Part XII. Michotubules and the Secretory Pocess: EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE...

15
PART XII. 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 in- stances 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 altera- tion 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 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, lo and to induce an increase in intracellular cAMP levels in target tissues,11* and cAMP mimics the effects of the hormone in these tis- sues.*? 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 vaso- pressin- 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, in- creasingly, 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) 5, * 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 Adminis- tration. 723

Transcript of Part XII. Michotubules and the Secretory Pocess: EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE...

Page 1: Part XII. Michotubules and the Secretory Pocess: EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE ACTION OF VASOPRESSIN

PART XII. 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 in- stances 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 altera- tion 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 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 tis- sues.*? 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 vaso- pressin- 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, in- creasingly, 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 )

5 ,

* 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 Adminis- tration.

723

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724 Annals New York Academy of Sciences

thus has intuitive appeal. It is now well recognized that microtubules are asso- ciated 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)

APICAL 7 BASAL

WA1

(URINARY)

CYCLIC

(BLOOD)

-VASOPRESSIN

proteln A T P phoephetaee

-08motIc Gradlrnt * FIGURE 1. Hypothetical diagram of vasopressin-sensitive epithelial cell. Vasopres-

sin 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 cellu- lar 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 trans- port can be studied in isolated (in vitro) systems, and can be compared in experimental and control halves of the same bladder; and (3) isolated prepara- tions of the hormone-sensitive epithelial cells can be easily obtained for bio- chemical 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 move- ment across the bladder is negligible even in the presence of a steep osmotic gradient across the tissue. In response to vasopressin or CAMP, the rate of osmotic water movement across the bladder wall increases some 20- to 80-fold and persists at a raised level for several 25

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Taylor et al. : Microtubules and Vasopressin 725

EFFECTS OF COLCHICINE, PODOPHYLLOTOXIN, AND THE VINCA ALKALOIDS ON WATER MOVEMENT AND ACTIVE SODIUM TRANSPORT

IN THE ISOLATED TOAD BLADDER

Colchicine, podophyllotoxin, and the vinca alkaloids are known to exert disruptive effects on microtubules in 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 hemi- bladders; 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 rou- tinely carried out at room temperature (24 & 2' C) .

In the absence of vasopressin, exposure of hemibladders to colchcine, vin- blastine, 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, vin- blastine, and podophyllotoxin on the response to vasopressin 20 mU/ml are depicted in FIGURE 2. Fifty percent inhibition of the response to the hormone occurred with approximately 6 X M podophyllo- 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, vin- blastine, or podophyllotoxin. The results of studies with colchicine and vin- blastine are shown in TABLE 1. I t 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

M colchicine, 1 X

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726

loo-

Annals New York Academy of Sciences

Colchicine- (n- 7-12) Vinblastine- (n- 6-12)

'4 0

-20 1 I I I I

10-8 10-7 10-6 10-5 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 2422°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 hor- mone; each point represents the mean 2 S.E. of n paired experiments.

TABLE 1 INHIBITORY EFFECT OF COLCHICINE AND VINBLASTINE ON WATER MOVEMENT

IN RESPONSE TO VASOPRESSIN AND TO CYCLIC AMP *

Alkaloid

Percent Inhibition (Mean -C S.E.) Vasopressin Cyclic AMP (20 mU/ml) (2-4 mM)

Colchicine 2x104M Vinblastine 2xlO-'M

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.

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Taylor et al. : Microtubules and Vasopressin 727

on water movement is calcium-sensitive, whereas its effect on sodium transport is not.31

EVIDENCE THAT INHIBITION OF THE VASOPRESSIN RESPONSE BY COLCHICINE AND OTHER ALKALOIDS Is DUE TO 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,2G these agents have also been shown to have cellular effects that do not appear to depend on micro-

33 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 vaso- pressin-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 blad- ders, (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 vasopressin- induced water movement.

To test the specificity of the colchicine effect, the effect of lumicolchicine, its structural isomer, was investigated. Lumicolchicine does not disrupt micro- tubules 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 move- ment 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,2G we 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 vaso- pressin response occurred in the absence of a period of preincubation with colchicine; the degree of inhibition increased with the period of preincubation

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728

70-

60-

50-

- 40-

E c 30.

s 2 0.

c .- P

-

1 0

0.

-10.

Annals New York Academy of Sciences

FIGURE 3. Time-dependence of the inhibitory effect of col- 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. Re- sults were calculated as de- scribed for FIGURE 2.

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 temperature- dependent, being greatly reduced at low temperature~,~~-37 we examined the temperature-dependence of the inhibitory effect of colchicine on the vaso- pressin 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 FIGURE 4, 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 trans- port.

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

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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 incuba- tion 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 high- molecular-weight component in the soluble fraction of the sonicated epithelial ~ e l l s . 3 ~ The 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~bul in ,~ . ' 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 move- 40 ment. 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 tem- peratures & 2" C. Results were cal- culated as described for FIGURE 2.

n=5-10 0-

I I I I I I I 0 5 10 15 20 25 30 35

Temperature ('C)

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730 Annals New York Academy of Sciences

1 C 0) " : E W a

P

.

E m 0

FIGURE 5 . Timedepen- dence of colchicine binding in the soluble fraction of iso- lated bladder epithelial cells. The 39,000 x g supernatant from a whole epithelial cell sonicate (1.36 mg total pro- tein/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 filtra- tion.'

