Pfliigers Archiv 286, 109--117 (1965)

Aus dem Physiologisehen Institut der Freien Universitgt Berlin

Microperfusion Study of Calcium Transport in the Proximal Tubule of the Rat Kidney * By

ANSELM FRICK~ (~ERHARD RUMRICH~KARL J. ULLRICH~ and WILLIAME. ~ASSITER"~'~ Wit 4 Figures in the Text

(Received June 18, 1965) Summary. Microperfusion experiments on the proximal convolution of the rat kidney in situ led to the following observations: 1. The concentration of Ca ++ in tubular fluid at which the net flux of Ca ++ is zero is about 2.66 mEq/L. This corresponds to a T F / P of 0.78, very similar to the equilibrium Na + T F / P of 0.76. The active pump potential for Ca ++ under these conditions is E t a ++ = 3 inV. 2. The rate of Ca ++ reabsorption in the proximal convolution varies linearly with concentration up to a tubular fluid concentration of 15 mEq/L. Parathyroid hormone deficiency or excess has no influence on Ca ++ reabsorption in the proximal convolution. 3. The ratio of net outward transport rates of Na + and Ca ++ at a T F / P of 1, is the same as the ratio of the concentrations of these ions in tubular fluid. 4. The ratio of net influxes of Na + and Ca ++ into Na+-frce and Ca++-free perfusion solutions is the same as tile ratio of the plasma concentrations of these ions. 5. Because of the striking similarities in the transport characteristics of Na + and Ca++, it is postulated that similar or identical mechanisms are involved in the transtubular transport of the two ions. C l e a r a n c e a n d m i e r o p u n c t n r e s t u d i e s h a v e d e m o n s t r a t e d t h a t Ca++ a n d N a + a r e r e a b s o r b e d b y t h e r e n a l t u b u l e in t h e s a m e r a t i o in w h i c h t h e y a r e p r e s e n t in plasma12, 2°, s u g g e s t i n g t h a t t r a n s p o r t o f t h e s e i o n s is i n t e r r e l a t e d . T r a n s p o r t p r o c e s s e s for N a +- a n d C I - h a v e b e e n c h a r a c t e r i z e d w i t h r e s p e c t t o 1. n e t reabsorption4,7,7a; 2. t h e e q u i l i b r i u m conc e n t r a t i o n s a t w h i c h n e t t r a n s p o r t is zero 9; a n d 3. i n f l u x o f t h e s e i o n s i n t o a N a + - a n d C l - - f r e e p e r f u s i o n solutionT,7a, s. T h e t u b u l a r fluid to * Supported by NIH-Grant AM06806-03 and the Deutsche Forschungsgemeinschaft. ** Present address: The University of Texas, Southwestern Medical School, Dallas, Texas. *** Established Investigator, Anlerican Heart Association, and Markle Scholar in Academic Medicine. Present address: Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, N.C.

110

A. FRICK, G. RvM~IcJ~, K. g. ULL~ICg, and W. E. LASSITER:

p l a s m a r a t i o ( T F / P ) of filtrable calcium r e m a i n s 1.0 along t h e p r o x i m a l c o n v o l u t i o n in free flow in n o n d i u r e t i c rats~2; this b e h a v i o r is similar to t h a t of sodium. I n t h e p r e s e n t studies m i c r o p e r f u s i o n and m i c r o p u n c t u r e t e c h n i q u e s were e m p l o y e d to o b t a i n f u r t h e r i n f o r m a t i o n concerning t h e m e c h a n i s m of t r a n s p o r t of Ca++, a n d its r e l a t i o n to Na+ t r a n s p o r t in the p r o x i m a l tubule. I t was f o u n d t h a t Ca++ a n d N a + fluxes, r e l a t i v e to t h e r e s p e c t i v e c o n c e n t r a t i o n s of t h e t w o ions, are identical. I n addition, p a r a t h y r o i d h o r m o n e was shown to h a v e no influence on r c a b s o r p t i o n of Ca++ in the p r o x i m a l tubule. Methods Nondiuretie male albino rats of the FW 49 strain, fed an Altromine Standard diet and weighing 150--250 g, were anesthetized by intraperitoneal injection of Inactin, 200 mg/kg body weight. The microperfusion and mieropuncture techniques have been previously described 1°. Blood was obtained from the inferior vena cava Table 1. Composition o/solutions used/or mieroper]usion Solution Solution Solution Solution

