PARTXII. MICROTUBULES AND

THE

SECRETORY PROCESS

EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE ACTION OF VASOPRESSIN * Ann Taylor, Roy Maffly, Leslie Wilson, and Eve Reaven Departments of Medicine and Pharmacology Stanford University School of Medicine Stanford, California 94305 and Veterans Administration Hospital Palo Alto, California 94304

Vasopressin (antidiuretic hormone) has two major physiological actions. Vasopressin induces the contraction or relaxation of certain types of smooth muscle and, in addition, promotes the movement of water (and in some instances of sodium and urea) across responsive epithelial tissues, most notably, the distal tubule of the mammalian kidney and amphibian urinary bladder and skin.’ The action of vasopressin on water movement in epithelial tissues has been extensively investigated; however, the cellular mechanisms involved in this action of the hormone are still to a large extent unknown.z The hormone appears to promote the osmotic movement of water across responsive cells by inducing an increase in the permeability of the membrane at their apical surface 3-6 (FIGURE 1); the permeability change is thought to result from alteration in the size 3, or number of aqueous channels in this rate-limiting barrier.? Under physiological conditions, the effect on the apical membrane is elicited only when vasopressin is present at the basal (i.e. the opposite) surface of the 5, epithelial It is generally accepted that the interaction of the hormone with specific receptors in the basal cell membrane leads to activation of adenyl cyclase and generation of cyclic 3’,5’-adenosine monophosphate (CAMP). Vasopressin has been shown to stimulate specific membrane-bound adenyl cyclase systems 9, l o and to induce an increase in intracellular cAMP levels in target tissues,11* and cAMP mimics the effects of the hormone in these tissues.*? l4 The cellular events that intervene between the generation of cAMP at the level of the basal cell membrane and the increase in permeability of the apical membrane have not been defined. Cyclic AMP-dependent protein kinases have been described in amphibian bladder15 and renal medulla,l6 and a vasopressin- and CAMP-dependent phosphoprotein phosphatase has been reported in toad bladder; l7 however the functional role of these enzymes in the response to the hormone is not known. Economy of mechanism at the cellular and molecular level appears, increasingly, to be a characteristic of biological systems. The possibility that the apparently discrete actions of vasopressin on smooth muscle contraction and transcellular water movement involve some common or analogous mechanism( s )

* These studies were supported in part by United States Public Health Service Grants AM 16327, AM 05678, and NS 09335, by American Cancer Society Grant CI-95,by a Grant-in-Aid from the American Heart Association, and by funds from the Santa Clara County Heart Association and the United States Veterans Administration. 723

Annals New York Academy of Sciences

724

thus has intuitive appeal. It is now well recognized that microtubules are associated with several types of cell movement and with the translocation of cell constituents.1s-21 Microtubules may be involved indirectly in the generation of such cellular movement or, in certain situations at least, they may actually participate in processes of mechanico-chemical transduction analogous to those occurring in 22, 23 These considerations led us to investigate the possibility that microtubules are associated with the action of vasopressin on transcellular water movement. Our studies have been carried out in the urinary bladder of the Colombian toad, Bufo marinus. As an experimental preparation the toad bladder has a number of biological and technical advantages: (1) it is a simple bilobed structure, lined on its inner surface by a single layer of epithelial cells; (2) 7

APICAL (URINARY)

BASAL (BLOOD)

CYCLIC -VASOPRESSIN

WA1 proteln phoephetaee

-08motIc

Gradlrnt

ATP

*

FIGURE1. Hypothetical diagram of vasopressin-sensitive epithelial cell. Vasopressin promotes transcellular water movement, in the presence of an osmotic gradient, when added to the basal (blood) surface of the cell. The hormone appears to induce an increase in the permeability of the rate-limiting apical cell membrane. The cellular events that intervene between generation of CAMP at the level of the basal cell membrane and the permeability change in the apical membrane are essentially unknown. hormone and drug effects on transepithelial water movement and sodium transport can be studied in isolated (in vitro) systems, and can be compared in experimental and control halves of the same bladder; and (3) isolated preparations of the hormone-sensitive epithelial cells can be easily obtained for biochemical studies by scraping the cells off the inside of the bladder. Vasopressin increases the rate of transfer of water and urea and, in addition, stimulates the rate of active sodium transport across the toad bladder epithelium, normally from the urine into the blood.K In the absence of vasopressin, net water movement across the bladder is negligible even in the presence of a steep osmotic gradient across the tissue. I n response to vasopressin or CAMP, the rate of osmotic water movement across the bladder wall increases some 20- to 80-fold 25 and persists at a raised level for several

