Reduction In Intracellular pH Research Articles

Vinyl acetate-induced intracellular acidification: implications for risk assessment.

Cancerbioassays have demonstrated the carcinogenic activity of vinyl acetate in rodents. Tumors appear only at the site of contact and mechanistic data suggest that the carcinogenic mechanism involves carboxylesterase-mediated metabolism of vinyl acetate to acetic acid. It has been hypothesized that intracellular formation of acetate causes a reduction of intracellular pH (pH(i)) at noncytotoxic levels, but that prolonged exposure to reduced pH(i) is cytotoxic and/or mitogenic and drives proliferative responses. Coupled with exposure to metabolically formed acetaldehyde at high administered concentrations, nonlinear dose-response curves for epithelial tumors are produced. Freshly isolated rat hepatocytes were used as a model system to test the concept that exposure of cells to vinyl acetate causes a reduction in pH(i). Quantitative fluorescence imaging ratio microscopy showed that exposure of hepatocytes to vinyl acetate concentrations ranging from 10 to 1000 microM caused rapid and sustained reductions of approximately 0.03 to 0.65 pH units. Cellular acidification was rapidly reversed to control pH(i) upon removal of vinyl acetate. There was minimal accumulation of protons during the exposure period, as suggested by minor differences in pH(i) of cells with or without prior exposure to vinyl acetate. The effect of vinyl acetate on pH(i) was attenuated by prior exposure to the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results support the concept that intracellular acidification is a sentinel pharmacodynamic response of cells to vinyl acetate exposure and that pH(i) is an appropriate metric dose for use in quantitative risk assessments of cancer and noncancer human health risk assessment.

Mode-of-action-based dosimeters for interspecies extrapolation of vinyl acetate inhalation risk.

Vinyl acetate is used in the manufacture of many polymers. The Clean Air Act Amendments of 1990 require that an inhalation risk assessment be conducted to assess risks to human health from ambient exposures. Vinyl acetate is a nasal carcinogen in rats and induces olfactory degeneration in rats and mice. Because of the many unique aspects of the rodent nasal cavity compared to that of humans, conventional means for extrapolating dosimetry between species are not appropriate. Physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling can address many of these unique aspects. A PBPK/PD model has been developed for vinyl acetate, but the choice of appropriate dosimeter(s) to use for interspecies extrapolation depends on a hypothesis regarding mode of action. This article summarizes the key studies that formulate a mode of action hypothesis for vinyl acetate. Dose-response relationships for vinyl acetate-induced nonneoplastic and neoplastic responses are highly nonlinear, suggesting complex kinetic processes. Carboxylesterase-dependent metabolism of vinyl acetate forms acetic acid, a potent cytotoxicant, and acetaldehyde, a weak clastogen. Cell death, proposed to be the result of intracellular acidification, results in restorative cell proliferation. In conjunction with sufficient genetic damage, induced by spontaneous mutation and acetaldehyde-induced DNA-protein cross-links (DPX), olfactory degeneration progresses to a state of elevated proliferation and eventually, at high vinyl acetate concentrations, to neoplastic transformation. Thus, reduction in intracellular pH (pHi) is proposed as the dosimeter most closely linked to the earliest stages of vinyl acetate toxicity. Consequently, risk assessments that are based on protection of nasal epithelium from intracellular acidification will be protective of all subsequent pathological responses related to vinyl acetate exposure. Proposing a reasonable mode of action is an important step in any risk assessment and is critical to the choice of dosimeter(s) to be used for interspecies dosimetry extrapolation.

The role of sodium/hydrogen exchange in the teratogenic response to acetazolamide

