dependent nature of the canine neutrophil induced contraction suggests that the 5-lipoxygenase product leukotriene A4 is taken up by endothelial cells for conversion to peptido-leukotrienes. Since SOD had no effect and FPL 55,712 totally blocked the vasoconstrictor effects of canine neutrophils, it appears that the vasoconstrictor effects of the latter are mediated primarily through peptido-leukotrienes. In contrast, the vasorelaxation by human neutrophils is mediated through release of NO.


Metabolic vasodilatation with glucose-insulin-potassium does not change the heterogeneous distribution of coronary blood flow in the dog. The heterogeneous distribution of coronary blood flow could represent regional differences in demand, or mismatching of regional O2 supply to demand, caused by regionally exhausted vasodilatation (anatomical/mechanical factors) or by regional arteriovenous diffusional O2 shunting. Regional coronary blood flow and global myocardial oxygenation and metabolism were measured during metabolic vasodilatation with glucose-insulin-potassium (GIK). Variables were studied before and 30 and 60 min after start of a 30 min infusion of GIK (50% glucose, 4 ml.kg-1, 8 mM KCl, and 3 U insulin.kg-1). Regional blood flows were measured by radioactive microsphere technique and cardiac output by thermodilution. Experimental subjects were six anaesthetised mongrel dogs, weighing 20-27 kg. GIK increased plasma osmolarity and lactate, decreased haemoglobin, and increased cardiac output by 67(29)% and systemic O2 supply by 32(13)%, at unchanged arterial and central venous pressures and heart rate. Coronary blood flow rose by 97(50)% and left ventricular O2 supply by 56(41)%. Although regional blood flows in small tissue samples of about 1 g in the left ventricle ranged from a factor 0.31 to 1.73 of mean flow, GIK did not change flow heterogeneity and regional flows significantly correlated in time. Left ventricular O2 uptake rose by 42(40)%, while venous PO2 increased and O2 extraction decreased. Global lactate uptake increased at unchanged extraction. Changes were reversed after GIK. GIK transiently increases myocardial O2 uptake following a raised cardiac output, caused by a hyperosmolarity induced rise in cardiac contractility rather than by haemodilution. Although myocardial O2 supply is distributed heterogeneously, the fractional rise with GIK is almost equal among regions. At constant lactate extraction, increased venous PO2 and decreased O2 extraction do not indicate overperfusion in some regions at the cost of underperfusion in others, are probably caused by a small, direct vasodilating effect of hyperosmolarity, and argue against diffusional O2 shunting. As for global O2 supply to demand, the increase in regional O2 supply is probably well adapted to regionally increased demand during GIK, so that the heterogeneous distribution of O2 supply can be explained by regional differences in demand and not by regionally exhausted vasodilatation or O2 shunting.


Echocardiographic determination of stroke volume during rapid atrial pacing and volume loading in normal rats. The aim was to validate echocardiographic assessment of acute changes of left ventricular stroke volume in normal rats. By transthoracic use of a 7.5 MHz ultrasonic transducer, the left ventricular dimensions were determined before and during rapid atrial pacing and saline infusion in seven Wistar rats weighing 310-470 g. Left ventricular volume was calculated from short axis dimensions according to a cube function formula. Echo stroke volume (SVE) was then compared with that obtained simultaneously using a pulsed Doppler flow meter placed around the ascending aorta (SVF). The SVE (ml) was decreased from 0.30(SD 0.12) to 0.13(0.06) by rapid pacing and increased from 0.27(0.12) to 0.63(0.16) by volume loading. Regression analysis showed high correlations between SVE and SVF during both pacing (r = 0.84) and infusion (r = 0.91) studies. Furthermore, correlations between SVE and SVF in individual animals were very close (r = 0.87-0.99 in the pacing study and 0.92-0.99 in the volume study). Interobserver and intraobserver variances were small, with close correlations (r = 0.96-0.99) and modest standard errors of the estimate (0.02-0.04 ml) between the two measurements. Echocardiography allows reliable in vivo measurement of cavity dimensions and assessment of acute alterations in stroke volume in normal rats.


Effects of exercise stress on left ventricular end diastolic pressure-length strain relations in dogs with and without coronary stenosis. The aim was to elucidate the alterations of left ventricular diastolic properties, taking into account changes in unstressed length during exercise stimuli with and without coronary stenosis. Left ventricular end diastolic pressure-length strain relations using segment length normalised to Lagrangian strain, and the rate of relaxation, were studied in seven open chest anaesthetised dogs with and without coronary stenosis on both left anterior descending and circumflex coronary arteries (approximately 30% resting flow reduction) during simulated dynamic exercise. Regional segment length was measured with ultrasonic crystals placed in the left anterior descending subendocardial region, and unstressed segment length at zero transmural pressure was obtained by occluding the vena cava. Peak negative dP/dt was decreased and isovolumetric left ventricular relaxation time constant increased by coronary stenoses; however neither changed significantly during simulated exercise. Left ventricular end diastolic pressure was significantly increased by coronary stenoses, from 3.1(SEM 0.8) to 7.0 (0.9) mm Hg (p < 0.05), and further increased to 15.5(1.1) mm Hg (p < 0.01) during simulated exercise, although right ventricular end diastolic pressure did not change. Unstressed length was increased in coronary stenoses from 9.03(0.08) to 9.89(0.13) mm (p < 0.01), and further increased to 10.34(0.14) mm (p < 0.01) during exercise, whereas it tended to decrease without coronary stenosis, from 9.03(0.08) to 8.79(0.11) mm during exercise. Left ventricular end diastolic pressure-length strain relations progressively shifted upward and leftward with coronary stenoses and subsequent exercise, but shifted downward and rightward during exercise without coronary stenosis. The increase in unstressed length or volume may contribute to exercise induced left ventricular dilatation observed in patients in effort angina. End diastolic distensibility decreases in both mild supply induced and exercise induced ischaemia, whereas in the normal heart, left ventricular end diastolic distensibility increases during exercise.


