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Expression of functional bradykinin receptors in Xenopus oocytes
E Phillips1, M J Conder, S Bevan
1Sandoz Institute for Medical Research, London, England, U.K.
Abstract:
mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.
Insights
Xenopus laevis oocytes injected with mRNA from various cells responded to bradykinin, revealing specific receptor expression patterns. This study identifies bradykinin receptor subtypes in human fibroblasts and NG108-15 cells.
Area of Science:
- Molecular Biology
- Neuropharmacology
- Xenopus Oocyte Expression Systems
Background:
- Bradykinin (BK) is a peptide involved in various physiological processes.
- Understanding the expression and function of BK receptors is crucial for pharmacology.
- Xenopus laevis oocytes are a well-established system for expressing exogenous proteins and studying receptor function.
Purpose of the Study:
- To investigate the expression of functional bradykinin receptors in different cell types.
- To characterize the subtypes of bradykinin receptors present in human fibroblasts and NG108-15 cells.
- To optimize mRNA isolation and fractionation for enhanced oocyte expression studies.
Main Methods:
- Injection of mRNA from various sources (NG108-15 cells, rat uterus, human fibroblasts, rat dorsal root ganglia, rat brain) into Xenopus laevis oocytes.
- Two-electrode voltage clamp electrophysiology to record membrane currents upon agonist application.
- Sucrose gradient fractionation of mRNA to identify optimal size fractions for expression.
- Pharmacological characterization using bradykinin and serotonin.
Main Results:
- Oocytes injected with mRNA from NG108-15, rat uterus, and human fibroblasts showed robust responses to bradykinin, characterized by inward currents and oscillations.
- Rat brain mRNA did not confer bradykinin responsiveness, but oocytes responded to serotonin, indicating successful expression of other functional receptors.
- Fractionation of mRNA led to a five-fold increase in response magnitude, with optimal expression observed for mRNA fractions around 4.5 kb.
- WI38 human fibroblasts express both B1 and B2 bradykinin receptors, while NG108-15 cells express only the B2 receptor subtype.
Conclusions:
- Xenopus laevis oocytes can functionally express bradykinin receptors from various mammalian tissues and cell lines.
- The study successfully identified distinct bradykinin receptor profiles in human fibroblasts (B1 and B2) and NG108-15 cells (B2).
- mRNA size fractionation is an effective strategy to enhance the sensitivity and efficiency of receptor expression studies in oocytes.