benzofurans and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

We have used microspectrofluorimetry with the pH-sensitive dye, BCECF, to examine the control of intracellular pH in the secretory endpieces of the sheep parotid gland. Unstimulated endpieces in HCO3(-)-free media have a cytosolic pH of 7.5 +/- 0.03 (n = 69) which is maintained by a Na(+)-dependent proton extrusion process that can be partially supported by Li+ but not by Cs+, and is not affected by changes in extracellular Cl-, HCO3- or K+. It is not blocked by SITS or DIDS, which inhibit Na(+)-(n)HCO3- co-transport and CL(-)-HCO3- exchange, nor is it sensitive to the amiloride analogs, MIA and EIPA, which inhibit Na(+)-H+ exchangers, although very high concentrations of amiloride itself (1 mmol/l) have a (probably non-specific) inhibitory effect. It seems likely that sheep parotid secretory endpieces do contain a Na(+)-H+ exchanger that drives secretion of a HCO3(-)-rich juice, and that its insensitivity to amiloride and its analogs explains why these drugs do not block fluid secretion by the intact sheep parotid gland.

Topics: Amiloride; Animals; Benzofurans; Biological Transport; Ethers, Cyclic; Fluoresceins; Fluorescent Dyes; Hydrogen; Hydrogen-Ion Concentration; Parotid Gland; Sheep; Sodium

Quantitation of Ca+ and H+ activities within cells using presently available fluorescent probes is optimal when the fluorescence signal is calibrated in situ after each experiment. Fura-2 and 2',7'-bis(2-carboxy-ethyl)-5,6-carboxyfluoroscein (BCECF) are difficult to calibrate in freshly dissociated adult cardiac myocytes because calibration procedures produce cellular hypercontracture. In situ calibration was accomplished in rat ventricular cells by saturating fura-2 with La3+, an agent known to produce myocardial relaxation. Since fura-2 has different spectral properties when complexed with La3+ than with Ca2+, scaling factors were defined in vitro and then verified by experiments in cultured neonatal myocytes. In adult rat myocytes using the La3+ method, intracellular Ca2+ concentration ([Ca2+]i) was 131 +/- 47 nM (n = 14) in quiescent cells; diastolic [Ca2+]i and systolic [Ca2+]i in myocytes stimulated at 1 Hz were 140 +/- 56 and 1,088 +/- 211 nM (n = 5), respectively. BCECF fluorescence was calibrated in situ by a method that prevented cellular hypercontracture and reported a pH value of 7.10 +/- 0.10 in cells stimulated at 1.5 Hz. An additional advantage of both methods is that the buffers employed prevented large changes in the redox state of intracellular pyridine nucleotides, thus preventing a change in cellular autofluorescence during the calibration procedure.

Topics: Aging; Animals; Animals, Newborn; Benzofurans; Calcium; Calibration; Cells, Cultured; Fluoresceins; Fluorescence; Fluorescent Dyes; Fura-2; Heart Ventricles; In Vitro Techniques; Lanthanum; Myocardium

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.

Topics: Animals; Benzofurans; Calcium; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Hydrolysis; Microscopy, Fluorescence; Mitochondria, Heart; Photochemistry; Protons; Rats; Rats, Inbred Strains

Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic H+ concentration [( H+]i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular H+ concentration in platelets, unstimulated ex vivo, from normotensive (n = 20) and hypertensive patients (n = 20). Cytosolic [H+] was 20% lower in hypertensive than in normotensive subjects (49.5 +/- 3.4 and 61.8 +/- 2.2 nmol/l cells, respectively, P less than 0.005; mean pHi values were 7.21 and 7.33, respectively). Platelet cytosolic H+ and free Ca2+ concentrations ([Ca2+]i) were determined in parallel in 15 normotensive and 15 hypertensive patients. [Ca2+]i was found to be 19% higher (P less than 0.01), and [H+]i 22% lower (P less than 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHi and [Ca2+]i were increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.

Topics: Adult; Benzofurans; Blood Platelets; Calcium; Cytosol; Female; Fluoresceins; Fura-2; Humans; Hydrogen; Hydrogen-Ion Concentration; Hypertension; Male; Middle Aged; Protons

The influx of Ca2+ and its subsequent intracellular increase are required for the acrosome reaction of sea urchin sperm to occur. Spermatozoa must undergo this reaction, which is triggered by the egg jelly, in order to fertilize the egg. Here, the egg jelly-induced Ca2+ influx mechanisms have been studied in sperm loaded with FURA-2 using Mn2+ under the assumption that this divalent ion is an indicator of Ca2+ influx through Ca2+ channels. Egg jelly induced the immediate entry of Ca2+ (mixing time 1 s), however; we found that the influx of Mn2+ increased after a lag time of 5 s. Nisol-dipine (a Ca2+ channel blocker) did not block the Mn2+ influx which was inhibited by 40 mM of external [K+], low Na+, and 5 mM of tetraethylammonium (a K+ channel blocker). These conditions also inhibited the alkalinization and the acrosome reaction. The inhibition of the Mn2+ influx could be overcome by increasing internal pH (pHi) with ammonium (10 mM). On the contrary the influx of Ca2+ during the first 5 s was not inhibited by any of the conditions indicated before, except by nisoldipine. These data could be explained by the activation of two different Ca2+ channels by egg jelly. The first one being a receptor-operator Ca2+ channel that opens when the receptor for egg jelly is occupied independently of the ionic conditions. The other one could be considered as a second messenger-operated Ca2+ channel that requires at least an increase in pHi to open.

