Potassium softens vascular endothelium and increases nitric oxide release.

In the presence of aldosterone, plasma sodium in the high physiological range stiffens endothelial cells and reduces the release of nitric oxide. We now demonstrate effects of extracellular potassium on stiffness of individual cultured bovine aortic endothelial cells by using the tip of an atomic force microscope as a mechanical nanosensor. An acute increase of potassium in the physiological range swells and softens the endothelial cell and increases the release of nitric oxide. A high physiological sodium concentration, in the presence of aldosterone, prevents these changes. We propose that the potassium effects are caused by submembranous cortical fluidization because cortical actin depolymerization induced by cytochalasin D mimics the effect of high potassium. In contrast, a low dose of trypsin, known to activate sodium influx through epithelial sodium channels, stiffens the submembranous cell cortex. Obviously, the cortical actin cytoskeleton switches from gelation to solation depending on the ambient sodium and potassium concentrations, whereas the center of the cell is not involved. Such a mechanism would control endothelial deformability and nitric oxide release, and thus influence systemic blood pressure.

Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina.

Apoptosis, a physiologically critical process, is characterized by a destruction of the cell after sequential degradation of key cellular components. Here, we set out to explore the fate of the physiologically indispensable nuclear envelope (NE) in this process. The NE mediates the critical nucleocytoplasmic transport through nuclear pore complexes (NPCs). In addition, the NE is involved in gene expression and contributes significantly to the overall structure and mechanical stability of the cell nucleus through the nuclear lamina, which underlies the entire nucleoplasmic face of the NE and thereby interconnects the NPCs, the NE, and the genomic material. Using the nano-imaging and mechanical probing approach atomic force microscopy (AFM) and biochemical methods, we unveiled the fate of the NE during apoptosis. The doomed NE sustains a degradation of both the mediators of the critical selective nucleocytoplasmic transport, namely NPC cytoplasmic filaments and basket, and the nuclear lamina. These observations are paralleled by marked softening and destabilization of the NE and the detection of vesicle-like nuclear fragments. We conclude that destruction of the cell nucleus during apoptosis proceeds in a strategic fashion. Degradation of NPC cytoplasmic filaments and basket shuts down the critical selective nucleocytoplasmic cross-talk. Degradation of the nuclear lamina disrupts the pivotal connection between the NE and the chromatin, breaks up the overall nuclear architecture, and softens the NE, thereby enabling the formation of nuclear fragments at later stages of apoptosis.

Aldosterone receptor sites on plasma membrane of human vascular endothelium detected by a mechanical nanosensor.

The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37 degrees C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 +/- 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 +/- 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.

Oscillating activity of a Ca(2+)-sensitive K+ channel. A prerequisite for migration of transformed Madin-Darby canine kidney focus cells.

Migration plays an important role in the formation of tumor metastases. Nonetheless, little is known about electrophysiological phenomena accompanying or underlying migration. Previously, we had shown that in migrating alkali-transformed Madin-Darby canine kidney focus (MDCK-F) cells a Ca(2+)-sensitive 53-pS K+ channel underlies oscillations of the cell membrane potential. The present study defines the role this channel plays in migration of MDCK-F cells. We monitored migration of individual MDCK-F cells by video imaging techniques. Under control conditions, MDCK-F cells migrated at a rate of 0.90 +/- 0.03 microns/min (n = 201). Application of K+ channel blockers (1 and 5 mmol/liter Ba2+, 5 mmol/liter tetraethylammonium, 100 mumol/liter 4-aminopyridine, 5 nmol/liter charybdotoxin) caused marked inhibition of migration, pointing to the importance of K+ channels in migration. Using patch-clamp techniques, we demonstrated the sensitivity of the Ca(2+)-sensitive 53-pS K+ channel to these blockers. Blockade of this K+ channel and inhibition of migration were closely correlated, indicating the necessity of oscillating K+ channel activity for migration. Migration of MDCK-F cells was also inhibited by furosemide or bumetanide, blockers of the Na+/K+/2Cl- cotransporter. We present a model for migration in which oscillations of cell volume play a central role. Whenever they are impaired, migration is inhibited.

Spontaneously oscillating K+ channel activity in transformed Madin-Darby canine kidney cells.

Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.

17beta-estradiol increases volume, apical surface and elasticity of human endothelium mediated by Na+/H+ exchange.

