Pfltigers Arch (1984) 400:300-305 Pflfigers Archiv

European Journal of Physiology

�9 Springer-Verlag 1984

Natriuretic factor exerts a ouabain-like activity in the rat colon in vitro

Blaise Martin and Herr6 Favre

Division de N~phrologie, D~partement de M~decine, H6pital Cantonal Universitaire, CH-1211 Gen~ve 4, Switzerland

Abstract. A previously described natriuretic factor (NF) found in urine from man receiving a high salt diet has been postulated to be of hormonal nature. This factor inhibits Na-K ATPase and binds to ouabain receptors. In order to investigate if NF exerts its physiological activity through the Na-K ouabain sensitive pump, its capacity for inhibiting sodium transport has been tested in an in vitro rat colon preparation. Colonic mucosa from rats fed 0.55, 3.55, and 6.55 mmol Na a day were mounted in an Ussing chamber. Inhibition of short-circuit current (SCC) and PD was ob- served only when NF was added to the serosal side of the membrane and was similar to that observed with ouabain. In rats fed a low or normal salt diet, inhibition of SCC and PD starts after a lag period of 10 rain and reaches its maximum inhibition (about 60 %) after 90 rain. By contrast colonic mucosa from rats receiving a high salt diet exhibits a higher SCC and PD (basal values) and inhibition of the sodium transport starts immediately after addition of NF to the bathing solution.

The data demonstrate the similarity of physiological action of ouabain and natriuretic factor on the sodium transport by the colon in vitro. Experiments with rats receiving a high salt diet suggest that by contrast to the kidney, NaCl-dependent cotransport is strongly stimulated by the salt intake in the colon and could be directly inhibited by ouabain or a ouabain-like substance i.e. NF.

Key words: Sodium content of diet - Sodium transport by the colon - Natriuretic factor - Short circuit current

Introduction

The existence of a natriuretic factor (NF) has been reported from several laboratories [20]. The evidence in favour of a hormone other than those already well-established, parti- cipating in the regulation of the sodium balance is based on physiological [16, 17] and pathophysiological data [7]. How- ever to date, the chemical nature of this putative hormone has yet to be defined. Nevertheless, there is agreement among several groups about the relationship between the natriuretic activity of partially purified factor obtained from the urine and the sodium status of the human subjects or animals providing this urine [3, 4, 8, 9]. There are also indications that this natriuretic factor acts on the distal segment of the

Offprint requests to: H. Favre at the above address

nephron [10], inhibits the Na-K ATPase [12] and reacts with digitalis antibodies [13].

In our laboratory, a natriuretic factor has been isolated from the urine of chronically salt loaded subjects. This factor was partially purified by Sephadex G25 chromatography. The fraction appearing immediately after the salt peak has been shown to possess a natriuretic activity when injected in a rat assay. The natriuretic activity is closely correlated with the sodium status and the natriuresis of the providers of the factor both in experimental animals [17] and in humans with diseases accompanied by sodium disorders [7]. More recently, this natriuretic fraction has been shown to inhibit Na-K ATPase and bind to ouabain receptors (unpublished data: personal observation).

On the assumption that this natriuretic factor is a circulating substance interfering with sodium transport by inhibiting the Na-K ATPase, one could envisage studies of organs other than the kidneys, in which ouabain dependent Na transport is present and where the natriuretic substance may be active.

In this regard, rat colon provides an organ of choice. It is known to transport sodium by different mechanisms includ- ing a Na-K ATPase dependent one which is suppressed by ouabain. This sodium transport by contrast to that occurring in the small intestine, is glucose independent and comparable to the sodium transport taking place in the distal segment of the nephron [5]. Sodium transport by the colon could be studied in vitro which would allow comparisons between the time courses of inhibition of sodium transport by ouabain and the natriuretic factor. It would give information on the cellular mode of action of the natriuretic factor.

Finally, compared to toad bladder preparations colon preparations have the advantage of providing information in the same species, the rat, in which most of the previous observations on the effects of the natriuretic factor have been obtained.

Therefore the present study was designed: a) to investigate a possible inhibition of sodium transport in the rat colon induced by the natriuretic factor, b) to compare the time courses of sodium transport inhibition by ouabain and natriuretic factor to determine if this natriuretic factor acts by binding to ouabain receptors and hence Na-K ATPase inhibition.

