Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation.

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts.

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells.

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.

An early transient increase of intracellular Na+ may be one of the first components of the mitogenic signal. Direct detection by 23Na-NMR spectroscopy in quiescent 3T3 mouse fibroblasts stimulated by growth factors.

23Na-NMR spectroscopy was designed to allow for continuous recording of intracellular Na+ in 3T3 fibroblasts stimulated by serum growth-factors in the presence of ion transport inhibitors. The metabolic state of cells at rest and following stimulation was monitored by 31P-NMR spectra of ATP and related high-energy phosphates. The study demonstrates that early activation of ion transporters by addition of serum is marked by the appearance of transient increase of the intracellular Na+, beginning 3 min after addition of serum to quiescent culture and lasting approx. 20 min. The initial rise in cellular Na+ results from an increased activity of the bumetanide-sensitive Na+/K+/Cl- cotransport and of the amiloride-sensitive Na+/H+ antiport. It is suppressed by any one of these inhibitors. Subsequent activation of the ouabain-sensitive Na+/K(+)-ATPase results in an increased Na+ efflux, leading to a return of intracellular Na+ to its initial baseline. Previous work had shown that the early activation of bumetanide-sensitive and amiloride sensitive ion-transporters by growth-factors was essential for induction of cell division, at least in some cell types. Preventing ion activation by adding ion-transport inhibitors lead to the inhibition of DNA synthesis 18 h later. This process was reversible upon elimination of these inhibitors. Even though alternative non-specific effects of these inhibitors cannot be ruled out, the observed transient peak in intracellular Na+ may be one of the earliest components of the mitogenic signal. On the basis of previous works, its effect seems to be related to the activation of Ca(2+)-dependent and cyclic AMP second messenger pathways. The different mechanisms whereby the activated Na+/K+/Cl- cotransport and the Na+/H+ antiport contribute to this signal need to be further investigated.

Basic characterization of an ouabain-resistant, bumetanide-sensitive K+ carrier-mediated transport system in J774.2 mouse macrophage-like cell line and in variants deficient in adenylate cyclase and cAMP-dependent protein kinase activities.

86Rb(K+) transport across the plasma membrane of macrophage-like cells was studied. The cells used were the wild-type J774.2 and its two variants, CT2 cells, deficient in adenylate cyclase, and J7H1 cells, deficient in cAMP-dependent protein kinase. In the three cell lines about 15% of the total 86Rb(K+) influx is transported by the K+ carrier-mediated transport system. The 86Rb(K+) efflux carried by the same transporter is negligible when measured in the absence of ouabain in the medium. Therefore this carrier conducts a net inward flux of K+ under the experimental conditions used. The transporter is sensitive to extracellular Na+ and inhibited by 'loop' diuretics; bumetanide inhibits ouabain-resistant 86Rb(K+) influx with IC50 of 0.1, 5.0, and 0.05 microM for J774.2, CT2 and J7H1 macrophages, respectively. The membrane potential of the three cells was measured, using the distribution of [3H]tetraphenylphosphonium [( 3H]TPP+) across the plasma membrane, and found to be -80.1, -108.5 and -105.1 mV for J774.2, CT2 and J7H1 cells, respectively. The addition of bumetanide to the cell medium does not alter [3H]TPP+ uptake indicating that the transporter is electrically silent. It is concluded that despite the differences in cAMP metabolism by the three macrophages, the basic characteristics of K+ carrier-mediated transport system of the three cells are very similar.

A mathematical analysis of the mortality kinetics of Drosophila melanogaster exposed to gamma radiation.

A mathematical model with nonlinear time-varying characteristics has been developed which describes the relationship between the kinetics of natural aging and radiation-induced delayed mortality. Based on this model, it appears that there is an immediate effect of radiation which is continuously, but nonlinearly, increasing in severity. Two phases appear in this variation, corresponding to the two phases (plateau and dying phase) of the mortality curves for control populations. Accordingly, S/E (survival time post-irradiation/further expectation of life) can best be interpreted as an increasing function during the plateau phase of normal mortality curves, which levels off during the ensuing dying phase.

Effects of temperature on the life span, vitality and fine structure of Drosophila melanogaster.