OBJECTIVE: 17beta-estradiol is known to delay the onset of atherosclerosis in women but cellular mechanisms are still unclear. Estrogens bind to specific receptors and initiate a signaling cascade that involves the activation of plasma membrane Na(+)/H(+) exchange. We hypothesized that estrogens interfere with ion transport across the plasma membrane and thus control endothelial structure and function. Therefore, we investigated the effects of the sex steroids 17beta-estradiol, progesterone, and testosterone on volume, apical surface and elasticity in human endothelium. METHODS: The atomic force microscope was used as an imaging tool and as an elasticity sensor. We applied the antiestrogen tamoxifen, the Na(+)/H(+) exchange blocker cariporide and the epithelial Na(+)channel blocker amiloride to elucidate the role of transmembrane ion transport in hormone-treated human umbilical vein endothelial cells (HUVEC). RESULTS: Incubation with 17beta-estradiol for 72 h led to a dose-dependent increase of endothelial cell volume (41%), apical cell surface (22%), and cell elasticity (53%) as compared to non-17beta-estradiol treated controls. Block of the 17beta-estradiol receptor by tamoxifen and of plasma membrane Na(+)/H(+) exchange by cariporide prevented the hormone-induced changes. Progesterone and testosterone were ineffective. CONCLUSIONS: 17beta-estradiol increases HUVEC water content and HUVEC elasticity mediated by activated estrogen receptors. The estrogen response depends on the activation of plasma membrane Na(+)/H(+) exchange. The increase in endothelial cell elasticity could be one of the vasoprotective mechanisms postulated for 17beta-estradiol.

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney.

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.

Rapid activation of calcium-sensitive Na+/H+ exchange induced by 20-hydroxyecdysone in salivary gland cells of Drosophila melanogaster.

Ecdysteroids play an important role in the larval moulting process of insects. 20-Hydroxyecdysone (20E) causes the induction of specific 'puffs' in polytene chromosomes of Drosophila melanogaster salivary gland cells. Although it is known that inorganic ions control pretranscriptional processes in the cell nucleus, the intracellular mechanisms of gene activation are still unclear. Therefore, we examined the effects of 20E on plasma membrane ion transport of Drosophila melanogaster salivary gland cells. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. The relative K+ conductance of the cell membrane (tK+) was measured with microelectrodes by performing ion substitution experiments. Under control conditions tK+ averaged to 0.16 + 0.02 (n = 15). Addition of 5 x 10(-6) M 20E increased tK+ within 2 min by 19.1 +/- 4.2% (n = 15). This rapid response to 20E was elicited only in the presence of calcium. Moreover, starting from a steady-state intracellular pH of 7.20-7.60, 20E induced a rise in cytoplasmic pH by 0.27 +/- 0.06 (n = 6) within minutes. Amiloride (10(-3) M), a blocker of plasma membrane Na+/H+ exchange, prevented the 20E-induced intracellular alkalinization. We conclude that 20E activates a calcium-sensitive plasma membrane Na+/H+ exchange leading to a rise of plasma membrane K+ conductance and intracellular alkalinization both being prerequisites for steroid hormone induced gene activation.

Chloride-dependence of the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

(1) Vasa deferentia obtained from reserpine-pretreated rats were exposed to 0.15 mumol l-1 3H-(-)noradrenaline (with monoamine oxidase and catechol-O-methyltransferase being inhibited) and initial rates of the neuronal 3H-noradrenaline uptake as well as IC50 values for inhibition of uptake by desipramine, cocaine or (-)metaraminol determined at various external Cl- concentrations (0-145 mmol l-1) and a fixed high Na+ concentration (145 mmol l-1). (2) When the Cl- concentration in the medium was decreased neuronal uptake fell. As far as Cl- concentrations ranging from 10 to 145 mmol l-1 are concerned, the dependence of uptake on Cl- obeyed Michaelis-Menten kinetics with an apparent Km and Vmax of 6.2 mmol l-1 and 116 pmol g-1 min-1, respectively. At Cl- concentrations below 10 mmol l-1, uptake was higher than expected from the values of Km and Vmax, and even in the nominal absence of Cl- from the medium a remainder of neuronal uptake was still detectable. Evidence is presented to show that, on incubation at Cl- concentrations below 10 mmol l-1, intracellular Cl- leaks out, so that the actual Cl- concentrations in the extracellular fluid are probably higher than in the medium. (3) The potencies of desipramine and cocaine for inhibition of neuronal uptake were markedly dependent on the Cl- concentration in the medium, but the type of Cl- -dependence differed. While the IC50 for desipramine decreased, that for cocaine increased with increasing Cl- concentration (2-145 mmol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone and nuclear signaling in kidney.

Initiation of transcription is an early step in steroid hormone action. We investigated by means of atomic force microscopy (AFM) and fluorescence imaging the role of nuclear pore complexes (NPCs) in mediating signal transduction of the mineralocorticoid hormone aldosterone from the extracellular space into the cell nucleus. With AFM, we imaged single NPCs of isolated nuclear envelopes under native conditions. We observed that individual NPCs contract in response to a Ca2+ signal, which is known to occur in seconds after aldosterone exposure. In living kidney cells in culture (MDCK cells), aldosterone led within seconds to the contraction of the whole nucleus measured by DNA-fluorescence imaging. Nuclear contraction was elicited at similar time scale and to similar extent by bradykinin, a peptide hormone known to mobilize Ca2+ from internal stores, and by ionomycin, a Ca2+ ionophore known to directly increase intracellular Ca2+. Nuclear contraction is explained by the individual contraction of calcium-sensitive NPCs that occur in high density in the nuclear envelope. We present a model in which nuclear pore complexes play a key role as barrier molecules of high plasticity in the control of aldosterone-induced gene expression.

