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SEASONAL VARIATIONS IN ENERGY METABOLISM AND NEUROHYPOPHYSEAL HORMONE ACTION ON WATER AND

SODIUM TRANSPORT IN FROGS (RANA TEMPORARZA) AND TOADS (BUFO MARZNUS)

DAE SUK HAN, YANG SAENG PARK and SUK KI HONG*

Departments of Physiology. Yonsei University College of Medicine, Seoul. Korea; University of Hawaii School of Medicine, Honolulu, HI 96822;

and State University of New York at Buffalo, Buffalo, NY 14214, U.S.A.

(Received 25 January 1978)

Abstract-l. Seasonal effects of neurohypophyseal hormone (oxytocin or vasopressin) action on water and Na transport across the skin and urinary bladder have been studied in frogs (R. trmporaria) inhabiting temperate zones and in toads (B. marinus) inhabiting subtropical zones.

2. The osmotic water transport across the skin in intact frogs immersed in distilled water and treated with oxytocin (lOi.u./lOOg body wt) was approximately 50% greater during cold months (October- March) than during warm months (April-September).

3. The glycogen content of the frog skin was significantly higher during cold months (86g/lOOg wet tissue) than during warm months (38g/lOOg wet tissue). However, the skin water, Na and K contents showed no seasonal variation.

4. Short-circuit current response to vasopressin (250 mu/ml) in isolated toad skin and osmotic water transport response to vasopressin (lOOmU/ml) in isolated toad urinary bladder showed no significant seasonal variation.

5. Glycogen content of liver and muscle and plasma levels of glucose, Na, K, and osmolality showed no seasonal variation in the toad.

6. These results taken together with our previous findings (Hong et ul., Am. J. Physiol. 215, 439-443. 1968) strongly suggest that variations in the effect of neurohypophyseal hormone on water and Na transport in amphibian preparation may be attributed to seasonal variations in tissue metabolic energy stores

INTRODUCTION

The stimulating effect of neurohypophyseal hormone on sodium transport across the frog skin undergoes marked seasonal variations. Herrera & Curran (1963) reported that vasoprqssin induced a sustained in- crease in sodium transport across the isolated frog (Runu pipiens) skin in cold months, whereas it had a transient effect in warm months. Similar observa- tions have been made by Hong et al. (1968) using a different species of frog, Rana temporaria. The latter studies have also indicated that the seasonal changes in vasopressin action are accompanied by parallel changes in the tissue glycogen store. Seasonal differ- ences in the magnitude of vasopressin-induced water transport across the skin have also been noted in the toad (Bufb hufi) (Jorgensen, 1950; Share & Ussing, 1965), although sufficient data are not available to define a clear pattern. Moreover, no attempt has been made in the past to examine seasonal variations in neurohypophyseal hormone actions in the amphibia inhabiting subtropical regions.

We therefore investigated in the present studies the seasonal characteristics of the action of neurohypo- physeal hormone in frogs (Rana temporaria) and toads (Bujii marinus). The results indicate that in the frog

*Send reprint requests to: Dr Suk Ki Hong, Depart- ment of Physiology, State University of New York at Buf- falo, 120 Sherman Hall, Buffalo, NY 14214, U.S.A.

of the temperate zone the magnitude of the water movement across the skin induced by neurohypophy- seal hormone is significantly higher during the cold months (October-March) than during warm months (AprilLSeptember), whereas in the subtropical toad there is no apparent seasonal variation in the effects of neurohypophyseal hormone either on the sodium transport across the skin or on the water permeability of the urinary bladder.

MATERIALS AND METHODS

A. Experiments on frogs

Frogs, Rana temporaria, of either sex were captured monthly in the field near Seoul, Korea, and were brought to the laboratory where they were kept fasting in tapwater at room temperature of approximately 2@ C. The experi- ments on these animals were performed within 2 weeks. The annual variation of the ambient temperature in the habitat of these animals is depicted in Fig. 1.