a b c d

139 mM/L NaC1; 15 mEq/L Ca++ (as CaC12) 150 mM/L NaC1; no Ca++ 100 mM/L NaC1; t00 mM/L marmitol; Polyethylene glycol solution 15.8°/0 (isotonic)

no Oa ++

immediately after each micropuncture. Composition of the solutions used for perfusion is given in Table 1. The perfusion rate was usually 1.6× 10-2/~l/min, but in one group of animals it was reduced to 0.85 × 10-2 #l/rain. At the end of each experiment, perfused tubules were injected with Neoprene and the kidney macerated 1, and the perfused segments isolated and measured. Calcium determination. Ca++ concentration in the mieropuncture samples was determined by an ultramicro modification of the murexide method of SIEGMUm) and Dunce 1~. Approximately 0.l #l of a standard solution or of tubular fluid was pipetted into 2.0 #I of doubly distilled water on a piece of Parafilm®. One #1 of 0.1 N NaOH and 1.0/A of a freshly prepared solution of murexide (45 mg-°/0) were added, and all constituents mixed well. The droplet was then placed at once into a plexiglass microcuvette is and the extinction measured at 480 m/~ in a Beckman spectrophotometer. Because the intensity of color development varies with time, eare was taken to insure that the elapsed time between the addition of murexide and the reading was kept constant. After each sample a similar standard concentration was measured for direct comparison. Extinction was a linear function of calcium concentration, and had a value of -l-0.700 at a [Ca++] of 15 mEq/L. The standard deviation was ± 0.24 mEq/L. Total calcium in plasma was measured on a Technicon Auto-Analyser® by Gitelman's modification 5 of the cresolphthalein complexone method of K]sssL~n and WoLF~.t~¢1°. Approximately 60--700/0 of plasma calcium has been shown to be ultrafiltrable in rats ~2, dogs s, and humansa,18,17. In addition, it has been demonstrated that in humans about 950/8 of the ultrafiltrable calcium is ionized~, 13. We have assumed that a similar relation exists in the rat, i.e., that plasma ionized calcium ([Ca++] p) is about 650/o of the total plasma calcium. Determination of radioactive Ca% Rats were given 250--500 #e Ca~5 intravenously in isotonic NaC1 (Caa5 specific activity 2--5 mc/mg Ca), and 1--2 hours

Calcium transport in the proximal tubule

111

allowed for equilibration before micropuncture. The radioaetivities of tubular fluid and plasma were measured in a liquid scintillation spectrometer. Radioactivity of the tubular fluid samples was approximately twice background; plasma radioactivity was roughly five times background. No quenching correction was necessary because of the small volume of the samples. Na + determination. Na + was measured both in the micro-samples (dilution 1 : 1000) and in plasma in a Beckman speetrophotometer with a flame attachment and phot~multipller and integrator equipment. Thyroparathyroidectomy. The tracheas of ether-anesthetized animals were exposed through a median incision in the neck, and the thyroid and parathyroid glands were totally extirpated, care being taken to remove all visible gland tissue. Subsequently, each rat was given 0.02 mg thyroxine daily intramuscularly, and micropuncture experiments were performed one to five days posgoperatively. For comparison with these hypoparathyroid rats, a group of normal rats was given large doses of parathormone (up to 20 Collip units per rat per day i.m.), either just before the experiment or over a period of several days.

Results

1. Ca++ e/flux/rom the proximal convolution D u r i n g perfusion of t h e p r o x i m a l c o n v o l u t i o n w i t h solution a (139 m M / L NaC1 + 15 m E q / L Ca ++) t h e Ca++ c o n c e n t r a t i o n d e c r e a s e d e x p o n e n t i a l l y along t h e p e r f u s e d s e g m e n t (Fig. 1). A t a p o i n t 900 # d i s t a l

[c~ ++] ~Eq/L 10"

•

5-

/'"

---

~

32-

1 500

PERVUSED

I

I

1

I 000

I 5OO

2O00

TUBULAR

jtl

SEGMENT

Fig. 1. ~cabsorption of Ca++ out of the lumen of the proximal convolution perfuscd with solution a (I39 m?,f/L NaCI q- 15 m E q / L Ca++). • Controls; • Normal rats receiving large doses of parathormone; • ttypoparathyroid rats

to t h e p o i n t of perfusion, c o r r e s p o n d i n g to a c o n t a c t t i m e of a p p r o x i m a t e l y 1.1 see, t h e Ca++ c o n c e n t r a t i o n in t h e perfusion solution h a d d e c r e a s e d f r o m 15 to 7.5 m E q / L . T h e lowest Ca ++ c o n c e n t r a t i o n s measu r e d in this series were 3 - - 4 m E q / L in p e r f u s e d s e g m e n t s 2000 # in length, b u t in a n o t h e r series of e x p e r i m e n t s c o n c e n t r a t i o n s as low as 2.0 m E q / L were observed.

