E. Lohrmann

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 arylamino-benzoates 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 μmol/l from the mucosal and at 0.7 μmol/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 Ω·cm2 to 136±21 Ω·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 with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 μmol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 μmol/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 μmol/l forskolin: −45±4mV 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 μmol/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.

Unmasking of the Apical Electrogenic H Pump in Isolated Malpighian Tubules (Formica polyctena) by the Use of Barium

In the present study evidence is given for the presence of an electrogenic, vacuolar type ATPase (V type ATPase) in the apical membrane of malpighian tubules of Formica. Barium (6 m M

Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon

Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (Vte) and resistance (Rte) were measured and the equivalent short-circuit current (ISC=Vte/ Rte) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 μmol/l) ISC was −71±11 (n = 118) μA/cm2. Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to + 32±4 (n=118) μA/cm2. In the presence of amiloride and indomethacin, prostaglandin E2 (1 μmol/l, s), known to induce Cl− secretion, generated an ISC of -143 ± 8 (n = 92) μA/cm2. The arylaminobenzoate and Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) reduced ISC reversibly with a half-maximal inhibition (IC50) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC50 to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-l-cysteine (m) preincubation. Pretreatment with N-acetyl-l-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and l-homocysteine (m) were more effective and reduced IC50 to approximately 50 μmol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/−1-phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl− conductance in the thick ascending limb of the loop of Henle (TAL). In the present study the (+) enantiomer inhibited significantly more strongly than the (−) form. This suggests that the inhibitory effect of NPPB, even though it requires rather high concentrations, is probably due to Cl− channel inhibition. For other arylaminobenzoates the sequence of potencies was different from that determined for the TAL. The present data indicate that substances that have been designed to block the Cl− conductance of the TAL segment also inhibit reversibly but with much lower affinity the PGE2-induced Cl− secretion in rabbit colon.

Properties of the potassium conductances of principal cells of rat cortical collecting ducts

In this study we examined by impalement techniques properties of the macroscopic K+ conductances in the luminal and basolateral membrane of principal cells from isolated perfused cortical collecting ducts (CCD) of the rat. Both membranes possess a dominating K+ conductance. Compared to their behaviour with K+, both membranes appear much less permeable to NH4+and Rb+, and the K+ conductances of both membranes are inhibited by these cations. In light of these findings, it is very unlikely that significant amounts of NH4+, which is secreted in the CCD, cross the principal cells as NH4+. Several inhibitors with known effects on K+ channels in patch-clamp studies have been examined. Tetraethylammonium, which inhibits the excised K+ channels of these cells, has no effect on the macroscopic K+ conductances of either membrane. Verapamil, which inhibits the K+ channels in the luminal membrane, acts predominantly on the basolateral membrane K+ conductance in the intact tubule. Therefore, some of the macroscopic properties of the K+ conductances are distinct from those of single channels thus far observed in patchclamp studies.

Intrinsic regulation of K+ transport in Malpighian tubules (Formica): Electrophysiological evidence

Intracellular basolateral (Vbl) and, indirectly, apical membrane potentials (Vap) have been measured in spontaneously secreting isolated Malpighian tubules of Formica. Vbl was sensitive to the bath K+ concentration, [K+]bl, (42 mV/decade) and very little to the Na+ and Cl− concentration. Vap was also sensitive to [K+]bl. The voltage changes in Vap were 60–70% of those in Vbl, so the transepithelial potential (Vte) also changed: it increased with [K+]bl (lumen became more positive). The overall result of an increase in bath K+ concentration was a facilitation of KCl secretion: the apical electrical gradient to be overcome for K+ extrusion was lowered and the transepithelial electrical gradient favouring Cl− movement to the lumen was increased. Transient changes in Vbl on varying [K+]bl also suggested dependence of the cellular K+ concentration on [K+]bl. When plotting fluid secretion rate, i.e. K+ transport, as a function of the calculated apical electrochemical gradient for K+, a highly significant inverse relationship was found. The gradient itself was very closely correlated with [K+]bl. In another series of experiments tubules were luminally perfused with symmetrical solutions (51 mM K+ 143 mM Cl−) and resistances were measured after current injection. We report a transepithelial length-specific resistance of 23 ± 3 kΩcm, a tissue specific resistance of 182 ± Ωcm2 and a length constant of 402 ± 29 μm (n = 38). From intracellular measurements and luminal injection of current the voltage divider ratio could be obtained. This gave an estimate of the ratio of apical over basolateral resistance of 44 ± 8 (n = 6). Analysis of the electrical equivalent model, taking into account the high apical over basolateral resistance, explained why changes in the basolateral electromotive forces were reflected on the apical membrane. Taken together the results of this study were consistent with a role for the high basolateral conductance (relative to the apical conductance) in the intrinsic regulation of K+ transport by bath [K+]. A second mechanism of regulation could be the fact that [K+]bl determines the amount of cellular K+ available for transport.

