SYNTHESIS AND SECRETION OF GROWTH HORMONE IN THE … · proteins in the rat somatotroph, together with their time cours and metabolie c requirements, ... Grain counts wer ae performet

J. Cell Sci. 12, 1-21 (1973)

Printed in Great Britain

SYNTHESIS AND SECRETION OF GROWTH

HORMONE IN THE RAT ANTERIOR PITUITARY

I. THE INTRACELLULAR PATHWAY, ITS TIMECOURSE AND ENERGY REQUIREMENTS

S. L. HOWELL AND MARGARET WHITFIELDSchool of Biological Sciences, University of Sussex, Fainter, Brighton, Sussex, England

SUMMARY

The intracellular processes involved in synthesis, transport and storage of newly synthesizedproteins in the rat somatotroph, together with their time course and metabolic requirements,have been investigated in a quantitative electron-microscopic radioautography study of thetissue following pulse labelling with tritium-labelled amino acids and chase incubations invarious conditions. Proteins are synthesized initially on the rough-surfaced elements of theendoplasmic reticulum and are transported within 10 min after their synthesis to transitionalareas between the rough-surfaced endoplasmic reticulum and Golgi complex. Transfer tothe Golgi lamellae is achieved, probably via transfer vesicles, within about 60 min after syn-thesis, while formation of mature storage granules occurs within 2 h following protein synthesis.Further experiments utilizing cycloheximide or ouabain during the chase incubations showedthat the intracellular transport of newly synthesized protein and its time course are not sig-nificantly affected by inhibitors of protein synthesis, or by inhibition of sodium-potassiumdependent ATPase by ouabain. Inhibitors of oxidative phosphorylation (250 /IM 2,4-dinitro-phenol) or of respiration (10 /IM antimycin A) markedly reduced intracellular ATP levels andinhibited the intracellular transport processes. The requirement for ATP appeared to beoperative at 2 stages: in the movement of transfer vesicles to the Golgi complex and in theformation of storage granules; possible roles of ATP in these processes are discussed.

INTRODUCTION

The intracellular pathways and metabolic requirement for the synthesis, storageand secretion of zymogens by the guinea-pig exocrine pancreas have been elucidatedin the classical studies of Jamieson & Palade (1967a, b; 1968a, b; 1971a, b). However,comparable studies have not to date been reported in other peptide-secreting celltypes and in particular in any endocrine tissues. The somatotroph of the rat anteriorpituitary provides a suitable model for such an investigation, since the ultrastructureof the somatotroph and aspects of the biosynthesis and secretion of growth hormonehave already been extensively investigated. In addition, methods for the isolation ofhormone storage granules in purified form are available (Hymer & McShan, 1963;Costoff & McShan, 1969) and some radioautographic studies of pituitary cells, and inparticular prolactin and MSH-cell function have already been reported (Racadot,Olivier, Porcile & Droz, 1965; Tixier-Vidal & Picart, 1967; Tixier-Vidal et al. 1971).

The first paper in this series reports electron-microscopic radioautographic studiesof the intracellular sites and time course for the transfer of newly synthesized protein

2 S. L. Howell and M. Whitfield

through the rat somatotroph. Requirements for continuing protein synthesis, for ATPand for activity of ouabain-sensitive adenosine triphosphatase in the transport andstorage processes have also been investigated. In a subsequent paper some propertiesof the isolated growth hormone storage granules are discussed (Howell & Ewart, 1972).

METHODS

Male rats of Sprague-Dawley strain weighing 200-250 g were utilized. They were killed bya blow on the neck, the pituitary being removed intact and rinsed immediately in bicarbonate-buffered saline solution (Gey & Gey, 1936) to remove excess blood. The 2 lateral portionscomprising anterior pituitary tissue were removed with a razor blade and each was cut into 3fragments of approximately equal size, each weighing about 1 mg. The fragments were pooledduring the subsequent incubations.

Incubation procedure in radioautographic experimentsA bicarbonate-buffered saline solution (Gey & Gey, 1936) containing 1 mg/ml albumin and

1 mg/ml glucose was utilized throughout, at 37 °C. The medium was gassed to pH 7-4 with95 % oxygen/5 % CO2 before use and gassing of the incubation flasks with a similar mixturewas maintained throughout the incubation procedure.

