Effects of Colchicine and on the Cellular Action of Vasopressin in Mammalian Kidney A POSSIBLE ROLE OF

Thomas P. Dousa, Larry D. Barnes

J Clin Invest. 1974;54(2):252-262. https://doi.org/10.1172/JCI107760.

Research Article

To evaluate the possible role of microtubules in the cellular action of vasopressin on the mammalian kidney, the effects of -disrupting agents were studied in vivo and in vitro.

In vivo studies were done in rats in mild to moderate water diuresis induced by drinking 5% glucose. Microtubule- disrupting alkaloids, colchicine (0.1 mg/day) or vinblastine (0.08 mg/day), given intraperitoneally, did not change water and solute excretion itself, but blocked or markedly inhibited the antidiuretic response (increase in urine osmolality and decrease in urine flow) to exogenous vasopressin. Total solute excretion was unaffected by these two alkaloids and there were no substantial changes in excretion of sodium, potassium, or creatinine. Lumicolchicine, a derivative of colchicine that does not interact with microtubules, did not alter the antidiuretic response to exogenous vasopressin. Activities of adenylate cyclase in the renal medullary plasma membrane, and cyclic AMP phosphodiesterase and protein kinase in renal medullary cytosol, were not influenced by 10-5—10-4 M colchicine or vinblastine in vitro. Studies on the subcellular distribution of microtubular protein (assessed as [3H]colchicine-binding protein) in renal medulla shows that this protein is contained predominantly in the cytosol. Particulate fractions, including plasma membrane, contain only a minute amount (less than 6%) of the colchicine-binding activity.

The results suggest that the integrity of cytoplasmic microtubules in cells of the distal nephron is required for […]

Find the latest version: https://jci.me/107760/pdf Effects of Colchicine and Vinblastine on the Cellular Action of Vasopressin in Mammalian Kidney

A POSSIBLE ROLE OF MICROTUBULES

THOMAS P. DOUSA and LARY D. B EmnS From the Nephrology Research Laboratories, Department of Medicine (Division of Nephrology) and Department of Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55901

A B S T R A C T To evaluate the possible role of micro- The results suggest that the integrity of cytoplasmic tubules in the cellular action of vasopressin on the microtubules in cells of the distal nephron is required mammalian kidney, the effects of microtubule-disrupting for the antidiuretic action of vasopressin, probably in agents were studied in vivo and in vitro. the sites distal to cyclic AMP generation in the mam- In vivo studies were done in rats in mild to moderate malian kidney. water diuresis induced by drinking 5% glucose. Micro- tubule-disrupting alkaloids, colchicine (0.1 mg/day) or vinblastine (0.08 mg/day), given intraperitoneally, did INTRODUCTION not change water and solute excretion itself, but blocked The mechanism by which vasopressin regulates water or markedly inhibited the antidiuretic response (in- permeability of certain epithelial membranes is only crease in urine osmolality and decrease in urine flow) to partially known. Considerable evidence accumulated in exogenous vasopressin. Total solute excretion was unaf- recent years indicates that cyclic AMP serves as an fected by these two alkaloids and there were no sub- intracellular mediator in the cellular action of vasopres- stantial changes in excretion of sodium, potassium, or sin (1, 2). The mechanism by which cyclic AMP formed creatinine. Lumicolchicine, a derivative of colchicine that under the influence of vasopressin increases water perme- does not interact with microtubules, did not alter the ability of collecting ducts remains unknown. Still hypo- antidiuretic response to exogenous vasopressin. Activi- thetical is the idea that cyclic AMP acts on the lumenal ties of adenylate cyclase in the renal medullary plasma plasma membrane through its influence on protein phos- membrane, and cyclic AMP phosphodiesterase and pro- phorylation (3-5). Taylor, Mamelak, Raven, and Maffly tein kinase in renal medullary cytosol, were not influ- (6) found that some drugs known to disrupt the struc- enced by 10'-10' M colchicine or vinblastine in vitro. ture of microtubules partially inhibit the increase in wa- Studies on the subcellular distribution of microtubular ter permeability in response to vasopressin and exoge- protein (assessed as ['H]colchicine-binding protein) in nous cyclic AMP in isolated toad urinary bladders. The renal medulla shows that this protein is contained pre- stimulatory effect of vasopressin and cyclic AMP on dominantly in the cytosol. Particulate fractions, including active sodium transport in the same tissue was not in- plasma membrane, contain only a minute amount (less fluenced by these drugs. This suggests that microtubules than 6%) of the colchicine-binding activity. are involved in the regulation of water permeability by vasopressin in steps subsequent to the cyclic AMP forma- This work was presented at the meeting of the Western Section of the American Federation for Clinical Research tion. in Carmel, California in February, 1974. (Clin. Res. 22: The regulatory effect of vasopressin on water perme- 206A, 1974). ability of the distal nephron in the mammalian kidney Dr. Dousa is an Established Investigator of the Ameri- appears to be analogous to the amphibian urinary bladder can Heart Association. Dr. Barnes is the recipient of a Mayo Research Fellowship. system, including the mediatory role of cyclic AMP Received for publication 21 November 1973 and in revised (1, 2). The presence of microtubules in cells of collecting form 28 January 1974. ducts has been described (7).

