Influence of mucosal and serosal pH on antidiuretic action in frog urinary bladder.

Mucosal acidification to pH 6.5 reduced by 88% the oxytocin- (2.2 x 10(-8) M) elicited increase of water permeability in frog urinary bladder. Mucosal alkalinization (pH 10.5) increased by as much as 200% the response to the same concentration of oxytocin. These effects were not observed when supramaximal concentrations of oxytocin were imployed. Similar changes were found when the serosal pH was modified. The hydrosmotic responses elicited by serosal hypertonicity or cyclic AMP plus theophylline were also affected by mucosal or serosal changes of the hydrogen in concentration, suggesting an effect at a post-cyclic AMP level. Important interactions were found between luminal pH and serosal hypertonicity when experimental conditions were employed similar to those observed in the collecting duct of mammalian nephron. Freeze-fracture studies showed that the number of intramembranous aggregates of particles induced by ADH in the luminal membrane was reduced by mucosal acidification and augmented by an increase in medium pH.     

Phorbol ester effect on the hydrosmotic response to vasopressin in frog skin

Serosal preincubation of frog skin with tetradecanoyl phorbol acetate, TPA, an activator of protein kinase C, inhibits the hydrosmotic response elicited by vasopressin (AVP) but not that induced by 8br-cAMP. This proves that serosal TPA primarily influences a pre-cAMP step. The TPA-induced inhibition of AVP response appears to be related to TPA-induced prostaglandin synthesis. The pretreatment with naproxen, in fact, prevents the inhibition induced by serosal TPA on the AVP response. On the contrary, mucosal TPA produces a more marked inhibition of the response to AVP and significantly diminishes the water flow induced by 8br-cAMP; this suggests that mucosal TPA interferes mainly with a post-cAMP step. Furthermore, naproxen is unable to completely prevent the inhibition induced by mucosal TPA on AVP response thus indicating that mucosal TPA may also activate a prostaglandin-independent mechanism able to inhibit one of the last steps of the hydrosmotic response to AVP.     

The isolated frog skin epithelium: Permeability characteristics and responsiveness to oxytocin,cyclic AMP and theophylline

Summary The combined treatment of the frog skin with collagenase and hydrostatic pressure enables complete separation of the epithelial layer from the supporting dermis. The separation entirely preserves the epithelium's passive permeability and active sodium transport capacity. The short circuit current, d.c. resistance, unidirectional fluxes of Na⁺ and Cl^– ions, and osmotic water permeability were found identical on series of isolated epitheliums and intact skins obtained from the same groups of animals. Furthermore, the isolated epithelium is fully responsive to oxytocin, cyclic AMP and theophylline—three agents known to enhance active sodium transport and water permeability of the intact skin.     

Does the gradual hydroosmotic response to antidiuretic hormone depend on intracellular cAMP accumulation or on the formation of intramembrane particle aggregates?

In experiments on frog urinary bladder the mechanisms behind the gradual development of a hydroosmotic reaction to antidiuretic hormone (ADH) were investigated. It was suggested that the velocity of hydroosmotic reaction may be limited by (a) formation and insertion of particle aggregates into the apical membrane or (b) by velocity of cAMP formation. The urinary bladders were exposed to 23 nM ADH for different times (from 1 to 20 min) and water flow was measured over a period of 40 min. It was found that the value of the full hydroosmotic response increased progressively with the time of exposure to the hormone; however, the enhancement of water flow was equal during each time interval before reaching the reaction maximum. A direct correlation between the value of ADH-stimulated water flow, cAMP content in bladder tissue and frequency of particle aggregates in the granular cell apical membrane was observed. The content of cAMP in ADH-treated bladders was higher by 80% in the absence than in the presence of an osmotic gradient. Pretreatment of urinary bladders with 50 M cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, significantly accelerated the development of the hydroosmotic reaction and increased the magnitude of water flow in comparison with the effect of ADH only. No changes in cyclic AMP phosphodiesterase activity were found in the urinary bladder homogenates under the action of ADH, so it seems likely that accumulation of cAMP depends only on the increase of adenylate cyclase activity. The data obtained allow one to conclude that the gradual hydroosmotic response to ADH depends on the accumulation of cAMP, which may be considered as the main limiting factor of the velocity of the hydroosmotic reaction.     

Gossypol interferes selectively with water and urea permeability of toad bladder

Summary Gossypol, a polyphenol compound with antifertility properties in human male, has been found to interfere with the response of toad bladder to vasopressin in terms of permeability to water and to urea; on the other hand, ion movement is spared for several hours as judged from short-circuit current and conductance data. Action of the drug lies distal to the generation of cyclic AMP since bladder hydrosmotic response to the latter was affected to the same extent as when the hormone was applied.     

