Why is sodium cocoyl isethionate (SCI) mild to the skin barrier? - An in vitro investigation based on the relative sizes of the SCI micelles and the skin aqueous pores.

Sodium cocoyl isethionate (SCI) is an important surfactant ingredient in mild, syndet (synthetic detergent) cleansing bars. In vitro and in vivo studies have demonstrated that SCI is mild and less damaging to the skin barrier than soaps and surfactants such as sodium dodecyl sulfate (SDS). We have recently shown that SDS forms small micelles in aqueous solutions contacting the skin relative to the aqueous pores in the stratum corneum (SC), and as a result, the SDS micelles can contribute to SDS skin penetration and induce skin barrier perturbation. In this paper, we attempt to explain the well-documented skin mildness of SCI by examining the size of the SCI micelles relative to that of the aqueous pores in the SC. For this purpose, we have conducted in vitro mannitol skin permeability and average skin electrical resistivity measurements upon exposure of the skin to an aqueous SCI contacting solution in the context of a hindered-transport aqueous porous pathway model of the SC. These in vitro studies demonstrate that an SCI micelle of radius 33.5 +/- 1 Angstrom (as determined using dynamic light-scattering measurements) experiences significant steric hindrance and cannot penetrate into the SC through aqueous pores that have an average radius of 29 +/- 5 Angstrom. We believe that this inability of the SCI micelles to contribute to SCI skin penetration and associated skin barrier perturbation is responsible for the observed skin mildness of SCI. Through in vitro quantitative skin radioactivity assays using (14)C-radiolabeled SCI and pig full-thickness skin (p-FTS), we also show conclusively that SCI skin penetration is dose-independent, an important finding that provides additional evidence that the larger SCI micelles cannot penetrate into the SC through the smaller aqueous pores that exist in the SC, and therefore, cannot induce skin barrier perturbation.

Isethionic acid and floridoside isolated from the red alga, Grateloupia turuturu, inhibit settlement of Balanus amphitrite cyprid larvae.

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml(-1)).

Probing an open CFTR pore with organic anion blockers.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.

Structural domains of chimeric dopamine-noradrenaline human transporters involved in the Na(+)- and Cl(-)-dependence of dopamine transport.

Catecholamine transporters constitute the biological targets for several important drugs, including antidepressants, cocaine, and related compounds. Some information exists about discrete domains of these transporters that are involved in substrate translocation and uptake blockade, but delineation of domains mediating the ionic dependence of the transport remains to be defined. In the present study, human neuronal transporters for dopamine and noradrenaline (hDAT and hNET) and a series of six functional chimeras were transiently expressed in LLC-PK1 cells. Substitution of Cl(-) by isethionate reveals that cassette IV (i.e., the region of the transporter encompassing transmembrane domain 9 through the COOH terminal) plays an important role in the Cl(-)- dependence of the uptake. Substitutions of Na(+) and NaCl by Tris(+) and sucrose, respectively, demonstrate that three different segments scattered across the transporter are involved in the Na(+)- dependence of the transport activity: cassette I (i.e., the region from the amino terminus through the first two transmembrane domains), cassette IV, and junction between transmembrane domains 3 to 5 and 6 to 8. Results of the present work also suggest that the use of Tris(+) as a substitute for Na(+) results in a biased estimate of the Hill number value for hDAT. This study provides useful clues for identifying specific residues involved in the uptake function of the catecholamine transporters.

Cytoprotective effect of taurine against hypochlorous acid toxicity to PC12 cells.

