Hiroshi Mochida

Water Adaptation Strategy in Anuran Amphibians: Molecular Diversity of Aquaporin

Most adult anuran amphibians except for the aquatic species absorb water across the ventral pelvic skin and reabsorb it from urine in the urinary bladder. Many terrestrial and arboreal species use a region in the posterior or pelvic region of the ventral skin that is specialized for rapid rehydration from shallow water sources or moist substrates. Periods of terrestrial activity can be prolonged by reabsorption of dilute urine from the urinary bladder. Aquaporin (AQP), a water channel protein, plays a fundamental role in these water absorption/reabsorption processes, which are regulated by antidiuretic hormone. Characterization of AQPs from various anurans revealed that the unique water homeostasis is basically mediated by two types of anuran-specific AQPs, i.e. ventral pelvic skin and urinary bladder type, respectively. The bladder-type AQP is further expressed in the pelvic skin of terrestrial and arboreal species, together with the pelvic skin-type AQP. In contrast, the pelvic skin-type AQP (AQP-x3) of the aquatic Xenopus has lost the ability of efficient protein production. The extra C-terminal tail in AQP-x3 consisting of 33 nucleotides within the coding region appears to participate in the posttranscriptional regulation of AQP-x3 gene expression by attenuating protein expression. The positive transcriptional regulation of bladder-type AQP in the pelvic skin and negative posttranscriptional regulation of pelvic skin-type AQP provide flexibility in the water regulation mechanisms, which might have contributed to the evolutionary adaptation of anurans to a wide variety of water environments.

VIP and PACAP stimulate tsh release from the bullfrog pituitary

We have recently shown that corticotropin-releasing hormone (CRH) is a major thyrotropin (TSH)-releasing factor in amphibians, but we have also found that, besides CRH, other hypothalamic substances stimulate TSH secretion in frog. In order to characterize novel TSH secretagogues, we have investigated the effect of frog (Rana ridibunda) vasoactive intestinal polypeptide (VIP) (fVIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) (fPACAP38 and PACAP27) on TSH release from bullfrog (Rana catesbeiana) pituitary cells in primary culture. Incubation of pituitary cells for 24h with graded concentrations of fVIP, fPACAP38 and PACAP27 (10(-9) to 10(-6)M) induced a dose-dependent stimulation of TSH release with minimum effective doses of 10(-9)M for fVIP and 10(-8)M for fPACAP38 and PACAP27. The PAC1-R/VPAC2-R antagonist PACAP(6-38) (10(-7) and 10(-6)M) dose-dependently suppressed the stimulatory effects of fVIP and fPACAP38 (10(-7)M each). Likewise, this antagonist (10(-6) and 10(-5)M) dose-dependently attenuated the stimulatory effect of PACAP27 (10(-7)M). On the other hand, the VPAC1-R/VPAC2-R antagonist [d-pCl-Phe(6), Leu(17)]VIP (10(-6) and 10(-5)M) dose-dependently inhibited the stimulatory effect of fVIP (10(-9)M) and PACAP27 (10(-8)M), but did not affect the response to fPACAP38 (10(-8)M). These data indicate that, in amphibians, the activity of thyrotrophs can be regulated by VIP and PACAP acting likely through VPAC2-R and PAC1-R.

Changes in the distribution of corticotropin-releasing factor (CRF)-like immunoreactivity in the larval bullfrog brain and the involvement of CRF in the cessation of food intake during metamorphosis

In submammalian vertebrates, corticotropin-releasing factor (CRF) acts as an anorexigenic neuropeptide as well as a potent stimulator of corticotropin and thyrotropin release from the pituitary. As a step for demonstrating the involvement of CRF in the feeding regulation of anuran larvae, which are known to stop feeding toward the metamorphic climax, we studied firstly the changes in the distribution of CRF-like immunoreactivity (CRF-LI) in the brain of metamorphosing bullfrog larvae. Neuronal cell bodies showing CRF-LI were invariably present in the thalamic regions throughout larval development. Cells with CRF-LI were also found in the hypothalamus. The number of cells with CRF-LI in the hypothalamus, but not in the thalamus, showed a significant increase as metamorphosis progressed. Immunoreactive nerve fibers were observed mainly in the median eminence, and became abundant as metamorphosis proceeded. The number of cells showing CRF-LI in the hypothalamus as well as the density of immunoreactive fibers in the median eminence decreased at the end of metamorphosis. Secondly, we examined the effect of intracerebroventricular (ICV) injection of CRF on the food intake in the premetamorphic larvae. ICV injection of CRF at 10 pmol/g body weight (BW) induced a significant decrease of food intake during 15 min. The CRF-induced anorexigenic action was blocked by the treatment with a CRF receptor antagonist [alpha-helical CRF(9-41)] at 100 pmol/g BW. The results suggest the involvement of CRF in the accomplishment of metamorphosis through the pituitary and in the feeding restriction that occurs during the later stages of metamorphosis through the central nervous system.