The effects of temperature on the aging process have been investigated in approximately 3500 imagoes of male Drosophila melanogaster (Oregon R), with focus on the following parameters: mortality, O2 utilization, vitality (as expressed by negative geotaxis and mating) and fine structural alterations in the abdominal organs and brain. The data on mortality kinetics of flies maintained continuously at 18 degrees, 21 degrees, 27 degrees or 30 degrees C or exposed in succession to 21 degrees and 27 degrees C or vice versa support the concept that life span is temperature dependent. Moreover, these data, together with the increased O2 utilization and accelerated loss of vitality at 21 degrees C as compared with 18 degrees C, suggest that, in agreement with the rate-of-living theory proposed by Alpatov and Pearl in 1929, the flies are living faster at the higher temperature. Fine structural aging changes, like ribosomal loss in the Malpighian tubules and lipofuscin-ceroid accumulation in the midgut cells, seem to be more intense at 27 degrees and at 29 degrees C than at 21 degrees C. Also, the low vitality exhibited through their lives by flies kept at previous exposure through many generations) to 21 degrees C. Flies maintained at 29 degrees C showed a striking degeneration of the brain with an almost complete loss of the cytoplasmic organelles of the nerve cells. This electron microscopic finding lends support to the view that nervous tissue injury (perhaps induced by thermal denaturation of membrane lipoproteins) may play a crucial role in life shortening induced by relatively high temperatures.

Thermodynamics of aging in Drosophila melanogaster.

The data on mortality kinetics and decline in functions reported in the preceding article are used to calculate temperature coefficients for the aging process(es) in Drosophila. Different values are found, according to the model chosen to account for the mortality kinetics. The respective implications of three equally suitable models are discussed. Thus, organization parameters on two different levels can be identified: rates of changes assumed to occur at the elementary molecular level, and redundancy factors at a more integrated level. Their temperature coefficients are compared with those of protein denaturations and lipid peroxidation pigment accumulation. It is suggested that elementary molecular processes responsible for aging can indeed be protein denaturations, whereas the known lipid peroxidation pigment accumulation is more likely to be a secondary effect, resulting from a failure of the overall cellular organization at a more integrated, supramolecular, level.

Nuclear magnetic resonance proton imaging of bone pathology.

Thirty-two patients with diversified pathology were examined with a supraconductive NMR imager using spin echo with different TR and TE to obtain T1 and T2 weighted images. They included 20 tumors (12 primary, eight metastasis), six osteomyelitis, three fractures, two osteonecrosis, and one diffuse metabolic (Gaucher) disease. In all cases except for the stress fractures, the bone pathology was clearly visualized in spite of the normal lack of signal from the compact cortical bone. Nuclear magnetic resonance (NMR) imaging proved to be at least as sensitive as radionuclide scintigraphy but much more accurate than all other imaging procedures including computed tomography (CT) and angiography to assess the extension of the lesions, especially in tumors extended to soft tissue. This is due both to easy acquisition of sagittal and coronal sections and to different patterns of pathologic modifications of T1 and T2 which are beginning to be defined. It is hoped that more experience in clinical use of these patterns will help to discriminate between tumor extension and soft-tissue edema. We conclude that while radionuclide scintigraphy will probably remain the most sensitive and easy to perform screening test for bone pathology, NMR imaging, among noninvasive diagnostic procedures, appears to be at least as specific as CT. In addition, where the extension of the lesions is concerned, NMR imaging is much more informative than CT. In pathology of the spine, the easy visualization of the spinal cord should decrease the need for myelography.

Nuclear magnetic resonance imaging of liver hemangiomas.

Nine patients with cavernous hemangioma of the liver were examined by nuclear magnetic resonance imaging (MRI) with a 0.5 T superconductive magnet. Spin-echo technique was used with varying time to echo (TE) and repetition times (TR). Results were compared with 99mTc red blood cell (RBC) scintigraphy, computed tomography (CT), echography, and arteriography. Four illustrated cases are reported. It was possible to establish a pattern for MRI characteristics of cavernous hemangiomas; rounded or smooth lobulated shape, marked increase in T1 and T2 values as compared with normal liver values. It is concluded that, although more experience is necessary to compare the specificity with that of ultrasound and CT, MRI proved to be very sensitive for the diagnosis of liver hemangioma, especially in the case of small ones which may be missed by 99mTc-labeled RBC scintigraphy.

