Tohoku J. exp. Med., 1973, 109, 281-296

A Comparison of Patterns of Changes in Urine Flow and Urine Electrical Conductivity Induced by Exogenous ADH in Hydrated Rats

TOKIHISA KIMURA* and RYUZO YOKOYAMA•õ

Department of Physiology, Tohoku University School of Medicine , S endai

KIMURA,T. and YOKOYAMA,R. A Comparisonof Patterns of Changesin Urine Flow and Urine Electrical ComductivityInduced by ExogenousADH in HydratedRats. Tohoku J. exp. Med., 1973, 109 (3), 281-296 Both urine flowrate and urine electrical conductivitywere recorded continuouslyin hydrated alcohol-anesthetizedrats, and the patterns of changesin these two induced by intravenous injection of ADH were compared. Although ADH-inducedchanges in urine flow rate and conductivity were reciprocallyrelated, significantdeviation from a simple reciprocal relation was found when a relatively high dose of ADH was given. Dose response curves as obtained by using the maximummagnitude or the time-integralof the response as the index of the response revealed that the urine-flowmethod has higher sensitivity to ADH in a relatively low dose range, whereas the conductivity method is superior for the assay of relatively high dose of ADH. Saluresis induced by NaCl-loadingor by administration of Furosemide produced parallel increases in both urine flow and conductivity, while a reduction of blood pressure caused parallel decreases. Asphyxia and pentobarbital sodium produced ADH-like (reciprocaltype) pattern of changes, but these changes were interpreted as the results of a liberation of endogenous ADH. Diuretic effect of a low dose of ADH, saluretic effect of a moderate dose of ADH, and vascular effect of a high dose of ADH werecharacterized by the dual recording of urine flow rate and conductivity. It is concludedthat the dual reocrdingof urine flow rate and conductivity is recommendablefor the assay of ADH of a wide range of dose since the renal effect of ADH is of compositenature, and the single recordingof either of these urinary factors cannot characterize the ADH action. urinary responses to ADH; ADH bioassay; flow rate measuring method; conductivity measuring method

Measurements of changes in either urine flow rate or urine conductivity in hydrated ethanol-anesthetized rats have currently been widely used in the bioassay of ADH (see Fitzpatrick and Bentley 1968). There seem to be controvertial views as to which method be more recommendable for the ADH bioassay, one measuring urine flow rate and the other measuring urine conductivity. Sawyer (1958) argued the superiority of the former, whereas Yoshida et al. (1963) and Bonjour and Malvin (1970) recommended to use the latter.

Received for publication, August 17, 1972. * Present address: The Second Department of Medicine, Tohoku University School of

Medicine, Sendai. Present•õ address: Department of Electronic Engineering, Faculty of Engineering, Iwate University, Morioka.

281 282 T. Kimura and R. Yokoyama

Principally, if ADH causes only a reduction of free water clearance of animals, these two methods should be entirely equivalent, since both measure reciprocally related phenomena. ADH, however, is known to have additional effects in rats, e.g., a diuretic effect in its low dose range (Kramar et al. 1966), natriuretic and kaliuretic effects over a wide range of dose (Chan 1965) and a vasopressor effect in a high dose range (Dekanski 1952). Furthermore, a conducti vity-measuring system usually has a small but significant dead space. All these factors may greatly modify the simple reciprocal relationship between changes in urine flow rate and urine conductivity and also affect the practical usefulness of these two methods in ADH bioassay. The aim of the present study is to compare the two methods above mentioned in an attempt to examine which is more reliable or useful as the procedure of ADH bioassay. Additionally, it was examined whether or not simultaneous recording of both changes in urine flow rate and conductivity has some advantages in detecting or measuring the urinary response to exogenous ADH given in rats under various diuretic conditions.