Taurine has been shown to be an effective scavenger of hypochlorous acid (HOCl). The role of HOCl is well established in tissue damage associated with reperfusion injury mediated by neutrophils. The role of HOCl in CNS injury and inflammatory reactions has not been well established. Myeloperoxidase activity is present in the CNS and it has been associated with ischemic injury. The aim of the present study was to determine the cytotoxicity of HOCl in a neuronal cell line (PC12) and the ability of taurine to prevent or reverse neurotoxicity. PC12 cells were grown in 96 well plates at a plating density of approximately 100,000 cells per well. HOCl was made up fresh from NaOCl for each experiment and the concentration verified spectrophotometrically. PC12 cells were exposed to HOCl for 1 hour in phosphate-buffered saline. Taurine was added at the time of HOCl treatment and in some experiments a post-treatment with taurine was performed by adding 1 or 10 mM taurine to the culture media (RPMI 1640). The cells were allowed 24 hours to recover and viability was determined using a tetrazolium-based (MTT) assay. The first series of experiments evaluated the toxicity of HOCl and the efficacy of taurine to protect PC12 cells. HOCl at 50 microM reduced PC12 cell viability by 50% and 150 microM reduced viability to <25% of control levels. Taurine (0.5-20 mM) was tested for cytoprotection against 150 microM HOCl and PC12 cells treated with 0.5 mM taurine exhibited only a 20% reduction in viability compared to untreated controls. Taurine concentrations of 1 mM or higher provided nearly 100% protection against HOCl. A second study was performed comparing taurine to beta-alanine, glutathione and isethionic acid. HOCl (100 microM) reduced viability to 25 +/- 1% of controls and taurine, beta-alanine and glutathione at 1 mM provided nearly complete protection. In contrast, isethionic acid, which lacks an amino group, failed to provide protection. Taurine (1 or 10 mM) added after 50 microM HOCl treatment did not provide any protection and PC12 cell viability was reduced to <39% of controls. In contrast, if taurine (50 microM) was present during the HOCl treatment and 1 mM taurine was added after the treatment, PC12 cell viability was 80 +/- 5% of controls. A combination of 250 microM taurine during the HOCl treatment and 1 mM taurine post-treatment produced 100% protection. These results clearly show that taurine is an efficient scavenger of HOCl and can prevent neuronal damage caused by HOCl. Since myeloperoxidase expression in the CNS is increased by ischemia, one function of taurine released during an ischemic event may be to scavenge HOCl and provide neuroprotection.

Chloride channel blockers attenuate the inhibition of renin secretion by angiotensin II.

The aim of this study was to assess the relevance of chloride channels to the inhibitory effect of angiotensin II (ANGII) on renin secretion. We thus examined the effects of the chloride channels blockers IAA-94 and niflumic acid, the Na-K-Cl cotransport blocker bumetanide and substitution of isethionate for extracellular chloride on the action of ANGII on renin secretion from isolated perfused rat kidneys. Renin secretion prestimulated by isoproterenol (10 nmol/l) was almost completely blocked by ANGII with a concentration yielding a half-maximal response (EC50) of around 150 nmol/l. In the presence of IAA-94 and niflumic acid the EC50 for ANGII was shifted to about 400 nmol/l. In the presence of bumetanide and isethionate renin secretion responded more sensitively to ANGII and the EC50 for ANGII was below 100 nmol/l. On the assumption that the chloride equilibrium potential in renin-secreting cells is more positive than the membrane potential, our findings would suggest that the inhibitory effect of ANGII is enhanced when chloride entry is blocked and attenuated when chloride efflux is impaired. Activation of chloride channels therefore probably contributes to the inhibitory action of ANGII on renin secretion.

The ethanol-induced open-field activity in rodents treated with isethionic acid, a central metabolite of taurine.

The effect of isethionic acid, a central metabolite of taurine, on ethanol-induced locomotor activity was investigated in rodents. Ten minutes following an (i.p.) simultaneous administration of ethanol (0.0, 1.5, 2.0, 2.5, 3.0, 3.5 g/kg) and isethionic acid (0.0, 22.5, 45.0, 90.0, 180.0 mg/kg), mice were placed in the open-field chambers and locomotor activity was measured during a ten-minute testing period. A significant interaction was found between isethionic acid and ethanol. Isethionic acid pre-treated mice (45.0, 90.0 and 180.0 mg/kg) showed a higher locomotor activity than the saline group at 2.5 and 3 g/kg of ethanol. In a second study, isethionic acid (45 mg/kg) and ethanol (1 g/kg) were simultaneously injected to rats. Ten minutes after the two treatments, rats were placed in the open-field chamber for a 30-minute period. The depressant effects that ethanol produced on rat locomotion were amplified by the same dose of isethionic acid as it affected ethanol-induced locomotion in mice (45 mg/kg). However, isethionic acid did not change the spontaneous locomotion at any of the doses tested in mice or rats. Since no differences in blood ethanol levels were detected in both mice and rats, the interaction between isethionic acid's action and ethanol-related locomotion does not seem to be due to different rates of absorption of ethanol or any other pharmacokinetic process related to ethanol levels. The current study displayed that isethionic acid, administered intraperitoneally, behaves in a similar way to its immediate precursor, taurine, by amplifying ethanol-induction of the locomotor activity.

Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles.

In apical membrane vesicles (AMV) prepared from late gestation fetal guinea pig lung we show that conductive 22Na+ uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G proteins. Intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl caused a significant stimulation (P<0. 05) of conductive Na+ uptake in AMV to 150+/-10% (n=10) of control, whereas GDPbetaS reduced uptake to 65+/-9% (n=4) of control. This contrasting response to GTPgammaS and GDPbetaS is characteristic of a G protein mediated pathway. GTPgammaS induced a significantly smaller stimulation, 125+/-8% (n=5) of control, in the presence of the relatively impermeant anion isethionate (Ise-). Taken together, these data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive 22Na+ uptake to 171+/-20% (n=13) of control in AMV resuspended in NaCl, but did not have a significant effect, 94+/-19% of control, in the presence of NaIse indicating the existence of tonic activation of Cl- channels in these AMV under resting conditions. As the combined effects of PTX and GTPgammaS diminished uptake, we propose that the G protein(s) responsible for Na+ channel activation in response to GTPgammaS is PTX-sensitive and that additional PTX-insensitive G proteins might also modulate 22Na+ uptake in these AMV. The presence of Gialpha1, Gialpha2, Gialpha3 and Goalpha in this apical membrane preparation was confirmed by PTX catalysed [32P]ADP-dependent ribosylation and Western blotting. Incubation of AMV with 200 microM DTT caused an inhibition of conductive Na+ uptake in AMV resuspended in NaCl or NaIse to 66+/-8% (n=11) and 64+/-8% (n=6) of control respectively. Pre-treatment with DTT did not affect the ability of GTPgammaS to stimulate conductive Na+ uptake suggesting that the regulation of 22Na+ uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein.

GABA-receptor-independent dorsal root afferents depolarization in the neonatal rat spinal cord.

Dorsal root afferent depolarization and antidromic firing were studied in isolated spinal cords of neonatal rats. Spontaneous firing accompanied by occasional bursts could be recorded from most dorsal roots in the majority of the cords. The afferent bursts were enhanced after elevation of the extracellular potassium concentration ([K+]e) by 1-2 mM. More substantial afferent bursts were produced when the cords were isolated with intact brain stems. Rhythmic afferent bursts could be recorded from dorsal roots in some of the cords during motor rhythm induced by bath-applied serotonin and N-methyl--aspartate (NMDA). Bilaterally synchronous afferent bursts were produced in pairs of dorsal roots after replacing the NaCl in the perfusate with sodium-2-hydroxyethansulfonate or after application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline with or without serotonin (5-HT) and NMDA. Antidromic afferent bursts also could be elicited under these conditions by stimulation of adjacent dorsal roots, ventrolateral funiculus axons, or ventral white commissural (VWC) fibers. The antidromic bursts were superimposed on prolonged dorsal root potentials (DRPs) and accompanied by a prolonged increase in intraspinal afferent excitability. Surgical manipulations of the cord revealed that afferent firing in the presence of bicuculline persisted in the hemicords after hemisection and still was observed after removal of their ventral horns. Cutting the VWC throughout its length did not perturb the bilateral synchronicity of the discharge. These findings suggest that the activity of dorsal horn neurons is sufficient to produce the discharge and that the bilateral synchronicity can be maintained by cross connectivity that is relayed from side to side dorsal to the VWC. Antagonists of GABAB, 5-HT2/5-HT1C, or glutamate metabotropic group II and III receptors could not abolish afferent depolarization in the presence of bicuculline. Depolarization comparable in amplitude to DRPs, could be produced in tetrodotoxin-treated cords by elevation of [K+]e to the levels reported to develop in the neonatal rat spinal cord in response to dorsal root stimulation. A mechanism involving potassium transients produced by neuronal activity therefore is suggested to be the major cause of the GABA-independent afferent depolarization reported in our study. Possible implications of potassium transients in the developing and the adult mammalian spinal cord are discussed.

