Subnormothermic preservation in somah: a novel approach for enhanced functional resuscitation of donor hearts for transplant.

Organ preservation at 4°C results in temporally irreversible injury to cellular structure and function. This study was designed to evaluate the possibility of storing hearts at ambient temperatures in novel organ preservation solution Somah to prevent damage and preserve optimum function by maintaining cellular energy over the temperature range of storage. Porcine hearts were stored in Celsior at 4°C and Somah at 4°C, 13°C and 21°C for 5 h thereafter reperfused and reanimated in vitro for 3 h. Heart weights, histopathology, ultrastructure and 2-dimensional echocardiography (2D-Echo) assessments showed preservation of structure in Somah groups. Tissue high-energy phosphate levels in Somah groups after storage were significantly greater than the Celsior hearts (p < 0.05) and highest in the 21°C Somah hearts. Upon reperfusion, myocardial O2 consumption and lactate levels quickly achieved steady state in 21°C hearts, but were delayed in Somah 4/13°C groups and severely depressed in the Celsior group. Inotrope and electroconversion requirements were inversely related to storage temperature. In vitro 2D Echo demonstrated a discordantly attenuated function in the Celsior group, moderate functionality in 4°C Somah group and superior reestablishment of performance in the Somah higher temperature groups. Hearts stored in Somah at 21°C were metabolically and functionally superior to any other groups.

Further evaluation of Somah: long-term preservation, temperature effect, and prevention of ischemia-reperfusion injury in rat hearts harvested after cardiocirculatory death.

OBJECTIVE: To identify and evaluate the ideal temperature for long-term storage of hearts from donation after cardiocirculatory death, in the novel organ preservation solution Somah. METHODS: DCD hearts from Sprague-Dawley rats were harvested after 30 minutes of euthanasia, preserved in Somah at 4°C, 10°C, 21°C, or 37°C for 24 hours and then reperfused with blood:Somah (3:1) perfusate at 37°C for 30 minutes. Myocardial biopsies were taken during storage and before and after reperfusion to assess the structural and functional viability of tissue using multiphoton imaging, biochemistry, and immunofluorescence. RESULTS: Myocyte viability, determined by Live-Dead and esterase assays, was similar at 4°C, 10°C, and 21°C (193, 198 and 217 normalized fluorescence counts [NFC]) with a significant decrease at 37°C (131 NFC). Upon reperfusion, esterase activity was enhanced in DCD hearts stored in Somah at 21°C but noticeably decreased at all other temperatures. High-energy adenosine triphosphate/creatine phosphate (ATP/CP) syntheses and the expression of structural/contractile proteins was well preserved at 21°C, both after 24-hour storage and upon reperfusion. In contrast, hearts stored at all other temperatures demonstrated variable degenerative changes, loss of protein expression, and/or deranged ATP/CP synthesis after 24 hours of storage and/or upon reperfusion. CONCLUSION: The robust maintenance of structural/functional integrity of cardiac tissue and the preservation of protein expression and cellular energy metabolism in DCD hearts after long-term preservation at subnormothermic temperature suggests that 21°C is ideal for long-term storage of DCD hearts in Somah solution.

Neural and anatomical abnormalities of the gastrointestinal system resulting from contusion spinal cord injury.

Gastrointestinal (GI) abnormalities resulting from spinal cord injury (SCI) are challenging disorders that have not been examined experimentally using clinically relevant models. In this study, female Sprague-Dawley rats (n=5/groupx4: T10-T11 contusion, laminectomy, or naïve) were fasted for 24 h before being submitted to dye recovery assays (Phenol Red solution, 1.5 ml/rat; per oral) on GI emptying/transiting at 48 h or 4 weeks postinjury (p.i.). Compared with controls, SCI significantly increased dye recovery rate (DRR, determined by spectrophotometry) in the duodenum (+84.6%) and stomach (+32.6%), but decreased it in the jejunum (-64.1% and -49.5%) and ileum (-73.6% and -70.1%) at 48 h and 4 weeks p.i., respectively (P

Nonthermal effects of radiofrequency-field exposure on calcium dynamics in stem cell-derived neuronal cells: elucidation of calcium pathways.

