Furosemide inhibits glucose transport in isolated rat adipocytes via direct inactivation of carrier proteins.

Furosemide inhibits 3-O-methyl-D-glucose equilibrium flux in isolated adipocytes. The inhibition is saturable with an increasing concentration of furosemide and shows a noncompetitive type of kinetics. Both basal and insulin-stimulated fluxes are equally affected by the inhibition. Hydrochlorothiazide and piretanide also inhibit the flux with a similar potency, whereas bumetanide, a more potent diuretic, is much less potent. To understand the molecular basis of this inhibition, effects of furosemide on the glucose-sensitive cytochaslasin B binding activities of adipocytes were studied. Furosemide inhibits the glucose-sensitive cytochalasin B binding of both microsomal and plasma membrane preparations. For both preparations, the inhibition is time dependent and only slowly reversible, is saturable with an increasing concentration of furosemide, shows a noncompetitive type of kinetics with apparent Ki (the inhibitor concentration that gives the half-maximum effect) of 3.5 and 0.7 mM after 2 and 18 h incubation, respectively, and is essentially identical between the basal and insulin-stimulated adipocytes. The inhibition develops with a first-order rate constant of approximately 0.12/h at 4 degrees C. These results indicate that furosemide inhibits glucose transport in adipocytes by directly inactivating transport carriers of both plasma membranes and microsomal reserve pool. This inactivation of glucose carrier may play a part in the diuretic-induced glucose intolerance frequently observed during diuretic therapy.

Mechanism of mitogen-induced stimulation of glucose transport in human peripheral blood mononuclear cells. Evidence of an intracellular reserve pool of glucose carriers and their recruitment.

The present study examines the effects of phytohemagglutinin stimulation of a population of human (h) PBMC enriched in lymphocytes (hPBMC) on D-glucose displaceable cytochalasin B binding sites or medium-affinity sites (M-sites) in relation to glucose transport. Previously we have shown that M-sites are glucose transporters in hPBMC (Mookerjee, B.K., et al. 1981. J. Biol. Chem. 256:1290-1300). Equilibrium exchange of 3-O-methyl D-glucose in unstimulated cells revealed two populations with fast and slow flux rates. Phytohemagglutinin stimulates flux rates by converting part of the slow flux population to the fast flux population. M-sites occur in two distinct pools, one in plasma membrane and the other in microsomal fraction. Phytohemagglutinin treatment increases the plasma membrane pool size of M-sites with a concomitant reduction in the microsomal pool size without affecting the binding affinities or the total number of M-sites/cell. Data presented in this paper demonstrate that there are two pools of glucose transporters in these cells and phytohemagglutinin stimulation induces an energy-dependent net translocation of glucose transporters from an intracellular reserve pool to the plasma membrane, which accounts for greater than 60% of the increment in glucose transport.

Human recombinant interleukin-2 is mitogenic to human lymphocytes.

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.

Chemiluminescence and lymphocyte proliferation: parallelism in collaboration between subpopulations of thymus cells for both types of responses.

Rat thymocytes respond to exposure to phytomitogens by oxidant generation as detected by chemiluminescence in presence of luminol. Maximal chemiluminescent response to a wide variety of plant lectins was obtained only after the cells had been incubated for 18 hours at 37 degrees C but not at 4 degrees C. This temperature dependence and the necessity for intact protein-and RNA-synthetic machinery during the incubation period indicate the occurrence of differentiation of thymocytes as they develop the capacity for chemiluminescent response. Furthermore, adherent-phagocytic cells play an essential collaborative role during this differentiation. A remarkable parallelism was shown to exist in the capacity of a cell subset to respond to concanavalin A by DNA synthesis and the ability of the same subset to respond by chemiluminescence. The latest-sedimenting small lymphocytes after velocity sedimentation of thymus cells develop the capacity for DNA-synthetic as well as chemiluminescent responses to concanavalin A only if allowed to collaborate with a population of early-sedimenting adherent-phagocytic cells.

Tuberous sclerosis with striking renal involvement in a family.