METHODS Preparation of animals: Male albino rats weighing 200-250g were used. Methods for anesthesiaand prehydrationwere almost the same as those reported by Czaczkes and Kleeman(1964). Briefly, 12% ethanol solution amounting 3% of body weight was given twice through stomach tube at an interval of 30 min. 20 min after the second infusion, warmed tap water amounting 2% of body weight was supplemented. The total amount of water, thus given, was 8% of body weight. The abdominalwall was openedby the mid-lineincision to exposethe urinary bladder. A short (2cm long)soft silicone-rubbertube of 1 mm inner diameter was inserted into the bladder through a cut made at the upper part of its ventral wall, then the tube was tied together with the bladder to fix it. The urethra was obstructed by ligating the penis. The jugular vein was catheterizedwith a thin polyethylene cannula, through which the maintenanceinfusion was carried out with a hypotonic saline (0.3% NaCl) containing2% ethanoland 1.6%glucose. The rate of infusionwas fixed at 0.1ml/minin most experiments. Atropine sulfate (0.01mg/kgbody weight) was administered subcutaneously in order to inhibit secretionof excesssputa during experiments. Tracheal cannulationwas made with a short polyethylene tube and pure oxygen gas was supplied onto the opening of the cannula. Declineof body temperature of animalswas prevented by warming their body with an electriclamp. Recordingof urine conductivityand urine flow rate: Conductivity-measuringsystem used is essentiallysimilar to those employedby Share (1961)and Yoshida et al. (1963). The conductivitywas measuredbetween two small stainless-steeltubes interposed along the courseof a polyethylenecannula which was connectedto the rubber tube inserted into the bladder. Each stainless-steelpipe had 1cm in length and 0.9mm in inner diameter, and both werefixed 8mm apart. The electrodeswere connectedto a Wheatstonebridge with fine lead wires, and the part of the electrodes was molded with epoxy resin. Alternating current of 1 kHz was supplied to the bridge from a oscillator, and the bridge output was amplifiedby a differentialamplifier. A devicewas made to obtain voltage outputs linear to fluid conductivity in the similar way to that reported by Rothe et al. (1967). The electrical circuit was shown in Fig. 1. Recording system for urine flow rate used is principally the same as those employedby Sawyer (1958), Tata and Gauer (1966) and Forsling et al. (1968). It consistsof a drop counter, a pulse generator and an integrator ADH Action on Urine Conductivity and Urine Flow Rate 283

Fig. 1. Electrical circuit of the apparatus for measuring urine electrical conductivity . Alternating current of 1 kHz is supplied to a Wheatstone bridge , and the bridge output is amplified by a differential amplifier so as to give voltage output linear to electrical conductivity of urine.

Fig. 2. Electrical circuit of the apparatus for recording urine flow rate continuously. Urine drops are converted to electrical pulses of constant voltage and constant duration, and the pulses thus generated are integrated by an integrator which is reset every minute with a timer (not illustrated in this figure) by short-circuiting S.

of pulses. Generation of pulses with constant duration and constant voltage was made synchronized with urine drops flowing out of the cannula from the bladder, and the integrator was reset every minute. The electrical circuit of the system was illustrated in Fig. 2. Both urine flow rate and conductivity were recorded on a two-pen ink-writing recorder (Towa Denpa Co., EPR-3T) or a multichannel ink-writing recorder (Watanabe Sokki Co., WA621-1).

Administration of ADH: In most experiments, arginine vasopressin (Sigma Co.) was used. Synthetic lysine vasopressin (Sandoz) was also used in some experiments. Both were dissolved in 0.9% NaCl solution containing 0.03% acetic acid. 0.1ml of diluted ADH solutions was slowly injected into the jugular vein in about 30 sec. During this interval, the infusion pump was stopped. An injection of 0.1ml isotonic saline had no detectable effect on either urine flow or conductivity.

Experiments in rats during osmotic diuresis: In experiments where ADH effects were observed during mannitol diuresis, maintenance infusion was carried out with 10% mannitol solution containing 2% ethanol. When necessary, systemic blood pressure was recorded from the common carotid artery by the use of an electrosphygmomanometer (Sanei Sokki Co., E-044). Other procedures were same as in experiments during water diuresis. 284 T. Kimura and R. Yokoyama

Chemical analyses of urine: Urine collection was made during three successive phases: during 10 min prior to ADH administration, during antidiuretic response, and during 10 min after the restoration of urine flow rate. For each urine sample, Na+ and K+ concentrations and volume were determined. Determination of Na+ and K+ concentrations were carried out by the use of a flame photometer (Elma).

RESULTS

1) Patterns of responses of urine flow and urine conductivity to ADH in water diu retic rats

In rats anesthetized with ethanol, the prehydration and the subsequent maintenance infusion of the hypotonic saline brought animals to a steady water diuresis within 30-60 min after the start of the maintenance infusion, when urine flow reached about the same rate as that of the infusion (100ƒÊl/min). At this stage, urine osmolality and urine conductivity were stabilized at 178•}27 mOsm/kg

H2O (mean•}S.E., n=4) and about 10mM equivalent NaCl, respectively.

