Specialized transduction of pro genes by coliphage P1: Structure of a partly diploid P1-pro prophage

Bacteria carrying P1-pro hybrid prophages have been isolated as rare (10—⁹Pro⁺ per pfu) Pro⁺ transductants of recA proA Escherichia coli. The proA gene is inserted into the P1 prophage since the transductants become Pro^? when the resident P1 is lost. Furthermore, when induced, these recA transductants produce HFT pro lysates which contain defective pro-transducing particles as well as plaque-forming particles which do not transduce pro. The number of PFUs obtained is about one-tenth the number obtained from normal lysogens. The density of the transducing particles was not distinguishable from that of the PFUs. The Pro⁺ transductants obtained by use of these HFT pro lysates (at low m.o.i.) carry only a small segment of the P1 genome, including cam and gene 2, and are not P1-immune. The number of Pro⁺ transductants increases about 20-fold when the recipient contains a helper P1 either as superinfecting phage or as prophage, The Pro⁺ transductants that arise in the presence of helper P1 are P1-pro lysogens formed by recombination between the DNA of the helper P1 and of the transducing particles. Like the original P1-pro lysogens, they produce lysates which contain plaque-forming and pro transducing particles. The circular P1-pro prophage is inferred to be oversized (at least as large in size as a P1 headful) and to have duplicate cam-gene 2 regions, one copy at each junction of P1 and Pro DNA. The DNA packaged after induction of P1-pro lysogens is not terminally redundant. However, after injection into recipient bacteria, recombination can occur between the redundant cam-gene 2 regions giving rise to either circular pro-cam-gene 2 transducing DNA or to circular P1 plaque-forming DNA. Evidence for this partly diploid prophage is the construction of P1-pro prophages which are heterozygous for gene 2.     

A kinetic analysis of the facilitatory action of adrenaline

The²²Na⁺-efflux from skeletal muscle cells of frog (Rana nigromaculata) was measured in Ringer solutions containing different concentrations of K⁺ (0.1 to 30 mM). The effects of adrenaline (30 M) and ouabain (10 M) on the²²Na⁺-efflux were investigated for the purpose to clarify the mechanism of the facilitatory effect of adrenaline on Na⁺–K⁺ pump. The rate coefficient for the ouabain-sensitive²²Na⁺-efflux increases with increasing extracellular K⁺ concentrations and adrenaline potently facilitates these rate coefficients. On the basis of Michaelis-Menten type kinetics assumed for the reaction between pump site and extracellular K⁺, it is concluded that adrenaline decreases the dissociation constant (K_m), and increases the maximum Na⁺-efflux.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Na+/H+ countertransport and calcium exchange in peripheral blood cells of healthy subjects and patients with chronic heart failure

A comparative study is performed of Na⁺/H⁺ exchange and Ca²⁻ mobilization in erythrocytes and platelets of patients with stage I–II chronic heart failure caused by dilative cardiomyopathy and ischemic heart disease. A significant rise in the Na⁺/H⁺ exchange rate is found in the cells of chronic heart failure patients, which correlates with an elevated erythrocyte and platelet concentration of Ca²⁺ and an increased “calcium” response of platelets to inductors. The findings testify to a certain functional relationship between various cation-transporting cellular systems whose change in properties upon chronic heart failure can play an important pathogenic role. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 572–575, December, 1994     

Isolation and characteristics of high-frequency genetic donors of a serotyped strain of Escherichia coli

As a result of crossing cells of donor strainEscherichia coli K-12 (P4X) with serotyped (group 0100) recipient cellsE. coli trp^–lac^–, nine recombinants possessing sex factor and ability to carry out chromosome transfer with high frequency were isolated. The isolated strains of donor cells carry sex factor in the integrated state and retain their membership of serogroup 0100.Patrice Lumumba People's Friendship University, Moscow. Department of Microbiology, Immunology, and Virology, Central Asiatic Pediatric Medical Institute, Ministry of Health of the USSR. Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 2, pp. 106–109, February, 1975.     

