Diisothiocyanatostilbene-2,2 ' - Disulphonate Research Articles

Biotic Control of Surface pH and Evidence of Light-Induced H+ Pumping and Ca2+-H+ Exchange in a Tropical Crustose Coralline Alga.

Presently, an incomplete mechanistic understanding of tropical reef macroalgae photosynthesis and calcification restricts predictions of how these important autotrophs will respond to global change. Therefore, we investigated the mechanistic link between inorganic carbon uptake pathways, photosynthesis and calcification in a tropical crustose coralline alga (CCA) using microsensors. We measured pH, oxygen (O2), and calcium (Ca2+) dynamics and fluxes at the thallus surface under ambient (8.1) and low (7.8) seawater pH (pHSW) and across a range of irradiances. Acetazolamide (AZ) was used to inhibit extracellular carbonic anhydrase (CAext), which mediates hydrolysis of HCO3-, and 4,4′ diisothiocyanatostilbene-2,2′-disulphonate (DIDS) that blocks direct HCO3- uptake by anion exchange transport. Both inhibited photosynthesis, suggesting both diffusive uptake of CO2 via HCO3- hydrolysis to CO2 and direct HCO3- ion transport are important in this CCA. Surface pH was raised approximately 0.3 units at saturating irradiance, but less when CAext was inhibited. Surface pH was lower at pHSW 7.8 than pHSW 8.1 in the dark, but not in the light. The Ca2+ fluxes were large, complex and temporally variable, but revealed net Ca2+ uptake under all conditions. The temporal variability in Ca2+ dynamics was potentially related to localized dissolution during epithallial cell sloughing, a strategy of CCA to remove epiphytes. Simultaneous Ca2+ and pH dynamics suggest the presence of Ca2+/H+ exchange. Rapid light-induced H+ surface dynamics that continued after inhibition of photosynthesis revealed the presence of a light-mediated, but photosynthesis-independent, proton pump. Thus, the study indicates metabolic control of surface pH can occur in CCA through photosynthesis and light-inducible H+ pumps. Our results suggest that complex light-induced ion pumps play an important role in biological processes related to inorganic carbon uptake and calcification in CCA.

Na+-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)

We investigated the mechanisms of pyruvate transport in the erythrocytes of an ancient marine agnathan, the Pacific hagfish (Eptatretus stouti), and a sedentary euryhaline teleost, the starry flounder (Platichthys stellatus). Uptake of [14C]pyruvate (50 µM, 10°C) by flounder erythrocytes was slow (t1/2(half-life) ~ 30 min), nonconcentrative, and mediated by the band 3 Cl-/HCO-3exchanger in combination with a process similar to the H+/monocarboxylate symporter present in freshwater teleosts and mammalian erythrocytes. In contrast, pyruvate uptake by hagfish erythrocytes (50 µM, 10°C) was rapid (t1/2~ 1.5 min) and, in 10 min, reached an intracellular concentration more than 20-fold higher than that present in the extracellular medium. Pyruvate accounted for almost 90% of the accumulated intracellular radioactivity, the remaining label being incorporated into tricarboxylic acid cycle intermediates and glutamate. Influx of pyruvate was saturable (apparent Km= 12 mM) and inhibited by p-chloromercuriphenylsulphonate (PCMBS) (Ki= 71 µM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (Ki= 0.49 mM). Transport was inhibited poorly by α-cyano-4-hydroxycinnamate (CIN) (Ki> 4 mM) and was not coupled to the movement of protons. Instead, the influx of pyruvate was Na+dependent. A sigmoidal relationship between pyruvate transport and extracellular Na+concentration was observed, suggesting a Na+:pyruvate coupling ratio greater than 1:1. In contrast with previously described Na+-dependent monocarboxylate transport activities in mammalian renal and intestinal epithelia, the hagfish erythrocyte system did not transport lactate.

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens.