ABSTRACT Administration of acetazolamide to pregnant rodents leads to postaxial forelimb ectrodactyly, generally right‐sided, in the offspring. We previously postulated that this uniquely localized malformation was caused by a reduction of intracellular PH(pHi) in the embryonic limb bud cells. The hypothesis is supported by numerous lines of evidence including the exacerbation of forelimb ectrodactyly by co‐administration of amiloride. One action of amiloride is to inhibit the activity of Na+/H+ exchangers (NHEs), the major mechanism by which most eukaryotic cells regulate their internal pH. A mouse without this exchange mechanism should theoretically be hypersensitive to acetazolamide‐induced postaxial forelimb ectrodactyly because the limb bud cells should be less able to recover from reduction of intracellular pH. To test this hypothesis we created a targeted knockout of NHE‐1 (Na+/H+ Exchanger‐1), to which we administered acteazolamide. The frequency and/or severity of forelimb ectrodactyly was no worse in NHE‐1 null fetuses than in wild type or heterozygous littermates. We interpret this negative result to suggest that there are compensatory pathways by which embryonic cells can rid themselves of excess protons. We examined this idea by co‐administration of acetazolamide and DIDS. This latter agent inhibits CI‐/HCO3 exchange, a common mechanism for regulation of pHi against acidosis. Although malformation frequency and severity were high after this combination treatment, NHE‐1 null embryos were not more affected with postaxial fore‐limb ectrodactyly than their heterozygous and wild type littermates. We presume that embryonic limb bud cells possess another pathway through which they can regulate cytoplasmic proton concentration under acidotic conditions.

The effects of pH on the interaction between capsaicin and the vanilloid receptor in rat dorsal root ganglia neurons.

1. The vanilloid receptor of sensory neurons is a polymodal nociceptor sensitive to capsaicin, protons, heat and anandamide. Although it is known that interaction occurs between these different mediators the mechanism by which this occurs is poorly understood. In this study capsaicin elicited currents were recorded from vanilloid receptors found in adult rat isolated dorsal root ganglia (DRG) neurons under conditions of varying pH and the mechanism whereby protons can modulate this capsaicin response investigated. 2. Under whole-cell voltage clamp, modulating extracellular pH shifted the position of the capsaicin log(concentration)-response curve. Acidification from pH 9.0 to pH 5.5 lowered the EC50 values from 1150+/-250 nM to 5+/-2 nM with coincident change in the mean apparent slope factor from 2.3+/-0.3 to 0.9+/-0.2 and no change in maximal response. 3. The magnitude of the potentiation seen on reducing extracellular pH was not significantly affected by changes in extracellular calcium and magnesium concentration. 4. The response to capsaicin was not potentiated by a reduction in intracellular pH suggesting a site of action more accessible from the extracellular than the intracellular side of the membrane. 5. Potentiation by low pH was voltage independent indicating a site of action outside the membrane electric field. 6. At the single channel level, reducing extracellular pH increased channel open probability but had no significant effect on single channel conductance or open time. 7. These results are consistent with a model in which, on reducing extracellular pH, the vanilloid receptor in rat DRG neurons, changes from a state with low affinity for capsaicin to one with high affinity, coincident with a loss of cooperativity. This effect, presumed to be proton mediated, appears to involve one or more sites with pK(a) value 7.4-7.9, outside the membrane electrical field on an extracellularly exposed region of the receptor protein.

MODE-OF-ACTION?BASED DOSIMETERS FOR INTERSPECIES EXTRAPOLATION OF VINYL ACETATE INHALATION RISK

Vinyl acetate is used in the manufacture of many polymers. The Clean Air Act Amendments of 1990 require that an inhalation risk assessment be conducted to assess risks to human health from ambient exposures. Vinyl acetate is a nasal carcinogen in rats and induces olfactory degeneration in rats and mice. Because of the many unique aspects of the rodent nasal cavity compared to that of humans, conventional means for extrapolating dosimetry between species are not appropriate. Physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling can address many of these unique aspects. A PBPK/PD model has been developed for vinyl acetate, but the choice of appropriate dosimeter(s) to use for interspecies extrapolation depends on a hypothesis regarding mode of action. This article summarizes the key studies that formulate a mode of action hypothesis for vinyl acetate. Dose-response relationships for vinyl acetate-induced nonneoplastic and neoplastic responses are highly nonlinear, suggesting complex kinetic processes. Carboxylesterase-dependent metabolism of vinyl acetate forms acetic acid, a potent cytotoxicant, and acetaldehyde, a weak clastogen. Cell death, proposed to be the result of intracellular acidification, results in restorative cell proliferation. In conjunction with sufficient genetic damage, induced by spontaneous mutation and acetaldehyde-induced DNA?protein cross-links (DPX), olfactory degeneration progresses to a state of elevated proliferation and eventually, at high vinyl acetate concentrations, to neoplastic transformation. Thus, reduction in intracellular pH (pHi) is proposed as the dosimeter most closely linked to the earliest stages of vinyl acetate toxicity. Consequently, risk assessments that are based on protection of nasal epithelium from intracellular acidification will be protective of all subsequent pathological responses related to vinyl acetate exposure. Proposing a reasonable mode of action is an important step in any risk assessment and is critical to the choice of dosimeter(s) to be used for interspecies dosimetry extrapolation.

Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pH(i)). It is a ubiquitous, amiloride-sensitive, growth factor-activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pH(i) than normal hematopoietic tissue. We then show that a direct correlation exists between pH(i) and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na(+)/H(+) exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pH(i) and induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pH(i) decreases, apoptosis-measured by annexin-V and TUNEL methodologies-rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents. (Blood. 2000;95:1427-1434)

Rational basis for use of sodium–hydrogen exchange inhibitors in myocardial ischemia

The cardiac sarcolemmal Na +/H + exchanger extrudes intracellular H + in exchange for Na +, in an electroneutral process. Of the 6 mammalian exchanger isoforms identified to date, the Na +/H + exchanger (NHE)-1 is believed to be the molecular homolog of the sarcolemmal Na +/H + exchanger. The exchanger is activated primarily by a reduction in intracellular pH (intracellular acidosis), although such activation is subject to modulation by a variety of endogenous mediators (e.g., catecholamines, thrombin, endothelin) through receptor-mediated mechanisms. A large body of preclinical evidence now suggests that inhibition of the sarcolemmal Na +/H + exchanger attenuates many of the unfavorable consequences of acute myocardial ischemia and reperfusion. Much of this evidence has been obtained with recently developed potent, selective inhibitors of the exchanger, such as HOE-642 (cariporide) and its structurally related congener HOE-694, in studies using both in vitro and in vivo models of ischemia and reperfusion in a variety of species. The data from these studies indicate that Na +/H + exchange inhibition leads to a decreased susceptibility to severe ventricular arrhythmia, attenuates contractile dysfunction, and limits tissue necrosis (i.e., decreases infarct size) during myocardial ischemia and reperfusion. Such protection is likely to arise, at least in part, from attenuation of “Ca 2+ overload,” which has been linked causally with all of these pathologic phenomena. The consistent and marked cardioprotective benefit that has been observed with cariporide and related compounds in preclinical studies suggests that Na +/H + exchange inhibition may represent a novel and effective approach to the treatment of acute myocardial ischemia in humans.

Effects of intra- and extracellular acidifications on single channel Kir2.3 currents.

1. The inward rectifier K+ channel Kir2.3 is inhibited by hypercapnia, and this inhibition may be mediated by decreases in intra- and extracellular pH. To understand whether Kir2.3 has two distinct pH sensors and whether cytosol-soluble factors are involved in the modulation of this channel during intracellular acidification, single channel currents were studied by expressing Kir2.3 in Xenopus oocytes. 2. In excised inside-out patches, Kir2.3 currents had a high baseline channel open-state probability (Po, at pH 7.4) with a strong inward rectification. Single channel conductance at hyperpolarizing membrane potential was about 17 pS with 150 mM K+ applied to both sides of the membrane. The channel showed a substate conductance of about 8 pS. 3. Reduction of intracellular pH (pHi) produced a fast and reversible inhibition of single channel Kir2.3 currents in inside-out patches. The extent of this inhibition is concentration dependent. A clear reduction in Kir2.3 currents was seen at pHi 7.0, and channel activity was completely suppressed at pHi 6.2 with mid-point inhibition (pK) at pH 6.77. 4. The effect of low pHi on Kir2.3 currents was due to a strong inhibition of Po and a moderate suppression of single channel conductance. The pK values for these single channel properties were pH 6.78 and 6.67, respectively. 5. The decrease in Po with low pHi resulted from an increase in the channel mean closed time without significant changes in the mean open time. Substate conductance was not seen during low pHi. 6. Decrease in extracellular pH (pHo) also caused inhibition of single channel activity of Kir2.3 currents in excised outside-out patches. This effect, however, was clearly different from that of pHi: the pK (pH 6.70) was about 0.1 pH units lower; more than 50 % channel activity was retained at pHo 5.8; and low pHo affected mainly single channel conductance. 7. These results therefore indicate that (1) there are two distinct pH sensors in Kir2.3, (2) different mechanisms are involved in the modulation of Kir2.3 through these two pH sensors, and (3) cytosol-soluble factors do not appear to be engaged in this modulation.

