Calcium handling by the cat carotid body--a pyroantimonate study.

Subcellular regulation mechanisms of calcium concentrations related to oxygen sensing in the carotid body are unclear. In the present study, we investigated the ultrastructural distribution patterns of calcium in carotid body cells and its changes evoked by hypoxia. Carotid bodies were dissected from anesthetized cats exposed in vivo to normoxic or acute hypoxic conditions. We used the oxalate-pyroantimonate technique that yields an electron-opaque calcium precipitate. X-ray microanalysis and appropriate controls confirmed the presence of calcium in the precipitate. Calcium precipitates were found in all types of cells in carotid body parenchyma: chemoreceptor cells, sustentacular cells, and nerve endings. In normoxic chemoreceptor cells, the precipitate was localized in dense core vesicles, mitochondria, and nuclei, but rarely in the cytoplasm. The most apparent effect of hypoxia was disappearance of the precipitate from dense core vesicles and was associated with its appearance in the cytoplasm. The amount of precipitate throughout the carotid body parenchyma was decreased overall due to hypoxia. These results indicate the involvement of subcellular calcium trafficking in hypoxia-sensing in the carotid body. The redistribution pattern of granular calcium deposits from organelles to the cytoplasm of chemoreceptor cells agrees with biochemical data of calcium release from intracellular stores during hypoxia.

Extracellular acidification decreases the basal motility of cultured mouse microglia via the rearrangement of the actin cytoskeleton.

The present study was undertaken to examine the effect of extracellular pH (pH(0)) on the locomotor function of murine microglial cells in vitro. We have found that basal motility of microglia, as measured by a computer-assisted video assay, decreased in an acidic, but not in an alkaline environment. Extracellular acidification affected the architecture of F-actin cytoskeleton, inducing bundling of actin and the formation of stress fibers. The change in intracellular pH (pH(i)) resulting from the change in pH(0) seems to be a prerequisite for the motility decrease since other means to decrease pH(i), namely Na(+)-free solution (in the absence of HCO(-)(3)) and nigericin-containing solution, mimicked the extracellular acidification. In contrast to its pronounced effect on basal motility of microglial cells, the motility increase, as induced by the chemoattractant complement 5a (C5a), was not affected by the acidic environment. The relationship of pH(0) to the locomotor function was also studied in a long-term microchemotaxis assay where microglia migrated within a pH gradient. Intracellular acidification induced by lowering pH(0) to 6.0 or removal of Na(+) from the assay medium decreased basal microglial cell migration. The C5a-induced chemotactic migration was moderately decreased by the acidic environment. In conclusion, our results suggest that acidification of the microglial extracellular milieu leads to a decrease in pH(i) and thereby reduces the basal microglial motility and C5a-induced chemotaxis via a rearrangement of the cytoskeleton. We would therefore like to speculate that changes in pH(i) constitute an important control mechanism in regulating the locomotor function of microglia in culture and probably also in the intact tissue.

Protein kinase C--a potential modifier of carotid body function.

This article deals with the potential role of protein kinase C (PKC) in signal transduction in the carotid body. The carotid body is a chemosensory organ which, by sensing reductions in arterial blood oxygen tension, is primarily responsible for the hyperventilation of hypoxia. The mechanisms of transduction of the hypoxic stimulus into a neural signal regulating respiration are not clear. Hypoxia increases the phosphoinositide-specific phospholipase C (PLC) activity in the carotid body. The PLC-derived signalling molecules are known to activate PKC. The enzyme might, thus, have the potential to interact with the process of chemoreception. This article demonstrates that PKC is present in the chemoreceptor cells of the cat carotid body and discusses the biology of the enzyme relevant to chemosensory function. This gives rise to the hypothesis that PKC-mediated mechanisms alter chemoreceptor cell function to a sufficient extent to metamorphose the hypoxic signal into an increased discharge frequency in the apposed sinus nerve endings.

Two modes of stimulation by ammonia of taurine release from cultured rabbit Müller cells.