After 15 min preincubation in basal medium, the excess medium was removed and replacedwith 10 ml of a medium containing 200 /tCi/ml L-[4,5-3H] leucine of specific activity 19 Ci/mM(Radiochemical Centre, Amersham, England), giving a final concentration of amino acid ofabout 10 /iM. This incubation was continued for 10 min, then the excess medium was removedand the tissue slices were rinsed 3 times with medium at 37 CC which contained 2 mM un-labelled L-leucine, the rinsing procedure requiring a total of 3 min. Tissue samples wereremoved at this time for fixation, the remaining fragments being incubated for further periodsof 20, 50, n o or 230 min in basal medium containing 20 fiM unlabelled L-leucine, beforeremoval of tissue samples for fixation in a similar way.

In experiments utilizing 50 /tg/ml cycloheximide (Actidione, Koch-Light Laboratories,Colnbrook, Bucks.), 0-25 mM 2,4-dinitrophenol (B.D.H. Ltd., Poole, Dorset), o-i mM ouabain(B.D.H. Ltd., Poole, Dorset) or 10 fiM antimycin A (Sigma Ltd., London), these agents wereadded to the rinsing medium immediately after pulse labelling and to all subsequent incubationmedia.

In a single experiment a mixture of 4 tritium-labelled amino acids (L-valine, 19 Ci/mM:L-tyrosine, 47 Ci/mM: L-histidine, 52 Ci/mM and L-leucine, 17 Ci/mM, all from RadiochemicalCentre, Amersham) were utilized, each at a concentration of ioo/<Ci/ml. The resulting graincounts suggested that a rate of incorporation was obtained in this system comparable to thatobtained by the use of L-[3H]leucine alone.

Preparation of tissue for electron microscopy

Tissue fragments were placed immediately after removal from the flasks into a 3 % glutaral-dehyde solution (Polysciences Inc., Warrington, Pa.) in Millonig's buffer at room temperaturefor 1 h. After rinsing the pieces were postfixed in buffered 2 % OsO4 (Johnson Matthey Ltd.,London) at pH 7-4 for a further 2 h. After dehydration in ethanol they were embedded in anepoxy resin by a standard procedure (Lockwood, 1964).

Radioautographic techniqueThe procedure employed for electron-microscopic radioautography has already been

described in detail (Howell, Kostianovsky & Lacy, 1969); the following modifications wereadopted in the present study. Sections were subjected to carbon evaporation and coated withIlford L-4 emulsion on glass slides without the prior staining with lead citrate which waspreviously employed. Exposure of sections to the emulsion was continued for 4-8 weeks at4 °C before development in Kodak D 19 developer and fixing in 20% sodium thiosulphate.

Intracellular processes in pituitary 3

Sections were mounted on copper grids and the excess collodion was removed by immersionfor 3 min in amyl acetate. Grain counts were performed at the microscope screen or fromprints, a total of at least 400 grains being assessed at each time interval. Sections were stainedin a saturated solution of uranyl acetate in 50 % ethanol for 5 min prior to micrography utilizingan AEI EM 6B electron microscope.

Rate of incorporation of [3H]leucine

Pituitary fragments of 2-5-4 mg wet weight were utilized to study rates of incorporation ofL-[3H]leucine into trichloroacetic acid (TCA) precipitable protein. Paired fragments fromindividual pituitaries were incubated for periods between 5 min and 4 h with 20 /tCi/mlleucine of specific activity 19 Ci/mM in the presence or absence of 50 /tg/ml cycloheximide orof 025 mM dinitrophenol. At the end of the incubation period, fragments were removed, rinsedbriefly in 2 mM unlabelled L-leucine, blotted on filter paper and homogenized in 10% TCA.After centrifugation the supernatants were removed for radioactivity determination and theprecipitates were centrifuge-washed 3 times with 5 % TCA. The washed precipitates were thendissolved in N NaOH for determination of their radioactivity in a liquid scintillation spectro-meter, utilizing a toluene:Triton X-ioo:2,5-diphenyloxazole (700:300:5, v:v:\v) scintillant.