Flk-1+Sca-1- mesenchymal stem cells: functional characteristics in vitro and regenerative capacity in vivo

Objectives: Mesenchymal stem cells (MSCs) represent a powerful tool in regenerative medicine because of their differentiation and migration capacities. We aimed to investigate the possibility of Flk-1⁺Sca-1^- mesenchymal stem cells (Flk-1⁺Sca-1^- MSCs) transplantation to repair erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED). Methods: In this study, we isolated Flk-1⁺Sca-1^- MSCs from bone marrow (bMSCs). Then, newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine for the purpose of tracking endogenous Flk-1⁺Sca-1^- MSCs. Eight weeks later, 8 of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group) while another 8 rats were subject to Flk-1⁺Sca-1^- MSCs treatment (DM+MSC group). All rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology. Results: Flk-1⁺Sca-1^- MSCs could differentiate into skeletal muscle cells and endothelial cells in vivo and in vitro. Engrafted Flk-1⁺Sca-1^- MSCs were shown to home to injured muscle, participate in myofibers repair and could partially reconstitute the sarcolemmal expression of myocardin and ameliorate the level of related specific pathological markers. Conclusion: Flk-1⁺Sca-1^- MSCs could be used in the treatment erectile function in diabetes mellitus associated erectile dysfunction by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis.     

Enterotoxic activity of living cultures of Shigella sonnei and enterotoxin formation in vivo

The overwhelming majority of virulent strains ofShigella sonnei caused the accumulation of fluid in the lumen of an isolated segment of rabbit small intestine; the fluid contained large quantities of mucus and sometimes blood; the mucous membrane of the segment was hyperemic and had petechial hemorrhages. Avirulent strains ofSh. sonnei as a rule did not cause exudation into the loop of intestine. The sterile and concentrated contents of the intestinal loops of rabbits responding to injection of the virulent strain ofSh. sonnei or a toxigenic strain ofShigella shigae invariably gave a positive reaction in other rabbits. The character of the exudate and the changes in the mucous membrane under these circumstances were indistinguishable from those following injection of living cultures.Microbiological Department, Moscow Research Institute of Epidemiology and Microbiology. (Presented by Academician of the Academy of Medical Sciences of the USSR P. N. Kosyakov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 703–705, December, 1977.     

„tet-gene pick up”︁ durch ein F-Plasmid in Proteus mirabilis

The Proteus mirabilis strains PG193 (his^- tet⁺ rec⁺), PG273 (thr^- tet⁺ rec⁺), and PG672 (thr^- tet⁺ rec⁺) are full resistant to 40 μg tetracyclin/ml, similar to other 513 wild type strains tested. The F-plasmid was introduced from Escherichia coli WF⁺ into these strains and into the tet^- P. mirabilis Pm2098 by conjugation. Pure F⁺ P. mirabilis strains are sensitive to the F-specific phage fr. The transfer of the F-plasmid from F⁺ P. mirabilis back to E. coli K12 strains occurs with high frequency. In several cases the F-plasmid is able to transfer the tet-gene from tet⁺ rec⁺ P. mirabilis strains (PG193, PG273) to E. coli recipients but not from F⁺ P. mirabilis tet⁺ rec^- (PG672) or from F⁺ P. mirabilis tet^- strains (Pm2098). E. coli tet⁺ recombinants are sensitive to the F-specific phage fr. They harbor F-plasmids stably linked to the tet-gene. Such recombinant plasmids are transferable with high frequency to other F^-R^- members of the Enterobacteriaceae but with reduced frequency of transmission to E. coli Hfr(H) indicating a superinfection immunity. These results coincide with the Campbell modell and point to the fact that the “gene pick up” may be the (or one) way of R-plasmid origin as proposed by Watanabe (1963).     

Cytopathogenic action of strains of Escherichia coli containing similar heterogenetic antigens of the AB0 type on human cell cultures

The character of interaction between different strains ofEscherichia coli serotype O26 and cells of continuous cultures of human strains HeLa, Tg-33, and RH was studied in vitro. The phenomenon of cytopathogenic action (CPA) of uropathogenic strains ofE. coli containing heterogenetic type O(H) and B antigens on human cell strains with the corresponding isoantigens was detected after interaction for 6 h. The number of dead cells in these cultures was 1.5–3 times greater than their number in control cultures to whichE. coli cells not containing heterogenetic antigens or containing dissimilar heterogenetic antigens of the human AB0 type were added. It is postulated that this phenomenon plays an important role in the development of chronic forms of colibacillary pyelonephritis.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 568–570, May, 1976.     

