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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. Introduction.

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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


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 argininevasotocin(AVT): causes fusion of vesicles containing AQPs with apical membrane of epithelial water absorption/reabsorption tissues


Introduction

  • Researchers used Real Time Polymerase Chain Reaction (RT-PCR) to identify 2 forms of AQP in epithelial tissues

  • AQP-h2 (isoform)

    • Termed “urinarybladder-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 andRanaspecies


Study Species

Hyla japonica

(treefrog)

Bufomarinus

(terrestrial toad)

Xenopuslaevis

(aquatic)

Ranacatesbaiana

aka bullfrog

(semi-aquatic)

Rana japonica

(semi-aquatic)

  • Rananigromaculata

  • (semi-aquatic)


Table 1. Phylogenetics of aquaporins in ventral pelvic skins of anuran species living in different habitats


Introduction

  • AQP-x3 mRNA homologous to AQP-h3 expressed in pelvic skin of aquatic species, Xenopuslaevis

    • but not translated toprotein

  • 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 Hylajaponica


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


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


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

III

II

kDa I II III

Protein

Molecular

Weight

Values

I


Materials and Methods: RT-PCR of Xenopus Ventral Skin AQP-x3

  • RNA extracted from ventral skin and reverse transcribed

  • Gel electrophoresis

  • DNA Sequenced


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


Materials and Methods: Water Permeability

  • Effect of AVT on hindlimb skin permeability compared with hydrins1 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-Dwass’s test using software


Results:Aquaporinsin 3 skin regions

Ranajaponica and Rananigromaculata:

  • AQP-h3 (skin-type) in hindlimb region only

  • Ranajaponica: inbasolateral, apical, and cytoplasm of FRC

  • Rananigromaculata: basolateral plasma membrane

Ranajaponica

Rananigromaculata


Results:Aquaporins in 3 skin regions

Ranacatesbeiana:

  • GreatestAQP-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


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

Pectoral

Hindlimb

Pelvis

Skin-type

AQP-h3

Bladder-type

AQP-h2


Results: aquaporinsin 3 skin regions

Xenopuslaevis:

  • Detected AQP-x3 mRNA expression in skin from pectoral, pelvic, and hindlimb regions but not dorsal skin

  • X. laevisskin not stimulated by AVT


Results: Water permeability and movement of AQPs after stimulation with AVT

Ranajaponica and Rananigromaculata:

  • Stimulation at hindlimb

  • AQP-h3 in apical plasma membrane in FRC layer

Ranajaponica

Rananigromaculata


Results: Water permeability and movement of AQPs after stimulation with AVT

Bullfrog:

  • Stimulation increased in order of pectoral, pelvic, hindlimbregions

  • 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


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

  • Translocationof AQP-h3 and AQP-h2 to apical plasma membrane of cells in FRC layer of hindlimb, pelvic, and pectoral regions


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


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 > R. nigromaculata > R. catesbeina > B. marinus

  • No differences among hormone response within species

  • Increased water flux rates (relative to controls):

    • 30–38 X in Ranajaponica

    • 15 X in Rananigromaculata

    • 8–12 X in Ranacatesbeina

    • 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)


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

  • RanaCatesbeiana: AQP-h3-like AQP observed in all skin regions

  • Ranajaponica and Rananigromaculata: AQP-h3-like AQP observed only in hindlimb

  • Greater response of Toadvs. Ranaspecies 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


Discussion: Physiological and behavioral variables that affect water absorption

  • Variable area-specific water flux across toadskin 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


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 RanaCatesbeiana, while only present in hindlimbs of Ranajaponica andRananigromaculata

      • “New World” Rana genus recently reclassified as Lithobates, including RanaCatesbeiana

      • Ranajaponica andRananigromaculataremain in “Old World” Rana genus


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 Xenopustropicalis, 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


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


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

  • Xenopuslaevis: secretes hydrin 1 and AVT but shows no hydro-osmotic response to either in skin

  • Xenopuslaevis: AVT and hydrin 1 stimulate water reabsorption from bladder

    • May be involved in water balance duringaestivation


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 toadand tree-frog species express AQP-h3-like AQPs and AQP-h2-like AQPs in skin

  • Semi-aquatic Ranids express onlyAQP-h3 in skin, primarily in ventral surface of hindlimbs

  • Aquatic Xenopuslaevistranscribes 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 phylogeneticrelationships associated with water balance adaptations


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