Mechanical restitution during alternans in guinea pig papillary muscles. The aim was to investigate alternate acceleration and retardation of mechanical restitution as a possible mechanism for mechanical alternans in isolated myocardium. Mechanical alternans was induced in papillary muscles from the right ventricles of 11 guinea pigs (200-300 g) by rapid pacing under hypothermic conditions (T = 27 degrees C). Mechanical restitution curves were constructed by measuring the force responses to stimuli applied following variable test intervals during steady state pacing. Curves were obtained under control conditions (steady state stimulation interval 3 s), and for the beats following the large and small contractions during mechanical alternans. Monoexponentials were fitted to the restitution curves. The mean rate constant for restitution following the large beat in alternans was found to be slightly but significantly smaller than that following the small. Both rate constants obtained during alternans were significantly larger than the control rate constant (restitution was faster in alternans). In addition, as the alternation widened, the restitution curve of the beat following the small contraction developed a higher plateau than that following the large. The results confirm that the small beat in alternans is followed by faster restitution than the large. This alone is insufficient to explain the observed extent of alternans. The restitution curve for the beat following the small contraction must also rise to a higher plateau. Both the amount of calcium available for intracellular release and the rate at which it is made available vary from beat to beat.


Haemodynamic and neurohumoral response in heart failure produced by rapid ventricular pacing. The aim was to determine the exact sequence of hormone changes during the progression of fluid retention in a canine model of "congestive cardiac failure" induced by rapid right ventricular pacing, and during recovery when pacing is stopped. Rapid ventricular pacing at a rate of 250 pulses.min-1 was used in six mongrel dogs with implanted right ventricular pacemakers. Right heart haemodynamics were measured by means of Swan Ganz catheterisation, allowing flow measurement by thermodilution and pressure measurement by external manometry. Plasma renin activity, arginine vasopressin, and atrial natriuretic factor were assayed on venous blood samples by radioimmunoassay. Noradrenaline was assayed by high pressure liquid chromatography. The onset of rapid pacing was accompanied by a fall in cardiac output and a rise in pulmonary arterial, pulmonary capillary wedge, and right atrial pressures. Noradrenaline and atrial natriuretic factor rose. Plasma renin activity showed an initial fall followed by a rise, and arginine vasopressin was unchanged in the first 8 h. When rapid pacing was continued for a further 35 d, clinical signs of fluid retention appeared by day 28, by which time cardiac output had fallen, and central pressures risen further. Atrial natriuretic factor peaked at around 14 d whereas plasma renin activity, arginine vasopressin, and noradrenaline tended to reach a plateau at about d 20 and then to show further increases as clinical signs of fluid retention appeared; this was most marked with plasma renin activity. Cessation of pacing at d 35 caused a rapid reversal (increase) of cardiac output but a more gradual reversal (decrease) of right heart pressures over 5 d; only wedge pressure returned to base line. Arginine vasopressin and plasma renin activity fell rapidly to around 40% of the final pacing levels and reached basal values after 8 h and 48 h respectively. Noradrenaline fell after 8 h and reached basal levels in 5 d. Atrial natriuretic factor fell quickly by 60% after 8 h but remained above basal levels for 5 d. At the end of pacing, body weight fell rapidly in conjunction with a large diuresis. These findings are compatible with a major role of one or more of renin, vasopressin, and noradrenaline in the pathophysiology of the fluid retention of heart failure; the manifestations are not counteracted by the rise in atrial natriuretic factor.


Myocardial oxygen requirements during experimental cardiopulmonary resuscitation. The aims were to determine myocardial oxygen requirements during cardiopulmonary resuscitation (CPR), and to test the hypothesis that endogenous catecholamines have a major effect on myocardial oxygen requirements in this setting. Myocardial oxygen consumption (MVO2) was measured during 20 minutes of CPR in eight anaesthetised dogs. Coronary blood flow was maintained at prearrest levels using an external pump to provide a permissive level of oxygen delivery during ventricular fibrillation. Oxygen content was measured in arterial and coronary sinus blood samples under prearrest conditions and at 5 min intervals during CPR. Four dogs were given propranolol (1 mg.kg-1) following the 5 min measurements. MVO2 averaged 108.7(SEM 12.8)% of the initial prearrest values after 5 min CPR (n = 8). After 10 min CPR, MVO2 fell to 53.8(13.3)% of the initial prearrest values in the subset of animals given propranolol after the 5 min measurements (n = 4), but remained at prearrest levels in untreated animals (p < 0.05 for an interactive effect between treatment and time). MVO2 subsequently tended to decrease with time in untreated animals, but remained a high percentage of prearrest values throughout the 20 min period of CPR. These findings suggest that endogenous sympathetic stimulation of the fibrillating heart results in high myocardial oxygen requirements during CPR.


