Biomedical subjects

M Paulmichl

Publications and source records attributed to M Paulmichl.

At least 19 recordsLinked to original sources

PubMed1992

New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.

Amino Acid Sequence↗

PubMed1991

Effect of BHT 920 on calcium-activated K+ channels in renal epithelioid MDCK cells.

In Madin Darby canine kidney (MDCK) cells, epinephrine has been shown to increase intracellular calcium, activate calcium-dependent K+ channels and hyperpolarize the cell membrane. The present study has been performed to test for the possible involvement of alpha 2-adrenergic receptors. To this end, the effects of alpha 2-adrenoceptor agonist BHT 920 have been studied on cell membrane potential, ion channel activity and intracellular calcium: Similar to epinephrine, BHT 920 hyperpolarizes the cell membrane, increases intracellular calcium and activates inwardly rectifying K+ channels (single channel slope conductances 30-80 pS). Half-maximal hyperpolarization is achieved at concentrations between 10 and 100 nmol/l. The hyperpolarizing effect of BHT 920 is abolished in the presence of alpha 2-adrenoceptor antagonist yohimbine (100 nmol/l) but not in the presence of alpha 1-adrenoceptor antagonist prazosin (100 nmol/l). At extracellular calcium activity below 100 nmol/l BHT 920 still leads to a transient hyperpolarization of the cell membrane but, in contrast to epinephrine, is unable to significantly increase intracellular calcium or significantly activate the calcium-sensitive K+ channels. The observations indicate that stimulation of alpha 2-receptors participates in the epinephrine-induced increase of intracellular calcium, channel activation and hyperpolarization.

Adrenergic beta-Agonists↗

PubMed1991

Cloning and expression of a rat cardiac delayed rectifier potassium channel.

We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2.

4-Aminopyridine↗

PubMed1991

Cellular mechanisms of bradykinin-induced hyperpolarization in renal epitheloid MDCK-cells.

Previous studies have demonstrated that bradykinin hyperpolarizes the cell membrane of subconfluent MDCK cells by increase of the potassium conductance. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of bradykinin on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate diester (TPA). In untreated cells, bradykinin leads to a transient increase of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, increase of Cai, activation of potassium channels and hyperpolarization of the cell membrane. The effects of bradykinin on PD and Cai are still present in the absence of extracellular calcium. In cells pretreated with pertussis toxin the effect of bradykinin on inositol trisphosphate formation is almost abolished but bradykinin still leads to a transient increase of Cai and PD in the presence and absence of extracellular calcium. In cells pretreated with TPA the bradykinin-induced increase of inositol trisphosphate formation is blunted, the bradykinin-induced increase of Cai abolished, but the bradykinin-induced hyperpolarization still present. The observations indicate that bradykinin increases Cai in part by phorbol ester and pertussis toxin sensitive activation of phospholipase C. In addition, bradykinin is capable of enhancing Cai by utilizing pertussis toxin insensitive mechanisms. Furthermore, bradykinin is able to transiently enhance the potassium conductance without a general increase of intracellular calcium.

Animals↗

PubMed1991

Effect of alpha-adrenoceptor agonists on cell membrane potential in renal epitheloid Madin Darby canine kidney cells.

In Madin Darby canine kidney (MDCK) cells epinephrine--via alpha-adrenoceptors--leads to sustained hyperpolarization of the cell membrane. The present study defined the subtype of alpha-adrenoceptor involved. Both the alpha 1-adrenoceptor agonist, phenylephrine, and alpha 2-adrenoceptor agonists (BHT 920, guanfacine, moxonidine, oximetazoline) were able to hyperpolarize the cell membrane. The effect of epinephrine was only transient in the presence of the alpha 2-adrenoceptor antagonist, yohimbine, whereas the effect persisted in the presence of the alpha 1-adrenoceptor antagonist, prazosin. The epinephrine-induced hyperpolarization was almost abolished in the presence of both prazosin and yohimbine. In conclusion, both, alpha 1- and alpha 2-receptors are involved in the hyperpolarizing action of epinephrine.

Adrenergic alpha-Agonists↗

PubMed1991

Cellular mechanisms of adrenaline-induced hyperpolarization in renal epitheloid MDCK cells.