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by alpha,beta-MeATP (3 microM), with IC50-values ranging from 1.1 to 100 microM. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8'-[Carbonylbis(imino-3, 1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate ) (NF023), suramin, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34-43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-4-amino-5-hydroxy-naphtha lene-1,3-disulphonate (NH01) and Evans blue even enhanced the response to ATP. For antagonists causing full or partial inhibition, the IC50-values against ATP were close to those against alpha,beta-MeATP. All antagonists attenuated the removal of ATP, with IC25%-values ranging from 0.8 microM to >320 microM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X1). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered.

Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR.

Vesicles enriched in a marker enzyme for apical membranes were isolated from HT29 cells. These vesicles contain an anion conductance with the selectivity gluconate approximately sulphate<F-<Cl-<Br-<NO3-<I-<SCN-. K+ diffusion potential-driven 36Cl- uptake was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB)>4, 4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS)>glibenclamide. The Cl- conductance was insensitive to Ca2+ and to extravesicular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inosine 5'-triphosphate (ITP). Using the reverse transcription polymerase chain reaction (RT-PCR) technique and sequencing of the amplified products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR), the putative Cl- channel or Cl- channel regulator pICln, and the Cl- channels ClC-2, ClC-3, ClC-5 and ClC-6 in HT29 cells. The properties of the vesicles' Cl- conductance resemble those of the intermediate conductance outwardly rectifying Cl- channel and tentatively exclude contributions of CFTR, pICln and ClC-2. Whether ClC-3, ClC-5, ClC-6 are involved in Cl- conductance remains to be determined.

Bicarbonate uptake and utilization in marine macroalgae

Several macroalgae from the Swedish West Coast (11 green, 5 red and 11 brown algal species) and two Ulva spp. from the Mediterranean were tested for mechanisms of HCO3 − utilization. In all except one (Palmaria palmata), photosynthesis supported by inorganic carbon (Ci) at pH 8.7 was rapidly and substantially (>50%) inhibited by acetazolamide (AZ), an inhibitor of extracellular carbonic anhydrase activity. This suggests that extracellular dehydration followed by uptake of the CO2 formed is an important component of the HCO3 − utilization mechanism(s) of these algae. In representatives of most of the green algal genera, e.g. Enteromorpha, Ulva, Chaetomorpha and Monostroma, a specific inhibitor response could be induced. This response, a sensitivity to the anion exchange inhibitor 4,4′-diisothiocyanatostilbene- 2,2′-disulphonate (DIDS) in the presence of AZ, is characteristic of an additional mechanism of HCO3 − utilization, a direct import of HCO3 − across the plasma membrane, which has been previously described only for some Ulva spp. and Enteromorpha intestinalis. The DIDS sensitivity could be induced in several ways, all involving a potential shortage of the supply of CO2 for photosynthesis: through photosynthesis at high pH, through growth in strong light and nutrient-rich seawater, or through photosynthesis in the presence of AZ. Induction of photosynthesis at high pH (which is a specific feature of green macroalgae) was not always accompanied by a development of this specific DIDS sensitivity ; it occurred in Chaetomorpha melagonium but not in C. linum. For Ulva lactuca cultivated in natural seawater (in continuous moderate irradiance), induction of DIDS sensitivity by high pH treatment was successful all year round, except during a period in late August. The results thus suggest that green macroalgae capable of developing photosynthesis at high pH are also capable of developing a DIDS-sensitive mechanism for HCO3 − uptake, but that some additional conditions (other than low CO2 availability) must be fulfilled. The ability to dehydrate extracellular HCO3 − still remained after induction of the direct HCO3 − uptake, and the two mechanisms for HCO3 − utilization may operate independently of one another. In fact the response to DIDS was more pronounced after the addition of AZ. The possibility that the results are related to a plasticity of the HCO3 − utilizing mechanisms of green macroalgae is discussed. Neither red nor brown macroalgae showed any sensitivity to DIDS in the presence of AZ.