Fig. 3 shows a typical example of responses to graded doses of ADH given during such steady water diuresis. In this particular experiment, 100, 75, 50 and

25ƒÊU of ADH were administered in sequence. Urine flow began to decrease within 2-3 min, and the maximum reduction occurred after 5-10 min, depending on the dose. The response in urine flow had a relatively simple time course, an initial rapid decrease followed by a gradual recovery. Urine conductivity began to increase with a time lag corresponding to 4 urine drops. The overall time

course of conductivity change was somewhat different from that of urine flow decrease, e.g., the peak of the increase appeared later than that of the decrease in

urine flow. Such a retardation was more exaggerated when higher doses of ADH were administered. At the recovery phase, the tracing of urine conductivity usually

had a prolonged tail, showing a sustained but slight increase in the total electrolyte

concentration even after the restoration of urine flow. Sometimes the decrease in urine flow was followed by an overshoot increase in flow rate, particularly when

a higher dose of ADH was administered. It was also noticed that repeated

injection of ADH caused gradual increases in both basic urine flow rate and conductivity.

Fig. 3. An example of responses of urine flow (lower tracing) and urine conductivity (upper tracing) to ADH injected during steady water diuresis. ADH of 100, 75, 50, and 25ƒÊU was injected intravenously in succession. ADH Action on Urine Conductivity and Urine Flow Rate 285

As seen in Fig. 3, patterns of both changes in urine flow and urine conductivity are nearly reciprocally symmetrical when dose of ADH is low. However, distortion of the mutual relation became marked when a high dose was given and urine flow was greatly reduced. In such cases an increase in conductivity at early phase was slowed down and the peak increase was markedly retarded (Fig . 4 a). Such deformation of pattern of conductivity response was largely dependent on both the basal urine flow and the extent of decrease in urine flow (compare Fig. 4 a and b). Response curves of conductivity usually assumed a tent-form or domeform, but rarely a plateau-forming type (Fig. 4 c).

Fig. 4. Various patterns of changes in urine flow rate and urine conductivity induced by ADH of various doses during water diuresis.

Fig. 5. Relationship between the duration of responses to ADH and the dose of ADH. Responses in urine conductivity (a) and in urine flow rate (b). 286 T. Kimura and R. Yokoyama

2) Dose-response relationships

A comparison was made of the dose-response relationships for three indexes of the response; the duration, the maximum magnitude and the time-integral of responses. The duration was estimated by measuring the time interval from the onset of response to the time when a tangential line of inflection point of recovery phase of response curve crossed the initial level. As shown in Fig. 5, log-dose response relation was approximately linear in both cases in the range from 10 to

100ƒÊU, but above this range, there was found a saturable tendency.

Fig. 6. Relationshipbetween the maximum magnitude of responseto ADH and the dose of ADH given. Responsesin urine conductivity (a) and in urine flow rate (b).

It is anticipated that ADH given disappears from circulating blood with an exponential time course. Blood level of given ADH, therefore, can be described by the following equation, as previously given by Lauson (1967): C=Coe-st (1) where Co is the initial blood level, C is the blood level at time t, and s is the rate constant of the disappearance. If we assume that the response disappears when blood level of ADH have decreased to a certain critical value , Cmin, the duration of response would coincide with the duration of time necessary for the decrease from Co to Cmin. Therefore, the relation between the duration of response (T) and COcan be described as: ADH Action on Urine Conductivity and Urin e Flow Rate 297

This implies that the duration of antidiuretic response , T, is linearly proportional t o the logarithm of the initial blood level which is determined by th e dose given. Th is was found to be nearly the case for doses up to 100ƒÊU . The half-life of ADH calculated from data presented in Fig . 5 was 3.46 min, the value being comparable to that obtained by Czaczkes and Kleeman (1964) in rats . The magnitude of response was estimated by measuring the diffe rence between the initial level and the peak value attained during antidiuretic response . As shown in Fig. 6, both curves were sigmoidal . The curve for conductivity change showed a sharp increase in the range of dose from 50 to 100ƒÊU , whereas the curve for urine flow had a nearly linear slope between 10 and 75ƒÊU .

Fig. 7. Relationship between the time integral of response to ADH and the dose of ADH given. Responses in urine conductivity (a) and in urine flow rate (b).

The time-integrals of responses in conductivity were estimated by measuring the area under the response curves from the onset (0 min) of response up to 15 min

and expressed in cm2. The integrated response in urine flow was estimated by

measuring the total amount of decrease during antidiuretic response as compared with basic flow and expressed in ƒÊl. Dose-response curves for both were also sigmoidal; the curve for conductivity had a much sharper inflection than for urine flow. The latter had a broad linear portion between 20 and 100ƒÊU. (Fig. 7).