Regression of left ventricular hypertrophy; what are appropriate therapeutic objectives?

Cardiac hypertrophy is a morphological adaptive increase in myocardial mass in response to chronic work overload and is a common clinical finding affecting 23% of men and 33% of women over the age of 59 years [1]. Pressure or volume overload on the myocardium results in an increase in myocardial wall stress and hypertrophy may be seen as an attempt to normalise wall stress and oxygen demand. Although initially protective, the increased myocardial mass requires an increase in coronary blood flow to maintain function; indeed, ventricular hypertrophy may be associated with myocardial ischaemia even with angiographically normal coronary arteries [2–4]. Left ventricular hypertrophy (LVH) significantly increases the risk of myocardial infarction, congestive heart failure and sudden cardiac death [5–7]. It is also associated with a greater prevalence of cardiac arrhythmias [8] and is an important risk factor for cardiac morbidity and mortality [9–10]. Understanding the potential mechanisms for these adverse effects has been the focus of much interest in recent years.

Effect of acidosis on tension and [Ca2+]i in rat cerebral arteries: is there a role for membrane potential?

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+ concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i (fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing PCO2 or reducing HCO3- of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2',7'-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]i is in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]i differs between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.

Bicarbonate therapy and intracellular acidosis.

1. The correction of metabolic acidosis with sodium bicarbonate remains controversial. Experiments in vitro have suggested possible deleterious effects after alkalinization of the extracellular fluid. Disequilibrium of carbon dioxide and bicarbonate across cell membranes after alkali administration, leading to the phenomenon of 'paradoxical' intracellular acidosis, has been held responsible for some of these adverse effects. 2. Changes in intracellular pH in suspensions of leucocytes from healthy volunteers were monitored using a fluorescent intracellular dye. The effect in vitro of increasing extracellular pH with sodium bicarbonate was studied at different sodium bicarbonate concentrations. Lactic acid and propionic acid were added to the extracellular buffer to mimic conditions of metabolic acidosis. 3. The addition of a large bolus of sodium bicarbonate caused intracellular acidification as has been observed previously. The extent of the intracellular acidosis was dependent on several factors, being most evident at higher starting intracellular pH. When sodium bicarbonate was added as a series of small boluses the reduction in intracellular pH was small. Under conditions of initial acidosis this was rapidly followed by intracellular alkalinization. 4. Although intracellular acidification occurs after addition of sodium bicarbonate to a suspension of human leucocytes in vitro, the effect is minimal when the conditions approximate those seen in clinical practice. We suggest that the observed small and transient lowering of intracellular pH is insufficient grounds in itself to abandon the use of sodium bicarbonate in human acidosis.

Valproate-induced limb malformations in mice associated with reduction of intracellular pH

Valproic acid (VPA) is a commonly used antiepileptic agent that recently has been found useful in the treatment of affective disorders and prophylaxis of migraine. VPA induces congenital malformations, especially spina bifida, in the offspring of women treated with this agent during early pregnancy. The mechanism by which VPA induces abnormal development remains unknown despite many studies in experimental animals in which VPA causes malformations similar to those seen in human infants. Because of its chemical structure as a weak organic acid and its capability to induce postaxial forelimb ectrodactyly in C57BL/6 mice, we postulated that VPA acts to perturb limb morphogenesis by reducing embryonic intracellular pH (pH i). We administered VPA, 200 to 400 mg/kg, to C57BL/6 mice on day 9 of gestation. A dose-dependent incidence of postaxial forelimb ectrodactyly was observed. Forelimb bud pH i was estimated by computer-assisted image analysis from the transplacental distribution of 14C-DMO. At the highest doses, 300 and 400 mg/kg, a decrease of pH i of 0.2 to 0.3 pH units was observed uniformly throughout the limb bud 1 h after VPA treatment. None of these changes were seen after treatment with 2-en VPA, a nonteratogenic analog of VPA. Furthermore, the capability of VPA to induce postaxial forelimb ectrodactyly was greatly enhanced by coadministration of agents that inhibit pH i regulatory processes. These data support the hypothesis that VPA-induced postaxial ectrodactyly in murine fetuses can be attributed to reduction in limb bud pH i.