Intracellular Ca(2+) spikes trigger cell proliferation, differentiation and cytoskeletal reorganization. In addition to Ca(2+) spiking that can be initiated by a ligand binding to its receptor, exposure to electromagnetic stimuli has also been shown to alter Ca(2+) dynamics. Using neuronal cells differentiated from a mouse embryonic stem cell line and a custom-built, frequency-tunable applicator, we examined in real time the altered Ca(2+) dynamics and observed increases in the cytosolic Ca(2+) in response to nonthermal radiofrequency (RF)-radiation exposure of cells from 700 to 1100 MHz. While about 60% of control cells (not exposed to RF radiation) were observed to exhibit about five spontaneous Ca(2+) spikes per cell in 60 min, exposure of cells to an 800 MHz, 0.5 W/kg RF radiation, for example, significantly increased the number of Ca(2+) spikes to 15.7+/-0.8 (P<0.05). The increase in the Ca(2+) spiking activities was dependent on the frequency but not on the SAR between 0.5 to 5 W/kg. Using pharmacological agents, it was found that both the N-type Ca(2+) channels and phospholipase C enzymes appear to be involved in mediating increased Ca(2+) spiking. Interestingly, microfilament disruption also prevented the Ca(2+) spikes. Regulation of Ca(2+) dynamics by external physical stimulation such as RF radiation may provide a noninvasive and useful tool for modulating the Ca(2+)-dependent cellular and molecular activities of cells seeded in a 3D environment for which only a few techniques are currently available to influence the cells.

Lower esophageal sphincter is achalasic in nNOS(-/-) and hypotensive in W/W(v) mutant mice.

BACKGROUND AND AIMS: It has been proposed that nitrergic nerves mediate lower esophageal sphincter (LES) relaxation with intramuscular interstitial cells of Cajal (ICC-IM) as an intermediary. Dysfunction of the nitrergic pathway has been shown to cause LES hypertension and impaired relaxation in achalasia. We determined whether mice with neuronal nitric oxide synthase gene disruption (nNOS(-/-)) and W/W(v) mice lacking ICC-IM have achalasia-like LES dysfunction. METHODS: Intraluminal manometry using a customized micro-sized catheter assembly was performed in anesthetized mice. Basal LES pressure and swallow- and vagal-evoked LES relaxations were quantified in wild-type, Nomega-nitro-L-arginine methyl ester HCl salt (L-NAME)-treated, nNOS(-/-), and W/W(v) mice. RESULTS: Wild-type mouse LES maintained a basal pressure (24 +/- 3 mm Hg; N = 8) and relaxed normally to swallow (87% +/- 3%; N = 8) and vagal stimulation (91% +/- 4% mm Hg; N = 6). Pretreatment with L-NAME (100 mg/kg, intravenously) attenuated LES relaxation to both stimuli (P < 0.05). The LES in nNOS(-/-) was significantly hypertensive (36 +/- 5 mm Hg; N = 10; P < 0.05) with a markedly impaired relaxation (P < 0.05). In contrast, W/W(v) mouse LES was significantly hypotensive (11 +/- 2 mm Hg; N = 6; P < 0.05) with normal relaxation that was blocked by L-NAME. CONCLUSIONS: nNOS(-/-) mice have LES hypertension with impaired relaxation resembling achalasia. In contrast, W/W(v) mice have hypotensive LES with unimpaired relaxation, suggesting that ICC-IM do not play a role in nitrergic neurotransmission.

Multi-photon microscopy in the evaluation of human saphenous vein.

BACKGROUND: The use of conventional fluorescence microscopy to image biological systems at the cellular level is limited by its inability to spatially resolve thick tissues. We have applied the technique of multi-photon fluorescence microscopy to study the structure and function of endothelial cells in living human saphenous vein taken from patients undergoing coronary artery bypass surgery. MATERIALS AND METHODS: Vein segments were preserved for 1-4 h to determine the temporal effects of storage. The effect of pH on endothelial and smooth muscle cell viability was examined by storing segments at pH 6.0, 7.4, and 8.0. Calcein-mediated green fluorescence and ethidium homodimer-mediated red fluorescence were used to differentiate cell viability. Increases in diaminofluorescein fluorescence were used to measure bradykinin activation of endothelial nitric oxide synthase (eNOS) with or without N-nitro-l-arginine (L-NNA). Multi-photon imaging was performed with the BioRad MRC1024ES system. RESULTS: Successful imaging of endothelial and smooth muscle cells of vein segments was achieved. Cell viability was well preserved up to 3 h of storage but dramatically decreased after 4 h. Cell viability was maintained at pH 7.4, diminished at pH 8.0, and was completely lost at pH 6.0. A two- to threefold increase in eNOS activity was observed upon activation by bradykinin which was completely inhibited in L-NNA-treated samples. CONCLUSIONS: We have demonstrated the successful application of multi-photon microscopy in imaging and quantifying nitric oxide production and cell viability under various storage conditions in human saphenous veins. This imaging technique allows for the functional imaging of cellular processes and may have diagnostic potential in cardiovascular surgery for patients undergoing bypass operations.

