A Target-Specific Cellular Assay for Screening of Topoisomerase I Inhibitors.

We have developed a cellular, target-specific high-throughput assay for the detection of topoisomerase I inhibitors. Topoisomerase I is a nonessential enzyme involved in controlling DNA topology. Topoisomerase I is the target of anticancer drugs such as camptothecin as well as a candidate target for new antifungal drugs. A wild-type Saccharomyces cerevisiae strain and its isogenic topoisomerase I deletion mutant (DeltatopI) were labeled with S65T and wild-type green fluorescent protein (GFP), respectively. We showed that the growth of such a pair of S. cerevisiae strains labeled with this GFP combination can be independently quantified after both strains were mixed. When growth of the mixture of wild-type and DeltatopI strain was monitored in the presence of compounds, only growth of the wild-type strain was inhibited by the topoisomerase I-specific drug camptothecin. In contrast, amphotericin B, a broad-spectrum antifungal drug, inhibited growth of both strains. The two strains were used to screen compound libraries. While 0.9% of all compounds inhibited growth of both strains, only 0.06% inhibited the wild-type but not the DeltatopI strain. Thus, by using a DeltatopI strain as internal control in the same assay mixture, the number of candidate topoisomerase I inhibitors to be retested could be reduced by more than 90%. Further applications of this type of S. cerevisiae-based cellular high-throughput assays will be discussed.

The effect of secretagogues on ion conductances of in vitro perfused, isolated rabbit colonic crypts.

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca2+ activity and are known to increase Cl- secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (Vte), transepithelial resistance (Rte), membrane voltage across the basolateral membrane (Vbl), and fractional basolateral membrane resistance (FRbl), were estimated. Basolateral prostaglandin E2 (PGE2, > or = 0.1 mumol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of Vbl. In the case of adenosine, the initial depolarization of Vbl was by 31 +/- 2 mV (n = 47). Rte fell significantly from 16.4 +/- 3.6 to 14.2 +/- 3.7 omega.cm2 (n = 6), and FRbl increased significantly from 0.11 +/- 0.02 to 0.51 +/- 0.10 (n = 6). In the second phase the repolarisation of Vbl amounted 11 +/- 2 mV (n = 47) and a steady-state (Vbl) of -51 +/- 2 mV (n = 47) was reached. Rte fell further and significantly to a steady-state value of 12.4 +/- 3.8 omega.cm2 (n = 6) and FRbl fell significantly to 0.42 +/- 0.13 (n = 6). In 30% of the experiments, a transient hyperpolarisation of Vbl by 8 +/- 2 mV (n = 14) was seen during wash out of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effects of dinitrophenol on active-transport processes and cell membranes in the Malpighian tubule of Formica.

In Formica Malpighian tubules KCl secretion is driven by a V-type H+ ATPase in the luminal membrane in parallel with a H+/K+ antiporter. The effect of the protonophore dinitrophenol (DNP) was investigated on the isolated, symmetrically perfused tubule. DNP was applied in two different concentrations: 0.2 mmol/l and 1 mmol/l. The effects were fast and rapidly reversible. The equivalent short-circuit current (Isc) was reduced significantly to respectively 25 +/- 3% Cn = 4) and -3 +/- 7% (n = 11) of the control value when 0.2 mmol/l or 1 mmol/l was added to the bath. When 1 mmol/l DNP was applied the transepithelial resistance (Rte) decreased significantly to 74 +/- 11% of the control value (n = 11), and the luminal over basolateral voltage divider ratio (VDR), providing an estimate of luminal over basolateral membrane resistance, decreased to 37 +/- 12% of the control (n = 6). A concentration of 1 mmol/l DNP was also applied from the lumen. The decrease in Isc was significant, but much less pronounced (74 +/- 5% of control; n = 6) and no significant changes in Rte and VDR were observed. It is argued that, when the concentration in the bath is high enough, DNP may cross the cell and have a protonophoric effect not only on the mitochondria but also across the luminal cell membrane explaining the drop in transepithelial and in relative luminal membrane resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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)

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.

Design, synthesis and biological activity of a series of torasemide derivatives, potent blockers of the Na+ 2Cl- K+ co-transporter: in-vitro study.

Pharmacomodulation of the torasemide molecule, a loop diuretic inhibiting Na+ 2Cl- K+ co-transport in the thick ascending limb of the loop of Henlé has been performed in order to obtain new long-acting diuretics. The aim of this study was to decrease the metabolism of the drug and to slow down its rate of excretion by increasing its hydrophobicity. The present study describes the synthesis and the inhibitory potency of new torasemide derivatives in the bioassay system of the cortical thick ascending limb of rabbit. A correlation between the lipophilicity (log P') of these substances and their activity as inhibitors of the Na+ Cl- K+ co-transporter was observed. The present design led to compounds more active than torasemide. Structure-activity relationships permit us to propose an interaction model between torasemide derivatives and the Na+ 2Cl- K+ co-transport system of the cortical thick ascending limb.

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 mumol/l) or isoprenaline (10 mumol/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 greater than ATP greater than ITP greater than GTP greater than TTP greater than 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 mumol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

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 NH+4 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 NH+4, which is secreted in the CCD, cross the principal cells as NH+4. 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 patch-clamp studies.

Action of diuretics at the cellular level.

Classification of diuretics is based on their site and mechanism of action in the nephron. The most frequently used substances comprise 1. the mostly proximally acting carbonic anhydrase inhibitors (CAI); 2. the loop diuretics (LD); 3. the early distally acting thiazides (TZ); and 4. the K+ sparing diuretics (KS) acting in the distal tubule. CAI such as acetazolamide inhibit the dehydration of H2CO3 at the luminal membrane, the hydration of CO2 within the proximal tubule cell and the exit of HCO-3 out of the cell. As a result of this proximal reabsorption of HCO-3 is reduced, a slight diuresis and saluresis is induced. The enhanced urinary excretion of HCO-3 will cause a metabolic acidosis. LD inhibit the Na+2Cl-K+ carrier in the thick ascending limb of the loop of Henle (TAL). This produces a marked diuresis and saluresis which is accompanied by enhanced Ca2+, Mg2+, K+ and acid excretion. TZ inhibit the Na+Cl- cotransporter in the early distal tubule. The diuresis is less marked than that induced by LD but the renal losses of K+ are comparable. KS inhibit Na+ channels present in the luminal membrane of the cortical collecting tubule. This leads to a very limited diuresis, but a marked attentuation of renal K+ losses. All diuretics act by inhibiting the admission of Na+ (LD, TZ, KS) or HCO-3 (CAI) into the cell. Their organotropy is merely due to the fact that they are concentrated in tubule fluid by volume reabsorption and by proximal tubule secretion.

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 mumol/l) Isc was -71 +/- 11 (n = 118) microA/cm2. Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to +32 +/- 4 (n = 118) microA/cm2. In the presence of amiloride and indomethacin, prostaglandin E2 (1 mumol/l, s), known to induce Cl- secretion, generated an Isc of -143 +/- 8 (n = 92) microA/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 mumol/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).(ABSTRACT TRUNCATED AT 250 WORDS)