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M Bleich

Publications and source records attributed to M Bleich.

At least 73 records · Page 4Linked to original sources
PubMed1996

The ion conductances of colonic crypts from dexamethasone-treated rats.

Whole-cell patch-clamp studies were performed in isolated colonic crypts of rats pretreated with dexamethasone (6 mg/kg subcutaneously on 3 days consecutively prior to the experiment). The cells were divided into three categories according to their position along the crypt axis: surface cells (s.c.); mid-crypt cells (m.c.) and crypt base cells (b.c.). The zero-current membrane voltage (Vm) was -56 +/- 2 mV in s.c (n = 34); -76 +/- 2 mV in m.c. (n = 47); and -87 +/- 1 mV in b.c. (n = 87). The whole-cell conductance (Gm) was similar (8-12 nS) in all three types of cells. A fractional K+ conductance accounting for 29-67% of Gm was present in all cell types. A Na+ conductance was demonstrable in s.c. by the hyperpolarizing effect on Vm of a low-Na+ (5 mmol/l) solution. In m.c. and b.c. the hyperpolarizing effect was much smaller, albeit significant. Amiloride had a concentration-dependent hyperpolarizing effect on Vm in m.c. and even more so in s.c.. It reduced Gm by approximately 12%. The dissociation constant (KD) was around 0.2 micromol/l. Triamterene had a comparable but not additive effect (KD = 30 micromol/l, n = 14). Forskolin (10 micromol/l, in order to enhance cytosolic adenosine 3', 5'-cyclic monophosphate or cAMP) depolarized Vm in all three types of cells. The strongest effect was seen in b.c.. Gm was enhanced significantly in b.c. by 83% (forskolin) to 121% [8-(4-chlorophenylthio)cAMP]. The depolarization of Vm and increase in Gm was caused to large extent by an increase in Cl-conductance as shown by the effect of a reduction in bath Cl-concentration from 145 to 32 mmol/l. This manoeuvre hyperpolarized Vm under control conditions significantly by 6-9 mV in all three types of cells, whilst it depolarized Vm in the presence of forskolin in m.c. and in b.c.. These data indicate that s.c. of dexamethasone-treated rats possess mostly a K+ conductance and an amiloride- and triamterene-inhibitable Na+ conductance. m.c. and b.c. possess little or no Na+ conductance; their Vm is largely determined by a K+ conductance. Forskolin (via cAMP) augments the Cl- conductance of m.c. and b.c. but has only a slight effect on s.c.

Amiloride↗
PubMed1996

Crypt base cells show forskolin-induced Cl- secretion but no cation inward conductance.

Whole-cell patch-clamp studies in base cells of isolated colonic crypts of rats pretreated with dexamethasone were performed to examine the effects of stimulation by forskolin (10 micromol/l). The experiments were designed in order to distinguish between two postulated effector mechanisms: the activation of a non-selective cation channel and the activation of Cl- channels. As shown in an accompanying report, forskolin depolarizes the membrane voltage (Vm) by some 40-50 mV and enhances the whole-cell membrane conductance (Gm) substantially in these cells. In this report all experiments were performed in the presence of forskolin. A reduction of the bath Na+ concentration from 145 to 2 mmol/l led to a hyperpolarization of Vm by some 20-30 mV. This hyperpolarization occurred very slowly suggesting that the hyperpolarization produced by the low-Na+ solution was caused indirectly and not by a change in the equilibrium potential for Na+, ENa+. A complete kinetic analysis of the effect on voltage of bath Na+ revealed a saturation-type relation with a high apparent affinity for Na+ of around 5-10 mmol/l. A reduction in bath Cl- concentration from 145 to 32 mmol/l caused a depolarization of Vm from -34 +/- 3 to -20 +/- 4 mV (n = 13) in the presence of a high bath Na+ concentration, but had the opposite effect at low (5 mmol/l) Na+ concentrations: Vm was hyperpolarized from -46 +/- 4 to -62 +/- 6 mV (n = 13). If the effect of Na+ on Vm was caused by a non-selective cation channel the opposite would have been expected. To test directly whether the Na+2Cl-K+ cotransporter was responsible for the effects of changes in bath Na+ on Vm, the effects of increasing concentrations of several loop diuretics were examined. Furosemide, piretanide, torasemide and bumetanide (up to 0.1-0.5 mmol/l) all hyperpolarized Vm, albeit only by less than 10 mV. Another subclass of loop diuretics containing a tetrazolate in position 1 [e.g. azosemide, no. 19A and no. 20A from Schlatter E, Greger R, Weidtke C (1983) Pflüger Arch 396: 210-217] were much more effective. Azosemide hyperpolarized Vm from -46 +/- 3 to -74 +/- 2 mV (n = 18) and reduced Gm from 11 +/- 1 to 4 +/- 1 nS (n = 14). These data indicate that forskolin stimulates Cl- secretion in these cells by a mechanism fully compatible with the current scheme for exocrine secretion involving the Na+2Cl-K+ cotransporter.