The effects of adrenaline on the potential difference across the cell membrane, on formation of inositol phosphates and on intracellular Ca2+ ([Ca2+]i) were analysed in cells without or with pretreatment with pertussis toxin or phorbol 12-myristate 13-acetate (PMA). In untreated cells, adrenaline leads to a sustained hyperpolarization, a stimulation of Ins(1,4,5)P3 and Ins(1,3,4,5,)P4 formation and a transient increase in [Ca2+]i from 78 +/- 7 to 555 +/- 43 nM, followed by a plateau of 260 +/- 23 microM. In the absence of extracellular Ca2+ the effect of adrenaline on both potential difference and [Ca2+]i is transient. In cells pretreated with pertussis toxin, the effects of adrenaline on InsP3 and [Ca2+]i are still preserved, but the effect on potential difference is transient. In cells pretreated with PMA, the effect of adrenaline on InsP3 formation is severely decreased and that on [Ca2+]i abolished, whereas a transient hyperpolarizing effect is still present. This transient hyperpolarization is abolished by additional pretreatment with pertussis toxin. The observations suggest that adrenaline hyperpolarizes the cell membrane of MDCK cells by several distinct mechanisms. First, adrenaline stimulates the formation of InsP3 and InsP4, which at least in part accounts for the release of intracellular Ca2+ and the entry of Ca2+ from the extracellular fluid. Stimulation of phospholipase C is not mediated by pertussis-toxin-sensitive G-proteins, but apparently is inhibited by activation of protein kinase C. Second, adrenaline hyperpolarizes the cell membrane by a mechanism independent from increase in [Ca2+]i which is sensitive to pertussis toxin but is, at least in part, insensitive to PMA.

Animals↗

PubMed1991

Activation of cell membrane potassium conductance by mercury in cultured renal epithelioid (MDCK) cells.

To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)-cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1 microM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by approximately 0.2 microM. Higher concentrations of mercury ions (greater than 10 microM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 microM, 1 microM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 microM, but not 1 microM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin- or pertussis toxin-sensitive G-proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.

Animals↗

PubMed1991

Cellular mechanisms of ATP-induced hyperpolarization in renal epitheloid MDCK-cells.

Previous studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)-cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoyl phorbol 13-acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5-trisphosphate (IP3), inositol 1,3,4,5-tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the cell membrane.

Adenosine Triphosphate↗

PubMed1991

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells.

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.

Animals↗

PubMed1991

Further characterization of volume regulatory decrease in cultured renal epitheloid (MDCK) cells.

In Madin Darby canine kidney (MDCK) cells volume regulatory decrease (VRD) is paralleled by a variable, transient hyperpolarization followed by a sustained depolarization of the cell membrane. In the depolarized cells, the cell membrane selectivity is decreased for potassium and increased for chloride. Without knowledge of the cell membrane resistance (Rm), these changes of cell membrane selectivity cannot be translated into conductances, i.e. the observed alterations of ion selectivity could have been due to inhibition of potassium conductance or activation of anion conductance. In the present study Rm has been determined by cellular cable analysis. To this end, three microelectrodes were impaled into three different cells of a cell cluster, current (up to 3 nA) was injected into one cell and the corresponding voltage deflections determined in the other two cells. As a result, exposure of the cells to hypotonic perfusates leads to a marked, sustained reduction of Rm. In the absence of chloride and in the absence of bicarbonate and chloride, the decrease of Rm is only transient. The data indicate that cell swelling leads to a transient increase of potassium conductance followed by a sustained increase of anion conductance. As evident from BCECF fluorescence, exposure of MDCK cells to hypotonic perfusates leads to a significant decrease of intracellular pH, which may in part be due to loss of bicarbonate through the anion conductive pathway.

Animals↗

PubMed1991

Further analysis of ATP-mediated activation of K+ channels in renal epithelioid Madin Darby canine kidney (MDCK) cells.

ATP activates K+ channels by increasing intracellular calcium activity in Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for the involvement of G-proteins and of protein kinase C in the intracellular transmission of these effects. To this end, the effect of ATP on intracellular calcium and K+ channel activity has been studied in cells pretreated with the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and/or pertussis toxin. The ATP-induced increase of intracellular calcium is not significantly affected by pretreatment with pertussis toxin, is significantly blunted by pretreatment with TPA and is abolished by pretreatment with both pertussis toxin and the phorbol ester. The ATP activation of K+ channels is similarly blunted by pretreatment with TPA, but is not abolished by pretreatment with both the phorbol ester and pertussis toxin. Furthermore, the ATP-induced hyperpolarization is not abolished in cells pretreated with both pertussis toxin and TPA. In those cells, ATP may activate K+ channels by calcium-dependent mechanisms or lead to localized increases of intracellular calcium sufficient to activate the K+ channels but escaping detection with fura-2 fluorescence.

