4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and ethylisopropylamiloride

Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils.. Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study.. We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI.. It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Ammonium Chloride; Benzamides; Bicarbonates; Cytochalasin B; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Ion Transport; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophils; Protein Isoforms; RNA, Messenger; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers; Sulfonamides

Anion secretion by colonic epithelium is dependent on apical CFTR-mediated anion conductance and basolateral ion transport. In many tissues, the NKCC1 Na(+)-K(+)-2Cl(-) cotransporter mediates basolateral Cl(-) uptake. However, additional evidence suggests that the AE2 Cl(-)/HCO(3)(-) exchanger, when coupled with the NHE1 Na(+)/H(+) exchanger or a Na(+)-HCO(3)(-) cotransporter (NBC), contributes to HCO(3)(-) and/or Cl(-) uptake. To analyze the secretory functions of AE2 in proximal colon, short-circuit current (I(sc)) responses to cAMP and inhibitors of basolateral anion transporters were measured in muscle-stripped wild-type (WT) and AE2-null (AE2(-/-)) proximal colon. In physiological Ringer, the magnitude of cAMP-stimulated I(sc) was the same in WT and AE2(-/-) colon. However, the I(sc) response in AE2(-/-) colon exhibited increased sensitivity to the NKCC1 inhibitor bumetanide and decreased sensitivity to the distilbene derivative SITS (which inhibits AE2 and some NBCs), indicating that loss of AE2 results in a switch to increased NKCC1-supported anion secretion. Removal of HCO(3)(-) resulted in robust cAMP-stimulated I(sc) in both AE2(-/-) and WT colon that was largely mediated by NKCC1, whereas removal of Cl(-) resulted in sharply decreased cAMP-stimulated I(sc) in AE2(-/-) colon relative to WT controls. Inhibition of NHE1 had no effect on cAMP-stimulated I(sc) in AE2(-/-) colon but caused a switch to NKCC1-supported secretion in WT colon. Thus, in AE2(-/-) colon, Cl(-) secretion supported by basolateral NKCC1 is enhanced, whereas HCO(3)(-) secretion is diminished. These results show that AE2 is a component of the basolateral ion transport mechanisms that support anion secretion in the proximal colon.

Topics: 1-Methyl-3-isobutylxanthine; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Amiloride; Animals; Animals, Newborn; Anion Transport Proteins; Anions; Antiporters; Bicarbonates; Bumetanide; Carbonic Anhydrase II; Carbonic Anhydrase III; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cation Transport Proteins; Cecum; Chlorides; Colforsin; Colon; Cyclic AMP; Electrophysiological Phenomena; Gene Expression; Ion Channels; Ion Pumps; Mice; Mice, Inbred Strains; Mice, Knockout; SLC4A Proteins; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2

Previously, we reported on the distinct effects of bioactive lysophospholipids, including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), and sphingosylphosphorylcholine (SPC), on membrane potentials in rat C6 glioma cells. In the present report we have tested lysophosphatidylserine (LPS), another bioactive lysophospholipid, on membrane potentials in the same cell line. Membrane potentials were estimated by measuring the fluorescence changes of DiBAC-loaded glioma cells. LPS largely increased membrane potentials in a concentration-dependent manner. The LPS-induced membrane potential increases were not affected by treatment with pertussis toxin, implying no involvement of Gi/o proteins. In contrast to other lysophospholipids, the LPS-induced membrane potential increase was not diminished by a Na(+)-free media but was enhanced by suramin. Furthermore, this change was blunted by EIPA, an inhibitor of Na(+)/H(+) exchanger, but not by SITS, a specific inhibitor of bicarbonate transporter. Our observations suggest that LPS acts on membrane potentials in a unique manner in the C6 glioma cells, although the precise action mechanism requires additional investigation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Cell Line, Tumor; Glioma; GTP-Binding Proteins; Lysophospholipids; Membrane Potentials; Rats; Receptors, G-Protein-Coupled