Under the standard diuretic conditions, urinary concentration of K+ was 5-10 times greater than that of Na+. ADH caused a marked increase in both

Na+ and K+ concentrations during antidiuretic response, but the increase in K+ was more marked (Table 1). During this period, the excretion rate of K+ was 288 T. Kimura and R. Yokoyama

TABLE 1. Changes in K+ and No+ concentrations in urine induced by administration of ADH of various doses during steady water diuresis in rats

Values are given as mean•}S.E. *n=4. •õn=2 . •ö n=3.

slightly increased, whereas no significant increase was seen in Na+ excretion rate . After the restoration of urine flow to the initial level, somewhat increased Na+ and K+ excretion rates were seen. Especially, in cases where a prolonged tail of response cruve of conductivity and an overshoot increase in urine flow were seen , an increase in Na+ excretion rate was invariably observed .

3) Diuretic effects of low doses of ADH

It was frequently observed that low doses of ADH, less than the minimum antitiduretic dose caused a significant diuretic response under the standard conditions. Fig. 8 shows an example of such cases, where 6.25, 12.5, 25 and 50ƒÊU of ADH were administered successively. 6.25ƒÊU of APR caused a signi ficant increase in urine flow, while urine conductivity remained almost unchanged. 12.5ƒÊU, however, had almost no effect on both. Further increase in dose produced dose-dependent antidiuretic responses as stated above.

Fig. 8. Responses of urine conductivity (upper tracing) and urine flow (lower tracing) to ADH in low doses. ADH Action on Urine Conductivity and Urine Flow Rate 289

Fig. 9. Responses in urine flow rate (upper tracing) and in urine conductivity (lower tracing) to intravenous injection of 1ml of 1.8% NaCl (a) and 1.25 mg Furosemide (b)

4) Patterns of responses different from ADH-induced type and ADH-type pattern induced by means other than ADH

In order to ascertain the specificity of the pattern of antidiuretic responses, observations were made of effects of various means affecting urine flow and conductivity. First, effects of an increase in Na+ excretion rate were examined.

Either intravenous injection of 1.0ml of 1.8% NaCl solution or of 1.0-1.25mg of

Furosemide, a potent diuretic agent, caused marked parallel increases in urine flow and conductivity, as shown in Fig. 9, a and b. On the other hand, a reduction of systemic blood pressure to about 70mmHg by acute withdrawal of blood (Fig. 10, a) or intravenous injection of 100ƒÊg of acetylcholine (Fig. 10, b) produced parallel decreases in both. These changes are clearly in contrast to the reciprocal type of ADH-induced responses.

Fig. 10. Changes in systemic blood pressure (upper), urine flow rate (middle) and urine conductivity (lower tracing) induced by acute withdrawal of 1ml blood via carotid artery (a) and by intravenous injection of 100ƒÊg acetylcholine (b) in water diuretic rat. 290 T. Kimura and R. Yokoyama

On the other hand, the reciprocal type of response similar to that of ADH- induced response could be observed under some other experimental conditions. Fig. 11, a shows the effect of transient obstruction of the trachea and Fig. 11, b shows the changes after an intraperitoneal injection of 1mg pentobarbital sodium. These changes could not be discriminated from the ADH-induced changes.

Fig. 11. Effects of partial obstruction of the trachea (acute asphyxia) (a) and intraperitoneal injection of 1mg pentobarbital sodium (b) on urine conductivity (upper tracing) and urine flow rate (lower tracing) in water diuretic rats.

5) Effects of ADH during mannitol diuresis

During mannitol diuresis, administration of ADH less than 1mU did not cause any change in both urine flow and conductivity and also in systemic blood pressure. A much higher dose, e.g., 10mU, caused distinct parallel increases in both urine flow and conductivity. In about half of animals tested, such parallel increases were preceeded by sharp parallel decreases lasting very short period of time. Blood pressure was elevated sharply and returned to the initial level within about 15-20 min, but the increases in urine flow and conductivity usually persisted more than 20-30 min (Fig. 12). The pattern of the initial changes in urine flow and conductivity resembled that of changes produced by a rapid reduction of systemic blood pressure, while the subsequent parallel increases resembled the pattern of saluretic response. Noradrenalin (5ƒÊg) caused a similar elevation of systemic blood pressure, but responses in urine flow and conductivity were different from those produced by ADH; namely, a slight parallel decrease persisted about 10 min.

