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Receptor-activated currents in mouse fibroblasts expressing transfected bombesin receptor subtype cDNAs
K Kusano1, H Gainer, J F Battey
1Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
BALB/c 3T3 cells do not normally express receptors for bombesin-like peptides [bombesin (Bn), gastrin-releasing peptide (GRP), and neuromedin B (NmB)]. Transfection of BALB/c 3T3 cells with complementary DNA-encoding GRP receptors or NmB receptors leads to stable expression of functional GRP receptors (GRP Rt) or NmB receptors (NmB Rt), respectively, which are coupled to cell membrane ion channels. Whole cell current analysis using patch electrodes shows that the activation of these newly expressed receptors induces cation conductance increases, most frequently a Ca(2+)-activated plasma membrane K+ conductance. The dose-response (peak-current) relations of both transfected receptor subtypes were sigmoidal and exhibited threshold activation concentration in the picomole range and the saturation of responses to higher concentrations than 10(-8) M. The GRP Rt responded about equally to GRP, NmB, and Bn when compared at equimolar levels, despite their known difference in binding affinity for the three peptides (GRP, Bn > NmB). In contrast, for the NmB Rt, the NmB was more potent than GRP or Bn. Among four GRP/Bn-receptor antagonists tested, the [D-Phe6]Bn(6-13) ethyl ester suppressed GRP Rt responses at low concentrations (10(-7) M). N-acetyl-GRP-(20-26) amide, [Leu13-psi(CH2NH)-Leu14]Bn, and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also blocked GRP Rt responses but at higher concentrations (10(-5) M). However, at these concentrations, these four antagonists had little effect on NmB Rt responses, thereby showing a specificity of these antagonists for the GRP receptors.
Insights
BALB/c 3T3 cells were engineered to express GRP or NmB receptors, activating ion channels and cation conductance. Specific antagonists selectively blocked GRP receptors, demonstrating their utility in studying these signaling pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Neuroendocrinology
Background:
- BALB/c 3T3 cells lack native receptors for bombesin-like peptides (Bn, GRP, NmB).
- Receptor expression is crucial for cellular responses to peptide signaling.
Purpose of the Study:
- To investigate the functional expression of GRP and NmB receptors in transfected BALB/c 3T3 cells.
- To characterize the ion channel activation and peptide sensitivity of these novel receptor systems.
- To evaluate the specificity of GRP/Bn receptor antagonists.
Main Methods:
- Cell transfection with GRP or NmB receptor cDNA.
- Whole-cell patch-clamp electrophysiology to measure ion channel activity.
- Dose-response analysis of peptide activation and antagonist blockade.
Main Results:
- Transfected cells stably expressed functional GRP or NmB receptors coupled to ion channels.
- Receptor activation induced cation conductance, notably Ca(2+)-activated K+ conductance.
- Dose-response curves were sigmoidal, with picomolar activation thresholds and nanomolar saturation.
- GRP receptors showed equal potency for GRP, NmB, and Bn, while NmB receptors were most sensitive to NmB.
- Specific antagonists ([D-Phe6]Bn(6-13) ethyl ester) selectively inhibited GRP receptor activity.
Conclusions:
- Engineered BALB/c 3T3 cells provide a model for studying GRP and NmB receptor function.
- Receptor activation leads to measurable ion channel activity.
- Developed antagonists exhibit specificity for GRP receptors, useful for further research.