Elimination Of Na Research Articles

Hamster spermatozoa incorporate hypotaurine via TauT for self-protection.

Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. β-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.

Diuretic activity assessment of an aqueous extract of Hibiscus sabdariffa (Malvaceae) leaves on Wistar rats

Hibiscus sabdariffa (Malvaceae) is a plant known as a diuretic. We undertook to evaluate the therapeutic effectiveness of this plant. Twenty Wistar rats aged between 6 and 8 weeks and weighing between 150 and 250 g were divided into five homogeneous batches of four rats. Batch 1 received only the saline overload, NaCl (50 ml/kg). Immediately after the saline overload, batches 2, 3, 4 and 5 received orally, furosemide, the standard diuretic (40 mg/kg bw), or single doses of 500, 1000 and 1500 mg/kg bw of the aqueous extract of H. sabdariffa leaves, respectively. The results showed that, 24 hours after the administrations, the urinary volumetric excretions of 123.06±11.07; 66.11± 6.19; 112.22±4.36 and 61.39±10.28% respectively. The increases in urinary volumetric excretion induced by the extract were statistically significant compared to the control rats (34.99±8.46%). The extract at a dose of 1000 mg/kg bw and furosemide showed statistically similar values of urinary volumetric excretion. This urinary excretion was accompanied by an elimination of Na+ and K+ ions similar to furosemide. The urinary measurement of creatinine and urea also revealed that the extract did not interfere with normal renal function during treatment. This study shows that the aqueous extract of H. sabdariffa leaves has diuretic and natriuretic activity similar to that of furosemide. This activity could be attributed to the presence of alkaloids, steroids and flavonoids, compounds known for their diuretic effects.
 Keywords: Hibiscus sabdariffa, Furosemide, urinary excretion, diuretic

Evaluation of the diuretic effects of crude stem bark extraction of Zanthoxylum heitzii (Rutaceae) in Wistar rats

ObjectiveZanthoxylum heitzii is a medicinal plant widely used in central Africa for the treatment of many diseases, especially cardiovascular diseases and hypertension. The diuretic effects of crude stem bark extraction were determined and its safety in rats was evaluated. MethodsThe diuretic effects of crude stem bark extraction of Z. heitzii were investigated in Wistar rats (250 g ± 10 g) of both sexes. The crude stem bark extraction of Z. heitzii at the doses of 225, 300 and 375 mg/kg was administered to rats at 5 mL/kg body weight. Urine volume was determined 1, 2, 3, 4, 5, 6 and 24 h after administration of the extract. Kinetics of electrolyte elimination in response to a single oral administration dose of acute treatment was measured. The experiments were performed under the same conditions with two synthetic pharmacological diuretics considered as reference (furosemide and hydrochlorothiazide). Urinary and plasma concentrations of sodium and potassium ions were determined using flame photometry. Concentrations of creatinine, urea, glucose, albumin and electrolytes in the plasma and urine samples were evaluated using a two-way digital bidirectional spectrophotometer. The osmolarity of plasma and urine samples was measured by cytometry using an osmometer. Aldosterone was measured by radioimmunoassay. ResultsThe plant extract accelerated the elimination of overloaded fluid and increased urine volume and the excretion of Na+, K+ and Cl− 24 h after administration (P<0.05). The increase in elimination of Na+, K+, and Cl− induced by the extract caused alkalinization of the urine, and showed a strong inhibitory effect on carbonic anhydrase and saluretic. These effects were mainly observed at the dose of 375 mg/kg. At the maximum diuretic response, urinary osmolarity decreased significantly (P < 0.05) when compared to controls. The stability of aldosterone level, the absence of correlation with the plasma levels of Na+, and increased clearance of free water in the animals treated with extract indicated that increased diuresis and natriuresis were tubular in origin. No significant (P > 0.05) changes were observed in the body temperature of the animals. ConclusionThe significant increase in urine volume 24 h after treatment followed a dose-response pattern. The excretion of Na+, K+ and Cl− caused a decrease in urine osmolarity. The stability of aldosterone, the absence of correlation with the plasma levels of sodium, and increased clearance of free water in animals treated with aqueous extract suggest that increased diuresis and moderate natriuresis elevation were of tubular origin.