In the first series, the effect of a neurohypophyseal hor- mone on the water transport across the sk;n was studied at 25°C in the whole free bv the method described earlier (Hong, 1957). At the beg&&g of each experiment the uri- nary bladder was emptied by abdominal pressure and the cloaca was ligated. The animal was then weighed to the nearest 0.1 g, and was placed for 3 hr in a covered beaker containing 1OOml of distilled water which was aerated. At the end of each hour the animal was taken out and weighed. The gain of weight expressed as a percentage of initial body weight was considered as the net water trans- port across the skin, since a frog does not usually drink

665

666

I I I I I,, , , , I 2 3 4 5 6 7 8 9 IO II 12

Month of the Year

Fig. 1. The annual variatton in the ambient temperature in Seoul. Korea and in Honolulu. HI.

water (Gordon. 1964). Half of the frogs were controls and the other half treated with oxytocin. which is known to have a greater effect on water transport than vasopressin (Sawyer rr ui., 1950). Ten international units of synthetic oxytocm (Nutritional Biochemicals Corp.) per 100 g body weight were injected into the dorsal lymph SX. This dose gives the maximal response (Sawyer (‘i rri., 1950).

In the second series, the glycogen content of the skin was determined by the methods of Hassid & Abraham (1957) and Nelson (1944). Na and K concentrations of the skin were also determined. The skins. excised from the abdomen, were weighed and dried in an oven (1 IO-Ci for 48 hr. After reweighing, each sample was digested in 0.5 ml of concentrated nitric acid and then diluted with distilled water to a constant volume before determinations of Na and K with a flame photometer.

Toads. BU$I tntrrin~rs. of either sex. captured monthly in the field near Honolulu. HI, IJ.S.A., were maintained in damp sand (23 25 C) and fasted for at least 3 days before use. All experiments on these ammals U?ere performed within 2 weeks. The annual variation of the ambient tem- perature in the habitat of these animals IS also shown in Fig. 1.

In the first series, the etfect of neurohypophyseal hor- mone on the sodium transport was studied at 25 C in the isolaied skin preparation. The abdominal skin was removed from the animal and mounted as a flat sheet between two Lucite chambers having a cross-sectional area of 3.14cm’. The short-circuit current (XC) technique of Ussing & Zerahn (1951) was used as the measure of active sodium transport. The potential difference across the skin was measured with a pair of calomel eiectrodes connected to the solution reservoirs bq cellulose gum -Ringer bridges. Current was driven through the skin \ia Ag--AgCI elec- trodes connected to the chambers by another pair of salt bridges. The solution bathing both sides of the skin was continuously stirred and aerated with a stream of air. The composition of the Ringer solution was as follows (in mM): NaCl 114. KCI 3.5. NaHCO, 2.X. CaCI, 0.89 and pH 7.8. After the baseline SCC was established, vasopressin (Pitres- sin; Parke. Davis & Co.) was added to the inside bathing medium at a concentration of 25OmlJ ml and changes in SCC were followed for the next 6Omin.

In the second series. the effect of neurohypophyseal hor- mone on the water permeability of the urinary bladder was estimated by measuring the net water transport across the bladder along an osmotic gradient at 25 C using a modification of BentIcy’s method (1958). Paired hemi-blad- ders were excised. and tied on to polyethylene tubes con- nected to syringe needles. forming sacs with a serosal sur- face outwards. Each sac was tilled with Zml of 1:5 Ringer solution (16.7 mM Nafl. 3.5 mM KCI. 2.X mM NaHCO,. and O.X9mM C~CI,I and suspended in 40ml of normal Ringer solution. The osmotic water transport across the

Figure 7 shows the aLerage rate of water tr;msport .

across the skin in control and oxytocin-treated frogs in each month of the year. The year has been divided into two portions: cold (October -March of the foi- lowing year) and warm (April September). In general, basal water transport in the control group was slightly higher (P < 0.05) during warm months (10.5”,, body wt!3 hr: N = 401 than during cold months (8.7: N = 48). The administr~ition of oxytocin resulted in a significant increase in the water transport in both seasons. However. the effect was distinctly greater during the fall and winter months. The average rates of water transport in ox~t~~cin-tr~atcd frogs were 27.9 + 7.0 (SE.) (N = 48) and IX.2 + 7.0 tzV = 40) I’/, body_wt:3 hr for cold and warm months. respectively (P < 0.005).

