Biomedical subjects

J F Dunne

Publications and source records attributed to J F Dunne.

4 recordsLinked to original sources

PubMed1992

AT1 angiotensin receptors mobilize intracellular calcium in a subclone of NG108-15 neuroblastoma cells.

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗

PubMed1991

Time window analysis and sorting.

Flow cytometric hardware and procedures were developed to continuously analyse and to sort a particular time window in a kinetic response. The technique uses balanced air pressure to drive a stimulus-bead mixture from a vial to a t-junction where it mixes passively with cells. The t-junction is distanced from the flow cell and air pressure regulated so that the stimulation occurs at a fixed and adjustable time before the cells are interrogated by the laser beam. Practical applications of the device demonstrate utility with cells whose responses are seen in seconds or minutes. The device is easily implemented on any sorting flow cytometer.

Animals↗

PubMed1991

Flow cytometric analysis of internal calcium mobilization via a B2-bradykinin receptor on a subclone of PC-12 cells.

Single cell Ca2+ mobilization was studied by nonparametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 microM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 microM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 microM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1-10 microM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.

Adrenal Gland Neoplasms↗

PubMed1989

Detection of mRNA in flow-sorted cells.

Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P-labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co-induction of IL-1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c-fos in U937 to demonstrate linearity. IL-1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS-induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c-fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.

Flow Cytometry↗