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 com- ponent. 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.

TABLE 2

OF SONICATED ,TOAD BLADDER EPITHELIAL CELLS * PROPERTIES OF COLCHICINE BINDING ACTIVITY IN SOLUBLE FRACTION

Incubation Conditions

Colchicine 2 . 6 ~ lo4 M

Percentage of Control Binding Activity

Toad Bladder Chick Brain

37" C Control

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

0" c

( 1 . 6 ~ 10-4 M)

100 14 21

184

150

88

100 6

10

152

- 89

* 39,000 x g supernatant fractions were prepared from sonicates of isolated blad- der epithelial cells and of a 13day old chick embryo brain; 0.5 ml aliquots were in- cubated 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.'

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Taylor et al. : Microtubules and Vasopressin

Eflect of Colchicine on Cytoplasmic Microtubules in Bladder Epithelial Cells

73 1

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 micro- tubules 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 gran- ules 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 photo- graphed 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 stereo- logical 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 frac- tional volume of microtubules relative to the cytoplasmic volume, was calcu- lated from (P) microtubules/[(P) cytoplasm X 1001, (P) being the total num- ber 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

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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 FIGURE 6. Microtubules (arrows) appear randomly distributed in the cytoplasm. One micro- tubule fragment appears to be associated with a group of granules (44,000 x).

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734 Annals New York Academy of Sciences

TABLE 3 VOLUME DENSITY OF MICROTUBULES IN CYTOPLASM OF

GRANULAR EP~THELIAL CELLS FROM PAIRED HEMIBLADDERS *

Animal 1 2 3 4 5 6

Mean f S.E.

Volume Density X lo' Control Colchicine 1.

6.4 0.4 8.5 0.8 2.8 1.2

14.8 3.2 10.0 0

0.8 - - 6.0 8 .1k 1.6 1.1k0.4 - -

Percent Difference

94 91 57 78 100 87

8 4 f 6 % - -

* Figures represent mean values for 5 cells from each hemibladder. t Hemibladders were exposed to colchicine 2 x lo-' M for 3 hours. % p <0.001.

was equal to 0.08% of the cytoplasmic volume). Although there was consider- able variation between the figures for individual bladders, the volume density of microtubules in the colchicine-treated hemibladders was in every instance lower than that in their paired controls, the mean percentage reduction being 84 2 6% (n = 6, p < 0.001); in contrast, no significant difference in the volume density of other cellular organelles was found. Possible errors inherent in the stereological method when applied to the counting of microtubules makes us cautious of the absolute figures for volume density; however, since any such errors apply equally to both control and experimental tissues, we are confident of the validity of the observed differences.

It is evident that exposure to a concentration of colchicine that leads to inhibition of the vasopressin response also results in disruption of cytoplasmic microtubules in the bladder epithelial cells.

CONCLUSIONS

We consider that our functional, biochemical and morphological studies, taken together, provide strong evidence in support of the view that the inhibi- tory effect of colchicine and other antimotitic agents on the vasopressin response is due to an effect on cytoplasmic microtubules. On the basis of this evidence, we conclude that cytoplasmic microtubules play a critical role in the action of vasopressin on transcellular water movement in the toad bladder.27

How microtubules are involved in the action of vasopressin is a matter for speculation. One possibility is depicted in FIGURE 8. Masur et al.43 have ob- tained evidence that CAMP stimulates the release of the membrane-limited granules at the apical surface of the granular cells; these workers have suggested that the membrane permeability change induced by the hormone is actually secondary to the release of these granules by exocytosis and the resultant incor- poration of the granule membrane into the existing apical surface membrane.

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Taylor et al. : Microtubules and Vasopressin 735

Microtubules have been implicated in the translocation and release of secretory products in several tissues.?'$ lr, It is therefore intriguing to consider the possibility that in the toad bladder, under the influence of vasopressin, micro- tubules play a role in the translocation of secretory material and new membrane components from the interior of the cells to their apical surface. The addition of secretory material to this surface and in particular the insertion of new membrane into the apical plasma membrane-with consequent alteration in surface architecture and function-may in turn be responsible for the membrane permeability change induced by the hormone.27. .*n

The possibility that the dual actions of vasopressin on smooth muscle con- traction and transcellular water movement involve a common mechanism remains open. Elucidation of the molecular mechanisms in which microtubules participate in vivo, and of the cellular processes whereby these are controlled, may provide evidence for such a concept.

FIGURE 8. Hypothetical diagram of granular epithelial cell of toad bladder, to il- lustrate a possible role of microtubules in the action of vasopressin. Microtubules may be involved in the translocation of granules from the interior of the cell to the apical surface prior to their release by exocytosis. The addition of secretory material to the cell surface, and the incorporation of the granule membrane into the apical plasma membrane, may be responsible for surface changes (e.g. microvillus forma- tion), which in turn may underlie the increase in permeability induced by the hormone.

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736 Annals New York Academy of Sciences

ACKNOWLEDGMENTS

We wish to record our gratitude to Mortimer Mamelak for his role in the initiation of our functional studies with colchicine. We thank Helen Golbetz, Yuen-Ling Lee, Anne Chamberlain, and Laura Hanahan for their skilled assist- ance.

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