Lack of effect of rat atrial natriuretic factor (rANF) on the renal function in frogs

Comp. Biochem.Physiol. Vol. 9OA,No. 3,pp.46W69, 1988

0300-9629/88 53.00+0.00 0 1988Pergamon Pressplc

Printed in Great Britain

LACK OF EFFECT OF RAT ATRIAL NATRIURETIC FACTOR (rANF) ON THE RENAL FUNCTION IN FROGS*? ANSELM FIUCK and AHMET TOYGAR Physiologisches Institut der Universitiit Mtlnchen, Pettenkoferstrasse 12, D-8000 Miinchen 2, FRG Telephone: (089) 5996 382 (Received

30 November

1987)

Abstract-l.

The aim of the present experiments was to examine the question whether the rat atria1 natriuretic factor (rANF l-28) could alter the fractional excretion of sodium (FE,,) and other solutes in the frog (Runa esculentn). 2. Although experiments were performed throughout the year possible seasonal changes in the animals were considered in particular. 3. In all frogs, a hypotonic diuresis was induced. 4. Under these conditions in winter frogs, the control FE, was 8.8 + 5.8% (15) [means f SD (n)], and during rANF administration 7.7 & 6.6% (13) (NS). 5. In summer frogs, the control and experimental FE,, was 5.2 + 2.8% (5) and 6.0 + 2.5% (5) respectively (NS). 6. These results show that there was no significant effect of this polypeptide on the fractional excretion of sodium in the frog.

INTRODUCTION Within the context of the current interest in recently defined atria1 natriuretic factors and their roles in the homeostasis of water and electrolytes and the blood pressure (De Wardener and Clarkson, 1985; Maack et al., 1985; Needleman et al., 1985) the question of

species differences and species cross-reactivity has arisen. De Bold and Salerno (1983) tested atria1 extracts of a series of different species such as man, cow, pig, rat, mouse, frog and chicken in bioassay rats. They found an increase in the excretion of sodium in response to all extracts, including the amphibian extract, with the exception of the chicken extract. On the other hand, Pamnani et al. (1984), using atria1 extracts from the rat, and Marumo and Sakamoto (1985), employing a synthesized human atria1 natriuretic peptide, both failed to demonstrate an effect on the sodium transport in the toad bladder in vitro.

The aim of the present study was to test the synthetic rat atria1 natriuretic factor (rANF l-28) on the renal function of intact frogs. In this in-vivo approach, a continuous intravenous infusion of rANF with different doses in clearance experiments was employed. The results in these studies document clearly that there was no significant effect of rANF on the renal function in the amphibian. Thus, both the former in-vitro (Pamnani et al., 1984; Marumo and Sakamoto, 1985) and our in-vivo results would suggest that there is a species difference in the receptors *This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Fr 239/g-2 and Fr 239/g-3). TDedicated to Professor Dr Eekehart Gerlach.

for the atria1 natriuretic peptides at least in the rat and these studied amphibians. MATERIALS AND

METHODS

Adult female frogs (Rana esculenra, about 100 g body wt) (R. Stein, Lauingen-Donau, FRG), kept in running spring water in tanks and fed once per week with sliced beef meat, were anesthetized percutaneously with MS 222 (Tricaine; Serva, Heidelberg, FRG) by immersing the animals in a bath containing this drug at a concentration of 200 mg/liter. In order to achieve adequate and stable urine flows, each frog was given a water load of 5 ml into the stomach and, throughout the experiment, an intravenous infusion of either water of frog Ringer’s solution (see below). The surgical procedure consisted of the insertion of catheters into a vein (V. brachialis), an artery (A. ischiadica) and both ureters by a dorsal approach (see Fig. 1). Water was infused intravenously (75 pl/min) for 1 hr, followed by frog Ringer’s solution (102.7 mM NaCl, 1 mM KCl, 0.9 mM CaCl,, 1.19 mM NaHCO,) for 1 hr at the same infusion rate, by which time urine flow generally matched infusion rate. The infusion of the frog Ringer’s solution was continued and the clearance collections were made. The amino acid sequence of the rat atria1 natriuretic factor used was: H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-GlyLeuGlyCys-Asn-Ser-Phe-Arg-Tyr-OH; M.W. 3062.30 g/ mol (Bachem, Bubendorf, Switzerland). Each batch of rANF was tested for diuretic activity by using a bioassay rat and found to be active. The polypeptide rANF was administered by priming dose and a prolonged intravenous infusion usually starting 45 min before the first clearance collection and continued throughout the experiment. This procedure was used because pilot experiments showed that intravenous bolus injection with different doses of rANF did not change either urine flow or sodium excretion. 465