112

A. F~IeK, G. RUMRZCH,K. J. ULL~ICI~,and W. E. LASSZTE~:

Regression lines of these results for controls, parathyroidectomized animals, and normal rats receiving large doses of par~thormone all had the same slope (Table 2; perfusion rate 1.6X 10-2 #l/rain). I t appears therefore that neither a deficiency nor an excess of parathormone had any influence on Ca++ reabsorption in the proximal convolution in the observed range of Ca++ concentrations. Table 2. Ca++ reabsorption in proximal convolution N u m b e r o f punctures

Perfusion rate × 10 -~/~l/min

b* X 10 6 ,u-~

-34.2 -79.8 -36.5 -34.2 Z(xy) - - ~ ~(x) * b ~- slope of regression lines for the single groups ~ Z(x2) - - 2 Z(x) '

Controls Controls Parathormone Parathyroideetomy

1.6 0.85 1.6 1.6

where y = log [Ca++l TF and x = length of perfused segment. To determine whether or not the perfusion rate influences Ca++ reabsorption, the rate was reduced from 1.6 to 0.85X 10-2 #l/min. The slope of the regression line changed by ~ factor of 2.3, not 1.9, as expected if the change in perfusion rate had no effect on calcium reabsorption and the surface area for reabsorption did not change (Table 2). Considering the experimental difficulties at the slower perfusion rate, however, the agreement between the observed and the predicted change in slope is reasonably close, and suggests that the perfusion rate had little, if any, effect on the absolute rate of calcium reabsorption.

2. Ca ++ influx into the lumen o/the proximal convolution To determine the rate of Ca ++ influx, tubules were perfused with Ca++-free solutions b and c. Results are shown in Fig. 2. At a point 1000 # distal to the site of perfusion, the Ca ++ concentration averaged [Ca**k

m E,:V'L 54321-

° ° .......

1000 PERFUSED

o~°,,,

2000 , TUBU~

•

i~

3C~

4000 ]~

SEGMENT

Fig. 2. Influx of Ca ++ into the l u m e n of the p r o x i m a l convolution. Perfusion w i t h Ca++-free solutions b and c

Calcium transpo1¢ in the proximal tubule

113

2.66 mEq/L. Since no further rise occurred in longer segments (up to 3,600#), one m a y assume t h a t this is the equilibrium concentration. This concentration is similar to t h a t observed in free flow b y LASSITEn, GOTTSC]~ALK,and MYLL~ 12 at the end of the proximal convolution during mannitol diuresis. Tubular segments shorter than 1000 # were not perfused because previous experiments had shown t h a t the method for Ca++ analysis is relatively inaccurate at very low concentrations. Measurements of Ca++ influx utilizing Ca 45, however, are in agreement with t h a t par~ of the curve indicated b y the dashed line.

3. Direct comparison o/relative Ca++ and Na+ influx into the lumen o/the proximal convolution Net influx of Ca 4~ and of Na+ into a solution containing no Ca++ or Na + (solution d) are compared in Fig. 3, in which TF/P of Ca 45 for each sample is plotted against T F / P Na+. I t is noted t h a t the regression line T F C a ++ PO.6--

•

/"

*/ / • /:/ •~ ./ •

0.5

0.40.3--

•

o" °o

•

O.]-

0.1

O.Z

t

0.3

I

0.6

J

0.5

I .......

0.6

p'~ Na ÷

Pig. 3. Influx of Ca 45 a n d Na + into the l u m e n of the proximal convolution. Perfusion with Ca ~+- a n d Na+-free solution d. On the ordinate is plotted TF]P for Ca++; on the abscissa, TF/P f o r / ~ a +. Solid line : l~cgression line. D a s h e d line : Line of equality

(solid) deviates only slightly from the line of equality (dashed) ; thus the relative net influxes of Na + and of Ca++ into the lumen of the proximal convolution were of similar magnitude.