DISCUSSION

The relationship between ADH-induced changes in urine flow rate and urine conductivity as measured by usual methods was found to be modified by several factors. The most important factor responsbile for the departure of the mutual relation from predicted simple reciprocal one may be the presence of a dead space in the conductivity-measuring system. In our system, the dead space, including the catheter, the urinary bladder, ureter and pelvis , was about 100ƒÊl, equivalent ADH Action on Urine Conductivity and Urine Flow Rate 291

Fig. 12. Effects of 10mU ADH (a) and 15ƒÊg noradrenalin (b) on the systemic blood pressure (upper), urine flow rate (middle) and urine conductivity (lower tracing) during mannitol diuresis.

to 4 drops of urine flowing out of the catheter. Owing to this dead space, an increase in urine conductivity associated with a reduction of CH2O is recorded later than urine flow changes, and such a delay is strongly exaggerated when urine flow is greatly and rapidly reduced. Consequently, the shape of response curves or the size of integrated response for the conductivity was more variable as compared with those of urine flow, as pointed out previously by Sawyer (1958).

Another important factor is the saluretic effect of ADH which becomes apparent on the tracings of both urine flow rate and conductivity at the recovery phase or immediately after the restoration of urine flow rate. Owing to this effect, the response curve for the conductivity usually exhibited a prolonged tail. Therefore the duration of the response or the integrated response had to be estimated by drawing a tangential line to the rapid falling phase of the response curve for the conductivity.

As to the dose-response relation, there was no significant difference when the duration of response was used as an index of response. The dose-response curve was approximately linear in the range of 10-100ƒÊU in both cases as expected from the assumption of the exponential decay of given ADH in circulating blood.

Above this range, the curves showed a level-off and a saturable tendency. The maximum duration obtained in the present study was about 20 min, the value being significantly shorter than the value reproted by Gauer and Tata (1966), who employed a feed-back controlled infusion technique in order to maintain constant 292 T. Kimura and R. Yokoyama hydration of animals throughout experiments. The constant infusion, as used in the present study, may cause a progressive overhydration when ADH was given repeatedly. The progressive increase in the basal urine flow and conductivity, and also slight diuresis and saluresis seen immediatly after the restoration of urine flow may probably be due to the accumulation of hypotonic fluid in the body.

There was a significant difference between the dose-response curves when the

maximum magnitude of responses was used as a measure of response. The curve for the conductivity showed a sharp increase in between 50 and 100ƒÊU, whereas the curve for urine flow was nearly linear over a relatively low dose range as shown in Fig. 7. Such a difference is largely due to the difference in the dose-dependent increase in changes in both parameters. It is clear that, the relation of urine

osmolality to urine volume under various diuretic and antidiuretic conditions is

hyperbolic, and that a relatively large reduction of urine flow from diuretic level is associated with a relatively small increase in osmolality or electrical conductivity.

On the other hand, in a range of low urine flow rate, a small decrease in flow rate results in a large increase in osmolality or conductivity. Therefore, the conductivity method has a higher sensitivity in a higher dose range, while the urine flow-method has a higher sensitivity in a relatively low dose range.

The dose-response curves for integrated response can be interpreted nearly as the products of the curves for both the duration and the maximum magnitude of responses. Thus, the sigmoidal nature was much more exaggerated for the conductivity, while an almost linear curve but with a slight tendency of sigmoidal nature was obtained for urine flow.

Practically, either of indexes above described can be employed for the purpose of ADH assay since the response was quantitatively related to the dose in all cases. However, it may be said that the conductivity-measuring method has an advantage for relatively higher doses, while urine flow method is superior for relatively low doses of ADH. Furthermore, ADH below the minimum dose producing the antidiuretic response has a diuretic effect, as reported by Kramar et al. (1966). Such a diuretic effect is only detectable on urine flow tracings. Therefore, it seems recommendable to employ two methods simultaneously or to use one of these interchangeably according to the dose range to be exami ned.