Methods

Male Wistar rats from Institute of Physiopathology (Bern, Switzerland) weighing 412 _+ 12 g were kept to one of the

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three different salt intakes for at least 10 days, in individual cages. Sodium deficient food containing 2.2 retool/100 g dry powder (C 1036 Altromine, Lage, FRG) and water contain- ing: a) 0 mmol sodium/l, b) 100 mmol sodium/l, c) 200 mmol sodium/l, were freely available. These different intakes pro- vided: 0.55, 3.55 and 6.55 mmol sodium a day as previously demonstrated in our laboratory [9].

Colon preparation. Rats were anaesthetized with an intra- peritoneal injection of 0.01 g/100 g BW of nembutal. After opening the peritoneal cavity, the ascending colon was clamped with artery clips at its origin, caecum excluded, and at the hepatic angle (first 1 - 2 cm). The colon was then opened beyond each clip and washed with 60 ml of Ringer's solution. Thereafter the mucosa was gently stripped off. The piece of tissue was then mounted in a conventional Ussing chamber of 1.13 c m 2 of aperture. Total preparation time was 5 - 8 rain after blood flow was interrupted.

The membrane was bathed on both sides with 20 ml of Ringer's solution containing in retool/l: 14 glucose, 118NaC1, 6KC1, 2.5 CaC12, 1.2MGSO4, 1.2H2PO4, 24 NaHCO3, pH 7.4 gassed with 95 % 02 and 5 To CO2 and maintained at a constant temperature of 25 ~ C.

The spontaneous transmural electrical potential differ- ences (PD) were measured by calomel electrodes connected to the bathing solution by 3 % Agar bridges attached to a digital voltmeter. The composition of the solution in the Agar bridges was a 3 M KC1. Except for periods of less than 30 s required to read the PD, the tissue was automatically and continuously short-circuited so that the PD was nullified. Current was passed via Ag-AgCI2 electrodes and Agar bridges. Before each experiment, the resistance of the Ringer's solution between the two electrodes was measured and later used in order to have a short circuit current (SSC) inducing a real null PD on the membrane. All the data were stored in a microprocessor and later transferred to a computer where they could be processed for statistical analyses.

Experiments were started after the membrane had stabi- lized for at least 20 rain. After stabilization, either Ringer's solution (control membrane), natriuretic factor or ouabain was added, according to the assigned protocol.

Natriureticfactor. This factor was isolated from a pool of 24 h urine provided by normal subjects receiving 300 mmol so- dium a day in addition to their normal diet during 10 consecutive days. This pool was lyophilized and reconstituted to obtain a solution equivalent to 5 h of urinary excretion per ml. It was chromatographed on an 80 cm/8 cm column packed with Sephadex G25 with an eluate of 0.01 M am- monium acetate, pH 6.8. A flow rate of 1 ml/min was maintained by the means of a Perpex pump and fractions of 10 ml each were collected. The natriuretic activity was recovered as previously described in the 10 tubes following the elution of the sodium and potassium [17]. This factor was lyophilized and reconstituted in distilled water such that 1 ml contained the equivalent of 8 h of urinary excretion. 1 ml of this fraction was added to one side of the membrane and the same volume of Ringer's to the other side. There was no change in the composition of the bathing milieu, the maximal difference in osmolality reaching 12.7 mOsm in favour of the side containing the natriuretic factor.

Ouabain. Cristallin G. Strophanthine was obtained from Merck (Darmstadt, FRG). 500 gl of an ouabain solution was

added to either both sides or the serosal side. Final con- centration of ouabain was 10- 3 M.

Statistical analyses. For each experiment, regression lines were fitted separately to the values observed during the pretreatment period and to those observed after the start of the treatment. The change in any pair of two consecutive regression slopes (b) reflects the treatment impact on the variables observed, i .e. tension, resistance and SCC. Measurements were done every 5 rain and the 'before-period' slope is defined with the 5 points - 2 0 to 0 rain, whereas the 'after-period' slope is defined with 12 points from 15 to 75 rain (linear part) excluding the last points from 75 to 90 rain which were not longer linear.

Appropriate statistical tests were applied to assess the significance of within experiment slope differences and the significance of the slope differences between groups of experiments.

Because of the heterogeneity of the variance usually observed between the 'before' and 'after' treatment slopes, a Behrens Fisher type test [14] was used to assess the difference.

