To develop and validate a vasopressin (AVP) receptor knockdown strategy, we infused an antisense oligodeoxynucleotide to the V1 subtype mRNA into the septum of male rats with osmotic minipumps and measured behavioral, cellular and molecular parameters. Compared to vehicle and scrambled-sequence oligo controls, chronic antisense administration for up to 4 d diminished the ability of the animals to distinguish a previously exposed juvenile from a novel one and to respond to exogenous AVP (1 ng/5 microliters, intracerebroventricular) with an improved social memory. Furthermore, anxiety-related behavior was reduced. As measured in the behaviorally tested rats, antisense treatment resulted in a reduced binding of radiolabeled AVP in the septum, but not in other limbic brain areas (receptor autoradiography), and an increased amount of V1 receptor mRNA (reverse transcriptase PCR), indicating translational arrest and ongoing transcriptional activity. In sense oligo-treated rats, on the other hand, both the social and the anxiety-related behavior scores lay between levels obtained in control and antisense-treated animals. These sense-treated rats showed a slightly reduced V1 receptor density in the septum and reduced receptor mRNA levels, indicating hybridization of the sense oligo to the DNA. The data show the potential of antisense targeting to further reveal relationships between local gene expression, neuropeptide-receptor interactions in distinct brain areas, and behavioral performance.
BACKGROUND AND PURPOSEThe β-receptor antagonist carvedilol blocks a range of ion channels. K2P2.1 (TREK1) and K2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K2P2.1 and K2P10.1 channels. Second, we sought to study ATI-dependent modulation of K2P K + current sensitivity to carvedilol.
EXPERIMENTAL APPROACHUsing standard electrophysiological techniques, we recorded currents from wild-type and mutant K2P2.1 and K2P10.1 channels in Xenopus oocytes and HEK 293 cells.
KEY RESULTSCarvedilol concentration-dependently inhibited K2P2.1 channels (IC50,oocytes = 20.3 μM; IC50,HEK = 1.6 μM) and this inhibition was frequency-independent. When K2P2.1 isoforms generated by ATI were studied separately in oocytes, the IC50 value for carvedilol inhibition of full-length channels (16.5 μM) was almost 5-fold less than that for the truncated channel variant (IC50 = 79.0 μM). Similarly, the related K2P10.1 channels were blocked by carvedilol (IC50,oocytes = 24.0 μM; IC50,HEK = 7.6 μM) and subject to ATI-dependent modulation of drug sensitivity.
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