453

Biochem. J. (1975) 148, 435-459 Printed in Great Britain

Colchicine Binding to Bovine Anterior Pituitary Slices and Inhibition of Growth-Hormone Release By PETER SHETERLINE,* J. GEORGE SCHOFIELD* and FANNY MIRAt * Department of Biochemistry, Medical School, University Walk, Bristol BS8 1 TD, U.K., and t Institute of Histology and Embryology, School ofMedicine, 1211, Geneva 4, Switzerland (Received 23 December 1974) 1. The uptake of [ring C-methoxyl-3H]colchicine into bovine anterior pituitary slices was studied. The data suggest that more than one site exists for the binding of colchicine. At low concentrations colchicine binds to saturable trypsin-sensitive site(s), with a dissociation constant of 3.1±0.69,UM. The binding capacity of these sites is 8.58±0.60pmol of colchicine/mg of wet pituitary. At higher colchicine concentrations binding occurs predominantly to sites which exhibit non-saturation kinetics. 2. Subcellular fractionation of colchicine-labelled slices shows that 90 % of the saturable sites are present in the fraction containing cytosol, where the binding protein has a molecular weight of about 11.9 x 104 and constitutes 0.7% of the protein present. The nuclear fraction contains 10% of the saturable sites, and the mitochondria and granule fraction contain only non-saturable sites. 3. The rate of colchicine uptake was studied at 0.84mM- and 2pM-colchicine. At both concentrations the colchicine space exceeded the total tissue water within 10min. Equilibration with the saturable binding sites was complete in 120min at 2flM-colchicine. 4. A concentration of colchicine (13.4pM) which would give 81 % maximum binding was found to decrease the length of observable microtubules in tissue fixed at 37°C in glutaraldehyde by 83±4%. 5. The colchicine-binding protein could be partially purified by using a standard procedure for isolation of brain tubulin. 6. Colchicine inhibits the release of growth hormone in the presence of 3-isobutyl-1-methylxanthine (0.1 mM), but does not alter basal release. The concentration-dependence of colchicine inhibition is similar to that of colchicine binding, but maximum inhibition is only 35%. A protein which binds the alkaloid colchicine is known to occur in many tissues (Taylor, 1965; Borisy & Taylor, 1967b; Creasey, 1967; Wilson & Freidkin, 1967), and this protein has been identified as tubulin, the subunit of microtubules (Weisenberg, 1972). Colchicine is also known to modify a variety of cellular processes, including mitosis (Inoue, 1952), movement of pigment granules (Malawista, 1965), hormone secretion (Lacy et al., 1968), axonal flow (Dahlstrom, 1968) and cell transformation (Hsei & Puck, 1972). It is assumed that colchicine modifies these processes by interacting specifically with tubulin and thus displacing the equilibrium between microtubules and their subunits. There is, however, evidence that at higher concentrations colchicine inhibits nucleoside transport (Mizel & Wilson, 1972) and aldose reductase (Gabbay & Tze, 1972) and modifies the behaviour of membrane proteins (Wunderlich et al., 1974). Colchicine has been used with conflicting results to evaluate the importance of microtubules in the release of growth hormone from rat pituitaries (Ewart & Taylor, 1971; Pelletier & Bornstein, 1972; Macleod et al., 1973; Sundberg et al., 1973), from rat pituitary tumour cells (Gautvi & Tashjian, 1973) and from Vol. 148

heifer pituitary slices (Schofield & Cole, 1971; Cooper et al., 1972). The variability in the results could arise from differences in experimental procedure, or from non-specific effects of the alkaloid at high concentrations. In the present studies we have attempted to characterize the binding of colchicine to pituitary slices, and to determine whether the effect of colchicine on growth-hormone release and on pituitary microtubules can be correlated with this binding. Materials and Methods

Chemicals Colchicine was purchased from BDH Chemicals Ltd., Poole, Dorset, U.K., and fromRalphEmmanuel Ltd., Wembley, Middx., U.K. Purity was monitored by using t.l.c. on silica gel and the u.v.-absorption characteristics in 95 % ethanol [imax. 244nm, 350nm, cmax. 16740M-1 cm'1; 5max. 30000Mm cm-I; (Horowitz & Ullyot, 1952)]. [ring-C-methoxyl3H]colchicine (2Ci/mmol) and [U-'4C]sorbitol (7Ci/ mmol) were purchased from The Radiochemical Centre, Amersham, Bucks., U.K. Enzymes, unless Lmax.