A role for chloride in the suppressive effect of acetylcholine on afferent vestibular activity.

Afferents of the frog semicircular canal (SCC) respond to acetylcholine (ACh) application (0.3-1.0 mM) with a facilitation of their activity while frog saccular afferents respond with suppression (Guth et al., 1994). All recordings are of resting (i.e., non-stimulated) multiunit activity as previously reported (Guth et al., 1994). Substitution of 80% of external chloride (Cl-) by large, poorly permeant anions of different structures (isethionate, methanesulfonate, methylsulfate, and gluconate) reduced the suppressive effect of ACh in the frog saccular afferents. This substitution did not affect the facilitatory response of SCC afferents to ACh. Chloride channel blockers were also used to test further whether Cl- is involved in the ACh suppressive effect. These included: niflumic and flufenamic acids, picrotoxin, 5-nitro-2-(-3-phenylpropylamino)benzoic acid (NPPB), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). As with the Cl- substitutions, all of these agents reduced the suppressive response to ACh in the saccule, but not the facilitatory response seen in the SCC. The suppressive effect of ACh on saccular afferents is considered to be due to activation of a nicotinic-like receptor (Guth et al., 1994; Guth and Norris, 1996). Taking into account the effects of both Cl- substitutions and Cl- channel blockers, we conclude that changes in Cl- availability influence the suppressive effect of ACh and that therefore Cl- may be involved in this effect.

Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia.

Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia. J. Neurophysiol. 78: 2372-2387, 1997. Numerous studies of plasticity in the feeding behavior of Aplysia have shown that substantial plasticity is due to peripheral neuromodulation of the feeding musculature. Extensive previous work focusing on the accessory radula closer (ARC) muscle has led to the realization that a major function of the modulation in that muscle may be to ensure efficient coordination between its contractions and those of its antagonist muscles. For a more complete understanding, therefore, we must study these muscles also. Here we have studied the radula opener muscles I7-I10. Using single isolated muscle fibers under voltage clamp, we have characterized ion currents gated by voltage and by the physiological contraction-inducing neurotransmitter acetylcholine (ACh) and the effects of the physiological modulators serotonin, myomodulins A and B, and FMRFamide. Our results explain significant aspects of the electrophysiological behavior of the whole opener muscles, as well as why the opener and ARC muscles behave similarly in many ways yet differently in some key respects. Opener muscles express four types of K currents: inward rectifier, A-type [IK(A)], delayed rectifier [IK(V)], and Ca2+-activated [IK(Ca)]. They also express an L-type Ca current [ICa] and a leakage current. ACh activates a positive-reversing cationic current [IACh(cat)] and a negative-reversing Cl current [IACh(Cl)]. The opener muscles differ from the ARC in that, in the openers, activation of IK(A) occurs approximately 9 mV more positive and there is much less IACh(Cl). In both muscles, IACh(cat) most likely serves to depolarize the muscle until ICa activates to supply Ca2+ for contraction, but further depolarization and spiking is opposed by coactivation of IK(A), IK(V), IK(Ca), and IACh(Cl). Thus the differences in IK(A) and IACh(Cl) may well be key factors that prevent spikes in the ARC but often allow them in the opener muscles. As in the ARC, the modulators enhance ICa and so potentiate contractions. They also activate a modulator-specific K current, which causes hyperpolarization and depression of contractions. Finally, in the opener muscles but not in the ARC, the modulators activate a depolarizing cationic current that may help phase-advance the contractions. Each modulator exerts these effects to different degrees and thus has a distinct effect on voltage and contraction size and shape. The overall effect then will depend on the specific combinations of modulators released in different behaviors. By understanding the modulation in the opener muscles, as well as in the ARC, we are now in a position to understand how the behavior of the two muscles is coordinated under a variety of circumstances.

Role of anions in the regulation of cell volume and intracellular pH in perfused rat mandibular salivary gland.

To investigate the role of Cl- in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pHi) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl- was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl- on the Na+-K+-2Cl- cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl- by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pHi consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO3- omission, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pHi was due to Cl-/HCO3- exchange while the secondary rise resulted from activation of Na+/H+ exchange. Although replacement of Cl- by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl- replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl- concentration contribute to the up-regulation of the exchanger following muscarinic stimulation.