B. G’I~crigc~. Na iirrti K ~0nfoni.s cif rhea jiq ?;!&I

As shown in Table I. the glqcopen content of the skin was significantly lower (P CI 0.005) during the spring and summer months (3X 1~: 4 mg, 100 g w-et tis- sue: Ri = 20) aa compared to cold months (86 I 7 mg,;lOO g wet tissue: N =- SW. However. there were no apparent seasonal changes in water. Na and K contents (Fig. 3). On average. water content of the skin was maintained at 75 @.I’,,. \+hile both Na and K concentrations were 40 50 m-eyuiv:kg wet tissue throughout the year.

Figure 4 shows both the basal SCC and peak SCC response to vasopressin in isolated toad skin. There

Gl! cogen contenl Month (mg I(W) 1 wet tlssut’l

_______ _I __ ~~.___ ._______._-_-. Warm season,

April XI.3 i 7.5* May ‘0.9 i 4.6 June 11.x _t_ 3.9 July 11.7 _t 3.s September 39.1 i 3.5

Mean + S.E. 17.9 t 3.9

(‘old \L’asons October 7x.x 5 3 6

November 9.2.6 _i~ 10.1 Mean + S.E. X6.2 * 7.0* ._

* Valuea arc means ( .~S.E.I of 10 frogs in each month. t Significantly higher than that of the ~:um season\

Neurohypophyseal hormone action in Amphibia 661

40 z i w-l

2 30 m ,-”

-:

d 20 z 3

5 IO

E

0

&j CONTROL (OXYTOCIN

Cold

Month of the Yeor

Fig. 2. The rate of water transport across the skin of the control and oxytocin-treated frog. Each value represents

the mean (*SE.) of eight frogs.

was no apparent seasonal variation in either the basal or the vasopressin-stimulated SCC. Annual means of the basal SCC and the peak SCC after vasopressin administration were 64 k 2.8 and 307 f 8.1 pAj3.14 cm2 (N = 181). respectively (P < 0.001). Similarly. osmotic water transport across the isolated urinary bladder in the absence (basal) and presence of vaso- pressin did not show any significant seasonal change (Fig. 5). Annual means of basal water transport rate and peak water transport rate in response to vaso- pressin were 1.8 f 0.12 and 16.5 + 0.59 pl/min (N = 38), respectively (P < 0.001).

D. Tissue glycogen content und plasma composition in toads

The glycogen contents of liver and muscle are shown in Fig. 6. For the purpose of comparison, the

Fig. 3. Water, Na and K contents of the frog skin. Each value represents the mean (kS.E.) of 10 frogs.

corresponding glycogen contents in the frog (Hong et ul., 1968) are also included. Frogs show dramatic seasonal variations in the tissue glycogen levels. How- ever, there were no clear seasonal changes in tissue glycogen contents in the toad. Regardless of the sea-

mc “E 14 3 IO 9 7 17 30302426lI N

0 ‘loo d _

T 300 c! 2.

7 8 9 IO II 12

Month of the year

Fig. 4. The SCC before (basal) and after vasopressin treat- ment in the isolated toad skin. Vertical bars represent

) S.E.

.c ,E 16 6 4 4 4 4 N i 20

t

:

Peak transfer after “asopressln

LCL- Basal transfer

7 IO 12

Month of the year

Fig. 5. Osmotic water transport across the isolated toad bladder before (basal) and after vasonressin treatment. Ver-

tical bars represent ‘kS.E.

a

0 I 2 3 4 5 6 7 a 9 IO II Ii

1234: i 6

R tempofah

“0 tl femporofio I

ll__dl ‘\ c.

7 a 9 to II I;

B1 morhs

Month of the Year

Fig. 6. Glycogen contents of liver and muscle of the frog (R. temporaria) and the toad (B. marinus). Each value for B. marinus represents the mean (+S.E.) of 610 animals. The values for R. temporaria are from Hong et al. (1968).

70

1 P glffcose

i50 y,‘\,/6-6 t +

6

- 4r

I 2 3 4 5 6 7 8 9 lo II 12

Month of the Year

Fig. 7. Plasma levels of glucose. Na. K and the osmolality in the toad (S. mwiu\). Each value represents the mean

son, the tissue glycogen stores of the toad were main- tained relatively high (approx;ma[cly 2-rl”,, in liver and O.lS-0.25”, in muscle). Similarly, the plasma levels of glucose, Na, K and osmolality revealed no apparent seasonal variations in toads (Fig. 7).