Reduction of intracellular ph by inhibitors of natural killer cell activity, nicardipine, methyl 2-( n-benzyl- n-methylamino)ethyl-2,6-dimethyl-4-(2-isopropyl-pyrazolo[1,5- a] pyridine-3-yl)-1,4-dihydro-pyridine-3,5-dicarboxylate (ahc-52), and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS)

Our previous study showed that nicardipine and its structural analog, methyl 2-( N-benzyl- N-methylamino)ethyl-2,6-dimethyl-4-(2-isopropyl-pyrazolo[l,5- a]pyridine-3-yl)-l,4-dihydro-pyridine-3,5-dicarboxylate (AHC-52), which is devoid of calcium channel blocking activity, were equally effective in inhibiting natural killer (NK) cell activity, perhaps through inhibition of P-glycoprotein. In this study, we confirmed this finding using a human NK-like cell line, YTN, which is highly cytotoxic to JY cells. The YTN cell-mediated cytotoxicity toward JY cells was inhibited by nicardipine and AHC-52 in a concentration-dependent manner, the concentrations required for 50% inhibition being 14 and 7 μM, respectively. We then examined by flow cytometry whether these reagents modulate the intracellular pH (pH i), since P-glycoprotein reportedly plays a role in pH i homeostasis, perhaps by altering chloride translocation. Both reagents reduced pH i at concentrations similar to those required for inhibition of the cytotoxicity. In addition, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), an inhibitor of anion exchangers, also inhibited NK cell activity, with an IC 50 value of 160 μM, and reduced pH i at a similar concentration, although it is not a P-glycoprotein blocker. Thus, the inhibitory activities of nicardipine, AHC-52, and DIDS toward NK cell activity paralleled their lowering activities of pH i, suggesting the possibility that disregulation of pH i is related to inhibition of NK cell activity.

Reduction of intracellular pH is not the mechanism for the synergistic interaction between photodynamic therapy and nigericin.

Previous studies showed that photodynamic therapy (PDT) sensitized by aluminum phthalocyanine can be dramatically potentiated by the K+/H+ ionophore nigericin. Nigericin equilibrates intracellular pH (pHi) and extracellular pH (pHe) and is most effective in potentiating PDT damage when cells are in an acidic environment (pH 6.5-6.7). We therefore hypothesized that the ability of nigericin to lower pHi is causally related to its ability to potentiate PDT. To test this, the pHi of A549 cells was reduced using pHe-adjusted growth medium, with or without addition of amiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibitors of the membrane-based exchangers responsible for regulating pHi. Using fluorescence ratio imaging, we found that pHi can be equilibrated to within +/- 0.05 pH unit, in the pH range of 6.0-6.8, for up to 1 h after pHe adjustment. Cells equilibrated to various pHi were subjected to PDT at various light fluences, then plated for clonogenic survival immediately after PDT treatment. There is no significant effect of lowering pHi, to values as low as 6.23, on the toxicity of PDT, regardless of whether pHi is lowered by adjustment of the medium alone or by addition of exchange inhibitors. However, cells equilibrated to pHi 6.0 are more sensitive to PDT, with survival reduced by 1 log at 20 kJ/m2 and 1.5 log at 30 kJ/m2, relative to cells treated at a pHi of 6.8 (controls). In contrast, 20 microM nigericin in medium at pHe 6.7 reduces pHi to 6.55, but reduces the surviving fraction at 20 kJ/m2 by nearly 3 logs relative to controls. These data conclusively demonstrate that the ability of nigericin to potentiate PDT is not directly related to its ability to lower pHi. Furthermore, they show that the expression of PDT damage is independent of pHi, except at the very low value of 6.0. Photodynamic therapy does not induce apoptosis in A549 cells, at surviving fractions of 0.1 to 0.01, under any of the treatment conditions used in this study.