Reciprocal changes in urine flow and conductivity seem to be characteristic to response to ADH in moderate doses. Changes in GFR or in tubular reabsorption of Na+ were found to produce either parallel increases or parallel decreases in both parameters. Besides ADH, only acutely induced asphyxia and administration of pentobarbital sodium were found to produce a similar type of urinary changes. Although the nature of effects of these could not be clarified in the present study, the effects seem to be related to liberation of endogenous ADH . Tata and Buzalkov (1966) showed that, even in animals anesthetized with ethanol, pain stimuli and hemorrhage caused considerable release of endogenous ADH . Asphyxia usually causes a strong vasoconstriction and an elevation of blood ADH Action on Urine Conductivity and Urine Flo w Rate 293

pressure, but the urinary changes cannot be accounted for by changes in GFR . Th erefore, it seems most probable that the release of endogenous ADH is st imulated b y asphyxia. As to the effects of pentobarbital sodium, Ginsberg (1968) obser ved similar effects in rats . However, Grindeland and Anderson (1964) and Bonjour and Malvin (1970) excluded a possibility of direct stimulation of liberation of endogenous ADH by this drug . As the drug usually depresses the respiration , a similar effect to that of asphyxia can be considered to be most probable . It seems of great importance , therefore, to prevent anoxia as possible and not to use pentobarbital sodium during ADH bioassay. Natriuretic and kaliuretic effects of ADH have long been noticed in dogs (Brooks and Pickford 1958; Chan and Sawyer 1961; Humphreys et al. 1970; Martinez-Maldonado et al. 1971), in rats (Kraanar et al. 1966; Chan 1965) and also in man (Jones et al. 1963). Chan and Sawyer (1961) showed that 8-arginine in the side chain of vasopressin molecule and 3-isoleucine in the oxytocin-ring were important for these effects. In the present study , kaliuresis was more marked during antidiuretic response, whereas natriuresis tended to occur at later phase. Saluresis cannot be detected on the dual record during antidiuretic phase, but could be clearly seen as the saluretic pattern after the restoration of the urine flow to the initial level. Accordingly, the dual tracing may be useful in some investigations with an intention to find out factors affecting saluretic response to ADH. The mechanisms of dieresis and saluresis induced by ADH is not well understood. Barer (1963) ascribed these to a vasodilating action of ADH by demonstrating an associated increase in the renal blood flow. However, saluresis occurs without changes in renal hemodynamics and GFR (Matinez-Maldonado et al. 1971). A possibility that ADH inhibits Na+ transprot by the proximal tubule is doubtful at present (Davis et al. 1967; Schnermann et al. 1969). Humphreys et al. (1970) postulated an inhibited Na+ transport by Henle's loop, but also this is not conclusive. Morel (1964, cited from Atherton 1969) postulated another possibility that inflow of Na+ into the collecting duct or the descending limb of Henle's loop from the medullary interstitium might increase as a result of increased medullary osmotic gradient due to ADH. During mannitol diuresis, much higher doses of ADH did not cause any change in urine flow and conductivity unless the systemic blood pressure was increased to a certain extent. This suggests that the natriuresis or saluresis may be related to the accumulation of Na+ in the medulla rather than the modification of tubular Na+ transport. The different time courses of appearance of kaliuresis and natriuresis suggest that kaliuresis may occur through a different mechanism from that for natriuresis. Cross and Sherrington (1965), Zain-Ul-Abedin (1967) and Saikia (1965) showed that K+ content of the papilla was decreased when ADH was administered to water diuretic animals, suggesting increased leak of K+ out of cells of the collecting duct. However, we could not confirm such a decrease in K+ content in the papilla in another series of experiments (unpublished). An alternative explanation may be 294 T. Kimura and R. Yokoyama that natriuresis is primarily produced by some mechanism above discussed and resultant increase in supply of Na+ to distal Na+-K+ exchange site may stimulate the secretion of K+ without a marked increase in Na+ excretion. Simultaneous recording of both urine flow and urine conductivity is also useful for observations of intrarenal vascular action of ADH and resultant urinary changes. For this purpose, the use of conditions of mannitol diuresis is suitable since during mannitol diuresis antidiuretic effect of ADH is minimum or absent (Koike and Kellogg 1957; Corcoran et al. 1956) mainly due to depletion of the corticomedullary osmotic gradient (Altherton et al. 1968, 1971). As suggested by Fisher et al. (1970), ADH causes an increase mainly in postglomerular resistance whereas noradrenalin acts on both the pre- and post-glomerular arterioles. Great difference between urinary responses to ADH and noradrenalin observed in the present study can be interpreted on the basis of the different sites of action of these two substances.

Acknowledgment We are indebted to Professor Takeshi Hoshi for his advice during this study and for reading the manuscript. We also thank Professor S. Yoshida, Department of Medicine, Jichi Medical School, Tochigi Prefecture, for his discussion on the conductivity method.

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