4. Characteristics o/Ca ++ transport in the proximal convolution% 7a Net outward transport of Ca++ from the lumen of the proximal convolution can be calculated from the data of Fig. 1 b y the formula : ~bCa++ out = - - ~1 b " r • v • ([Ca++]x -- [Ca++JE ) , in which b is the slope of the reabsorption line, r the radius of the proximal convolution (10 #), v the velocity of flow of the perfusion fluid down the Pfliigers Arch. ges. Physiol., Bd. 286

8

114

A. FRIOI;,G. I ~ U M R I C t t , K. J.

ULLRICtt,

and W. E. LASSI~EE:

tubule, [Ca++]x the Ca++ concentration in the perfusion solution at the end of a perfused segment of length X, and [Ca++]E the Ca ++ concentration in tubular fluid at equilibrium. Ca++ concentrations were corrected for net transtubular water movement, determined with C14-inu]in. Net outward transport of Ca++ at the beginning of any perfusion ([Ca++Jx = 15 mEq/L) was: ~bCa++ out : 4.03 • 10-~ # E q / m m ~ sec. Net Ca ++ influx into the lumen of the proximal tubule was calculated from measurements of CM5 influx into perfused segments with lengths less than 250 #, using the following formula:

~bCa++ in

In

r =

• v"

~-

[Ca++]P

[Ca++]~- - [Ca++]z X " [Ca++] P "

Assuming [Ca++]~ to be 3.41 m E q / L (650/0 of 5.24 mEq/L), the calculated q~Ca++in was 2.09 × 10-5 # E q / m m ~ sec. ]NFLUX

(~ Ca +* ( x l 0 "5 .~LIEcl/rnrn 2 s e c ) --+3'

--÷2

-4

i

-2

1

*

÷

+

*

+2

-1-

-3-

-4-

~5

OUTFLUX F i g . 4 . T r a n s p o r t characteristics of Ca ++. N e t fluxes (ordinate) are plotted as a function of the difference in Ca ++ concentrations b e t w e e n i n t r a l u r a i n a l fluid a n d p l a s m a . P o s i t i v e v a l u e s on the ordinate indicate influx; n e g a t i v e values, efflux

The complete characterization of Ca++ transport is illustrated in Fig. 4, in which net flux rates are plotted as a function of the difference between intraluminal and plasma Ca++ concentrations. Since the equilibrium Ca++ concentration, a~ which net transtubular flux is zero, was 2.66 ttEq/L, the equilibrium T F / P was 2.66/3.41 = 0.78.

Calcium transport in the proximal tubule

115

Discussion 1. Outward transport o / C a ++

These studies confirm the previously reported observation of LASSITEI~, GOTTSCHALKand MYLLE 1~ t h a t Ca++ m a y be rcabsorbed from the proximal tubule against an electrochemical gradient, and thus provide further evidence for the importance of active transport in the proximal reabsorption of calcium. The p u m p potential for this process can be RT % calculated b y means of the Nernst equation, E = =~ ~ - l n ~ , in which ci is the equilibrium concentration in the tubular fluid, at which net transport of Ca++ is zero (in this instance 2.66 mE@L), and co is the Ca++ concentration in plasma (in this instance assumed to be 3.41 mEq/L). I f the measured transtubular potential is zero ~a, the calculated p u m p potential Eca++, is: Eca ++ = 30.7 m V

(

log 2~6-] = 3.3 mV.

Net outward transport increases linearly with increasing tubular fluid [Ca++] at concentrations between 2.6 and 15 m E q / L , and there is no evidence of saturation of the transport system at 15 m E q / L (see Fig. 4). 2. Influence o/parathyroid hormone on Ca ++ reabsorption

Several investigators have shown t h a t parathyroid extract (PTE) promotes tubular reabsorption of calcium in dogs 11 and rats2,15,16. WIDRow and L ~ v ~ s ~ : v ~ concluded from stop-flow studies in dogs t h a t P T E enhanced Ca++ reabsorption primarily in the distal tubule. The results of the present study, which demonstrate t h a t parathormone does not influence Ca ++ reabsorption in the proximal convolution do not contradict these stop flow findings since we have not yet studied distal tubular Ca++ transport. 3. Relations between Ca ++ and N a + transport Influx. Comparison of the T F / P ratios for Ca++ and Na+ (Fig.3) reveals t h a t the relative net influxes of Na+ and Ca++ into the proximal tubule are of similar magnitude. The absolute net Na+ influx (¢Na+in) in these experiments, calculated in the same way as the simultaneous absolute Ca ++ influx, was 80.07 × l0 -5/~Eq/mm ~. see ; thus the ratio of Na+in to ~b Ca++ m equaled 38.5 (Table 3, a). The ratio of plasma concentrations of Na+ to Ca ++ was 41.3 (Table 3, a). The agreement between the flux ratio and the concentration ratio suggests the existence of similar influx mechanisms for Na + and Ca ++. Efflux. GE~Tz ~ and HIEgI~OLZm~7,7a have found the net Na + transport out of the lumen of the proximal convolution at a T F / P of 1 to be 8.9 × 10 -5 # E q / m m 2. see. The net efflux of Ca++ under similar circumstances was 0.25 × 10 -~ # E q / m m ~" see (Fig.4) ; thus the ratio of Na+ to 8*

