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R Greger

Publications and source records attributed to R Greger.

At least 145 records · Page 8Linked to original sources
PubMed1994

pH regulation in HT29 colon carcinoma cells.

The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1994

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique.

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca2+ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10(-5) mol/l)-induced intracellular Ca2+ transient, (2) a reduced fura-2 Mn(2+)-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca2+ as a function of membrane voltage, we measured simultaneously the intracellular Ca2+ activity ([Ca2+]i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT29 cells. Ca2+ influx was either stimulated by ATP (10(-5) mol/l) or thapsigargin (TG, 10(-8) mol/l). After [Ca2+]i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca2+]i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca2+]i (ATP: 27.5 +/- 10 nmol/l per 10 mV, n = 6; TG: 19 +/- 7.9 nmol/l per 10 mV, n = 12). The summarised data show a linear relationship between the delta fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca2+]i (n = 9). Under extracellular Ca(2+)-free conditions [Ca2+]i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca2+ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Adenosine-induced hyperpolarization of the membrane voltage in rat mesangial cells in primary culture.

1. The effect of adenosine on membrane voltage and ion currents was studied in rat mesangial cells in primary culture. Membrane voltage was measured with the patch clamp technique in the slow- or fast whole cell configuration. The resting membrane voltage of mesangial cells was -48 +/- 0.5 mV. Adenosine (10(-8)-10(-3) M) induced a sustained and concentration-dependent hyperpolarization of membrane voltage (ED50 approximately 6 x 10(-7) M). Adenosine (10(-5) M) hyperpolarized the membrane voltage by 14 +/- 0.5 mV. During the hyperpolarization ion currents were monitored simultaneously. An increase of the outward current by 51 +/- 11% was observed. 2. An increase of the extracellular K+ concentration (from 3.6 to 18.6 M) caused a depolarization of membrane voltage to -34 +/- 2 mV. In the presence of increased K+ the hyperpolarization of membrane voltage induced by adenosine was significantly attenuated by 61 +/- 5%. The K(+)-channel blocker, Ba2+ (5 x 10(-3) M) depolarized membrane voltage to -24 +/- 2 mV. In the presence of Ba2+ the adenosine-induced hyperpolarization was significantly inhibited by 72 +/- 8%. 3. Preincubation of the adenosine antagonist, 8-phenyltheophylline (10(-4) M) significantly inhibited the adenosine (10(-5) M) mediated membrane voltage response by 67 +/- 8%. The adenosine agonists 5-N-ethylcarboxamidoadenosine (NECA), R-(-)N6-(2-phenylisopropyl)adenosine (R-(-)-PIA), S-(+)-N6-(2-phenylisopropyl)adenosine (S-(+)-PIA), N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), and 2-chloroadenosine (2-CA) also hyperpolarized membrane voltage of mesangial cells. The rank order of potency of the agonists at 10-5 M was NECA> adenosine = > R-(-)-PIA = DPMA = 2-CA > S-( + )-PIA.4. Stimulation of cyclic AMP by forskolin induced a concentration-dependent hyperpolarization of membrane voltage (ED50 ~2 x 10-7 M). Application of forskolin (10-5 M) in the presence of adenosine(10-4 M) had no additive hyperpolarizing effect on the membrane voltage.5. Activation of protein kinase C by phorbol 12,13 dibutyrate (PDBu) induced a sustained depolarization of membrane voltage (ED50~ 5 x 10-9 M). In the presence of PDBu, adenosine (10-5 M) still hyperpolarized membrane voltage of mesangial cells.6. The data indicate that adenosine activates K+-conductance via an A2 receptor in mesangial cells; the activation of the K+-conductance, which is probably mediated by cyclic AMP led to a hyperpolarization of membrane voltage.

Adenosine↗
PubMed1994

Tubular effects of the diuretic torasemide.

Torasemide is a lipophilic loop diuretic which is largely metabolized in the liver and has an almost neutral pKa. Experiments were designed to address the questions of whether torasemide is secreted by the proximal tubule, and hence accumulates in tubular fluid, whether torasemide paralyses active transport in the cortical thick ascending limb of the nephron and hence reduces its ATP requirement, and finally whether torasemide is active in its protonated or unprotonated form. Intravenous torasemide, 10 mg/kg, induced a marked diuresis and natriuresis and a moderate kaliuresis in antidiuretic rats. All effects were dose-dependently suppressed by intravenous probenecid, 20-80 mg/kg, indicating that torasemide is secreted by the anion secretory system of the proximal tubule. A time-dependent depolarization of the basolateral membrane of in vitro perfused rabbit cortical thick ascending limb segments was observed after removal of metabolic substrates and after addition of ouabain. This effect, caused by Na+ entry, K+ loss and cell swelling, was prevented when torasemide was added to the luminal perfusate before substrate removal or addition of ouabain, indicating that torasemide significantly reduced ATP consumption of the cortical thick ascending limb. To test whether protonated or unprotonated torasemide was the biologically active compound, cortical thick ascending limb segments were perfused over the pH range 6-8 and torasemide was added in the concentration range 0.01-10 mumol/l; active transport was measured as the equivalent short-circuit current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

[The principles of studying ion transport in the kidney tubules].