A previous study revealed that a 10-min ('acute') treatment of cultured Müller glia with ammonium ions (further referred to as 'ammonia') at 0.5-5 mM concentration stimulated the release of newly loaded taurine (Tau) by a cAMP-dependent, osmoresistant mechanism. Here we showed that a 24 h treatment of the cells with 1 mM ammonia increased both Tau release and intracellular cAMP content in a degree similar to acute treatment with 5 mM ammonia, and the effects were similarly resistant to an increase of medium tonicity by addition of 50 mM sucrose. A 65 min superfusion of the cells with a guanylate cyclase inhibitor [methylene blue (MB)], a protein kinase inhibitor (H7) and a calcium-free buffer containing 10 mM Mg2+ (OCa-10Mg) also increased Tau release and cAMP level in the cells. Acute treatment with 5 mM ammonia of cells pretreated for 24 h with 1 mM ammonia or for 65 min with MB, H7 or OCa-10Mg produced additional significant stimulation of Tau release, without further increasing the cAMP level in the cells. By contrast, a 10-min treatment with 65 mM KCl, which is a potent, cAMP-independent stimulus of Tau release in untreated Müller glia, produced no further enhancement of Tau release in ammonia-, MB-, H7 or OCa-10Mg-pretreated cells. The results indicate that acute treatment with ammonia, on top of treatments that evoke Tau release associated with an increase of cAMP, produces an extra Tau release that is cAMP-independent. Tau released by this extra ammonia treatment possibly originates from a different pool than Tau liberated by the pretreatments or 65 mM KCl.

Intracellular pH regulation in cultured microglial cells from mouse brain.

Intracellular pH (pHi) and the mechanisms of pHi regulation have been investigated in cultured microglial cells from mouse brain using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). Cells were acidified by a pulse of NH4+ (4-5 min; 20 mM) and the subsequent pHi recovery from an acidification was studied. In HCO3(-)-free saline, pH regulation was dependent on extracellular [Na+] and sensitive to amiloride, indicating the involvement of the Na+/H+ exchanger. In HCO3(-)-containing solution 2 mM amiloride slowed but did not block pHi recovery; the recovery however was dependent on extracellular [Na+] and sensitive to 0.3 mM DIDS, suggesting the presence of Na+/HCO3 cotransporter and/or Na(+)-dependent Cl-/HCO3-exchanger. The involvement of a Na-dependent Cl-/HCO3-exchanger was inferred from the observation that removal of Cl- or application of 1 mM furosemide decreased but did not block the recovery rate. Increasing [K+]0 resulted in an alkalinization by a process that was neither HCO3- nor Na(+)-dependent, nor DIDS- and amiloride-inhibitable. In conclusion, microglial cells express a distinct set of pH regulatory carriers which control for a defined level of pHi. An increase in [K+]0 can offset this level.

Ammonia-induced taurine release from cultured rabbit Müller cells is an osmoresistant process mediated by intracellular accumulation of cyclic AMP.

A previous study demonstrated the release of newly loaded radiolabelled taurine (Tau) from cultured rabbit Müller glia not only following typical cell volume-increasing treatments with high (65 mM) potassium ions or hypotonic media, but also with ammonium chloride (further referred to as ammonia), in a dose-dependent manner, at doses ranging from physiological (0.25 mM) to those accompanying hyperammonemic coma (5 mM) (Faff-Michalak et al., Glia 10:114-120, 1994). Stimulation of Tau release by ammonia, but not by 65 mM potassium, was correlated with a dose-dependent increase of intracellular cAMP levels. The release, as measured at 5 mM ammonia, was abolished by compounds that prevented cAMP increase: an adenylate cyclase inhibitor, miconazole, a protein kinase A inhibitor HA 1004, an anion channel blocker, niflumic acid, and a Tau transport site agonist, beta-alanine. The release by ammonia differed from potassium-induced release in its resistance to 1) increase of medium tonicity by addition of 50 mM sucrose; 2) addition of the anion/cation cotransport blocker, furosemide; and 3) removal of calcium from the superfusion medium. The results suggest that ammonia-induced Tau release is mediated by intracellular accumulation of cAMP and may occur either via an osmoresistant, cAMP-controlled channel or a cAMP-activated Tau transporter. The release observed at the physiological concentration of ammonium chloride suggest a role for ammonia as a signal molecule.

Rat cerebral mitochondrial glutaminase activity is unaffected by moderate hyperammonemia in two models.

The phosphate-dependent (PAG) and phosphate-independent (PIndG) glutaminase activities were measured in cerebral perikaryal mitochondria derived from rats subjected to ammonium acetate- induced "simple" hyperammonemia (SHA) or thioacetamide-induced hepatic encephalopathy (HE). These two moderately hyperammonemic conditions were previously found to be accompanied by pronounced changes in virtually all the enzyme activities coupling the tricarboxylic acid cycle to the synthesis and metabolism of the excitatory neurotransmitter glutamate. Both PAG and PIndG remained unaffected by SHA or HE, indicating that they do not contribute to the cerebral glutamine/glutamate imbalance associated with both conditions.