Thyroid parafollicular (PF) cells are neural crest-derived endocrine cells that secrete serotonin and calcitonin. The secretory vesicles of PF cells acidify when secretion is induced by increased extracellular Ca2+ or TSH. We tested the hypothesis that acidification is regulated by secretogogue-gated Cl- channels in vesicular membranes. Cl- channel (p64) immunoreactivity was enriched in purified PF vesicles. X-Ray microanalysis showed a change in chlorine level in PF vesicles in response to secretogogue-stimulation of isolated cells. Secretogogue stimulation also altered the degree of p64 channel phosphorylation. Protein kinase and phosphatase inhibitors antagonized secretogogue-induced vesicle acidification and secretion; however, secretion could occur even when acidification was blocked. We conclude that acidification of PF vesicles is regulated by a gatable Cl- channel in vesicle membranes and that protein phosphorylation and dephosphorylation are involved in channel activation. Acidification of vesicles is not required for exocytosis.
We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization . Particularly useful were methods in which intracellular sites of calcium uptake were detected by incubation of saponin-treated isolated retinas in calciumcontaining media, with oxalate present as a trapping agent . With these procedures, cell compartments accumulate deposits, which can be shown to contain calcium by x-ray microanalysis . Calcium accumulation was prominent in the rough endoplasmic reticulum in the myoid region . In addition, deposits were observed in agranular reticulum and in certain Golgiassociated compartments of the myoid region, in mitochondria, in axonal reticulum, and in agranular reticulum of presynaptic terminals . Calcium was also detected in the endoplasmic reticulum of retinas fixed directly upon isolation, by a freeze-substitution method . The factors influencing accumulation of calcium in the endoplasmic reticulum were evaluated by a semiquantitative approach based on determining the relative frequency of calcium oxalate crystals under varying conditions. Calcium accumulation was markedly enhanced by ATP. Studies with a nonhydrolyzable ATP analogue (adenylyl-imidodiphosphate) and with inhibitors of the sarcoplasmic reticulum Ca t+-Mg2+ ATPase (mersalyl and tetracaine) indicated that this ATP-dependent calcium uptake reflects an energy-dependent process roughly comparable to that in the sarcoplasmic reticulum .Calcium ions play regulatory roles in a variety of cell types . Most extensively studied is skeletal muscle, in which Ca" regulates the contraction-relaxation cycle (21, 69) . In skeletal muscle (30,67,69) and also in cardiac (17,23,25,81,98) and smooth muscle (18, 71), the sarcoplasmic reticulum, which is a specialized form of the smooth endoplasmic reticulum, is largely responsible for the control of concentrations of calcium ions free in the sarcoplasm . By means ofan energydependent Ca"-Mg 2+ ATPase, the sarcoplasmic reticulum can transport calcium ions into its interior (38,85) where the ions are sequestered by binding to calsequestrin and other binding proteins.Much less is known about the regulation of calcium concentration in cells of the nervous system . There is reason to suspect that the situation may resemble that in muscle, at least to some extent. Though few details are understood, there is good evidence that Ca 2+ plays a focal regulatory part in neurons, participating, for example, in regulation of transmitter release (20,40,47,57) . Neuronal mitochondria can THE JOURNAL OF CELL BIOLOGY " VOLUME
BackgroundPhenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, is a chemopreventive agent. It blocks initiation and post-initiation progression of carcinogenesis. Mechanism study in human prostate cancer cells revealed that PEITC is a dual inhibitor of aberrant DNA hypermethylation and histone deacetylases, reactivating silenced genes and regulating the androgen-mediated growth of tumor cells. The identity of the cellular organelle that initially interacts with PEITC has not been fully described.MethodsHuman prostate cancer LNCaP cells were exposed to PEITC and the effects on cellular fine structure examined by transmission electron microscopic studies. Alteration of mitochondrial membrane potential and cytochrome c release were evaluated as early events of apoptosis, and the TUNEL method for quantifying apoptotic cells. Mitochondria were isolated for determining their protein expression.ResultsUltrastructural analyses have revealed condensed mitochondria and a perturbed mitochondrial cristae structure, which assumed a rounded and dilated shape within 4-hours of PEITC contact, and became more pronounced with longer PEITC exposure. They presented as the most prominent intracellular alterations in the early hours. Mitochondria structure alterations were demonstrated, for the first time, with the isothiocyanates. An increase in the number of smooth endoplasmic reticulum and vacuoles were also noted that is consistent with the presence of autophagy. Early events of apoptosis were detected, with cytochrome c released along with the appearance of mitochondrial alteration. Mitochondrial membrane potential was disrupted within 18 hours of PEITC exposure, preceding the appearance of apoptotic cells with DNA strand breaks. In parallel, the expression of the mitochondrial class III ß-tubulin in the outer membrane, which associates with the permeability transition pore, was significantly reduced as examined with isolated mitochondria.ConclusionMitochondria may represent the organelle target of the isothiocyanates, indicating that the isothiocyanates may be mitochondria-interacting agents to inhibit carcinogenesis.
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