Bicarbonate uptake and utilization in marine macroalgae

Several macroalgae from the Swedish West Coast (11 green, 5 red and 11 brown algal species) and two Ulva spp. from the Mediterranean were tested for mechanisms of HCO3 − utilization. In all except one (Palmaria palmata), photosynthesis supported by inorganic carbon (Ci) at pH 8.7 was rapidly and substantially (>50%) inhibited by acetazolamide (AZ), an inhibitor of extracellular carbonic anhydrase activity. This suggests that extracellular dehydration followed by uptake of the CO2 formed is an important component of the HCO3 − utilization mechanism(s) of these algae. In representatives of most of the green algal genera, e.g. Enteromorpha, Ulva, Chaetomorpha and Monostroma, a specific inhibitor response could be induced. This response, a sensitivity to the anion exchange inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) in the presence of AZ, is characteristic of an additional mechanism of HCO3 − utilization, a direct import of HCO3 − across the plasma membrane, which has been previously described only for some Ulva spp. and Enteromorpha intestinalis. The DIDS sensitivity could be induced in several ways, all involving a potential shortage of the supply of CO2 for photosynthesis: through photosynthesis at high pH, through growth in strong light and nutrient-rich seawater, or through photosynthesis in the presence of AZ. Induction of photosynthesis at high pH (which is a specific feature of green macroalgae) was not always accompanied by a development of this specific DIDS sensitivity ; it occurred in Chaetomorpha melagonium but not in C. linum. For Ulva lactuca cultivated in natural seawater (in continuous moderate irradiance), induction of DIDS sensitivity by high pH treatment was successful all year round, except during a period in late August. The results thus suggest that green macroalgae capable of developing photosynthesis at high pH are also capable of developing a DIDS-sensitive mechanism for HCO3 − uptake, but that some additional conditions (other than low CO2 availability) must be fulfilled. The ability to dehydrate extracellular HCO3 − still remained after induction of the direct HCO3 − uptake, and the two mechanisms for HCO3 − utilization may operate independently of one another. In fact the response to DIDS was more pronounced after the addition of AZ. The possibility that the results are related to a plasticity of the HCO3 − utilizing mechanisms of green macroalgae is discussed. Neither red nor brown macroalgae showed any sensitivity to DIDS in the presence of AZ.

On the suitability of adenosine 3′-phosphate 5′-phosphosulphate as a selective P2Y receptor antagonist in intact tissues

Agonist and antagonist effects of the putative P2Y 1 receptor antagonist adenosine 3′-phosphate 5′-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC 50 3.3 μM). The response was attenuated by the P2 receptor antagonists 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) and reactive red 2 with apparent K d values (0.27 and 0.29 μM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of α, β-methylene ATP ( α, β-MeATP) in the taenia coli. Incubation with PAPS (10–100 μM) attenuated the P2Y receptor-mediated relaxation caused by 5′- O-(2-thiodiphosphate) (ADP βS); PAPS (100 μM) also attenuated the relaxation caused by α, β-MeATP, as well as the α 1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC 50 24.7 μM), which was reduced by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonate ( iso-PPADS; 1 μM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 μM) attenuated the P2Y receptor-mediated relaxation caused by ADP βS; PAPS (100 μM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 μM) attenuated the P2X receptor-mediated contraction elicited by α, β-MeATP but did not alter the α 1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

