The water-absorption region of ventral skin of several semi-terrestrial and aquatic amphibians identified by aquaporins

1
The water-absorption region of ventral skin of
several semi-terrestrial and aquatic amphibians
identified by aquaporins

• Yuji Ogushi, Azumi Tsuzuki, Megumi Sato, Hiroshi
  Mochida, Reiko Okada, Masakazu Suzuki, Stanley D.
  Hillyard and Shigeyasu Tanaka

2
Introduction

• Semi-terrestrial water balance strategy
• Used by many tree frog and toad species
• Use ventral pelvic patch to absorb water
  cutaneously
• Capillaries contact basement membrane beneath
  epithelium
• Store dilute urine in bladder for re-absorption
  while foraging far from water
• Aquaporins (AQPs) plasma membrane proteins
  forming water channels into cells (present in
  almost all organisms)
• Control water permeability across membranes
• Stimulated by arginine vasotocin (AVT) causes
  fusion of vesicles containing AQPs with apical
  membrane of epithelial water absorption/reabsorpt
  ion tissues

3
Introduction

• Researchers used Real Time Polymerase Chain
  Reaction (RT-PCR) to identify 2 forms of AQP in
  epithelial tissues
• AQP-h2 (isoform)
• Termed urinary bladder-type AQP
• Found in urinary bladder of all study species
• Found in pelvic skin region of toad and tree frog
• AQP-h3 (isoform)
• Termed ventral skin-type AQP
• Found in skin but not bladder of tree frogs,
  toads and Rana species

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Study Species
Hyla japonica (tree frog)
Bufo marinus (terrestrial toad)
Xenopus laevis (aquatic)
Rana catesbaiana aka bullfrog (semi-aquatic)
Rana japonica (semi-aquatic)
Rana nigromaculata (semi-aquatic)
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Table 1. Phylogenetics of aquaporins in ventral
pelvic skins of anuran species living in
different habitats
Pelvic Skin Pelvic Skin Bladder
Habitat Species AQP-h2-like Protein ( Bladder-Type) AQP-h3-Like cDNA (Ventral Pelvic-Type) AQP-h2-Like Protein (Bladder-Type)
Arboreal Hyla japonica
Terrestrial Bufo japonica
Semi-aquatic Rana catesbeiana -
Semi-aquatic Rana nigromaculata -
Semi-aquatic Rana japonica -
Aquatic Xenopus laevis - (but not expressed)
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Introduction

• AQP-x3 mRNA homologous to AQP-h3 expressed in
  pelvic skin of aquatic species, Xenopus laevis
• but not translated to protein
• Hydrins intermediate peptides derived from a
  provasotocin-neurophysin precursor
• Stimulate osmotic water movement across skin and
  bladder
• Only present in anurans
• Have stimulatory effects on water permeability
  across pelvic skin in Hyla japonica

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Objectives

• Examine relationship between AQP distribution in
  apical membranes and ATV stimulation of water
  permeability in hindlimb, pelvic and pectoral
  zones of ventral skin
• Examine expression of AQP-x3 mRNA in skin of
  hindlimb, pelvic, pectoral, dorsal regions
• Different patterns of regional specialization
  present in terrestrial, arboreal, and semiaquatic
  species
• Extend observations and compare them with
  response of Ranid and toad species to AVT

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Materials and Methods Immunohistochemistry

• 4-mm sections of ventral skin mounted on slides
• Reacted with fluorescent labeled anti-bodies
• Nuclei stained with DAPI (appear blue)
• Pelvic skin type AQP proteins (AQP-h3) stained
  using Alexa Fluor 488 (appears green)
• Urinary bladder-type AQP proteins (AQP-h2)
  stained using Cy3 (appears red)
• Specimens examined with microscope equipped with
  fluorescence attachment

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Materials and Methods Western Blot Analysis

• Skin from hind-limb (I), pelvic (II) and pectoral
  (III) regions removed and homogenized
• Proteins separated via gel electrophoresis,
  transferred to membrane, and probed (detected)
  using antibodies

kDa I II III
Protein Molecular Weight Values
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Materials and Methods RT-PCR of Xenopus Ventral
Skin AQP-x3

• RNA extracted from ventral skin and reverse
  transcribed
• Gel electrophoresis
• DNA Sequenced

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Materials and Methods Water Permeability