Estimation of A TP content

Individual paired pituitaiy fragments weighing 2-5—40 mg were incubated in the presenceor absence of 2,4-dinitrophenol (025 mM) for periods of between 5 min and 4 h. Immediatelyafter the end of the appropriate incubation period, the fragments were homogenized in i'4Nperchloric acid at 4 °C. After centrifugation at 700 g for 10 min at 4 °C, the supernatant wasremoved and neutralized to pH 7 with 3 M potassium carbonate. The resultant precipitate ofpotassium perchlorate was removed by centrifugation and the supernatant stored at — 20 °Cfor assay at a later date. ATP concentrations were determined by the method of Lowry,Passonneau, Hasselberger & Schulz (1964) utilizing enzymes and intermediates obtained fromBoehringer Corp., London.

Protein estimation

Protein concentrations were estimated by the method of Lowry, Rosebrough, Farr &Randall (1951), using crystalline albumin standards.

RESULTS

The ultrastructure of the rat somatotroph

The somatotroph may readily be identified from the other cell types in the anteriorpituitary by the diameter of its storage granules, which lie in the range 320-390 nm(Hedinger & Farquhar, 1957), and by the frequency of its occurrence in thin sections,since in the male rat the somatotroph constitutes 30-40% of the pituitary cellpopulation. Very recently direct confirmation of the nature of these cells has beenobtained in a demonstration of the growth hormone content of their storage granulesby an immunochemical technique (Baker, Midgely, Gersten & Yu, 1969; Nakane,1970). The ultrastructure of the rat somatotroph has already been described in detail(Hedinger & Farquhar, 1957; Farquhar, 1961) and only additional observationsrelevant to the secretory process within the cell will be noted here.

The elements of the rough-surfaced endoplasmic reticulum run approximatelyparallel to each other and terminate in areas of cytoplasm which are rich in freeribosomes and polysomes and contain in addition numerous small vesicles varying in


diameter between 40 and 70 nm. Microtubules are also frequently observed in thesame areas of cytoplasm as the vesicles but are seldom found in direct contact withthem; microtubules are occasionally seen in contact with storage granules (Howell &Whitfield, in preparation); these features are evident in several of the radioautographs(Figs. 3-7). The vesicles are also frequently seen in association with the Golgi complexand it seems possible by analogy with similar organelles identified in the exocrinepancreas and elsewhere that they might represent carrier vesicles which are transitionalbetween the rough-surfaced endoplasmic reticulum and the Golgi complex. In theinvestigation of their role which follows, therefore, the areas of cytoplasm foundbetween the rough-surfaced reticulum and the Golgi complex have been termed'transitional areas', and the 50-nm vesicles have been tentatively described as 'transfervesicles'.

Polarization of pituitary cells to give a concentration of secretory function at theirvascular poles has previously been reported (Farquhar, 1961) and was also noted inthe present study. In addition, numbers of granules were frequently observedindividually lying very close to the plasma membrane, although not in contact with it.There was no evidence in this study of any alteration in ultrastructure of the intactcells during 4 h incubation in vitro (Figs. 3-7).

Metabolic characteristics of the rat anterior pituitary in vitro

The metabolic characteristics of the isolated rat anterior pituitary have beendocumented in a number of recent studies, although ultrastructural observationsfollowing incubation of the tissue in vitro for short periods have only recently beendescribed (Tixier-Vidal et al. 1971). In our investigations incorporation of L-[3H]leu-cine into TCA-precipitable protein was shown to be linear over a 4-h period ofincubation, and to be inhibited by more than 90% during periods of incubation inthe presence of cycloheximide (so/^g/ml) (Fig. 1); this effect was demonstrable within5 min, the earliest time examined. By contrast, 2,4-dinitrophenol (DNP) had aneffect on incorporation of amino acids into protein which was less marked than that ofcycloheximide and of slower onset, approximately 15 min being required to attain50% inhibition (Fig. 1). This effect of dinitrophenol was presumably secondary to alack of availability of ATP for the maintenance of protein biosynthesis. The rate ofincorporation in pituitaries obtained from animals previously treated with insulin andincubated in the presence of 0-2 mg/ml glucose during the 10-min labelling periodwas not significantly different from that seen in untreated animals.

