Biomedical subjects

L Wojnowski

Publications and source records attributed to L Wojnowski.

28 records · Page 2Linked to original sources

PubMed1994

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells.

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca2+ oscillations from an inositol 1,4,5-triphosphate-sensitive cytoplasmic Ca2+ store. In this study, Ca2+ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca2+ video imaging and the Fura-2/Mn2+ quenching technique. Oscillations were dependent on extracellular Ca2+ concentration and were inhibited by extracellularly applied La3+, Co2+ and Ni2+. Depolarization of the cell membrane with high K+ concentrations and the L-type Ca2+ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca2+ channels. Mn2+ quenching experiments disclosed significant Ca2+ influx into MDCK-F cells. The rate of this influx was constant between Ca2+ spikes, but markedly increased during the spontaneous Ca2+ spikes. Similar transient increases in Ca2+ entry could be mimicked by agents triggering intracellular Ca2+ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca2+ permeability permitting Ca2+ entry into the cytoplasm. The rate of Ca2+ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca2+ concentration.

Animals↗

PubMed1994

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1992

Renal potassium bicarbonate release in humans exposed to an acute volume load.

Cells of the renal medulla regulate their volume by transmembrane ion movements when exposed to large changes in osmolality. Since renal cells in culture release KHCO3 in response to hypotonic stress [11], we investigated the effect of an acute water load on urinary KHCO3 excretion in 5 healthy individuals. Water diuresis was induced by the ingestion of 1.5 l hypoosmolal fluid (22 mosm/kg H2O) over 15 min. The rate of urinary volume excretion increased from an initial value of 1.4 ml/min to 9.3 ml/min after 75 min. Urinary osmolality dropped from an initial value of 940 +/- 32 mosm/kg H2O to 74 +/- 4 mosm/kg H2O (n = 5). The decrease of osmolality was accompanied by the transient release of potassium and bicarbonate. Peak values of KHCO3 excretion were observed between 30 and 45 min after the onset of the experiment corresponding to the drop of urinary osmolality. The magnitude of renal potassium release correlated significantly (r = 0.93; P less than 0.05) with endogenous plasma aldosterone concentrations measured prior to the experiment in the 5 volunteers. We conclude that medullary epithelial cells release KHCO3 when exposed to hypotonic stress. The volume regulatory response is upregulated by aldosterone.

Adult↗

PubMed1992

Oscillations: a key event in transformed renal epithelial cells.

Intracellular pH (pHi) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pHi may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form "foci" in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca2+ flux activates plasma membrane K+ channels and Na+/H+ exchange. This leads to periodic changes of membrane voltage and pHi at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.

Alkalosis↗

PubMed1992

Endothelin-1 blunts transepithelial transport and differentiation of Madin-Darby canine kidney cells.

We investigated the effects of endothelin-1 (ET-1) on Madin-Darby canine kidney (MDCK) cells, a cell line originating from the renal collecting duct. The activity of transepithelial transport was assessed as the rate of dome formation in monolayers grown on solid support. The pH value of the dome fluid (dome pH) was measured by means of pH-selective microelectrodes. Differentiation of monolayer cells was estimated as the peanut-lectin(PNA)-binding capacity of the apical membrane. Confluent monolayers were incubated for 12-72 h in serum-free medium at various concentrations of ET-1. Exposure to 1 nmol/l ET-1 reduced dome formation by a maximum of 41 +/- 8% (n = 4; P less than 0.02) after 24 h. ET-1 (10 nmol/l; 24 h) decreased dome pH from 7.52 +/- 0.02 (n = 53) to 7.36 +/- 0.03 (n = 51; P less than 0.02). Apical application of amiloride (1 mmol/l) reduced dome pH in both ET-1-treated and non-treated domes to essentially the same level, 7.25 +/- 0.03 (n = 19) and 7.23 +/- 0.03 (n = 17) respectively. ET-1 (10 nmol/l; 24 h) reduced PNA-binding capacity by 19 +/- 3% (n = 5; P less than 0.02). Moreover, ET-1 prevented the increase in PNA binding (+ 53 +/- 7%; n = 5) induced by 0.1 mumol/l aldosterone. We conclude that ET-1 inhibits transepithelial transport and PNA binding via inhibition of apical Na+/H+ exchange, thus antagonizing aldosterone action in MDCK cells.

Animals↗

PubMed1992

Spontaneous membrane potential oscillations in Madin-Darby canine kidney cells transformed by alkaline stress.