Taylor et al. : Microtubules and Vasopressin

725

EFFECTSOF COLCHICINE,PODOPHYLLOTOXIN, AND THE VINCA ALKALOIDS ON WATERMOVEMENT AND ACTIVESODIUMTRANSPORT IN THE ISOLATED TOAD BLADDER Colchicine, podophyllotoxin, and the vinca alkaloids are known to exert disruptive effects on microtubules i n vivo and to interact with tubulin in vitro.2G In our initial studies we tested the effects of these agents on osmotic water movement and active sodium transport in isolated toad bladder preparations.?' Osmotic water movement was measured gravimetrically by the method of Bentley.2s Paired hemibladders were mounted as bags on glass tubing, filled with hypotonic fluid and suspended in baths of Ringer's solution. The test alkaloids were added to the outer bathing medium of one of each pair of hemibladders; 0 to 4 hours later vasopressin or cAMP was added to the outer bathing medium of both members of each pair. Each hemibladder was removed from its bath and weighed at 30 minute intervals; the rate of water movement out of the bladders, before and after addition of vasopressin or CAMP, was thus estimated as weight loss of the bags in mg/minute.Z7 Active sodium transport was measured in paired hemibladders mounted in glass chambers, using the short-circuit current technique of Ussing and Zerahn.28 The studies were routinely carried out at room temperature (24 & 2' C) . I n the absence of vasopressin, exposure of hemibladders to colchcine, vinblastine, vincristine, or podophyllotoxin had no effect on the baseline rate of water movement across the tissue. However, the increase in water movement in response to vasopressin was reduced in a concentration-dependent manner in hemibladders that had been exposed to each of these agents. The dose-response relationships of the inhibitory effect of 4 hours exposure to colchicine, vinblastine, and podophyllotoxin on the response to vasopressin 20 mU/ml are depicted in FIGURE 2. Fifty percent inhibition of the response to the hormone M colchicine, 1 X M podophyllooccurred with approximately 6 X toxin, and 2.5 X lo-' M vinblastine. The reduced response to vasopressin was not associated with a reduction in the osmotic gradient across the bladder wall. The inhibitory effect of each of the alkaloids on the response to the hormone appeared to be reversible.?', 3O The increase in water movement in response to cAMP was also found to be markedly reduced in bladders that had been exposed to colchicine, vinblastine, or podophyllotoxin. The results of studies with colchicine and vinblastine are shown in TABLE 1. It is apparent that the inhibitory effect of the alkaloids on the response to cAMP was equal to or greater than their effect on the response to vasopressin (these studies were performed using physiologically equivalent concentrations of the nucleotide and hormone). Thus it appears that the alkaloids exert their effects at a cellular site distal to the action of vasopressin on cAMP synthesis. In contrast to the inhibitory effects of the alkaloids on vasopressin- and CAMP-induced water movement, colchicine, vinblastine, and podophyllotoxin were found to have no effect on active sodium transport across the bladder. Neither the baseline rate of sodium transport, nor the rise in sodium transport induced by vasopressin, were significantly influenced by 4 hours exposure of the bladders to these agents.?' This finding is consistent with other evidence that the effects of vasopressin on water and sodium movement involve separate cellular mechanisms, in particular with evidence that the effect of vasopressin

Annals New York Academy of Sciences

726

loo-

ColchicineVinblastine-

(n- 7-12) (n- 6-12)

'4 0

-20 1 10-8

I

10-7

I

10-6

I

10-5

I

10-4

Moles per Liter

FIGURE 2. Dose-response relationships of the inhibition of vasopressin-induced water movement by colchicine, vinblastine and podophyllotoxin. The alkaloids were added 4 hours before the addition of vasopressin 20 mU/ml; the studies were carried out at 2 4 2 2 ° C . Percent inhibition of the response to vasopressin was calculated from the difference between the weight loss of the experimental hemibladders and that of their paired controls over the 30-minute period following addition of the hormone; each point represents the mean 2 S.E. of n paired experiments.