The present overview indicates that the various nephron segments take part in the Na+Cl- reabsorption, the primary driving force for it is the (Na+, K+)-ATPase, which is localized in the basolateral membrane. The various segments have different modalities of Na+ uptake: in is by Na+/H+ exchange in the proximal tubule, by Na+/2Cl-/K+ co-transport in TAL, Na+Cl- cotransport in the distal tubule, and via Na+ channels in the principal cell of the collecting duct. In the proximal tubule bulk reabsorption occurs, but very small ionic gradients are built up, there for the transport here is so economical. In the TAL transport is already less economical, however ionic gradients are built up by this nephron segment inasmuch as Na+Cl- is reabsorbed and water cannot follow (the urinary concentrating mechanism). The distal tubule is concerned with defined control of Na+ and K+ excretion. Transport at this side is expensive, but very steep ionic gradients can be built up. The control is mediated by several hormones amongst which ADH and aldosterone are the most important ones.

Absorption↗
PubMed1994

[Patch-clamp methods in research on the kidney tubules].

Patch-clamp studies in renal tubule cell have been very helpful in increasing our understanding of the tubule transport function. However, it has been a disappointing experience that the properties of cultured cells deviate grossly from the physiology of the intact tubule segment. Also, several important conductive pathways have not been characterized thus far on single channel level. Several ion channels, which have been found in the patch-clamp studies, such as the non-selective cation channel, do not find their equivalent in the intact tubule preparation. Therefore, much more efforts are required not only to characterize individual ion channels, but rather to integrate this knowledge for the understanding of tubule function.

Animals↗
PubMed1993

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling.

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca2+]i to a stable plateau of 10-20 nmol/l above resting [Ca2+]i was observed. Lower osmolalities resulted in a triphasic increase of [Ca2+]i to a peak value. In a first phase after the volume change, lasting 20-40 s, [Ca2+]i increased slowly by about 30 nmol/l. Thereafter [Ca2+]i increased more rapidly within 20-30 s to a peak value. This peak was 189 +/- 45 nmol/l (190 mosmol/l, n = 9) and 243 +/- 41 nmol/l (160 mosmol/l, n = 20) above resting [Ca2+]i. The peak was then followed by a decline of [Ca2+]i over the next 2-3 min to a stable plateau value of 28 +/- 6 (n = 6) and 32 +/- 11 nmol/l (n = 11) above resting [Ca2+]i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca(2+)-free bath conditions the peak value for the cell-swelling-induced [Ca2+]i transient was reached significantly later (60-100 s, compared to 40-60 s under control conditions). The peak values under Ca(2+)-free conditions were not significantly lower. This indicates that the [Ca2+]i peak was mostly of intracellular origin. No [Ca2+]i plateau phase was observed under Ca(2+)-free bath conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1993

Effect of bradykinin and histamine on the membrane voltage, ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture.

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (Vm), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37 degrees C. The mean value of Vm was -41 +/- 0.5 mV (n = 189). BK (10(-6) mol/l, n = 29) and Hist (10(-5) mol/l, n = 55) induced a rapid transient hyperpolarization by 15 +/- 1 mV and 18 +/- 1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6 +/- 1 mV (BK 10(-6) mol/l) and 7 +/- 1 mV (Hist 10(-5) mol/l). The ED50 was about 5 x 10(-8) mol/l for BK and 5 x 10(-7) mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K+ concentration from 3.6 to 30 mmol/l depolarized Vm by 8 +/- 1 mV and completely inhibited the hyperpolarizing effect of both agents (n = 11). Reduction of extracellular Cl- concentration from 145 to 30 mmol/l led to a depolarization by 2 +/- 1 mV (n = 25). In 30 mmol/l Cl- the depolarizations induced by BK (10(-7) mol/l) and Hist (10(-6) mol/l) were augmented to 9 +/- 2 mV (n = 14) and to 10 +/- 2 mV (n = 11), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin↗
PubMed1993

Isolated perfused rabbit colon crypts: stimulation of Cl- secretion by forskolin.