454

P. SHETERLINE, J. G. SCHOFIELD AND F. MIRA

otherwise stated, were obtained from Boehringer Corp. (London) Ltd., London W.5, U.K. Butyl-PBD [5 - (4 - biphenylyl) - 2 - (4 - t -butylphenyl)-1 -oxa-3,4-diazole] was obtained from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K., and other reagents were obtained from BDH Chemicals Ltd. Methods Tissue incubation procedures. Pituitary glands were removed from heifers within 5min of death, and the anterior pituitary was sliced as described previously (Schofield, 1967). Slices were incubated at 37°C with shaking (100 strokes/min) in a bicarbonate-buffered medium (Krebs & Henseleit, 1932) equilibrated with 02+CO2 (95:5) and containing 5.5mM-D-glucose and 5.5mM-DL-3-hydroxybutyrate as substrates. The slices (10-15mg wet wt.) were preincubated in 1 ml of medium for 40min. Subsequent incubations involving colchicine were performed in the dark. At the end of the incubation, media were removed and stored at -14°C before determination of growth-hormone concentrations, and in binding experiments portions were removed for radioactivity determination. Tissue slices for electron microscopy were fixed by addition of 2% (w/v) glutaraldehyde in 100mM-sodium phosphate, pH7.2, at 37°C for 2h. Determination of microtubule length. The glutaraldehyde-fixed tissue was post-fixed with OS04, stained with uranyl acetate, dehydrated through graded alcohols and propylene oxide and embedded in epon. Sections were counterstained with lead citrate and examined in a Philips 300 or AEl EM6B electron microscope. To ensure a random sample the microscope operator did not know the conditions represented by each grid, and to eliminate variations in magnification a complete series of conditions was photographed in each session. Between 18 and 31 photographs were taken with and without coichicine at 3.4uM and 13.4/uM, the length of microtubule present was determined with an opisometer and the volume of cytoplasm containing the microtubules determined by planimetry. For each photograph the length of microtubule was calculated per pm3 of cytoplasm, excluding the nucleus, assuming a thickness of section of 60nm. Kinetics ofcolchicine binding to slices. To determine the kinetics of colchicine binding, slices were incubated in Krebs-Henseleit (1932) buffer containing ["4C]sorbitol (0.1 ,Ci/ml) and [3H]colchicine (1 .0uCi/ ml) at the concentrations and for the times given in the legends of the Tables. At the end of incubations, the slices were blotted and thoroughly homogenized in 0.5ml of 3% (v/v) HC104; insoluble material was removed by centrifugation. Samples (0.2ml) of incubation media were also added to 0.3 ml of 5% HC104. The radioactivity present in 0.2ml samples of supematant or acidified medium was determined in

the presence of 10mI of scintillator (600ml of toluene, 400ml of methoxyethanol containing 80 g of naphthalene and 6g of butyl-PBD) by using the dual setting on a Nuclear-Chicago Isocap 300 liquid-scintillation counter. An external standard was used for quench correction. The intracellular volume was derived from the total tissue water (78.3±0.2% wet wt., n = 32) and the sorbitol space (Schofield, 1971). The difference between the total tissue colchicine content and the colchicine contained in the total tissue water was assumed to represent bound colchicine. Intracellular location of bound colchicine. Slices were incubated in Krebs-Henseleit medium containing ['4C]sorbitol (0.1 #Ci/ml) and [3H]colchicine (1 .0uCi/ml), at the concentrations given in the legends of the Tables, for 120min, the time required for equilibration. At the end of the incubation period the slice was blotted and homogenized in 1 ml of ice-cold histidine buffer (5mM-histidine-HCl, 5 uM-EDTA and 0.25M-sucrose, pH6.4) in a loose-fitting Kontes all-glass conical grinder. The homogenate was centrifuged at 275g.,. for 10min (r,. 16cm) to sediment nuclei and cell debris. The supernatant was further centrifuged for 2min at 15000ga,. in an Eppendorf 3200, which sedimented most of the mitochondria and secretory granules. The two pellets were washed once and resuspended in 1 ml of histidine buffer. Samples of each fraction and of the medium were assayed for radioactivity as above. Fractions were also assayed for lactate dehydrogenase (EC 1.1.1.27), glutamate dehydrogenase (NAD+) (EC 1.4.1.2) activities and for DNA and growth hormone. Fixed and stained sections of both sedimented fractions were also examined in an AEI EM6B electron microscope. Estimation of molecular weight of the colchicinebinding protein. The molecular weight of the binding site(s) in the cytosol-containing fraction was deter-mined on a column (1 cm x 50cm) of Sephadex G-100 equilibrated with 20mM-potassium phosphate and 2mi4-MgCI2, pH6.8 (Andrews, 1964). Lactate dehydrogenase, alkaline phosphatase, malate dehydrogenase and haemoglobin were used as markers. Assay methods. (i) Protein concentrations were estimated with bovine serum albumin as standard (Lowry et al., 1951). (ii) Lactate dehydrogenase was assayed by following the disappearance of NADH at 340nm in 50mM-potassium phosphate, pH7.5, containing 0.31mM-sodium pyruvate and 0.13mMNADH (Bergneyer et al., 1965). (iii) Glutamate dehydrogenase (NAD+) activity was determined in fractions which had been frozen and thawed three times to disrupt mitochondria. The assay followed disappearance of NADH at 340nm in 50mM-triethanolamine, pH8.0, containing 3mM-EDTA, 10mM-2-oxoglutarate, 1.6mM-ADP, 120mm-ammonium acetate and 0.1 mM-NADH (Martin & Denton, 1970). (iv) To determine DNA concentrations the fractions were extracted twice with ice-cold 10% 1975