Effect of 5-aminosalicylic acid on myocardial capillary permeability following ischaemia and reperfusion. The aim was to evaluate the effect of 5-aminosalicylic acid on myocardial capillary permeability for small hydrophilic molecules after ischaemia and reperfusion. Open chest anaesthetised dogs were subjected to a 20 min occlusion of the left anterior descending coronary artery followed by 1 h reperfusion. 5-Aminosalicylic acid (bolus injection 12 mg.kg-1, followed by 105 micrograms.kg-1.min-1) (n = 10) or saline (control, n = 12) was given intravenously for 1 h, starting 20 min before ischaemia. The myocardial plasma flow rate, myocardial capillary extraction fraction, and myocardial capillary permeability-surface area (PS) product for 99mTc-DTPA were determined before ischaemia, and 5 and 60 min after the start of reperfusion by employing the single injection residue detection method. Immediately after reperfusion, the reactive hyperaemic plasma flow was measured by the 133Xe washout method. Four dogs (two untreated and two treated with 5-aminosalicylic acid) were eliminated due to ventricular fibrillation at the time of reperfusion. In the remaining animals (10 controls and eight treated) the plasma flow rate, capillary extraction fraction and PS were similar before myocardial ischaemia. After 5 min reperfusion, the plasma flow rate and PS were significantly increased in control animals (p < 0.02 and p < 0.008, respectively), but were unchanged in dogs treated with 5-aminosalicylic acid. In addition, after 5 min reperfusion, PS was significantly higher in the control group than in treated animals (p < 0.005). Microcirculatory variables returned to preocclusive values in both groups by 60 min after reperfusion. 5-Aminosalicylic acid had no significant effect on haemodynamics or on reactive hyperaemic plasma flow. The results suggest that 5-aminosalicylic acid can attenuate the microvascular changes after reversible myocardial ischaemia. The effect is potentially beneficial, and may be mediated by well recognised anti-inflammatory actions of 5-aminosalicylic acid (ie, scavenging of oxygen free radicals and neutrophil inhibition).


Imipramine induced heart failure in the dog: a model to study the effect of cardiac assist devices. The value of intravenous imipramine in creating a reversible model of short term heart failure was evaluated in anaesthetised dogs. Acute effects of imipramine were studied in 11 dogs using invasive haemodynamic pressure measurements and two dimensional echo evaluation. After a 30 min imipramine infusion (7.5 mg.kg-1.h-1), positive left ventricular dP/dtmax decreased from 1368(SEM 108) to 909(119) mm Hg.s-1 (p < 0.05), left ventricular end diastolic pressure increased from 8(1) to 12(2) mm Hg (p < 0.05), while left ventricular pressure decreased from 106(4) to 87(6) mm Hg (p < 0.05). Cessation of imipramine administration resulted within 60 min in partial restoration of cardiac function. This deterioration and subsequent recovery was also demonstrated with echocardiographic measurements, which showed a decrease in ejection fraction from 54(3)% to 28(2)% (p < 0.05). During administration of imipramine neither significant electrophysiological changes nor supraventricular/ventricular arrhythmias were seen. Repeated infusions of imipramine in three anaesthetised dogs with a two week interval showed the reproducibility of the haemodynamic effects and the recovery of ventricular function. Since the model was developed to evaluate the use of cardiomyoplasty in heart failure, the effect of imipramine was also evaluated on latissimus dorsi muscle contraction. Administration of imipramine did not affect skeletal muscle force development at the dosage used to create heart failure. This model can be used to produce short term reversible heart failure in anaesthetised animals to test the efficacy of supportive interventions like dynamic cardiomyoplasty, intra-aortic balloon pumping, and mechanical cardiac assist devices.


Adenosine induced chest pain--a comparison between intracoronary bolus injection and steady state infusion. Adenosine may induce chest pain in at least two ways, either by direct stimulation of sensory afferents before actual ischaemia occurs or secondary to ischaemia. The aim was to study if the mechanism of pain induction may depend on the method of adenosine administration. Increasing doses of adenosine were given to seven male patients with ischaemic heart disease referred for coronary angiography: first as a bolus intracoronary injection (2.5-50 mumol), second as a 1 ml.min-1 steady state infusion (0.01-20 mumol.min-1) and third as an intravenous steady state infusion (0.076-0.76 mumol.kg-1 x min-1). Pain, rate-pressure product, coronary sinus blood flow, and ECG were monitored. Lactate was analysed in coronary sinus and arterial blood. After intracoronary bolus injection there were no signs of myocardial ischaemia, whereas during intracoronary steady state infusion, and in spite of a lower, but definite, degree of pain, 5/7 patients showed myocardial lactate production and three patients showed ST depression. During the intravenous steady state infusion 6/6 patients showed ST depression. These findings suggest that when using adenosine for studies on the mechanisms of chest pain in patients with ischaemic heart disease it is preferable to use an intracoronary bolus injection technique rather than a steady state infusion, as the risk of inducing ischaemia with the latter model cannot be ignored.


