Biomedical subjects

E K Asem

Publications and source records attributed to E K Asem.

At least 55 records · Page 3Linked to original sources

PubMed1993

Acanthamoeba binds to extracellular matrix proteins in vitro.

PURPOSE: To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro. METHODS: A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba. RESULTS: Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner. CONCLUSION: In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.

Acanthamoeba↗

PubMed1992

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC.

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Fibronectin production by chicken granulosa cells in vitro: effect of follicular development.

In vitro studies were conducted to investigate the role of chicken ovarian granulosa cells in the production of fibronectin, a component of the basal lamina of ovarian follicles. Collagenase dispersed granulosa cells obtained from the first (F1; about 35 mm in diameter) and third (F3; 15-20 mm in diameter) largest preovulatory follicles, as well as from a pool of small yellow follicles (SF; 6-10 mm in diameter), were incubated in serum-free medium-199 for 24 to 96 h in the absence and presence of luteinizing hormone (LH) or forskolin. Fibronectin secreted in the medium was quantitated by enzyme linked immunosorbent assay. Basal fibronectin production (which increased with the duration of incubation) was significantly greater (p < 0.001) in granulosa cells derived from mature follicle (F1) than in F3 or SF cells. Both LH and forskolin stimulated fibronectin production in SF and F3 cells in a dose-dependent manner; however, they were without effect in F1 cells. The magnitude of increase in fibronectin production elicited by LH or forskolin was greater in SF cells than in F3 cells. The cytoplasm of cultured granulosa cells taken at all stages of follicular development stained positively for fibronectin. These findings indicate that chicken granulosa cells produce fibronectin. This ability is acquired early in follicular development and the stimulatory effect of the gonadotropin (LH) diminished as the follicle approached ovulation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Calcium ionophores increase intracellular pH in chicken granulosa cells.

Several hormone agonists exert their physiological actions by triggering an inositol phospholipid-Ca2+ signalling cascade and cytosolic alkalinization. Although calcium ionophores have been used extensively to probe the role of Ca2+ in the regulation of steroidogenesis in granulosa cells, the precise relationship between changes in intracellular Ca2+ (Ca2+i) and pH (pHi) is unclear. In the present study we have used a fluorescent pH indicator, 2'7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, to examine the influence of two Ca2+ ionophores, ionomycin and 4-Bromo-A23187 (4-Br-A23187), on pHi in chicken granulosa cells. Chicken granulosa cells from the largest preovulatory follicle were incubated with Ca2+ ionophores (0-2 microM) and/or inhibitors of Na+/H+ antiport (amiloride, dimethylamiloride and ethylisopropyl amiloride; 0.5, 5 and 50 microM respectively) in the presence of Na+ (or choline+; 0-144 mM) and/or Ca2+ (0-10 mM). Ionomycin or 4-Br-A23187 elicited a rapid and sustained cytosolic alkalinization. The magnitude of increase in pHi was dependent on the concentration of the Ca2+ ionophore and the presence of extracellular Ca2+ but independent of extracellular Na+. Pretreatment of the cells with amiloride or its analogues failed to affect the increase in pHi induced by the Ca2+ ionophores significantly. These findings demonstrate that, in addition to their widely reported effects on Ca2+i redistribution in granulosa cells, 4-Br-A23187 and ionomycin cause Ca(2+)-dependent cytosolic alkalinization. This action of the Ca2+ ionophores is independent of the Na+/H+ antiport. Caution must be exercised in using Ca2+ ionophores as probes to define the role of Ca2+ in the regulation of granulosa cell function.

Amiloride↗

PubMed1991

Epidermal growth factor elevates intracellular pH in chicken granulosa cells.