Animals↗
PubMed1996

The amiloride inhibitable Na+ conductance of rat colonic crypt cells is suppressed by forskolin.

Previously we have shown that mid crypt cells of corticoid treated rats possess an amiloride inhibitable Na+ conductance (NAC) and show an increased Cl- conductance when stimulated by prostaglandin or the second messenger cAMP. The NAC is supposed to determine the magnitude of NaCl absorption. The Cl- conductance defines the magnitude of NaCl secretion. In the present whole cell (WC) patch clamp study we have examined whether the amiloride (3 "mu"mol/l) inhibitable NAC is downregulated when the Cl- conductance is increased by forskolin (5 "mu"mol/l, n=20) or the phosphodiesterase inhibitor IBMX (1 mmol/l, n=5). Under control conditions the amiloride inhibitable NAC was 2.7+/-0.4 nS. Forskolin depolarized the voltage from -58+/-2.0 to -48+/-1.9 mV and enhanced the WC conductance by 3.25+/-0.6 nS in these cells. The amiloride inhibitable NAC was reduced to 0.38+/-0.2 nS. These data confirm that forskolin enhances the Cl- conductance in these cells and they show for the first time that the Na+ conductance is reduced simultaneously. Thus the cells are able to change the direction of NaCl transport from absorption to secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1996

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells.

We have shown previously that secretagogues acting via the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl- conductance, a K+ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K+ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 micromol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K+ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency (fc) of 308 +/- 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K+ selective. 293B reduced the power density of the noise (So) to 46 +/- 8.7% of its control value and shifted fc from 291 +/- 26 to 468 +/- 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5'-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba2+ and was completely absent when K+ in the bath was replaced by Na+. These data suggest that forskolin activates a K+ channel of very small conductance which can be inhibited directly and reversibly by 293B.

Animals↗
PubMed1996

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells.

We have previously shown that a new type of K+ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K+ channel of 16 +/- 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0. 1 mmol/l, n = 26). Reduction of extracellular Ca2+ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SKCa). It was also inactivated by forskolin (5 micromol/l, n = 38), whilst the K+ channel noise caused by the very small K+ channel increased. Activity of non-selective cation channels (NScat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NScat, with a mean conductance of 49 +/- 1.0 pS (n = 96), was Ca2+ activated and required >10 micromol/l Ca2+ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3',5-dichloro-diphenylamine-2-carboxylate (10-100 micromol/l, n = 5). SKCa was also Ca2+ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 micromol/l (cs) and then fell sharply to almost zero at 0.1 micromol/l Ca2+ (cs, n = 12). SKCa was inhibited by Ba2+ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca2+ activity ([Ca2+]i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K+ concentration to 30 mmol/l, reduced [Ca2+]i markedly (n = 8-10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca2+-sensitive K+ channel. This channel is activated promptly by very small increments in [Ca2+]i and is inactivated by a fall in [Ca2+]i induced by forskolin.

Adenosine Triphosphate↗
PubMed1995

Effect of NH4+/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop.