COLCHICINE EFFECTS IN BOVINE PITUITARY SLICES

455

(w/v) trichloroacetic acid and the DNA was removed from the precipitate with 5 % trichloroacetic acid at 85°C for 20min. The DNA concentration was measured colorimetrically at 600nm after incubation overnight with a diphenylamine acetaldehyde reagent (Burton, 1956). (v) Fractions were frozen and thawed to liberate granule-bound growth hormone, and the growth-hormone concentration was determined by a single antibody radioimmunoassay, as described earlier (Schofield, 1967) but with the addition of dextran-coated charcoal to separate bound and free 1251-labelled growth hormone (Gottlieb et al., 1965).

cine in the internal space was rapid at 0.84mM-colchicine (ti, 15min), at which concentration binding occurs mainly to the non-saturable sites. Equilibration was slower at 24uM-colchicine (t*, 50min), and an equilibration time of 120min was therefore adopted in subsequent binding experiments. The uptake of colchicine, studied at various colchicine concentrations, is shown in Fig. 2. The data can be resolved into two components, one exhibiting saturation kinetics and the second showing first-order uptake at concentrations of colchicine up to 0.84mM. The binding can thus be expressed by the formula:

Results Colchicine binding to anterior pituitary slices Kinetic analysis of colchicine binding to protein in tissue extracts is complicated by the rapid denaturation of the binding site (t*, 2.5h) compared with a relatively long equilibration time. Studies on intact tissue provide data on a native and stable site (Hemminki, 1973), and minimize alteration of the site during equilibration. To measure the entry of colchicine in heifer anterior pituitary cells, the extracellular space was determined by using sorbitol which does not penetrate bovine anterior pituitary cells (Schofield, 1971). Both the equilibration of sorbitol with the extracellular space and the attainment of external colchicine concentration in the internal space were complete in 10min (Fig. 1). Equilibration of colchi-

(SI Btotal = k[S]+Bma+. K1 + [5] where Bto,ai is the total colchicine bound, Bmax. is the amount ofcolchicine required to saturate one binding component, [S] is the free colchicine concentration which is assumed to equal the extracellular concentration, Kd is the dissociation constant and k is related to the first-order constant for colchicine uptake to the second component. By using colchicine concentrations much greater than Kd, a plot of Btotai against [S] gave a value of k of 0.38,p1/mg wet wt. Correction of Btotai for this binding at low colchicine concentrations gave data which conformed with Michaelis-Menten kinetics, with a Kd of 3.1 ±0.7 uM and a Bn,ax. of 8.6± 40

700

30

600

0o

US ,

a 4)

| 400

:-2

300

0

o

8!