The coronary artery bypass conduit: I. Intraoperative endothelial injury and its implication on graft patency.

Prevention of intraoperative injury to the vascular endothelium is of primary importance in maintaining viability and patency of the aorto-coronary saphenous vein graft. Surgical manipulation, ischemia, storage conditions, and distension before anastomosis can abnormally alter the antithrombogenic property of the endothelium leading to vasospasms, thrombogenesis, occlusive intimal hyperplasia, and stenosis. Endothelial injury can also form an initiation site for the formation of later-stage atheromas and graft failure. A multifactorial strategy aimed at prevention of endothelial injury and graft failure should include improved surgical techniques, optimal preservation conditions, avoidance of nonphysiologic distension pressures, and use of specific pharmacologic agents as the primary form of intervention. The successful application of this strategy, and the development of newer and more efficacious strategies that may impact on long-term graft patency, can now be aided by assessment of the structural and functional integrity of bypass conduits using multiphoton imaging techniques.

Integrin-dependent human macrophage migration induced by oscillatory electrical stimulation.

Electrical stimulation has been used to promote wound healing. The mechanisms by which such stimulation could interact with biological systems to accelerate healing have not been elucidated. One potential mechanism could involve stimulation of macrophage migration to the site of a wound. Here we report that oscillatory electric fields induce human macrophage migration. Macrophages exposed to a 1 Hz, 2 V/cm field show an induced migration velocity of 5.2+/-0.4 x 10(-2) microm/min and a random motility coefficient of 4.8+/-1.4 x 10(-2) microm2/min on a glass substrate. Electric field exposure induces reorganization of microfilaments from ring-like structures at the cell periphery to podosomes that are confined to the contact sites between cell and substrate, suggesting that the cells are crawling on glass. Treatment of cells with monoclonal antibodies directed against beta2-integrins prior to field exposure prevents cell migration, indicating that integrin-dependent signaling pathways are involved. Electric fields cause macrophage migration on laminin or fibronectin coated substrates without inducing podosome formation or changes in cellular morphology. The migration velocity is not significantly altered but the random movement is suppressed, suggesting that cell movements on a laminin- or fibronectin-coated surface are not mediated by cell crawling. It is suggested that electric field-induced macrophage migration utilizes several modes of cell movement, including cell crawling and possibly cell rolling.

Calcium-independent activation of endothelial nitric oxide synthase by ceramide.

The endothelial isoform of NO synthase (eNOS) is targeted to sphingolipid-enriched signal-transducing microdomains in the plasma membrane termed caveolae. Among the caveolae-targeted sphingolipids are the ceramides, a class of acylated sphingosine compounds that have been implicated in diverse cellular responses. We have explored the role of ceramide analogues in eNOS signaling in cultured bovine aortic endothelial cells (BAEC). Addition of the ceramide analogue N-acetylsphingosine (C(2)-ceramide; 5 microM) to intact BAEC leads to a significant increase in NO synthase activity (assayed by using the fluorescent indicator 4,5-diaminofluorescein) and translocation of eNOS from the endothelial cell membrane to intracellular sites (measured by using quantitative immunofluorescence techniques); the biologically inactive ceramide N-acetyldihydrosphingosine is entirely without effect. C(2)-ceramide-induced eNOS activation and translocation are unaffected by the intracellular calcium chelator 1, 2-bis-o-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA). Using the calcium-specific fluorescent indicator fluo-3, we also found that C(2)-ceramide activation of eNOS is unaccompanied by a drug-induced increase in intracellular calcium. These findings stand in sharp contrast to the mechanism by which bradykinin, estradiol, and other mediators acutely activate eNOS, in which a rapid, agonist-promoted increase in intracellular calcium is required. Finally, we show that treatment of BAEC with bradykinin causes a significant increase in cellular ceramide content; the response to bradykinin has an EC(50) of 3 nM and is blocked by the bradykinin B(2)-receptor antagonist HOE140. Bradykinin-induced ceramide generation could represent a mechanism for longer-term regulation of eNOS activity. Our results suggest that ceramide functions independently of Ca(2+)-regulated pathways to promote activation and translocation of eNOS, and that this lipid mediator may represent a physiological regulator of eNOS in vascular endothelial cells.

Chloramphenicol-induced mitochondrial dysfunction is associated with decreased transferrin receptor expression and ferritin synthesis in K562 cells and is unrelated to IRE-IRP interactions.