Amiloride inhibits the vasopressin-induced increase in epithelial water permeability

The vasopressin (VP)-induced increase in water permeability in high-resistance, amphibian epithelia is not altered by the abolition of net Na⁺ flux caused by amiloride added to the apical bathing medium. In this work we looked at the effects on water transport of amiloride added to the serosal medium at a concentration (10^–3 M) known to inhibit Na⁺/H ⁺ exchange. In urinary bladders of Bufo marinus, amiloride partially blocked the hydrosmotic response to VP. A similar inhibition was found with cyclic adenosine 5-monophosphate (cAMP) or serosal hypertonicity. We hypothesized that this effect of amiloride could be due to an inhibition of Na⁺/H⁺ and/or Na⁺/Ca²⁺ antiporters present in the epithelial basolateral membrane and looked at the effects of the diuretic in Na⁺-free media. A similar degree of inhibition of water flow was still found, thus showing that amiloride acts on a cell target other than the antiporters. In toad skin, amiloride did not inhibit the hydrosmotic response to VP and to isoproterenol; however the response to high K⁺ was significantly reduced. Among the amiloride cell targets described so far, adenylate cyclase and protein kinase A appear to be the best candidates to explain the inhibition of the hydrosmotic response reported here. Direct measurements of intracellular cAMP are needed however to substantiate this hypothesis.     

Endogenous prostaglandins, adenosine 3':5'-monophosphate and sodium transport across isolated frog skin.

1. Sodium transport across isolated frog skin, as measured by the short-circuit current, was decreased by acetylsalicylic acid, mefenamic acid, paracetamol and phenylbutazone. Indomethacin (6 X 10(-6) M) had a biphasic effect on the short-circuit current: a transient increase followed by a sustained decrease. 2. The release of prostaglandin-like material from the skin was reduced by acetylsalicylic acid and indomethacin. Paracetamol caused a significant reduction in the short-circuit current response of the skin to low doses of arachidonic acid, but the response to the highest dose tested was not significantly altered. 3. Indomethacin (6 X 10(-6) M) increased the sensitivity of the skin to applied prostaglandin E1. The other prostaglandin synthetase inhibitors did not have this effect. Indomethacin (6 X 10(-6) M) also enhanced the effect of antidiuretic hormone on the short-circuit current. 4. Indomethacin (30 X 10(-6) M) increased the short-circuit current and diminished the response to applied prostaglandin E1. 5. In sulphate Ringer, theophylline increased the short-circuit current and diminished the response to prostaglandin E1. 6. Prostaglandin E1 increased the levels of cyclic AMP in frog skin and these increases preceded the increases in short-circuit current. There was a seasonal variation in the level of cyclic AMP in the skin: the levels in winter exceeded those in summer. There was also a seasonal variation in the cyclic AMP response to prostaglandin E1: the winter response was greater than that in summer. 7. Indomethacin (6 X 10(-6) M) had a biphasic effect on cyclic AMP levels in the skin, an initial increase followed by a decrease. Indomethacin also potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 8. Theophylline increased cyclic AMP levels in the skin and potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 9. Pre-treatment of the skin with theophylline reversed the effects of cyclic AMP on the short-circuit current and open-circuit potential. 10. It is concluded that endogenous prostaglandins help to maintain sodium transport across isolated frog skin and that the effects of E-type prostaglandins on the short-circuit current are mediated by increased cyclic AMP levels. The transient increase in short-circuit current and the increased skin sensitivity caused by indomethacin (6 X 10(-6) M) are attributed to inhibition of phosphodiesterase activity. The failure of theophylline to potentiate the short-circuit current response of the skin to prostaglandin E1 is attributed to alteration of cyclic AMP action on the skin by theophylline.     