INHIBITORY

Alkaloid Colchicine 2x104M Vinblastine 2xlO-'M

TABLE1 EFFECTOF COLCHICINE AND VINBLASTINE ON WATER MOVEMENT IN RESPONSE TO VASOPRESSIN AND TO CYCLIC AMP * Percent Inhibition (Mean -C S.E.) Vasopressin Cyclic AMP (20 mU/ml) (2-4 mM) 65.026.8 (n=7) 83.023.0 (n=12)

75.923.5 (n=6) 82.023.8 (n=5)

* The alkaloids were added 4 hours prior to the addition of vasopressin or cyclic AMP; all studies were carried out at 2422" C. Percent inhibition was calculated from the difference between the weight loss of the experimental hemibladders and that of their paired controls over the 30 minute period following addition of the hormone or nucleotide.

Taylor et al. : Microtubules and Vasopressin

727

on water movement is calcium-sensitive, whereas its effect on sodium transport is not.31 THATINHIBITIONOF THE VASOPRESSIN RESPONSEBY EVIDENCE COLCHICINE AND OTHERALKALOIDS Is DUETO AN EFFECT ON CYTOPLASMIC MICROTUBULES

While the effects of the antimitotic agents on microtubules in vivo, and their interaction with tubulin in vitro, are well established,2Gthese agents have also been shown to have cellular effects that do not appear to depend on micro3 3 For example, colchicine and podophyllotoxin inhibit nucleoside transport in cultured cells, and this effect has been attributed to interaction of the drugs with a cell membrane component.3* It was clearly important for us to attempt to establish whether the inhibitory effect of the alkaloids on vasopressin-induced water movement is in fact due to their interaction with tubulin and thus to an effect on cytoplasmic microtubules. We therefore sought to obtain evidence that would allow us to define the locus of action of colchicine and other alkaloids in the bladder. Accordingly we have carried out functional, biochemical and electron microscopic studies: ( 1) to characterize further the effects of colchicine on vasopressin-induced water movement in isolated bladders, (2) to quantitate and characterize the binding of colchicine in subcellular fractions of the hormone-sensitive epithelial cells, and (3) to determine the morphological effect of colchicine on cytoplasmic microtubules in the epithelial cells. Characteristics of the Inhibitory Effect of Colchicine on Vasopressin-Induced Water Movement

Through functional studies in isolated bladders we proceeded to examine further the characteristics of the inhibitory effect of colchicine on vasopressininduced water movement. To test the specificity of the colchicine effect, the effect of lumicolchicine, its structural isomer, was investigated. Lumicolchicine does not disrupt microtubules in vivo, does not interact with tubulin in vitro, and does not interfere with the binding of colchicine to tubulin.3' On the other hand, this agent, like colchicine itself, inhibits nucleoside uptake in cultured cells.32 Exposure of hemibladders to 2 X M lumicolchicine for 4 hours was found to have no effect on the response to vasopressin: The rate of hormone-induced water movement in lumicolchicine-treated and control hemibladders differed by 13.8 & 13.9% ( n = 6, n.s.), whereas after 4 hours exposure to the same concentration of colchicine, the response to vasopressin was inhibited by 65 k 7% ( n = 7, p < 0.001). The failure of lumicolchicine to inhibit the vasopressin response strongly supports the view that the inhibitory effect of colchicine is related to its specific ability to bind to tubulin. Since the binding of colchicine to tubulin i n vitro is a slow process,2Gwe examined the time-dependence of the inhibitory effect of 2 X 1 W M colchicine on the vasopressin response. As seen in FIGURE 3, no inhibition of the vasopressin response occurred in the absence of a period of preincubation with colchicine; the degree of inhibition increased with the period of preincubation

