Naoji Fujishiro

Hypoxia and cyanide induce depolarization and catecholamine release in dispersed guinea‐pig chromaffin cells

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 Ca2+ ions or by application of Cd2+ or methoxyverapamil (D‐600). 4 Cyanide and muscarine produced depolarizations with generation of action potentials and increased intracellular Ca2+ concentrations determined using the acetoxymethyl (AM) ester form of fluo‐3 in the presence of external Ca2+ 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‐D‐glucamine for 134 mM 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 Ca2+ channels and that this depolarization is due to opening of cation channels, which are possibly identical to muscarinic cation channels.

Homogeneous Ca2+ stores in rat adrenal chromaffin cells

The localization and function of Ca(2+) stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP(3)), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP(3), and the majority of BODIPY-Ry-binding area was bound by fluorescent IP(3). Under Ca(2+)-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca(2+) increases, as observed with fluo-3, lasted for a few tens of seconds under Ca(2+)-free conditions, whereas a caffeine-induced Ca(2+) transient diminished rapidly with a half decay time of 3s and this spike-like Ca(2+) transient was then followed by a sustained increase with a low level. These results indicate that IP(3) receptors and Ry receptors (RyRs) are present in common ER Ca(2+) storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.

Pituitary adenylate cyclase-activating polypeptide may function as a neuromodulator in guinea-pig adrenal medulla

1 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. 2 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. 3 Pretreatment with PACAP augmented the muscarine‐induced non‐selective cation current, but did not affect the muscarine‐induced outward current or nicotine‐induced current. 4 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. 5 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. 6 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. 7 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.

Role of ATP decrease in secretion induced by mitochondrial dysfunction in guinea‐pig adrenal chromaffin cells

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.

Na^+ pump inhibition and non-selective cation channel activation by cyanide and anoxia in guinea-pig chromaffin cells

1 Hypoxia and metabolic inhibition with cyanide (CN) evoke catecholamine secretion in adrenal chromaffin cells through depolarization. We elucidated mechanisms for a CN‐ or anoxia‐induced inward (depolarization) current, using the perforated patch method. 2 Bath application of Ba2+ induced a dose‐dependent inhibition of a muscarine‐induced current (IMUS) and part of the CN‐induced current (ICN) with an IC50 (concentration responsible for 50 % inhibition) of 1.3 mM. The Ba2+‐sensitive component was estimated to comprise 58 % of the total ICN. 3 The Ba2+‐resistant component of ICN tended to increase with shifts of membrane potential from ‐40 to 40 mV and was markedly suppressed by exposure to a K+‐free solution or 200 μm ouabain, indicating that the majority of the Ba2+‐resistant component of ICN is due to suppression of the Na+ pump current (Ipump). 4 The non‐Ipump component of ICN diminished progressively in K+‐free solution. Substitution of glucose for sucrose in a K+‐free CN solution further diminished the CN potency to produce the non‐Ipump component. 5 The I‐V relationship for the non‐Ipump component of ICN had a reversal potential of ‐3 and ‐47 mV at 147 and 5.5 mM Na+, respectively, and showed an outward rectification, indicating that the non‐Ipump component of ICN is due to activation of non‐selective cation channels. 6 Exposure to anoxia induced a current with an amplitude comparable to that of ICN, and the anoxia‐induced current apparently occluded development of ICN. The anoxia‐induced current diminished by ca 60 % in the absence of K+ and reversed polarity at 5 mV under K+‐free conditions. 5 It is concluded that exposure to CN and to anoxia induces suppression of the Na+ pump and activation of non‐selective cation channels, probably due to an ATP decrease resulting mainly from consumption by the Na+ pump.

Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells

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 Ca2+ 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‐InsP3 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 Ca2+ store sites. This close association of the α2β2 isozyme with peripheral Ca2+ store sites may account for the facilitation of mobilization‐dependent secretion in the presence of 1 μm ouabain.

Role and Source of ATP for Activation of Nonselective Cation Channels by AlF Complex in Guinea Pig Chromaffin Cells

Abstract. Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (INS) at −55 mV in chromaffin cells. Amplitudes of INS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of INS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of INS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC50 of 0.265 mm. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of INS by the AlF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating INS. Bath application of the kinase inhibitor, H-7 (100 μm), suppressed AlF-induced INS in a manner depending on intracellular Mg2+. We conclude that ATP is a prerequisite for generation of INS as a phosphoryl donor and that mitochondria is the main source of ATP.

Tetanus responses under rapid bath solution change: Electrotonic depolarization of transverse tubules may release Ca2+ from sarcoplasmic reticulum of Rana japonica skeletal muscle

1. Single skeletal muscle fibers were transferred from a normal Ringer solution to Na+ ion free solution, and vice versa, and tetanus responses were recorded immediately after the transfer. 2. Fractional tetanus tension recorded immediately after the displacement from the Na+ ion free solution to normal Ringer solution was dependent on fiber diameter. 3. Diffusion of Na+ ions along the transverse tubules was simulated [apparent diffusion constant was 3.11 x 10(-6) (cm2/s)]. 4. Our results suggest that the electronic spreading of membrane potential, caused by an action potential in the transverse tubules, could release Ca2+ ions from sarcoplasmic reticulum.

A program for calculation of the proton activity coefficient.

A program for calculation of the proton activity coefficient at any given ionic strength and temperature was written in Quick Basic. The calculation of proton activity coefficient was confirmed by titration of HEPES buffer.

Wavelength dependence of the optically recorded action potentials in guinea pig atrial muscles

The optically recorded action potentials of the frog atrial muscles which lack transverse tubules showed different features from those reported by Heiny and Vergara (Heiny JA, Vergara J. Optical signals from surface and T system membranes in skeletal muscle fibers. J Gen Physiol 1982;80:203-230) in skeletal muscles (Fujishiro N, Kawata H. The wavelength dependence of optically recorded action potentials in the atrial muscles of the bullfrog (Rana catesbeiana). Comp Biochem Physiol 1996;114A:153-157). We examined whether or not the differences were consistent in other atrial muscles which lack transverse tubules with guinea pig atrial muscles. Two dyes (merocyanine rhodanine and merocyanine oxazolone) were used, and the dependence of the maximum rising phase of the optical signals on the wavelength of the incident beam was analyzed. No dependence was observed between them, and this finding was consistent with the structure of the membrane system of the guinea pig atrial muscles. The optical signals recorded at 718 nm of the incident beam from the guinea pig atrial muscles which stained with merocyanine oxazolone showed a more prominent second rising phase after the initial rapid rising phase of the optical signal than that recorded in the frog atrial muscles. This phase was not observed in the optical signals recorded at other wavelengths. The features of the optically recorded action potentials in guinea pig atrial muscles were consistent with those recorded in frog atrial muscles. Nifedipine did not affect the second rising phase.