We describe five cases of tuberous sclerosis in members of one family, all having renal involvement but with differences in age of onset and mode of presentation. Clinical, laboratory, and pathologic features helpful in diagnosing this condition and in distinguishing it from polycystic kidney disease and renal neoplasms are stressed. Tuberous sclerosis should always be considered in differential diagnosis of patients with multiple cystic renal lesions, particularly when age of onset of symptoms ranges from infancy to adult life in different members of one family. Absence both of specific glomerular or tubular lesions in ultrastructure and of major abnormalities in renal tubular function supports the existing concept that replacement of nephrons by hamartomatous lesions is the cause of progressive renal failure.

Renal prostaglandins and the regulation of blood pressure and sodium and water homeostasis.

In addition to its well known prohypertensive role in various states of experimental and human hypertension, the kidney has also been shown to exert an antihypertensive "endocrine" function. According to this hypothesis, certain forms of experimental and human hypertension might not solely be the result of an excess in the activity of such renal pressor systems as the renin-angiotensin system and the sympathetic nervous system, but might also result from an absolute or relative deficiency of intra-renal vasodilator antihypertensive factors which might allow pressor systems to act unopposed to produce peripheral arteriolar vasoconstriction and sustained hypertension. At least four factors have been characterized in the kidney of various animal species and man which might be responsible for such an antihypertensive function. These are (1) the renomedullary prostaglandins (PGs), (2) the renomedullary antihypertensive neutral lipid, (3) antirenin phospholipid and (4) the renal kinins. This review is restricted to an examination of the possibility that the vasodepressor renomedullary prostaglandins (PGA and/or PGE) may, at least in part, mediate the so-called antihypertensive function of the kidney and participate in the regulation of renal blood flow and natriuresis by physiologic antagonism of various renal vasoconstrictor stimuli such as the renal renin-angiotensin and the sympathetic nervous systems.

Influence of separation techniques on the distribution and function of lymphocyte subpopulations. A comparison of three techniques.

The effect, if any, of three different lymphocyte separation techniques on the composition and functional characteristics of the purified cell suspensions has been studied. Each separation technique was shown to yield a cell population with highly specific and reproducible characteristics. The Ficoll-Hypaque technique led to good lymphocyte yields but low yields of sheep erythrocyte-rosetting (E-rosetting) T cells, and the separated cell population responded least well to phytohemagglutinin. The glass sand filtration technique led to lowest overall yield of small lymphocytes and of EAC-rosetting cells. There was significantly lower total yield of E-rosetting T cell as well, but the separated lymphocyte suspension had excellent purity, had relatively high percentage of E-rosetting T cells, and they responded extremely well to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). The Technicon separation involving magneticremoval of phagocytic cells by exposure to iron particles consistently led to large yields of small lymphocytes with good purity, the largest total harvests of E-rosetting T cells, as well as EAC-rosetting cells while the separated population had the highest percentage of E-rosetting cells and responded very well to PHA and PWM. These results show that lymphocyte losses during purification are not nonspecific and that the choice of the separation technique profoundly affects the characteristics of the purified lymphocyte population obtainable.

Functional characteristics of monocytes. 1. Essential role in the transformational response of human blood lymphocytes to phytomitogens.

Phagocytic glass-adherent mononuclear cells in peripheral human blood (morphologically monocytes) have been shown to play an essential role in lymphocyte transformational response to phytomitogens. Lymphocytes from peripheral blood have been exhaustively depleted of monocytes by successive treatments, first with opsonized iron particles with eventual magnetic elimination of phagocytic cells, followed by filtration of the same population through packed glass-sand columns. This subpopulation, enriched in T lymphocytes and containing around 4% B lymphocytes, does not transform in response to concanavalin A, phytohemagglutinin, or pokeweek mitogen unless viable monocytes are added to the culture. Supernatant fluids from monocyte-enriched human blood leukocyte cultures can substitute for monocytes in restoring response. Interaction of the phytomitogen with lymphocyte, while essential, is not sufficient to trigger transformation, requiring a second signal provided by the monocyte probably via release of soluble mediators.

Functional characteristics of monocytes. II. Further observations on lymphocyte-monocyte collaboration in the response to phytomitogens.

