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

We studied the spontaneously active in vitro tadpole brainstem and recorded whole nerve respiratory activity while simultaneously visualizing intracellular pH (pHi) dynamics using the pH-sensitive dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, AM). The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. We generated a calibration curve to correlate the emitted fluorescence of BCECF to pHi. In addition, we demonstrated that the dye loading protocol that we established labeled an adequate number of cells and did not disrupt spontaneous respiratory rhythmogenesis or the respiratory response to central chemoreceptor stimulation. Validation of the use of the pH sensitive dye BCECF in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors and allow correlation between the changes in pHi in central chemoreceptors and respiratory motor output recorded from cranial nerves.

Topics: Animals; Brain Stem; Chemoreceptor Cells; Cranial Nerves; Fluoresceins; Fluorescent Dyes; Gills; Hydrogen-Ion Concentration; Hypercapnia; In Vitro Techniques; Larva; Models, Animal; Rana catesbeiana; Respiratory Mechanics

The chemosensitive response of locus coeruleus (LC) neurones to changes in intracellular pH (pH(i)), extracellular pH (pH(o)) and molecular CO(2) were investigated using neonatal rat brainstem slices. A new technique was developed that involves the use of perforated patch recordings in combination with fluorescence imaging microscopy to simultaneously measure pH(i) and membrane potential (V(m)). Hypercapnic acidosis (15 % CO(2), pH(o) 6.8) resulted in a maintained fall in pH(i) of 0.31 pH units and a 93 % increase in the firing rate of LC neurones. On the other hand, isohydric hypercapnia (15 % CO(2), 77 mM HCO(3)(-), pH(o) 7.45) resulted in a smaller and transient fall in pH(i) of about 0.17 pH units and an increase in firing rate of 76 %. Acidified Hepes (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid)-buffered medium (pH(o) 6.8) resulted in a progressive fall in pH(i) of over 0.43 pH units and an increase in firing rate of 126 %. Isosmotic addition of 50 mM propionate to the standard HCO(3)(-)-buffered medium (5 % CO(2), 26 mM HCO(3)(-), pH(o) 7.45) resulted in a transient fall in pH(i) of 0.18 pH units but little increase in firing rate. Isocapnic acidosis (5 % CO(2), 7 mM HCO(3)(-), pH(o) 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH(i) preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH(o). In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH(i), indicating that a decrease in pH(i) is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.

Topics: Acidosis; Animals; Animals, Newborn; Carbon Dioxide; Electrophysiology; Extracellular Space; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; Kinetics; Locus Coeruleus; Membrane Potentials; Microscopy, Fluorescence; Neurons; Patch-Clamp Techniques; Pons; Propionates; Rats; Rats, Sprague-Dawley; Tetrodotoxin

The hydrogen ion is an important factor in the alteration of vascular tone in pulmonary circulation. Endothelial cells modulate vascular tone by producing vasoactive substances such as prostacyclin (PGI2) through a process depending on intracellular Ca2+ concentration ([Ca2+]i). We studied the influence of CO2-related pH changes on [Ca2+]i and PGI2 production in human pulmonary artery endothelial cells (HPAECs). Hypercapnic acidosis appreciably increased [Ca2+]i from 112 +/- 24 to 157 +/- 38 nmol/l. Intracellular acidification at a normal extracellular pH increased [Ca2+]i comparable to that observed during hypercapnic acidosis. The hypercapnia-induced increase in [Ca2+]i was unchanged by the removal of Ca2+ from the extracellular medium or by the depletion of thapsigargin-sensitive intracellular Ca2+ stores. Hypercapnic acidosis may thus release Ca2+ from pH-sensitive but thapsigargin-insensitive intracellular Ca2+ stores. Hypocapnic alkalosis caused a fivefold increase in [Ca2+]i compared with hypercapnic acidosis. Intracellular alkalinization at a normal extracellular pH did not affect [Ca2+]i. The hypocapnia-evoked increase in [Ca2+]i was decreased from 242 +/- 56 to 50 +/- 32 nmol/l by the removal of extracellular Ca2+. The main mechanism affecting the hypocapnia-dependent [Ca2+]i increase was thought to be the augmented influx of extracellular Ca2+ mediated by extracellular alkalosis. Hypercapnic acidosis caused little change in PGI2 production, but hypocapnic alkalosis increased it markedly. In conclusion, both hypercapnic acidosis and hypocapnic alkalosis increase [Ca2+]i in HPAECs, but the mechanisms and pathophysiological significance of these increases may differ qualitatively.

Topics: Acidosis, Respiratory; Alkalosis, Respiratory; Calcium; Endoplasmic Reticulum; Endothelium, Vascular; Epoprostenol; Extracellular Space; Fluoresceins; Fluorescent Dyes; Fura-2; Humans; Hydrogen-Ion Concentration; Hypercapnia; Hypocapnia; Pulmonary Artery; Triglycerides

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.

Topics: Acidosis; Animals; Bicarbonates; Calcium; Carbon Dioxide; Cerebral Arteries; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; In Vitro Techniques; Male; Membrane Potentials; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Wistar

Hypercapnia and hypoxia both relax airway smooth muscle, but the mechanisms responsible are poorly understood. Because hypercapnia and hypoxia can each decrease intracellular pH (pHi) and acidosis can inhibit Ca2+ channels, we hypothesized that decreased pHi mediates relaxation of trachealis muscle by each of these respiratory gases. To examine the relationship between pHi and tone, we measured isometric tension, bath pH, and fluorescence intensity (540 nm) in porcine tracheal smooth muscle strips loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and excited alternately with 440- and 500-nm light. Strips equilibrated in Krebs-Henseleit solution bubbled with 95% O2-5% CO2 were contracted with carbachol and then relaxed with either 95% N2-5% CO2 or 93% O2-7% CO2. The ratio of fluorescence intensity at 500 nm to 440 nm was calibrated vs. pHi with use of nigericin. Baseline pHi was 7.19 +/- 0.03 (n = 13). Hypoxia decreased active tension by approximately 60% but did not change pHi. Hypercapnia induced decreases in tension that were associated with substantial decreases in pHi. Thus, decreased pHi does not mediate hypoxic relaxation, but the relaxation during physiologically relevant increases in CO2 concentration is associated with significant cellular acidification.

Topics: Animals; Female; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; Hypoxia; Intracellular Membranes; Male; Muscle Relaxation; Muscle, Smooth; Swine; Trachea