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Spiking of intracellular calcium ion concentration in single cultured pig aortic endothelial cells stimulated with ATP or bradykinin.

ABSTRACT: Single pig aortic endothelial cells in culture loaded with the Ca(2+)-sensitive fluorescent dye Indo-1 were stimulated with ATP (0.1-100 microM) or bradykinin (0.1-5.0 nM). Spiking or oscillations of [Ca2+]i were seen in approx. 50% of cells stimulated with either agonist. Non-spiking or transient responses in which [Ca2+]i returned to pre-stimulation levels rapidly 9120-250 s), or sustained responses in which [Ca2+]i remained elevated for many minutes, were seen in a further 20% of cells in each case, stimulated with either agonist. There was a marked variation between individual cells in the latency, magnitude, frequency and overall pattern of oscillations induced by ATP and bradykinin, although the patterns of response to bradykinin were less variable. In cells where repetitive spikes were seen, a relation between concentration of ATP and the latency of the response and the frequency of spiking was evident. Effects of removal of extracellular Ca2+, elevation of extracellular K+ concentration (35 or 70 mM) or exposure to phorbol 12,13-dibutyrate or 1,2-dioctanoyl-sn-glycerol were tested on the spiking Ca2+ responses. Each of these procedures reversibly slowed or prevented Ca2+ spiking evoked by ATP or bradykinin. In contrast, the inactive phorbol ester 4 alpha-phorbol didecanoate had no effect on Ca2+ spiking evoked by these hormones. Our results thus indicate that the responses of single cells to ATP or bradykinin exhibit marked heterogeneity, and suggest that secretory events driven by extracellular Ca2+ may be regulated by repetitive spikes or oscillations of Ca2+.

SUBMITTER: Carter TD

PROVIDER: S-EPMC1151402 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Project description:The hypotheses that PKC epsilon is necessary for: 1) PGF2 alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKC epsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKC epsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2 alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2 alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKC epsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKC epsilon expression was ablated by 75%, the inhibitory effect of PGF2 alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKC epsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKC epsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2 alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 mumol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2 alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKC epsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2 alpha.

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Spiking of intracellular calcium ion concentration in single cultured pig aortic endothelial cells stimulated with ATP or bradykinin.

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