Many bioregulators, such as epidermal growth factor (EGF), induce intracellular alkalinization by activating a membrane bound Na+/H+ antiporter. The present studies were designed to examine the influence of EGF on intracellular pH (pHi) in chicken granulosa cells. pHi in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the dye 2',7'-bis(carboxyethyl-5(6)-carboxyfluorescein. The resting pHi was 6.81 +/- 0.006 (n = 30) when the extracellular pH and sodium concentration (Na+o) were 7.3 and 144 mM, respectively. EGF (5-100 ng/ml) induced a concentration-dependent increase in pHi, which reached a maximum of 0.217 +/- 0.009 pH units at a concentration of 100 ng/ml EGF. Cytosolic alkalinization was observed within 10 min of the addition of EGF and lasted over the 60 min observation period. The increase in pHi was dependent upon the presence of Na+o, since the EGF effect was attenuated when Na+o was substituted with equimolar concentrations of nonpermeant choline chloride. The EGF-induced pHi change was also inhibited by amiloride, dimethyl amiloride, and ethylisopropyl amiloride, inhibitors of the Na+/H+ antiporter. The alkalinization effect of EGF was mimicked by transforming growth factor-alpha but not by insulin, insulin-like growth factor-I, or transforming growth factor-beta. These studies suggest for the first time that intracellular alkalinization resulting from activation of the Na+/H+ antiporter may be a part of the transmembrane signaling pathway in the action of EGF on chicken granulosa cells.

Animals↗

PubMed1989

Na+/H+ exchange in granulosa cells of the three largest preovulatory follicles of the domestic hen.

Sodium-dependent intracellular pH (pHi) regulation was compared in granulosa cells from the three largest avian ovarian follicles by monitoring the pHi with biscarboxyethylcarboxyfluorescein, a dye whose fluorescence increases with alkalinity. Collagenase-dispersed granulosa cells obtained from the largest (F1), second largest (F2), and third largest (F3) preovulatory follicles about 2-3 hr prior to expected ovulation of F1 were used in the present study. The resting pHi measured in nominally bicarbonate free buffer with extra-cellular Na+ (Nao+ = 144 mM) and external pH (pHo) of 7.3 was about 6.8 in cells from F1, F2, and F3. There was no correlation between the stage of follicular development and the pHi whether the follicles were removed in the early or late preovulatory period. After acute cytoplasmic acidification by exposure of cells to nigericin in choline+ buffer, or by the abrupt removal of ammonium chloride, complete recovery of pHi occurred in 4-5 min. The rate and magnitude of the recovery were dependent upon the concentration of Nao+ and were abolished when Nao+ was replaced completely by choline+. Recovery in the presence of Nao+ was inhibited dose-dependently by amiloride (sodium-hydrogen exchange inhibitor). There was no difference between the rate and the extent of pHi recovery in acid-loaded cells obtained from F1, F2, and F3. Furthermore, by varying the concentration of Nao+ between 0 and 144 mM both young and matured granulosa cells extruded acid at the same rate. In addition, amiloride inhibited the Nao+ dependency of pHi recovery to a similar degree in F1, F2, and F3 cells. Our observations demonstrate in avian granulosa cells the existence of a Nao+-dependent, amiloride-sensitive pHi regulatory system that is equally effective in cells obtained from the three largest yolk-filled follicles.

Amiloride↗

PubMed1989

Effect of sodium on progesterone production in granulosa cells of rapidly growing chicken ovarian follicles.

The importance of extracellular sodium (Nao+) in the progesterone production in hen granulosa cells from the first (F1), second (F2), and third (F3) largest preovulatory follicles was investigated in short term incubations. Progesterone synthesis in the absence or presence of the gonadotropin increased with increasing Nao+ concentration. Luteinizing hormone (LH) caused an additional and significant increase in steroidogenesis at every Nao+ concentration tested. However, no significant difference in the ED50 of Nao+ among F1, F2, and F3 cells was observed whether in the absence or the presence of the gonadotropin. Furthermore, although LH provoked steroidogenesis dose dependently in F1, F2, and F3 granulosa cells, no significant change in the ED50 of LH was observed among F1, F2, and F3 cells. The Na+/H+ exchange inhibitor, amiloride, attenuated both basal and LH-stimulated steroidogenesis in a concentration-dependent manner in the presence of Nao+. Amiloride was ineffective in the absence of Nao+. The present studies indicate the importance of Nao+ in the modulation of steroidogenesis in hen granulosa cells. Since steroidogenesis was suppressed by amiloride only in the presence of sodium, it is suggested that Nao+ is important for the maintenance/regulation of intracellular pH by an exchange with intracellular H+. Our studies also suggest that the sodium modulatory mechanism may not be a determinant in the differential progesterone production observed among the three largest preovulatory follicles.