Monocytes play a crucial role in the response of lymphocytes to phytomitogens. This role is evident during the very early events after exposure to the mitogen. Thus, significant lymphocyte transformation does occur when monocytes are removed from the culture within a few hours after exposure to mitogen, and the presence of monocytes after the initial period is no longer necessary. Monocytes appear to be required for the early increase in protein synthesis and burst in RNA synthesis following exposure to the mitogen. Monocytes may help in lymphocyte responses partly by longer range, by way of release of soluble humoral factors into culture supernatant fluids. Temperature dependence of the effect of pulse exposure of lymphocytes to these supernatants raises the possibility that membrane receptors on these cells to humoral factors released by monocytes may exist. Moncytes do not appear to act primarily by preserving the viability of lymphocytes in culture.

Effects of diphtheria toxin and other exotoxins on oxidant generation by human and murine phagocytes.

Bacterial exotoxins such as diphtheria toxin (D.T.), staphylococcal alpha toxin and Leucocidin can powerfully activate granulocytes and macrophages as detected by production of chemiluminescence in presence of Luminol. Production of superoxide by granulocytes and of prostaglandin E2 in macrophages is also stimulated by D.T. In contrast with the known resistance of rodent parenchymal cells to the diphtheria toxin, human and rodent leucocytes have similar sensitivities to D.T.

Acute renal failure requiring dialysis after allogeneic blood and marrow transplantation identifies very poor prognosis patients.

We examined the incidence, risk factors and associated mortality of acute renal failure requiring dialysis (Renal Bearman Grade [BG] 3) in a 3-year cohort of 97 consecutive allogeneic blood and marrow transplantation (alloBMT) patients. In all, 20 (21%) developed Renal BG3 (all died by day +132) and 77 (79%) developed renal insufficiency (Renal BG1-2). Renal BG3 was a contributing or primary cause of death in 18 (90%) patients who continued to require dialysis at time of death. The two Renal BG3 patients whose deaths were not related to renal failure died on day +103 of hemorrhage and day +132 of underlying disease. By univariate analysis, age, unrelated donor, veno-occlusive disease (VOD) and grade III-IV acute graft-versus-host disease with hepatic involvement were significantly associated with Renal BG3. The multivariate model of time to Renal BG3 determined only a prior diagnosis of severe acute GVHD (RR=4.1, 95% CI 1.6-10.3, P=0.003) and VOD (RR=9.1, 95% CI 3.5-23.7, P<0.001) as significant independent predictors. Renal BG3 is generally considered a conditioning regimen-related toxicity. This study demonstrates that Renal BG3 is most commonly a complication of hepatic co-morbidities after allogeneic blood and marrow transplantation and identifies patients with a very poor prognosis.

Case report: mesangial IgA-IgG deposition in mixed cryoglobulinemia.

Glomerular deposits of immunoglobulin A (IgA) (and immunoglobulin G [IgG]) has been described in IgA-nephropathy (Berger's disease), in anaphylactoid purpura, and systemic lupus erythematosus. The pathogenesis of mesangial IgA deposits in these disease states is unclear. Circulating immune complexes have been speculated to be involved although their existence in blood in IgA nephropathies have never been reported. We describe a 53-year-old man who presented with a petechial rash in lower extremities accompanied by painful swelling of left ankle and wrist. Polyclonal elevation of IgA and IgG in serum was found, along with cryoglobulins containing IgA and IgG (rheumatoid factor positive). After two years, hematuria, mild proteinuria, and active urinary sediment developed and renal biopsy revealed focal proliferative changes. Immunofluorescence revealed predominantly mesangial deposits of IgA and IgG but no IgM nor complement. This experience suggests that circulating complexes consisting of IgA and IgG may lead to renal lesions presenting clinically and histologically in a way similar to that commonly seen in IgA nephropathies.

Hypertension in the elderly.

Successful control of blood pressure through drug therapy can reduce morbidity and mortality in the elderly population. However, age-related factors also make this group prone to adverse side effects. A cautious approach is therefore recommended in the effort to achieve ideal control of blood pressure.

Effects of cytochalasins on lymphocytes: mechanism of inhibition of rosette formation.