High pH is known to be associated with normal cell growth and neoplastic transformation. We observed that Madin-Darby canine kidney (MDCK) cells grown under sustained alkaline stress (pH 7.7) develop "foci" composed of spindle-shaped cells lacking contact inhibition and exhibiting only poor adhesion to the culture support. Foci-developing (F) cells were cloned and grown in control medium (pH 7.4), where they maintained their neoplastic features indicating a stable pH-induced genetic transformation. After F cells had been fused to giant cells with polyethylene glycol, the cell membrane potential (Vm) was measured by means of microelectrodes. In contrast to non-transformed MDCK cells, Vm of F cells showed spontaneous biorhythmicity caused by periodic opening of Ca2(+)-activated K+ channels. Spiking activity was blunted by the Ca2+ channel blocker nifedipine, by the K+ channel blocker Ba2+, by the Na+/H+ exchange blocker amiloride and its analogue ethylisopropylamiloride, and by an extracellular pH of 7.6 and 6.8. We conclude that MDCK cells transformed by sustained alkaline stress have lost their stable plasma membrane potential but, instead, exhibit endogenous Ca2(+)- and pH-sensitive oscillations.

Animals↗

PubMed1991

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation.

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium+ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84 +/- 2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na+ and HCO3- concentrations was observed. It was followed by a sustained increase in intracellular K+ and Cl- concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K+ accumulation significantly, whereas the H+/K(+)-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19 +/- 2 mV, owing to the decrease of the ratio of Cl- conductance to K+ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na+/H+ exchange in MDCK cells; (b) transient alteration of intracellular Na+ and pH stimulates Na+/K(+)-ATPase and Cl-/HCO3- exchange, exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl-/K+ conductance ratio of the plasma membrane.

Adenosine Triphosphatases↗

PubMed1991

Hypotonic stress-induced release of KHCO3 in fused renal epitheloid (MDCK) cells.

Mechanisms of cell volume regulation induced by the reduction of the osmolality of the Ringer solution by one-third were studied in fused Madin-Darby canine kidney (MDCK) cells. Intracellular HCO3-, K+ and Cl- concentrations [ion]i in parallel with cell membrane potential (PD), cell membrane conductance (Gm) and conductances of individual ions (Gmion) were evaluated with microelectrode techniques. Fused cells regulate their cell volume by about 50%. Gm increased from 0.43 +/- 0.03 mS/cm2 in isotonic Ringer solution to 4.3 +/-0.3 mS/cm2 in the steady state phase of cell swelling. GmCl was 0.31 +/- 0.03 mS/cm2 in isotonic Ringer solution and thus was the dominant individual ion conductance. In the initial phase of cell swelling GmK increased transiently 64-fold to 0.32 +/- 0.03 mS/cm2, and consequently PD hyperpolarized. At peak hyperpolarization GmCl transiently decreased by 15%. Cell swelling increased GmCl 11-fold and GmHCO3 28-fold to 0.95 +/- 0.1 mS/cm2 in the steady state phase of cell swelling. In this phase GmCl and GmHCO3 were dominating, whereas GmK was only slightly increased compared to isotonic conditions. The hyperpolarization of PD was paralleled by cytoplasmic acidification. At peak acidification [HCO3-]i decreased by 6.4 mmol/kg H2O. Cl- extrusion was not detectable in the initial phase of cell swelling. In isotonic Ringer solution [K+]i was 125 +/- 5 mmol/kg H2O. During the initial phase of cell swelling 23 +/- 5 mmol/kg H2O K+ was extruded, indicating that yet unknown anions participated in cell volume regulation in this phase of cell swelling. In the steady state phase of cell swelling [pH]i was normalized by replenishing [HCO3-]i, whereas Cl- was extruded. We conclude that fused renal epitheloid cells acutely release KHCO3 in response to hypotonicity, but then regain pH homeostasis in the steady state phase of cell swelling.

Animals↗

PubMedDate not supplied

[The importance of combining ultrasonography of the carotid artery bifurcation and Doppler imaging of cerebral arteries].

The USG of carotid bifurcation and Doppler imaging of cranial arteries were performed in 74 patients with ischaemic stroke. The Doppler image as well as USG were found to be helpful for prognosis. Arteriosclerotic USG changes corresponded more often with the neurological syndrome in severely ill patients (at discharge from hospital). Also reduction of flow in medial cerebral artery was significantly more often in these patients in the first hours of the illness. In thrombosis of the first part of the internal carotid artery only the succeeding image can indicate it (2 cases). Arteriography only can demonstrate artery tortuosity (1 case).

Adult↗