Amiloride↗

PubMed1989

Sodium-dependent regulation of steroidogenesis in rat granulosa cells: possible involvement of a Na+/H+ antiport.

The possible role of Na+/H+ antiport in the gonadotropic regulation of steroidogenesis was examined in rat granulosa cells incubated for up to 6 h in a chemically defined medium in the absence or presence of Na+ (128 mM), gonadotropin (FSH or LH; 0-500 ng/ml), dibutyryl cyclic AMP [Bu)2cAMP; 2 mM) and amiloride (0-1 mM). Replacement of Na+ (Na+0) in the incubation medium with choline chloride resulted in a marked decrease in basal and LH-, FSH- and (Bu)2cAMP-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) synthesis in vitro. The Na+/H+ exchange inhibitor, amiloride significantly suppressed basal and hormone-stimulated progestin production dose-dependently in the presence of Na+0. However, it was without effect in Na+-deficient medium. The effect of the inhibitor on progestin production appeared to be directed at specific step(s) involved in the synthesis of pregnenolone, as concentrations of amiloride which inhibited progesterone production failed to influence the metabolism of exogenous pregnenolone to progestins. Cell viability and the incorporation of [3H]leucine into acid-precipitable material were not affected by amiloride. Our findings support the contention that extracellular sodium is important for steroidogenesis in rat granulosa cells. The inhibition by amilordie indicates an involvement of the Na+/H+ exchange in the regulation of this granulosa cell function.

20-alpha-Dihydroprogesterone↗

PubMed1989

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells.

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.

Animals↗

PubMed1989

Phorbol ester inhibits luteinizing hormone-, forskolin-, and dibutyryl cyclic AMP-induced progesterone production in chicken granulosa cells.

The possible role of protein kinase C in avian granulosa cell steroidogenesis was studied in vitro by examining the effect of tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on progesterone synthesis in chicken granulosa cells in short-term (3h) incubations. TPA (1-100 nM) caused a marginal but nonsignificant increase in progesterone production in granulosa cells isolated from the largest preovulatory follicle. When incubated in combination with luteinizing hormone (5-100 ng/mL), TPA suppressed the stimulatory effects of submaximally and maximally effective doses of the gonadotropin in a concentration-related manner. Similarly, the phorbol ester inhibited the steroidogenic responses to forskolin and dibutyryl cyclic AMP. By comparison, TPA had no appreciable effect on the metabolism of exogenous pregnenolone substrate to progesterone. Our data indicate that the tumor-promoting phorbol ester influences steroidogenic steps distal to cyclic AMP generation but at or before pregnenolone formation, and that protein kinase C may be a negative regulator of steroid biosynthesis in chicken granulosa cells.

Animals↗

PubMed1989

T- and L-calcium channels in steroid-producing chicken granulosa cells in primary culture.

Steroidogenesis and other functions in granulosa cells are calcium dependent. Using the patch-clamp technique to record single ion channel activity, we have identified for the first time two kinds of calcium channels through which the divalent ion may enter chicken granulosa cells. The cells were maintained in primary culture whose basal and hormone-stimulated progesterone production was evaluated at different times in culture and at different temperatures. A small channel, with a conductance of 4-5 picosiemens (pS), had short openings and inactivated rapidly. A large channel had a conductance of 20-30 pS, a high activation threshold, and slow inactivation kinetics. The dihydropyridine compound Bay K-8644, a L-calcium channel agonist, significantly increased the activity of the large channel, and nifedipine, a dihydropyridine calcium channel blocker, inhibited it completely. Both types of channels were seen in functionally competent signal-responsive granulosa cells cultured for up to 48 h. Whether these channels are involved in steroidogenesis, protein production, and/or secretion remains to be established.

Animals↗

PubMed1989

Influence of A23187 and dibutyryl cyclic AMP on progestagen production by rat granulosa cells in vitro.

The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP [Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) but not 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20 alpha-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20 alpha-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH. We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

20-alpha-Dihydroprogesterone↗

PubMed1988

Ionic currents in avian granulosa cells.