252 The Journal of Clinical Investigation Volume 54 August 1974- 252-262 In the present study, colchicine and vinblastine, two tected after administration of alkaloids (Tables I and III). blocked the anti- At termination of in vivo experiments, animals were sacri- microtubule-disrupting agents (8), ficed and the abdominal cavity was opened and macro- diuretic effect of exogenous vasopressin in mammalian scopically examined. No signs of ascites or other macro- kidney. Colchicine or vinblastine did not inhibit adenylate scopic pathological changes were observed. Kidneys were cyclase, cyclic AMP phosphodiesterase, or cyclic AMP- cut coronally and inspected macroscopically. No differences dependent protein kinase in subcellular fractions from in either cortical or medullary tissues were observed be- renal medulla. The results the view that micro- tween control and experimental animals. support Additional experiments were conducted to test for non- tubules play a role in the cellular action of vasopressin specific toxic effects of colchicine. A group of five rats was in steps distal to cyclic AMP formation in the mam- treated with colchicine, as described in Table I and com- malian kidney. pared with the same number of control animals. No differ- ence was observed between control and colchicine-treated METHODS rats in levels of serum creatinine, blood urea nitrogen, calcium, potassium, sodium, or in urinary pH measured at Materials. [PH]colchicine and [(H]cyclic AMP were the end of experiment. Kidneys were examined histologi- products of New England Nuclear, Boston, Mass. ('P]a- cally and no pathological changes were observed in glomer- ATP and [P]'y-ATP were products of ICN Corp., Chemi- uli, tubuli, or other renal structures. In another control cal & Radioisotopes Div., Irvine, Calif. All chemicals and experiment, animals were again treated with the colchicine biochemicals used were of highest purity grades. Nonradio- as described in Table I and the drug was then discontinued active colchicine and vinblastine were gifts of Eli Lilly & for 3 days. After this rest period, the rats were injected Co., Indianapolis, Ind. For in vitro stimulation of adenylate with exogenous vasopressin. The animals treated previously cyclase, synthetic [8-argininel-vasopressin, Sigma Chemical with colchicine did not differ in the antidiuretic response Co., St. Louis, Mo., was employed. In animal experiments, from the control animals. Urine osmolality (Uo.m) ' was vasopressin tannate (Pitressin tannate in oil, Parke Davis measured with a Fiske osmometer (Fiske Associates, & Co., Detroit, Mich.) was used. The dose of 0.5 U was Inc., shown to Uxbridge, Mass.), and sodium (UN.) and potassium (UK) elicit supramaximal antidiuretic effect in rats were determined with a Technicon Autoanalyzer II (Tech- (9). Lumicolchicine was prepared by irradiation of colchi- nicon Instruments Corp., Tarrytown, N. Y.). Urinary and cine with a long wavelength ultraviolet lamp as described by Mizel and Wilson (10). Measurement of the decreased serum creatinine was measured spectrophotometrically (12). at nm in 24-h urinary output (urine flow), total solute excretion absorbancy 350 100% ethanol established the (UVo.m), sodium (UVN.), potassium (UVK), and crea- conversion of colchicine to lumicolchicine. The ethanol was tinine (UVc,) were calculated and expressed per 100 grams evaporated under a stream of nitrogen and the lumicolchi- of body weight. Colchicine, vinblastine, and lumicolchicine cine was dissolved for injection into animals. were dissolved in a solution of 70 mM NaCl and 5 mM In vivo methods. The effect of colchicine, vinblastine, and Tris-HCI, pH 7.4, for intraperitoneal injections. Each in- lumicolchicine on the action of vasopressin in vivo was dividual dose of drugs was contained in 0.2 ml of solvent, studied in adult male rats of Sprague-Dawley strain weigh- and control animals received the same volume of solvent ing 250-300 g. Body weight was measured daily and did without drug. not change significantly during the experimental period either in control animals or in animals receiving studied Dosages of alkaloids utilized in the present study were drugs. The animals were allowed to adjust to individual based on estimation from other studies (13) and preliminary metabolic cages for several days before the experiment. experiments. Dosages lower than those employed in present The animals were kept in air-conditioned animal rooms experiments produced no or only borderline effect. with a temperature of 25°C and were fed dry food ad lib In vitro methods. The content of microtubular protein (Purina Laboratory Chow, Ralston Purina Co., St. Louis, was determined by utilizing its ability to bind colchicine Mo.). Mild to moderate water diuresis was induced in rats specifically (8). The colchicine-binding protein was assayed by feeding 5% glucose (11). Animals developed diuresis of by the method described by Weisenberg, Borisy, and Taylor different degrees, which was nevertheless relatively con- (14) with DEAE filter paper disks. When particulate stant in each individual animal. Therefore, based on ob- fractions of renal medullary homogenate were studied and servations from control periods durng the first two days, in compared with soluble proteins, double-layer disks of each experiment the animals were divided into two groups DEAE cellulose paper and GF/C Whatman glass fiber having, on the average, a similar degree of diuresis. On papers were used to assure retention of membrane frag- days 3-6, one group of animals served as a control and ments. GF/C glass fiber paper itself retained neither ['H]- received solvent without drugs, while the other group, ex- colchicine nor colchicine-binding protein. The tissue extracts perimental, received the test drugs. On days 4-6, both were incubated with 0.6 X 1lo M ['Hicolchicine (10 cpm/ groups received the same dose of vasopressin. The animals tube) at 370C, in 0.3 ml of a buffer containing 10 mM receiving the indicated doses of colchicine, vinblastine, or MgCl,% 10 mM potassium phosphate, and 5 X 10' M EDTA lumicolchicine did not show any apparent clinical signs of (pH 6.8). The radioactivity of [(H]colchicine retained in toxicity. No diarrhea was observed. The urines were col- filters was counted in Bray's solution. lected daily under mineral oil and qualitatively examined Adenylate cyclase was assayed by the method described in for the presence of glucose and proteinL; no glucosuria or our previous study (15). For the chromatographic separa- proteinuria was detected. No change in urinary excretion tion of cyclic AMP from other nucleotides, we used a of creatinine or in serum creatinine concentration was de- modification described recently (16). 'The glucose was determined with Clinitest and protein 'Abbreviations used in this paper: Cr, creatinine; GFR, was estimated with Albustix Reagent Strips, Ames Co., glomerular filtration rate; Uom, urine osmolality; UV, total Div. of Miles Lab., Inc., Elkhart, Ind. solute excretion. Microtubules in the Action of Vasopressin 253 TABLE I Inhibition of Renal Response to Vasopressin by Colchicine