Effect of specific ion channel blockers on cultured schwann cell proliferation

Mitogenesis in a variety of tissues is known to be inhibited by K+ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Using radiolabeled thymidine as a proliferation index we have examined what role, if any, specific K+ channels have in cultured Schwann cells that have been induced to proliferate by pre-exposure to mitogens. TEA and 4-AP are "broad-spectrum" in that they block a variety of different types of K+ channel. In contrast, we found that alpha-dendrotoxin (alpha-DTX), a specific blocker of the type 1 fast delayed rectifier current (the largest component of Schwann cell K+ current) does not affect proliferation, suggesting that type 1 current may not be involved in mitogenesis. This suggestion is supported by our finding that the values of the KD for the mitogenic effect (722 nM, 4-AP; 13 mM, TEA) are much larger than the corresponding electrophysiological values for type 1 channels (0.1 mM, 4-AP; 0.2 mM, TEA). Charybdotoxin (200 nM) and iberiotoxin (100 nM), inhibitors of Ca2+-activated K+ channels, cesium (5 mM), an inhibitor of inward rectifier channels, and furosemide (100 pM), which blocks Na+/K+/Cl- cotransport, all had no effect on proliferation. Interestingly, 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS), which blocks voltage-gated Cl- channels, reduced proliferation. In summary, broad-spectrum K+ channel blockers inhibit Schwann cell proliferation, but inhibitors specific for type 1, Ca2+-activated, and inward rectifier K+ channels do not. Whether the inhibition is mediated by type 2 K- channels, by an as yet unidentified Schwann cell K+ channel, or by another mechanism remains unclear.

Two Types of Relaxation-Mediating P2 Receptors in Rat Gastric Circular Muscle

Effects of purinoceptor antagonists on the relaxant responses to adenine nucleotides were examined to characterize the subtypes of P2-receptor in rat gastric circular muscle. In tissues contracted by acetylcholine, a P2-receptor antagonist, suramin (100 μM), inhibited the relaxant responses to ATP, adenosine 5'-O-(2-thiodiphosphate) (ADPβS) and α,β-methylene ATP but not that to adenosine, while a P1-receptor antagonist, 8-phenyltheophylline (3 μM) did vice versa. The inhibitory effect of suramin was more potent for the relaxant responses to α,β-methylene ATP than those to ATP or ADPβS. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (3–30 μM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (30 and 100 μM) inhibited the relaxation caused by α,β-methylene ATP but not by ATP, ADPβS or adenosine. These results suggest that ATP and ADPβS cause relaxation via the classical P2Y receptors resistant to PPADS and DIDS. In addition, α,β-methylene ATP causes relaxation via the distinct P2 receptors sensitive to PPADS and DIDS in rat gastric circular muscle.

The intrinsic Cl- conductance of mouse kidney cortex brush-border membrane vesicles is not related to CFTR.

Brush-border membrane vesicles (BBMV) were prepared from whole Balb/c mice kidneys by a Mg2+ precipitation technique. The presence of an intrinsic Cl- conductance co-expressed with Na+/glucose cotransport was inferred by the anion dependence of [14C]glucose uptake and overshoot with inward Na(+)-anion gradients. In Na(+)-equilibrated conditions, an inside-negative membrane potential difference (p.d.) produced by an inward Cl- gradient alone was capable of driving intravesicular [14C]glucose accumulation. The apparent anion conductance had a selectivity of Br- = I- = Cl- > F- > > gluconate, was inhibited by 0.5 mM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) but was unaffected by 0.5 mM 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS). BBMV were isolated from mice in which the CFTR gene had been disrupted by a termination mutation (-/-) and compared with normal litter mates (+/+) and heterozygotes (-/+)[18]. [14C]Glucose uptake in NaCl media was significantly greater than glucose uptake in Na gluconate media for all three genotypes measured at 20 s: for homozygous -/- animals [14C]glucose uptake was increased by 2.80 +/- 0.53 fold in Cl- media compared to gluconate media, n = 6; for wild-type +/+, by 2.16 +/- 0.53 fold, n = 8; and for heterozygous +/- animals, by 2.17 +/- 0.45 fold, n = 8. The observation of a Cl-(-)dependent component in BBMV isolated from homozygous -/- mutant animals shows that the chloride conductance in these vesicles cannot be due to CFTR expression.

Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis.