Chloramphenicol is an antibiotic that consistently suppresses the bone marrow and induces sideroblastic anemia. It is also a rare cause of aplastic anemia. These toxicities are thought to be related to mitochondrial dysfunction, since chloramphenicol inhibits mitochondrial protein synthesis. We hypothesized that chloramphenicol-induced mitochondrial impairment alters the synthesis of ferritin and the transferrin receptor. After treating K562 erythroleukemia cells with a therapeutic dose of chloramphenicol (10 microg/ml) for 4 days, there was a marked decrease in cell surface transferrin receptor expression and de novo ferritin synthesis associated with significant decreases in cytochrome c oxidase activity, ATP levels, respiratory activity, and cell growth. Decreases in the transferrin receptor and ferritin were associated with reduced and unchanged message levels, respectively. The mechanism by which mitochondrial dysfunction alters these important proteins in iron homeostasis is not clear. A global decrease in synthetic processes seems unlikely, since the expression of the cellular adhesion proteins VLA4 and CD58 was not significantly decreased by chloramphenicol, nor were the message levels of beta-actin or ferritin. The alterations were not accompanied by changes in binding of the iron response protein (IRP) to the iron-responsive element (IRE), although cytosolic aconitase activity was reduced by 27% in chloramphenicol-treated cells. A disturbance in iron homeostasis due to alterations in the transferrin receptor and ferritin may explain the hypochromic-microcytic anemia and the accumulation of nonferritin iron in the mitochondria in some individuals after chloramphenicol therapy. Also, these studies provide evidence of a link between mitochondrial impairment and iron metabolism in K562 cells.

Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase.

Although estrogen is known to stimulate nitric oxide synthesis in vascular endothelium, the molecular mechanisms responsible for this effect remain to be elucidated. Using quantitative immunofluorescence imaging approaches, we have investigated the effect of estradiol on the subcellular targeting of endothelial nitric oxide synthase (eNOS) in bovine aortic endothelial cells. In unstimulated endothelial cells, eNOS is predominantly localized at the cell membrane. Within 5 min after the addition of estradiol, most of the eNOS translocates from the membrane to intracellular sites close to the nucleus. On more prolonged exposure to estradiol, most of the eNOS returns to the membrane. This effect of estradiol is evident at a concentration of 1 pM, and a maximal estradiol effect is seen at a concentration of 1 nM. Neither progesterone nor testosterone has any effect on eNOS distribution. After estradiol addition, a transient rise in intracellular Ca2+ concentration precedes eNOS translocation. Both the Ca2+-mobilizing and eNOS-translocating effects of estradiol are completely blocked by the estrogen receptor antagonist ICI 182,780, and the intracellular Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevents estradiol-induced eNOS translocation. Use of the nitric oxide-specific dye diaminofluorescein shows that estradiol treatment increases nitric oxide generation by endothelial cells; this response is blocked by ICI 182,780 and by the eNOS inhibitor Nomega-nitro-L-arginine. These results show that estradiol induces subcellular translocation of eNOS by a rapid, Ca2+-dependent, receptor-mediated mechanism, and they suggest a nongenomic role for estrogen in the modulation of NO-dependent vascular tone.

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.

Receptor-regulated translocation of endothelial nitric-oxide synthase.

The endothelial nitric-oxide synthase (eNOS) is activated by transient increases in intracellular Ca2+ elicited by stimulation of diverse receptors, including bradykinin B2 receptors on endothelial cells. eNOS and B2 receptors are targeted to specialized signal-transducing domains in the plasma membrane termed plasmalemmal caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation, but in resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. We used a quantitative approach exploiting immunofluorescence microscopy to investigate regulation of the subcellular distribution of eNOS in endothelial cells by bradykinin and Ca2+. In resting cells, most of the eNOS is localized at the cell membrane. However, within 5 min following addition of bradykinin, nearly all the eNOS translocates to structures in the cell cytosol; following more protracted incubations with bradykinin, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The bradykinin-induced internalization of eNOS is completely abrogated by the intracellular Ca2+ chelator BAPTA; conversely, Ca2+-mobilizing drugs and agonists promote eNOS translocation. These results establish that eNOS targeting to the membrane is labile and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Reorganization of microfilament structure induced by ac electric fields.