116

A. FRICK, G. I{UIIIRICtt, K. J. ULLRICtI, and W I E. LASSlTER:

Ca++ outflux is 35.6 (Table 3, b). This ratio is similar to the ratio of the i n t r a t u b u l a r c o n c e n t r a t i o n s of the two ions, which is 44.0 (Table 3, b). Consequently, characteristics of the o u t w a r d t r a n s p o r t of b o t h Na+ a n d Ca ++ are v e r y similar. Table 3. Comparison o] Ca ++ and Na + transport rates a) Ket Influx Na + Influx Ca++ Influx

[Na+]p [Ca++]p"

80.07 - - --38.5 2.09 141.0 -- 3.4~ - - 4 1 . 3 -

-

b) Ne~ Efflux at TF/P = i Na+ Outward Transport Ca++ Outward Transport [Na+] TF 150 [Ca++]TF -- 3.41

-

-

8.9 -- 35.6 0.25

44.0

a) Ratios of net influxes of l~a+ and Ca++, and of simultaneous concentrations of these ions in plasma. (Experiments of Fig. 3.) b) Ratios of net outward transport rates of Na + and Ca++, and of simultaneous intratubular concentrations. (Experiments of Fig. 1.) (Transport rates are expressed as × 10-5 #Eq/mm e sec; concentrations as mEq/L.) E q u i l i b r i u m concentration. A t e q u i l i b r i u m (net flux = zero) the 110 T F / P N a + - - 145 - - 0.76; this ratio is equal to t h a t for Ca++, which 2.66 is 3.4~ ~ 0.78. T h e s t r i k i n g s i m i l a r i t y i n the b e h a v i o r of Ca++ a n d Na+ i n t e r m s of o u t w a r d t r a n s p o r t , influx i n t o the l u m e n of the p r o x i m a l c o n v o l u t i o n , a n d the c o n c e n t r a t i o n ratio a t e q u i l i b r i u m , s t r o n g l y suggests a c o m m o n t r a n s p o r t m e c h a n i s m for Ca ++ a n d Na+, as originally proposed b y WALSm¢ ~° because of the s i m i l a r i t y b e t w e e n Na+ a n d Ca++ clearances i n a v a r i e t y of conditions. Adcnowledgements. The authors would like to express their gratitude to Dr. Dr. G. Fllcws for his assistance in calculation of the ion fluxes, and to Dr. H. HOLZGI~V~ for his help in determination of [Na+] in the micro-samples.

References 1 BOTT,P. A.: Renal excretion of creatinine in necturus. A reinvestigation by direct analysis of glomerular and tubule fluid for creatinine and inulin. Amer. J. Physiol. 168, 107 (1952). e C~lCTEI~,N., D. CALl)am, R. STE~L~, and D. W. S~LI)I~: Effect of parathyroidectomy on calcium excretion in normal and acidotic rats. Clin. Res. 10, 62 (1962). a FANCOXI,A., and G. A. Rosin: The ionized, complexed, and protein-bound-fractions of calcium in plasma. Quart. J. lVled. 27, 463 (1958). aa Fl~5~m]m,E., u. U. ttEG~L: Potentialmessungen am proximalen Tubuhis der Rattenniere. Pfliigers Arch. ges. Physiol. 288, R 23 (1965). 4 Gv.l~TZ,K.-tt. : Transtubul~ire Natriumchloridfliisse und Permeabilit~t ffir l~ichtelektrolyte im proximalen und distalen Konvolut der Rattenniere. Pfliigers Arch. ges. Physiol. 276, 336 (1963). 5 GITELI~AI%H. : Personal communication (1964).

