tretinoin and fura-2-am

When treated with low doses of retinoic acid (RA), cephalic chondrocytes of the chick embryonic sternum mature and express phenotypic characteristics of postmitotic hypertrophic cells. In concert with these maturation-dependent changes, cells release adenine nucleotides into the culture medium. To ascertain if these compounds modulate chondrocyte function, we challenged chondrocytes with nucleotides and measured one determinant of the signal transduction pathway, intracellular Ca2+ concentration ([Ca2+]i). In the presence of micromolar concentrations of ATP, there was a dose-dependent elevation in chondrocyte [Ca2+]i; ADP caused a small but significant rise in the peak [Ca2+]i response. We found that the change in the [Ca2+]i response is linked to retinoid-dependent maturation of chondrocytes. Thus the [Ca2+]i rise was dependent on the RA concentration and treatment time. Immature caudal chondrocytes, cells that were not affected by RA, were used as control cells for this study. When treated with ATP, these cells did not exhibit a [Ca2+]i response. Although the purinergic subtype receptor was not characterized, the observation that cells responded to ATP and ADP but were refractory to AMP and adenosine suggested that P2 purinoceptors were expressed by chondrocytes. Because, during the same culture period, chondrocytes exhibited many of the unique characteristics of the terminally differentiated cell, the acquisition of purinergic receptors represents a new feature associated with expression of the mature phenotype. Finally, to ascertain if the ATP-dependent response was due to release of Ca2+ from intracellular stores, cells were treated with thapsigargin. Since this compound significantly reduced the [Ca2+]i signal, we concluded that the ATP response is mediated by release of cation, from the endoplasmic reticulum.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Cartilage; Cells, Cultured; Cellular Senescence; Chick Embryo; Dose-Response Relationship, Drug; Extracellular Space; Fluorescent Dyes; Fura-2; Intracellular Membranes; Osmolar Concentration; Tretinoin

A promyelocytic leukemia cell line, HL-60, was induced to differentiate into monocyte-macrophage lineage cells by treatment with active vitamin D3 and phorbol esters, and into granulocyte lineage ones by retinoic acid and dimethylsulfoxide. The changes in intracellular concentration of free calcium ions ([Ca2+]i) were measured and analyzed by calcium-imaging analysis with Fura 2-AM. A significant and transient increase in [Ca2+]i was observed in active vitamin D3 and phorbol ester systems; however, no change was detected with retinoic acid and dimethylsulfoxide. This increase was due to the influx of calcium ions from outside of the cells, and L-type calcium channels were shown to mainly contribute to this influx. Protein kinase C was also shown to be involved in the increase in [Ca2+]i.

Topics: Calcitriol; Calcium; Cell Differentiation; Dimethyl Sulfoxide; Fura-2; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; Macrophages; Monocytes; Protein Kinase C; Spectrophotometry, Ultraviolet; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Extracellular calcium concentrations markedly affect the pattern of proliferation and differentiation in cultured keratinocytes. When medium contains 0.1 mM calcium or above, the cells lose their proliferative ability, rapidly stratify, and terminally differentiate. Because 1,25(OH)2D3 (a modulator of Ca++ homeostasis) enhances the differentiation of keratinocytes, we investigated whether a link exists between 1,25(OH)2D3-induced release of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) from PtdIns 4,5-P2 and intracellular calcium [Ca++]i release from keratinocytes. Specifically, primary culture of keratinocytes were loaded with fluorescence dye Fura-2AM (10 microM) and changes in fluorescence intensity were monitored at the excitation wavelengths of 340 and 380 nm and emission wavelength of 505 nm. Additions of two agonists, 1,25(OH)2D3 (1.2 x 10(-9) M) and 13-Cis retinoic acid (0.2 x 10(-9) M), to dye-loaded keratinocytes induced rapid release of [Ca++]i, respectively, followed by gradual return to the prestimulated state. Addition of Ins(1,4,5)P3 (10 microM) to saponin-treated (leaky) keratinocytes also resulted in a rapid release of [Ca++]i. In contrast, the addition of inositol-1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P4 at similar concentrations exerted negligible effect. Taken together, these results support the view that 1,25(OH)2D3-induced [Ca++]i release in keratinocytes may be via the Ins(1,4,5)P3-induced early release of intracellular [Ca++]i. This may explain, at least in part, 1,25(OH)2D3-enhanced keratinocyte differentiation.

Topics: Animals; Animals, Newborn; Calcitriol; Calcium; Cell Differentiation; Cells, Cultured; Egtazic Acid; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Keratinocytes; Kinetics; Mice; Mice, Inbred BALB C; Spectrometry, Fluorescence; Tretinoin