20

I0

,.c 200

0

mq

0-100 0

0 0

20

40

60

80

100

120

140

Time (min) Fig. 1. Time-course of colchicine uptake by pituitary slices Slices were incubated for given times in 1 ml of KrebsHenseleit (1932) buffer containing 0.1 uCi of [14CJsorbitol/ml and l.O,Ci of [3H]colchicine/ml at 2#M- (@) and 0.84mi- (o) colchicine. The netcolchicine space is the difference between the tottl colchicine space and the sorbitol space. Data are expressed as a means±s.E.M. for between four and eight slices incubated in several different experiments. Vol. 148

10

20

30

40

50

[Colchicine] (uM)

60

70

80

90

Fig. 2. Colchicine binding to pituitary slices incubated in vitro Slices were incubated in quadruplicate for 120min in Krebs-Henseleit buffer containing 0.1 pCi of [4C]sorbitol/ ml and 1.OpCi of [3H]colchicine/ml at various colchicine concentrations. Bound colchicine was calculated as described in the Materials and Methods section. *, Total colchicine bound; A, colchicine binding to the colchicinebinding protein corrected for the non-saturable binding. Data are expressed as a mean for four slices in several different experiments.

456

P. SHETERLINE, J. G. SCHOFIELD AND F. MIRA

Table 1. Marker analysis of cell fractions Subcellular fractions were prepared from bovine anterior pituitary and analysed as described in the Materials and Methods section. All values are the means of at least duplicate determinations on one homogenization. One unit is defined as 1 umol of substrate oxidized/min. N.S., Not measureable. Fractions ... Pellet (10min at 275g) Pellet (2min at 15000g) Final supernatant 56 44 43 Protein (mg/g wet wt.) 14900 1220 800 Lactate dehydrogenase (units/g wet wt.) N.S. 450 54 Glutamate dehydrogenase (units/g wet wt.) 12.0 4.4 13.6 DNA (mg/g wet wt.) 9.6 18.3 11.8 Growth hormone (mg/g wet wt.)

0.6pmol/mg wet wt. The dissociation constant obtained agrees closely with estimates for the Kd of tubulin (Taylor, 1965; Wilson & Freidkin, 1967; Borisy & Taylor, 1967a; Owellen et al., 1972; Wolff & Williams, 1973). Intracellular location of bound colchicine The fractionation procedure used was designed to produce a rapid separation of nuclei, secretory granules with mitochondria, and microsomal preparations with cytosol. Analysis of markers showed that some nuclei were damaged since DNA was present in the cytosol, but otherwise the fractions were as described (Table 1). The composition of the fractions was confirmed by electron microscopy. The experimental design was to incubate tissue with radioactive colchicine at various concentrations, and determine the amount of radioactivity present in each of the fractions. The colchicine-tubulin complex is relatively stable at 4°C (Weisenberg et al., 1968; Wilson, 1970), and the time taken from homogenization to assay of radioactivity was only 15min so that dissociation of the complex was at a minimum. The results (Fig. 3) show that more than 90% of the low-Kd-binding activity was in the fraction containing the cytosol, and that the mitochondrial and granule fraction contained only non-saturable sites. Characteristics of the colchicine-binding site Incubation of the cytosol fraction (460ug of protein) and colchicine (2,UM) at 37°C for 60min with trypsin (5,ug) or Sigma proteinase type VI (10,ug, Sigma (London) Chemical Co., Kingston-uponThames, Surrey KT2 7BM, U.K.) completely prevented subsequent detection of colchicine binding by the DEAE-Sephadex filter disc method (Weisenberg et al., 1968). However, the binding site was not destroyed by similar incubation with phospholipase A (lO,ug), Sigma a-amylase type IA (6,pg) or Sigma ribonuclease type IA (6,ug).

.3la

14

a

612

I

0 o

70

60.5 0-0 50 c 4o .

*=0.-.°. 8 0

30t 0

6 4

20=

=

2

I0m X

a o

o

8c.) c2 8

'

100

200

300

-L 400 r 103

104

0

Bound colchicine (pmol/mg of protein) Fig. 3. Scatchardplots of colchicine binding topituitary cell fractions Slices were incubated for 120min in Krebs-Henseleit buffer containing ['4C]sorbitol and [3H]colchicine at 1.2, 2.0, 3.7, 8.9, 13.8, 84 and 840p1M. Fractions were prepared and assayed for radioactivity as described in the Materials and Methods section. Binding values for each fraction were corrected for extracellular contamination by using [14C]sorbitol and the final supernatant was assumed to contain an intracellular space equivalent of colchicine and was duly corrected. 0, Cytosol; o, nuclei; A, mitochondria and granules. Each point represents the mean of duplicate pairs of slices fractionated in two experiments.