Adenosine Triphosphate↗

PubMed1990

Cobalt activates potassium conductance in the plasma membrane of cultured renal epithelioid (MDCK)-cells.

Cobalt has been shown to stimulate sodium transport across the distal nephron of the newt kidney. The mechanism of this action remained elusive. The present study has been performed to test for effects of cobalt on electrical properties of cultured subconfluent kidney (MDCK)-cells: cobalt (10 microM) leads to a rapid, sustained and reversible hyperpolarization of the cell membrane, paralleled by an increase of the potassium selectivity and a decrease of the resistance. Thus, cobalt increases the potassium conductance of the cell membrane. The half-maximal effect is elicited by approx. 1 microM. At extracellular calcium concentration reduced to less than 0.1 microM, cobalt (10 microM) leads to a transient hyperpolarization, which can be elicited only once. Thus, cobalt enhances the potassium conductance in a calcium dependent way. At higher concentrations (100 microM) cobalt hyperpolarizes the cell membrane only transiently even in the presence of extracellular calcium. Furthermore 100 microM cobalt interferes with ATP-induced hyperpolarization, which is known to result from calcium mediated activation of K+ channels. Thus, 100 microM cobalt may inhibit ATP-stimulated calcium entry into the cell.

Adenosine Triphosphate↗

PubMed1990

Cadmium enhances potassium conductance in cultured renal epitheloid (MDCK) cells.

The kidney is a main target organ for cadmium toxicity. The present study has been performed to test for effects of cadmium on electrical properties of cultured subconfluent kidney (MDCK) cells. Cadmium leads to a rapid, sustained and reversible hyperpolarization of the cell membrane, paralleled by an increase of the potassium selectivity and a decrease of the resistance. Thus, cadmium increases the potassium conductance of the cell membrane. The half maximal effect is elicited congruent to 0.2 microM, a concentration encountered during chronic cadmium intoxication. At extracellular calcium concentration reduced to less than 0.1 microM, 5 microM cadmium leads to a transient hyperpolarization, which can be elicited only once. High concentrations (50 microM) of cadmium lead to a sustained hyperpolarization even at extracellular calcium concentrations of less than 0.1 microM. According to fluorescence measurements cadmium leads to an increase of intracellular calcium activity, which is sustained at 1 mM and transient at less than 1 microM extracellular calcium activity. In conclusion, cadmium at low concentrations enhances the potassium conductance in a calcium dependent way. The observations suggest that cadmium enhances intracellular calcium both by recruitment from intracellular stores and by modification of calcium transport across the cell membrane. At high concentrations cadmium enhances the potassium conductance independently from enhanced intracellular calcium activity.

Animals↗

PubMed1989

The effect of hypoosmolarity on the electrical properties of Madin Darby canine kidney cells.

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cells is 1,892 +/- 16 fl (n = 8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269 +/- 18 fl (n = 4) and subsequent volume regulatory decrease to 2,052 +/- 22 fl (n = 4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 mumol/l). The potential difference across the cell membrane averages -53.6 +/- 0.9 mV (n = 49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7 +/- 0.8 mV (n = 67), reduces the apparent potassium selectivity of the cell membrane, from 0.55 +/- 0.07 (n = 9) to 0.09 +/- 0.01 (n = 26), and increases the apparent chloride selectivity from close to zero to 0.34 +/- 0.02 (n = 21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2 +/- 1.1 mV (n = 13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0 +/- 1.4 mV (n = 12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by -6.7 +/- 2.2 mV (n = 11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4 +/- 1.7 mV (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Effect of extracellular adenosine triphosphate on electrical properties of subconfluent Madin-Darby canine kidney cells.