Alterations in ATP levels within the intact pituitary during prolonged incubationsare shown in Fig. 2; cellular ATP levels obtained after comparable incubations per-formed in the presence of dinitrophenol or of cycloheximide are also shown in thesame figure. There was no significant alteration of ATP levels in control tissue during4 h of incubation, but it is clear that DNP was effective in lowering intracellular con-centrations of ATP within a half-time of about 5 min after its addition to the incuba-tion medium, and of maintaining them at low levels in the succeeding 4-h period ofincubation. Addition of cycloheximide resulted in a lowering of ATP levels only after120 min of incubation, possibly as a result of depletion of enzymes or cofactors whose

Intracellular processes in pituitary

137 000)120 -

5 10Time, min

Fig. i. Incorporation of [3H]leucine into TCA-precipitable protein during incubationof the rat pituitary. Pituitary fragments were incubated with 20 /<Ci/ml [3H]leucinein basal medium ( # — • ) , medium containing 50 fig /ml cycloheximide (x — x ), or0-25 mM 2,4-dinitrophenol (O—O)- Means ± S.E.M. of 4 observations at each timeare shown.

synthesis is required for continuing ATP production during longer periods ofincubation.

The pathway and time course for the intracellular transport of newly synthesized proteins

The distribution of silver grains over somatotrophs which were fixed for electronmicroscopy at 3, 20, 50, n o or 230 min after the end of pulse labelling is shown inTable 1. At least 400 individual grains were counted at each time interval, grains beingassigned to those organelles over which > 50% of the grain area was superimposed.Typical grain distributions observed at each time interval are illustrated in Figs. 3-7;the principal observations are described below. The distribution of grains over eachorganelle was assessed independently by 2 investigators and was expressed as apercentage of the total number of grains assessed at each time. There appeared to belittle variation in the degree of labelling of individual somatotrophs studied, or in thelabelling of somatotrophs compared with other endocrine cell types.

S. L. Howell and M. Whitfield

-§. 6

"o

% 12

i i i5 10 30 60 120

Time, min

Fig. 2. ATP levels in rat pituitary fragments during incubation in vitro. Incubationswere completed in basal medium (#—#) , medium containing 50 /tg/ml cycloheximide( x — x ), or 0-25 mM 2,4 dinitrophenol (O—O)- Means ± S.E.M. of 4 observations ateach time are shown.

Table 1. Distribution of silver grains over organelles of the rat somatotrophat various times after pulse labelling with [3H]leucine

Tl i l fo f iAn /"VTI 7 U I d.HUH KJi.

incubationafter 10-min

pulse labelling,min

32 0

5°n o2 3 0

Rough-surfaced

endoplasmicreticulum

35231 0

13

14

Percentage

Transitional Golgiareas

29

4537252 1

complex

1 2

16

25

95

of grains over

Storagegranules

74

14

3742

Nuclei

138

1 2

1 1

1 2

Mito-chondria

+ lysosomes

442

56

Pituitary fragments were incubated for 10 min with L-[3H]leucine (200 /iCi/ml) and afterrinsing with excess unlabelled L-leucine were reincubated in basal medium for a furtherperiod of up to 230 min. Results are expressed as the percentages of the total number of grainsassessed at each time which were present over each organelle.

3 min. Tissue fixed immediately after rinsing with excess unlabelled leucine follow-ing a 10-min pulse-labelling period, and therefore in contact with the radioactiveamino acid for not longer than 13 min in all, showed a high proportion (35%) ofgrains over the rough-surfaced endoplasmic reticulum. A considerable proportion ofthe radioactivity observed at the earliest intervals studied was also present over thetransitional areas and in peripheral regions of the Golgi complex (Table 1).Labelling of other organelles and in particular of storage granules was at a low level.

20 min. By 20 min after the end of pulse labelling a high proportion of the radio-


Table 2. Distribution of silver grains over organelles of the rat somatotroph after pulselabelling followed by 4 h of incubation in the presence of dinitrophenol, cycloheximide,ouabain or antimycin A.

t

Additionto incubation

medium

None2,4-dinitro-phenol (0-25 imi)

Cycloheximide(50 /*g/ml)

Rough-surfaced

endoplasmicreticulum

1 2

1 21

15

Ouabain (o-1 ITIM) I IAntimycin(10 fiM) 25

Transi-tionalareas

23

37

1726

32

Percentage

Golgicomplex

6

16

8

15

of grains overA

Storagegranules

46

1 2

3639

1 0

Nuclei

9

7

1 2

1 0

14

Mito-chondria

+ lysosomes

4

4

46

4

Pituitary fragments were incubated for 10 min with L-[3H]leucine (200 /«Ci/ml) and afterrinsing with excess unlabelled L-leucine were reincubated in basal medium or medium con-taining inhibitor at the concentration shown for a further period of 230 min. Results areexpressed as the percentages of the total number of grains assessed which were present overeach organelle.

activity was present in the transitional areas, associated with the transfer vesiclesand also over the lamellae of the Golgi complex. There was a marked reduction in thegrains present over elements of the rough-surfaced endoplasmic reticulum (Fig. 4).