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole-cell patch-clamp technique. Under voltage-clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than -50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.

Animals↗

PubMed1988

Evidence for two distinct potassium channels in avian granulosa cells.

Single-channel potassium currents were recorded in avian granulosa cells using the patch-clamp technique. Two types of channel were observed. The smaller of the two channels, gK1, had a conductance of 15 to 30 picosiemens (pS) and was voltage- and calcium-independent. Its null-current potential was -50 mV in the cell-attached recording mode. The other channel, gK2, was infrequently observed in the cell-attached configuration. Its conductance was between 160 and 195 pS. It could be activated by calcium on the cytoplasmic side of the membrane patch in the inside-out configuration. It was also voltage-dependent. These results suggest that fast transmembrane potassium movements may be involved in the membrane voltage regulation of granulosa cells, which in turn may play an important role in the modulation of steroidogenesis and other metabolic activities.

Animals↗

PubMed1988

The effect of kaurenol on steroidogenesis and cyclic adenosine monophosphate production in rat granulosa cells.

The effect of kaurenol (ent-kaur-16-en-15 beta-ol) on steroidogenesis and cyclic AMP production was examined in rat granulosa cells in short-term incubations (6 h). Kaurenol alone significantly augmented the production of progesterone in time- and concentration-dependent manner but attenuated the accumulation of the progesterone metabolite 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P). The steroidogenic effect of kaurenol is due to the acute stimulation of pregnenolone production from endogenous cholesterol and an inhibition in 20 alpha-hydroxysteroid dehydrogenase which catalyzes the metabolism of progesterone to 20 alpha-OH-P. Kaurenol had no appreciable effect on conversion of exogenous pregnenolone to progesterone. Although kaurenol was without effect on basal cyclic AMP generation, it inhibited the actions of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and forskolin on the production of the cyclic nucleotide. Kaurenol also significantly attenuated the LH-, FSH- and forskolin-stimulated progesterone and 20 alpha-OH-P production in a concentration-dependent manner. Because kaurenol induced steroidogenesis without increased cyclic AMP accumulation, it is concluded that its action on basal steroidogenesis is mediated by a mechanism independent of the cyclic nucleotide. Kaurenol may serve as a useful tool for elucidating cyclic AMP-independent action(s) of hormones in intact tissue/cells.

Animals↗

PubMed1988

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells.

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.

Animals↗

PubMed1988

Cyclic adenosine monophosphate (cAMP) production in avian theca cells during follicular maturation.

LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2-T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation.

Androstenedione↗

PubMed1988

Sodium-dependent intracellular pH regulation in granulosa cells of the domestic hen (Gallus domesticus).

The existence and importance of the Na+/H+ exchanger in intracellular pH (pHi) regulation in ovarian cells was studied in acid-loaded avian granulosa cells by monitoring the recovery of normal pHi using a trapped fluorescein derivative as an indicator. The resting pHi of freshly isolated granulosa cells from preovulatory follicles was 6.80 +/- 0.08 when the extracellular pH (pHo) and sodium concentration (Na+o) were 7.3 and 144 mmol/l respectively. While exposure of granulosa cells to high pHo (pHo greater than 7.45) medium shifted the pHi upward with time, incubation of the cells in low pHo (pH less than 6.80) buffer resulted in a significant decrease in pHi. In contrast, pHi remained constant when pHo was varied between the broad range of 6.8-7.4. When the cytoplasm was acidified by treatment with nigericin in choline+ buffer, both the magnitude and rate of recovery of normal pHi was suppressed significantly with decreasing pHo, but increased in high pHo medium. The recovery of pHi was dependent upon the concentration of extracellular sodium, in that the recovery rate and magnitude increased concomitantly with increases in Na+o concentrations, while the recovery was abolished when Na+o was completely replaced with choline+. In addition, the sodium ionophore monensin enhanced the recovery rate of normal pHi in a concentration-dependent manner. This action of monensin was observed only when sodium was present in the incubation medium, indicating that Na+o entry is important for the recovery of normal pHi. Monensin also evoked further cytoplasmic alkalinization in fully recovered cells, with a relative net effect dependent upon the level of Na+o present.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