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (Vbl) was -65 +/- 1 mV (n = 240). Bath Ba2+ (1 mmol/l) and verapamil (0.1 mmol/l) depolarized Vbl by 21 +/- 2 mV (n = 7) and 31 +/- 1 (n = 4), respectively. Lowering of bath Cl- concentration hyperpolarized Vbl from -69 +/- 3 to -75 +/- 3 mV (n = 9). Lowering of luminal Cl- concentration did not change Vbl. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vbl in non-stimulated crypts. Forskolin (10(-6) mol/l) in the bath depolarized Vbl by 29 +/- 2 mV (n = 54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vbl was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vbl significantly and concentration dependently with a potency sequence of bumetanide > piretanide > or = furosemide. Lowering bath Cl- concentration hyperpolarized Vbl. Lowering of luminal Cl- concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vbl by 7 +/- 2 mV (n = 10). We conclude that Vbl of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl- conductance. Basolateral uptake of Cl- occurs via a basolateral Na+:2Cl-:K+ cotransport system.

Animals↗
PubMed1993

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells.

There is increasing evidence that some agonists not only induce intracellular Ca2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca2+ efflux. We have investigated the agonist-stimulated response on the intracellular Ca2+ activity ([Ca2+]i) in the presence of thapsigargin (10(-8) mol/l, TG) in HT29 and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10(-7)-10(-3) mol/l, n = 9), carbachol (10(-6)-10(-3) mol/l, n = 5) and neurotensin (10(-10)-10(-7) mol/l, n = 6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 cells. This decrease of [Ca2+]i could be caused by a reduced Ca2+ influx, either due to a reduced driving force for Ca2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca2+ permeability. Using the fura-2 Mn2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca2+ into the cell, but rather due to stimulation of Ca2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of -49 +/- 3.6 mV (n = 9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Small and intermediate conductance chloride channels in HT29 cells.

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT29 cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g(o)) is caused by inhibition of small non-resolvable channels, which are impermeable to Na+ and SO4(2-), but permeable to Cl-. The regulation of these small Cl(-)-conducting channels (g(o)) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (Po) of the ICOR or on the g(o)) were observed. The Po of the ICOR was reduced by 22% in a Ca(2+)-free solution. g(o) was insensitive to changes in the Ca2+ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT29 cells contain ICOR and small non-resolvable Cl(-)-conducting channels which are similarly inhibited by C. I.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Agonist-induced intracellular Ca2+ transients in HT29 cells.

In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1-40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10(-10) to 10(-7) mol/l) in most cases only a [Ca2+]i spike lasting 2-3 min. The [Ca2+]i plateau induced by ATP (10(-6) mol/l) and CCH (10(-5) mol/l) was abolished by reducing the Ca2+ activity in the bath from 10(-3) to 10(-4) mol/l (n = 7). In Ca(2+)-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23 +/- 1.5 s (n = 15) indicating a similar mechanism in each case. Verapamil (10(-6)-10(-4) mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10(-3) mol/l) reduced the plateau value by 70%.

Adenosine Triphosphate↗
PubMed1993

Cation specificity and pharmacological properties of the Ca(2+)-dependent K+ channel of rat cortical collecting ducts.

The luminal membrane of principal cells of rat cortical collecting duct (CCD) is dominated by a K+ conductance. Two different K+ channels are described for this membrane. K+ secretion probably occurs via a small-conductance Ca(2+)-independent channel. The function of the second, large-conductance Ca(2+)-dependent channel is unclear. This study examines properties of this channel to allow a comparison of this K+ channel with the macroscopic K+ conductance of the CCD and with similar K+ channels from other preparations. The channel is poorly active on the cell. It has a conductance of 263 +/- 11 pS (n = 36, symmetrical K+ concentrations) and of 139 +/- 3 pS (n = 91) with 145 mmol/l K+ on one side and 3.6 mmol/l K+ on the other side of the membrane. Its open probability is high after excision (0.71 +/- 0.03, n = 85). The channel flickers rapidly between open and closed states. Its permeability in the cell-free configuration was 7.0 +/- 0.2 x 10(-13) cm3/s (n = 85). It is inhibited by several typical blockers of K+ channels such as Ba2+, tetraethylammonium, quinine, and quinidine and high concentrations of Mg2+. The Ca2+ antagonist verapamil and diltiazem also inhibit this K+ channel. As is typical for the maxi K+ channel, it is inhibited by charybdotoxin but not by apamin. The selectivity of this large-conductance K+ channel demonstrates significant differences between the permeability sequence (pK > pRb > pNH4 > pCs = pLi = pNa = pcholine = 0) and the conductance sequence (gK > gNH4 > gRb > gLi = gcholine > gCs = gNa = 0). The only other cations that are significantly conducted by this channel besides K+ (gK at Vc = infinity is 279 +/- 8 pS, n = 88) re NH+4 (gNH4 = 127 +/- 22 pS, n = 10) and Rb+ (gRb = 36 +/- 5 pS, n = 6). The K+ currents through this channel are reduced by high concentrations of choline+, Cs+, Rb+, and NH+4. These properties and the dependence of this channel on Ca2+ and voltage classify it as a "maxi" K+ channel. A possible physiological function of this channel is discussed in the accompanying paper.