Na+/H+ exchange and pH regulation in the control of neutrophil chemokinesis and chemotaxis

Large proton fluxes accompany cell migration, but their precise role remains unclear. We studied pH regulation during the course of chemokinesis and chemotaxis in human neutrophils stimulated by attractant peptides. Activation of cell motility by chemoattractants was accompanied by a marked increase in metabolic acid generation, attributable to energy consumption by the contractile machinery and to stimulation of the NADPH oxidase and the ancillary hexose monophosphate shunt. Despite the increase in acid production, the cytosol underwent a sizable alkalinization, caused by acceleration of Na(+)/H(+) exchange. The development of the alkalinization mirrored the increase in the rate of cell migration, suggesting a causal relationship. However, elimination of Na(+)/H(+) exchange by omission of external Na(+) or by addition of potent inhibitors was without effect on either chemokinesis or chemotaxis, provided the cytosolic pH remained near neutrality. At more acidic levels, cell motility was progressively inhibited. These observations suggest that Na(+)/H(+) exchange plays a permissive role in cell motility but is not required for the initiation or development of the migratory response. Chemokinesis also was found to be exquisitely sensitive to extracellular acidification. This property may account for the inability of neutrophils to access abscesses and solid tumors that have been reported to have inordinately low pH.

Analysis of polypropyleneglycols using electrospray ionization mass spectrometry. Effects of cationizing agents on the mass spectra.

In electrospray ionization mass spectrometry (ESI-MS) of polypropyleneglycol (PPG), effects of cationizing agents were examined. When NaI was used as a cationizing agent, the distribution of multiply-charged ions in the spectra was greatly affected by the ratio of cationizing agent and PPG. However, the distribution was not affected by the use of CH(3)COONH(4). With an increase of cone voltage, fragmentation occurred by in-source collision-induced dissociation (CID) when CH(3)COONH(4) was used. On the contrary, no decomposition of the PPG backbone was observed with NaI. Instead, the intensity of the lower-charged ions, whose mass-to-charge (m/z) ratios are larger, increased because of the elimination of Na(+) with increase of cone voltage. Under optimum conditions for ESI-MS analysis, PPGs that have different molecular weights, different initiators or end groups were easily and accurately characterized. A tandem mass spectrometry (MS/MS) study of NH(4)(+) adduct ions of PPG indicated that a vinyl-terminated linear structure is formed at the end group during the fragmentation.

Elimination of Na+-dependent bile acid transporter from small intestine by ileum resection increase colonic tumorigenesis in the rat fed deoxycholic acid

Ileal Na+-dependent bile acid transporter (ISBT) constituting a gateway to enterohepatic circulation of bile acids occurs exclusively at the distal site of the small intestine. In the present study, we examined colonic tumorigenesis promoted by deoxycholic acid in relation to the expression of the ISBT. For this purpose, the small intestine of a Fischer-344 rat was resected a length of 20 cm above the ileo-cecal valve (ileal resection) or below the duodenum (jejunal resection). Then, rats were treated with an intraperitoneal injection of azoxymethane (15 mg/kg body wt.) once a week for 3 weeks and fed a 20% casein diet supplemented with 0.2 % deoxycholate for 39 weeks. Northern blot analysis demonstrated that the ISBT mRNA was hardly detectable in ileum-resected rats. The excretion of fecal bile acids was 1.5-fold higher in the ileum-resected group than in the jejunum-resected group (P<0.05). On the contrary, the serum bile acids concentration of ileal-resected rats was about one-half of that of jejunum-resected animals (P<0.05). The tumor incidence and the total tumor number were significantly higher in the ileum-resected group than in the jejunum-resected one (P<0.05). Interestingly, no tumor was found at the proximal colon in the jejunum-resected group while tumors developed frequently at the proximal site as well as mid and distal colon in the ileum-resected group. These observations demonstrate that malabsorption of bile acids owing to the lack of ISBT enhanced colon tumorigenesis.