• Skin from pectoral, pelvic, and hindlimb regions
  mounted between two chambers connected by a small
  opening
• Chamber on serosal (inner) side of skin filled
  with Ringer (salt) solution
• Mucosal (outer side) chamber filled with water
• Water movement from mucosal to serosal side
  recorded over 30 min with Ringer solution in
  mucosal chamber
• Followed by 30 min of Ringer solution with AVT
• Skins examined by immuno-fluorescence microscopy
  to evaluate incorporation of AQPs into apical
  membrane of First Reacting Cell (FRC) layer
• FRC layer continuous barrier between outside and
  inside of body

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Materials and Methods Water Permeability

• Effect of AVT on hindlimb skin permeability
  compared with hydrins 1 and 2
• Skins pretreated with AVT to increase number of
  AQPs inserted in apical plasma membrane
• Skins treated with HgCl2
• Water movement with continued AVT treatment
  measured for additional 30 min
• Results from 5 or 6 individuals expressed as
  means
• Statistical Analysis data compared by
  Steel-Dwasss test using software

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Results Aquaporins in 3 skin regions

• Rana japonica and Rana nigromaculata
• AQP-h3 (skin-type) in hindlimb region only
• Rana japonica in basolateral, apical, and
  cytoplasm of FRC
• Rana nigromaculata basolateral plasma membrane

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Results Aquaporins in 3 skin regions

• Rana catesbeiana
• Greatest AQP-h3 in hindlimb
• Present in small number pelvic skin cells
• In hindlimb and pelvic skin, localized in
  basolateral plasma membrane in FRC layer
• In pectoral region, dot spot only in cytoplasm of
  few cells in FRC layer
• Intensity of labeling decreased from hindlimb to
  pectoral skin

Pelvic
Pectoral
Hindlimb
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Results Aquaporins in 3 skin regions

• B. marinus
• AQP-h3 and AQP-h2 in all regions
• Predominantly in cytoplasm just beneath apical
  membrane
• Number of cells varied among toads (less in
  pectoral skin of some)
• Western Blot Intensity of bands decreased from
  hindlimb to pectoral skin

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Results aquaporins in 3 skin regions

• Xenopus laevis
• Detected AQP-x3 mRNA expression in skin from
  pectoral, pelvic, and hindlimb regions but not
  dorsal skin
• X. laevis skin not stimulated by AVT

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Results Water permeability and movement of AQPs
after stimulation with AVT

• Rana japonica and Rana nigromaculata
• Stimulation at hindlimb
• AQP-h3 in apical plasma membrane in FRC layer

Rana japonica
Rana nigromaculata
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Results Water permeability and movement of AQPs
after stimulation with AVT

• Bullfrog
• Stimulation increased in order of pectoral,
  pelvic, hindlimb regions
• Translocation of AQP-h3 protein to apical plasma
  membrane of FRC layer greater in hindlimb region
  and decreased in pelvic and pectoral region

hindlimb
pectoral
pelvic
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Results Water permeability and movement of AQPs
after stimulation with AVT

• B. marinus
• Stimulation variable depending on individuals and
  regions of skin but above controls
• ½ of toads response greatest in hindlimb,
  declined in pelvic and pectoral skin
• Other ½ response greatest in pelvic skin
• Translocation of AQP-h3 and AQP-h2 to apical
  plasma membrane of cells in FRC layer of
  hindlimb, pelvic, and pectoral regions

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Results Water permeability and movement of AQPs
after stimulation with AVT
For Bufo marinus
Hindlimb
Pelvic
Pectoral
Skin-type AQP-h3
Bladder- type AQP-h2
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Results Water permeability and dynamic movement
of AQPs after stimulation of AVT and hydrins

• AVT and hydrin 1 and 2 increased water
  permeability of hindlimb skin in
• R. japonica gt R. nigromaculata gt R.
  catesbeina gt B. marinus
• No differences among hormone response within
  species
• Increased water flux rates (relative to
  controls)
• 3038 X in Rana japonica
• 15 X in Rana nigromaculata
• 812 X in Rana catesbeina
• 3 or 4 X in Bufo marinus
• When hindlimb skin from each species stimulated
  with AVT following HgCl2 treatment, ratio of
  water flux decreased (compared with AVT
  stimulation groups)

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Discussion Importance of AQP-rich hindlimbs for
water absorption

