Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoarchitecture of the fetal murine soleus muscle

The organogenesis of the soleus muscle of the 129 ReJ mouse (a mixed muscle, which in the adult contains approximately equal numbers of slow-twitch oxidative and fast-twitch oxidative-glycolytic myofibers) was studied in spaced, serial transverse, and longitudinal sections of muscles of 14-, 16-, and 18-day in utero and 1- and 5-day postnatal mice. A discrete soleus muscle was distinguished by 14 days in utero. It consisted of groups of closely apposed primary myotubes displaying junctional complexes and a pleomorphic population of mononucleated cells. Between 14 and 16 days in utero there was little de novo myotube formation. At 16 days in utero, basal lamina surrounded groups of primary myotubes; and primitive motor endplates were found on these myotubes. At 18 days in utero, the basal-lamina-enclosed groups of primary myotubes were no longer present. At this stage, basal lamina surrounded clusters (consisting of one primary myotube and one or more secondary myotubes) or independent myotubes (single myotubes surrounded by their own basal lamina). Cluster formation and cluster dispersal occurred concurrently, beginning at 18 days in utero and extending until birth. At birth, there was still a substantial population of immature, secondary myotubes that interdigitated with larger, more mature primary myofibers. At this stage, intermuscular axons had begun to myelinate, and postsynaptic specialization of the motor endplates had begun. Cluster dispersal and myonuclear migration was completed during the first 5 days postnatally with the muscle taking on adult characteristics. Beginning at 16 days in utero and extending into the neonatal period, there was evidence of myotube death in the soleus muscle.     

Swelling and ion uptake in cat cerebrocortical slices:

Cat cerebrocortical slices incubating in medium containing normal K+ concentrations were exposed to a number of different transmitters. Norepinephrine, histamine and adenosine or 2-chloroadenosine caused increased swelling of the slices associated with an increased Na+ and Cl- content. These effects were seen only when both Cl- and HCO3- were present in the medium, and were inhibited by a number of anion transport inhibitors. These characteristics were identical to those of the HCO3(-)-dependent component of the swelling induced by high K+ levels in the medium. Other transmitters, namely 5-hydroxytryptamine, dopamine, and gamma-amino butyric acid, were ineffective. The effects of norepinephrine, histamine and 2-chloroadenosine were antagonised by propranolol and phentolamine, chlorpheniramine and diphenhydramine, and theophylline respectively. These antagonists also inhibited HCO3(-)-dependent, K+-stimulated swelling. The transmitters which induced swelling also stimulated the carbonic anhydrase activity of cerebrocortical slices. We conclude from these data that the HCO3(-)-dependent component of K+-stimulated swelling may be due to K+-stimulated release of transmitters. Furthermore, the fact that the transmitters which induce swelling have also been reported to be most effective in increasing cAMP content in both brain slices or cultured astrocytes is consistent with the swelling response being mediated via cAMP-induced changes and being predominantly localized to astrocytes.     

THE INTERACTION OF AUXIN AND ETHYLENE IN THE MAINTENANCE OF LEAF BLADE FORM IN PHASEOLUS VULGARIS L. VAR. PINTO

Auxin treatment results in hyponastic curvature of the primary leaves of Phaseolus vulgaris L. var pinto. Ethylene production by hyponastic leaves is detected within 1 hr after treatment with IAA in concentrations at or above 1 μm. The amount of ethylene detected is proportional to the concentration of auxin applied. Untreated control leaves and leaves treated with 2,3,5-tri-iodobenzoic acid or gibberellic acid did not produce ethylene detectable by our equipment. The hyponastic curvature induced by auxin treatment can be inhibited by exogenous application of ethylene or ethylene-generating compounds, and these treatments produce epinasty in auxin-treated leaves. Treatment with inhibitors of ethylene synthesis or action, such as aminoethoxy-vinylglycine, carbon dioxide, or heat treatment, prolong hyponasty. The planar form, therefore, appears to be affected by both hyponastic auxin effect and an epinastic ethylene effect.     

Properties and localization of bicarbonate-stimulated ATPase activity in rat brain

Abstract— A 20–30 per cent stimulation of ATPase activity by added NaHCO₃ was found in homogenates of a variety of mammalian tissues. The subcellular distribution of this (HCO₃^-)—stimulated activity was examined in detail using rat cerebral cortex. The stimulation was specific for the HCO₃^- ion and was predominantly localized in the mitochondrial subcellular fraction, in which a 2-fold stimulation by HCO₃^- was found. The effect of inhibitors supported the identification as the mitochondrial ATPase. Both sodium azide and thiocyanate were inhibitory. The effects of varying the Mg²⁺ concentration, HCO₃^- concentration, and pH were also studied. In the presence of HCO₃^- the K_m for ATP was reduced approximately 3-fold. There was no effect of HCO₃^- on the ma + K) ATPase or Mg²⁺ ATPase from the microsomal fraction of rat cerebral cortex. Our findings have been discussed in relation to previous work on HCO₃^- stimulation of ATPae activity in subcellular fractions from other tissues, as well as its possible relevance to the known effects of HCO₃^- and carbonic anhydrase on active ion transport.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.