50 min. One quarter of all the silver grains were present over the lamellae of theGolgi complex at this time and a small increase in the proportion of labelled granuleswas evident for the first time. High levels of radioactivity remained over transitionalelements of cytoplasm peripheral to the Golgi complex (Fig. 5).

n o and 230 min. The growth hormone storage granules were the most heavilylabelled organelles at each of these times. Labelling over the Golgi complex fell pro-gressively to very low levels (5 %) and there appeared to be some return of radioactivityto the elements of the rough-surfaced endoplasmic reticulum between n o and 230 min.

Grains over lysosomes and mitochondria remained at consistently low levels at eachof the time periods studied. A feature of some interest was a fairly constant degree oflabelling of the nucleus at every time interval studied, amounting to up to 13 % of thetotal grains present. Similar nuclear labelling has been noted in the prolactin secretorycell of the duck anterior pituitary after incubation with labelled amino acids (Tixier-Vidal & Picart, 1967) but has not been reported in studies of other endocrine secretorycell types (Figs. 6, 7).

Requirement for continuing protein synthesis

The effects of inhibition of protein synthesis on the intracellular translocation ofnewly synthesized hormone through the rat somatotroph was investigated by incuba-tion of the tissue in the presence of 50/tg/ml cycloheximide for 4h after a 10-min


pulse-labelling period. The grain counts obtained by radioautography of the tissueafter such a 4-h period of chase incubation in the presence of cycloheximide areshown in Table 2. Although fewer grains appeared to be present over the granulesthan in control tissue incubated for the same period, there were no significant differ-ences between the grain distributions seen after incubation in the presence or absenceof cycloheximide. Furthermore, the presence of cycloheximide in addition to anti-mycin A in a further series of experiments showed no significant differences from thegrain distribution seen when antimycin A alone was used.

Role of ATP

A possible requirement for ATP, derived from oxidative phosphorylation or fromrespiration, in the intracellular translocation of newly synthesized protein was investi-gated by pulse labelling the tissue with subsequent 4-h incubation in the presence of2,4-dinitrophenol (0-25 mM) or of antimycin A (10 fiM). Grain counts obtained in thepresence of either of these agents were very similar and are shown in Table 2. It isclear that granule formation was almost completely prevented during incubation witheither of these agents, but that some transfer of radioactivity as far as the lamellae ofthe Golgi complex was not completely prevented by the use of either agent. Incubationin the presence of dinitrophenol or antimycin A for periods as short as 30 min, how-ever, resulted in dilation of the elements of the endoplasmic reticulum and some dis-organization and distension of the lamellae of the Golgi complex (Figs. 8, 9), whichin some cells led to a pronounced vacuolation in this organelle. In addition there wasa marked aggregation of transfer vesicles in the transitional areas adjacent to theendoplasmic reticulum.

Requirement for ouabain-sensitive ATPase

In a further experiment incubation was continued at the end of the pulse-labellingperiod in the presence of ouabain in the incubation medium at a concentration (o-1 mM)known to inhibit specifically sodium-potassium ATPase activity (Post, Merritt,Kinsolving & Albright, i960). The results obtained after 4 h incubation (Table 2)show no significant differences between the grain distribution seen in control tissueand that incubated in the presence of ouabain. However, interpretation of this resultmay be complicated by uncertainty as to whether ouabain will penetrate the cells toinfluence intracellular ATPase. No major ultrastructural alterations were seen afterincubation in the presence of ouabain or of cycloheximide except for increased forma-tion of lytic bodies after some 4 h of incubation in the presence of o-i mM ouabain.