The mechanism of lithium accumulation in the isolated frog skin epithelium

Summary The intracellular concentration of Li ([Li]c) of epithelia isolated from frog skins was determined after a one hour exposure to external Na free Choline Ringer containing from 1 to 25 mM of LiCl. In both open and short circuit conditions, [Li]c was found to have values up to ten times that of the outside Li concentration, indicating accumulation of Li in the epithelia. A loss of an approximately equivalent amount of cellular K was associated with this Li accumulation, whereas no significant change in epithelial content of Na was observed. This accumulation of Li is reduced if Na is present together with Li in the outer solution. Preincubation of the epithelia with Amiloride (10^–4 M) suppressed short circuit current and potential difference changes consecutive to externali solutions exposure, prevented Li accumulation and K loss and reduced transepithelial movements of Li. The entry of Li into the cell was speeded up by oxytocin (20 mU/ml). In cyanide (10^–4 M) pretreated epithelia, Li uptake was reduced and accumulation failed to occur. 2,4 dinitrophenol (5×10^–4 M) also lowered the Li uptake. It is concluded that the main mechanism for monovalent cations entry into the epithelium is a process for which Na and Li compete. The existence of an active transport step at the level of the outward facing membrane of the frog skin epithelium is proposed.     

Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH

With the single-channel patch-clamp technique we have identified Ca²⁺-sensitive, high-conductance (maxi) K⁺ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K⁺ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K⁺. In excised inside-out patches and with symmetrical 140 mmol/l K⁺, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K⁺ over Na⁺. Depolarization of the BLM increased maxi K⁺ channel activity. Increasing cytosolic free Ca²⁺ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca²⁺ directly activated channel activity in a voltage-dependent manner. Maxi K⁺ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K⁺ channel outward current was reversibly blocked by micromolar concentrations of Ba²⁺ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.     

Intracellular pH changes during neutrophil activation: Na+/H+ antiport

Activation of the Na+/H+ antiport mechanism was studied in human neutrophils by monitoring intracellular pH with a carboxyfluorescein derivative. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phospholipase C (PLC) induced biphasic pH changes. Amiloride, which inhibits the antiport, completely blocked alkalinization but enhanced acidification. Polymyxin B, which inhibits protein kinase C, only blocked alkalinization. Activation with phorbol 12-myristate 13-acetate (PMA) led to alkalinization only; this was inhibited by amiloride or polymyxin B. Thus, during polymorphonuclear leukocyte (PMN) activation, intracellular alkalinization appears to be mediated by an amiloride-sensitive Na+/H+ antiport. Antiport activity can also be blocked indirectly by inhibition of protein kinase C activity. Early intracellular acidification does not appear to require kinase activity but is observed when phospholipids are remodeled with PLC. The antiport was also activatable by hypertonic buffered media. This response did not appear to be mediated by protein kinase C because it was unaffected by polymyxin B. Finally, superoxide generation was investigated. It is affected by, but not soley controlled by, either antiport or protein kinase C activity.     

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na⁺ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3–4 days or 7–10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na₂SO₄ (25 mmol/l) solutions modified the Na⁺ transport rate and the morphology of the epithelium. The highest Na⁺ transport rates were found for the animals adapted to the Na⁺ free solutions and were correlated with an increase in the total MR cell pit-surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO₂ increase and HCO ₃ ^– decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10^–6 M) or during acidification of the epithelium by serosally applied CO₂. Na⁺ transport was enhanced by AVT (10^–6 M) or oxytocin (100mU/ml) when the skin was bathed on its apical side with a high Na⁺ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na⁺ transport when the apical solution was low in Na⁺ (0.5mmol/l). It is concluded that MR cells play a key role in Na⁺ and H⁺ transport through the frog skin epithelium when bathed on its apical side with a low Na⁺ containing solution. Distinct pathways for sodium transport through two cell types (MR cells and granular cells) are proposed depending on the Na⁺ concentration of the solution bathing the apical side of the epithelium.     

Amiloride blockage of Na+ channels in amphibian epithelia does not require external Ca2+

Noise analysis was used to study the influence of external Ca²⁺ on the blockage of Na⁺ transport by amiloride. Experiments were done using frog skin (Rana temporaria and Rana catesbeiana), toad urinary bladder (Bufo marinus) and epithelia of A6 cells. In non-depolarized skins and bladders, removal of Ca²⁺ from the mucosal bath diminished markedly the inhibitory effect of amiloride. Ca²⁺ depletion also gave rise to the appearance of an additional noise component related to cation movement through the poorly selective cation channel in the apical membrane [Aelvoet I, Erlij D, Van Driessche W (1988) J Physiol (Lond) 398:555–574; Van Driessche W, Desmedt L, Simaels J (1991) Pflügers Arch 418:193–203]. The amplitude of this Ca²⁺-blockable noise component was elevated by amiloride and markedly exceeded the amiloride-induced Lorentzian noise levels as recorded in the presence of Ca²⁺. On the other hand, in K⁺-depolarized skins and bladders as well as in non-depolarized epithelia of A6 cells, the Ca²⁺-blockable noise was absent or of much smaller amplitude. Depolarization of frog skin and toad urinary bladder apparently inactivated the poorly selective channels, whereas in A6 cells they were not observed. Under these conditions the typical amiloride-induced blocker noise could also be analysed in the absence of Ca²⁺ and demonstrated that the on and off rates for amiloride binding were not significantly altered by external Ca²⁺. We conclude that (a) external Ca²⁺ per se does not affect the inhibitory potency of amiloride, and (b) that the observed differences between frog skin, toad urinary bladder and A6 cells originate from the presence or absence of a poorly selective cation channel rather than from a different amiloride receptor structure.     