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.

The water-absorption region of ventral skin of several semiterrestrial and aquatic anuran amphibians identified by aquaporins

Regions of specialization for water absorption across the skin of Bufonid and Ranid anurans were identified by immunohistochemistry and Western blot analysis, using antibodies raised against arginine vasotocin (AVT)-stimulated aquaporins (AQPs) that are specific to absorbing regions of Hyla japonica. In Bufo marinus, labeling for Hyla urinary bladder-type AQP (AQP-h2), which is also localized in the urinary bladder, occurred in the ventral surface of the hindlimb, pelvic, and pectoral regions. AQP-h2 was not detected in any skin regions of Rana catesbeiana, Rana japonica, or Rana nigromaculata. Hyla ventral skin-type AQP (AQP-h3), which is found in the ventral skin but not the bladder of H. japonica, was localized in the hindlimb, pelvic, and pectoral skins of Bufo marinus, in addition to AQP-h2. AQP-h3 was also localized in ventral skin of the hindlimb of all three Rana species and also in the pelvic region of R. catesbiana. Messenger RNA for AQP-x3, a homolog of AQP-h3, could be identified by RT-PCR from the hindlimb, pectoral, and pelvic regions of the ventral skin of Xenopus laevis, although AVT had no effect on water permeability. In contrast, 10(-8) M AVT-stimulated water permeability and translocation of AQP-h2 and AQP-h3 into the apical membrane of epithelial cells in regions of the skin of species where they had been localized by immunohistochemistry and Western blot analysis. Finally, water permeability of the hindlimb skin of B. marinus and all the Rana species was stimulated by hydrins 1 and 2 to a similar level as seen for AVT. The present data demonstrate species differences in the occurrence, distribution, and regulation of AQPs in regions of skin specialized for rapid water absorption that can be associated with habitat and also phylogeny.

Pro-Opiomelanocortin-Containing Neurons in Rat Median Eminence

A significant number of pro-opiomelanocortin (POMC)-containing cells were detected in the rat median eminence (ME) by immunocytochemistry using an antibody raised against a synthetic peptide corresponding to the cleavage site between adrenocorticotropin and beta-lipotropin moieties. Distribution of POMC-positive cells was restricted to the internal zone of the anterior parts of the ME. Such cells were observed as early as the 14th day of gestation in the area of the primitive ME, long before glial fibrillary acidic protein-positive cells appeared postnatally in this structure. Dissociated cell cultures obtained from the ME of 1-day-old rats produced cells immunoreactive for neurofilaments and the POMC moiety. Such cells displayed a neuronal morphology: the cell body was oval (13-18 microns) with long and fine beaded fibers. These findings clearly demonstrate the early appearance of POMC neurons in the developmental ME, a target organ of the hypothalamic infundibular neurons.

Inhibitory effect of corticotropin-releasing factor on food intake in the bullfrog, Aquarana catesbeiana

Corticotropin-releasing factor (CRF) and CRF-related peptides exert hypophysiotropic and anorexigenic effects in mammals and teleost fish. In anuran amphibians, CRF acts as a potent stimulator of thyrotropin release from the pituitary. According to our recent study, CRF also acts as an anorexigenic factor for the cessation of food intake in the metamorphosing bullfrog larvae. However, the anorexigenic action of CRF has not been confirmed in adult bullfrogs. In this context, we examined the effect of feeding status on the expression level of the CRF transcript in the hypothalamus of the adult bullfrog. Levels of CRF mRNA in the hypothalami from bullfrogs fasted for 7 days were lower than in those from the bullfrogs that had been fed normally. Subsequently, we developed a method for measuring food intake in adult bullfrogs, and then investigated the effect of CRF on their food consumption in these animals. Intracerebroventricular (ICV) administration of CRF at 1 and 10pmol/g body weight (BW) induced a significant decrease of food intake during 60min. The CRF-induced anorexigenic action was blocked by treatment with a CRF receptor 1/CRF receptor 2 antagonist, α-helical CRF((9-41)), at 100pmol/g BW. These results provide direct evidence for the inhibitory effect of CRF on food intake, and suggest the involvement of CRF in the regulation of feeding through a CRF receptor-signaling pathway in the adult bullfrog.