[Na+] i /[K+] i -independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression

The cytotoxic effect of long-term exposure of renal epithelial cells to ouabain and other cardiotonic steroids (CTS) is mediated by the interaction of these compounds with Na⁺,K⁺-ATPase but is independent of the inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes. Sustained application of CTS also leads to Na⁺,K⁺-ATPase endocytosis and its translocation into the nuclei that might trigger the cell death machinery via the regulation of gene expression. This study examines the role of Na⁺,K⁺-ATPase internalization and de novo gene expression in the death of ouabain-treated C7-Madin–Darby canine kidney (MDCK) cells derived from distal tubules of the MDCK. In these cells, 6-h exposure to 3 μM ouabain led to the internalization of ∼50% of plasmalemmal Na⁺,K⁺-ATPase. Prolonged incubation in a K⁺-free medium abolished ouabain-induced Na⁺,K⁺-ATPase internalization but did not affect the cytotoxic action of ouabain seen after 18-h incubation. Previously, it was shown that CTS-induced Na⁺,K⁺-ATPase internalization is mediated by its interaction with Src within caveolae. Neither caveolae damage by cholesterol depletion with methyl-β-cyclodextrin nor Src inhibition with 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyridine affected the death of ouabain-treated C7-MDCK cells. Actinomycin D at the 0.1-μg/ml concentration almost completely abolished ribonucleic acid synthesis but did not protect C7-MDCK cells from the cytotoxic action of ouabain. Our results show that neither Na⁺,K⁺-ATPase endocytosis nor de novo gene expression contributes to -independent cell death signaling evoked by prolonged exposure to CTS.     

Characteristics of Expanded CD4+CD28null T Cells in Patients with Chronic Hepatitis B

Expanded CD4⁺CD28^null T cells have not been described in the circulation of patients with chronic hepatitis B (CHB). The aim of the present was to detect and characterize the surface phenotype and functional capacity of these cells in CHB patients. Expanded CD4⁺CD28^null T cells were detected in the circulation of CHB patients with high viral load and elevated aminotransferase levels. Most CD4⁺CD28^null T cells showed a CD27?CD45RA^?CD45RO⁺ surface phenotype. The markers CD56, CD57 and killer immunoglobulin-like receptor (KIR) were detected on CD4⁺CD28^null T cells, but the majority were positive for CD57. Functionally, CD4⁺CD28^null T cells were found to be potent cytotoxic T lymphocytes with perforin and granzyme B secretion profiles. These findings indicate that the expanded CD4⁺CD28^null T cells are cytotoxic memory T cells and display a distinct functional phenotype in comparison with CD4⁺CD28⁺ T cells. The presence of these cells appears to be associated with inflammatory conditions, suggesting that these elevated CD4⁺CD28^null T cells might be involved in the pathogenesis of CHB.     

Interspersed normoxia during live high, train low interventions reverses an early reduction in muscle Na+, K+ATPase activity in well-trained athletes