The present study on the intact frog indicates that the oxytocin-induced water transport across the skin was considerably greater in cold months than in warm months (Fig. 2). This suggests that the stimula- tory effect of oxytocin on water transport across the skin undergoes seasonal changes similar to those of vasopressin on Na transport across the isolated frog skin (Hong CC ul., 1968). However. the nature of these seasonal variations in the action of neurohypophyseal hormone is not clearly understood.

In a previous study (Hong of af., 1968) we observed that there is a striking correlation between the gly- cogen store of the tissue (liver, muscle and skin) and the Na transport response of the skin to vasopressin, and, furthermore, that the summer frog skin which is ordinarily refractory to vasopressin becomes re- sponsive to this hormone if the skin is fortified with pyruvate or fi-hydroxybutyrate. However, the gly- cogen content of the skin was not directly measured, although its level. estimated by the periodic acid- Schiff (PAS) staining, appeared to be much lower in summer frogs than in winter frogs. In the present study, the glycogen content of the skin was signifi- cantly higher in cold months than in warm months (Table 1). It thus appears that seasonal changes in the metabolic substrate content of the skin may be in part responsible for corresponding changes in the neurohypophyseal hormone action in the frog skin,

Neurohypophysea~ horm one action in Amphibia

tents in liver and muscle in the subtropical B&J pntir- inus, as observed in the present study (Fig. 6), would suggest that there are no such seasonal variations in metabolism in tropical amphibia.

Seasonal variations in blood glucose levels have been reported in &&I bufi, a species of toad inhabit- ing temperate zones. Hermansen & Jerrgensen (1969) observed that the blood glucose of these animals was high during summer and low during winter. showing the peak tevel at the breeding time. lnter~stingl~ enough. they found no seasonal variation of blood glucose in toads kept in the laborqtory at a constant temperature (2~,22-C). Furthermore, in toads kept in the refrigerator at about 2’C, the blood ghicose vahie remained at the level of hibernating animals. They concluded that, outside the breeding period, the sea- sonal variations in blood glucose are mainly due to variations in temperature. If this is true. then one would expect no seasonal variation of blood ghxose in the toad of s~ibtro~i&aI zones. Indeed. in the present study the blood glucose of Bt& ~iar~~us, which has no specific breeding season in Nawaii, showed no apparent seasonal change (Fig. If.

iayiz_ Vol. 3 (Edited by COLOWICK s. P. & K.u’LAN

N. 0.). p. 37. Academic Press, New York. HERMANSEN 13. & JBRGENS~N B. (1969) Blood glucose in

male toids (~uf;l huj‘o): annual variation and hormonal regulation. Gun. camp. Endocr. 12, 313-321.

HERRERA F, C’. & CURRAN P. F. (1963) The effect of Ca and antjdiuFet~c hormone across frog skin. 1. Examin- ation of interrelationship between Ca and hormone. J. gen. Physiot. 46, 999- 1010.

HUNG S. K. (1957) Effects of pituitrin and cold on wdtcr CXChdngCS in frogs. .~ittt. f. Pltysioi. 188, 43% 447.

HONG S. K., PARK t. S.. PARK Y. S. & KIM J. K. (~9t%) Seasonal changes of antidiuretic hormone action on sodium transport across frog skin. &r. J. ~~~si~~/. 215, 439-443. .

JURGENSEX C. B. (1950) The ampbib~an water economy, with special regard to the effect of neurohypophyseal extracts. Acre physiol. .scund. 22 (Suppl. 78). 7 71.

JUNC;REIS A. M. (1970) The effects of long-term starvation and acclimation temperature on glucose regulation and nitrogen anabotism in the frog. Run(z ~~~j~~.~ Ii. Sum- mer animals. frump. 3j~i~~~~?~. Ph_~sit~i. 32, 433-444.

JUNGREIS A. M. (1974) Seasonal effects of dehydr~~ti~~n in air on urea production in the frog, R~rntr pip&s. Camp. Biochum. Ph,wiol. 47, 39. 50.

JUPJGREIS A. M. d HOOPE:K A. B. (19’70) The effects of long- term starvation and acclimation temperature on ply- cogen regulation and nitrogen anabolism in the frog. Rlrnu pip&is- I. Winter animals. (I‘ctnzp. Bir&zPm. Phy- siol. 32, 4 1 i-432.

AcknowleriUmlents--This investigation was supported in part by a China Medical Board of New York Grant No. 65-847 (Project 3).

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