Synthesis of BEA by dry gel conversion and its characterization

Zeolite BEA with SiO 2/Al 2O 3 ratios ranging from 30 to 740 were crystallized at 413 and 453 K by the dry gel conversion technique. The particle size of the products increased with increasing SiO 2/Al 2O 3 ratio as well as with increasing crystallization temperature. In as-synthesized BEA, the TEA + ions interacting with Si-O − decomposed between 473 and 673 K and those interacting with Al-O − were cleaved between 673 and 798 K. During their elimination, the TEA + ions interacting with Al-O − underwent higher degradation than those interacting with Si-O −. Chemical compositions showed the least influence on the fractions of decomposition product during the elimination between 473 and 673 K. The framework of H-BEA and NH 4-BEA zeolites with SiO 2/Al 2O 3 ratios ranging from 400 to 740 were stable even at 1373 K, whereas H-BEA with SiO 2/Al 2O 3 = 30 was stable only up to 1173 K and became amorphous when calcined at 1373 K. After calcination at 1373 K, Na-BEA with SiO 2/Al 2O 3 = 400 and 730 were transformed to tridymite (dense phase) whereas that with SiO 2/Al 2O 3 = 30 became amorphous. The elimination of Na + ions is necessary to have a BEA remain stable above 1173 K.

Inositol-1,4,5-trisphosphate induces responses in receptor neurons in rat vomeronasal sensory slices.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents and putative second messenger-activated currents in receptor neurons in the vomeronasal sensory epithelium of female rats. The resting membrane potential and input resistance were -45.5 +/- 2.5 mV (mean +/- SEM, n = 39) and 1.5 +/- 0.2 G omega (mean +/- SEM, n = 37). Current injection of 1-3 pA induced overshooting action potentials. The firing frequency increased with increasing current injections linearly from 1 to 10 pA and reached a plateau at 30 pA, suggesting that rat vomeronasal receptor neurons sensitively elicit action potentials in response to a small receptor potential. Under voltage clamp, voltage-dependent Na+ inward current, inward Ca2+ current, sustained outward K+ current and Ca-(2+)-activated K(+)-current were identified. Dialysis of D-inositol-1,4,5-trisphosphate (D-IP3) induced inward currents with an increase in membrane conductance in approximately 54% of the cells and inward current fluctuations in 15% of the cell. L-IP3 also induced inward currents and current fluctuations in 53 and 13% of the cells respectively. The mean amplitude of inward currents induced by 100 microM D-IP3 and L-IP3 were 84.6 +/- 14.0 pA (SEM, n = 82) and 66.1 +/- 9.4 pA (SEM, n = 100) respectively. The IP3-induced responses were blocked by elimination of Na+ and Ca2+ in the external solution or application of 10 microM ruthenium red. The present study suggested that IP3-mediated transduction pathways exist in rat vomeronasal receptor neurons.

Amiloride-sensitive Na+/H+ antiporter in basolateral membrane of hamster ascending thin limb of Henle's loop.

The mechanisms of intracellular pH (pHi) regulation were investigated in the in vitro microperfused hamster ascending thin limb (ATL) of Henle's loop with the fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. pHi of ATL cells was 7.05 +/- 0.02 (n = 30) when microperfused with a CO2/HCO(3-)-buffered solution. In HEPES-buffered solution, pHi was 7.10 +/- 0.02 (n = 16), which was significantly higher than the value in CO2/HCO(3-)-buffered solution (P < 0.05, n = 16). In HEPES-buffered solution, elimination of Na+ and addition of 1 mM amiloride to basolateral solution decreased the pHi by 0.12 +/- 0.03 (n = 6) and 0.11 +/- 0.02 (n = 5) at 1 min, respectively. The same manipulations in the luminal solution had no effect on pHi. One millimolar of N-ethylmaleimide (NEM) added to either side of ATL caused no significant change in pHi. Elimination of K+ on either side of ATL did not alter pHi. After adding 20 mM NH4Cl to basolateral solution, pHi instantaneously increased from 7.17 +/- 0.01 to 7.51 +/- 0.03 (n = 3), and then returned to steady-state level of 7.21 +/- 0.05 (n = 15) in 3 min. Removal of NH4Cl from basolateral solution then caused a rapid fall in pHi to 6.31 +/- 0.05 (n = 15), followed by spontaneous recovery at a rate of 0.43 +/- 0.06 unit/min (n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)