Left ventricular volumes and function during atrial pacing in coronary artery disease: a radionuclide angiographic study.

This study set out to determine the pathophysiologic changes in the left ventricle during atrial pacing in 22 patients with coronary artery disease. Graduated right atrial pacing to a rate of 160 beats/min, or the induction of angina pectoris or significant ST depression was undertaken. Ventricular volumes were measured at rest and at rates of 100, 120, 140 and 160 beats/min using radionuclide angiography. The volumes at a pacing rate of 100 beats/min were used as a reference standard (100%). In the 22 patients with coronary artery disease, left ventricular end-diastolic volume decreased from 118 +/- 3% at rest to 80 +/- 5% at a rate of 160 beats/min; stroke volume from 121 +/- 3% to 54 +/- 5%; and ejection fraction (EF) from 49 +/- 3% to 37 +/- 5%. End-systolic volume decreased from 118 +/- 4% at rest, reached its minimal value of 94 +/- 5% at a rate of 120 beats/min and then increased slightly to 106 +/- 9% at 160 beats/min. Cardiac output and blood pressure did not change significantly. Compared to the control group of 10 normal subjects, the patients had a significantly smaller decrease in end-diastolic volume and end-systolic volume than in normal control subjects. EF in the normal subjects did not change. Blood pressure, cardiac output and stroke volume were similar in both groups. Atrial pacing tachycardia induced reversible ventricular dysfunction with a decrease in EF. Stroke volume was maintained because of relative ventricular dilatation.

The use of thallium 201 myocardial imaging to exclude myocardial infarction after dissection in congenital coarctation of the aorta.

The use of a mobile gamma camera with thallium 201 myocardial imaging is described to exclude myocardial infarction in a patient admitted to the coronary care unit in shock and with clinical, enzyme, and ECG changes consistent with infarction. The patient suffered from acute aortic dissection associated with congenital coarctation of the aorta. The myocardial scan excluded transmural myocardial injury.

Diagnosis of coronary artery disease by multigated radionuclide angiography during right atrial pacing.

Right atrial pacing in conjunction with radionuclide angiography was used as a method to augment gradually myocardial oxygen demand in seven normal subjects and 11 patients with angiographically documented coronary artery disease (CAD). In five of the seven normal subjects the ECG during atrial pacing was normal and falsely pathologic in the two others; in all seven the ejection fraction (64 +/- 8 percent) did not change during the pacing. The atrial pacing was positive in seven of the eleven patients with CAD and falsely negative in four. In comparison, nine of these patients had more than 15 percent reduction in ejection fraction, while only two were regarded as negative. A maximal decrease of 31 percent in the average ejection fraction was observed in CAD patients at a pacing rate of 160/min. Wall motion pattern remained normal in all seven normal subjects, while new wall motion abnormalities developed during the atrial pacing in nine of the 11 CAD patients. We concluded that changes in ejection fraction induced by atrial pacing are sensitive indicators of myocardial ischemia. In patients with CAD, ejection fraction falls during pacing compared with normal subjects in whom ejection fraction remains unchanged.

Radionuclide assessment of aortocoronary bypass surgery.

Rest and exercise thallium 201 myocardial scintigraphy and multiple gated radionuclide angiography were correlated with the results of clinical status, exercise electrocardiography, coronary arteriography, and contrast left ventriculography in a series of 12 prospectively studied consecutive patients before and after aortocoronary bypass operation. Patients were divided into two groups based on a comparison between preoperative and postoperative 201T1 scintigrams. Group 1 (6 patients) demonstrated improved or normal postoperative perfusion scintigrams and excellent correlation between the site of a patent graft and the improvement in myocardial perfusion on the postoperative exercise scintigrams. Regional wall motion remained normal in 3 patients and improved in 3. In Group 2 (6 patients) the postoperative 201T1 scintigrams were unchanged or worse. Each patient demonstrated graft occlusion, graft stenosis, distal disease, or a perioperative myocardial infarction. No improvement in regional wall motion occurred in 4 of these 6 patients. Neither the symptomatic response to aortocoronary bypass operation nor the response to exercise testing successfully predicted graft patency.