Hypoxia and exercise each modulate muscle Na⁺, K⁺ATPase activity. We investigated the effects on muscle Na⁺, K⁺ATPase activity of only 5 nights of live high, train low hypoxia (LHTL), 20 nights consecutive (LHTLc) versus intermittent LHTL (LHTLi), and acute sprint exercise. Thirty-three athletes were assigned to control (CON, n = 11), 20-nights LHTLc (n = 12) or 20-nights LHTLi (4 × 5-nights LHTL interspersed with 2-nights CON, n = 10) groups. LHTLc and LHTLi slept at a simulated altitude of 2,650 m (F_IO₂ 0.1627) and lived and trained by day under normoxic conditions; CON lived, trained, and slept in normoxia. A quadriceps muscle biopsy was taken at rest and immediately after standardised sprint exercise, before (Pre) and after 5-nights (d5) and 20-nights (Post) LHTL interventions and analysed for Na⁺, K⁺ATPase maximal activity (3-O-MFPase) and content ([³H]-ouabain binding). After only 5-nights LHTLc, muscle 3-O-MFPase activity declined by 2% (P < 0.05). In LHTLc, 3-O-MFPase activity remained below Pre after 20 nights. In contrast, in LHTLi, this small initial decrease was reversed after 20 nights, with restoration of 3-O-MFPase activity to Pre-intervention levels. Plasma [K⁺] was unaltered by any LHTL. After acute sprint exercise 3-O-MFPase activity was reduced (12.9 ± 4.0%, P < 0.05), but [³H]-ouabain binding was unchanged. In conclusion, maximal Na⁺, K⁺ATPase activity declined after only 5-nights LHTL, but the inclusion of additional interspersed normoxic nights reversed this effect, despite athletes receiving the same amount of hypoxic exposure. There were no effects of consecutive or intermittent nightly LHTL on the acute decrease in Na⁺, K⁺ATPase activity with sprint exercise effects or on plasma [K⁺] during exercise.     

Effect of Clostridium perfringens type a toxin and metabolic products of Clostridium butyricum on liver lysosomes in vitro

During incubation of the lysosomal fraction of the albino mouse liver withClostridium perfringens type A toxin and also with the toxin and filtrate of a broth culture ofClostridium butyricum, and increase in the specific acid phosphatase activity was observed. The action ofC. perfringer toxin on the lysosomal membrane was potentiated under the influence of metabolic products ofC. butyricum. Potentiation of the action ofC. perfringens toxin on the lysosomes was due to thermostable substances in theC. butyricum filtrate.Department of Microbiology, N. I. Pirogov Odessa Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 8, pp. 181–183, August, 1979.     

Intracellular pH regulation in cultured rat astrocytes in CO2/HCO 3 − -containing media

We studied the regulation of intracellular pH (pH_i) and the mechanisms of pH_i regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2,7-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pH_i was 7.00±0.02 in HCO ₃ ^- containing solutions at an extracellular pH of 7.35. Addition of 4, 4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) or amiloride decreased pH_i, as did removal of extracellular Na⁺, while removal of extracellular Cl^- was followed by an increase in pH_i. Following exposure to an acid transient induced by increasing the CO₂ content from 5 to 15%, pH_i rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min^-1 (corresponding to a mean acid extrusion rate of 6.3±0.36 mmolo 1^-1 min^-1). Regulation of pH_i was impaired when either amiloride or DIDS was added or Cl^- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pH_i regulation was completely blocked in the absence of extracellular Na⁺. The rapid regulation of pH_i normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na⁺, but was partially inhibited by DIDS and by the absence of extracellular Cl^-. The results are compatible with the presence of at least three different pH_i-regulating mechanisms: a Na⁺/H⁺ antiporter, a Na⁺-dependent HCO ₃ ^- /Cl^- exchanger (both regulating pH_i during a transient acidification), and a passive Cl^-/HCO ₃ ^- exchanger (regulating pH_i during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na⁺/HCO ₃ ^- symporter.     

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis

Objective: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4⁺CD25⁺ T cells in experimental autoimmune myocarditis (EAM).

Methods: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4⁺CD25⁺ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4⁺CD25⁺ T cells in vitro. Relative mRNA level of Foxp3 and TGF-β was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4⁺CD25⁺ T_regs.

Results: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad–CMV–CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4⁺CD25⁺ T_regs significantly. T cell proliferation was greatly inhibited in the CTLA4Ig group compared with the untreated and EGFP-treated groups in vitro. CTLA-4 and B7-2 proteins were down-regulated in the CTLA4Ig group, Foxp3 and TGF-β mRNA was up-regulated significantly by CTLA4Ig treatment.

Conclusions: Adenovirus vector-mediated CTLA4Ig gene transfer alleviated inflammation in EAM, one of the potential mechanisms is up-regulation of CD4⁺CD25⁺ T_regs.     