466

ANSELM FRICKand AHMET TOYGAR

Fig. I. Surgical procedures (a-c) in a dorsal approach for the insertion of the catheters (d) in the ureters of a frog (R. esculenta).

Experiments with shorter infusion of rANF were also carried out. In order to examine the effect of this atria1 factor on renal function in frogs, three series of experiments were performed: Series 1

In these studies, we increased the priming dose of rANF from 0 (control), to 0.05, 0.4 (see Hammond et al., 1985), 0.85, 1.7 and 3.4!cg per frog. (Assuming a volume of distribution of 25% of the body weight, the approximate concentrations of rANF in the extracellular Ruid would have been about 10e9 to lo-‘M.) This was in order to observe any possible dose-response relationship between this drug and the renal parameters. Each of these six groups of animals respresents a different dose of rANF. Finally, furosemide (Lasix, Hoechst, Frankfurt/M., FRG) in a clinical dose (0.03 mg per frog), was employed in a separate group. In each experiment the priming dose and the continuous infused dose per hr were identical and ANF and furosemide were administered 45 min before the first collection and throughout the experiment (five collections every 30 min). These groups of experiments were performed throughout all seasons in 1986 not considering any possible renal differences in the frogs between the seasons. Series 2

In these experiments control and experimental frogs were matched in an even more close seasonal relation: thus, “winter frogs” and “summer frogs” were studied at different doses of rANF (both priming and continuous dose per hr were either 0.4 or 3.4pg per animal). The rest of the

experimental protocol was identical to that of Series 1 and again furosemide was administered in a different group. Series 3

In this series each frog was studied under both control and experimental conditions in summer or autumn. The dose of rANF (priming and continuous dose per hr) was 0.4 or 0.05pg per frog and similarly that of furosemide was 0.03 mg per frog as in the other series. Finally, time control experiments were performed. Usually the collection period in these clearance experiments was 30 min; in Series 3 it was 10 min. During each period, urine was collected in small plastic tubes (microtubes 3810, Eppendorf, Hamburg, FRG) and weighed and a sample of IOOjtl blood from the arterial catheter was withdrawn~ and the blood was heparinized. The analytical methods for these experiments are standard techniques and are described elsewhere (Frick and Durasin, 1986): Inulin (Inutest, Laevosan-Co., Linz/Donau, Austria), sodium, potassium, calcium, chloride, inorganic phosphate, osmolarity and hematocrit values were measured in urine, plasma and blood, respectively. Osmolarity was determined by using a micro-osmometer (Knauer, BerlinWest, FRG)._The blood pressure was measured with a Hellige Servomed (Hellige, Freiburg, FRG) and a Gould-P 23ID-USA (Gould Electronics BV, Bilthoven, The Netherlands). The results are expressed as mean values & standard deviations (SD). Significances of the differences were evaluated by Student’s ~-test using unpaired analysis.

Lack of effect of rANF on renal function in frogs

50 z

RANA

ESCULENTA

( Frog’s

Ringer-

467

( CJ, b.w. lC’Jg1

infusion:

50

75yllmin)

20

10 B 0

0

Fig. 2. Control and experimentat in different animals: lack of effect of rANF (different doses) on the fractional excretion of sodium, and a dmmatic effect of furosemide (P < 0.001). Means rf SD, n = number of animals; in each animal about five observations were made (Series I). Summer frogs. In the months of May to July using the higher dose of rANF (3.4pg per frog) again no significant change, either in the blood values or in the renal function was observed. Blood pressure was not decreased nor could an increase in the glomerular filtration rate, urine flow or fractional excretion of sodium be seen (Table 2). However, the administration of furosemide resulted in significant alterations in both blood values and renal parameters except urine flow (Table 2).