Negative bone scans in impending tibial stress fractures. A report of three cases.

Three highly motivated military recruits who presented with tibial pain on exertion are reported. Their initial bone scan assessments to rule out stress fracture were normal, and the recruits were returned to demanding training. One month later, because of persistent and increasing tibial pain, they were rescanned and focal activity representative of tibial stress fractures was found in each case. Until now it has been assumed that a negative bone scan ruled out a stress fracture unequivocally. Our reported cases show that bone pain may in fact precede scintigraphic evidence of a stress fracture. Persistent and increasing bone pain during demanding physical activity, even in the presence of a prior normal bone scan, may represent stress fracture and repeat bone scan may be indicated.

Assessment of proton magnetic resonance spectroscopy of blood serum as a diagnostic tool in bone marrow transplantation.

The use of proton high-resolution magnetic resonance spectroscopy (MRS) allows the rapid detection and quantitation of modification in the blood serum metabolic profiles in haematooncological patients. This study examines the feasibility of using proton MRS as a diagnostic tool in predicting the outcome of bone marrow transplantation (BMT) at the earliest possible date. Proton spectra of serum samples from 18 BMT patients (11 autologous-BMT and seven allogeneic-BMT), six hematooncological patients that did not undergo BMT and six normal individuals were recorded at 400 MHz. A longitudinal MRS study was carried for these groups and the data were evaluated for statistical significance. It was determined that the MRS results, taken at different time points before and after the BMT treatment, are statistically significant. However, no significant difference was observed in the MRS parameters between the transplanted patients and the control patients. We could not obtain significant correlation between the MRS results and the immunoglobulin level, engraftment parameters or the age, sex, stage of basic disease, conditioning protocols, transplant type, post transplant complications (including death) and outcome.

Cinderella--a clinical nuclear medicine retrieval and filing on-line stand-alone system for patient data.

We describe a microcomputer-based, stand-alone, on-line scheduling data entry and retrieval clinical data base system, specially tailored for a nuclear medicine department. It includes provision for free format clinical diagnostics text answers to each radioisotope scan. Such a system is currently installed at the Department of Medical Biophysics and Nuclear Medicine at the Hadassah University Hospital, and handles all the patient tests performed in this department. We feel that the design of an independent microprocessor-based system customized for one's department needs has indeed improved the quality of the clinical services, both in ensuring that the patient records are kept up-to-date and complete, and in providing, through fast interactive retrieval transactions, immediate answers for a given patient status.

The multislice cylinder: a new approach to quantification of cardiac single photon emission tomography.

A new method of displaying SPECT (single photon emission tomography) thallium-201 images is described in which cardiac activity is constructed as a cylindrical map. Exercise thallium-201 scintigrams from 52 patients with known or suspected coronary artery disease are analysed by this technique and a simplified severity score devised.

Left ventricular function during atrial pacing: a radionuclide angiographic study.

Multigated radionuclide angiography was performed at rest and during atrial pacing in 10 normal subjects. Left ventricular volumes in diastole and systole were measured by total counts in the region of interest of the left ventricle. Stroke volume, cardiac output, ejection fraction, and rates of ejection for each heart rate were calculated. As heart rate increased, there was a gradual decrease in end-diastolic, end-systolic, and stroke volumes without a significant change in cardiac output and ejection fraction. Ejection fraction rate increased and the uncorrected ejection rate decreased with the increase in heart rate. The normal response to tachycardia induced by atrial pacing was a decrease in ventricular volumes without a change in ejection fraction or cardiac output.

Brain SPECT imaging with Tc-99m HM-PAO in Creutzfeld-Jakob disease.

A Tc-99m HM-PAO brain SPECT study of a patient with biopsy-proven Creutzfeld-Jakob disease demonstrated perfusion defects in the left frontal and right temporoparietal areas. A brain CT was normal. The clinical potential of this imaging modality in the evaluation of dementia is discussed.