DISCUSSION

Validation of in vitro preparation

Isolated anterior pituitary fragments have previously been utilized in severalstudies of the regulation of the synthesis and secretion of pituitary hormones (Rao,Robertson, Winnick & Winnick, 1967; Schofield, 1967; MacLeod & Abad, 1968;Burek & Frohman, 1970); in general, the responses observed have been consistent


with the known characteristics of the tissue in vivo. The release of growth hormonefrom the rat anterior pituitary in response to theophylline has been shown to be linearduring a 4-h period of incubation (Ewart & Taylor, 1971), and the incorporation of3H-labelled amino acids into TCA-precipitable protein was also essentially linear overthis period (Fig. 1, see also Rao et al. 1967; MacLeod & Abad, 1968; Burek & Froh-man, 1970; Samli, Lai & Barnett, 1971; Labrie, Beraud, Gauthier & Lemay, 1971).

In our experiments utilizing male animals of a size previously demonstrated to showmaximal rates of incorporation of radioactivity into growth hormone there was anegligible lag period before incorporation of amino acids commenced, and incorpora-tion over a 10-min labelling period was not significantly enhanced by pre-incubationof the tissue at 4 °C to allow penetration of the label into the tissue (data not shown).Furthermore, attempts to increase rates of incorporation of label into growth hormoneby pretreatment of the animals with insulin, which might be expected to provokehypoglycaemia-induced growth hormone secretion, during 1-6 h before sacrifice,resulted in only small changes in rates of growth hormone synthesis. Addition oftheophylline (5111M) or dibutyryl cyclic 3',5'-AMP (1 HIM) to isolated pituitary frag-ments at concentrations known to provoke growth hormone secretion did not signifi-cantly alter rates of incorporation of [3H]leucine during 10-min periods of labelling,although it has been reported that dibutyryl cyclic AMP may induce a 20-40%increase in incorporation of labelled amino acids into growth hormone after at least2-3 h of incubation in vitro (MacLeod & Lehmeyer, 1970; Labrie et al. 1971). Thestimulatory effect of aminophylline on growth hormone secretion in similar conditionsoccurs within 3 min (Steiner et al. 1970), and it seems possible that the enhanced rateof synthesis observed after relatively lengthy periods of incubation may be a secondaryeffect resulting from hormone depletion during this prolonged period of secretoryactivity.

It is clear that the ultrastructure of the cells secreting growth hormone may remainentirely normal during periods of incubation in vitro of up to 4 h duration, and thisisolated pituitary preparation therefore represents a viable surviving system by all thebiochemical and ultrastructural criteria which we have tested. However, throughoutthe incubation period a small but consistent proportion of cells of all types is seen withdiscontinuities of their plasma membranes, and the organelles in these cells haveappearance similar to those seen in homogenates, with rounded up mitochondria andmicrosomal spheres in evidence. Storage granules also remain in the 'ghosts' of theruptured cells and the slow dissolution of their hormone contents into the incubationmedium may account for the basal rate of hormone release which does not appear tobe subject to metabolic regulation, but which is a feature common to most in vitroincubation systems employing product-storing secretory tissues.

Limitations of radioautographic studies of intracellular transport processes

Technical aspects of the radioautographic procedure utilized here and its validationhave been discussed elsewhere (Howell et al. 1969). The major advantage of a tech-nique of this type is its capacity to identify the exact intracellular localization ofradioactivity within intact cells. In particular, it is possible to identify positively

io S. L. Howell and M. Whitfield

organelles such as the Golgi complex which are not readily isolated in an intact andhighly purified form by conventional subcellular fractionation techniques. However,electron-microscopic radioautography is limited at present by the lack of emulsionsof small grain size which still retain high sensitivity, and it is thus impossible tolocalize tritium to a sphere of less than 80 nm by present techniques; this limitationof resolution has recently been discussed by Salpeter, Bachman & Salpeter (1969).A further serious drawback for the cytologist is the lack of identification of the natureof the radioactively labelled protein under study in radioautographic experiments.For this purpose it is essential to determine the major biosynthetic products of thetissue under examination and its distribution in characterized subcellular fractions.

The role of ATP in the intracellular transport of growth hormone

The ATP requirements for the intracellular transport of newly synthesized growthhormone through the somatotroph are in general similar to those already reported forthe exocrine pancreas (Jamieson & Palade, 19686, 1971a) and for the B cells of theislets of Langerhans (Howell, 1972). Thus there appears in all these cell types to bean energy-requiring step or lock involved in the transfer of newly synthesized proteinfrom the endoplasmic reticulum via the transfer vesicles to the Golgi complex. Thenature of this ATP-requiring step and the mechanisms involved in the movement ofthe vesicles are, however, completely unknown.