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Urine flow, ml/100 g body wt Control 16.4 24.07 20.55 5.42 4.50 4.40 per 24 h4SEM ±2.65 :14.93 ±5.44 40.55 40.42 ±0.33 Experimental 20.65 19.24 19.35 21.16 20.41 18.77 ±3.61 ±5.06 -±-4.97 ±5.55 44.97 ±4.23 P value* NS NS NS <0.02 <0.01 <0.01 Uoum, mosmol/kg H204SEM Control 657 533 640 1,466 1,707 1,717 ±89 ±163 ±189 ±t69 ±101 ±96 Experimental 586 745 724 684 688 722 ±126 4201 ±210 ±-225 ±222 4212 P value NS NS NS <0.01 <0.005 <0.005 UVo.., posmol/JOO g body wt Control 9,773 9,260 8,730 7,772 7,528 7,443 per 24 h±SEM ±841 ±946 ±:974 ±511 ±500 ±490 Experimental 9,019 9,907 9,348 8,865 9,041 9,439 ±487 ±487 ±402 ±686 ±t721 ±797 P value NS NS NS NS NS NS UVNa, ,.ueq Na/100 g body wt Control 920 907 622 616 688 664 per 24 h±SEM 454 ±43 ±45 i16 ±476 ±53 Experimental 859 897 764 914 737 697 ±20 ±43 -±-86 ±67 ±68 ±-85 P value NS NS NS <0.005 NS NS UVK, ,ueq K/100 g body tControl 1,324 1,404 1,211 1,043 924 910 wt per 24 h±SEM +68 ±73 ±102 ±70 ±83 463 tExperimental 990 1,461 1,372 1,364 1,385 1,315 ±65 ±108 ±125 ±57 ±82 ±123 P value <0.01 NS NS <0.01 <0.005 <0.2 UVCr mg/100 g body wt per Control 7.05 6.42 5.63 5.84 6.35 6.00 24 h±SEM ±0.35 ±0.47 ±0.20 ±0.18 i0.70 ±t0.47 Experimental 6.20 8.09 6.31 7.26 7.58 6.83 ±0.26 ±t0.47 ±0.47 ±2.12 ±0.71 ±0.48 P value NS NS NS NS NS NS

* For significance of the difference between control and experimental groups. NS denotes P > 0.05 (t test). $ There were no significant changes in UVK between consecutive days in either control or experimental groups of animals (P > 0.05, paired t test), with the exception between days 1 and 2 for the experimental group. Rats from experimental group received 0.1 mg colchicine intraperitoneally on days 3, 4, 5, and 6; the control group received only solvent. On days 4, 5, and 6 both groups were administered 0.5 U of pitressin tannate in oil subcutaneously. Each group con- sisted of six rats. At the end of experiment serum creatinine levels in control and experimental group were 0.789±0.059 and 0.727±0.038 mg/100 ml±SEM, respectively. These values were not significantly different from each other (t test).