Intracellular pH can have profound effects on tissue function, but little is known about how pH is regulated, buffered or affects the function of gastric smooth muscle. As the pH of gastric myocytes may alter with pathophysiological disturbance of the gastric lining, or reduction in blood flow to the stomach, these parameters were investigated. Intracellular pH was measured in strips of corpus from rats and guinea-pigs and pH perturbed by the addition of Na butyrate. pH regulation was investigated using pharmacological inhibitors and ionic substitutions. Resting pH was found to be around 7.0, and buffering power relatively high, compared to other muscles in both species. In the guinea-pig amiloride, EIPA and HOE694 prevented pH regulation from an acid load, but amiloride- and EIPA-insensitive pH-regulating mechanisms were found in the rat. The pH-regulatory mechanism present in the rat was also insensitive to DIDS, SITS and removal of external Cl-, but inhibited by Na+ substitution and HOE694. Acidification reduced gastric tone in both species. We conclude that pH alteration will significantly affect gastric contractility, despite a high capacity of the tissue to buffer and regulate pH change. The sensitivity to NHE inhibitors differs between rat and guinea-pig, suggesting that Na+/H+ exchanger isoform expression differs between gastric tissue.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acid-Base Equilibrium; Amiloride; Animals; Anti-Arrhythmia Agents; Buffers; Butyrates; Diuretics; Gastric Emptying; Gastric Mucosa; Guanidines; Guinea Pigs; Hydrogen-Ion Concentration; Methylamines; Muscle, Smooth; Rats; Sodium; Sulfones

The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Aorta; Humans; Hydrogen-Ion Concentration; Muscle, Smooth, Vascular; Pulsatile Flow; Regional Blood Flow; Signal Transduction; Stress, Mechanical

Intracellular pH (pH(i)) was measured in human platelets using fluorescent probes. Basal pH(i) was higher in HC(O3-)- buffered solutions (7(7.33 +/- 0.01) than in nominally HCO3- free, Hepes buffered solutions (7.16 +/- 0.01, P < 0.05). Addition of EIPA caused to fall in Hepes, but did not inhibit the increase of pH(i) when platelets maintained in Hepes were transferred to a CO2/HCO3- buffer. After an intracellular acidosis induced by an NH4Cl prepulse, the initial velocity of recovery (d(pH)/dt(i), in pH units/min) was 3.32 +/- 0.69 in Hepes-buffered solution and 2.85 +/- 0.88 in HCO3- media. Taking into account the differences in buffer capacity, the efflux of acid equivalents after 1.2 min was twice as much in the presence of bicarbonate. The addition of 30 mumol/1 EIPA effectively blocked acid efflux (d(pH)/dt(i) = 0.08 +/ 0.04) in a nominally HCO3- free solution, whereas the recovery was reduced but not abolished (d(pH)/dt(i) = 0.37 +/- 0.10, P < 0.05) in the presence of bicarbonate. The stilbene derivative SITS further inhibited the EIPA-resistant pH(i) recovery. Removal of external Na+ inhibited the HCO(3-)-dependent recovery whereas depletion of internal Cl-, did not suppress it. Depolarization of the membrane had no effect on this recovery. The results suggest the contribution of an electroneutral Na+/HCO3- cotransport in the recovery of pH(i) following an acid load. Both the Na+/H+ antiport and the HCO(3-)-dependent mechanism contribute approx. 50% each to the total acid equivalent efflux during the recovery from a pH(i) 6.46 +/- 0.14 to the basal pH(i) in human platelets.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Bicarbonates; Biological Transport; Blood Platelets; Carrier Proteins; Chlorides; Humans; Hydrogen-Ion Concentration; Potassium; Sodium; Sodium-Bicarbonate Symporters

The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms.. Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques.. In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA.. At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adenosine Triphosphate; Amiloride; Animals; Antiporters; Bicarbonates; Carrier Proteins; Cells, Cultured; Chloride-Bicarbonate Antiporters; Hydrogen-Ion Concentration; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers

Experiments were performed in cat papillary muscles in order to explore the possible existence of an electrogenic Na+/HCO3- cotransport. Developed tension (DT), intracellular pH (pHi) with the pH-sensitive dye 2'-7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and resting membrane potential (Vm) with 3M KCl filled glass microelectrodes were measured. A change from HEPES to HCO3(-)-buffered superfusate induced an immediate decrease in pHi and DT followed by a recovery in which pHi and DT stabilized at values slightly higher than in HEPES buffer. Introduction of HCO3- hyperpolarized Vm by 8 +/- 2.3 mV (P < 0.05). SITS (0.1 mM) completely abolished the hyperpolarization and attenuated the recovery of both pHi and DT. Under steady-state conditions in HCO3- buffered media, SITS induced a depolarization compatible with the suppression of the entry of negative charges. Depolarization by high Ko+ (45 mM) elicited a rise in pHi of 0.07 +/- 0.02 (P < 0.05), that was reversed by returning Ko+ to normal. The depolarization-induced rise in pHi proved to be Na(+)-dependent, SITS sensitive and still occurred after EIPA (microM) blockade. All the evidence strongly supports the existence of an electrogenic Na+/HCO3- cotransport mechanism that participates in the regulation of myocardial pHi. At pHi of 6.94 this mechanism seems to contribute almost equally to the Na+/H+ exchanger to pHi regulation. However, acid equivalent extrusion is potentiated when both the Na+/H+ exchanger and the HCO3-dependent mechanism are simultaneously regulating pHi.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Anti-Arrhythmia Agents; Bicarbonates; Carrier Proteins; Cats; Homeostasis; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Membrane Potentials; Myocardium; Papillary Muscles; Potassium; Sodium; Sodium-Bicarbonate Symporters; Time Factors

This study aimed to assess the suitability of sheep tracheal epithelium as a model for studies of human airway ion transport. Ovine and human airway epithelium were mounted in Ussing chambers under short circuit conditions. Bumetanide (100 microM) reduced short-circuit current (Isc) by a mean of 21.3% +/- SEM 2.0, n = 8, in sheep, and 30.4% +/- 9.7, n = 3, in human airway epithelium. Acetazolamide (100 microM) decreased Isc by 10.6% +/- 1.2, n = 18, in sheep, and 5.8% +/- 2.9, n = 3, in human airways. Phloridzin (200 microM) reduced Isc by 4.7% +/- 0.8, n = 7, and 3.1% +/- 5.1, n = 3 in sheep and human tissue respectively. Amiloride (100 microM) decreased Isc by 42.9% +/- 3.5, n = 12, in sheep airways, whilst bathing the mucosal surface with Na(+)-free solutions reduced Isc by 67.4% +/- 4.2, n = 18. The sequential addition of acetazolamide, bumetanide, phloridzin, amiloride and mucosal Na(+)-free solutions totally inhibited the basal Isc in both sheep and human tissues, suggesting that Cl- and HCO3- secretion, Na(+)-glucose co-transport and amiloride-sensitive and -insensitive Na+ absorption contribute to the Isc. The similarities between the species suggest that sheep tracheal epithelium is a useful model for basal studies of airway ion transport, and may prove a valuable tool for further regulatory studies.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Action Potentials; Amiloride; Animals; Bronchi; Bumetanide; Epithelium; Humans; Ion Transport; Ouabain; Phlorhizin; Sheep; Trachea

The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na(+)-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3- plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady-state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2 = 5 min) and was dependent on external Na+ (EC50 = 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- antiports, blocked recovery of pHi in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/micrograms of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Antiporters; Bicarbonates; Carrier Proteins; Cells, Cultured; Chlorides; Choroid Plexus; Epithelium; Hydrogen-Ion Concentration; Male; Rabbits; Sodium