Topics: Acrosome; Animals; Benzofurans; Calcium Channels; Female; Fluoresceins; Fluorescent Dyes; Fura-2; Kinetics; Male; Manganese; Models, Biological; Ovum; Potassium; Sea Urchins; Sperm-Ovum Interactions; Spermatozoa; Tetraethylammonium; Tetraethylammonium Compounds

Living outer hair cells were irradiated under conditions of fluorescent microscopy (epi-illumination through the objective) with UVR (waveband 340-380 nm), blue light (waveband 450-490 nm) or green light (waveband 515-560 nm) in the intracellular presence or absence of the fluorescent dyes fura-2 or 2',7'-bis-(carboxyethyl) 5-(and 6-)carboxyfluorescein (BCECF). In response to UVR with or without intracellular fura-2 and to blue light in the presence of BCECF (irradiation intensities of 2-12 x 10(5) W/m2), the cells shortened and swelled within 15-30 s, accompanied by the formation of numerous cytoplasmic granulations. The cellular reactions were significantly delayed to 3 min by the addition of 1 mM of the radical scavengers p-phenylenediamine or n-propyl gallate. This protection suggests that free radicals, produced under UVR or under blue light irradiation in the presence of the sensitizer BCECF, are possible causative agents of this cell damage. The response of the photo-damaged cells, namely shortening and increase in volume, resembled the characteristics of hair cells exposed to an hypo-osmotic shock. This suggests that structural alterations of the cytoplasmic membrane and the sub-membrane cortex occurred under photo-irradiation, and that these structures can be implicated in the maintenance of the elongated cylindrical shape of the outer hair cells, possibly by maintaining intracellular hyperosmolarity.

Topics: Animals; Benzofurans; Fluoresceins; Fluorescence; Free Radicals; Fura-2; Guinea Pigs; Hair Cells, Auditory; In Vitro Techniques; Light; Phenylenediamines; Propyl Gallate; Ultraviolet Rays

1. Intracellular calcium concentration [( Ca2+]i) and pH (pHi) were measured in single, isolated rat ventricular myocytes using, respectively, the fluorescent indicators Fura-2 and BCECF (2',7'-bis(carboxyethyl-5(6)-carboxyfluorescein). Contraction was measured simultaneously. The intracellular calibration of BCECF is demonstrated. In a HEPES-buffered bathing solution of pH 7.4, pHi had a mean value of 7.16 +/- 0.05 (mean +/- S.E.M.). 2. Addition of NH4Cl (5-20 mM) produced an intracellular alkalosis that was associated with an increase of contraction amplitude. Removal of NH4Cl produced an acidosis and decrease of contraction. 3. The addition of 2 mM-cyanide (CN-) to inhibit oxidative phosphorylation had variable effects on contraction amplitude. Changes of contraction amplitude could largely be accounted for by changes in the systolic Ca2+ transient. 4. CN- addition increased lactic acid production. However, in the majority of experiments, this was not accompanied by an intracellular acidosis. 5. Anaerobic glycolysis was inhibited by either removal of glucose, addition of deoxyglucose, or addition of iodoacetate. Under these conditions the application of CN- decreased systolic [Ca2+]i and contraction amplitude. This was sometimes preceded by a transient increase of systolic [Ca2+]i and contraction amplitude. 6. When glycolysis was inhibited, the subsequent addition of CN- always increased diastolic [Ca2+]i and produced a contracture. The increase of [Ca2+]i occurred before the contracture. However, once the contracture had developed, decreasing [Ca2+]i (by removal of external Ca2+) did not cause relaxation. 7. With glycolysis inhibited, addition of CN- resulted in a large (0.51 +/- 0.05 pH unit) acidosis that was sometimes preceded by an alkalosis. This acidosis was unaffected by removal of external Ca2+ or external alkalinization. Calculations show that some of this acidosis may result from protons released by ATP hydrolysis. 8. If the acidosis produced by metabolic blockade was partly reversed by adding NH4Cl then a contracture immediately developed. This suggests that the acidosis delays the onset of the contracture. 9. We conclude that metabolic inhibition increases diastolic [Ca2+]i. The accompanying acidosis prevents contraction. Once the contracture has developed it is maintained by factors other than increased [Ca2+]i, possibly by a fall of [ATP].

Topics: Action Potentials; Ammonium Chloride; Animals; Benzofurans; Calcium; Cyanides; Fluoresceins; Fura-2; Glycolysis; Heart; Hydrogen-Ion Concentration; In Vitro Techniques; Iodoacetates; Iodoacetic Acid; Muscle Contraction; Myocardium; Rats; Time Factors

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.

Topics: Animals; Benzofurans; Calcium; Fluoresceins; Fura-2; In Vitro Techniques; Indicators and Reagents; Microscopy, Fluorescence; Myocardium; Rats; Video Recording

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.

Topics: Amiloride; Benzofurans; Blood Platelets; Calcium; Carrier Proteins; Fluoresceins; Fluorescent Dyes; Fura-2; Humans; Hydrogen-Ion Concentration; Nigericin; Sodium-Hydrogen Exchangers; Spectrometry, Fluorescence; Thrombin

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.

Topics: Animals; Benzofurans; Calcium; Digitonin; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mitochondria, Heart; Rats; Spectrometry, Fluorescence