Swelling and damage to nerves and glial cells by acidosis

Development of acidosis is a prominent pathophysiological factor in acute cerebral disorders, such as ischaemia or severe brain trauma. The impairment of the acid-base state in brain parenchyma among others is involved in the development of brain oedema, eventually leading to irreversible damage of neurons and glial cells. In the present study the pathophysiological role of acidosis for cytotoxic cell swelling and damage of glial and neuronal cells was investigated in vitro under conditions found in the ischaemic penumbra in vivo--the still viable perifocal border zone surrounding an infarct with elevated interstitial K(+)- and H(+)-concentrations. Assessment of cell swelling by acidosis was combined with experiments on underlying mechanisms as a basis for therapeutical interventions to inhibit cytotoxic brain oedema in vivo. C6 glioma cells, astrocytes from primary culture, as well as Neuro-2A cells were cultivated, harvested and suspended as single cells under continuous control of pH, pO2, and temperature according to a standard procedure. Cell volume and cell viability were quantified by flow cytometry. Acidosis was induced by isotonic sulfuric- or lactic acid, respectively. Acidification of the medium led to cell swelling once pH fell below 7.0. Cell viability, however, was not affected by the increasing acidosis down to pH 6.2, while pH 5.6 or below was associated with cell death dependent on the duration of exposure. Acidosis-induced cell swelling was attenuated or completely inhibited by blocking of ion exchange mechanisms, such as the Na+/H(+)-antiporter, or elimination of Na+ ions from the medium. The present results provide new information on the nature of cytotoxic cell swelling and damage in central nervous system by acidosis under consideration of underlying mechanisms. Accordingly, acidosis-induced cell swelling is attributable to activation of ion exchange mechanisms, such as the Na+/H(+)- and Cl-/HCO3(-)-antiporter, in order to maintain a normal cellular acid-base state. This compensation process, however, is associated with the loss of cell volume control by net uptake of osmotic active solutes. Consequently, cell swelling occurring under these conditions is a result of regulatory mechanisms to defend homoeostasis rather than a consequence of cytotoxic cell damage. If cell swelling is inhibited by appropriate treatment, care should be exercised not to enhance the vulnerability of the nerve and glial cells.

Effects of prostaglandin E2 on membrane voltage of the connecting tubule and cortical collecting duct from rabbits.

1. Effects of prostaglandin E2 (PGE2) on ion transport were examined by observing the transmural (VT) and basolateral membrane voltage (VB) in the in vitro perfused rabbit connecting tubule (CNT) and the cortical collecting duct (CCD). 2. Addition of 1 microM PGE2 to the bath induced a biphasic response of transmural voltage (VT), with initial negative VT deflection followed by positive deflection in the CNT, but monophasic negative deflection in the CCD. Because PGE2 had no affect on the basolateral membrane voltage (VB), PGE2 mainly causes changes in the apical membrane voltage. 3. Elimination of Na+ from the lumen abolished the PGE2-induced VT response in the CNT. In the presence of 10 microM luminal amiloride, PGE2 caused only an initial negative deflection without causing later positive deflection. The positive VT deflection induced by PGE2 in the CCD was also blocked by luminal amiloride. 4. Addition of ouabain (0.1 mM) to the bath completely abolished the PGE2-induced VT changes in the CNT, indicating that an intact Na(+)-K+ pump is a prerequisite for the VT response to PGE2. 5. Addition of 2 mM Ba2+ to the lumen did not affect biphasic VT response to PGE2, indicating that Ba(2+)-sensitive K+ conductance is not involved. 6. Basolateral addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP inhibited only the negative VT deflection induced by PGE2. 7. The positive VT deflection was blocked by basolateral addition of 50 microM 8-(N,N-diethylamino)octyl 3,4,5-trimethoxy benzoate hydrochloride (TMB-8), an inhibitor of intracellular Ca2+ release. But elimination of luminal Ca2+ did not affect the biphasic response to PGE2. 8. These findings suggest that the initial negative VT deflection is caused by an increase in Na+ influx across the luminal membrane through an amiloride-insensitive Na+ conductive pathway, whereas the later positive deflection is caused by the inhibition of Na+ influx through the amiloride-sensitive Na+ conductive pathway. The cAMP messenger system may be responsible for the initial negative deflection, whereas an increased intercellular Ca2+ release from the store is necessary for the later positive deflection caused by PGE2. The response in the CCD is comparable to the later response in the CNT.

