Abstract
A DNA, cloned after screening a rat genomic bank with probes derived from the sequence of a putative dog histamine H2 receptor [Gantz, I., Schäffer, M., Delvalle, J., Logsdon, C., Campbell, V., Uhler, M. & Yamada, T. (1991) Proc. Natl. Acad. Sci. USA 88, 429-433], was used to prepare a probe for Northern blot analysis and to transfect Chinese hamster ovary (CHO) cells. Distribution of the gene transcripts in guinea pig tissues was consistent with that of H2 receptors. Transfected CHO cells expressed a high density of sites binding [125I]iodoaminopotentidine, a selective H2-receptor ligand. These sites were characterized as typical H2 receptors by using a series of competing agents that displayed apparent dissociation constants closely similar to corresponding values at a reference biological system. In transfected cells, histamine stimulated, with high potency and large receptor reserve, the accumulation of cAMP. In addition, in the same cells, histamine potently inhibited the release of arachidonic acid induced either by stimulation of constitutive purinergic receptors or by application of a Ca2+ ionophore. This inhibition was independent of either cAMP or Ca2+ levels. The results suggest that a single H2 receptor may be linked not only to adenylyl cyclase activation but also to reduction of phospholipase A2 activity. Because H1 receptors have been reported to stimulate arachidonic acid release, inhibition of this release, an unexpected signaling pathway for H2 receptors, may account for the opposing physiological responses elicited in many tissues by stimulation of these two receptors subtypes.
Highlights
A DNA, cloned after screening a rat genomic bank with probes derived from the sequence of a putative dog histamine H2 receptor [Gantz, I., Schiffer, M., Delvalle, J., Logsdon, C., Campbell, V., Uhler, M. & Yamada, T. (1991) Proc
In Chinese hamster ovary (CHO)(H2) cells labeled by incubation with [3H]arachidonic acid (A4Ach), HA inhibited [3H]A4Ach release evoked by stimulating constitutive purinergic receptors with 0.1 mM ATP (22); inhibition was maximal at 0.5 AM HA (38 ± 3%) and competitively antagonized by ranitidine (1 AM) (Fig. 5)
Northern blot analysis of guinea pig tissues revealed a single band of size similar to that obtained in rat tissues (12)
Summary
Cells (24-well plates) were labeled by incubation for 2 h at 370C with 0.5 tkCi of [3H]A4Ach in 1 ml of DMEM supplemented with 0.2% bovine serum albumin to trap the released radioactivity. After washing twice with 0.75 ml of DMEM plus bovine serum albumin, cells were preincubated for 10 min in 0.5 ml of the same medium containing appropriate drugs. Cells (35-mm dishes) were incubated in 3 ml of DMEM containing 0.2% bovine serum albumin and [3H]A4Ach at 0.05 ,uCi/ml for 30 min at 37C. Cells were trypsinized, washed twice with DMEM plus bovine serum albumin (0.2%), and incubated for 90 min at 37°C in 2 ml of the same medium containing fura-2 acetoxymethyl ester (2 ,uM). Cells were washed and resuspended in Tyrode's solution, and [Ca2+i was measured by using a Hitachi F-2000 fluorescence spectrophotometer (19)
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