We previously demonstrated that formate and oxalate stimulate Cl-transport in the rat proximal tubule in accord with a model involving NaCl uptake across the luminal membrane via Cl-/organic anion exchange in parallel with Na+/H+ exchange and organic acid recycling. The purpose of the present study was to test whether similar mechanisms contribute to Cl- transport in the rat distal tubule. In distal tubules microperfused in situ with an isotonic solution, addition of 0.5 mM formate to the luminal perfusate increased Cl- (JCl) and fluid (Jv) absorption by 77 and 93%, respectively. Addition of 5 microM oxalate increased JCl and Jv by 52 and 108%, respectively. In distal tubules perfused with a hypotonic solution, formate stimulated JCl and Jv by 85 and 98%, respectively, and oxalate stimulated JCl and Jv by 80 and 115%, respectively. Addition of 0.5 mM acetate caused no significant change in JCl and Jv. The stimulation of JCl and Jv by formate was largely abolished by addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM) or ethylisopropyl-amiloride (10 microM) to the luminal perfusate. Chlorothiazide (0.5 mM) inhibited baseline JCl and Jv but caused no inhibition of the increments in Jv and JCl induced by formate. Formate stimulation of JCl and Jv was confined to early segments of the distal tubule. We conclude that formate and oxalate markedly enhance JCl and Jv in the early distal tubule by a thiazide-insensitive mechanism involving NaCl entry across the apical membrane by Cl-/organic anion exchange in parallel with Na+/H+ exchange.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Absorption; Amiloride; Animals; Anti-Arrhythmia Agents; Biological Transport; Carrier Proteins; Chlorides; Formates; Kidney Tubules, Distal; Male; Oxalates; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchangers

Cell membrane-associated ion transporters, Na+/H+ exchanger and Na(+)-dependent HCO3-/Cl- antiport, were shown to be important in the regulation of acidic intracellular pH in different cell types. This study investigated the role of the ion exchangers and their inhibitors in the serum-induced proliferation of two murine tumour cell lines, P815 and L929. The presence of Na+/H+ exchanger [inhibited by amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA)] and Na(+)-dependent HCO3-/Cl- antiport [inhibited by 4,4'-diisothiocyanostilbene-2,2-disulphonic acid (DIDS)] was shown on the tumour cell line tested. EIPA suppressed tumour cell proliferation more strongly than amiloride, and its effect was further increased after intracellular acidification by nigericin. DIDS slightly inhibited proliferation of L929 cell line and did not influence proliferation of P815 cells. However, in nigericin acidified cells DIDS had a dose dependent antiproliferative effect. Furthermore, DIDS significantly increased antiproliferative effects of amiloride and EIPA, suggesting the activity of Na(+)-dependent HCO3-/Cl- antiport in tumour cell proliferation. These results demonstrate the importance of Na(+)-dependent HCO3-/Cl- exchange in addition to Na+/H+ antiport, in tumour cell proliferation and indicate the possibility that ion exchange inhibitors could act as antitumour reagents.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Biological Transport, Active; Carrier Proteins; Cell Division; Chloride-Bicarbonate Antiporters; Mice; Mitosis; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured

Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Bicarbonates; Cells, Cultured; Chlorides; Dogs; Female; HEPES; Hydrogen-Ion Concentration; Male; Muscle Contraction; Muscle, Smooth, Vascular; Serotonin; Sodium; Stimulation, Chemical

This study investigated developmental changes in Na(+)-H+ exchange and HCO3(-)-Cl- exchange activities in newborn and adult rabbit hearts. pHi was measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in isolated myocytes. Myocardial mechanical function was measured in the isolated ventricular preparation. Intracellular acidosis with normal pHo was induced by an NH4Cl (10 mM) prepulse technique. Upon removal of NH4Cl, pHi fell transiently and then recovered toward the control level. In the HCO3-/CO2-buffered solution, the rate of recovery of pHi in the newborn was greater than in the adult. In the HCO3-/CO2-buffered solution, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na(+)-H+ exchange, inhibited the recovery of pHi completely in the adult. In the newborn, however, significant recovery of pHi was observed in the presence of EIPA. In the presence of both EIPA and 4-acetamido-4'-isothiocyanatostilbene-2',2'-disulfonic acid (SITS), an inhibitor of HCO3(-)-Cl- exchange, the recovery of pHi was not observed in the two age groups. In the HEPES-buffered solution that did not contain HCO3-/CO2, the rate of recovery of pHi after NH4Cl removal was similar in the two age groups. In the HEPES-buffered solution, the recovery of pHi was completely inhibited by EIPA in the two age groups. In the presence of EIPA in the HCO3-/CO2-buffered solution, contractile function decreased during acidosis after NH4Cl removal and did not recover in the adult. In the newborn, significant recovery of contractile function was observed after NH4Cl removal in the presence of EIPA. The recovery of mechanical function observed in the presence of EIPA in the newborn was inhibited by SITS. These data suggest that, although there is no developmental change in the Na(+)-H+ exchange activity, HCO3(-)-Cl- exchange is more active in the premature myocardium. The presence of the HCO3(-)-Cl- exchanger is important in maintaining myocardial contractile function during acidosis, especially when Na(+)-H+ exchange is inhibited and may partly explain the greater resistance of the premature myocardium to acidosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acidosis, Respiratory; Age Factors; Amiloride; Animals; Animals, Newborn; Bicarbonates; Chlorides; Heart; Hydrogen-Ion Concentration; In Vitro Techniques; Myocardium; Rabbits; Sodium