Pulmonary artery compliance: its role in right ventricular-arterial coupling. The aim was to investigate the ventricular/vascular coupling of the intact right heart under conditions of normal operation and acute pulmonary hypertension. Right ventricular contractility was obtained by calculating the end systolic pressure-volume relationship (Ees) and the effective pulmonary arterial elastance (Ea), applying the Windkessel parameters of the pulmonary arterial input impedance. Coupling between the ventricle and its load could be determined in terms of Ees and Ea. Acute pulmonary hypertension was induced by injecting glass microspheres into the pulmonary vascular bed until a mean pulmonary arterial pressure of more than 35 mm Hg had been reached. Experimental subjects were Landras/Large white pigs (n = 11), studied under general anaesthesia. Ees was obtained by normalising the right ventricle pressure-diameter equivalent of Ees to stroke volume. The lumped element parameters of the Windkessel analogue were calculated from the pulmonary artery pressure and blood flow. Stroke work was calculated from the pressure-volume loop and oxygen consumption derived from the pressure-volume area. Efficiency was taken to be the ratio between stroke work and oxygen consumption. Ea increased significantly as mean pulmonary artery pressure rose, while Ees remained linear and constant. Stroke work, as well as efficiency, increased, with the maximum of the stroke work curve lying to the right of the efficiency maximum. At the control step (before pulmonary artery hypertension), Ees = 1.71 Ea (n = 11). Under control conditions, the right ventricle operates at maximum efficiency and submaximal work output. Compliance of the pulmonary artery is a significant factor in decoupling the right ventricle from its vascular load. As the compliance decreases with acute pulmonary hypertension, the maximum stroke work against load point shifted in such a manner that the right ventricle changed its operational status from a flow to a pressure pump, resulting in a decreased stroke volume.


Functional changes in the right and left ventricle during development of cardiac hypertrophy and after its regression. The aim was to determine left and right ventricular functional reserves and collagen concentration during the development of cardiac hypertrophy and after its regression. Two experimental models of cardiac hypertrophy (chronic thyroxine or isoprenaline treatment of adult rats) were compared 24 h and five weeks after the agent was last given. Pressure changes in the left (right) ventricle before and after acute aortic (pulmonary artery) ligation were recorded in open chest anaesthetised animals. The difference in dP/dtmax after and before ligation was regarded as the functional reserve. The total collagen concentration was determined in both ventricles separately by means of hydroxyproline. Left and right ventricular weight increased by 20% and 30% respectively in the two models employed. In the thyroxine treated group, the functional reserve of the left ventricle rose very noticeably, whereas in the isoprenaline treated group it decreased. The right ventricular functional reserve did not differ from that in the controls in either of the two groups. The collagen concentration rose in the left ventricle in the isoprenaline group only. Five weeks after the last administration of the agent, cardiac mass and ventricular function did not differ from the control values in either of the models studied; the only exception was the incomplete regression of left ventricular hypertrophy and persistent structural and functional impairment of the left ventricle in the isoprenaline treated group. Our results indicate that hearts undergoing a comparable degree of experimental hypertrophy may have different functional and structural properties; significant differences were found between the right and left ventricular response. Regression of hypertrophy together with a reversal of ventricular function usually occurs unless the myocardium has received severe structural damage.


Effects of adenosine on cardiac performance and phosphate compounds in microembolised guinea pig hearts. The excessive release of adenosine has been reported to cause hyperaemia and to attenuate ischaemic injury in microembolised hearts. The direct effects of exogenous adenosine on cardiac performance were investigated in microembolised guinea pig hearts with the simultaneous measurement of phosphate compounds. 21 male guinea pig hearts were perfused according to the Langendorff technique and microembolism was induced by injecting microspheres. Hearts were then treated as follows: group A, adenosine 20 microM and theophylline 60 microM; group B, theophylline 60 microM; group C, saline (control). Cardiac performance was monitored and phosphate compounds were measured by 31P magnetic resonance spectroscopy. Group A showed a significant increase in the left ventricular developed pressure and coronary flow, and a modest restoration of ATP content. Pi/(Pi+Pcr) remained virtually constant. There were no significant changes in left ventricular developed pressure or coronary flow in groups B and C. There was a gradual decrease in tissue ATP content and slight increase in Pi/(Pi+Pcr). Adenosine improved cardiac performance without adverse effect on energy metabolism in microembolised hearts. Adenosine also restored the ATP stores in microembolised hearts. (Pi = inorganic phosphate; Pcr = phosphocreatine.)


Myocardial stretch alters twitch characteristics and Ca2+ loading of sarcoplasmic reticulum in rat ventricular muscle. The aim was to determine the influence of diastolic muscle length on force development and timing parameters of cardiac muscle twitch contraction and to determine whether a length dependency exists for the calcium loading capacity of the sarcoplasmic reticulum. Right ventricular papillary muscles and trabeculae were isolated from hearts of female Wistar rats weighing 220-280 g. Papillary muscles were stretched to diastolic lengths of 90, 95, and 100% Lmax and paced at 1.0 Hz. Individual twitch profiles were characterised by their peak force and the maximum rate (dF/dt) of the positive and negative force changes. Intrinsic timing was identified through waveform analysis that divided the twitch profile into time domains for the ascending limb (T0-T1; T1-T2) and the descending limb (T2-T3; T3-T4). Each domain was compared at three muscle lengths. The sarcoplasmic reticular calcium content at short (1.88 microns) and long (2.11 microns) sarcomere lengths was characterised by rapid cooling contractures after 1 s and 60 s of diastolic rest. Peak developed force and the maximum rate of positive and negative force development decreased as diastolic muscle length was reduced from Lmax to 90% Lmax. The intrinsic timing for the segment that reflects the relaxation phase of the twitch (T1-T4) was shortened as muscle length was reduced. The time domain that reflects the combined effects of calcium release and the early phase of contraction (T0-T1) was insensitive to diastolic muscle length. The fractional release of sarcoplasmic reticular calcium at different muscle lengths was approximately 32-35% of the total sarcoplasmic reticulum calcium pool. The data on the intrinsic timing of the twitch characteristics coupled with rapid cooling contracture analysis suggests a fractional calcium release that is approximately 32-35% of the total sarcoplasmic reticular capacity at either long or short muscle lengths. However, the loading capacity of the sarcoplasmic reticulum is greater when the muscle operates at a shorter diastolic length. This can be interpreted as meaning that diastolic muscle length differentially influences sarcoplasmic reticular calcium storage and release processes.