Furthermore, as the amount of information available to define the 'before-slope' was limited, the normality of the slope distribution remained uncertain; hence we also used a non-parametric test [19]. The significance level presented in Fig. 1 was based on this latter test, which is more conservative than the Behrens Fisher type test.

Statistical analysis of differences between groups of experiments was based on a Mann-Whitney test applied to the set of slopes measured within the groups compared.

Within each group the differences between observations made at 0 and 10 rain were evaluated with a paired Wilcoxon test, while differences between sets were assessed with a Mann-Whitney rank sum test.

Results

Figure 1 shows the changes in the slopes before and after addition of test substances. For control membranes (group 1) after addition of I ml of Ringer's both sides, there is no change in the slope of the SCC. However, there is a small significant difference (P < 0.05) in the slope of the PD. When natriuretic factor is added to the mucosal side of the membrane (group 3) no effect could be detected. By contrast, ouabain or natriuretic factor added to the serosal bath (groups 2, 4, 5, 6) produced a significant change in the slope of both SSC and PD at a statistical level o fP < 0.001. In none of these experimental settings is the resistance significantly altered.

Figure 2 gives the results in both absolute and relative terms obtained after addition of natriuretic factor to colonic mucosa from rats receiving 0.55, 3.55 and 6.55 mmol sodium a day. These curves are compared to control experiments done with colonic mucosa from rats receiving 3.55 mmol sodium a day. Basal values clearly depend on the salt intake of the animals. The highest sodium transfer by the colon in vitro is observed with membranes from rats receiving 6.55 mmol sodium a day and the lowest with those from rats receiving a salt deprived food. Relative values shown on the right panel of Fig. 2 allowed comparison between the different slopes obtained both in control and in the different groups of membranes to which NF was added to the serosal side. All these slopes are different from each other (P < 0.05) and from

302

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Fig. 1. This figure summarizes the differences observed in the slopes (-b) of PD, resistance and SSC before (close bars) and after (hatched bars) manipulation. Group 1 is the control (n = 6), group 2 represents membrane receiving ouabain at the serosal side (n = 9), group 3 membranes receiving natriuretic factor at the mucosal side (n = 7); rats from groups I, 2 and 3 received a salt intake of 3.55 mmol Na a day. Groups 4 (n = 5), 5 (n = 5), 6 (n = 7) are membranes from rats fed 3.55, 0.55 and 6.55 mmol sodium a day respectively, to which natriuretic factor was added to the serosal side. Asterisks indicate the statistical significances: *P < 0.05, * * P < 0.01, ***P< 0.001

controls (P < 0.001). Table i and Fig. 5 show the existence of a lag per iod of 10 rain after addi t ion of NF , characterized by no change (group 4) or a marginal change ( P < 0.043; group 5) in SCC and PD. By contrast there is an immediate decrease in PD both in absolute terms (2210 gV) and with a high degree of significance (P < 0,018) but a non-significant drop in SCC of 9.4 gA in group 6.

Figure 3 compares the slopes obtained after adding the natriuretic factor either on serosal or mucosal side. On the left panel, it could be seen that there are statistical differences (P < 0.01) between the two slopes for SCC, PD and re- sistance. That is demonstra ted on the right panel of the same figure which shows the time course of the inhibition. The natriuretic factor is active only when added to the serosal side while values obtained when it is added to the mucosal side do not differ from controls.

Figure 4 demonstrates the perfect similitude between the slopes obtained after addi t ion of ouabain or natriuretic factor on the serosal side. There is no statistical differences between the slopes as shown on the left panel of the figure.

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Fig. 2. This figure illustrates in absolute terms (left panel) and relative terms (right panel) the changes in PD, resistance and SCC induced by addition of natriuretic factor to the serosal side of the membranes compared to control experiments (hatched area) which is mean + SEM. The curves are given as mean (solid and dashed lines) + SEM (dotted lines) of 5 to 9 experiments. The slopes differ from each other at the statistical level of at least P < 0.01. Significant inhibition starts after 10 min for membranes from rats fed 0.55 and 3.55 mmol sodium a day (groups 5 and 4), By contrast, a first phase of inhibition is immediate for membranes from rats receiving 6.55 mmol sodium a day (group 6)

Table 1. Mean differences + SEM between brackets for tension and SCC from 0 to 10 m after addition of substances according to the group appartenance as defined in 'Method'. Two tailed P value of the Wilcoxon test are given when significant. The only important change both in absolute value and level of significance is observed in tension measured in colon mucosa for rats fed 6.55 mmol Na/day (group 6)

