Receptor-mediated internalization of bradykinin. DDT1 MF-2 smooth muscle cells process internalized bradykinin via multiple degradative pathways.
This study was undertaken to evaluate the role of internalization in the action of the peptide autacoid bradykinin (BK). At 4 degrees C [3H]BK binds to an apparently single class of B2 kinin receptors on DDT1 MF-2 smooth muscle cells (C. M. Munoz, S. Cotecchia, and L. M. F. Leeb-Lundberg, manuscript submitted). At this temperature the [3H]BK binding was confined exclusively to the cell surface. On the other hand, at 37 degrees C the B2 receptor-specific cell surface [3H]BK binding was rapidly followed by a receptor-specific internalization of [3H]BK (t1/2 approximately 9 min). The internalization reached a steady-state level after 30-40 min that was 80-100% of the level of specifically bound [3H]BK on the cell surface at 4 degrees C, and this level was maintained for greater than or equal to 2 h. Internalized [3H]BK was routed via at least two intracellular degradative pathways which were distinguished primarily based on subcellular localization but also on a small but significant difference in the rate of [3H]BK degradation. One pathway was localized in a plasma membrane-enriched fraction and had a relatively high degradative capacity. Another pathway was localized in a microsomal fraction and had a relatively low degradative capacity. The internalized [3H]BK activity was rapidly released into the media (t1/2 approximately 24 min). Following a single round of internalization, the released activity consisted almost exclusively of small [3H]BK fragments (less than [3H]BK(1-5)). In contrast, at steady-state [3H]BK represented 30-40% of the released activity. While chloroquine (100 microM) did not alter the rate of [3H]BK internalization or release or the intracellular distribution of [3H]BK, this agent significantly decreased the rate of [3H]BK degradation in both pathways. In all, these results show that B2 kinin receptor-mediated internalization of BK is a process integral to the interaction of BK with DDT1 MF-2 smooth muscle cells and may be a mechanism for terminating BK actions by rapidly removing extracellular free and receptor-bound BK and accessing various intracellular BK degradative pathways.
Munoz
C M
CM
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Leeb-Lundberg
L M
LM
D000595:Amino Acid Sequence; D001920:Bradykinin / Q000378:metabolism*; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D002738:Chloroquine; D001920:Bradykinin