Potassium and sodium ions in the nerve tissue of emotionally overstrained rabbits

Measurement of K⁺ and Na⁺ concentrations in samples of individual brain nuclei and in ganglia of the autonomic nervous system from rabbits subjected to severe emotional stress (ES) through aperiodic stimulation of ventromedial hypothalamic nuclei and electrocutaneous stimulation revealed significantly altered levels of these ions in locus ceruleus samples from animals predisposed to ES-induced cardiovascular disorders and in samples of neurons of the caudal part of the brainstem from those resistant to such disorders. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, No 8, pp. 129–131, August, 1994 Presented by K. V. Sudakov, Member of the Russian Academy of Medical Sciences     

Rhythmic Cl− current and physiological roles of the intestinal c-kit-positive cells

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit ⁺) cells when studied immuno-histochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c -kit ⁺ cells, we studied the excitability of intestinal c -kit ⁺ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltageclamp at –40 mV, the majority of c -kif ⁺cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263±24 pA and 2.30±0.25 cycles/min (mean±SEM), respectively. Intracellular perfusion of the c -kit ⁺ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca²⁺-free external solution or low holding voltage (<-60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl^–(E _Cl ) Moreover the rhythmic current was depressed by a Cl^– channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2-disulphonic acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca²⁺-activated K⁺current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca²⁺-activated K⁺nor rhythmic Cl^– currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c -kit ⁺ cells, but not the smooth muscle cells, possess a rhythmic Cl^– current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.     

Independent integration of genes controlling the invasive properties and streptomycin resistance of enteropathogenic strain Escherichia coli 0124

CrossingEscherichia Coli K12 Hfr AB313 with an enteropathogenic strain ofE. coli of the serological group 0124 yielded recombinants which had lost their invasiveness. The loss of invasiveness of these recombinants was not due to the acquisition of genes controlling resistance to streptomycin.I. P. Pavlov First Leningrad Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1144–1145, September, 1976.     

Dependence of the electrogenic pump current of Xenopus oocytes on external potassium

The membrane potential of Xenopus oocytes showed a variable response to an increase of the K⁺ concentration in the bathing solution, [K⁺]_e, from 2.5 mM to 20 mM. In 54% of the cases (n=52) the cells hyperpolarized (by max. 70 mV). In the presence of 10^–5 M ouabain, all cells depolarized suggesting that the hyperpolarization was caused by an electrogenic Na⁺/K⁺ pump. In cells stored overnight in a Na⁺-free solution the transition from 2.5 to 20 mM [K⁺]_e always caused depolarization indicating that the stimulation of the pump requires high internal sodium, [Na⁺]_i. Cells stored overnight in a Na⁺-rich solution had a [Na⁺]_i of 30.7±7 mM, i.e. the Na⁺/K⁺ pump was saturated with sodium (Lafaire and Schwarz 1986). With 9 such cells we determined the K⁺ activation of the Na⁺/K⁺ pump. The activation follows Hill kinetics with I_max=90.5 nA, K_s=2.3 mM, and n=1.68.     

NPPB block of Ca++-activated Cl− currents in Xenopus oocytes

NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), a mentoer of the novel class of Cl^– channel blockers related to diphenylamine-2-carboxylate, was studied for its effect on the Ca⁺⁺-activated Cl^– current (I_Cl(Ca)) in frog (Xenopus laevis) oocytes. I_Cl(Ca) was activated by bath application of 5 mM Ca⁺⁺ to oocytes that had been Ca⁺⁺ permeabilized with the Ca⁺⁺ ionophore A23187. In order to prevent the incativation of I_Cl(Ca)) that occurs with repetitive applications of Ca⁺⁺, oocytes were also treated with H-7 (1,5-isoguinolinesulfonyl-1,2 methylpiperazine), an inhibitor of protein kinases. NPPB reversibly blocked both the fast transient (I_fast) and slow (I_slow) components of I_Cl(Ca) with inhibition constants or 22 M and 68 M, respectively. NPPB block of I_Cl(Ca) was voltage dependent and potentiated by depolarization of the oocyte membrane. Our results indicate that NPPB is a potent blocker of the oocyte endogenous I_Cl(Ca) and may prove useful in the study of exogenously expressed Cl channels.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.