Relations between intracellular ions and energy metabolism: a study with monensin in synaptosomes, neurons, and C6 glioma cells

Treatment of rat brain synaptosomes with 10 microM monensin stimulated activity of the Na/K pump, which enhanced oxygen consumption and lactate production. Glycolytic flux was also increased independently of the pump activation by a fall in [H+]i. Under such conditions, glycolysis provided 26% of ATP for the ouabain-sensitive ATPase, a value substantially greater than the 4% obtained in veratridine-treated preparations (Erecińska and Dagani, 1990). In C6 glioma cells, a glia-derived line endowed with high rates of aerobic lactate synthesis, the cytosolic and mitochondrial ATP generation contributed 50% each for the support of the pump in the presence of 10 microM monensin. The fraction of energy utilized by the pump was greater in synaptosomes than in C6 cells. Enhancement of ion movements was accompanied by changes in the levels of high-energy phosphate compounds. Measurements with ion-sensitive microelectrodes in C6 cells and cultured neurons showed that monensin caused an increase in pHi by 0.4-0.5 unit and a parallel rise in [Na+]i. The increases in [Na+]i were about twofold in both types of cells, but the absolute values attained were much higher in neurons (40-50 mM) than in C6 cells (10-12 mM). Membrane potentials transiently declined by less than 10 mV and returned to their original values after 20 min of treatment. Rises in [Ca2+]i were small in neurons as well as in C6 cells. These changes could be explained by the known mechanism and/or consequences of monensin action. In contrast, in synaptosomes monensin caused an internal alkalinization of 0.1-0.15 pH unit, a large depolarization of the plasma membrane, and massive leakage of potassium into the external medium. The decrease in plasma membrane potential was accompanied by an increase in [Ca/+]i and release of the neurotransmitter amino acids GABA, aspartate, and glutamate. The depolarization and loss of K+ were unaffected by calcium withdrawal, replacement of chloride with gluconate, and addition of 1 mM 4-acetamido-4'-isothiocyanostilebene-2,2'-disulfonic acid (SITS), but was markedly attenuated by elimination of Na+. It is proposed that in synaptosomes monensin and/or the consequences of its action open a nonspecific cation channel that allows Na+ entry and K+ exit, with a consequent decrease in membrane potential.

Interaction of trichlormethiazide or amiloride with PTH in stimulating Ca2+ absorption in rabbit CNT

To determine the renal tubular site and mechanism of the anticalciuric effects of trichloromethiazide (TCM) and amiloride, we studied the effects of these diuretics on net Ca2+ absorption (JCa) in isolated rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). TCM (100 microM) in the lumen increased JCa in the CNT without affecting lumen-negative transepithelial voltage (VT). This effect was dependent on the amount of parathyroid hormone (PTH, 0.1 or 1 nM) in the bath. TCM had no detectable effect on JCa in the absence of PTH. Addition of 100 microM ouabain to the bath decreased PTH-stimulated JCa and abolished the TCM-stimulated JCa. Elimination of Na+ from the lumen increased PTH-stimulated JCa. Under this condition the ability of TCM to increase JCa was abolished, suggesting that the inhibition of Na+ entry from the lumen with TCM may account for the increase in PTH-stimulated JCa. Elimination of Na+ from the bath in the presence of PTH decreased JCa and abolished the stimulatory effect of TCM on JCa in the presence of PTH. Changes in VT caused by amiloride may not account for the increase in JCa, because JCa was not changed when VT was deflected to more positive direction by increasing bath K+ from 5 to 15 mM. Neither TCM nor amiloride affected JCa in the CCD. From these observations, we speculate that the inhibition of Na+ entry across the apical cell membrane by either thiazides or amiloride may stimulate PTH-induced JCa. The intact Na(+)-Ca2+ antiporter in the basolateral cell membrane is essential for the anticalciuric effect of thiazides and amiloride.