Dietary fat modulation of left ventricular ejection fraction in the marmoset due to enhanced filling. The aim was to investigate the influence of long term dietary fish oil consumption on cardiac function in a non-human primate, to elucidate further the basis of the apparently reduced cardiovascular disease mortality associated with its consumption in man. Adult male marmoset monkeys (Callithrix jacchus) were fed diets supplemented with polyunsaturated fatty acids (PUFA) of marine (tuna fish oil) or plant (sunflower seed oil) origin, saturated animal fat (sheep perirenal fat), or a low fat reference diet for 24 months. Cardiac function was assessed using radionuclide angiography under pentobarbitone anaesthesia with a counts based adaptation for ventricular volume estimations. Measures were made at rest and during infusion of adrenaline. The mean left ventricular ejection fraction was greater in the tuna fish oil group [55.0(SEM 1.1)% n = 7] and the sunflower seed oil group [58.1(2.4)% n = 8] than in the reference group [48.5(1.4)% n = 9] and the sheep fat group [47.6(1.8)% n = 8]. This was associated with a more than 25% greater end diastolic volume and 40-70% increases in stroke volume in tuna fish or sunflower seed oil fed animals. There was no evidence of cardiac hypertrophy. In contrast, adrenaline increased stroke volume and ejection fraction by increasing emptying, thus reducing residual end systolic. Tuna fish oil fed animals had a low resting heart rate. When this was raised to comparable levels by adrenaline, lower pressure-rate indices and greater cardiac minute work suggested higher myocardial energy efficiency in PUFA fed animals compared with the reference and sheep fat groups. Dietary fish oil and sunflower seed oil increased the left ventricular ejection fraction in the marmoset monkey by enhancing ventricular filling, thus providing an energy sparing promotion of diastolic relaxation.


Mechanical determinants of myocardial oxygen consumption with special reference to external work and efficiency. The aim of the study was to investigate the ambiguous effect of left ventricular afterload on myocardial work, oxygen consumption, and efficiency. Myocardial oxygen consumption and mechanical parameters of the left and right ventricle were measured in situ in a modified heart-lung preparation in the rat. Left ventricular afterload was adjusted arbitrarily by means of a Starling resistor mounted in a shunt circuit between the left ventricle and the caudal caval vein. Left and right ventricular pressure and aortic pressure as well as pulmonary flow and the flow in the shunt circuit were measured. The left ventricular pressure and volume values were converted into wall stress and length data assuming a thick walled sphere, and external work was calculated from left ventricular force and shortening. Left ventricular external work ran through a maximum with decreasing aortic pressure. Left ventricular oxygen consumption per gram and beat correlated linearly with left ventricular peak wall stress, tension-time integral, and maximum rate of stress development. Left ventricular force and shortening, the two components of external work, acted differently: force determined left ventricular oxygen consumption, whereas shortening had no direct effect on myocardial oxygen consumption, but was important in determining left ventricular efficiency. The interplay between left ventricular afterload and coronary perfusion pressure is of special significance for the heart in situ. The decrease in shortening and external work as well as the diminution in efficiency, observed at low aortic pressure values, can be attributed to impaired coronary perfusion. The coronary perfusion pressure must therefore be taken into consideration for the critical examination of the efficiency of the heart in situ.


Failure of mannitol to reduce myocardial infarct size in the baboon. The aim was to determine whether mannitol, previously shown to have several myocardial protective properties, could reduce the myocardial infarct size after coronary occlusion in the baboon. Anaesthetised baboons underwent a 2 h transient coronary artery occlusion. Each was randomised into one of two groups receiving either mannitol (n = 6) or no adjunct (n = 8). Mannitol (20%) was given at a rate of 0.4 ml.min-1 x kg-1 starting at 105 min postocclusion until reperfusion was allowed at 2 h, and at a rate of 0.2 ml.min-1 x kg-1 thereafter, until a total of 500 ml had been delivered. Changes in the ST segments were recorded with epicardial wires. The animals were killed at 24 h postocclusion and the hearts excised. Silicone microvascular dye was injected into the previously occluded coronary artery to delineate the perfusion bed. The hearts were fixed in formalin, sliced, and mounted on slides. Using planimetry the ratios of the mean volume of infarct to the mean volume of the perfusion bed (VI/VPB) were calculated and compared. The VI/VPB for the mannitol treated group was 71.7(SEM 14.0)% and for the control group, 65.6(6.9)% (NS). No significant difference was noted in the mean summated ST segment elevations between the two groups. Mannitol does not reduce myocardial infarct size or ischaemia in the baboon.