Annals New York Academy of Sciences

728

7060-

FIGURE3. Time-dependence of the inhibitory effect of col-

50-

-.-

40-

E c

30.

chicine on vasopressin-induced water movement. Colchicine (final concentration 2 x lo-' M) was added 0 to 4 hours prior to addition of vasopressin 20 mU/ml; the studies were carried out at 24 f 2" C. Results were calculated as described for FIGURE2.

c

P

s

2 0. 10

0. -10.

i

1

A

i

Time In Hours

with the alkaloid, the slope curving off after 1 to 2 hours. This time-dependence is similar to that previously reported for the binding of colchicine to tubulin in vitro;34*35 moreover, as will be seen, it shows a striking parallel to the time-dependence of the binding of colchicine to tubulin in the soluble fraction of sonicated bladder epithelial cells. It is relevant to note that the effect of colchicine on nucleoside transport is not time-dependent at drug concentrations similar to those used in this Since the rate of binding of colchicine to tubulin in vitro is also temperaturedependent, being greatly reduced at low temperature~,~~-37 we examined the temperature-dependence of the inhibitory effect of colchicine on the vasopressin response. The response to the hormone was itself found to be markedly temperature-dependent and at 0" C the response was negligible or very slight and variably delayed; our studies were therefore carried out over the range 5" to 35" C. As seen in FIGURE4, the inhibitory effect of 2 X M colchicine did in fact vary with temperature; inhibition was reduced by approximately one half with every decrease of 10°C. In contrast, inhibition of nucleoside transport by colchicine has been found to be independent of temperat~re.~? It is evident that the characteristics of the inhibitory effect of colchicine on vasopressin-induced water movement are similar to the characteristics of its interaction with tubulin, and differ from those of its effects on nucleoside transport. Colchicine Binding Activity and Tubulin Content of Isolated Bladder Epithelial Cells In an attempt to define more precisely the locus of action of colchicine in the bladder, the binding of tritium-labeled colchicine in subcellular fractions of isolated epithelial cells was quantitated and characterized. Isolated epithelial cells were obtained by scraping the cells from the inside of freshly excised

Taylor et al. : Microtubules and Vasopressin

729

bladders with the edge of a glass cover slip. The cells were suspended in ice-cold buffer (20 mM sodium phosphate, 100 mM sodium glutamate, pH 6.75), sonicated and centrifuged at 39,000 X g for 45 minutes at 0" C, prior to incubation with labeled colchicine. Colchicine binding activity in the soluble fraction was quantitated by gel filtration; 3G the colchicine binding activity associated with the particulate fraction was determined after washing the pellet three times by resuspension and centrifugation. Ninety-eight percent of the total colchicine binding activity (estimated as initial colchicine binding capacity 38) was found to be associated with a highmolecular-weight component in the soluble fraction of the sonicated epithelial ~ e l l s . 3The ~ characteristics of the colchicine binding activity in this fraction were similar to those of colchicine binding to tubulin in chick embryo brain extracts (TABLE 2). Colchicine binding activity was markedly reduced on incubation at O'C, and it was inhibited by the addition of podophyllotoxin, which competes with colchicine for the same binding site; 2G on the other hand, the binding activity was stabilized (and thus apparently increased) in the presence of vinblastine and vincristine, which interact with tubulin at separate binding In the presence of lumicolchicine, which does not bind to t ~ b u l i n , ~colchicine .' binding activity was not appreciably altered. It is clear from these findings that the colchicine in the soluble fraction of the bladder cells is binding to tubulin. FIGURE 5 shows the time-dependence of the colchicine binding activity in the soluble fraction of the epithelial cells at 23" C (the temperature at which our functional studies were performed). It is apparent that the rate of binding of the drug was slow. This time-dependence is very similar to that observed for colchicine binding to other t ~ t i u l i n s . ~ ~ - ~ ~ The binding constant for the binding of colchicine to tubulin in the bladder cells was found to be 1 X los liters per mole at 37" C; this value is identical to that obtained for chick embryo brain tubulin.zs The identity of the binding constants permitted the tubulin content of the soluble fraction of the bladder epithelial cells to be quantitated using the time-decay assay method of Bamburg, Shooter and Wilson; 38 tubulin was found to constitute 5 % of the soluble protein of the cells.3D Since the effect of vasopressin is apparently dependent on a change in membrane structure or function, we were particularly interested to determine