ATP may play an additional role in storage granule formation in the pancreatic Bcell and in the somatotroph since this process also seems to be inhibited in the presenceof reduced levels of the nucleotide. The presence of significant quantities of radio-activity over the Golgi complex, together with the relatively long delay betweensynthesis and incorporation of newly synthesized protein into the granule (at least75 min), suggests that the inhibition of granule formation may result not from the lackof available newly synthesized protein as a consequence of the reduced transfer ofprotein to the Golgi complex imposed by the fall in ATP levels, but rather that ATPis itself required in some aspect of the process of granule formation. Further experi-ments, in which transfer of newly synthesized protein to the Golgi complex is com-pleted before the application of a metabolic block, will be required to clarify this point.

Finally, ATP is known to be essential for the secretion of growth hormone (Ewart& Taylor, 1971) as well as of many other exportable proteins; possible roles of thenucleotide in this aspect of the intracellular transfer process have been discussedelsewhere (Jamieson & Palade, 1971a).

Requirements for protein synthesis in the intracellular transport process

The lack of significant effect of cycloheximide, an inhibitor of protein synthesis, onthe intracellular transport process is consistent with results already reported in theexocrine pancreas (Jamieson & Palade, 1968a), and in the pancreatic B cell (S. L.Howell, unpublished observations). The results suggest that the intracellular transportand secretion of newly synthesized growth hormone does not require the synthesis ofother non-secretory proteins at any stage of the secretory cycle. This appears to be


compatible with the demonstration of Amsterdam et al. (1971) of the simultaneoussynthesis in the rat parotid of the proteins of the granule membrane together with thegranule content, which would clearly obviate the need for synthesis of membraneproteins during storage granule formation. Finally, it can be excluded that the effectsof inhibitors of oxidative phosphorylation or respiration already reported above couldresult from inhibition of protein synthesis secondary to lowering of ATP levels, sinceprotein synthesis does not itself appear to be required in the subsequent intracellulartranslocation processes.

The basic intracellular pathway for the translocation of newly synthesized proteins

The pattern of grain distribution and the changes in this distribution with theduration of chase incubation shown in Tables 1 and 2 is similar to that reported in theprolactin cell of the duck by Tixier-Vidal & Picart (1967), and in qualitative studiesof the rat adenohypophysis by Racadot et al. (1965). It appears to be consistent withthe following description of the intracellular processes involved in the passage ofradioactively labelled proteins through the somatotroph:

(a) The rapid incorporation of amino acids into protein in the rough-surfacedendoplasmic reticulum followed by a very rapid transfer of the newly synthesizedprotein through the cisternae of the reticulum to the transitional areas; this veryrapid synthesis is consistent with the 1-2 min estimated time for albumin synthesisin mammalian liver (Peters, Fleischer & Fleischer, 1971). The transfer of newlysynthesized growth hormone to the transitional areas was not inhibited or delayedduring chase incubation in the presence of dinitrophenol or antimycin A in ourexperiments. This may be either because this phase of the transfer process is inde-pendent of metabolic energy derived from ATP, as was shown by Jamieson & Palade(1968&) in the pancreatic exocrine cell, or alternatively because the time required forthis transfer (probably of the order of 5 min) is less than that needed to induce aneffective lowering of intracellular ATP levels.

(b) The transfer of protein to the Golgi lamellae across the transitional areasmay be achieved via the transfer vesicles already described. The small size of thesevesicles makes it impossible to identify them positively as the carriers of newlysynthesized protein by radioautography alone, but attempts to separate and charac-terize them in subcellular fractionation experiments are at present in progress.

(c) The formation of storage granules within the lamellae of the Golgi complexappears to require metabolic energy, and may also involve the utilization of ATPin stabilizing and concentrating the growth hormone prior to its precipitation withinthe granules (Howell & Ewart, 1972). The nucleotide does not appear to be requiredas a substrate for ouabain-sensitive ATPases, although ATPase systems of this typemay be important in mediating sodium and potassium fluxes and thus in acting assolvent pumps in many biological membrane systems.