Association of increased pHi with calcium ion release in skeletal muscle.

The pH difference across the cell membrane of frog sartorius muscle cells was measured with the distribution of 5,5-dimethyl-2,4-oxazolidine-dione (DMO) as the marker. Depolarization of the muscles to values at or below the contraction threshold caused by elevating external potassium up to approximately 20 mM resulted in an internal alkalinization. The change was smaller with superthreshold depolarization (20--30 mM [K+]). The alkalinization was blocked by agents that block calcium release from the sarcoplasmic reticulum (procaine and dantrolene sodium). Other agents that cause calcium release (caffeine, theophylline, and quinine) were found to give alkalinization when tested at concentrations just below the contracture threshold. Increased acidification of the extracellular medium was associated with the internal alkalinization. The data were interpreted as indicating the presence of a calcium-stimulated H+ and/or OH- ion transport system in the muscle membrane.     

Extracellular pH dynamics of retinal horizontal cells examined using electrochemical and fluorometric methods

Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.     

Release of cyclic AMP by toad urinary bladder.

Cyclic AMP accumulates in the Ringer solution bathing the toad urinary bladder in vitro. At least 4 times more cyclic AMP is released into the solution bathing the serosal surface than into the solution bathing the mucosal surface. Most of the cyclic AMP originates in the epithelial cells rather than the stroma. Vasopressin increased the content of cyclic AMP in the epithelial cells and increases the amount of cyclic AMP in the Ringer solution. Since there is not an increase in medium cyclic AMP when cell cyclic AMP levels are increased by theophylline, it is suggested that theophylline may reduce the permeability of the cell membrane to cyclic AMP. Finally, it is demonstrated that 10 mM NaF increase the amount of cyclic AMP in the epithelial cells and in the solution bathing the bladder, but block the effect of vasopressin on water permeability, presumably at a step subsequent to the formation of cyclic AMP.     

The effect of ouabain, insulin, and cyclic AMP on the acidification of luminal fluid in the proximal tubule of bullfrog kidney

A transient urinary acidification was induced in the bullfrog proximal tubule by the peritubular administration of ouabain (10(-4) M). Insulin (200 mIU/ml) provoked a prolonged decrease of tubular fluid pH (TFpH), whereas dibutyryl cyclic AMP (10(-4) M) produced a transient increase of TFpH. The above data support the view that the urinary acidification in the proximal tubule is not explained by a simple mechanism, such as Na+/H+ exchange.     

Evidence for a two-site model of forskolin action in frog urinary bladder

In the present study several aspects of the osmotic water-flow-regulation mechanism in frog urinary bladder were examined, utilizing forskolin either as a direct hydrosmotic agent or in association with vasopressin. It was found that forskolin induces a hydrosmotic effect similar to the one induced by vasopressin. This effect is rapid, reversible and dose-dependent. The half-maximally effective concentration (Ec ₅₀ ^FSK ) is 1.37 M forskolin. No additional effect on the osmotic water flow was observed when maximal concentrations of forskolin and vasopressin were given simultaneously. Moreover, forskolin can also markedly potentiate vasopressin-induced hydrosmotic response. This potentiation was maximal with submaximal doses of vasopressin, whereas it disappeared when the hormonal concentration was increased to very high levels. Therefore, forskolin increases vasopressin potency without affecting vasopressin efficacy. The Ec ₅₀ ^FSK for the forskolin-induced increase in vasopressin potency was 11 nmol, about two orders of magnitude lower than the Ec ₅₀ ^FSK for the direct effect of forskolin on the osmotic water transport. On the whole, our results are compatible with a two-site model of forskolin action in frog urinary bladder: a low affinity site (Ec ₅₀ ^FSK =1.37 M) that mediates the direct action of forskolin on the osmotic water flow and a high affinity site (Ec ₅₀ ^FSK =11 nmol), which mediates the synergic effect of forskolin with the antidiuretic hormone.Part of this work has been presented at the 2nd meeting on Membrane Plasmatiche e Fisiologia Cellulare, 1985 (Lecce, Italy)     

A role for endogneous prostaglandins in the short-circuit current responses to osmolal changes in isolated frog skin.

