Bullfrog tadpoles retain water when injected with vasotocin. This response is very small in young tadpoles but increases later, especially just prior to metamorphorsis. Vasotocin also increases the osmotic permeability of the skin in such tadpoles. The quantities of neurohypophysial peptides stored in the tadpoles’ pituitary are about one-fifth of those seen in adult bullfrogs.
Most adult Amphibia respond to injected neurohypophysial peptides by retaining water (‘(water balance effect,” Heller, 1941). In anurans this response is due to an increased water uptake through the skin, a decreased rate of urine formation by the kidney, and an increased water reabsorption from the urinary bladder. Urodele Amphibia generally only exhibit the renal response (Bentley and Heller, 1964). Water retention in response to injected neurohypophysial peptides is greater in terrestrial than in aquatic species; it is, for instance, absent in the aquatic anuran Xenopus 1aevi.s and is poor in the aquatic urodele Necturus maculosus (see Heller and Bentley, 1965). Anuran tadpoles also exhibit little or no response to such peptides until they reach a stage when they begin to metamorphose (Howes, 1940; Alvarado and Johnson, 1966) * The present experiments have reexamined the “water balance effect” in the tadpoles of the bullfrog (Runa catesbeiana) and have measured the amount of neurohypophysial peptides present in the pituitaries of these larvae. Bullfrog tadpoles weighing 7 to 19 gm were either taken from ponds or obtained from a commercial supplier. They were kept in aquaria in the laboratory for at least a week before they were used for experiments. They were fed on algae or let412
tuce. Injections of synthetic %argininevasotocin or control solutions were made by puncturing the skin at the base of the tail with a 26-gauge needle and threading this under the dorsal skin to a region just behind the head where the injection was made. The tadpoles were then weighed at hourly intervals by draining them of water and placing them into a tared beaker of water. The water transfer across the skin was measured in vitro by tying the skin from the animals ventral surface onto the end of a piece of glass tubing to make a diaphragm with an area of 2 cm2. The outside (epidermal surface) was bathed with a dilute solution (10 mM choline chloride) while the inside was bathed with Ringer solution which was aerated. The 8-argininevasotocin was added to the inner solution. Water transfer was measured by weighing the tube and attached skin and recording the change in weight. The hormonal contents of the neurohypophyses were assayed in extracts of pooled glands (from 14 to 32) dissected from decapitated tadpoles. These were dried in acetone and extracted (twice) in hot 0.25% acetic acid for 5 min and the supernatant separated by centrifugation. The activities of the extracts were measured by recording change in osmotic permeability of isolated urinary bladders from toads (Bufo ma&us) using a 2 + 2 assay design (Sawyer, 1960; Bentley, 1969)
-2.9 +O.l +0.2 +0.9
f + f f
Control 0.6 (8) 0.6 (6) 0.6 (6) 0.7(8)
-0.2 +6.9 +9.5 +11.3 BW
f k f f = body
0.1 0.7 2.6 1.6
Vasotocin (10-S M/kg)
balance effect” in BW 3 hr)
a The results are as means +SE No. of experiaents in parentheses. a Activity equivalent to that of these amounts of oxytocin. E From Bentley (1969).