Cyclic AMP phosphodiesterase was assayed by the method medulla. All preparations were done at 0-2°C. The prepara- described in another of our studies (17). None of the tion of plasma membranes followed in principle the method tested drugs interfered with the breakdown of 5'-AMP to described by Fitzpatrick, Davenport, Forte, and Landon nucleosides and phosphate by snake venom 5'-nucleotidase. (18) except for -the following modification: all solutions For the assay of cyclic AMP-dependent protein kinase, we contained 10 mM MgCl2, 10 mM potassium phosphate, 2X used the same method as in our earlier experiments (4) 10-4 M GTP and 5 X 10- M EDTA, and were adjusted to with the exception that the initial trichloroacetic acid pH 6.8. These conditions stabilize colchicine-binding pro- (TCA) precipitate was dissolved in 0.1 ml of 1 N NaOH tein (19). Other subcellular fractions were prepared as and reprecipitated by 5%7 TCA before the final washing on follows: The pellet from the first centrifugation in the Millipore filters (Millipore Corp., Bedford, Mass.). All above-mentioned modified procedure was resuspended in 2 M assays were run in triplicate. sucrose, centrifuged at 13,000 g for 10 min, and the pellet In studies on subcellular localization of colchicine-binding was taken as the "nuclear" fraction. The supernate from protein, the fractions were prepared from bovine kidney the first centrifugation in the above-mentioned modified 254 T. P. Dousa and L. D. Barnes procedure for plasma membrane preparation was further VASOPRESSIN centrifuged for 20 min at 10,000 g. The sediment was 20CDO- 0.5 U/day (--) resuspended in homogenizing medium and recentrifuged as ~~~~~~~~r before. The resulting pellet represented the "mitochondrial" fraction. The supernate from the first centrifugation at 10,000 g for 20 min was further centrifuged at 100,000 g for 60 min and the supernate was taken as the "cytosol." 15CDO The sediment was resuspended in homogenizing medium and centrifuged again at 100,000 g for 60 min and the pellet was then taken as the "microsomal" fraction. The fractions were quickly frozen in dry ice and kept in dry ice until 1,cDO assayed for colchicine-binding activity. Fractions for studies of adenylate cyclase, cyclic AMP phosphodiesterase, and protein kinase activities were prepared in a slightly different way. The plasma membrane fraction was prepared accord- ing to the method of Fitzpatrick et al. (18) as employed 5cDo - in our previous studies (4). The supernate from the first L- centrifugation in this procedure was further centrifuged for F 0.1 mg/day (---) I 60 min at 100,000 g, and the supernate (cytosol) was used OL I I I I I as a source of protein kinase and cyclic AMP phospho- 1 23 45 6 diesterase activities. The protein content in preparations Days was determined by the method of Lowry, Rosebrough, Farr, FIGURE 1 Response of urine osmolality to vasopressin in and Randall (20). Radioactivity was determined by liquid control rats (0 O) and in rats treated with colchicine scintillation counting. Student's t test was used for statis- on 3-6 experimental days (0 - - - 0). Each group con- tical evaluation of results for both group and paired com- sisted of six animals. * value significantly different from parisons (21). controls at P < 0.05 or more (t test). RESULTS In vivo studies ences in UVK on days 4-6 between the two groups are due to a decrease in potassium excretion in controls Effect of colchicine. The effect of colchicine was rather than to any change in the experimental group. tested on 12 rats divided into two equal groups. The The effect of lumicolchicine was tested in the same first 2 days were the control period, and on the 3rd day manner described for colchicine. This derivative, given the experimental group received 0.1 mg of colchicine in- at the same dosage as colchicine, (0.1 mg/day), had no traperitoneally divided into two daily doses. On the 4th, blocking effect on the antidiuretic response to exogenous 5th, and 6th days the experimental group received the vasopressin in terms of an increase in U08m and a de- same dose of colchicine as on the 3rd day, and both con- crease in urine flow (Table II). trol and experimental groups received 0.5 U of Pitressin Effect of vinbiastine. The effect of vinblastine was subcutaneously. The administration of colchicine itself evaluated in an experiment like that for colchicine, as (day 3) did not produce any change in urine flow Uo.r, described above, except that only five rats were used per (Fig. 1) UVosm, sodium, potassium, or creatinine ex- group. Vinblastine sulfate was administered intraperi- cretion (Fig. 1 and Table I). As expected, the control toneally to rats in the experimental group on days 3, 4, group responded to exogenous vasopressin by a marked 5, and 6 in a single daily dose of 0.08 mg. Results are increase in Uosm (Fig. 1) and a corresponding decrease summarized in Table III and Fig. 2. Vasopressin on the in urine flow, which continued for 2 subsequent days. 4th day produced only a very small decrease in urine On the other hand, the same dose of vasopressin did not flow and a slight increase in Uosm (Fig. 2) in animals on change Uosm (Fig. 1) and urine flow in animals treated vinblastine, while controls developed marked antidiure- with colchicine either on the 4th or on the following sis. This increase in U08m (+ A317+55 mosmol±SE) experimental days. Despite a striking difference in the in experimental group was much less than in the control response to exogenous vasopressin in water excretion group (+ A1,057±200 mosmol+SE) and difference be- parameters (Uosm and urine flow) there were no sig- tween magnitude of the responses in these two groups nificant differences between control and experimental was significant at P < 0.025. Moreover, on the following groups in UVosm or UVcr (Table I and Fig. 1). The days (5th and 6th), while still receiving the same dose serum concentration of creatinine, measured at the end of vasopressin, the Uo0m in the experimental group of experiment, was not different between control and dropped (Fig. 2), urine flow (Table III) increased, and colchicine-treated groups (Table I). With the exception the difference in these parameters between control and of a slight increase in sodium excretion in experimental experimental group increased further. There were no sig- animals on day 4, there was no difference in UVNa be- nificant differences between the two groups in UVoum tween control and experimental animals. Colchicine does or in serum creatinine measured at the end of experi- not increase potassium excretion. The apparent differ- ment (Table III). Vinblastine did not increase the ex-

Microtubules in the Action of Vasopressin 255 TABLE I I Failure of Lumicolchicine to Influence Renal Response to Vasopressin