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro

To examine the mechanisms of H+ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pHi) in the in vitro perfused tubules by microscopic fluorometry using 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pHi was 6.74 +/- 0.04 (n = 45) in HCO3(-)-free modified Ringer solution. Either elimination of Na+ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pHi. After acid loading with 25 mM NH4Cl, pHi spontaneously recovered with an initial recovery rate of 0.096 +/- 0.012 (n = 23) pH unit/min. In the absence of ambient Na+, after removal of NH+4, the pHi remained low (5.95 +/- 0.10, n = 8) and showed no signs of recovery. Subsequent restoration of Na+ only in the lumen had no effect on pHi. However, when Na+ in the bath was returned to the control level, pHi recovered completely Amiloride (1 mM) in the bath completely inhibited the Na(-)-dependent pHi recovery. Furthermore, elimination of Na+ from the bath, but not from the lumen, decreased pHi from 6.97 +/- 0.07 to 6.44 +/- 0.05 (n = 12) in the HCO3-/Ringer solution or 6.70 +/- 0.03 to 6.02 +/- 0.5 pH unit/min in the presence of CO2/HCO3-, whereas it did not recover in the absence of CO2/HCO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of parathyroid hormone on the connecting tubule from the rabbit kidney: biphasic response of transmural voltage

The effect of parathyroid hormone (PTH) on ion transport was examined by observing transmural (VT) and basolateral membrane voltage (VB) in the in vitro perfused rabbit connecting tubule. Addition of 10 nmol/l PTH to the bath induced a biphasic response of VT, with hyperpolarization followed by depolarization. Chlorophenylthioadenosine cyclic 3',5'-monophosphate mimicked the effect of PTH, which did not change the VB in the connecting tubule cell, but mainly caused changes in the apical membrane voltage. The VT of distal convoluted tubule and the cortical collecting duct were not affected by PTH. Elimination of Na+ from the lumen abolished the PTH-induced VT responses in the connecting tubule. In the presence of 10 mumol/l amiloride, PTH caused an initial hyperpolarization but did not induce the late depolarization. The same was seen in the absence of luminal Ca2+. Either addition of 0.1 mmol/l ouabain to the bath or elimination of bath Na+ completely abolished the PTH-induced VT changes. The presence of 5 mmol/l Ba2+ in the lumen did not affect the response to PTH. These findings indicate that the initial hyperpolarization may be caused by an increase in Na+ influx across the luminal membrane through an amiloride-insensitive Na+ conductive pathway and that the late depolarization may be caused by the decrease in Na+ influx through the amiloride-sensitive Na+ conductive pathway. Luminal Ca2+ is necessary for the late depolarization caused by PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Elimination of Na + and GTP regulation of α 2-adrenoceptor-agonist interactions in rat kidney membranes by 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS)

Pretreatment of rat kidney membranes with 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) did not affect the B max but reduced the affinity of the receptors for antagonist by less than 2-fold. However, such treatment reduced the affinity of the receptors for agonist by several fold by preventing the formation of agonist-receptor-G i-protein complex. The modulatory effects of GTP and Na + on α 2-adrenoceptor-agonist interactions were also abolished. These results suggest that DIDS acts at multiple sites on α 2-adrenoceptor-adenylate cyclase complex.

Effects of anion transport inhibitors and ion substitution on Cl- transport in TAL of Henle's loop.

To identify the mechanism of Cl- transport across the thin ascending limb of Henle's loop (TAL), we examined effects of anion transport inhibitors and ionic substitution in the isolated segments of hamsters using the in vitro microperfusion technique. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at 10(-3) M changed the NaCl diffusion voltage (Vt) to the orientation that corresponds to the decrease in the Cl(-)-Na+ permeability ratio when it was added either to the bath or to the lumen. DIDS, added to the bath or to the lumen decreased the lumen-to-bath flux coefficient for 36Cl, KCl-(1----b) (X10(-7) cm2/s), from 74.5 +/- 2.1 to 17.9 +/- 3.6 and from 77.7 +/- 3.6 to 51.1 +/- 5.4, respectively, whereas it had little effect on the flux coefficient for 22Na, KNa+(1----b). Elimination of HCO3- slightly increased rather than decreased both NaCl diffusion potential and KCl-(1----b), indicating that Cl- transport is independent of HCO3- transport. Phloretin at 10(-3) M inhibited both diffusion potential and KCl-(1----b) to a similar extent as DIDS did. KNa+(1----b) was also changed little. The inhibitory effect of phloretin was rapid and reversible. Phloridzin was ineffective. Furosemide added to the bath at 10(-3) M decreased Vt and KCl-(1----b) from 29 +/- 0.9 to 5.3 +/- 0.7 mV and from 75.7 +/- 3.9 to 62.8 +/- 4.1 X 10(-7) cm2/s, respectively; but it was ineffective when added to the lumen. Elimination of Na+ caused only a small decrease in KCl-(1----b) from 119.2 +/- 6.9 to 107.3 +/- 8.5 X 10(-7) cm2/s. Elimination of K+ and addition of Ba2+ did not change KCl-(1----b). From these observations, we conclude that Cl- transport across the TAL is distinct from Na+ and is not coupled with Na+, K+, or HCO3-.

