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Characterization of platelet-activating factor receptors on canine T lymphocytes.
J Immunol. 1993 Nov 15; 151(10):5310-8.JI

Abstract

The ability of purified canine T lymphocytes to selectively bind platelet activating factor (PAF) was characterized. Authentic radiolabeled PAF rapidly and selectively bound to T lymphocytes and reached saturation within 1 min. This binding was reversible and highly selective for (R) PAF because (S) PAF, lyso-PAF, and diacyl PAF did not displace the bound (R) PAF probe. Only increasing quantities of chemically pure (R) PAF displaced the radiolabeled (R) PAF probe. The binding maximum of PAF was determined to be 35 pM per 2 x 10(6) lymphocytes. Competitive radioligand binding studies and Scatchard analysis indicated a single class of high affinity receptors with a dissociation constant of 0.077 nM and a receptor density of 6419 receptors per cell. The ability of purified canine T lymphocytes to hydrolyze PAF to the biologically inactive metabolite lyso-PAF was also studied. Over a 30-min incubation period, about 5% of PAF was metabolized to lyso PAF. This rate of PAF hydrolysis was the same as the rate observed with the media without cells, suggesting a small degree of nonenzymatic hydrolysis. The effects of varying concentrations of authentic PAF on intracellular free Ca2+ release in purified T lymphocytes was evaluated using the fluorescent probe Fura-2 and excitation-emission spectrofluorometry. PAF below the concentration of 1.0 nM did not significantly increase intracellular Ca2+ in T lymphocytes. More than 1 nM PAF, intracellular-free Ca2+ modestly, but significantly, increased in T lymphocytes. In other experiments, canine PBMC proliferated in response to Con A and in the one way MLR. These proliferative responses were abolished when the selective PAF receptor antagonist SC-47014A was added to the culture medium. In the MLR, this inhibitory effect was dependent on the length of time that the antagonist was in the culture. Specifically, inhibition of proliferation was incrementally reversed when the PAF antagonist was introduced progressively later into the 7-day MLR stimulation period, suggesting that PAF receptor blockade prevents an MLR response from occurring, but is unable to suppress an existing MLR response. Although the Con A-induced mononuclear cell proliferation was abolished with PAF receptor antagonists, the addition of authentic biologically active PAF or PAF analogs did not alter the proliferative response to Con A. In conclusion, canine T lymphocytes possess high affinity receptors for PAF. These binding sites are highly selective and reversible. PAF binding slightly increases intracellular free Ca2+ in T lymphocytes and appears to be involved in lymphocyte proliferation in response to soluble plant mitogen and alloantigen.

Authors+Show Affiliations

Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

8228226

Citation

Mangino, M J., et al. "Characterization of Platelet-activating Factor Receptors On Canine T Lymphocytes." Journal of Immunology (Baltimore, Md. : 1950), vol. 151, no. 10, 1993, pp. 5310-8.
Mangino MJ, Murphy MK, Weiss A, et al. Characterization of platelet-activating factor receptors on canine T lymphocytes. J Immunol. 1993;151(10):5310-8.
Mangino, M. J., Murphy, M. K., Weiss, A., & Anderson, C. B. (1993). Characterization of platelet-activating factor receptors on canine T lymphocytes. Journal of Immunology (Baltimore, Md. : 1950), 151(10), 5310-8.
Mangino MJ, et al. Characterization of Platelet-activating Factor Receptors On Canine T Lymphocytes. J Immunol. 1993 Nov 15;151(10):5310-8. PubMed PMID: 8228226.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of platelet-activating factor receptors on canine T lymphocytes. AU - Mangino,M J, AU - Murphy,M K, AU - Weiss,A, AU - Anderson,C B, PY - 1993/11/15/pubmed PY - 1993/11/15/medline PY - 1993/11/15/entrez SP - 5310 EP - 8 JF - Journal of immunology (Baltimore, Md. : 1950) JO - J Immunol VL - 151 IS - 10 N2 - The ability of purified canine T lymphocytes to selectively bind platelet activating factor (PAF) was characterized. Authentic radiolabeled PAF rapidly and selectively bound to T lymphocytes and reached saturation within 1 min. This binding was reversible and highly selective for (R) PAF because (S) PAF, lyso-PAF, and diacyl PAF did not displace the bound (R) PAF probe. Only increasing quantities of chemically pure (R) PAF displaced the radiolabeled (R) PAF probe. The binding maximum of PAF was determined to be 35 pM per 2 x 10(6) lymphocytes. Competitive radioligand binding studies and Scatchard analysis indicated a single class of high affinity receptors with a dissociation constant of 0.077 nM and a receptor density of 6419 receptors per cell. The ability of purified canine T lymphocytes to hydrolyze PAF to the biologically inactive metabolite lyso-PAF was also studied. Over a 30-min incubation period, about 5% of PAF was metabolized to lyso PAF. This rate of PAF hydrolysis was the same as the rate observed with the media without cells, suggesting a small degree of nonenzymatic hydrolysis. The effects of varying concentrations of authentic PAF on intracellular free Ca2+ release in purified T lymphocytes was evaluated using the fluorescent probe Fura-2 and excitation-emission spectrofluorometry. PAF below the concentration of 1.0 nM did not significantly increase intracellular Ca2+ in T lymphocytes. More than 1 nM PAF, intracellular-free Ca2+ modestly, but significantly, increased in T lymphocytes. In other experiments, canine PBMC proliferated in response to Con A and in the one way MLR. These proliferative responses were abolished when the selective PAF receptor antagonist SC-47014A was added to the culture medium. In the MLR, this inhibitory effect was dependent on the length of time that the antagonist was in the culture. Specifically, inhibition of proliferation was incrementally reversed when the PAF antagonist was introduced progressively later into the 7-day MLR stimulation period, suggesting that PAF receptor blockade prevents an MLR response from occurring, but is unable to suppress an existing MLR response. Although the Con A-induced mononuclear cell proliferation was abolished with PAF receptor antagonists, the addition of authentic biologically active PAF or PAF analogs did not alter the proliferative response to Con A. In conclusion, canine T lymphocytes possess high affinity receptors for PAF. These binding sites are highly selective and reversible. PAF binding slightly increases intracellular free Ca2+ in T lymphocytes and appears to be involved in lymphocyte proliferation in response to soluble plant mitogen and alloantigen. SN - 0022-1767 UR - https://www.unboundmedicine.com/medline/citation/8228226/Characterization_of_platelet_activating_factor_receptors_on_canine_T_lymphocytes_ L2 - https://www.jimmunol.org/lookup/pmidlookup?view=long&pmid=8228226 DB - PRIME DP - Unbound Medicine ER -