The mechanisms operative in the inhibition by cytochalasins of human peripheral blood T lymphocytic rosette formation with sheep erythrocytes remain obscure in the light of the multiplicity of biologic effects of cytochalasins. Moreover, we have shown the existence of three distinct classes of cytochalasin-binding sites (H-, M-, and L-sites) in such lymphocytes (J. Biol. Chem. 256:1290-1300, 1981). We have, therefore, explored the mechanism of rosette inhibition and present evidence that shows: a) Inhibition of rosetting is not caused by inhibition of glucose transport in lymphocytes; b) cytochalasin binding to the H- and M-sites, both integral plasma membrane proteins, is not involved in the effect; c) nonspecific partitioning of cytochalasins in the plasma membrane lipids of lymphocytes appears unlikely to explain the effect; d) evidence presented in this paper strongly suggests that cytochalasin binding to the actin associated L-site mediates the inhibition of rosetting. We conclude that cytoskeletal microfilaments play a critical role in the normal functioning of cell surface receptors for binding to sheep erythrocytes.

Hemodialysis for phenformin associated lactic acidosis.

A patient who attempted suicide by ingesting a large amount of phenformin developed a severe lactic acidosis. After receiving hemodialysis on two successive days, she recovered without sequela. On both occasions, the dialyses temporarily improved the degree of acidosis. The amount of phenformin extracted during these two procedures was measured and found to be only 1.5% of the total amount of drug ingested. Therefore, it is felt that hemodialysis does not remove the offending drug but does play a supportive role in the management of these patients by temporarily ameliorating some of the biochemical abnormalities.

Inhibitory effect of furosemide on glucose transport.

The carrier-mediated glucose transport function in the intact human erythrocytes and also that of their isolated membranes is markedly inhibited in the presence of furosemide in vitro. A noncompetitive process is believed to be involved since the transport inhibition was not affected by the presence of high concentrations of glucose in the medium. The inhibition developed slowly over 2 hours. Most of the inhibition was slowly reversed during a 3-hour incubation in medium free of the drug. It is concluded that furosemide inhibits the glucose transport function directly in absence of insulin-mediated transduction mechanism and independently of any interference with intracellular glycolysis.

Effects of cytochalasins on lymphocytes: some distinctive features of cytochalasin-E.

Cytochalasin-E (CE) has specific properties that distinguishes it from other cytochalasin congeners. We have taken advantage of these in investigating the mechanisms operative in the effects of cytochalasins on lymphocyte proliferative responses to phytomitogens. Like the other cytochalasins, CE inhibits these responses only when present during the early phases of exposure of lymphocytes to mitogens, but not when added later on. The effects of CE are irreversible since prior incubation of lymphocyte in CE renders them incapable of response. Unlike the effects of cytochalasin A, the only other irreversibly active congener, lymphocytes preincubated in CE completely recover the ability to respond if they are cultured in cytochalasin-free medium for 48 hours. Unlike cytochalasins A and B, cytochalasin-E does not inhibit glucose transport into lymphocytes and all. We have shown that human lymphocytes bind cytochalasins at 3 distinct classes of sites named L, M, and H (J. Biol. Chem. 256:1290-1300, 1981). CE binds irreversibly to the L and H-sites on the short to medium term but does not bind to the glucose displaceable M-site at all. CE may have potential usefulness as affinity label towards isolation of specific binding sites since the chemical structure offers feasible approaches towards isotopic labelling.

The effects of cytochalasins on lymphocytes: V. Interaction of trifluoperazine and cytochalasin B in inhibition of human lymphocyte proliferation.

Trifluoperazine (TFP), a phenothiazine derivative, is known to inhibit calmodulin-mediated phenomena. We report here that TFP reversibly inhibited lymphocyte proliferative responses to mitogenic lectins. This inhibition was observed only when TFP was added during the early stages of exposure of lymphocytes to the stimulus. Furthermore, at suboptimally inhibitory concentrations of each compound, effects of TFP on lymphocyte proliferation were additive to those of cytochalasin B (CB). Incubation of lymphocytes in TFP (10(-5)-10(-4) M) markedly inhibited cytochalasin B binding to the actin associated, low affinity binding site without affecting its binding to the high affinity site or to the medium affinity site. This effect developed gradually during incubation with TFP, becoming demonstrable after 30 minutes reaching maximum after 30-60 min of incubation at 37 degrees. The findings suggest the occurrence of an interaction of TFP with the lymphocyte cytoskeleton, which may play a role in the impairment in the transmission of the mitogenic signal.