Factors affecting the microclimate pH in rat jejunum.

1. Single-barrelled pH-sensitive microelectrodes filled with liquid ion exchanger were used to study the layer of microclimate pH in the vicinity of the surface of rat jejunum in vitro. 2. During perfusion with a Na+-containing solution of pH 7.30, a layer having a pH gradient ranging from 7.30 (pH of the luminal bulk phase) to 6.05 +/- 0.03 (pH of the deepest region) was detected in eighteen different animals. The thickness of the layer was estimated to be 600-700 microns. No regional difference was seen along the height of the villus. 3. The addition of D-glucose to the perfusion solution significantly augmented the acidity of the deepest region without changing the thickness of the layer. On the other hand, the elimination of Na+ from the perfusion solution caused a significant reduction of the pH gradient. The lowest pH changed from a control value of 6.18 +/- 0.15 (n = 13 measurements from three animals) to 6.46 +/- 0.06 (n = 13). The gradient was sensitive to amiloride in the presence of Na+, K+, Ca2+ and Cl- had no significant effect on the microclimate pH. 4. Depletion of the surface mucus by treatment with dithiothreitol significantly raised the pH of the deepest region. 5. Glycylglycine and L-carnosine were found to reduce the microclimate pH gradient significantly, while glycine did not. 6. These results indicate that H+ secretion by the Na+-H+ antiport and the formation of mucus layer are important factors for maintaining the microclimate pH layer, and that H+-coupled co-transport, such as H+-dipeptide co-transport, causes a significant diminution of the microclimate pH gradient.

Modification of single cardiac Na+ channels by DPI 201-106

In inside-out patches from cultured neonatal rat heart cells, single Na+ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na+ channels studied at -30 mV have one open state which is rapidly left with a rate constant of 826.5 sec -1 at 20 degrees C during sustained depolarization. Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3 X 10(-6) m DPI in the pipette solution, stepping to -30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na+ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na+ channels exhibit a sustained high, time-independent open probability. Similar to normal Na+ channels, is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na+ channel inactivation as it may follow from an interaction of DPI with Na+ channels.

Renal transport and metabolism of nicotinic acid.

Renal metabolism and brush-border transport of nicotinic acid were studied in renal cortical slices and brush-border membrane vesicles exposed to a physiological concentration of vitamin (2.2-3.5 microM). Vesicle transport of [3H]nicotinic acid was found to be Na+ dependent and concentrative. The presence of a Na+ gradient resulted in a fivefold increase in the rate of nicotinic acid uptake over that observed with mannitol and caused a transient nicotinic acid accumulation two- to fourfold above the equilibrium value. The effects of membrane potential, pH, and elimination of Na+-H+ exchange were also studied. Cortical slices and isolated tubules exposed to 2.2 microM [14C]nicotinic acid took up vitamin and rapidly metabolized most of it to intermediates in the Preiss-Handler (J. Biol. Chem. 233: 488-493, 1958) pathway for NAD biosynthesis; little free nicotinic acid was detectable intracellularly. The replacement of Na+ with Li+ in the bathing medium reduced total accumulation of 14C label primarily as a result of reduced nicotinic acid uptake. Cortical tissue concentrated free nicotinic acid only when the involved metabolic pathways were saturated by levels of nicotinic acid far in excess of what occurs in vivo.