FIGURE 4. Temperature-dependence of the inhibitory effect of colchicine on vasopressin-induced water movement. Colchicine (final concentration 2 x lo4 M) was added 1 hour prior to addition of vasopressin 20 mU/ml. In the studies at 5 " , 15" and 35" C, paired hemibladders were incubated in a water bath maintained at these temperatures & 2" C. Results were calculated as described for FIGURE 2.

40

n=5-10

0-

0

I

I

I

I

I

5

10

15

20

25

Temperature ('C)

I

I

30 35

Annals New York Academy of Sciences

730

FIGURE 5 . Timedependence of colchicine binding in the soluble fraction of isolated bladder epithelial cells. The 39,000 x g supernatant from a whole epithelial cell sonicate (1.36 mg total protein/ml) was incubated at 23" C with 1.5 x lo-' M [rnethowy-JHIcolchicine. At the times indicated 0.5 ml aliquots were removed, cooled to 0" C, and the quantity of bound colchicine determined by gel filtration.'

1

C 0) "

:

. E W

a

P

E 0

m

Time in Hours

the colchicine binding activity of the particulate fraction of the bladder cells. Colchicine binding activity in the particulate fraction was found to amount to only 2% of the total binding activity.39 Preliminary data suggest that most of this binding is not to tubulin but rather to a second colchicine binding component. The low binding activity suggests that there are few such particulate binding sites and/or their affinity for colchicine is low. These studies indicate that tubulin accounts for an appreciable quantity of the soluble protein of the bladder epithelial cells, and they suggest that the inhibitory effect of colchicine (and other alkaloids) on the vasopressin response is indeed attributable to binding to tubulin. TABLE2 PROPERTIES OF COLCHICINE BINDING ACTIVITYIN SOLUBLE FRACTION OF SONICATED ,TOAD BLADDER EPITHELIAL CELLS* Incubation Conditions Colchicine 2 . 6 lo4 ~ M 37" C Control

0" c 37" C plus podophyllotoxin (l.OxlO-bM) 37" C plus vinblastine (5 x M) 37" C plus vincristine ( 8 . 2 lo-' ~ M)

37" C plus lumicolchicine

Percentage of Control Binding Activity Toad Bladder Chick Brain 100 14 21

100 6 10

184

152

150

-

88

89

( 1 . 6 10-4 ~ M)

* 39,000 x g supernatant fractions were prepared from sonicates of isolated bladder epithelial cells and of a 13day old chick embryo brain; 0.5 ml aliquots were incubated with 2.0 x lo4 M [rnetho~y-~H]colchicine for 2 hours at 37" C under the conditions shown above. Bound colchicine was determined by gel filtration.'