1. There is evidence that defective submucosal gland secretion contributes to the airway pathology of cystic fibrosis (CF). Using a capacitance probe technique, we have compared fluid transport across submucosal gland cultures from individuals with and without CF. 2. Under baseline conditions, approximately 60% of non-CF cultures secreted fluid; the rest absorbed. In secreting tissues, amiloride increased secretion, whereas in absorbing tissues it reduced or reversed absorption. 5-Nitro-2(3-phenylpropylamino)-benzoate (NPPB) a blocker of the CF transmembrane conductance regulator (CFTR), converted secretion to absorption. Thus, the direction and magnitude of baseline fluid movement depended on a balance between active absorption of Na+ and cAMP-dependent secretion of Cl-. 3. 8-(4-Chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), methacholine and luminal uridine 5'-triphosphate (UTP) all induced or increased fluid secretion across non-CF cultures. Results with NPPB and with 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), a blocker of Ca(2+)-activated Cl- channels, suggested that fluid secretion induced by CPT-cAMP was mediated primarily by CFTR; UTP acted entirely via Ca(2+)-activated Cl- channels, and methacholine activated both pathways. 4. All CF cultures showed baseline fluid absorption, which was abolished by amiloride. 5. CF cultures showed a normal secretory response to UTP, a reduced response to methacholine, and no response to CPT-cAMP. 6. Thus, the absorptive processes of airway glands are retained in CF, but the cAMP-dependent secretory process is lost. This would markedly reduce the water content of gland secretions. The resulting change in viscosity would contribute to the accumulation of airway mucus which is characteristic of this disease.

Discrimination between UTP- and P2-purinoceptor-mediated depolarization of rat superior cervical ganglia by 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and uniblue A.

1. Using a grease-gap recording technique we have investigated the effects of some antagonists of P2-purinoceptors on the depolarization of the rat isolated superior cervical ganglion evoked by 100 microM alpha, beta-methylene-adenosine 5'-triphosphate (alpha,beta-MeATP) or uridine 5'-triphosphate (UTP). The effects of the putative P2Z-purinoceptor antagonist, coomassie brilliant blue G, putative P2X-purinoceptor antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A (an analogue of the P2Y- and P2X-purinoceptor antagonist reactive blue 2) were investigated. 2. At the highest concentration examined uniblue A (300 microM) depressed alpha,beta-MeATP-induced depolarization and at 100 and 300 microM enhanced UTP-evoked depolarizations. Coomassie brilliant blue G (1 and 10 microM) did not affect depolarizations evoked by alpha,beta-MeATP or UTP. Depolarizations evoked by potassium (5 mM) or muscarine (100 nM) were unaltered by either coomassie brilliant blue G or uniblue A. Uniblue A (100 and 300 microM) produced a concentration-dependent depression of hyperpolarizations evoked by adenosine (100 microM) whereas coomassie brilliant blue G at up to 10 microM, did not alter adenosine-induced hyperpolarizations. 3. DIDS (30 and 100 microM) did not alter adenosine-evoked hyperpolarizations, or depolarizations evoked by potassium or UTP. DIDS at 100 microM did not alter depolarizations evoked by muscarine. In contrast DIDS produced a concentration-dependent depression of alpha,beta-MeATP-evoked depolarizations. 4. These results are consistent with the proposal that uniblue A and DIDS but not coomassie brilliant blue G are antagonists of P2-purinoceptors and that uniblue A is also an antagonist at P1-purinoceptors present on the rat superior cervical ganglion. 5. The ability of uniblue A and DIDS to distinguish between depolarizations evoked by UTP and alpha,beta-MeATP provides further justification for the proposal that these nucleotides activate separate receptors present on the rat superior cervical ganglion, i.e. pyrimidinoceptors and P2-purinoceptors respectively.