(d) Evidence which will be discussed in detail in a future paper suggests that thenewly formed storage granules may be transferred from the Golgi complex to thecytoplasmic granule pool via a microtubular system of a type similar to that whichhas already been shown to mediate the intracellular movement of granules in


several types of cell (Malawista, 1965; Lacy, Howell, Young & Fink, 1968; Gillespie,Levine & Malawista, 1968; Poisner & Bernstein, 1971).

(e) The final secretion of growth hormone involves the movement of the granulesfrom the cytoplasmic granule pool to the plasma membrane, where their contents areextruded by exocytosis (Farquhar, 1961).

We thank Drs K. W. Taylor and R. B. L. Ewart for encouragement and advice, and MrColin Atherton for skilled photographic work. Financial assistance from the Medical ResearchCouncil, British Insulin Manufacturers and Hoechst Pharmaceuticals is gratefully acknow-ledged.

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synthesis of membrane protein and exportable protein of the secretory granule in the ratparotid. J. Cell Biol. 50, 187-200.

BAKER, B. L., MIDGELY, A. R., GERSTEN, B. E. & Yu, Y. Y. (1969). Differentiation of growthhormone and prolactin containing acidophils with peroxidase labelled antibody. Anat. Rec.164, 163-172.

BUREK, C. L. & FROHMAN, L. A. (1970). Growth hormone synthesis by rat pituitaries in vitro:effect of age and sex. Endocrinology 86, 1361-1367.

COSTOFF, A. & MCSHAN, W. H. (1969). Isolation and biological properties of secretory granulesfrom rat anterior pituitary glands. J. Cell Biol. 43, 564-574.

EWART, R. B. L. & TAYLOR, K. W. (1971). Regulation of growth hormone secretion from therat anterior pituitary in vitro. Biochem. J. 124, 815-826.

FARQUHAR, M. G. (1961). Origin and fate of secretory granules in cells of the anterior pituitary.Trans. N.Y. Acad. Sci. 23, 346-351.

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{Received 30 May 1972)

5. L. Howell and M. Whitfield

if

•?,Fig. 3. Radioautograph of rat somatotroph showing distribution of silver grains intissue fixed 3 min after the end of a io-min labelling period with [3H]leucine. Grainsare present over elements of the rough-surfaced endoplasmic reticulum and overtransitional areas between the rough endoplasm and the Golgi complex. Transfervesicles are clearly seen (arrows), x 23000 approx.


Fig. 4. Radioautograph of rat somatrotroph showing distribution of silver grains intissue fixed 20 min after the end of a pulse labelling with [3H]leucine. Grains arepresent over the rough-surfaced endoplasmic reticulum, transitional areas and overthe lamellae of the Golgi complex, x 23 000 approx.


Figs. 5, 6. Radioautograph of rat somatotroph showing distribution of silver grains intissue fixed 50 (Fig. 5) or 110 min (Fig. 6) after a io-min pulse-labelling period. Fewgrains are now associated with the endoplasmic reticulum or transitional areas butthey are present over the lamellae of the Golgi complex and over storage granules.Numerous microtubules are present (arrows). Fig. 5, x 22000; Fig. 6, x 32000approx.



Fig. 7. Radioautograph of rat somatotroph showing distribution of silver grains intissue fixed 230 min after a io-min pulse-labelling period. Silver grains are nowpresent predominantly over the storage granules, x 22000 approx.Fig. 8. Radioautograph of rat somatotroph showing distribution of silver grains intissue incubated for 230 min in the presence of 2,4-dinitrophenol following a io-minpulse-labelling period. Grains are present over distended elements of the endoplasmicreticulum and over the disorganized lamellae of the Golgi complex. Aggregation oftransfer vesicles is seen (arrow), x 18000 approx.

Intracelliilar processes in pituitary

' • *

zo 5. L. Howell and M. Whitfield

Fig. 9. Radioautograph of rat somatotroph showing distribution of silver grains intissue incubated for 230 min in the presence of 2,4-dinitrophenol following a io-minpulse-labelling period. Grains are again present over distended elements of theendoplasmic reticulum, and a marked aggregation of transfer vesicles is evident(arrow), x 28000 approx.


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SYNTHESIS AND SECRETION OF GROWTH HORMONE IN THE … · proteins in the rat somatotroph, together with their time cours and metabolie c requirements, ... Grain counts wer ae performet