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Both cultured bovine and human vascular endothelial cells possess histamine receptors but express them differently
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Both cultured bovine and human vascular endothelial cells possess histamine receptors but express them dff|erently Williams, P.B., Blackmore, P,F. and Rosenthal, M.D. Departments of Pharmacology and Biochemistry, Eastern Virginia Medical School, Box 1980. Norfolk, VA 23501, U.S.A.
Activation of histamine receptors on vascular endothelium stimulates synthesis and release of vasoactive substances, such as prostacyclin and endothelium-dependent relaxing factor. This study sought to characterize endothelial histamine receptors. Radioligand binding studies with the histamine H 1 antagonist, [3H]pyrilamine, were performed to estimate both affinity (KD) and maximal binding capacity (Bmax) in both calf pulmonary artery (CPAE) and human umbilical vein (HUVE) endothelial cells. Concomitant studies were performed to assay histamine-mediated changes in intracellular metabolism. They included histamine-stimulated release of [I-14C]arachidonate and increase intracellular free calcium [Ca i ]Radioligand binding with [3H]pyrilamine was highly specific ( > 95%). Scatchard analysis indicated that CPAE and HUVE possess similar high and low affinity forms of the Hrreceptor (Table 1). In competition studies the Hi-antagonists, pyrilamine and diphenhydramine, rapidly displaced [3H]pyrilamine from both cell types. For CPAE the ICS0's were 4.1 =t=1.5 uM and 5.5 + 1.5 uM respectively; for HUVE the IC50's were 7.3 :t= 3.0 uM and 5.9 + 2.7 uM. H~ antagonists had little or no effect. After prelabelling with [I-14C]arachidonate, the washed cells were incubated with histamine or bradykinin in medium containing 50 uM fat-free albumin. Release of free [I-14C]arachidonate was quantitated by liquid scintillation spectroscopy. In HUVE, histamine (1-10 uM) consistently stimulated release of [I-14C]arachidonate. In CPAE, the release of [I-14C]arachidonate by histamine was sporadic; bradykinin, however, consistently released [I14C]arachidonate. To investigate further these differences in functional responses between HUVE and CPAE, subsequent studies examined the ability of histamine to increase [Ca i]. Endothelial cells were incubated with Fura2-AM (6 uM) and [Ca i] measured in a SPEX Flurolog spectrofluorometer. Histamine (1-100 uM) increased [Cai] in HUVE in a dose dependent manner. H 1- but not H2-antagonists inhibited the response to histamine (pA a for pyrilamine= 8.2). Although bradykinin (0.1 ug/ml) significantly increased [Ca i] in CPAE, histamine was without effect. Thus, both CPAE and HUVE possess two populations of Hi-receptors with similar binding capacities, but the expression of these receptors is different. Furthermore, the amount of pyrilamine required to inhibit receptor function in HUVEC was much less than that required for maximum receptor occupancy. Supported in part by the AHA/Virginia Affiliate and NIH-RR05771. Table 1 Binding Characteristics of the H 1 receptor. High affinity B~ax (fmole/cell) KD(10-tM)
HUVE 0.032 +0.022 0.87 +0.50
Low affinity CPAE 0.113 + 0.011 2.01 +1.32
HUVE 1.48 + 0.22 47.0 +14.6
CPAE 1.24 + 0.18 14.6 +21.8
Both cultured bovine and human vascular endothelial cells possess histamine receptors but express them dff|erently Williams, P.B., Blackmore, P,F. and Rosenthal, M.D. Departments of Pharmacology and Biochemistry, Eastern Virginia Medical School, Box 1980. Norfolk, VA 23501, U.S.A.
Activation of histamine receptors on vascular endothelium stimulates synthesis and release of vasoactive substances, such as prostacyclin and endothelium-dependent relaxing factor. This study sought to characterize endothelial histamine receptors. Radioligand binding studies with the histamine H 1 antagonist, [3H]pyrilamine, were performed to estimate both affinity (KD) and maximal binding capacity (Bmax) in both calf pulmonary artery (CPAE) and human umbilical vein (HUVE) endothelial cells. Concomitant studies were performed to assay histamine-mediated changes in intracellular metabolism. They included histamine-stimulated release of [I-14C]arachidonate and increase intracellular free calcium [Ca i ]Radioligand binding with [3H]pyrilamine was highly specific ( > 95%). Scatchard analysis indicated that CPAE and HUVE possess similar high and low affinity forms of the Hrreceptor (Table 1). In competition studies the Hi-antagonists, pyrilamine and diphenhydramine, rapidly displaced [3H]pyrilamine from both cell types. For CPAE the ICS0's were 4.1 =t=1.5 uM and 5.5 + 1.5 uM respectively; for HUVE the IC50's were 7.3 :t= 3.0 uM and 5.9 + 2.7 uM. H~ antagonists had little or no effect. After prelabelling with [I-14C]arachidonate, the washed cells were incubated with histamine or bradykinin in medium containing 50 uM fat-free albumin. Release of free [I-14C]arachidonate was quantitated by liquid scintillation spectroscopy. In HUVE, histamine (1-10 uM) consistently stimulated release of [I-14C]arachidonate. In CPAE, the release of [I-14C]arachidonate by histamine was sporadic; bradykinin, however, consistently released [I14C]arachidonate. To investigate further these differences in functional responses between HUVE and CPAE, subsequent studies examined the ability of histamine to increase [Ca i]. Endothelial cells were incubated with Fura2-AM (6 uM) and [Ca i] measured in a SPEX Flurolog spectrofluorometer. Histamine (1-100 uM) increased [Cai] in HUVE in a dose dependent manner. H 1- but not H2-antagonists inhibited the response to histamine (pA a for pyrilamine= 8.2). Although bradykinin (0.1 ug/ml) significantly increased [Ca i] in CPAE, histamine was without effect. Thus, both CPAE and HUVE possess two populations of Hi-receptors with similar binding capacities, but the expression of these receptors is different. Furthermore, the amount of pyrilamine required to inhibit receptor function in HUVEC was much less than that required for maximum receptor occupancy. Supported in part by the AHA/Virginia Affiliate and NIH-RR05771. Table 1 Binding Characteristics of the H 1 receptor. High affinity B~ax (fmole/cell) KD(10-tM)
HUVE 0.032 +0.022 0.87 +0.50
Low affinity CPAE 0.113 + 0.011 2.01 +1.32
HUVE 1.48 + 0.22 47.0 +14.6
CPAE 1.24 + 0.18 14.6 +21.8