Characterisation of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots

Aluminium (Al) stimulates the efflux of malate from the apices of wheat (Triticum aestivum L.) roots (Delhaize et al. 1993, Plant Physiol. 103, 695–702). The response was five to tenfold higher in Al-tolerant seedlings than Al-sensitive seedlings and the capacity for Al-stimulated malate efflux was found to co-segregate with Al tolerance in a pair of near-isogenic wheat lines differing in Al-tolerance at a single dominant locus. We have investigated this response further using excised root apices. Half-maximal efflux of malate from apices of Al-tolerant seedlings was measured with 30 μM Al in 0.2 mM CaCl2, pH 4.2, while saturating rates of 2.0 nmol·apex−1·h−1 occurred with concentrations above 100 μM Al. The stimulation of malate efflux by Al was accompanied by an increase in K+ efflux which appeared to account for electroneutrality. The greater stimulation of malate efflux from Al-tolerant apices compared to Al-sensitive apices could not be explained by differences in the activities of phosphoenolpyruvate carboxylase or NAD-malate dehydrogenase. Several other polyvalent cations, including gallium, indium and the tridecamer Al13, failed to elicit malate efflux. Aluminium-stimulated efflux of malate was correlated with the measured concentration of total monomeric Al present, and with the predicted concentrations of Al3+ and AlOH2+ ions in the solution. Several antagonists of anion channels inhibited Al-stimulated efflux of malate with the following order of effectiveness: niflumic acid≈NPPB>IAA-94≈A-9-C>ethacrynic acid. Lanthanum, chlorate, perchlorate, zinc and α-cyano-4-hydroxycinnamic acid inhibited malate release by less than 30% at 100 μM while 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) had no effect. These results suggest that the Al3+ cation stimulates malate efflux via anion channels in apical cells of Al-tolerant wheat roots.

Two relaxation-mediating P2-purinoceptors in guinea-pig taenia caeci.

2-Methylthio ATP, noradrenaline, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), alpha, beta-methylene ATP, ATP and adenosine caused relaxation of guinea-pig isolated taenia caeci precontracted by carbachol, with potency decreasing in the order indicated. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonate (DIDS) 10, 32 and 100 microM shifted the concentration-response curve of alpha, beta-methylene ATP increasingly to the right, by the factor 141 at DIDS 100 microM. Concentration-response curves of the other agonists were not shifted to the right by DIDS 100 microM, except for the curve of ADP beta S which was shifted by the factor 2.4. The relaxation produced by 2-methylthio ATP faded rapidly. When the fade was complete, further addition of 2-methylthio ATP or ATP did not elicit relaxation, whereas the relaxant effect of alpha, beta-methylene ATP was unchanged. The results indicate that there are at least two relaxation-mediating P2-purinoceptors in guinea-pig taenia caeci, the P2Y-purinoceptor which is relatively insensitive to DIDS and a distinct receptor for alpha, beta-methylene ATP which is very sensitive to DIDS.

Blockade by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonate (DIDS) of P2X‐purinoceptors in rat vas deferens

1. The possibility of an antagonist effect of 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) at P2X-purinoceptors was studied in rat vas deferens. 2. DIDS reduced contractions elicited by alpha,beta-methylene ATP 3 microM, IC50 1.6 microM, but did not change contractions elicited by K+ 35 mM. DIDS 3.2 microM slightly shifted the concentration-response curve of alpha,beta-methylene ATP to the right and reduced the maximum. DIDS 10 microM markedly decreased and DIDS 32 microM abolished contractions over the entire range of the alpha, beta-methylene ATP concentration-response curve. DIDS 32 microM also abolished contractions elicited by ATP but did not change contractions elicited by noradrenaline. The antagonist effect of DIDS was only slowly reversible. 3. The presence of either suramin 320 microM or alpha,beta-methylene ATP 10 microM during the exposure to DIDS protected the tissue from the long-lasting blocking effect of DIDS. 4. 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulphonate (H2DIDS) was equipotent with DIDS whereas several analogues in which one or both of the isothiocyanate residues were replaced were less effective or without effect against alpha,beta-methylene ATP. 5. DIDS attenuated the purinergic component of neurogenic contractions elicited by electrical field stimulation, IC50 3.9 microM, but did not change the adrenergic component. 6. It is concluded that DIDS causes a selective, long-lasting, non-equilibrium blockade of P2X-purinoceptors in rat vas deferens. Due to this effect it also selectively blocks the purinergic component of neurogenic contractions.