Animals↗
PubMed1993

Localization of NaPi-1, a Na-Pi cotransporter, in rabbit kidney proximal tubules. I. mRNA localization by reverse transcription/polymerase chain reaction.

We have recently isolated from a rabbit cortex cDNA library a cDNA clone (NaPi-1), which, after in vitro transcription (cRNA) and injection into Xenopus laevis oocytes, expresses Na-dependent Pi uptake [Werner A, et al. (1991) Proc Natl Acad Sci USA 88:9608-9612]. The aim of the present work was to study the nephron location of the NaPi-1-related mRNA(s) by combining nephron microdissection procedures, reverse transcription (RT) and amplification of the resultant cDNA by the polymerase chain reaction (PCR). RT-PCR using NaPi-1-specific primers (different combinations) and either total kidney cortex RNA or microdissected proximal tubule segments resulted in two PCR products, both of approximately the expected length (but differing by about 30 base pairs). Restriction-enzyme analysis and nucleotide sequencing confirmed that both PCR products are related to NaPi-1 and that the "longer" PCR product has an insert of 26 base pairs containing an AluI restriction site. Nephron microdissection documents expression of NaPi-1-related mRNA(s) in superficial and deep proximal tubules (S1, S2 and S3 segments) and their absence in glomeruli, thin descending limb and thick ascending limbs of Henle's loop, distal convoluted tubules and cortical and inner medullary collecting ducts. These experiments suggest a "microheterogeneity" of NaPi-1-related mRNA(s) (which is not detected in Northern blot analysis) and proximal tubular expression of NaPi-1.

Animals↗
PubMed1993

Increase in cytosolic Ca2+ regulates exocytosis and Cl- conductance in HT29 cells.

Increases of cytosolic Ca2+, as occur with agonists such as ATP, neurotensin (NT), hypotonic cell swelling and ionomycin, enhance the membrane conductance (GM) and hence the input conductance (GI) of HT29 cells. In the present study we have examined whether these increases in GM are paralleled by exocytosis. To this end the membrane capacitance (CM) of HT29 cells was measured by patch clamp techniques. Two methods to monitor CM were used: a direct method (DM) and a phase tracking method (PTM). With the DM the following results were obtained. NT (10(-8) mol/l, n = 9) increased GM and CM significantly from 2.4 +/- 0.3 nS and 23.5 +/- 3 pF to 32 +/- 8 nS and 27.3 +/- 3.1 pF respectively. ATP (10(-4) mol/l, n = 29) had a very similar effect. GM and CM were increased from 5.7 +/- 1 nS and 36 +/- 4.4 pF to 111 +/- 21 nS and 44 +/- 5.4 pF respectively. Hypotonic cell swelling (160 mosmol/l, n = 18) had a comparable effect: GM and CM were increased from 4.9 +/- 1 nS and 30 +/- 4.1 pF to 46 +/- 10 nS and 37 +/- 4.9 pF respectively. Ionomycin (10(-7) mol/l, n = 4) gave similar results. With the PTM it was possible to monitor the rapid changes in GM and CM, as they were induced by ATP (n = 42) and NT (n = 29), with high time resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture.

1. The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2. Addition of extracellular ATP (10(-5) and 10(-4) M) to MC led to a cell contraction which was independent of extracellular calcium. 3. Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 x 10(-6) M). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4. In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mM) ATP induced a depolarization augmented to -4 +/- 4 mV. 5. ATP-gamma-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas alpha,beta-methylene-ATP (all 10(-5) M) had no effect on Vm. 6. The Ca2+ ionophore, A23187, depolarized Vm transiently from -51 +/- 2 to -28 +/- 4 mV and caused an increase of the inward current. 7. The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 x 10(-6) M). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free buffer, whereas the plateau was abolished. Verapamil (10(-4) M) did not inhibit the [Ca2+]i increase induced by ATP. 9. The data indicate that extracellular ATP contracts MC and is able to increase [Ca2+]i by the release of Ca2+ from intracellular stores and recruitment from the extracellular space. In addition ATP depolarizes Vm of MC by activating a Cl- conductance. The ATP-induced depolarization is mediated by a P2y receptor.

Adenosine Triphosphate↗