Day I Day 2 Day 3 Day 4 Day 5 Day 6 Urine flow, ml/100 g body wt Control 18.28 19.72 24.'59 4.18* 4.76 5.39 per 24 h/±SEM ±3.08 44.72 44.60 ±0.47 ±0.37 40.48 Experimental 17.12 16.80 24.05 3.84* 4.12 4.82 43.50 42.62 ±5.13 ±0.72 ±0.61 40.64 Uo.mmosmol/kg H20OSEM Control 496 510 434 1,3224 1,421 1,370 ±106 ±123 ±100 ±21 ±104 ±156 Experimental 590 532 445 1,473+ 1,436 1,450 ±129 ±132 ±t129 ±:167 ±163 ±158 * Urine flow decreased in response to vasopressin from day 3 to day 4 in both control and experimental groups (A 20.41 ±4.68 and A 20.21±4.56, respectively). These decreases were both significant (P < 0.01) but not significantly different from each other. I Uo. increased in response to vasopressin from day 3 to day 4 in both control and experimental groups (A 888±108 and A 1,028±74 mosmol/kg H20, respectively). The increases in both groups were significant (P < 0.001) but not significantly different from each other. Rats from experimental group received 0.1 mg of lumicolchicine on days 3, 4, 5, and 6; control group received only solvent. On days 4, 5, and 6 both groups received 0.5 U of Pitressin tannate in oil subcutaneously. Number of animals in each group was six. There were no significant differences in urine flow or Uo.m between groups in any experimental day. cretion of either sodium or potassium. The apparent evaluated in preparations from bovine renal medulla. differences in UVNa and UVK on days 4-6 are due to de- Basal or vasopressin-stimulated adenylate cyclase ac- creased excretion of sodium and potassium in controls tivity was not inhibited by colchicine or vinblastine rather than an increased excretion in experimental (Table IV). Since, as shown below, binding of colchicine animals. to proteins is a rather slow process (Fig. 3), the cytosol was incubated with either colchicine or vinblastine for In vitro studies 60 min before the assay for cyclic AMP phosphodiester- Cyclic AMP metabolism. The effects of vinblastine ase. No change in cyclic AMP phosphodiesterase ac- and colchicine on the plasma membrane adenylate cy- tivity was observed due to preincubation with these two clase and cytosolic cyclic AMP phosphodiesterase were drugs (Table IV). Protein kinsue. Protein kinase was assayed in cytosol VASOPRESSIN and the effects of preincubation with colchicine and vin- 0.5 U/day (:--) blastine (similar to the experiments with cyclic AMP M T T 20010 _ 40

Q, ze ,# 30 th 1S0

b220 14 10 Et 100 10 I

ILL... 0.08 mg/day (---) l 2 3 4 5 6 Days FIGURE 2 Response of urine osmolality to vasopressin in control rats (0-O) and in rats treated with vinblastine on 3-6 experimental days (0 --- 0 ). Each group con- FIGURE 3 Time-course of the binding of [8H]colchicine to sisted of five animals. * values significantly different from the proteins of renal medullar cytosol. Protein content was controls at P < 0.05 or more (t test). 87 ,ug/tube; for -incubation conditions see Methods. 256 T. P. Dousa and L. D. Barnes TABLE I I I Inhibition of the Renal Response to Vasopressin by Vinblastine

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Urine flow, ml/10() g body wt Control 9.45 9.11 9.22 3.52 3.62 3.11 per 24 h±SEM ±1.87 41.69 ±1.96 ±0.21 ±0.37 ±0.28 Experimental 10.21 8.63 8.06 6.15 7.78 9.21 i1.75 ±1.48 ±1.34 ±0.38 ±1.57 ±1.54 P value* NS NS NS <0.001 <0.02 <0.005 U0im, mosmol/kg H204SEM Control 778 1,176 1,080 2,137 1,946 1,989 ±179 ±268 ±262 i89 ±139 ±151 Experimental 792 1,011 1,006 1,323 1,003 878 ±77 ±84 ±-95 ± 119 ±98 ± 146 P value NS NS NS <0.001 <0.001 <0.001 U\V0sm, isosmol/)100 g body wt Control 6,241 9,585 8,051 7,456 6,878 6,045 per 24 h±SEM ±955 ±1,764 ±527 ±284 ±451 ±416 Experimental 7,625 8,362 7,670 7,977 7,447 7,372 ±866 ±-966 ±509 ±319 ±876 ±802 P value NS NS NS NS NS NS UVNa.,uIeq K/100 g body wt Control 588 611 760 638 635 523 per 24 h±SEM ±58 +45 ±40 ±38 ±-49 ±i38 Experimental 648 752 747 787 775 786 ±t50±)53 ±47 ±79 ±25 ± 123 P value NS NS NS <0.02 <0.05 NS UVK, ,ueq K/100 g body wt tControl 919 1,007 1,153 774 709 673 per 24 h±SEM ±45 ±115 ±48 ±42 ±84 ±66 TExperimental 1,073 1,187 1,298 1,207 1,274 1,317 ±52 ±t43 ±45 ±44 ±69 ± 184 P value NS NS NS <0.005 <0.02 <0.05 UVcr, mng/100 g body wt Experimental 5.27§ 5.48 4.70 5.44 6.57 4.55 per 24 h±KSEM ±0.32 ±0.48 ±0.70 ±1.07 ±0.29 ±0.96

* For significance of the difference between control and experimental groups, NS denotes P > 0.05 (t test). Therer were no significant changes in UVK between consecutive days *P > 0.05, paired t test); except between days 3 and 4 for the control group. § UVc, value for control group was 5.06±0.37. Rats in experimental groups received 0.08 mg vinblastine intraperitoneally on days 3-6; controls received only solvent. On days 4-6 both control and experimental were injected with 0.5 U of pitressin tannate in oil subcutaneously. Each group consisted of five rats. At the end of experiment, serum creatinine levels in control and experimental animals were 1.118±0.110 and 1.124 ±0.041 mg/100 ml, ±SEM. These values were not significantly different from each other (t-test). phosphodiesterase) were studied (Table V). Both col- distribution of microtubular (colchicine-binding) pro- chicine and vinblastine produced a very small decrease tein among subcellular fractions of renal medulla indi- in the enzyme activities when the cytosolic proteins cated that cytosol (100,000-g supernate) far exceeds all (endogenous substrate) themselves served as a sub- other fractions in its ability to bind [8H]colchicine (Fig. strate for phosphorylation or when added histones were 4). If the colchicine-binding activity of cytosol is taken used as a substrate (Table V). as 100%, the ability of particulate fractions to bind Subcellular distribution of colchicine-binding protein. [PH]colchicine was less than 6%, plasma membranes Colchicine binds to cellular proteins from renal medulla, being the least active. Colchicine-binding protein in renal indicating the presence of microtubular protein (8). medullary cytosol showed some characteristic properties This binding is proportional to time (Fig. 3), and is described for analogous protein from brain (19): the linear with the protein content up to 1.2 mg protein/ colchicine-binding activity was labile after prolonged tube under conditions employed. Determination of the incubation of 00C or after short incubation at 60'C, and