Role of membrane-permeable ions in renin secretion by renal juxtaglomerular cells.

In this study we examine the role of membrane-permeable ions in renin secretion from renal juxtaglomerular (JG) cells. To this end, extracellular Cl- (100 mmol/l) in the culture medium of isolated mouse renal JG cells was replaced by the permeable anion NO3- or by the membrane-impermeable anion isethionate. Alternatively, extracellular Na+ (100 mmol/l) was substituted by the membrane-impermeable cation choline. The effects of these ion substitutions on basal and stimulated renin secretion were then examined. Renin secretion was stimulated by the adenylate cyclase activator forskolin (10 microM), the NO donor sodium nitroprusside (SNP, 100 microM), the calmodulin antagonist calmidazolium (10 microM), by lowering extracellular Ca2+ concentration ([Ca2+]e) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (2 mM), and by increasing [Ca2+]e from the normal value of 0.5 to 3 mM. Substitution of extracellular Cl- by isethionate, but not by NO3-, inhibited basal renin release over 20 h of incubation. NO3- also did not change renin secretion stimulated by forskolin, SNP, calmidazolium, EGTA, or by increased [Ca2+]e. Isethionate, on the other hand, markedly attenuated the effects of EGTA and of increased [Ca2+]e, but not the stimulatory effect of forskolin, calmidazolium, or SNP. Substitution of Na+ by choline also attenuated basal renin secretion and renin secretion stimulated by lowering or raising [Ca2+]e. These findings suggest that, with respect to the dependency on permeable ions, at least two different pathways of regulated renin secretion from JG cells exist: a cation- and anion-dependent Ca(2+)-related pathway and a less ion-sensitive pathway for renin secretion activated by adenosine 3',5'-cyclic monophosphate and NO.

Basolateral transport of taurine in epithelial cells of isolated, perfused Mytilus californianus gills.

We found that the basolateral surface of the gill epithelium of the marine mussel Mytilus californianus possesses a carrier-mediated process capable of concentrating taurine within epithelial cells. We used retrograde perfusion of gill sections to demonstrate the kinetics, specificity and ion-dependence of taurine transport. [3H]taurine was concentrated relative to a space marker ([14C]mannitol); this accumulation was blocked by the inclusion of 10 mmol l-1 unlabeled taurine in the perfusate. The drop in [3H]taurine uptake at increasing concentrations of unlabeled taurine was fitted to Michaelis-Menten kinetics and indicated a basolateral process with a taurine concentration at which transport is half-maximal (Kt) of 35.3 mumol l-1 and a maximal flux (Jmax) of 0.35 mumol g-1 wet mass h-1. Taurine accumulation on the apical surface had a higher affinity (Kt = 9.5 mumol l-1) and a higher maximum rate of transport (Jmax = 1.23 mumol g-1 h-1). Basolateral transport was inhibited by inclusion in the perfusate of 1 mmol l-1 of another beta-amino acid (beta-alanine), but not by inclusion of alpha-alanine, glutamic acid or betaine. The dependence of basolateral taurine transport on Na+ (when replaced with N-methyl-D-glucamine) was sigmoidal with an apparent Hill coefficient of 2.3, indicating that more than one Na+ is necessary for the transport of each taurine molecule. Complete substitution of Cl- in bathing media reduced taurine accumulation by 90% and 70% on the apical and basolateral surfaces, respectively. Taurine accumulation on both surfaces was reduced by only 20% when Cl- was reduced from 496 to 73 mmol l-1, suggesting that taurine uptake is not significantly influenced by the changes in Cl- concentration accompanying the salinity fluctuations normally encountered by mussels. We estimate that the various Na+ and Cl- gradients naturally encountered by epithelial cells are capable of providing ample energy to maintain a high intracellular concentration of taurine. We suggest that the ability of epithelial cells to accumulate taurine across the basolateral surface from the hemolymph plays a significant role in the intracellular regulation of this important osmolyte and may effect osmolality-dependent changes in the intracellular concentration of taurine.

Differential effects of extracellular anions on renin secretion from isolated perfused rat kidneys.