“Limb bud” (stages III-V) “Premetamorphic” (stages XI-XVIII) “Metamorphic” (stages XX-XIII) Adult
f IL f +
1.1 0.69 0.54 13.7c
4.7 4.9 5.0 61
f 1.0 (12) f 0.6 (12) f 0.53 (12) f 3.5
3.2 2.3 3.3 13
1 IN BULLFROQ
3.8 1.6 1.9
f 1.3 f 0.45 f 0.54 -
39 61 42 280
* 4 (9) f 14 (10) f 9 (9) f 36~ (9)
Activity in neurophysis, U (oxytocin)b/kg BW
and oxytocin (Syntocinon, Sandoz) as a standard. The stage of development of the larvae is described according to the method of Taylor and Kollros (1946). When vasotocin (synthetic, Sandoz, 245 mU rat pressor/mpmole) was injected into bullfrog tadpoles which were at a stage of their development when only limb buds were present, there was no measurable increase in water retention, but the water loss normally observed in the control larvae was reduced, indicating a small action (Table 1). When such experiments were carried out on tadpoles in the “premetamorphic stages” (developing hind limbs), there was a prominent retention of water, and this was even greater in tadpoles whose forelimbs had also appeared (“metamorphic stages”). These results confirm those of Alvarado and Johnson (1966). Isolated skin preparations were made from tadpoles in each of these three groups, and the osmotic permeability to water was measured. In the “limb bud” group no change in permeability of the skin to water could be measured after exposing it to vasotocin. In the other two groups, however, there was an increase of about 100% in permeability to water which is, however, far less than that seen in adult bullfrogs (Table 1). The skin of the youngest tadpoles was fragile so the possibility of damage altering the response cannot be excluded. The permeability of the tadpole skin is much less than that seen in adult frogs and conforms to the pattern seen in aquatic, rather than terrestrial, Amphibia (Bentley, 1969). The hydro-osmotic activity of the stored neurohypophysial peptides present in the tadpole neurohypophyses was also measured in the three groups of tadpoles. The concentration (oxytocin-hydro-osmotic U/kg body weight) in all of the tadpole neurohypophyses was similar, and about one-fifth of that observed in adult bullfrogs (Table 1). As this assay is relatively insensitive to oxytocin or mesotocin, this, presumably, substantially reflects the amounts of vasotocin which are present. The latter peptide has an activity equivalent to 90 hydro-osmotic (oxytocin) U/mpmole
(Bentley, 1969) so that the concentration, *I in mpmole/kg body weight, of vasotocin in the tadpoles varies from 0.4 to 0.7, compared to 3.1 in adult bullfrogs. It is interesting that a similar hydro-osmotic activity of these peptides is also observed in the aquatic urodele Necturus maculosus (Follett and Heller, 1964; Bentley, 1969) which is a neotenous species. The attainment of adult levels of neurohypophysial hormones in bullfrogs must occur after metamorphosis possibly in response to the drier conditions experienced in a terrestrial life.
REFERENCES ALVARADO, R. H., AND JOHNSON, S. R. (1966). The effects of neurohypophysial hormones on water and sodium balance in larval and adult bullfrogs (Rana catesbeiana). Comp. Biochem. Physiol. 18, 549-561. BENTLEY, P. J. (1969). Neurohypophyseal hormones in Amphibia: A comparison of their actions and storage. Gen. Comp. Endocrinol. lsi
39-44. BENTLEY. P. J.. AND HELLER, H. (1964). The actions of neurohypophysial hormones on t.he water and sodium metabolism of urodele amphians. J. Physiol. London 171, 434-253. FOLLETT. B. K.. AND HELLER, H. (1964). The neurohypophysial hormones of lung-fishes and amphibians. J. Physiol. London 172, 92-106. HELLER, H. (1941). Differentiation of an (amphibian) water balance principle from antidiuretic principle of the posterior pituitary gland. J. Physiol. London 100, 125-141. HELLER, H.. AND BENTLEY, P. J. (1965). Phylogenetic distribution of the effects of neurohypophysial hormones on water and salt metabolism. Gen. Comp. Endcrinol. 5,96-108. HOWES, N. H. (1940).The response of the water regulating mechanism of developmental stages of the common toad Bufo bufo bufo CL.) to treatment with extracts of the posterior lobe of the pituitary body. J. Ezp. BioZ. 17, M-138. SAWYER, W. H. (1960). Increased water permeability of the bullfrog (Rana catesbeiana) bladder in vitro in response to synthetic oxytocin and arginine vasotocin and to neurohypophysial extracts from non-mammalian vertebrates. Endocrinology 66, 112-120. TAYLOR, A. C., AND KOLLROS, J. J. (1946). Stages in the development of normal Ranu [email protected]
~ larvae. Anat. Rec. 94, 7-23.
P. J. BENTLEYI L. GREENWALD~ Departments of Ophthalmology and Pharmacology Mt. Sinai Medical School of the City University of New York New York, New York, 10029, and ‘Supported
Deparbment of Zoology Duke University Durham, North Carolina 27706 Received October 10, 1969
by NIH predoctoral fellowship No. 657-03 (General Medical Sciences).