RESULTS Series 1: Dose-response relationship between rANlF and the~actionaf excretion of sodium (Fig. 2)

Increasing the dose of rANF from 0.05 to 3.4pg per frog did not result in any significant differences between the experimental and the control in the fractional excretion of sodium in these animals (Fig. 2). However, a low dose of furosemide (0.03 mg per frog) induced a dramatic increase in the fractional excretion of sodium (Fig. 2; P < 0.001). Series 2: Seasonal aspects during rANF administration on the renal function (Tables 1 and 2) Winter frogs. In the lower range of the dose of rANF (0.4pg per frog) during the months January and February, the administration of the atria1 natriuretic peptide did not change either blood values or renal parameters (Table 1).

Series 3

In this group of experiments it was possible to test the effects of rANF (0.4 pg/hr) and furosemide under control conditions in each animal (Fig. 3). Again there was no effect of rANF and again a dramatic effect of furosemide, on the fractional excretion of sodium. The effect of furosemide was not altered when rANF was administered before (Fig. 3). Similar

Table 1. Blood values and renal function during rANF infusion in diuretic frogs studied in winter Control Body weight(9)

Blood values Blood pressure (mmHg)

128+19(n=15)

rANF (0.4 &hr) 115+i5fn=13)

P values NS

28 If: 3 98.9 r?:1.2 86.2 rt 8.1 1.69ttO.12 0.92zfrO.16 217 + 17

21+4 99.2 + 5.5 84.6 & 6.2 1.65 j: 0.21 0.87 +0.12 216 2 12

NS NS NS NS NS NS

2.29 & 0.41 I .79 + 0.35 8.7 f 5.8

2.37 + 0.54 I .88 * 0.47 7.7 + 6.6

NS NS NS

FE0 (%) FE, (%)

13.8 rt 8.4 33.4 & 18.9

11.3k9.9 26.8 + 16.3

NS NS

FE, (%) FEo,,,, PO) @JP),,

82.3 i: IS.3 16.6 f 6.4 0.20 f 0.06

74.2 +- 20.0 14.6 & 7.8 0.17 f 0.08

:: NS

[Nab (mEqi0 PI,, Ws/U I% (mEs/Q @Q&, (mmoW> Psmlp, (mosmol~) Renal functiott: GFR (ml/30 min) V, (ml/30 min) FE,, W)

Means f SD. Abbreviations are as follows: ma],, =concentration of sodium in plasma; Gsm = osmotically active particles; GFR = glomerular filtration rate; Vu= urinary flow; FE,, = fractional excretion of filtered load of Na; = mine-to-plasma concentration ratio for osmotically active particles; w/p),, NS = not significant.

468

APEELM

FRICKand AHMETTOYGAR

Table 2. Blood values and renai function during rANF or furosemide infusion in diuretic frogs studied in summer

Control __l_“__ Body weight (g)

CC) 87k lZ(n =5)

~-

rANF (3.4 ygihr) (I) ___.~____.___. 92i9(n=5)

P values

Furosemide (0.03 mg/hr) (II) 109*16(n=5)

K-I) NS

(C-II) <0.05

3load u&es

Blood pressure (mm Hg)

WI,, WW) [CIIPI (mJW) IUP, @WI) PWl (mmolil) [OsmJpl (mosmol/l)

24 5 3 104.1 i 4.0 89.3 + 2.4 2.04 _t 0.27 0.93 5 0.17 216k9

24 + 2 101.2 + 5.8 84.0 + 9.7 2.06 + 0.23 1.16~0.14 213 + 15

26 & 5 93.7 f 5.8 72.3 f 3.7 2.26 f 0.21 1.15 +0.31 193 + 10

NS NS NS NS co.05 NS

NS <0.025
2.51 * 0.24 1.93+0.13 5.2 + 2.8 5.9 + 5.3 22.2 * 13.4 13.2 f 8.9 9.8 f 4.6 0.13 ao.05