Calcium transport in the proximal tubule

117

6 GREENE, C. H., and M. H. P o w E r : The distribution of electrolytes between serum and the in vivo dialysate. J. biol. Chem. 91, 183 (1931). 7 HIERItOLZER,K. : Analyse der Natrium-TransportstSrung in der Niere adrenalektomierter Ratten. Untersuchungen am Einzelnephron. Habilitationsschrift, B e r l i n 1964. 7a _ M. WIEDERHOL% H. STOLT~, and G. Rv~mIc~: Transtubul~re Na-Str5me im proximalen und distalen Tubulus adrenalektomierter Ratten. (In preparation.) s HOLZGREVE,H., A. PRICK, G. ~UMRICtt, ~ . WIXEDERHOLT U. K . J . ULLRICH: Wirkungsweise yon Diuretica auf den transtubularen Transport yon Natriumchlorid. In: Normale und pathologische Punktionen des Nierentubulus. 3. Symposium der Ges. fiir Nephrologie, Berlin 1964. Hrsg. yon K. J. ULLRIC~ U. K. HIER~OLZV.R. Bern: Huber 1965. 9 KAS~GARIA~, M., H. STSOKLE, C. W. GOTTSCHALK, and K. J. ULLRICH: Transtubular electrochemical potentials of sodium and chloride in proximal and distal renal tubules of rats during antidiuresis and water diuresis (Diabetes insipidus). Pfliigers Arch. ges. Physiol. 277, 89 (1963). 10 K~ssL]sR, G., and M. WOLF~AN: An automated procedure for the simultaneous determination of calcium and phosphorus. Clin. Chem. 10, 686 (1964). n KLEEMAN, C. R., D. BER~ST~nV, R. ROCKier, J. T. DOWLrSG, and M. H. MAXW~LL : Studies on the renal clearance of diffusible calcium and the role of the parathyroid glands in its regulation. In: The Parathyroids, Proceedings of a symposium on advances in parathyroid research, edit. by R. C. G~EE~ and R. V. TAL~AGE, p. 353. Springfield, Ill. : Ch. C. Thomas 1961. 12 LASSITER, W. E., C. W. GOTTSCHALK, and M. iV~YLLE: i~cropuncture study of renal tubular reabsorption of calcium in normal rodents. Amer. J. Physiol. 204, 771 (1963). 13 RosE, G. A. : Determination of the ionised and ultrafilterable calcium of normal human plasma. Clin. chim. Acta 2, 227 (1957). 14 SIEG~VND, P., u. H. J. DOLCE: Einflul3 des Carboanhydratase-Inhibitors 2-Acetamino-l,3,4-thiodiazol-sulfonamid-(5) (Diamox) auf den Caleinmstoffweehsel yon Legehennen. Hoppe-Seylers Z. physiol. Chem. 820, 149 (1960). 1~ TALMAGS,,R. V., and F. W. KRArSTZ: Progressive changes in renal phosphate and calcium excretion in rats following parathyroidectomy or parathyroid administration. Proc. Soc. exp. Biol. (N.Y.) 87, 263 (1954). 16 _ _ and G. D. BUCHANA~: Effect of parathyroid extract and phosphate salts on renal calcium and phosphate excretion after parathyroidectomy. Proe. Soc. exp. Biol. (N.Y.) 88, 600 (1955). 17 TORIBA~A,T. Y., A. R. TER~PKA, and P. A. DEWEY: The ultrafiltrable calcium of human serum. I. Ultrafiltration methods and normal values. J. elin. Invest. 36, 738 (1957). is ULLRmH, K. J., u. A. HAMPET.: Eine einfaehe Mikrokfivette fiir Monochromator Zeiss und Beckman Modell DU. Pfliigers Arch. ges. Physiol. 268, 177 (1958). G. R u ~ I C H u. G. P~cHs: Wasserpermeabilit~t und transtubul~rer Wasserflu~ corticaler Nephronabsehnitte bei verschiedenen Diuresezust~nden. Pflfigers Arch. ges. Physiol. 280, 99 (1964). 20 W A n s ~ , l~I. : Calcium clearance as a function of sodium clearance in the dog. Amer. J. Physiol. 20@, 1099 (1961). 21 WIDROW, S. H., and N. G. LEVINSKY: The effect of parathyroid extract on renal tubular calcium reabsorption in the dog. J. olin. Invest. 41, 2151 (1962). ~9

_

Dr. A. PRICK, Physiologisches Institut der Freien Universitiit, 1 Berlin 33, Arnimallee 22