1. The present study has been performed to test for an influence of extracellular ATP on the potential differences across the cell membrane (PD) in subconfluent MDCK cells utilizing conventional microelectrodes. 2. In the absence of ATP, the mean measured PD was -47.5 +/- 0.3 mV (+/- S.E.M., n = 320). Application of 10 mumol/l ATP leads to rapid (less than 2 s) hyperpolarization of the cell membrane by -18.5 +/- 0.4 mV (n = 221), reduction of input resistance by 14 +/- 1 M omega (n = 106) and increase of the sensitivity of PD to alterations of extracellular potassium. 3. The concentration needed for half-maximal effect (K1/2) of ATP is approximately 0.5 mumol/l. ATP-gamma-S (K1/2 approximately 0.4 mumol/l) aand ADP (K1/2 approximately 0.9 similarly effective, whereas up to 1 mmol/l AMP or adenosine does not significantly alter PD. Application of 10 mumol/l theophylline, 1 mumol/l phentolamine and 10 mumol/l indomethacin does not blunt the hyperpolarizing effect of ATP. 4. The ATP-induced hyperpolarization is completely abolished in the presence of 1 mmol/l quinidine but only incompletely by 0.1 mmol/l quinidine or 1 mmol/l barium. In calcium-free extracellular fluid (1 mmol/l EDTA added) PD is 18.5 +/- 1.7 mV (n = 18). With reduced extracellular calcium, the hyperpolarizing effect of ATP is blunted (-12.3 +/- 1.6 mV, n = 18) and only transient. 5. In conclusion, ATP hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium.

Adenosine Diphosphate↗

PubMed1989

Activation of potassium channels in renal epithelioid cells (MDCK) by extracellular ATP.

Extracellular ATP has been shown to stimulate transepithelial chloride transport in confluent Madin-Darby canine kidney (MDCK) cell layers and to enhance potassium conductance in subconfluent MDCK cells. The present study has been performed to test for the effect of extracellular ATP on channel activity in patches from subconfluent MDCK cells. Within 8 s, addition of extracellular ATP (10 mumol/l) leads to a sustained, but fully reversible, appearance of potassium-selective channels in cell-attached patches [increase of open probability from 0.03 +/- 0.02 (n = 10) to 0.50 +/- 0.07 (n = 6)]. With the use of pipettes filled with 145 mmol/l KCl, inwardly rectifying property of the channels is disclosed with a single-channel conductance of 65.7 +/- 3.1 pS (n = 9) at zero potential difference between pipette and bath and with a reversal potential of 75.4 +/- 2.0 mV (n = 5; pipette negative vs. reference in the bath). The open probability of the channels is not significantly modified by altering pipette potential from -50 mV, pipette positive, to 50 mV, pipette negative. At extracellular calcium activities of less than 10 nmol/l, ATP leads to a transient activation of channels. In conclusion, extracellular ATP activates inwardly rectifying potassium channels in the cell membrane of subconfluent MDCK cells. A sustained activation of the channels requires the presence of extracellular calcium and is probably mediated by increases in intracellular calcium.

Adenosine Triphosphate↗

PubMed1988

Enhancement of intracellular calcium concentration by extracellular ATP and UTP in Madin Darby Canine Kidney cells.

Fura2 - fluorescence was utilized to test for the effect of extracellular nucleotides on intracellular calcium concentration of subconfluent Madin-Darby Canine Kidney (MDCK)-cells. Extracellular ATP (10 mumol/l) and UTP (10 mumol/l) lead to rapid (within seconds), sustained, and fully reversible enhancement of intracellular calcium concentration from 138 +/- 9 nmol/l (n = 27), to 1561 +/- 260 nmol/l (n = 10) and 3435 +/- 949 nmol/l (n = 5), respectively. Half maximal effects are observed at some 1 mumol/l. In the absence of extracellular calcium the effect of ATP is transient, pointing to release of intracellular calcium. The sustained effect in the presence of extracellular calcium indicates that the nucleotides in addition recruit calcium from extracellular space.

Adenosine Triphosphate↗

PubMed1988

Effects of extracellular nucleotides on electrical properties of subconfluent Madin Darby canine kidney cells.

ATP and ADP but not AMP lead to sustained hyperpolarization of Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for an influence of other nucleotides on the potential difference across the cell membrane (PD) in subconfluent MDCK cells. PD has been continuously monitored with conventional microelectrodes during rapid exchange of extracellular fluid. Application of 1 mumol/1 UTP leads to a rapid (less than 2 s) hyperpolarization of the cell membrane by -17.0 +/- 0.4 mV (from -50.1 +/- 0.6 mV), a reduction of cell membrane resistance and an increase of the sensitivity of PD to alterations of extracellular potassium. The concentration needed for half maximal effect of UTP is approximately equal to 0.2 mumol/1. ITP is similarly effective, whereas UDP, GTP and GDP are less effective. Up to 1 mmol/1 UMP, GMP, TTP or CTP do not significantly alter PD. In calcium-free extracellular fluid the hyperpolarizing effect of UTP is blunted (-11.6 +/- 2.3 mV) and only transient. In conclusion, UTP similar to purine triphosphates hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium, which is apparently recruited from both intra- and extracellular sources.

Animals↗