• Area-specific rate of AVT-stimulated water flow
  across hindlimb skin similar for moist and
  dry-adapted species
• Toad AVT-stimulated water flow correlated with
  presence of AQP-h2-like water channel in all skin
  regions
• Rana Catesbeiana AQP-h3-like AQP observed in all
  skin regions
• Rana japonica and Rana nigromaculata AQP-h3-like
  AQP observed only in hindlimb
• Greater response of Toad vs. Rana species in vivo
  could result from relative area of skin that
  contains AQPs rather than an area-specific
  response
• HgCl2 inhibited water flux across hindlimb skin
  under AVT-stimulation.
• AQP proteins are mercury sensitive, so this
  proves waterflux was mediated by AQPs

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Discussion Physiological and behavioral
variables that affect water absorption

• Variable area-specific water flux across toad
  skin could result from greater dependence on
  vascular perfusion relative to thinner frog skin
• Behavioral water absorption response
• Skin pressed to moist surface
• Large increase in blood flow to absorbing area of
  seat patch
• Insertion of AQPs into apical membranes of FRC
  skin layer

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Discussion Phylogenetic significance of AQPs in
ventral pelvic skin

• Largest superfamilies of anurans are Hyloidea
  (includes modern tree frog and toad species) and
  Ranoidea (includes Ranids (typical frogs)
• AQP-h2-like proteins not only in bladder, but in
  skin of tree frog and toad species, which also
  have more pelvic patches
• Apomorphic (only these lineages have this
  character)
• AQP-h3 found in toad, tree frog, and Ranid
  species
• Pleisiomorphic (likley shared with common
  ancestors)
• Present in all ventral skin regions of Rana
  Catesbeiana, while only present in hindlimbs of
  Rana japonica and Rana nigromaculata
• New World Rana genus recently reclassified as
  Lithobates, including Rana Catesbeiana
• Rana japonica and Rana nigromaculata remain in
  Old World Rana genus

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Discussion Expression of 2 AVT-stimulated AQPs
in skin of toad and tree frog species

• AQP-h2 homolog detected in bladder of all species
  examined but in skin of only toad and tree frog
  species
• mRNA encoding AQP-h3 homolog identified in skin
  but not bladder of all species examined
• Based on genetic analyses of Xenopus tropicalis,
  likely that h2- and h3-like AQPa2 genes were
  generated by local gene duplication of AQP2 in
  anuran lineage
• For contemporary anurans h2-like AQPa2 occurs in
  bladder, while h3-like AQPa2 is expressed in
  ventral skin
• In toad and tree frog species, h2-like AQPa2
  gene may have undergone a change to express this
  gene in the ventral skin, not just the bladder
• Might give terrestrial species an advantage
  cutaneous water absorption / adaptiaton to drier
  environments

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Discussion A unique AQP in aquatic Xenopus

• No hydro-osmotic response to AVT
• Identified mRNA for AQP-x3 in pelvic skin
  homologous to that for AQP-h3, but contains extra
  C-terminal tail preventing translation
• AQP-x3 present in all 3 skin regions
• Data lacking on possibility of expression during
  dry periods

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Discussion Regulation of AQP expression by AVT
and related peptides

• Hydrin 1 and 2 stimulated water permeability of
  hindlimb skin of toad and tree frog species at
  level equivalent to AVT
• Km values for cAMP production by tree frog
  V2-type AVT receptor suggests hydrin 1 and 2
  share a common receptor
• Both peptides generated from down-regulation in
  post-translational processing
• Xenopus laevis secretes hydrin 1 and AVT but
  shows no hydro-osmotic response to either in skin
• Xenopus laevis AVT and hydrin 1 stimulate water
  reabsorption from bladder
• May be involved in water balance during
  aestivation

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Perspectives and Significance

• Anurans have 2 AQP isoforms stimulated by AVT to
  increase water absorption across ventral skin and
  re-absorption from bladder
• All species examined express AQP-h2-like AQPs in
  bladder
• Only semi-terrestrial toad and tree-frog species
  express AQP-h3-like AQPs and AQP-h2-like AQPs in
  skin
• Semi-aquatic Ranids express only AQP-h3 in skin,
  primarily in ventral surface of hindlimbs
• Aquatic Xenopus laevis transcribes mRNA for
  homologs of both isoforms but a C-terminal
  sequence prevents translation
• Future studies needed to examine species
  differences in expression of AQP-h2 and AQP-h3 to
  examine phylogenetic relationships associated
  with water balance adaptations