Microtubules in the Action of Vasopressin 257 TABLE IV Effects of Colchicine and Vinblastine on Adenylate Cyclase and Cyclic AMP Phosphodiesterase

Activity Condition Basal activity with 2 X 10-6 M [8-arginine]-vasopressin

Pmol/min per mg protein* Adenylate cyclase of renal medullary plasma membranes assayed in presence or absence of colchicine and vinblastine control 4.91±0.22 (3) 38.4±0.56 (3) with 10-5 M colchicine 4.98±0.33 (3) 42.434±2.26 (3) with 10-5 M vinblastine 4.86±0.12 (3) 39.1 1 ±2.35 (3) Cyclic AMP phosphodiesterase after preincubationj with colchicine or vinblastine nmol/min per mg protein* control 1.61±0.11 (6) Preincubated with colchicine 1.62±i0.11 (6) Preincubated with vinblastine 1.58±0.16 (6) * Mean±SE; number of experiments in parentheses. t Bovin~e renal medullary cytosol was incubated at 37°C in a medium containing 8 X 10-3 M MgC92, 8 X 10-3 M potassium phosphate, and 1 X 10-6 M EDTA (pH 6.8). Concentration of colchicine was 5 X 10-5 M, of vinblastine 1.3 X 10-4 M. After a 60-min incubation, tubes were cooled in ice and samples of preparations assayed in triplicate for cyclic AMP phospho- diesterase activity (see Methods). colchicine-binding protein was completely precipitated different mechanisms: (a) interference with the action from the cytosol by incubation with vinblastine (Table of vasopressin on cells of distal nephron to increase VI). water permeability and (b) washout of the cortico- medullary gradient of solutes. Although our results can- DISCUSSION not decide completely between these two possibilities, In our in vivo experiments, two structurally unrelated several features favor the interpretation that colchicine microtubule-disrupting agents (8) prevented or mark- and vinblastine interfered with the cellular action of edly inhibited the antidiuretic action (decrease in urine vasopressin and not primarily by producing medullary volume and increase in Uo.m) of exogenous vasopressin. solute washout. Since no changes in UVoum occurred, Lumicolchicine, a derivative of colchicine that does not solute washout due to osmotic diuresis is not likely. interact with microtubules (22), did not block the anti- UVNa did not increase (with the exception of a slight diuretic response to exogenous vasopressin. transitory increase on day 4 in the presence of colchicine) Action of any drug which inhibits the antidiuretic ac- and this indicates that no major depression of sodium tion of vasopressin could be attributed to at least two reabsorption (such as occurs after administration of