A portion of the cytosol fraction containing bound radioactive colchicine was subjected to gel filtration on Sephadex G-100 (Andrews, 1964), and its elution position corresponded to a molecular weight of 119000. This is close to the values obtained for tubulins (Borisy & Taylor, 1967b; Wilson & Friedkin, 1967; Weisenberg et al., 1968). Gel filtration of the same fraction was performed on a column (25cm x 1.5cm) of Sephadex G-150 (Fig. 4). The ratio of bound/free colchicine corresponds closely to that predicted by the kinetic data. 1975

COLCHICINE EFFECTS IN BOVINE PITUITARY SLICES

Attempts to purify the colchicine-binding protein by methods described for tubulin from brain (Weisenberg et al., 1968) met with only limited success. This may be due to the large differences in the amount of the protein present in the two tissues: in brain 10% of the protein is tubulin, but in the pituitary it may be calculated from the Bmax. that only 0.7 % of the total protein is colchicine-binding protein. Most of the pituitary colchicine-binding protein was recovered

). 8

0.7 0.6 l O. 5 E

E a *_

04....a0 +-

0.-i

0. 3 °

0)

x

13

20

30 40 50 Fraction no. (1 ml) Fig. 4. Gelfiltration of cytosol-containingfraction Slices were incubated with 2.6 pM-[3H]colchicine (specific radioactivity 384Ci/mol) for 120min then rapidly homogenized in ice-cold Mes buffer [2-(N-morpholino)ethanesulphonic acid] (10mM), MgCl2 (5mM), pH6.8, containing 0.24M-sucrose. The homogenate was centrifuged for 2min at 15000gav. in an Eppendorf 3200 centrifuge and a portion of the supmrnatant subjected to gel filtration on a column (1.Scmx25cm) of Sephadex G-150 equilibrated with the same buffer at 4°C. Fractions (1 ml) were collected and assayed for radioactivity and protein content as described in the Materials and Methods section. 0, [3H]Colchicine radioactivity, o, protein concentration (from E280). The void volume (Dextran Blue) was 15 ml.

457

in the fraction precipitated between 40 and 60% satd.-(NH4)2SO4 and could only be eluted from DEAE-Sephadex by 0.6M-NaCl (Table 2). However, this fraction which is more than 90 % tubulin in brain is only an estimated 10% tubulin in pituitary preparations.

Length of microtubules in fixed tissue If it is assumed that colchicine binds to the subunit of microtubules, then the total length of microtubule should decrease in direct proportion to the increase in colchicine binding. This point was investigated by using tissue fixed after 3 h incubation in the presence of colchicine at 3.4 or 13.4,UM. The total lengths of microtubules observed are shown in Table 3. It can be seen that the percentage fall in microtubule length is very close to the binding of colchicine predicted on the basis of the observed Kd of 3.1 /M. Thus it would appear that the colchicine binding observed in the whole tissue can be related to the disappearance of microtubules from secretory cells in the tissue. Inhibition ofgrowth-hormone release by colchicine To determine the effect of colchicine on growthhormone release, pituitary slices were preincubated with various concentrations of colchicine for 120min to allow binding to reach equilibrium. Release of growth hormone was then stimulated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, which increases ox pituitary cyclic AMP concentrations (Schofield & McPherson, 1974). The results are shown in Fig. 5. Increasing the colchicine concentration from 3 to 20AuM progressively inhibited growthhormone release. However, this inhibition reached a maximum value of only 35 % at 20uM-colchicine, a concentration at which the binding protein is more than 85 % saturated. Further increases in colchicine

Table 2. Fractionation of colchicine-binding protein by (NH4)2SO4 or DEAE-Sephadex A-50 Fresh pituitary glands were minced with scissors and homogenized in cold sodium phosphate (10mM), MgCI2 (2mM), GTP (0.1 mM), sucrose (0.24M), pH7.0, in a Kontes all-glass tissue grinder. The homogenate was centrifuged at 23000gav. for 30min in an MSE High-Speed 18 centrifuge. The supernatant was removed and either (method a) treated with increasing concentrations of (NH4)2SO4, the precipitate being retained after each increase, or (method b) treated with DEAE-Sephadex A-50 for 10min on ice. The DEAE-Sephadex was washed in the same buffer without sucrose, and subjected to increasing ionic strength buffers indicated below. Colchicine-binding activity in the precipitate or eluate was analysed by the disc method of Weisenberg et al. (1968). The data are the means of triplicate determinations expressed as a percentage of the original supernatant activity in two separate experiments. Method (a) (NH4)2S04 Colchicine-binding activity (% supernatant) (NH4)2SO4 (% saturation)

...