Following the observation that interleukin 1 beta (IL-1 beta) production in lipopolysaccharide (LPS)activated monocytes increases in concert with a rise in intracellular pH (pHi), the role of ion transport in IL-1 beta production was investigated. The amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)-H+ antiporter, inhibited extracellular IL-1 beta. The replacement of Na+ in the culture medium with sucrose or choline chloride also prevented monocyte activation. The sodium ionophore monensin, in doses from 100 pM to 1 microM, potentiated LPS-stimulated extracellular IL-1 beta when compared with LPS alone. In the absence of LPS activation, monensin by itself at 10 nM stimulated IL-1 beta production to 63%. EIPA at 10 microM inhibited the Na+ influx, the rise in pHi, and intra- and extracellular IL-1 beta production in activated monocytes; this inhibition was reversed by 10 nM monensin. In the absence of bicarbonate, or in the presence of 10 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, the pHi of activated monocytes and the total protein synthesis did not change, but the production of IL-1 beta was inhibited. The data suggest that the stimulated influx of Na+ via the Na(+)-H+ antiporter regulates both pHi and IL-1 beta production in LPS-activated monocytes. The requirement for bicarbonate indicates an additional mechanism(s), separate from the modulation of pHi and intracellular Na+.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Bicarbonates; Chlorides; Culture Media; Extracellular Space; Humans; Hydrogen-Ion Concentration; Infant, Newborn; Interleukin-1; Intracellular Membranes; Lipopolysaccharides; Monensin; Monocytes; Protein Biosynthesis; Sodium

To investigate intracellular pH (pHi) in human resistance arteries in essential hypertension, vessels were obtained from small biopsies of skin and subcutaneous fat from 14 untreated patients, and the results were compared with those from 14 matched normotensive control volunteers. Segments of isolated resistance arteries were mounted in a myograph and loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Fluorescence signals were monitored using a series of barrier filters and chromatic beam splitters. In this way both resting pHi and the changes in pHi observed during isometric contractions initiated by agonists could be recorded. Resting pHi was not different in vessels from hypertensive patients (hypertensive, 7.24 +/- 0.06 versus control, 7.25 +/- 0.04 pH units). The application of ethylisopropylamiloride (EIPA) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) demonstrated that both Na(+)-H+ exchange and bicarbonate-dependent membrane mechanisms contributed to pHi homeostasis but that neither system was overactive in hypertension (pHi change with EIPA in vessels from hypertensive versus control subjects was -0.11 +/- 0.02 and 0.13 +/- 0.03 pH units, respectively, and pHi change with DIDS in vessels from hypertensive versus control subjects was -0.097 +/- 0.05 and -0.091 +/- 0.03 pH units, respectively). The application of norepinephrine or 125 mM K+ solution induced contraction in the arterial segments with an accompanying fall in pHi. With norepinephrine this fall was significantly attenuated in vessels from hypertensive patients. These results fail to provide evidence for raised pHi in resistance arteries in human essential hypertension, and contrary to previous reports in circulating blood cells, Na(+)-H+ exchange is not overactive in the vessels of such patients.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adipose Tissue; Amiloride; Anti-Arrhythmia Agents; Arteries; Bicarbonates; Biological Transport; Calibration; Carrier Proteins; Female; Fluoresceins; Homeostasis; Humans; Hydrogen-Ion Concentration; Hypertension; Male; Middle Aged; Norepinephrine; Potassium; Skin; Sodium-Hydrogen Exchangers; Vascular Resistance