TABLE V Effects of Colchicine and Vinblastine on the Protein Kinase

Endogenous substrate Histones as substrate (150 jig/assay) (40 jg/assay) with with 5 X 10-6M 5 X 10-6M Condition Basal cyclic AMP Basal cyclic AMP pmol Pi/l mg protein/min Control 0.2340.01* (7) 0.84±0.07 (7) 0.54±0.09 (7) 2.2540.206 (7) Preincubated with colchicine 0.21±0.02 (6) 0.78±0.03 (6) 0.46±0.04 (6) 2.16±0.65 (6) Preincubated with vinblastine 0.2140.02 (6) 0.79±0.07 (6) 0.44±0.07 (6) 2.2140.22 (6) Preincubation conditions as in Table IV. Activities of enzymes incubated with alkaloids were not significantly different (P > 0.05) from controls when evaluated by t test for paired observations. * Mean ±i:SEM; number of experiments in parentheses. 258 T. P. Dousa and L. D. Barnes saluretics) occurred. There was no significant increase TABLE VI in urinary potassium due to administration of colchicine Some Properties of Colchicine-Binding Protein of and vinblastine, and excessive gastrointestinal loss of Bovine Renal Medulla potassium is unlikely since no diarrhea was observed. Also in control experiments, no decrease in serum po- Condition ['H]-colchicine bound tassium was observed. These results suggest that the cOM X 102/mg protein per 90 min* observed unresponsiveness to exogenous vasopressin is Stability of the colchicine-binding protein of renal medullary cytosol not caused by depletion of potassium (23). Cytosol 1,2834±3 Cytosol incubated S min at 600C 197+5 A large alteration in glomerular filtration rate (GFR) Cytosol incubated 15 min at 600C 36 ±9 could produce changes in urine flow and urine concen- Cytosol incubated 24 h at 00C 171±2 tration. Total creatinine excretion did not change mark- Precipitation of the colchicine-binding activity from the renal medullary edly in the course of experiments in either control ani- cytosol by vinblastinet mals or animals treated with alkaloids, and this obser- Cytosol 1,926±43 Vinblastine supernate 266 ± 14 vation, in conjunction with finding of normal plasma Vinbiastine precipitate 74,150±187 levels of creatinine at the end of the experiment, indi- cates that GFR did not change due to the action of drugs * Mean±SE. Bovine renal medullary cytosol was incubated in a medium contain or hormone (Tables I and III).- It is unlikely that the 8 X 10-1 M MgCli; 8 X 10' M potassium phosphate, 1.8 X 10-4 M GTP alkaloids damaged the renal tubules since the excretion and 2 X 101 M vinblastine sulfate (pH = 6.8) at 00C for 60 min. Precipi- of sodium, potassium, and creatinine was unaffected and tate was obtained by centrifugation at 27,000 g for 60 min at 0'C. Concen- tration of vinblastine in all samples was adjusted to 10-4 M and the [EH]- no glucosuriaor proteinuria was detected. Hypercalcemia colchicine-binding was assayed as described in Methods. Protein in prepa- can inhibit the response to vasopressin (24). In our ration was precipitated by TCA and vinblastine removed by methanol (35) control experiment, no changes in serum calcium were before protein determination by Lowry's (20) method. observed in colchicine-treated rats; Henneman, Mon- vasopressin on water flow in spite of the fact that the tierth, and Nichols reported that colchicine caused hy- osmotic pocalcemia in rats is an gradient across the epithelium was artifically (13). Thus, hypercalcemia un- maintained. The fact that the likely explanation for the unresponsiveness to exogenous colchicine or vinblastite vasopressin in rats treated with the alkaloids. did not inhibit the active sodium transport (basal or stimulated the The view that colchicine and vinblastine by vasopressin) in amphibian bladder blocked the also suggests, although indirectly, that the tested effect of exogenous vasopressin by interfering with its agents do not interfere with active sodium transport across cellular -action on the distal nephron finds further sup- analogous epithelia of mammalian port in reported observations on the toad bladder nephron. (6). Colchicine and lumicolchicine have been reported to In that system the tested alkaloids blocked the effect of inhibit some membrane transport phenomena in mam- malian cells (10). In the present studies colchicine in- hibited the antidiuretic response to exogenous vasopres- sin while lumicolchicine had no effect. Since only colchi- cine and not lumicolchicine can 3 interact with micro- tubular protein both in vivo and in vitro (22), the vaso- pressin-blocking effect of colchicine is probably related to its interaction with microtubular protein rather than to its direct effect on membranes in renal cells. ~* The of 5% inhibition antidiuretic action of vasopressin Vt was not complete, since urine osmolality remained always above osmolality of plasma. This feature also is similar to the findings in toad bladder (6). Although the alka- loids clearly prevented the response to exogenous vaso- pressin, they apparently did not reverse the effect of endogenous vasopressin under our experimental condi- N Mt Ms PM C tions (urine osmolality on day 3 remained higher than serum osmolality; Tables I and III). A possible ex- planation for this finding may be related- to the FIGURE 4 Subcellular distribution of [$H]colchicine-bind- mecha- ing protein in bovine renal medulla. Fractions denoted: N nism by which these alkaloids interfere with microtu- = nuclear; Mt = mitochondria; Ms = microsomes ; PM = bules in this tissue. Microtubule-disrupting effects of plasma membranes; C=cytosol. Values represent means colchicine and vinblastine may be due to their ability to ±SE for triplicate determinations. prevent polymerization of microtubular subunits. Several