0-20 69

20-30 56

30-40 118

40-50 297

50-60 278

60-70 0

Method (b) Elution from DEAE-Sephadex A-50

Eluent [NaCl] (M)

Vol. 148

...

0.01 51

0.2 56

0.4 75

0.6 407

0.8 0

P. SHETERLINE, J. G. SCHOFIELD AND F. MIRA

458

Table 3. Decrease of total microtubule length by colchicine Microtubule length was determined as described in the Materials and Methods section. The data are given as means±S.E.M. and the numbers in parentheses refer to the number of prints examined and were derived from sections obtained from two different incubations. Percentage of tubulin unbound at the given colchicine concentration was calculated from the observed Kd of 3.1±0.7pmol litre-1. Length of microtubule/ volume of cytoplasm Tubulin unbound to colchicine [Colchicine] (/IM) (% of control) (,um/,pm3) (% of Bmax.) 0 4.73 ±0.51 (22) 100 100 3.4 2.10+0.25 (31) 44 48 13.4 0.81 ±0.18 (18) 17 19

130 "--'120

80F

s. . '0

60

40 o

,c20

0

10-6

10-5

10-'

[Colchicine] (mol-litre-') Fig. 5. Inhibition of3-isobutyl-1-methylxanthine-stimulated growth-hormone release by colchicine Groups of four separate slices were incubated for 60min in Krebs-Henseleit (1932) buffer as described in the Materials and Methods section. The buffer contained various concentrations of colchicine with and without 0.1 mM-3-isobutyl-1-methylxanthine. Portions of the incubation media were assayed in duplicate for growth hormone and the results expressed as a percentage of the stimulated release in the absence of colchicine in each experiment. The data are expressed as a means±s.E.M. for between six and 20 slices in several experiments.

concentration, which increase binding to non-saturable sites, did not increase the amount of inhibition. Discussion The data here demonstrate the existence of colchicine-binding sites in bovine anterior pituitary slices. These trypsin-sensitive sites have a similar dissociation constant for colchicine and exhibit a similar apparent molecular weight to those reported for tubulins. On purification they behave in a manner characteristic of tubulins from brain (Weisenberg et al., 1968) and thyroid (Bhattacharya & Wolff, 1974). It is reasonable to conclude that the colchicinebinding sites are tubulin-like proteins. The determination of total microtubule length in fixed growthhormone-containing cells by electron microscopy

shows, moreover, that the binding of colchicine to these tubulin-like proteins is correlated to a disappearance of microtubules. Thus the incubation of pituitary tissue in the presence of 13.4pM-colchicine leads to 81 % saturation of the binding protein and a loss of 83 % of the microtubules. The effects of colchicine on pituitary-hormone release as reported in the literature are varied. Ewart & Taylor (1971) found that colchicine (0.1 mM) did not inhibit release of rat growth hormone in the presence of dibutyryl cyclic AMP. Temple et al. (1972), Kraicer & Milligan (1971) and Sundberg et al. (1973) reported that colchicine did not inhibit the action of hypothalamic releasing hormones, although Kraicer & Milligan (1971) found that lOpM-colchicine inhibited the release of adrenocorticotrophin in response to high K+. In the ox pituitary system, the stimulation of growth-hormone release by prostaglandin E2 was inhibited by 0.1 mMcolchicine although 10,uM-colchicine was without effect (Cooper et al., 1972). The data presented in the present paper indicate that colchicine also inhibits the release of ox growth hormone induced by 3isobutyl-1-methylxanthine. The inhibition observed reached about 35% at 10.5 uM-colchicine, and did not increase at higher concentrations. Since the effect was small it was not possible to determine an accurate KL for the inhibition of release by colchicine, but inhibition was maximum at concentrations of colchicine that almost saturate the binding protein. From a mechanistic point of view it is interesting that 84puM-colchicine, a concentration expected to disaggregate more than 96 % of the cytoplasmic microtubules, gave only 35% inhibition of release. It is unlikely therefore that intact microtubules are essential for stimulation of growth-hormone release, although the dependence of growthhormone release on microtubules could change during the course of a 60min incubation. The data could mean that the microtubule subunit, tubulin, is itself involved in the release process and that bound colchicine decreases its subsequent activity. 1975

COLCHICINE EFFECTS IN BOVINE PITUITARY SLICES This research was supported in part by a grant from the British Diabetic Association to Professor P. J. Randle and by Grant no. 3.8080.72 from the Fonds National Suisse de la Recherche Scientifique, Bern, Switzerland. P. S. is a recipient of an M.R.C. award for training in research methods, J. G. S. thanks the M.R.C. for the award of a Travelling Fellowship.