Polymorphism of somatolactin-producing cells in the goldfish pituitary: immunohistochemical investigation for somatolactin-α and -β

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin family of adenohypophyseal hormones. In teleost fish, SL is encoded by one or two paralogous genes, namely SL-α and -β. Our previous studies have revealed that pituitary adenylate-cyclase-activating polypeptide stimulates SL release from cultured goldfish pituitary cells, whereas melanin-concentrating hormone suppresses this release. As in other fish, the goldfish possesses SL-α and -β. So far, however, no useful means of detecting the respective SLs immunologically in this species has been possible. In order to achieve this aim, we raised rabbit antisera against synthetic peptide fragments deduced from the goldfish SL-α and -β cDNA sequences. Using these antisera, we observed adenohypophyseal cells showing SL-α- and -β-like immunoreactivities in the goldfish pituitary, especially the pars intermedia (PI). Several cells in the PI showed the colocalization of SL-α- and -β-like immunoreactivities. Then, using single-cell polymerase chain reaction with laser microdissection, we examined SL-α and -β gene expression in adenohypophyseal cells showing SL-α- or -β-like immunoreactivity. Among cultured pituitary cells, we observed three types of cell: those that possess transcripts of SL-α, -β, or both. These results suggest a polymorphism of SL-producing cells in the goldfish pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates release of somatolactin (SL)-α and SL-β from cultured goldfish pituitary cells via the PAC1 receptor-signaling pathway, and affects the expression of SL-α and SL-β mRNAs

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that stimulates the release of adenohypophyseal hormone from the pituitary in fish. In the goldfish, PACAP induces the release of somatolactin (SL), in particular, from cultured pituitary cells. SL belongs to the growth hormone and prolactin family, and comprises two molecular variants termed SL-α and SL-β in goldfish. However, there is no information about the involvement of PACAP in the regulation of SL-α and SL-β release and the expression of their mRNAs. Therefore, we examined the effect of PACAP on SL-α and SL-β release from cultured goldfish pituitary cells. Treatment with PACAP (10(-10)-10(-7)M) increased the release of both SL-α and SL-β. The stimulatory action of PACAP (10(-9)M) on SL-α and SL-β release was blocked by treatment with a PACAP-selective receptor (PAC1R) antagonist, PACAP(6-38) (10(-6)M). We also examined whether PACAP affects the expression of SL-α and SL-β mRNAs in cultured pituitary cells. Treatment with PACAP (10(-9) and 10(-8)M) for 6h decreased the expression level of SL-α mRNA but increased that of SL-β mRNA. The action of PACAP (10(-8)M) on SL-β mRNA expression was blocked by treatment with PACAP(6-38) (10(-6)M), whereas PACAP(6-38) elicited no change in the expression of SL-α mRNA. These results indicate that in cultured goldfish pituitary cells, PACAP stimulates the release of SL-α and SL-β, and expression of SL-β mRNA, via the PAC1R-signaling pathway. However, the mechanism whereby PACAP inhibits the expression of SL-α mRNA does not seem to be mediated by PAC1R signaling.

Production and Characterization of Specific Anti-peptide Antiserum Against Free α-subunit of Rat Pituitary Glycoprotein Hormones

To obtain an antibody specific for the α-subunit of rat pituitary glycoprotein hormones, we synthesized a peptide corresponding to the sequence 37–53 (ST-7: Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) of the rat α-subunit. The polyclonal antiserum against this peptide was generated in rabbits. This region is hydrophilic and highly conserved among several mammalian species. Noncompetitive binding tests showed that the ST-7 antiserum had specific affinity for the rat free α-subunit, but not for rat intact LH, FSH, and TSH. The ST-7 antiserum immunostained two types of cells in the rat anterior pituitary, i.e., gonadotrophs and thyrotrophs. This was also the case in mouse, cattle, sheep, and pig, which have an identical sequence of ST-7 in their α-subunit. The pituitary cells of horse (Arg substituted for Lys as residue 48 of the rat α-subunit), human, and eel (Leu for Ala at residue 45), chicken (Met for Ala at residue 45), and bullfrog (Tyr for Phe at residue 37 and Met for Ala at residue 45) were not stained with the ST-7 antiserum. This study indicated that the ST-7 antiserum is sequence-specific for the α-subunit and is therefore useful for immunohistochemical studies on the secretory pathway of the free α-subunit.