The role of Ca 2؉ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca 2؉ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca 2؉ signals that are important for the functional response of a cell. Using the RBL-2H3 mucosal mast cell as a model, we have studied the temporal relationship between changes in intracellular Ca 2؉ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry. Secretion in response to antigen never occurs until intracellular Ca 2؉ is elevated, nor is it seen during the first few oscillations in Ca 2؉ . Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca 2؉ . Ca 2؉ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca 2؉ from stores is prevented with thapsigargin, Ca 2؉ influx can still trigger secretion, suggesting that store-associated microdomains of Ca 2؉ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca 2؉ signal and Ca 2؉ influx for stimulus-secretion coupling in these nonexcitable cells.Although it is generally agreed that an increase in Ca 2ϩ is both necessary and sufficient for the initiation of secretion in most excitable cells, the role of Ca 2ϩ in nonexcitable cells is less clear (1). The individual Ca 2ϩ responses of nonexcitable cells are often extremely heterogeneous, and many models have been proposed for the generation of these complex and often oscillatory patterns (2). Both amplitude-encoded and frequency-encoded Ca 2ϩ signals have been proposed (3, 4), and the availability of both mechanisms would allow multiple signaling pathways to be activated by Ca 2ϩ in a single cell (5). Hepatocytes, with their repetitive oscillations of constant amplitude but variable frequency are prime candidates for frequencymodulated Ca 2ϩ signaling (6), and it has now been shown that mitochondrial NAD(P)H production in these cells is indeed regulated by the frequency of Ca 2ϩ oscillations (7,8). In contrast, it seems clear that the secretory response of single rat salivary acinar cells is tightly coupled to the amplitude of the Ca 2ϩ response (9), as is ciliary beating in tracheal epithelial cells (10). Furthermore, it has recently been shown that differential activation of transcription factors in B lymphocytes is achieved via non-oscillatory Ca 2ϩ signals of different amplitudes and durations (11).In most cases, however, it has not been easy to determine the specific features of the Ca 2ϩ signal that are important for a physiological response. This is because sensitive methods for detecting function at the single-cell level and with high temporal resolution are not readily available. Furthermore, it now seems clear that additional signals, such as the activation of protein kinase C (12) ...
In this research, laser-excited fluorescence was examined for sensitive detection of aqueous dityrosine. Samples were excited with a 6.3-mW, 325-nm helium-cadmium laser focused into a small volume-fluorescence cell with a 10-cm lens. The resulting fluorescence emission was collected perpendicular to the excitation and detected with two different schemes. An optical bandpass filter was used with a photomultiplier tube for sensitive quantitative measurement, while a photodiode array detector was used in conjunction with a spectrograph for qualitative characterization of fluorescence emission spectra. Dityrosine detection on the order of 2 × 10−11 M was obtained with the use of the photomultiplier tube with bandpass optical filter. The dityrosine fluorescence yield is found to vary with the solution pH, the relative concentrations of ferric and ferrous iron, and the amount of dissolved oxygen. A maximum fluorescence yield is observed for iron-free, oxygen-free alkaline solutions. Fluorescence quenching by oxygen is a cumulative photolysis effect that diminished fluorescence yield with increased irradiation time. Flowing the solutions minimized photolysis effects in oxygenated solutions. Quenching by ferrous and ferric iron is found to be due primarily to complex formation. The ferrous iron complex appears to have a fluorescence efficiency of ∼20% of the free dityrosine. The ferric iron dityrosine complex appears to have two ferric ions per molecule at low iron concentration. Other complexes may form at different concentrations. Solvent effects on dityrosine absorption and fluorescence spectra were also investigated. A red shift in dityrosine fluorescence maximum was observed in 1 M trichloroacetic acid and in N, N-dimethylformamide. The fluorescence emission maximum was shifted to the blue in acetonitrile and glacial acetic acid. These shifts were attributed to typical solvochromic behavior.
Slo gene-encoded BK potassium channels are prominent in both adrenomedullary and pituitary tissues. At one alternative splicing site, both tissues express variants with and without the optional 'STREX' exon. In adrenal chromaffin cells, this splicing choice, which has important ramifications for cell excitability, has been shown to be regulated by steroid hormones, including glucocorticoids and adrenal androgens. Moreover, striking sex differences are seen between male and female tree shrews. Here, we test the hypothesis that gonadal testosterone regulates splicing in these tissues. No significant sex differences were found in rats, in either adrenals or pituitaries. In the adrenal medulla, prepubertal castration in male rats increased the relative abundance of STREX transcripts slightly, but not significantly, as measured several weeks after puberty. However, castration substantially decreased STREX representation in the rat pituitary. Silastic implants of testosterone inserted at castration prevented this STREX decline. In postpubescent males, castration or T implants had less effect. Thus, we report (i) steroidal regulation of Slo splicing in the pituitary and (ii) participation of the male gonads in this regulation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.