Group N U (gV) I (~tA)

1 6 70 (240) 1.8 (4.3) 2 9 210 (210) 0.7 (3.4) 3 7 -120 (370) --1.1 (9.1) 4 4 400 (270) --12.4 (8.1) 5 5 820 (200) e ----- 0.043 5.4 (4.7) P = 0.043 6 7 2210 (280) P = 0.018 9.4 (4.7)

Discussion

Colonic mucosa is a tissue difficult to handle by comparison to toad bladder preparat ions. One of the greatest difficulties is to maintain a stable membrane over a long per iod of time. Preliminary studies indicated that it was possible to achieve a reasonable stability of the membrane at a temperature of 25 ~ C but not at 37 ~ C, presumably because at body tempera- ture, membranes are altered by the bacteria contaminat ion. However even at 25 ~ C, absolute stability of the membrane cannot be achieved. Therefore validation of the experiment requires well-defined criteria. The membrane was considered stable if the slope of PD was lower than 0.2 mV over 5 rain on

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Fig. 3. This figure compares the inhibitory effect of natriuretic factor on PD, resistance and SCC when added either to the serosal or mucosal sides (group 3). On the righ panel, it could be seen that the slopes (-b) are significantly different at P < 0.01. On the right panel, the time-course of the inhibition is shown. Each curve represents the mean of 7 experiments (_+ SEM) on the mucosal side and 5 on the serosal (_+ SEM)

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Fig.4. This figure compares the effects of ouabain (t0 -3 M) (final concentration) and natriuretic factor added to the serosal side of the membrane from rats fed 3.55 mmol sodium a day (group 2). Both the slopes (-b) for PD and SCC (left panel) and the time-course (right panel) are identical. The increase of the SSC during the first 10 rain is not significant

four consecutive periods. At that time SCC should be at least of 30 gA. If these criteria were not met within 2,5 h of preparing the colon, the experiment was not continued. Because the stabilization of the membrane prior to addition of test substances was not absolute, it was mandatory to use

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Fig. 5. The left panel demonstrates the absence of significance (except for groups 5 and 6) of the variation of tension and SCC during the first 10 min after addition of the test-substance. The extent of change in tension is however significantly different (P < 0.01) when groups 4 and 5 are compared to group 6 demonstrating the difference of comportement of these groups during the first 10 min of the ex- periment

accurate statistical analyses to judge the results, taking into account the differences of slopes before and after manipu- lation. As can be seen in Figs. 2 - 4, use of the statistics only supports the data and conclusion but does not create significant differences unrelated to physiological pheno- menon. It is clear from these figures, which show absolute data, that the slopes are different before and after addition of natriuretic factor or ouabain. On the other hand the logical comparison with a nonactive eluate has not been done as global differences between solutions were too important (Osm, Na, K, etc).

Sodium transport through the colon depends on at least two systems. The first is an electrogenic transport, Na-K ATPase dependent which can be inhibited by ouabain; the second is a sodium chloride dependent transport which is inhibited by furosemide [2]. Natriuretic factor should act on the first system, as suggested by its inhibitory effect on SCC and PD which is identical to that observed with ouabain both in terms of time-course and maximal inhibition (Fig. 4).

An important finding from the point of view of gastroin- testinal physiology is the demonstration of a relationship between the salt intake and the capacity of the ascending colon in vitro to reabsorb sodium: the higher the salt content of the diet, the higher the basal SCC and PD spontaneously created by the colonic mucosa once mounted in Ussing chamber. The present data contrast with the in vivo results obtained by Edmonds who showed an increase in PD across the colon in salt depleted rats. However the setting used here cannot be compared to in vivo experiments where submucosa, blood and lymphatic drainages could contribute to transfer of ions [6].

When natriuretic factor is added to the serosal side of membranes from rats fed 0.55 and 3.55 mmol sodium a day, substantial inhibition of the SCC and PD is observed after a

304

lag period of 10 min during which there are no, or marginal changes in slopes compared to the control period. Almost complete maximal inhibition (60 %) is achieved after 90 min, as the curves reach a plateau (indicating a new steady state) after 9 0 - 1 2 0 rain (not shown in the figures). The residual values at 120 min can be suppressed by cutting oxygen flow. The natriuretic factor is active only when added to the serosal side. Addition of this factor to the mucosal side has no effect on SCC and PD. All these characteristics are similar to that previously described in toad bladder preparations when the same natriuretic factor was added to the serosal bathing solution at the same final concentration [8].