Interdependence of intracellular taurine and sodium in guinea pig heart. The aim was to investigate the effects of raising intracellular taurine on the intracellular sodium activity (aNa1) in isolated guinea pig ventricular myocytes, and the effect of procedures that raise intracellular sodium on taurine concentration in the perfused guinea pig ventricular tissue. Taurine was introduced into the sarcoplasm of isolated ventricular myocytes, either during cell isolation or by diffusion from a penetrating micropipette, and the effect on aNai was measured using an ion sensitive microelectrode. Guinea pig hearts, mounted on a Langendorff apparatus, were perfused with a variety of physiological media and the level of taurine in the ventricles determined using high pressure liquid chromatography. An increase in intracellular taurine caused by its presence during cell isolation or by diffusion from a micropipette significantly reduced the aNai of isolated myocytes at rest, during perfusion with Ca depleted solutions, or on inhibition of the Na pump. In the guinea pig ventricles, taurine at 13.0(SEM 0.6) mmol.kg-1 wet weight comprised up to 45% of the free amino acids; since plasma taurine was 64(13) mumol.litre-1, this means that in vivo a large outwardly directed gradient for taurine exists (equivalent to a free energy of 13.7 KJ.mol-1). Upon perfusion with Ca,Mg free Tyrode solution (which raises intracellular sodium markedly), a time dependent loss of taurine occurred. Both the rate of loss and the total amount lost were increased when the Na pump was also inhibited. This loss of tissue taurine was not due to release from dead or lysed cells, as it was antagonised by procedures known to reduce the rise of aNai during Ca depletion, was inhibited by beta alanine (an inhibitor of taurine transport), and the fall in tissue taurine was not correlated with the appearance of lactate dehydrogenase in the effluent. The data from isolated myocytes and perfused guinea pig hearts were consistent with the presence of a Na/taurine symport which is activated to cause efflux of Na and taurine when either rise above their physiological level.


Digoxin and digoxin derivative induced arrhythmias: in vitro binding and in vivo abolition of arrhythmias by digoxin immune Fab (DIGIBAND). The aim was to compare the binding characteristics of a highly purified digoxin specific antigen binding fragment (digoxin immune Fab: DIGIBIND) with digoxin and with two commonly used derivatives of digoxin, beta methyl digoxin and beta acetyl digoxin, and to assess its ability to abolish the arrhythmogenic effects of these digitalis glycosides. The binding characteristics of DIGIBIND with digoxin, beta methyl digoxin, and beta acetyl digoxin were assessed in vitro by measuring their ability to inhibit the binding of DIGIBIND to 3H-digoxin. From these studies the affinities of the interactions between DIGIBIND and these glycosides, and the binding capacity of DIGIBIND for each of these glycosides, could be measured. The ability of DIGIBIND to abolish the arrhythmogenic effects of digoxin, beta methyl digoxin, and beta acetyl digoxin was assessed using an in vivo anaesthetised guinea pig model (n = 36, weight 300-400 g), in which these glycosides were infused intravenously (50 micrograms.kg-1 x min-1) until the onset of ventricular arrhythmias, at which point the total amount of glycoside given was calculated. A single bolus dose of either vehicle or DIGIBIND was then given intravenously, and the time to restoration of normal cardiac rhythm noted. After the administration of DIGIBIND, a second infusion of the same glycoside was given to reinitiate the ventricular arrhythmias. The time to onset of the arrhythmias was noted, and the additional amount of glycoside given calculated. In vitro studies showed the binding of DIGIBIND to 3H-digoxin to be inhibited by digoxin and by the two derivatives. The affinities of these interactions with DIGIBIND were significantly different, that for digoxin being some twofold greater than that for beta methyl digoxin and beta acetyl digoxin. The ED50 concentrations were 14.1 (95% CI 12.2, 15.2), 29.2(26.1, 32.7), and 36.2(33.0, 39.8) nM, respectively. However, there were no significant differences between these glycosides in their binding capacities. The in vivo studies showed that intravenous infusion of digoxin, beta methyl digoxin, or beta acetyl digoxin induced similar ventricular arrhythmias. The onset of the arrhythmias was clearly discernible, and required a significantly lower dose of digoxin compared with that of beta methyl digoxin and beta acetyl digoxin. These doses were 667(SEM 55), 868(33), and 854(40) nmol.kg-1, respectively. Termination of the infusion had no effect on the arrhythmias, and in those animals which received a bolus intravenous injection of saline there was no return to normal cardiac rhythm. By contrast, in animals which received a bolus intravenous injection of DIGIBIND, there was complete abolition of the arrhythmias within 4-6 min. Although the dose of DIGIBIND given to abolish digoxin induced arrhythmias was approximately 25% less than that given to abolish beta methyl digoxin and beta acetyl digoxin induced arrhythmias (p < 0.05), the time to restoration of normal cardiac rhythm after DIGIBIND was not significantly different for digoxin compared with beta methyl digoxin and beta acetyl digoxin, at 4.6(0.9), 4.9(0.8), and 5.7(0.8) min, respectively. To reinitiate the arrhythmias in those animals which had received DIGIBIND, a dose of glycoside was required which was not significantly different from that given prior to the DIGIBIND. This observation therefore confirmed the stoichiometric relationship between DIGIBIND and each of the glycosides in respect of the neutralising action of DIGIBIND in abolishing the arrhythmogenic effects of these agents. Although there is some small difference in the affinities of the binding interactions, there is no difference in the binding capacities of DIGIBIND for digoxin, beta methyl digoxin, or beta acetyl digoxin in vitro. These binding interactions are manifest as the ability of DIGIBIND to abolish the arrhythmogenic effects of digoxin and the two derivatives in vivo.