Microtubudes in the Action of Vasopressin 259 studies indicate that monomers and polymers of micro- tained mostly in cytosol. Colchicine or vinblastine did tubular protein exist in the in dynamic equilibrium not produce appreciable inhibition of cytosolic protein (8). It has been demonstrated that these alkaloids bind kinase in our experiments. This may indicate that col- to the microtubular protein subunit (8), but do not chicine or vinblastiIke do not act by inhibition of protein interact with polymerized microtubules (25, 26). In ad- kinase in this tissue. Protein kinase activity in the crude dition, colchicine prevents the polymerization of micro- unfractioned cytosol may conceivably represent a mix- tubular protein (27, 28). If the cellular action of vaso- ture of enzymes differing in some properties, such as pressin promotes polymerization of microtubular pro- relative substrate specificity and others (36). Therefore, tein, colchicine and vinblastine may prevent the action the possibility still remains that colchicine and vin- of vasopressin by binding to microtubular protein, thereby blastine might act on some specific protein kinase that interfering with microtubule formation. On the other represents a very small fraction of the total protein kin- hand, under our experimental conditions the alkaloids ase activity in the crude cytosol or that the inhibition is may not have interacted with existing polymerized mi- apparent only if the enzyme acts on a natural protein crotubules, and hence could not reverse the effect of substrate that has not yet been identified. endogenous vasopressin. The ultimate site of vasopressin action appears to Since microtubule-disrupting alkaloids inhibited the be the change of water permeability of lumenal plasma response to both exogenous vasopressin and cyclic AMP membranes (37). According to some reports, appreciable in the isolated toad bladder, the authors concluded that amounts of microtubular protein were found to be tightly the alkaloids acted on a site distal to cyclic AMP gen- associated with cellular membranes (38, 39). In renal eration (6). Renal response to exogenous cyclic AMP medulla, microtubular protein, estimated by ['H]colchi- is difficult to evaluate in the whole organism because cine binding (14, 40), was found almost exclusively in exogenous cyclic AMP or its derivatives could possibly cytosol (Fig. 4), a finding similar to that described for mimic not only the action of vasopressin on distal tu- the tissue culture cells (40). No major portion of colchi- bules but also of a host of other hormones, all mediated cine-binding protein was found to be tightly associated by cyclic AMP. Administration of cyclic AMP or its with plasma membranes or other membrane particles in derivatives to the whole organism produced various ef- our in vitro system. The minimal amount of colchicine- fects, and findings varied from antidiuresis to no re- binding activity detected in plasma membranes (less sponse to a clearly diuretic response (29-32). than 4% of the activity in cytosol) may well be a con- In view of this, and also because colchicine and es- tamination by cytosolic proteins. Nevertheless, we do pecially vinblastine were reported to influence enzymes not exclude the possibility, suggested by the morphologi- of cyclic AMP metabolism from organs other than the cal findings in other cell systems (41), that cytoplasmic kidney (33), we tested the effects of these two drugs on microtubules may be in contact and act on the plasma the adenylate cyclase and cyclic AMP phosphodiesterase membrane in the intact cells. from renal medulla in vitro. The known components of The role of cytoplasmic microtubules in the cyclic the cellular action of vasopressin are qualitatively simi- AMP action to increase water permeability is rather a lar in all mammalian renal medulla examined (15), and matter of speculation at the present time. Two major bovine renal medulla was chosen for our in vitro studies possibilities may be considered. First, cyclic AMP may because it was available in sufficient quantities for act directly or indirectly (e.g. via phosphorylation) to subcellular preparations. change some properties of microtubules (e.g. ability to Lack of inhibition of renal medullary adenylate cyclase assemble or turnover rate). Such cyclic AMP-induced and cyclic AMP phosphodiesterase (Table IV) supports change in microtubules may influence the lumenal plasma the view that neither drug interferes with the cyclic membrane permeability, e.g. by release of secretion AMP metabolism itself in this tissue. As mentioned granules (6, 8) or by direct change of topographic dis- above, a possible role of cyclic AMP action in the mem- tribution of membrane proteins (42). The degree of brane permeability regulation currently under investiga- polymerization of microtubules in a submembranal layer tion is the action involving phosphorylation of membrane of might also conceivably be associated with proteins (4, 5). Reports differ whether microtubular pro- changes of water permeability and some mechanical tein contains cyclic AMP-stimulated protein kinase or properties of lumenal plasma membrane, elicited by vaso- not ($, 34); findings showing that microtubule protein pressin and cyclic AMP (43). can serve as a substrate for the phosphorylation, at least An alternative possibility is that cyclic AMP does in the brain, appear to be more consistent (8, 34, 35). In not interact with microtubules at all, but that the func- bovine renal medulla, the protein kinase activity' as tion of microtubules in cyclic AMP action is rather a well as the colchicine-binding protein (Fig. 4) are con- passive or permissive one. Cytoplasmic microtubules may 3L. D. Barnes and T. P. Dousa. Unpublished results. serve as structural elements that hold cyclic AMP-de- 260 T. P. Dousa and L. D. Barnes pendent enzymes (e.g. cyclic AMP-dependent protein 10. Mizel, S. B., and L. Wilson. 1972. Nucleoside transport kinase or protein phosphatase) acting on membranes in mammalian cells. Inhibition by colchicine. Biochem- in istry. 11: 2573-2578. proper localization with their respective substrates (the 11. Andriole, V. T., and F. H. Epstein. 1965. Prevention of membrane proteins). Microtubules may also provide a pyelonephritis by water diuresis: evidence for the role structural basis for compartmentalization of pools of of medullary hypertonicity in promoting renal infection. substrates and modulators such as cyclic AMP, ATP, Ca, J. Clin. Invest. 44: 73-79. or Mg within the cell. In the latter situation, microtubule- 12. Levinson, S. A., and R. P. MacFate. 1969. Clinical Laboratory Diagnosis. Lea & Febiger, Philadelphia. dissociating alkaloids may not interact and interfere with 7th edition. 413. any of the individual components of cyclic AMP-medi- 13. Henneman, D. H., E. Montierth, and G. Nichols, Jr. ated system (enzymes, membranes), but by disrupting 1973. Colchicine blockade of parathyroid (PTH) ef- the microtubular framework, they can render this system fects on collagen and calcium metabolism in bone. inoperative in the intact cells. Abstract 55th Annual Meeting of the Endocrine So- ciety. 162. ACKNOWLEDGMENTS 14. Weisenberg, R. C., G. G. Borisy, and E. W. Taylor. 1968. The colchicine-binding protein of mammalian We are indebted to Dr. Hosley of Eli Lilly Co., Ltd. for brain and its relaltion to microtubules. Biochemistry. 7: supplies of colchicine and vinblastine sulfate. Dr. Keith E. 44664483. Holley, Department of Experimental and Anatomical Pa- 15. Dousa, T. P., 0. Hechter, I. L. Schwartz, and R. thology, Mayo Clinic and Foundation kindly performed the Walter. 1971. Neurohypophyseal hormone-responsive morphological examination of kidneys. We thank Carma adenylate cyclase from mammalian kidney. Proc. Natl. Jean Fink for secretarial assistance and Mr. Christopher A cad. Sci. U. S. A. 68: 1693-1697. Wilson and Mrs. Yvonne S. F. Hui for expert technical 16. McKenzie, S. G., and H. P. Bar. 1973. On the mecha- assistance. nism of adenyl cyclase inhibition by adenosine. Can. J. This study was supported by U. S. P. H. S. research Physiol. 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262 T. P. Dousa and L. D. Barnes