We investigated the relevance of anions for the regulation of renin secretion from the kidneys. For this purpose we measured renin release from isolated rat kidneys that were perfused with medium containing either 120 mmol/l (normal) chloride or 95 mmol/l of isethionate, acetate, or nitrate anions in exchange for equimolar amounts of chloride. Lowering the extracellular chloride concentration by either of these maneuvers significantly enhanced renin secretion rates (RSR) at a perfusion pressure of 100 mmHg. Increasing pressure above 100 mmHg inhibited renin release in the presence of isethionate and acetate but not with nitrate anions. The renin stimulatory effects of isethionate and acetate but not that of nitrate anions disappeared in the presence of bumetanide (100 mumol/l), an inhibitor of macula densa chloride transport. Activation of renin secretion by isethionate and acetate was blunted with 100 pmol/l angiotensin II (ANG II), whereas tenfold higher concentrations of ANG II were required to attenuate the effect of nitrate ions. The amount of renin released in the presence of nitrate was fully additive to RSR values obtained with maximally effective doses of isoproterenol. These findings are consistent with the idea that impermeant anions such as isethionate and acetate enhance renin secretion from the kidneys predominantly via the tubular macula densa mechanism. The stimulatory influence of membrane-permeable nitrate anions appears to involve additional pathways and is mediated by a decreased calcium sensitivity of the renin secretory process rather than resulting from an adenosine 3',5'-cyclic monophosphate-dependent action.

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.

In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.

Cl- channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites.

A unique property of basolateral membrane Cl- channels from the mTAL is that the Cl- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (Po). The K1/2 for maximal activation of Po by Cl- facing intracellular domains of these channels is 10 mM Cl-. The present experiments evaluated the nature of these Cl(-)-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl- channel faces, could augment Po with a K1/2 in the range of 10 mM isethionate without affecting conductance (gCl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mM phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mM), prevented the activation of Po usually seen when the Cl- concentration of solutions facing intracellular channel domains was raised from 2 to 50 mM. However, when the Cl- channel activity was increased by first raising the Cl- concentration bathing intracellular channel faces from 2 to 50 mM, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl- channel faces had no effect on Po. We conclude that the intracellular aspects of these Cl- channels contain Cl(-)-interactive loci (termed [Cl]i) which are accessible to impermeant anions in intracellular fluids and which contain arginine- and lysine-rich domains which can be inactivated, at low ambient Cl- or isethionate concentrations, by interactions with PGO or TNBS.

Ionic mechanisms underlying the depolarizing and hyperpolarizing afterpotentials of single spike in guinea-pig cingulate cortical neurons.

Intracellular recordings and hybrid single-microelectrode voltage-clamp techniques were used to study the ionic mechanisms underlying the afterdepolarization and the subsequent slow afterhyperpolarization that followed a single action potential in layers V/VI neurons of the guinea-pig anterior cingulate cortex in in vitro slices. Both the afterdepolarization and afterhyperpolarization were markedly suppressed in size by addition of Co2+ or Cd2+, reduction in extracellular Ca2+, and intracellular EGTA injection. On the other hand, elevation of extracellular Ca2+ concentration augmented the amplitudes of the afterpotentials. The afterdepolarization amplitude was selectively depressed by the stilbene derivatives, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate, disodium 3H2O, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, disodium salt. Reduction in external Cl- and intracellular Cl- injection enhanced the afterdepolarization amplitude without affecting the afterhyperpolarization. The null potentials for the afterdepolarizations recorded with K acetate- and Cs acetate-electrodes were -68 and -63 mV, respectively. The slope of the null potential obtained with K acetate electrodes or Cs acetate electrodes was 49 and 53 mV, respectively, per log unit of the external Cl- concentration. Reduction in external K+ markedly depressed the afterdepolarization and augmented the afterhyperpolarization in size, whereas rise in external K+ markedly augmented the afterdepolarization and depressed the afterhyperpolarization. The null potential for the afterhyperpolarization recorded with K acetate electrodes was -94 mV. The slope of the null potential was 57 mV per log unit of the external K+ concentration. Reduction in extracellular Na+ concentration slightly depressed both the amplitudes of the afterdepolarization and afterhyperpolarization. A hybrid voltage-clamp analysis revealed a slow decaying inward current and a subsequent outward current that followed an action potential. Both the amplitudes of the inward current corresponding to afterdepolarization and the outward current corresponding to afterhyperpolarization were suppressed by addition of Co2+. Reduction in extracellular Cl- concentration augmented the inward current amplitude without significantly affecting the outward current. These results indicate that the afterdepolarization is mainly due to an increase in a Ca(2+)-activated Cl- conductance, while the afterhyperpolarization is mainly generated by an activation of Ca(2+)-mediated K+ conductance.