AC electric fields induce redistribution of integral membrane proteins. Cell-surface receptor redistribution does not consistently follow electric field lines and depends critically on the frequency of the applied ac electric fields, suggesting that mechanisms other than electroosmosis are involved. We hypothesized that cytoskeletal reorganization is responsible for electric field-induced cell-surface receptor redistribution, and used fluorescence video microscopy to study the reorganization of microfilaments in human hepatoma (Hep3B) cells exposed to low-frequency electric fields ranging in strength from 25 mV/cm to 20 V/cm (peak to peak). The frequency of the applied electric field was varied from 1 to 120 Hz and the field exposure duration from 1 to 60 min. In control cells, cytoplasmic microfilaments were aligned in the form of continuous parallel cables along the longitudinal axis of the cell. Exposure of cells to ac electric fields induced alterations in microfilament structure in a manner that depended on the frequency of the applied field. A 1 or 10 Hz ac field caused microfilament reorganization from continuous, aligned cable structures to discontinuous globular patches. In contrast, the structure of microfilaments in cells exposed to 20-120 Hz electric fields did not differ from that in control cells. The extent of microfilament reorganization increased nonlinearly with the electric field strength. The characteristic time for microfilament reorganization in cells exposed to a 1 Hz, 20 V/cm electric field was approximately 5 min. Applied ac electric fields could initiate signal transduction cascades, which in turn cause reorganization of cytoskeletal structures.

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.

ATP depletion causes translational immobilization of cell surface transferrin receptors in K562 cells.

We have used quantitative fluorescence microscopy and fluorescence photobleaching recovery to examine the role of metabolic energy in the translational movement of transferrin receptors in the plasma membrane of K562 erythroleukemia cells. Cellular ATP depletion caused a significant decrease in the translational mobility of cell surface transferrin receptors and a significant increase in the number of receptors on the cell surface. ATP repletion restored receptor translational mobility and cell surface expression to control values. Inhibition of ATP hydrolases by orthovanadate also immobilized cell surface transferrin receptors and altered cell surface receptor expression, in a concentration-dependent manner. Vanadate-induced changes in receptor mobility and cell surface expression were reversible upon washing out the drug. Cellular ATP depletion did not affect the translational mobility of plasma membrane glycophorins or a fluorescent phospholipid analogue. We conclude that the translational movement of cell surface transferrin receptors specifically requires metabolic energy and ATP hydrolysis.

Microtubule inhibitors differentially affect translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 cells.

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleaching parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37 degrees C and 21 degrees C were calculated to be 0.10-0.15 min-1 and 0.02-0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by beta-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtubule inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells.

Induced redistribution of cell surface receptors by alternating current electric fields.

The molecular mechanisms that underlie the biological effects of low frequency sinusoidal electric fields may involve induced changes in the physical state of charged cell surface receptors. We have used intensified fluorescence video microscopy to study the redistribution of cell surface receptors, including transferrin receptors (TFR) and low density lipoprotein receptors (LDL-R), in response to externally applied alternating current electric fields in the 3 to 23 V/cm range (peak to peak). Redistribution of both TFR and LDL-R was prominent at frequencies of 1 and 10 Hz but negligible at frequencies of 60 and 120 Hz. Application of a 1 Hz, 23 V/cm field for 15 min caused a twofold change in local TFR surface density, whereas application of a 60 Hz, 23 V/cm field resulted in no significant TFR redistribution. The extent of TFR redistribution induced by a 1 Hz field changed by only 20% over the field strength range from 3.5 to 23 V/cm. AC field-induced cell surface receptor migration did not consistently follow electric field lines, suggesting that mechanisms more complex than classical electrophoresis and electroosmosis mediate receptor redistribution. Joule heating and plasma membrane calcium channel activation were shown not to be involved in the mechanism of receptor redistribution. Applied external electric fields may reorganize cytoskeletal and plasma membrane structures, providing pathways for cell surface receptors to migrate anharmonically.

Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.

Schistosoma mansoni: membranes from adult worms reversibly perturb shape, volume, and membrane organization of intact human red blood cells.

Adult forms of Schistosoma mansoni ingest host (human) red blood cells (RBCs). To elucidate potential mechanisms by which contact with adult parasites perturbs RBC membranes, we studied the effects of the membrane fraction of isolated schistosomes on RBC shape, volume, potassium ion content, and phospholipid and transmembrane protein lateral mobility. S. mansoni-treated RBCs exhibited rapid but spontaneously reversible shape change from discocytes to spheroechinocytes, reversible decrease in cell volume, and rapid loss of intracellular potassium ions. Treated RBCs also showed rapid but spontaneously reversible immobilization of membrane phospholipids and of band 3, the major transmembrane protein. These data suggest that components of adult S. mansoni membranes can perturb host RBC volume and membrane organization. In the absence of RBC lysis, RBC metabolic and repair mechanisms can reverse these effects.