2.23 + 0.27 l.67k 0.18 6.0 f 2.5 10.5 + 5.0 36.1 zk6.8 71.4 1 25.8 13.5 * 2.0 0.18 * 0.02

2.51 + 0.70 2.12 + 0.57 44.3 f 8.0 61.8 + 8.9 118.5 + 7.6 87.7; 5.9 54.6 & 9.8 0.63 + 0.07

NS

NS

<0.05 NS NS NS NS NS NS


Renal function

GFR (m1/30 min) V,, cm1/30 min)

FE;, (oio, FE, (%) FE, (%) F& (%)

FE,, W) w/%m Means + SD. Abbreviations are as in Table 1.

results were obtained using a lower dose of rANF (0.05 pg/hr). Furosemide, however, did not increase the urine flow under these conditions (as in Series 2). DISCUSSION

The present results clearly document that there are no significant effects of rat atria1 natriuretic peptide in the renal function of frogs at least approximately equivalent to 10-9-10-7 mmol/l of rANF. There was no decrease of the blood pressure and no increase in either glomerular filtration rate or in the urinary flow and the fractional excretion of sodium and chloride, parameters which change significantly when rANF is

RANA (Frog’s

I-

administered to the rat (Beasley and Malvin, 198.5; Maack et al., 1985; Needleman et al., 1985). On the other hand, in these studies a dramatic effect of furosemide on the fractional excretion of sodium was observed, which documents the potency of the known high-ceiling natriuretic effect of this drug also in this species and furthermore confirms the adequacy of our experimental model. A number of reasons may be advanced to explain the lack of effect of rANF in the present studies: First, the rat atria1 polypeptide administered may be inactivated by cleavage in the blood of the frog during the experiments by as-yet undefined enzymes, or may be transformed into an inactive form, thus

ESCULENTA (0. b.w.lOOg1 Ringer-Infusion:75@lminl

T

-y

, I

T

I-

NS

j , ,

60

50

LO

I-

30

,-

20

I.

10

d -

rANF_ 0

IIHHY

XYltmn PERIODS

Fig. 3. Control and experimental in the same animal: lack of effect of rANF (0.4 yg/hr) on the fractional excretion of sodium, and a high effect of furosemide.Means f SD; n = number of animals (series 3).

Lack of effect of rANF on renal function in frogs

preventing any natriuretic response. Recently, Briggs et al. (1984) and Veress et al. (1985) suggested an inactivation of atria1 natriuretic peptides in the blood of rats. It is possible that similar biochemical processes occurred in the present experiments with frogs. However, in our studies, rANF was infused continuously and at varying rates. Thus, if a significant cleavage of the rat polypeptide was occurring, and was the reason for the lack of effect of rANF, then it must be assumed that the rate of cleavage matched the highest infusion rate of 3.4pg/hr per frog. Although the rate of cleavage is unknown, this seems improbably high. A further possibility would be that the intact rat atria1 polypeptide was circulating without biochemical alteration in the blood of the frog; however, there was no binding of rANF at the renal receptors of the frog because of the specificity of the receptors in rat and frog. Finally, it could be that there is an absence of renal receptors for natriuretic factors in the frog, including its own peptides, although both atria1 and ventricle extracts from frog induce natriuretic responses in the bioassay rat (De Bold and Salerno, 1983). Thus, it could be that the atria1 (and ventricle) substances of the frog (not yet defined) are not natriuretic factors in this species as expected. The granules in the heart of frogs found could play an important role in the blood pressure regulation of this species. Although the exogenous ANF used in the present experiments did not change the blood pressure (see Tables 1 and 2), the endogenous cardiac substances of the amphibian may still influence the blood pressure in this species. Thus, the natriuretic effects of both the atria1 and ventricular extracts of the frog in experiments in the rat (De Bold and Salerno, 1983) may be unspecific effects with no renal physiological relevance for the frog. The rat atria1 natriuretic factor has also been tested in in-vitro studies using amphibian skin (R. esculenta or R. temporaria) in a modified Ussing type chamber. At a rANF concentration of 10m6M in Ringer’s fluid (in mmol/l: NaCl 115, KHCO, 2.5, CaCl, 1; pH 8.1 after saturation with air), rANF at either the serosal or the mucosal side of abdominal skin did not significantly change the transepithelial sodium transport (short circuit current) (W. Nagel, personal communication). This result is consistent with the lack of effect of rANF in our in-viva experiments. In summary, the present in-uiuo experiments clearly demonstrate the lack of effect of the rat atria1 natriuretic factor on the renal function in the frog. Further investigation is necessary and may elucidate this lack of effect of rANF in amphibians. For instance, biochemical studies could be carried out to clarify the role of possible specific peptidases in the inactivation of the circulating exogenous (or endogenous) ANF in the blood of the frog; and binding