1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre-treatment with 200 microM-DIDS of a pre-acidified cell in Na(+)-free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM-EIPA. We conclude, that in the presence of CO2-HCO3-, a Na(+)- and HCO3-dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Animals, Newborn; Bicarbonates; Buffers; Carotid Body; Hydrogen-Ion Concentration; Meglumine; Rats; Sodium; Spectrometry, Fluorescence

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. These studies were performed to determine the identity and relative importance of the sarcolemmal transport systems that mediate net acid efflux in primary cultured vascular smooth muscle cells from canine femoral artery. In HEPES- or HCO3(-)-buffered physiological salt solution (HEPES-PSS, HCO3(-)-PSS), recovery from an acute acid load was totally dependent on external Na+. 5-[N-ethyl-N-isopropyl]amiloride (EIPA, 50 microM) inhibited pHi recovery 100 and 68% in HEPES-PSS and HCO3(-)-PSS, respectively. EIPA-insensitive pHi recovery in HCO3(-)-PSS was inhibited 48% by 4,4'-diisothyocyanostilbene-2,2'-disulfonic acid (DIDS). An outwardly directed H+ gradient stimulated amiloride-sensitive 22Na+ uptake, and an inwardly directed HCO3- gradient stimulated amiloride-insensitive 22Na+ uptake. The latter was inhibited by DIDS or prior depletion of cell Cl-. In HEPES-PSS, resting pHi was 7.17 +/- 0.03, was not affected by DIDS, but was lowered by EIPA or by removing extracellular Na+. In HCO3(-)-PSS, resting pHi was 7.25 +/- 0.02 (P less than 0.05) and was not affected by EIPA. Removing extracellular Na+ in the presence of EIPA decreased pHi in HCO3(-)-PSS but not in HEPES-PSS. DIDS lowered resting pHi in HCO3(-)-PSS, after which EIPA further lowered pHi. We conclude that acid efflux from these cells is mediated by a Na(+)-H+ exchanger and a Na(+)-dependent Cl(-)-HCO3- exchanger. In HEPES-PSS, acid efflux via the Na(+)-H+ exchanger maintains resting pHi. In HCO3(-)-PSS, additional acid efflux via the Na(+)-dependent Cl(-)-HCO3- exchanger results in a higher pHi. Although the Na(+)-H+ exchanger is primarily responsible for acid efflux after an acute acid load, the Na(+)-dependent Cl(-)-HCO3- exchanger is responsible for acid efflux under physiological conditions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Bicarbonates; Cells, Cultured; Chlorides; Dogs; Female; Femoral Artery; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Male; Muscle, Smooth, Vascular; Sodium; Spectrometry, Fluorescence

We investigated in a physiological salt solution (PSS) containing HCO3- the intracellular pH (pHi) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and 22Na+ influx rates. The recovery of pHi from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO2/HCO3(-)-buffered PSS than in HEPES-buffered PSS. Recovery of pHi was dependent on extracellular Na+ (Km, 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO3(-)-dependent mechanisms. Recovery instead appeared mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism. Differentiation between this HCO3(-)-dependent mechanism and Na+/H+ exchange was achieved after depletion of cellular ATP. Under these conditions, the NH4Cl-induced 22Na+ influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO2/HCO3(-)-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na+, the EIPA inhibition curve for the intracellular acidosis-induced 22Na+ influx was monophasic (IC50, 39 nM), whereas in an identical CO2/HCO3(-)-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 microM). Taken together, the results indicate that Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism play an important role in regulating the pHi of human vascular smooth muscle. The involvement of the latter mechanism depends on the severity of the intracellular acidosis, varying from approximately 25% in severe intracellular acidosis up to 50% at lesser, more physiological, levels of induced acidosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acid-Base Equilibrium; Adenosine Triphosphate; Aged; Amiloride; Ammonium Chloride; Bicarbonates; Buffers; Cells, Cultured; Energy Metabolism; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Mammary Arteries; Middle Aged; Muscle, Smooth, Vascular; Protons; Sodium; Sodium Radioisotopes