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K+. In excised membrane patches the luminal K+ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (Vm) in slow-whole-cell (SWC) recordings and the open probability (Po) of K+ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of Vm can be recorded. The pipette solution contained K+ 125 mmol/l and Cl- 32 mmol/l. Intracellular pH was determined by 2',7'bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH4+/NH3 to the bath. In the presence of NH4+/NH3 intracellular pH acidified by 0.53 +/- 0.11 units (n = 7). Inhibition of the Na+2Cl-K+ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62 +/- 0.14 units (n = 7). In SWC experiments Vm of TAL cells was -72 +/- 1 mV (n = 70). NH4+/NH3 depolarised Vm by 22 +/- 2 mV (n = 25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH4+ from 24 +/- 3 mV to 7 +/- 3 mV. Under control conditions the single-channel conductance of TAL K+ channels in CAN experiments was 66 +/- 5 pS and the reversal voltage for K+ currents was 70 +/- 2 mV (n = 35). The Po of K+ channels in CAN patches was reduced by NH4+/NH3 from 0.45 +/- 0.15 to 0.09 +/- 0.07 (n = 7). NH4+/NH3 exposure depolarised the zero current voltage of the permeabilised patches by -9.7 +/- 3.6 mV (n = 5). The results show that TAL K+ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH4+/NH3 exposure at concentrations which are physiologically relevant because Na+2Cl-K+(NH4+) cotransporter-mediated import of NH4+ exceeds the rate of NH3 diffusion into the TAL. K+ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH4+ uptake occurs by the Na+2Cl-K+ cotransporter. The findings that, in the presence of NH4+/NH3 and furosemide, Vm is not completely repolarised and that K+ channels are not activated suggest that the respective K+ channels may in addition to their pH regulation be inhibited directly by NH4+/NH3.

Ammonia↗
PubMed1995

A new class of inhibitors of cAMP-mediated Cl- secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance.

Previously we have shown that arylaminobenzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (Isc) induced by prostaglandin E2 (n = 7), vasoactive intestinal polypeptide (VIP, n = 5), adenosine (n = 3), cholera toxin (n = 4) and cAMP (n = 6), but not by ionomycin (n = 5) in distal rabbit colon half maximally (IC50) at 2 mumol/l from the mucosal and at 0.7 mumol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116 +/- 16 omega.cm2 to 136 +/- 21 omega.cm2 (n = 5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound within this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mumol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mumol/l 293 B had no effect on the membrane voltage across the basolateral membrane (Vbl) in non-stimulated crypt cells: -69 +/- 3 mV versus -67 +/- 3 mV (n = 10), whilst in the same cells 1 mmol/l Ba2+ depolarised Vbl significantly. However, 293 B depolarised Vbl significantly in the presence of 1 mumol/l forskolin: -45 +/- 4 mV versus -39 +/- 5 mV (n = 7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised Vbl from -40 +/- 5 mV to -30 +/- 4 mV (n = 19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mumol/l 293 B: -75 +/- 6 mV versus -75 +/- 6 mV (n = 6). Also 293 B had no effect on basal K+ conductance (n = 4). Hence, we conclude that 293 B inhibits the K+ conductance induced by cAMP. This conductance is apparently relevant for Cl- secretion and the basal K+ conductance is insufficient to support secretion.

Amiloride↗
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↗
PubMed1990

The luminal K+ channel of the thick ascending limb of Henle's loop.

In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n = 260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60 +/- 6 pS (n = 24) and 31 +/- 7 pS (n = 4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72 +/- 4 pS (n = 37) at a clamp voltage of 0 mV. The permeability was 0.33 +/- 0.02 . 10(-12) cm3/s. The selectivity sequence of this channel was: K+ = Rb+ = NH4+ = Cs+ greater than Li+ much greater than Na+ = 0; the conductance sequence was K+ much greater than Li+ much greater than Rb+ = Cs+ = NH4+ = Na+ = 0. In excised patches Rb+, Cs+ and NH4+ when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5 +/- 1.5 ms, n = 18) and by two closed (1.4 +/- 0.1 and 14 +/- 2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mumol/l -0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10-100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities greater than 10(-7) mol/l and by adenosine triphosphate greater than or equal to 10(-4) mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by less than or equal to 0.2 units).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