1. Reduction in osmolality of the Ringer solution bathing the morphological inside of frog skin (by lowering the NaCl concentration) caused a significant increase in sodium transport as measured by the short-circuit current. Pretreatment of the skin with acetylsalicylic acid (2.5 x 10(-4)M) abolished the short-circuit current and open-circuit potential responses to osmolal change.2. The output of prostaglandin-like material from isolated frog skin was increased by incubating the skin in hypotonic Ringer solution.3. The cyclic AMP levels of isolated frog skin were also increased by a reduction in the osmolality of the Ringer fluid bathing the skin.4. Prostaglandin-like material was released both by the separated epithelial and dermal layers of frog skin and the output from both layers, on a unit wet weight basis, did not differ.5. The output of prostaglandin-like material from the separated layers of the skin was substantially greater than from whole skin. Indomethacin (6 x 10(-6)M) reduced the output of this material by more than 90% from both layers.6. The release of prostaglandin-like material from the separated layers of the skin was not altered by a reduction in osmolality of the bathing medium.7. It is concluded that a reduction in the osmolality of the solution bathing frog skin stimulates prostaglandin production and that the increased level of prostaglandins stimulates transepithelial sodium transport by stimulating cyclic AMP accumulation. It is also concluded that the response to osmolal change can only occur in intact skin since separation of the epithelial and dermal layers abolished the increase in the release of prostaglandin-like material to osmolal change. The site of the increased prostaglandin production and the exact nature of the stimulus remain to be determined.     

Volume regulation of frog skin epithelium

Previous results (MacRobbie & Ussing 1961) in combination with published values for cellular chloride concentration and for intracellular potentials show that the chloride concentration in frog skin epithelium cells is higher than predicted for equilibrium with the inside bathing solution. Both the apical and the basolateral membrane of these cells are normally almost tight to chloride, so that the maintenance of the high chloride concentration requires little work. A basolateral permeability to chloride is, however, activated by cell swelling, and the cells lose KCI. It is now shown that the KCI thus lost cannot be regained neither in the absence of sodium in the inside bath nor in the presence of furosemide. The volume regulation reactions are, however, independent of the composition of the outside bath. It is concluded that the recovery of KCI by the epithelium is due to a basolateral co-transport of NaCl from medium to cells, combined with return of Na to the medium via the Na-K pump. The co-transport mechansism thus restores the high chloride concentration of the cells, but seems to be virtually dormant unless the cells have lost chloride.     

Extracellular pH responses in CA1 and the dentate gyrus during electrical stimulation, seizure discharges, and spreading depression

Since neuronal excitability is sensitive to changes in extracellular pH and there is regional diversity in the changes in extracellular pH during neuronal activity, we examined the activity-dependent extracellular pH changes in the CA1 region and the dentate gyrus. In vivo, in the CA1 region, recurrent epileptiform activity induced by stimulus trains, bicuculline, and kainic acid resulted in biphasic pH shifts, consisting of an initial extracellular alkalinization followed by a slower acidification. In vitro, stimulus trains also evoked biphasic pH shifts in the CA1 region. However, in CA1, seizure activity in vitro induced in the absence of synaptic transmission, by perfusing with 0 Ca(2+)/5 mM K(+) medium, was only associated with extracellular acidification. In the dentate gyrus in vivo, seizure activity induced by stimulation to the angular bundle or by injection of either bicuculline or kainic acid was only associated with extracellular acidification. In vitro, stimulus trains evoked only acidification. In the dentate gyrus in vitro, recurrent epileptiform activity induced in the absence of synaptic transmission by perfusion with 0 Ca(2+)/8 mM K(+) medium was associated with extracellular acidification. To test whether glial cell depolarization plays a role in the regulation of the extracellular pH, slices were perfused with 1 mM barium. Barium increased the amplitude of the initial alkalinization in CA1 and caused the appearance of alkalinization in the dentate gyrus. In both CA1 and the dentate gyrus in vitro, spreading depression was associated with biphasic pH shifts. These results demonstrate that activity-dependent extracellular pH shifts differ between CA1 and dentate gyrus both in vivo and in vitro. The differences in pH fluctuations with neuronal activity might be a marker for the basis of the regional differences in seizure susceptibility between CA1 and the dentate gyrus.