Mitogenic factors regulate ion channels in Schwann cells cultured from newborn rat sciatic nerve.

1. Patch clamp studies were carried out in Schwann cells cultured from newborn rat sciatic nerve to determine the effects of mitogens on voltage-gated currents without the confounding influences of axonal contact and myelin present in vivo. The relevance of the various Schwann cell currents to proliferation was assessed using assays of [3H]thymidine incorporation. 2. Treatment of cultured Schwann cells with known mitogens, namely axon fragments (AF), myelin fragments (MF), or glial growth factor in combination with forskolin (GGF+F), increased the magnitudes of delayed rectifying potassium (K+) and sodium (Na+) currents. 3. In both control and mitogen-treated cells, the magnitude of net outward current paralleled clearly the magnitude of the cells' proliferative response. 4. The K+ channel-blocking quaternary ammonium ions, tetrabutylammonium (TBuA), tetrapentylammonium (TPeA) and tetrahexylammonium (THeA), but not the Na+ channel blocker tetrodotoxin (TTX), reduced proliferation in a dose-dependent fashion offering further evidence for a role for K+ channels in Schwann cell proliferation. 5. Voltage-gated chloride (Cl-) currents were observed in both control and mitogen-treated cells. Addition of the Cl- channel blockers, 4-acetamido-4'-isocyanatostilbene-2,2'-disulphonate (SITS) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), to the culture media enhanced proliferation. 6. The possible intermediary role of the Schwann cell resting potential was explored in ion substitution experiments by increasing the K+ concentration of the media and by adding ouabain. Both manipulations inhibited Schwann cell mitosis. 7. Comparison of the expression of functional ion channels in vitro with that previously described for Schwann cells in vivo suggests a difference in the Schwann cell response to the membrane fragment mitogens and their intact counterparts in regard to the regulation of ion channels. MF up-regulates the number of functional channels, whereas the elaboration of myelin (or a factor related to its presence) in vivo appears to down-regulate channel expression, at the cell soma of myelinating Schwann cells. In addition, axonal contact may be required for normal expression of functional inwardly rectifying K+ channels.

The effect of hypo-osmolarity upon transepithelial ion transport in cultured renal epithelial layers (MDCK)

Regulatory volume decrease after exposure to hypo-osmotic media in MDCK epithelial cells results from activation of both K+ and Cl- conductances. Swelling-stimulated 86Rb(K) losses were observed only across the basal-lateral membrane and were relatively insensitive to 10 mM Ba2+. The effect of hypo-osmotic media upon MDCK epithelia mounted in Ussing chambers has been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (Isc) followed by inhibition of inward Isc in both control layers and in layers where inward current (due to transepithelial Cl- secretion) was first stimulated by 5 microM prostaglandin E1 (PGE1). The transient stimulation of inward current by hypo-osmotic media was not markedly attenuated by 10 mM Ba2+ in PGE1-stimulated layers. After stimulation of inward (Cl(-)-secretory) current to high levels by 10 microM adrenaline, the predominant effect of basal-lateral exposure to hypo-osmotic media was an inhibition of the inward current. This inhibition was partially reversed by 40 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS). The stimulation, then inhibition, of inward Isc is likely to be the result of separate swelling-induced K+ and Cl- conductances (respectively) at the basal-lateral membrane. The swelling-stimulated Cl- conductance is distinct from the apical Cl- conductance regulated by PGE1 or adrenaline.