References Andrews, P. (1964) Biochem. J. 91, 222-233 Bergmeyer, H. U., Bernt, E. & Benno, H. (1965) Methods of Enzymatic Analysis, p. 736-743, Academic Press, New York Bhattacharya, B. & Wolff, J. (1974) Biochemistry 13, 2364-2368 Borisy, G. G. & Taylor, E. W. (1967a) J. Cell Biol. 34, 335-348 Borisy, G. G. & Taylor, E. W. (1967b) J. Cell Biol. 34, 525-534 Burton, K. (1956) Biochem. J. 62, 315-323 Cooper, R. H., McPherson, M. & Schofield, J. G. (1972) Biochem. J. 127, 143-154 Creasey, W. A. (1967) Pharmacologist 9, 192-197 Dahlstrom, A. (1968) Eur. J. Pharmacol. 5, 111-113 Ewart, R. B. L. & Taylor, K. W. (1971) Biochem. J. 124, 815-826 Gabbay, K. H. & Tze, W. J. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 1435-1439 Gautvi, K. M. & Tashjian, A. H. (1973) Endocrinology 93, 793-799 Gottlieb, C., Lan, K. S., Wasserman, L. R. & Herbert, V. (1965) Blood 25, 875-881 Hemminki, K. (1973) Biochim. Biophys. Acta 310, 285-288 Horowitz, R. M. & Ullyot, G. E. (1952) J. Am. Chem. Soc. 74, 587-592 Hsei, A. W. & Puck, T. T. (1972) Proc. Natl. Acad. Sci. U.S.A. 68, 358-361 Inoue, S. (1952) Exp. Cell Res. Suppl. 2, 305-3 18

Vol. 148

459

Kraicer, J. & Milligan, J. V. (1971) Endocrinology 89,408412 Krebs, H. A. & Henseleit, A. (1932) Hoppe-Seyler's Z. Physiol. Chem. 210, 33-66 Lacy, P. E., Howell, S. L., Young, D. A. & Fink, C. J. (1968) Nature (London) 219, 1177-1178 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Macleod, R. M., Lehmeyer, J. E. & Bruni, C. (1973) Proc. Soc. Exp. Biol. Med. 144, 259-267 Malawista, S. E. (1965) J. Exp. Med. 122, 361-384 Martin, B. R. & Denton, R. M. (1970) Biochem. J. 117, 861-877 Mizel, S. B. & Wilson, L. (1972) Biochemistry 11, 25732578

Owellen, R. J., Owens, A. M. & Donigian, D. W. (1972) Biochem. Biophys. Res. Commun. 47, 685-691 Pelletier, G. & Bornstein, M. B. (1972) Exp. Cell Res. 70, 221-223

Schofield, J. G. (1967) Biochem. J. 103, 331-341 Schofield, J. G. (1971)Biochim. Biophys. Acta252, 516-525 Schofield, J. G. & Cole, E. N. (1971) Mem. Soc. Endocrinol. 19, 185-199 Schofield, J. G. & McPherson, M. (1974) Biochem. J. 142, 295-300 Sundberg, D. K., Krilich, L., Fawcett, C. P., Illner, P. & McCann, S. M. (1973) Proc. Soc. Exp. Biol. Med. 142, 1097-1100 Taylor, E. W. (1965) J. Cell Biol. 25, 145-160 Temple, R., Williams, J. A., Wilber, J. F. & Wolff, J. (1972) Biochem. Biophys. Res. Commun. 46, 1454-1461 Weisenberg, R. C. (1972) Science 177, 1104-1105 Weisenberg, R. C., Borisy, G. G. & Taylor, E. W. (1968) Biochemistry 4466-4479 Wilson, L. (1970) Biochemistry 9, 4999-5007 Wilson, L. & Freidkin, M. (1967) Biochemistry 6, 31263135 Wolff, J. & Williams, J. A. (1973) Recent Progr. Horm. Res. 29, 229-278 Wunderlich, F., Muller, R. & Spelth, V. (1974) Science 182, 1136-1138