Recovery from NMDA-induced intracellular acidification is delayed and dependent on extracellular bicarbonate.

A 30-s exposure to N-methyl-D-aspartate (NMDA) produced a dose-dependent and long-lasting (10-20 min) reduction in intracellular pH in cultured cortical neurons, detected by the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This intracellular acidification could be blocked by addition of the NMDA antagonist, D-(-)-2-amino-5-phosphonovalerate, or by removal of extracellular Ca2+. Removal of extracellular HCO3- markedly impaired recovery from NMDA-induced intracellular acidification. Recovery was also impaired when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, inhibitors of HCO3- transport, were added to the cultures immediately after NMDA exposure. In contrast, the Na+/H+ exchange blocker, 5-(N-ethyl-N-isopropyl)amiloride, did not affect pH recovery. Removal of extracellular Cl- partially prevented pH recovery after NMDA stimulation. In addition, extracellular HCO3- increased intracellular Na+ after NMDA exposure, consistent with HCO3- activation of a Na(+)-dependent exchanger. These results demonstrate that stimulation of cortical neuronal NMDA receptors is followed by long-lasting intracellular acidification and that the presence of extracellular HCO3- is important in the subsequent recovery of normal intracellular pH, likely acting at least in part via the Na(+)-dependent Cl-/HCO3- exchanger.

The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte.

The relationship between factors external to the human oocyte and the developmental potential of the female gamete after meiotic maturation in vivo is discussed. The findings indicate very different intrafollicular oxygen contents exist among follicles from the same and different patients, and marginally hypoxic conditions in some follicles are associated with a comparatively low adenosine triphosphate content and a reduction in intracellular pH for the corresponding metaphase II-stage oocyte. For such oocytes, the finding of an increased frequency of cytoplasmic pathology and chromosomal scattering (aneuploidy) suggests a compromised developmental potential. Three distinctly different patterns of cumulus cell attachment and proliferation during the first 24 hours of culture after insemination are described. As reported earlier, different cumulus cell phenotypes observed in vitro are independent of the presence of the oocyte or fertilized egg and unrelated to the degree of expansion of the cumulus at retrieval. However, developmental viability and implantation potential after in vitro fertilization-embryo transfer appear to be related to an intrinsic pattern of cumulus cell behavior in vitro. The results are discussed with respect to the role of factors external to the human oocyte that may influence or determine the ability of the oocyte to develop progressively after fertilization.

Reduction of intracellular pH by tenidap: Involvement of cellular anion transporters in the pH change

Tenidap [5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1 H-indole-1-carboxamide], a novel antirheumatic agent, produces a rapid and sustained intracellular acidification when applied to cells in culture. To investigate the mechanism by which this change in ionic homeostasis is achieved, the acidification activities of structural analogs of tenidap were determined, and the movements of [ 14C]tenidap into and out of cells were explored. The acidification activity of tenidap was enhanced by lowering extracellular pH, suggesting that the free acid species was required for this process. Consistent with this requirement, a non-acidic analog of tenidap did not produce a change in intracellular pH (pHi). In contrast, multihalogenated derivatives of tenidap produced greater changes in pHi than did tenidap, and one analog produced a transient acidification from which the cell recovered; this recovery, however, was blocked by an inhibitor of the Na + H + antiporter. Fibroblasts incubated with [ 14C]tenidap achieved within 5 min a level of cell-associated drug that remained constant during longer incubations. Simultaneous addition of the electrogenic ionophore valinomycin or the P-glycoprotein inhibitor 4-(3,4-dihydro-6,7-dimethoxy-2(1 H)-isoquinolinyl)- N-[2-(3,4-dimethoxyphenyl)ethyl]-6,7-dimethoxy-2-quinazolinamine (CP-100,356) caused a time- and concentration-dependent increase in the level of cell-associated [ 14C]tenidap; other agents tested did not promote this enhanced cellular accumulation. [ 14C]Tenidap accumulated by fibroblasts in the presence of CP-100,356 subsequently was released when these cells were placed in a tenidap- and CP-100,356-free medium. Importantly, several agents that are known to inhibit anion transport processes, including a-cyano-β-(1-phenylindol-3-yl) acrylate, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and meclofenamic acid, inhibited efflux of [ 14C]tenidap. In contrast, ethacrynic acid and 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid did not impair the efflux process. Likewise, tenidap analogs that produced a sustained intracellular acidification blocked the efflux of [ 14C]tenidap, but non-acidifying species did not. These data suggest that movements of tenidap into and/or out of cells is a facilitated process subject to pharmacological intervention. Together, the structural selectivity of the acidification response and the evidence of facilitated transport suggest that the pHi modulating activity of tenidap is dependent on its unique physicochemical properties. Due to the dependence of these physicochemical properties on environmental and cellular conditions, in vivo expression of the acidification activity is likely to occur only within restricted environments that favor this tenidap-induced process.