Colons from rats fed 6.55 mmol sodium a day behave differently. Their basal activity in transporting sodium is higher than that observed in membranes from rats belonging to the two other groups. Addition of natriuretic factor produces an inhibition of the SCC and PD characterized by a different time-course. There is an immediate fall in the SCC and PD with statistical significance only in PD during the first 10 rain followed by a continuing inhibition, the slope of which is similar to those observed in the two other groups of rats (Fig. 2). It is unlikely that the first phase of the curve is artefactual because of its remarkable reproducibility. There- fore the entire curve of inhibition observed of a physiological phenomen which may be related to NaCl-dependent cotransport.

Heintze et al., in experiments performed in rabbit colon accumulated evidence which permitted a model for sodium and chloride transports by the colonic mucosa to be pro- posed. There is an ouabain sensitive Na-K exchange process at the basolateral membranes of both absorptive and sec- retory epithelia [15] that provides a link through which metabolic energy is coupled to NaCl-dependent cotransport. Accordingly, ouabain or ouabain-like substances could abolish Na-K pump activity, NaCl-dependent cotransport or both. NaCl-dependent cotransport is inhibited specifically by loop diuretics which induce an immediate and reversible drop in PD [14] by contrast to the delayed effect (10 min) of ouabain on Na-K pump. Therefore, the kinetics of the inhibition of PD by the natriuretic factor observed in the group of rats ingesting a high salt diet is compatible with a stimulation of NaCl-dependent cotransport by the high salt diet and its inhibition by the natriuretic factor which behaves as an ouabain-like substance. In this case, Na-K ouabain sensitive pump would be a specific, low capacity system by comparison to NaCl-dependent cotransport which would represent a system of low specificity but high capacity.

Considering the global maintenance of the sodium bal- ance in vivo, it has long been known that the kidney is the major organ responsible for eliminating a sodium load, while the contribution of the GI tract is negligible [I]. The well- established salt retaining hormone, aldosterone, acts on the rectum and has no action on the ascending colon [11]. However, as its circulating level is expected to be low in salt loaded animals, the increased sodium reabsorption by the GI tract required to maintain constant the Na elimination by the stools, when large amounts of sodium are given orally should take place in the ascending colon and be independent of aldosterone. As demonstrated in this study, ouabain and natriuretic factor are active in this location. Clearly the ascending colon is less sensitive to the natriuretic factor than the kidney, as we demonstrated previously that rats ingesting a 6 mmol sodium a day diet exhibit a natriuresis and have a high level of natriuretic factor in their urine [17] whereas

sodium absorption by the ascending colon is not yet inhibited. This phenomenon could be explained by a difference in the number and/or the binding affinity of receptor for natriuretic factor in the kidney and in the ascending colon. As the ascending colon responds to presumably large doses of natriuretic factor by inhibiting sodium reabsorption, one may assume that if the salt intake is further increased, N F would participate in vivo in the sodium regulation inducing an increase in the fecal sodium elimination. By contrast, in salt depleted animal the sodium delivery from the caecum is reduced by 70 % [6] and therefore the total sodium available for reabsorption by the ascending colon would be diminished which could explain the lower PD measured under this dietary condition at the ascending colon, the descending colon and the rectum being responsible for the conservation of the sodium under the control of aldosterone [6]. As previously demonstrated, the conservation of the sodium by the GI tract under these dietary conditions takes place in the rectum under the control of aldosterone. Thus, in GI tract, the natriuretic factor may have a significant role only when the sodium intake is largely increased to a level where diarrhea appear.

Acknowledgements. The authors are indebted to Mr. Jean-Claude Bost~d6ch6, Mr. Eric Perrin and Mr. Silvio Bergamaschi for their expert technical assistance, to Dr. Pierre Vuagnat for help with the statistical analysis, to Mr. Constantin Jornot and Mr. Albert Rieben for their help with programming, to Dr. Richard James for reviewing the English, and to Mrs. Carmen Meylan for her secretarial assistance.

This work was supported by a grant (Nr. 3.122.081) from the Swiss National Science Foundation and from the Montus Foundation.

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Received July 6/Accepted November 28, 1983