No abstract

1. The perforated patch method and amperometry were used to determine whether the adrenal medullary cell itself is capable of sensing hypoxia and, if so, how such sensation is transduced to secretion of catecholamines (CA). 2. Exposure to hypoxia, cyanide (CN), or muscarine facilitated CA secretion from dissociated chromaffin cells. The CN-induced secretion was not affected by removal of glucose, indicating that the CN release is due to chemical hypoxia. 3. The secretions induced by CN and muscarine were markedly diminished by removal of Ca¥ ions or by application of Cd¥ or methoxyverapamil (D_600). 4. Cyanide and muscarine produced depolarizations with generation of action potentials and increased intracellular Ca¥ concentrations determined using the acetoxymethyl (AM) ester form of fluo-3 in the presence of external Ca¥ ions, but not in their absence. 5. Hypoxia and CN produced inward currents at an equilibrium potential for Cl¦ ions, irrespective of whether or not Na¤ ions were present in the cells, and substitution of N_methyl-ª_glucamine for 134 mÒ Na¤ ions in the perfusate inhibited the CN current by 71%. The reversal potential for the CN current was −24 mV in the standard perfusate. 6. The hypoxia-, CN-and muscarine-induced currents decreased in parallel with hyperpolarizations, and exposure to CN prevented muscarine, but not nicotine, from inducing a further inward current. 7. We conclude that hypoxia and CN induce CA secretion through depolarization and the subsequent activation of voltage-dependent Ca¥ channels and that this depolarization is due to opening of cation channels, which are possibly identical to muscarinic cation channels.

The mechanism related to mitochondrial dysfunction‐induced catecholamine (CA) secretion in dispersed guinea‐pig adrenal chromaffin cells was investigated using amperometry and confocal laser microscopy. Application of CCCP, which does not stimulate generation of reactive oxygen species (ROS), reversibly induced CA secretion, whereas application of either cyanide or oligomycin (OL), a stimulator for ROS, enhanced CA secretion to a smaller extent. The CCCP‐induced secretion was abolished by removal of external Ca2+ ions and was markedly diminished by D600. The mitochondrial membrane potential, measured using rhodamine 123, was rapidly lost in response to CCCP, but did not change noticeably during a 3 min exposure to OL. Prior exposure to OL markedly facilitated depolarization of the mitochondrial membrane potential in response to cyanide. The mitochondrial inhibitors rapidly produced an increase in Magnesium Green (MgG) fluorescence in the absence of external Ca2+ and Mg2+ ions, an increase that was larger in the cytoplasm than in the nucleus. The rank order of potency in increasing MgG fluorescence among the inhibitors was similar to that in increasing secretion. Thus, mitochondrial inhibition rapidly decreases [ATP] and the mitochondrial dysfunction‐induced secretion is not due to ROS generation or to mitochondrial depolarization, but is possibly mediated by a decrease in ATP.

The role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in catecholamine secretion from dissociated adrenal chromaffin cells of the guinea‐pig was investigated using amperometry, the patch clamp technique and immunochemistry. Pretreatment of adrenal chromaffin cells with 0·3–10 nm PACAP for 2 min resulted in enhancement of nicotine‐ and muscarine‐induced secretions in either the presence of external Ca2+ ions or nominally Ca2+‐free solution, with no change in basal secretion or the holding current at −60 mV in most of the cells tested. Pretreatment with PACAP augmented the muscarine‐induced non‐selective cation current, but did not affect the muscarine‐induced outward current or nicotine‐induced current. PACAP‐induced enhancement of nicotine‐ and muscarine‐induced secretions was suppressed by the simultaneous application of PACAP and the protein kinase inhibitors 100 μm HA1004 or 2 μm H89. Application of forskolin enhanced both muscarine‐ and nicotine‐induced secretions, whereas application of a phorbol ester augmented the nicotine‐induced secretion, but suppressed the muscarine‐induced secretion in a reversible manner. Immunohistochemical analysis of adrenal medullae revealed that PACAP‐like immunoreactivity was present in nerve fibres surrounding putative chromaffin cells. PAC1R‐like immunoreactivity was distributed diffusely in the plasma membrane, whereas nicotinic ACh receptor‐like immunoreactivity was concentrated at the plasma membrane near the nucleus, where the synapses were mainly localized. These observations suggest that PACAP in the guinea‐pig adrenal medulla functions as a neuromodulator to facilitate ACh‐induced secretion through a cAMP‐protein kinase A‐dependent pathway.

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na + ,K + -ATPase α and β subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 µM ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca 2+ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na + ,K + -ATPase α1 subunit-like and β3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas α2-and β2-like IR materials were present in restricted parts of the cell periphery. The α1 and α2 subunits were mainly immunoprecipitated from AM preparations by anti-β3 and anti-β2 antisera, respectively. Peripheral BODIPY-FL-InsP 3 binding sites were localized below membrane domains with α2-and β2-like IR materials. The results indicate that in AM cells, α1β3 isozymes of Na + ,K + -ATPase were present ubiquitously in the plasma membrane, while α2β2 isozymes were in the membrane domain closely associated with peripheral Ca 2+ store sites. This close association of the α2β2 isozyme with peripheral Ca 2+ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 µM ouabain.