Uptake of DTP, the Herbicidal Entity of Pyrazolate, by Corn Root Protoplasts

The uptake of DTP [4-(2,4-dichlorobenzoyl)-1, 3-dimethyl-5-hydroxypyrazole], the herbicidal entity of pyrazolate, by corn ( Zea mays L. cv. Pioneer 3377) root protoplasts increased as the external pH (pH°) decreased. The ratio of the DTP concentration inside to outside the protoplasts (c i/c o) reached ca. 0.4, 2.0, 9.0, and 35 to 40 at pH° 7.5, 6.5, 5.5, and 4.5, respectively, within 75 min of incubation. These data quantitatively agreed with the theoretical values expected from the weak acid hypothesis. Five metabolic inhibitors with different types of action inhibited DTP accumulation by the protoplasts and the inhibition was closely correlated with the reduction in intracellular pH regardless of the type of action of the inhibitors. These results suggest that a predominant component of DTP absorption and retention is the diffusion of undissociated DTP molecules, which is indirectly driven by the pH gradient across the plasmalemma. The calculation based on the diffusion equilibrium predicted that if a typical plant cell is exposed to paddy water (pH 5.7) containing 1 ppm DTP, the DTP concentrations accumulated in the cytoplasm and the vacuole would be 75 and 0.6 ppm, respectively. This suggests that the absorbed DTP would be highly accumulated in the cytoplasm thereby effectively performing its herbicidal activity.

Intracellular Calcium and pH Alterations Induced by Tri- n-butyltin Chloride in Isolated Rainbow Trout Hepatocytes: A Flow Cytometric Analysis

The effects of tri- n-butyltin chloride (TBT) on ionic homeostasis on isolated trout hepatocytes were investigated by flow cytometry (FCM), using the Ca 2+-sensitive and pH-sensitive fluorescent probes Indo-1 and SNARF-1, respectively. Cell viability was monitored concurrently. Treatment of hepatocytes with 1 and 5 μM TBT caused a rapid and sustained elevation of cytosolic free Ca 2+ concentration [Ca 2+] i and an important cytoplasmic acidification. These changes were dependent upon TBT concentration and were maintained over 60 min, the maximum exposure period investigated. At 0.5 μM TBT, there was a slight but not significant increase in [Ca 2+] i and a significant reduction in intracellular pH (pH i) only after 60 min of exposure. A rise in [Ca 2+] i and cytoplasmic acidification were observed before loss of viability was detectable. Experiments carried out in Ca 2+-free medium suggest that TBT mainly mobilizes Ca 2+ from intracellular stores in trout hepatocytes. The cytoplasmic acidification following TBT exposure seems to be caused by the combination of intracellular Ca 2+ mobilization and by direct action of TBT. The present results suggest that ionic homeostasis perturbations could be early events in the mechanism of cell injury by TBT.

Reduction of electrical coupling between Necturus taste receptor cells, a possible role in acid taste.

Cytoplasmic acidification in taste receptor cells is thought to be involved, at least in part, in acid taste transduction. Since in taste buds about 20% of the receptor cells are electrically coupled, we have tested whether reduction in intracellular pH affects these lateral synaptic interactions. By applying the patch clamp technique to a slice preparation of Necturus lingual epithelium, we found that electrical coupling between taste receptor cells was strongly reduced by cytoplasmic acidification. Therefore, electrical coupling in taste buds might be modified during acid stimulation.