Taylor et al. : Microtubules and Vasopressin

73 1

Eflect of Colchicine on Cytoplasmic Microtubules in Bladder Epithelial Cells

Having obtained functional and biochemical evidence that the inhibitory effect of colchicine on the vasopressin response is due to its interaction with tubulin, we sought to ascertain in morphological studies whether exposure to an inhibitory concentration of colchicine in fact leads to disruption of microtubules in the bladder epithelial cells. The toad bladder epithelium is made up of four types of cells; of these approximately 85% are granular cells, named for the membrane-limited granules that are present in their cytoplasm and that frequently appear to be lined up under the apical surface of the cells (FIGURES 6 and 7).40 Microtubules are present in the cytoplasm of all four epithelial cell types;27 however, the movement of water induced by vasopressin appears to be confined to the granular cells.41 Therefore, the distribution and content of microtubules in the granular cells of both untreated (control) and colchicine-treated bladders was examined. Paired hemibladders were prepared as bags, filled with isotonic Ringer's solution, and suspended in this solution, with or without 2 X M colchicine, for 3 hours prior to preparation for electron microscopy; the tissues were mounted on rings, fixed overnight in 1% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.2), and postfixed in 1 % osmium tetroxide followed by 0.5% uranyl acetate. All fixation procedures were carried out at room temperature. Thin sections (400 A) were stained with uranyl acetate and Reynold's lead citrate. On electron microscopic examination of untreated bladders, microtubules appeared to be randomly distributed in the cytoplasm of the granular cells (FIGURES 6 and 7 ) ;27 however, fragments of microtubules were occasionally seen to be closely associated with specific cellular organelles, including both mitochondria (FIGURE 6) and granules (FIGURES 6 and 7 ) . Preliminary studies of bladders that had been exposed to colchicine revealed that microtubules were still present in the granular cells, although they appeared to be reduced in number. It thus became apparent that a quantitative analysis of the microtubule content of cells from paired hemibladders would be required in order to establish the extent of the colchicine effect. Accordingly, portions of 5 granular cells, containing both apical and lateral surfaces, were randomly selected from thin sections from one tissue block from each of the paired control and colchicine-treated hemibladders. The cells were selected and were photographed at a magnification at which microtubules could not be identified by the microscopist; microtubules in both transverse and longitudinal profile were subsequently identified on the enlarged photographic prints. Estimations of the volume density of microtubules were made by the point-counting stereological method of Weibel..12 A transparent grid with a 2 mm lattice was placed over each print and used to estimate microtubule volume, while a grid with a 2 cm lattice was used to estimate cytoplasmic volume; volume density, or fractional volume of microtubules relative to the cytoplasmic volume, was calculated from ( P ) microtubules/[(P) cytoplasm X 1001, ( P ) being the total number of lattice points falling over microtubules and the cytoplasm, respectively. All steps in these studies were performed without knowledge of the origin of the section being evaluated. The results of the estimations are given in TABLE 3. The volume density of microtubules in the cytoplasm of granular cells from untreated bladders was found to be 8.1 X 10-' (i.e. the volume of microtubules

of a representative granular cell from toad bladder epithelium (untreated). The majority of granules ( G ) appear to be lined up under the apical surface of the cell. Fragments of microtubules (arrows) are seen in longitudinal and transverse profile; these may be associated with specific cellular organelles, including mitochondria (M) and granules. (47,000 x )

FIGURE6. Electron micrograph of apical portion

c) v1 CD

B

% P.

'3

b*

.c

8

3 3

g s

Taylor et al. : Microtubules and Vasopressin

733

FIGURE 7. Electron micrograph of apical portion of granular cell from toad bladder epithelium (untreated). Membrane-limited granules (G) are conspicuous, but are not obviously oriented in relation to the apical plasma membrane as in FIGURE6. Microtubules (arrows) appear randomly distributed in the cytoplasm. One microtubule fragment appears to be associated with a group of granules (44,000 x).

Annals New York Academy of Sciences

734

TABLE 3

VOLUMEDENSITYOF MICROTUBULES IN CYTOPLASM OF GRANULAR EP~THELIAL CELLSFROM PAIREDHEMIBLADDERS * Animal

Volume Density X lo' Control Colchicine 1.

1

6.4

0.4

2 3

8.5 2.8

4

14.8 10.0

0.8 1.2 3.2

5

6

Mean f S.E.

6.0 8 . 1 k 1.6

* Figures represent mean values for 5

Percent Difference 94 91 57 78

0

100

0.8 -

87 -

1.1k0.4

84f6 %

cells from each hemibladder.

t Hemibladders were exposed to colchicine 2 x lo-' M for 3 hours. % p

Evidence for involvement of microtubules in the action of vasopressin.

PARTXII. MICROTUBULES AND THE SECRETORY PROCESS EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE ACTION OF VASOPRESSIN * Ann Taylor, Roy Maffly, Les...
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