Life on biomembranes viewed with the atomic force microscope.

Since its invention in 1986, the atomic force microscope (AFM) has become one of the most widely used near-field microscopes. Surfaces of hard samples are imaged almost routinely with atomic resolution. Soft biological surfaces, however, are still challenging. In this brief review, the AFM technique is introduced to the experimental biologist. We discuss recent data on imaging molecular structures of biomembranes, and give detailed information on the application of the AFM with three representative examples. One is imaging plasma membrane turnover of transformed renal epithelial cells during migration in vivo, another is visualizing a cloned and isolated potassium channel usually located in kidney, and a third is imaging macromolecular pore complexes of the nuclear envelope of aldosterone-sensitive kidney cells and of Xenopus laevis oocytes. The review ends with the conclusion that nuclear pores can serve as birthday candles on a Guglhupf.

Aldosterone remodels human endothelium.

AIM: In response to aldosterone endothelial cells swell and stiffen. Although amiloride-sensitive sodium and water uptake is known to be involved, the underlying mechanisms are yet unclear. We tested the hypothesis whether the intracellular accumulation of water or organic matter is responsible for the structural and functional alterations. METHODS: Atomic force microscopy was used as an imaging tool and a mechanical nanosensor. Cell water, organic cell matter and cell pressure was measured at single cell level in human umbilical vein endothelial cells (HUVEC). Furthermore, we tested by means of a miniature perfusion chamber in vitro the physical robustness to blood flow of the aldosterone-treated endothelium. RESULTS: In response to a three-day treatment with 1 nM aldosterone HUVEC swell. To our surprise, cell water decreased from 82+/-6% to 71+/-5% while intracellular organic matter increased from 18+/-1.8% to 29+/-3.0%. These changes were paralleled by a rise in cell pressure of 114%, measured in living HUVEC in vitro. Blood flow across the endothelium was found significantly altered after aldosterone treatment. Imaging the endothelial monolayer after blood perfusion disclosed large gaps between cells treated with aldosterone. The mineralocorticoid receptor blockers, spironolactone and eplerenone could prevent the aldosterone actions. CONCLUSION: Mild aldosteronism causes intracellular accumulation of organic matter at the cost of cell water. This makes endothelium stiff and vulnerable to shear stress. The measurements could explain clinical observations that high blood pressure combined with high plasma aldosterone concentration may damage the endothelium of blood vessels.

Bradykinin shifts endothelial fluid passage from para- to transcellular routes.

The signalling peptide bradykinin (BK) is implicated in inflammation and angiogenesis. It promotes fluid transport from blood vessels to interstitial space, and thus facilitates oedema formation. To clarify whether paracellular or transcellular pathways mediate this effect, we investigated the BK-stimulated fluid transport across endothelial monolayers in vitro by comparison of electrical and fluorescence methods. Electrical cell impedance sensing monitored a biphasic response of cell layers to BK with high time resolution: a short decrease (18%, 1 min) was followed by a more sustained increase in paracellular resistance (30%, 10 min). The two phases can be assigned to second messengers of the BK-signalling pathway: Ca(2+) for the decrease and cyclic adenosine monophosphate for the rise of resistance, respectively. Despite tightening of the intercellular clefts, BK increased the fluid permeability by 39%, indicating transcellular fluid transport. Additionally, BK stimulated both in- and outwardly directed membrane trafficking as assessed by vesicular fluid uptake (by 49%) and secretion of von Willebrandt factor (by 85%). In conclusion, the combination of electrical and fluorescence data suggests that BK induces a shift from para- to transcellular fluid transport across endothelium.

Intracellular pH in diluting segment of frog kidney.

Chronic exposure to high potassium (K+ adaptation) stimulates H+ net secretion in the diluting segment of the frog kidney. In order to investigate the cellular mechanism of the H+ secretory process intracellular pH (pHi) measurements were performed in cells of the diluting segment of the isolated doubly-perfused kidney of K+ adapted Rana esculenta. pHi changes were monitored by pH-sensitive microelectrodes while the tubule lumen was rapidly perfused with various solutions. With control solutions (extracellular pH = 7.80) pHi averaged 7.60 +/- 0.05. Luminal application of furosemide (5 X 10(-5) mol/l) or reduction of luminal Cl- (from 104 mmol/l to 9 mmol/l) hyperpolarized the cell membrane potentials but pHi was not altered. Reduction of luminal Na+ (from 98 mmol/l to 3 mmol/l) depolarized the cell membrane potentials but pHi remained constant. Complete removal of luminal Na+, however, led to a significant decrease of pHi from 7.61 +/- 0.08 to 7.18 +/- 0.08. Luminal application of amiloride (1 X 10(-3) mol/l) also decreased pHi significantly (delta pHi = 0.15 +/- 0.02). The results indicate that an amiloride-sensitive H+ extrusion mechanism exists in the luminal cell membrane of the K+ adapted frog diluting segment. The data are consistent with Na+/H+ exchange which maintains a constant pHi even at extreme experimental conditions.