469

studies with endogenous factors from the frog heart and exogenous atria1 natriuretic peptides could be carried out to clarify whether specific receptors for these substances in the renal tubules of the frog are present or not. Acknowledgements-The

authors gratefully acknowledge

Dr M. Marin-Grez for discussions and for the rANF bioassays in his laboratory (see Materials and Methods); Dr W. Nagel for the electrophysiological experiments (see Discussion): and Dr J. M. Davis for the heloful comments in preparaiion of the manuscript. We also’gratefully acknowledge the expert secretarial assistance of Mrs K. SchaippMetzner and the excellent technical assistance of Mrs Regine Bleicher and Mrs Elvira Gela. Port&s of these results have been presented at the Spring Meeting of the German Society of Physiology in Homburg 1987 and published in abstract form (Frick and Toygar, 1987).

REFERENCES Beasley D. and Malvin R. L. (1985) Atria1 extracts increase glomerular filtration rate in vivo. Am. J. Physiol. 248, F24-F30. Briggs J. P., Marin-Grez M., Steipe B., Schubert G. and ~hne~ann J. (1984) Inactivation of atria1 natriuretic substance by kallikrein. Am. J. Physiol. 247, F480-F484. De Bold A. J. and Salerno T. A. (1983) Natriuretic activity of extracts obtained from hearts of different species and from various rat tissues. Can. J. Physiol. Pharmacol. 61, 127-130.

De Wardener H. E. and Clarkson E. M. (1985) Concept of natriuretic hormone. Physiol. Rev. 65, 658-759. Frick A. and Durasin I. (1986) Regulation of the renal transport of inorganic sulfate: effects of metabolic changes in arterial blood pH. Pfltigers Arch. 407,541-546. Frick A. and Toygar A. (1987) Lack of effect of rat atria1 natriuretic factor on the fractional excretion of sodium in frogs during water diuresis. PJlii;sers Arch. 408, R40. Hammond T. G., Haramati A. and Knox F. G. (1985) Synthetic atria1 nat~uretic factor decreases renal tubular phosphate reabsorption in rats. Am. J. Physiof. 249, F3lS-F318. Maack T., Camargo M. J. F., Kleinert H. D., Laragh J. H. and Atlas S. A. (1985) Atria1 natriuretic factor: structure and functional properties. Kidney Int. 27, 607?615. Marumo F. and Sakamoto H. (1985) The absence of any effect of a-human atria1 natriuretic peptide on sodium transport and osmotic water flow in the toad bladder. Endocrinol. Japan_ 32,921-927.

Needleman P., Adams S. P., Cole B. R., Currie M. G., Geller D. M., Michener M. L., Saper C. B., Schwartz D. and Standaert D. G. (1985) Atriopeptins as cardiac hormones. Hypertension 7, 469-482. Pamnani M. B., Clough D. L., Chen J. S., Link W. T. and Haddy F. J. (1984) Effects of rat atria1 extract on sodium transport and blood pressure in the rat. Proc. Sot. exp. Biol. Med. 176, 123-131. Veress A. T., Chong C. K. and Sonnenberg H. (1985) Inactivation of atria1 natriuretic factor in blood. Can. J. Physiol, Pharmacol. 63, 1615-1617.