Anatomical identification of glomeruli in the antennal lobes of the male sphinx moth Manduca sexta. Computer-assisted neuroanatomical methods have been used to demonstrate unique identities of the glomeruli of the antennal lobes (ALs) in males of the sphinx moth Manduca sexta. The glomerular neuropil consists of the male-specific macroglomerular complex, which comprises two closely apposed bulky subunits, and 64 +/- 1 "ordinary" glomeruli arrayed in a shell around a central region of coarse neuropil. Computer-generated maps show the exact locations of all glomeruli and adjacent groups of neuronal somata in a constant Cartesian coordinate system, such that these can be accurately identified in any individual. The glomeruli belong to three classes according to the number and type of identification criteria they satisfy. The larger class comprises glomeruli (n = 44) identified only in the computer-generated maps on the basis of their relative positions. The other two classes include glomeruli that were also identified in sections, either directly from their proximity to readily identifiable structures and their shape and size (n = 10, including the labial-palp-pit-organ (LPO) glomerulus), or indirectly from their positions relative to the former (n = 9). Two very small glomeruli were present in only one AL, demonstrating the existence of anomalous glomeruli, whereas another glomerulus had no homologue in both ALs of one individual. The true number of ordinary glomeruli (per male AL) was thus estimated to be 64. The uncertainty in delineating some glomeruli might affect this number without implying modification of the homologies proposed. The locations of tracts and cell groups, both within and near the AL, are also invariant with respect to glomeruli, as shown in the computer maps. The methods employed are general and might be useful to researchers in related fields. The results obtained call for more attention to the precise geometry of neural structures.


Distribution of laminin, type IV collagen, and fibronectin in the cell columns and trophoblastic shell of early macaque placentas. The cytotrophoblastic cell columns and trophoblastic shell of macaque placentas accumulate progressively greater amounts of intercellular material during early gestation. We studied the composition of this material in placentas collected from 22-34 days of gestation by using immunoperoxidase techniques directed to the extracellular matrix molecules fibronectin, type IV collagen, and laminin. These antigens co-localized within the intercellular deposits at all stages studied. At day 22 the proximal cell columns were composed of cells with narrow interstices and which lacked immunoreactivity for the 3 antigens. Distally the cells were vacuolated and the intercellular spaces increased in size and contained dense matrix deposits. The trophoblastic shell consisted of closely packed, non-vacuolated cytotrophoblast cells with only a delicate meshwork of matrix. By day 27 the matrix deposits of the distal cell columns increased markedly in size. The trophoblastic shell contained larger numbers of vacuolated cells and was occupied by accumulations of matrix. By 34 days the matrix deposits of the cell columns expanded substantially along the longitudinal axes of the columns. These deposits were often continuous with a matrix-dense, cell-deficient layer in the trophoblastic shell. This matrix-rich zone lay between a cellular layer adjacent to the intervillous space and a similar, but discontinuous, cell layer that formed the junctional zone with the endometrium.


Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis. Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immunoreactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.


Characterization of macrophage-like cells in the external layers of human small and large intestine. In the external layers of human small and large intestine macrophage-like cells were characterized by immunohistochemical, histochemical and electron-microscopical methods. Using immunohistochemistry and a number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of macrophages were evaluated. In all locations macrophage-like cells were identified with antibody EBM11, which recognizes CD68 antigen, C3bi which recognizes CD11b, and partly with an antibody which recognizes protein 150,95 (CD11c). Macrophage-like cells in the external muscle layer were HLA-DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role in gastrointestinal motility and/or has an immunological function.


Postsmolt change in numbers of acetylcholinesterase-positive cells in the pineal organ of the Pacific coho salmon. We have examined the occurrence of acetylcholinesterase (AChE)-positive cells in the pineal organ of different developmental stages of the Pacific coho salmon. Large numbers of AChE cells were present in freshwater living alevins, in all stages of presmolts (n = 307-544), and in adult spawners (n = 696-1774), whereas seawater-living postmolts displayed a total lack of labeled cells. The AChE-reactive cells were evenly distributed within the pineal end-vesicle and stalk of the presmolts and adults. However, the AChE-positive cells that occurred in the pineal stalk were of a smaller type and more uniform in shape than the cells of the pineal end-vesicle. The dense populations of AChE-stained cells in the alevins were all situated in the caudal part of the pineal end-vesicle. We conclude that changes in pineal metabolism occur in postsmolt salmon that live in salt-water. It is not clear whether the observed change in pineal AChE expression is an "unspecific" change caused by the life in the sea, reflecting alterations that are related to aspects of osmoregulation, and/or is involved in the visual function of the pineal organ resulting from changes in the environmental lighting conditions, e.g., photoperiod, light-intensity, or spectral composition. This study adds to our previous findings of changes that occur in the central nervous system of the salmon during the time of the parr-smolt transformation and migration between limnic and marine environments, and indicates a possible central role of the pineal organ in the control of these events.


Peptidergic neurons of the crab, Cardisoma carnifex, in defined culture maintain characteristic morphologies under a variety of conditions. Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, "superlarge," found occasionally, has a soma diameter greater than 40 microns and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with L-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26 degrees C instead of 22 degrees C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K+]o medium ([K+]o = 15-110 mM; standard = 11 mM) has no long-term effect, but growth is arrested by [K+]o greater than 30 mM. Cultures were also grown in media in which [Ca2+]o ranged from 0.1 microM to 26 mM (standard = 13 mM). Outgrowth occurred from all neuronal types in all [Ca2+]o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.


Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin. Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.