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 (Vb1) was −65±1 mV (n=240). Bath Ba2+ (1 mmol/ l) and verapamil (0.1 mmol/l) depolarized Vb1 by 21±2 mV (n=7) and 31±1 (n=4), respectively. Lowering of bath Cl− concentration hyperpolarized Vb1 from −69±3 to −75±3 mV (n=9). Lowering of luminal Cl− concentration did not change Vb1. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vb1 in non-stimulated crypts. Forskolin (10−6 mol/l) in the bath depolarized Vb1 by 29±2 mV (n=54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vb1 was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vb1 significantly and concentration dependently with a potency sequence of bumetanide > piretanide ≥ furosemide. Lowering bath Cl− concentration hyperpolarized Vb1. Lowering of luminal Cl− concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vb1 by 7±2 mV (n=10). We conclude that Vb1 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.

A Chromanol Type of K+ Channel Blocker Inhibits Forskolin- but Not Carbachol-Mediated Cl- Secretion in Rat and Rabbit Colon

In a previous study [Lohrmann et al: Pflugers Arch Eur J Physiol 1995;429:517-530] we have shown that chromanol K+ channel blockers inhibit Cl– secretion in rabbit colon. Their effect was easily demonstrable after stimulation by hormones acting through increases of cytosolic cAMP. The present study was undertaken to test in more detail the mechanism of action of one of these compounds (293 B). Two types of studies were performed: Ussing chamber experiments in rabbit distal colon and whole cell patch clamp studies in the isolated in vitro perfused rat colonic crypts. Carbachol (CCH, 100 µmol/l) enhanced Cl– secretion, quantified as equivalent short circuit current (Isc), in rabbit colon significantly more than did prosta-glandin E2 (PGE2). In whole cell patch clamp studies in rat colonic crypt cells from the base, CCH hyperpolarized the membrane voltage (Vm) and enhanced whole cell conductance (Gm). In agreement with previous impalement studies, 10 µmol/l 293 B depolarized Vm in forskolin-treated rat colonic crypt base cells even further and reduced Gm. In Ussing chamber experiments in rabbit colon, 293 B abolished the Isc induced by PGE2. CCH, in the continued presence of 293 B, still induced a large Isc. These data indicate that 293 B specifically inhibits the forskolin- but not the CCH-induced Cl– secretion, and supports our previous conclusion that this class of substances inhibits a cAMP-regulated K+ conductance.

Transmitter-induced changes of the membrane voltage of HT29 cells

The colonic carcinoma cell line HT29 was used to examine the influence of agonists increasing cytosolic cAMP and Ca2+ activity on the conductances and the cell membrane voltage (Vm). HT29 cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements Vm was −51±1 mV. An increase in bath K+ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba2+ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl− concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 μmol/l) or isoprenaline (10 μmol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP > ATP > ITP > GTP > TIP > CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 μmol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT29 cells possess Cl−- and K+-conductive pathways. The Cl− conductance is regulated by intracellular cAMP level, cytosolic Ca2+ activity, and cell swelling. The K+ conductance in HT29 cells is regulated by intracellular Ca2+ activity.

Characteristics of the luminal proton pump in malpighian tubules of the ant.

The active pump mechanisms involved in K+ secretion of the malpighian tubules of the ant and present in the luminal membrane were investigated on isolated, luminally perfused tubules of Formica. The specific blocker for vacuolar type ATPases, bafilomycin A1, was found to half-maximally inhibit secretion at a concentration of 10(-5) mol/l when added to the lumen. N-Ethylmaleimide reduced the calculated short circuit current (Isc) to 78 and 21% of control value when added at 5 x 10(-4) mol/l, respectively, to the lumen and the bath. Reducing luminal pH inhibited Isc with a half-maximal inhibition at a luminal pH of 4.5. Acidified omeprazole, Schering compound 28080 and vanadate (both 10(-3) and 10(-4) mol/l) inhibited Isc only partially. The present data suggest that the luminal membrane of ant malpighian tubules contains a H+ pump. This pump is only poorly bafilomycin-sensitive. Furthermore, additional active transport systems responsible for secretion may be present. Part of these results have been published as abstracts.

Electrical Properties of the Isolated, in vitro Perfused Malpighian Tubule of the Ant, the CI– Pathway

An electrical characterization of the isolated, perfused malpighian tubule of Formica polyctena and F. rufa is reported. Some specific problems of the perfusion method as applied to malpighian tubules of the ant are discussed and a practical solution presented. Estimates for the individual membrane resistances are derived. The shunt is investigated by basolateral biionic replacements and found to be anion specific. The Cl– pathway is further investigated by the addition of Cl– channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4’-diisothiocyano-2,2’-stilbenedisulfonate (DIDS), to the lumen. The lack of effect suggests the absence of DIDS- and NPPB-sensitive Cl– channels.