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)     

Desensitization to Nicotinic Cholinergic Agonists and K+, Agents That Stimulate Catecholamine Secretion, in Isolated Adrenal Chromaffin Cells

Desensitization of catecholamine (CA) release from cultured bovine adrenal chromaffin cells was studied to characterize the phenomenon of desensitization and to attempt an elucidation of the mechanism(s) involved in this phenomenon at the level of the isolated chromaffin cell. Prior exposure of chromaffin cells to nicotinic cholinergic agonists [acetylcholine (ACh) or nicotine] caused a subsequent depression or desensitization of CA release during restimulation of the cells with the same agonists. Rates of development of and recovery from nicotinic desensitization were in the minute time range and the magnitude of nicotinic desensitization of CA release was greater at 37 degrees C than at 23 degrees C. ACh- (or nicotine)-induced desensitization was shown to be the result of two processes: (1) a Ca2+-dependent component of desensitization, possibly due to a depletion of intracellular CA stores and (2) a Ca2+-independent, depletion-independent component of desensitization. Prior exposure of cultured chromaffin cells to an elevated concentration of K+ also resulted in desensitization of K+-induced CA release in these cells. K+-induced desensitization was completely Ca2+-dependent and was shown to be the result, at least in part, of a mechanism that is independent of depletion of CA stores.     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

The Quaternary eolian sequence of Madeira: stratigraphy, chronology, and paleoenvironmental interpretation

A thick (ca. 40 m) sequence of coastal eolian sediments occurs on a narrow peninsula on the eastern end of the island of Madeira, located in the Eastern Atlantic at 33°N latitude. The sediments consist of black volcanic sands (with or without bioclasts) as well as clay units up to 2 m thick. A series of inceptisols (Eutrochrepts) and one alfisol (a Hapludalf) are developed in these sediments. Land snail shells and secondary carbonates, in the form of well-developed rhizoliths, calcretes, fissure-fills, and soil nodules, are present in abundance. The chronology of the sequence was determined by ¹⁴C and U---Th analyses of land snail shells and secondary carbonates and amino acid epimerization analysis of land snail shells. All sediments, including the clay units, are originally of eolian origin, derived from the beach to the south of the deposit, but some have been redeposited by colluviation. Temporal variation in the lithology of the sediments relates to variations in sea-level, with black sands being deposited during lower sea level stands and clays at the lowest. It is suggested that fine marine sediments, exposed during low sea-level stands, may also be the dominant source of silty or clayey units in other coastal eolian deposits in the subtropical Atlantic and Mediterranean.

The sequence spans from 200,000–300,000 years ago up to the 20th century. Sedimentation was discontinuous and often rapid; erosional hiatuses are present. During the Holocene, eolian sands started accumulating at 8200 yr B.P. during a transgressive phase and stopped at 4500 yr B.P. as sea level approached its present height. Colluviation increased dramatically following the first human settlement of the island in the 15th century and continued up to the 20th century, as dated by amino acid epimerization analysis of land snails. Earlier periods of colluviation were identified from the age distribution of land snail shells redeposited in younger colluvium.

Paleoenvironmental reconstruction was based mainly on soil and sediment features (including rhizolith morphology) and land snail faunas but also on stable isotope variations (¹³C, ¹⁸O) in land snails and secondary carbonates, pollen (generally not well preserved), and phytoliths. Most of the portion of the Middle Pleistocene represented in the sequence was characterized by moderately dry conditions, in comparison to the late Pleistocene and Holocene. During the last interglacial, relatively wet conditions occurred, wetter than during the Holocene interglacial. Moderately moist conditions were present during the accumulation of the thick unit dating to ca. 80,000 yr B.P. As sea level fell subsequent to this period, conditions appear to have become drier. Starting ca. 50,000–55,000 yr B.P., conditions were especially wet, but prior to the last glacial maximum, markedly arid conditions ensued. Toward the end of the last glacial, wet conditions returned and produced the best-developed soil preserved in the sequence. Moderately moist conditions occurred during the early to middle Holocene but apparently become slightly drier after 4500 yr B.P. The impact of human settlement can be seen in the loss of woody vegetation and enhanced gullying and colluviation during the last ca. 500 years.     

On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

Functional and morphological characterization of isolated bovine adrenal medullary cells

Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% collagenase in Ca++-free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 X 10(6) cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min. Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenaline-containing and (b) noradrenaline-containing cells. Biochemical analysis showed that the cells contained catecholamines and dopamine-beta-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca++ have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

Production and Release of Acetylcholinesterase by a Primary Cell Culture of Bovine Adrenal Medullary Chromaffin Cells

Abstract: Bovine adrenal chromaffin cells were isolated and maintained as primary culture monolayers. Total acetylcholinesterase (AChE) activity in the cells increased during the culture period, and AChE activity appeared in the culture medium. We have examined the role of the AChE synthesized by the cells on ACh-evoked release of catecholamine from the cells. A progressive decrease in the efficacy of ACh (5 × 10-⁵m ) to evoke release of [³H] norepinephrine from day 3–15 cultures suggests that exogenously applied ACh is hydrolyzed by the nascent AChE synthesized by the cells. These findings provide evidence that chromaffin cells produce AChE and release it into their immediate environment.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.