Gonadotropin-releasing hormone neurons and pathways in the brain of the female mink (Mustela vison). The distribution of gonadotropin-releasing hormone-immunoreactive neurons and processes was mapped in the female mink brain using coronal, horizontal and sagittal sections. Perikarya were found along a ventral continuum including the olfactory tubercle, the diagonal band of Broca, the lateral septum, the preoptic and anterior hypothalamic area and the mediobasal hypothalamus; 80% of the perikarya were counted in the mediobasal hypothalamus. Fibres were mainly observed in the organum vasculosum of the lamina terminalis and the median eminence. A few processes terminated in the ependymal cells lining the third and lateral ventricles. The total number of immunoreactive perikarya was the highest in the brains of females sacrificed in July; it then significantly decreased until December. This variation is discussed in relation to the annual breeding cycle.


Glycophorin A interacts with interleukin-2 and inhibits interleukin-2-dependent T-lymphocyte proliferation. Sialoglycolipids shed by tumor cells have been implicated in tumor-induced inhibition of T-lymphocyte responses to interleukin-2 (IL-2). In the present study, we have used glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, to investigate whether shedding of glycoproteins might also contribute to immunosuppression. Glycophorin A inhibited IL-2-stimulated proliferation of the IL-2-dependent cell lines HT-2 and CTLL-2 in a dose-dependent manner. Time course studies on synchronized cell populations indicated that the glycoprotein acted early in the activation process. On the other hand, glycophorin A had essentially no effect on IL-1-mediated stimulation of the IL-1-sensitive thymocyte cell line EL-4 NOB-1. Gel filtration FPLC demonstrated that IL-2 was able to bind to glycophorin aggregates under physiological conditions. Reconstituted vesicles containing glycophorin were also shown to bind IL-2. In addition, both soluble glycophorin aggregates and lipid vesicles containing glycophorin blocked binding of IL-2 to high-affinity cellular IL-2 receptors. Taken together, these results suggest that shedding of tumor sialoglycoproteins with oligosaccharide chains similar to glycophorin A might contribute to negative modulation of IL-2-mediated immune responses.


Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression. Published reports indicate that CD45RO-CD45RAbright T cells are native T cells, CD45RObrightCD45RA- T cells are memory T cells, and that concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status. Thus, following in vitro activation of CD45RO- CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed. Interestingly, all three of these phenotypic subsets are represented in the circulating human lymphocyte pool. We thus used dual-color flow cytometry to phenotypically characterize CD45RObrightCD45RA-, CD45ROdimCD45RAdim, and CD45RO- CD45RAbright lymphocytes. Both the CD45RObrightCD45RA- and CD45ROdimCD45RAdim subsets consisted almost entirely of T cells, whereas the CD45RO-CD45RAbright subset contained T cells plus essentially all of the B and natural killer cells. Additional studies used three-color flow cytometry to assess activation markers on T cells within the three subsets defined by CD45RO/CD45RA expression. CD25 expression increased with conversion from naive to memory status (5% of CD45RO-CD45RAbright, 24% of CD45ROdimCD45RAdim, and 42% of CD45RObrightCD45RA- T cells), whereas CD38 expression decreased during conversion (76, 53, and 27%, respectively). We also assessed the fluorescent intensities of CD11a, CD2, and CD44, shown by others to be increased on memory, compared to naive T cells. Visual inspection of fluorescence cytograms confirmed these findings, and further showed that transitional T cells express these markers at levels indistinguishable from those for naive T cells. These findings suggest that acquisition of CD25 and loss of CD38 occur relatively early in the naive-to-memory transition process, being evident in the transitional cell subset. In contrast, increased expression of CD11a, CD2, and CD44 appear to represent late events, occurring after loss of CD45RA and gain of CD45RO has been completed.


Down-regulation of cell surface CD4 molecule expression induced by anti-CD4 antibodies in human T lymphocytes. Antigenic modulation was defined as the down-regulation of a cell surface antigen expression induced by exposure to specific antibody. We investigated the modulation of CD4 surface expression in human peripheral blood lymphocytes incubated in vitro with anti-CD4 monoclonal antibodies (mAbs). Modulation of surface CD4 was achieved at 37 degrees C, but not at 4 degrees C, with five different murine anti-CD4 mAbs of IgG1 and IgG2a subclasses, with different epitope specificities. Modulation was dose dependent with a maximum at nonsaturating mAb concentration. It was reversible upon culture in mAb-free medium. It was accelerated and amplified in the presence of monocytes or after cross-linking of anti-CD4 mAbs. It could be induced with solid phase anti-CD4 mAbs, but not with soluble F(ab')2 fragments. Its magnitude was identical on all CD4+ lymphocytes. It was associated with a moderate down-regulation of CD2 and CD3 but not of CD8 and HLA class I surface expression. Modulation was slightly augmented by addition of inhibitors of the endosome/lysosome pathway but not by protein synthesis inhibitors. The anti-CD4 mAb initially bound to cell surface was no longer detectable after 24 hr of culture. Most of surface CD4 proteins complexed with antibody were rapidly internalized and transiently replaced by CD4 from an intracytoplasmic pool and then no longer were expressed. CD4 mRNA was moderately decreased in cells incubated with anti-CD4 mAb while beta-actin and beta 2-microglobulin mRNAs remained at stable levels. It was concluded that down-regulation of CD4 surface expression induced by anti-CD4 mAb concerned only a part of CD4 molecules and was associated with a decreased synthesis. The delay required to achieve maximal modulation is likely to reflect exhaustion of the intracytoplasmic recycling pool of CD4 molecules.