The trophectoderm of the mouse blastocyst is a fluid transporting epithelium that is responsible for generating a fluid-filled cavity called the blastocoel. Vectorial transport of ions from the medium into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. We report here that substitution of Na+ or Cl-, but not K+, in the medium halves the rate of blastocoel expansion in the mouse blastocyst. Entrance of Na+ into the trophectoderm may involve several routes, since both blastocoel expansion and 22Na+ uptake are decreased in the presence of the highly specific Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, and to a lesser extent with the amiloride-sensitive Na+-channel blocker, benzamil. Uptake of 22Na+ manifests saturation kinetics as a function of extracellular Na+ concentration, whereas uptake of 36Cl- is linear. Furthermore, neither 4,4-diisothiocyanostilbene-2,2-disulfonic acid, which is an inhibitor of the Cl-/HCO3- exchanger, nor 2-(3,4-dichlorobenzyl)-5-nitrobenzoic acid, which is a Cl- -channel blocker, affect either blastocoel expansion or 36Cl- uptake. These results suggest that Na+ entry into the mouse blastocyst is carrier-mediated and probably involves several routes that include the Na+/H+ exchanger and possibly the Na+-channel. Chloride entry, however, may not be carrier-mediated and may occur through a paracellular route, i.e., between the trophectodermal cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Amiloride; Animals; Biological Transport; Blastocyst; Carrier Proteins; Chloride Channels; Chloride-Bicarbonate Antiporters; Chlorides; Cytochalasin D; Cytochalasins; Ectoderm; Epithelium; Kinetics; Membrane Proteins; Mice; Nitrobenzoates; Potassium; Sodium; Sodium Channels; Sodium-Hydrogen Exchangers

Measurements of intracellular pH (pHi) and of apical cell membrane potential (Va) were made in principal cells of frog skin (Rana pipiens) with double-barrel microelectrodes under open-circuit conditions. The tissues were pretreated with stilbenes (10(-3) M) and bathed in HCO3- -free NaCl Ringer solution that was buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.8). Substitution of extracellular Na+ on both sides of the epithelium with N-methyl-D-glucamine caused intracellular acidification by 0.27 pH units. Restoration of Na+ on the apical side alone or on both sides caused a pHi recovery of 0.24 and 0.28 pH units, respectively, whereas return of Na+ on the basolateral side caused no recovery. Recovery of pHi on restoration of Na+ to the apical side was prevented by 10(-5) M 5-(N-ethyl-N-isopropyl)-amiloride. In individual preparations there was no correlation between pHi recovery due to return of apical Na+ and changes in Va. The average change in pHi was several times greater than the one expected from voltage clamp-induced changes in Va at constant extracellular Na+. The results suggest the presence of a Na+-H+ exchange on the apical side of principal cells. Such a process could be part of a negative feedback mechanism for regulation of Na+ entry via apical Na+ channels into principal cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Cell Membrane; Dimethyl Sulfoxide; Hydrogen-Ion Concentration; Membrane Potentials; Protons; Rana pipiens; Skin; Skin Physiological Phenomena; Sodium

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Ammonia; Animals; Bicarbonates; Cells, Cultured; Glomerular Mesangium; Hydrogen-Ion Concentration; Kinetics; Rats; Spectrometry, Fluorescence

We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Alanine; Amiloride; Animals; Carrier Proteins; Cations, Divalent; Cells, Cultured; Chloride-Bicarbonate Antiporters; Chlorides; Glomerular Mesangium; Glucose; Hydrogen-Ion Concentration; Kinetics; Membrane Proteins; Rats; Sodium