Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors

Abstract

The neurohypophysial nonapeptides vasotocin (VT) and mesotocin (MT) are the amphibian counterparts of arginine vasopressin (AVP) and oxytocin (OT). We have here reported the cloning and functional characterization of the receptors for vasotocin (VTR) and mesotocin (MTR) in two species of frog, Rana catesbeiana and Rana esculenta. The frog VTR and MTR cDNAs encode proteins of 419 and 384 amino acids respectively. Frog VTR exhibits a high degree of sequence identity with the mammalian AVP-1a (V1a) receptor while the frog MTR possesses a high degree of sequence identity with the mammalian OT receptor. Activation of VTR induced both c-fos promoter-and cAMP-responsive element (CRE)-driven transcriptional activities, while activation of MTR induced c-fos promoter-driven transcriptional activity but failed to evoke CRE-driven transcriptional activity, suggesting differential G protein coupling between VTR and MTR. The VTR exhibited the highest sensitivity for VT followed by OT.AVP<MT, whereas the MTR showed preferential ligand sensitivity for MT.OT.VT.AVP. A V1a agonist but not V2 and OT agonists substantially activated both VTR and MTR with a similar sensitivity. V1a, V2 and OT antagonists inhibited MT-induced MTR activation but not VT-induced VTR activation. In the frog brain, VTR and MTR mRNAs were found to be widely expressed in the telencephalon, diencephalon and mesencephalon, and exhibited very similar regional distribution. In the pituitary, VTR and MTR were expressed in the distal and intermediate lobes but were virtually absent in the neural lobe. Taken together, these data indicated that, although the distribution of VTR and MTR largely overlaps in the frog brain and pituitary, VT and MT may play distinct activities owing to the ligand selectivity and different signaling pathways activated by their receptors.

Vasotocin and Mesotocin Stimulate the Biosynthesis of Neurosteroids in the Frog Brain

“…Conversely, the stimulatory effect of MT was blocked by the OTR antagonist [d(CH 2 (Richard et al, 1991) was inactive. However, the fact that MT is ϳ30 times more potent on MTR-transfected cells than on VT1R-or V1aR-transfected cells (Acharjee et al, 2004) supports the notion that the effect of MT on neurosteroidogenesis is actually mediated through MTR. Furthermore, the V1b agonist (Schwartz et al, 1991) provoked a significant stimulation of neurosteroid formation.…”

“…Oligonucleotides were designed from partial VT receptor (VTR) and MT receptor (MTR) cDNA sequences cloned previously in the frog R. esculenta (Acharjee et al, 2004) using the following primers: VTR forward, 5Ј-GGC ATG TTT GCG TCT ACC TAC-3Ј; VTR reverse, 5Ј-CAG ATG TGG TGC GTT TGG GAT-3Ј; MTR forward, 5Ј-GGG ATG TTT GCT TCT ACC TAC-3Ј; MTR reverse, 5Ј-CAG ATG TGG TCA GTC TGG GAC-3Ј.…”

“…We recently cloned a V1a-type VTR and the MTR in R. esculenta (Acharjee et al, 2004), so that we are now able to compare the distribution of the mRNAs encoding these two receptors with the localization of steroidogenic neurons in the frog diencephalon (Table 1). Intense expression of VTR and/or MTR mRNAs was seen in most hypothalamic nuclei that contain 3␤-HSDand/or P450 C17 -positive neurons.…”

African lungfish,Protopterus annectens, possess an arginine vasotocin receptor homologous to the tetrapod V2-type receptor

“…First-strand kidney cDNA was reverse-transcribed from DNase I-treated total RNA using the PrimeScript TM 1st strand cDNA Synthesis Kit (TaKaRa Bio, Otsu, Shiga, Japan). Degenerate primers (Table 1) for AVT receptors were designed based on the alignment of the three subtypes (V1a, V2, V1b/V3) of the mammalian AVP receptors Morel et al, 1992;Sugimoto et al, 1994), amphibian AVT receptors (Kohno et al, 2003;Acharjee et al, 2004;Hasunuma et al, 2007) and teleost V1a-type receptors (Mahlmann et al, 1994;Warne, 2001). Polymerase chain reaction (PCR) was performed using BIOTAQ DNA polymerase (Bioline, London, UK) as follows: 94°C for 2 min, 35 cycles at 94°C for 1 min, 55°C for 30 s, 72°C for 40 s, and finally 72°C for 10 min.…”

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream

“…It is unsurprising that AQP1a is regulated by AVTRV2 given that PKA is the preferential signaling pathway associated with this receptor, and PKC signaling seems to play a minor role (Wargent et al, 1999;Warne, 2001). However, previous studies in frog demonstrated the V2-type receptor could also function via stimulation of PLC pathways, one of the alternative pathways for PKC (Acharjee et al, 2004;Zhu et al, 1994). Further studies will be required to establish whether sea bream AVT V2-type receptors could also function via PKC signaling.…”