Chloride and depolarization by acetylcholine in canine airway smooth muscle.

The role of chloride channels has been examined in canine tracheal smooth muscle by recording mechanical responses to field stimulation and to acetylcholine (ACh) and by sucrose gap recording of excitatory junction potentials and ACh-induced electrical changes. The results of substitution studies using isethionate for chloride provided evidence that a chloride conductance contributes to the resting potential. The extrapolated reversal potential for ACh-induced depolarization was positive to the resting potential. Isethionate substitution inhibited ACh-induced depolarization, consistent with a contribution from increased Cl- conductance to the depolarization induced by ACh. However, closure of K+ channels and opening of a non-specific cation channel could also contribute to depolarization. Further study of the effects of isethionate substitution during prolonged tissue exposure to chloride-free medium showed that retention or the accumulation of Ca2+ in intracellular stores was impaired. We conclude that effects of chloride deprivation on responses to ACh may reflect an early increase in Cl- conductance, but longer term changes reflect the requirement for this anion to maintain internal Ca2+ stores.

Regulation of glycogen synthesis and glycolysis by insulin, pH and cell volume. Interactions between swelling and alkalinization in mediating the effects of insulin.

The effects of changes in cell volume and pH on glycogen synthesis and glycolysis and their control by insulin were investigated in hepatocyte cultures. 1. Cell acidification, by increasing [CO2] from 2.5% to 5%, inhibited glycolysis and stimulated glycogen synthesis. The inhibition of glycolysis was also observed in Na(+)-free media and when K+ uptake was inhibited, but the stimulation of glycogen synthesis was abolished under these conditions, suggesting that it is secondary to ionic or volume changes. Alkalinization had converse effects on glycolysis and glycogen synthesis. 2. In HCO3(-)-containing media, replacement of NaCl with sodium acetate or potassium acetate, like acidification with CO2, inhibited glycolysis and stimulated glycogen synthesis. The latter correlated with an increase in cation content. Amiloride, an inhibitor of Na+/H+ exchange, inhibited both the increase in cation content and the stimulation of glycogen synthesis, suggesting that the latter is secondary to cell swelling. 3. Hypo-osmotic swelling increased glycogen synthesis in HCO3(-)-containing media, in both the absence and the presence of Na+ and at both 2.5% and 5% CO2, but it increased glycolysis in the presence of Na+ and at 2.5%, but not at 5%, CO2. In HCO3(-)-free media, during acidification and swelling, glycogen synthesis correlated with pH and not with cell volume, indicating that inhibition by acidification over-rides stimulation by swelling. 4. Stimulation of glycolysis by insulin was not additive with stimulation by alkalinization. The stimulation of glycogen synthesis by insulin was partially suppressed under alkaline conditions; it was markedly suppressed in isosmolar Na(+)-free media and restored by hypo-osmotic swelling. In hypo-osmolar Na(+)-free media insulin prevented the decrease in glycogen synthesis with decreasing [HCO3-], suggesting that it counteracts inhibition by acidification. 5. It is concluded that glycogen synthesis and glycolysis are both stimulated by cell swelling and inhibited by acidification, under certain conditions, but glycolysis is more sensitive to inhibition by acidification and glycogen synthesis to stimulation by swelling. Consequently, simultaneous swelling and acidification is associated with inhibition of glycolysis and stimulation of glycogen synthesis. Stimuli that cause swelling and alkalinization activate both glycogen synthesis and glycolysis, alkalinization being more important in control of glycolysis and swelling in control of glycogen synthesis. Both cell swelling and alkalinization are components of the mechanism by which insulin controls glycogen synthesis and glycolysis.