Selected hormonal effects on protein secretion and amino acid uptake by acini from bovine mammary gland

0020-71 Ix~79~IM)I-088910?.~/0

Int. I Biochrm, Vol. IO. pp. 889 lo X94 0 Pergamon Press Ltd 1979. Printed in Great Britain

SELECTED HORMONAL EFFECTS ON PROTEIN SECRETION AND AMINO ACID UPTAKE BY ACINI FROM BOVINE MAMMARY GLAND C. S. PARK, J. J. SMITH, W. N. EIGEL and T. W. KEENAN Mammary Biology Laboratory, Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, U.S.A. (Received 3 April 1979)

Abstract-l. Release of newly synthesized proteins from acini freshly isolated from lactating bovine (Bos taurus) mammary tissue was inhibited by cycloheximide. Millimolar concentrations of the cyclic AMP phosphodiesterase inhibitor theorphylline also inhibited release of protein from acini. 2. Addition of either insulin or dibutyryl cyclic AMP to incubation medium stimulated release of newly synthesized protein from acini. Combinations of insulin and dibutyryl cyclic AMP were more effective than either alone. 3. Cycloheximide inhibited uptake of the nonmetabolizable amino acid, 1-aminocyclopentane carboxylic acid (cycloleucine), by bovine mammary acini. Uptake of this amino acid was stimulated by inclusion of insulin and/or dibutyryl cyclic AMP in incubation medium.

The present study was designed to investigate protein secretion in response to cycloheximide, CAMP and theophylline and to determine the influence of insulin, cycloheximide and CAMP on uptake of an amino acid analog by acini from bovine mammary gland.

INTRODUCTION Several investigators have reported relationships between adenyl cyclase activity and hormones during lactation (Bar, 1973; M.ajumder & Turkington, 1971; Sapag-Hagar & Greenbaum, 1974). The role of, and interrelationships among, various endocrine glands and their hormone secretions in the regulation of

mammary functions have been reviewed (Cowie, 1971). Louis & Baldwin (1975), in an attempt to understand the role of 3’,5’-cyclic adenosine monophosphate (CAMP) as a mediator of hormone action, examined intracellular CAMP concentrations and activities of adenyl cyclase. CAMP phosphodiesterase and protein kinase during pregnancy and lactation in rats. While the role of CAMP in mediating cellular growth and development has been extensively studied, the action of CAMP on formation of milk constituents has received little investigaton. Olivier-Bousquet & Denamur (1975) reported that CAMP accelerated intracellular transport and release of proteins from rabbit and ewe mammary tissue slices. However, addition of CAMP to explants of mid-pregnant mouse mammary gland decreased casein synthesis (Rillema, 1976) and inhibited lactose synthesis in guinea-pig mammary tissue slices (Loizzi et al., 1975). Previously we demonstrated that acini isolated from lactating.mammary gland could be used to advantage in studies of milk protein synthesis and secretion; protein synthesis by bovine mammary acini was stimulated by CAMP (Park et al., 1979). Recently there has been considerable research on amino acid transport in a variety of mammalian tissues. Amino acid availability plays a central role in milk protein synthesis and secretion but, in spite of the obvious importance, relatively little is known regarding transport of amino acids by mammary alveolar epithelial cells. Recently the uptake of the nonmetabolizable amino acid analog a-aminoisobutyric acid was examined with diced mouse mammary tissue (Lobitz & Neville, 1977).

MATERIALS AND

METHODS

Eagle’s minimal essential medium (MEM), Hank’s balanced salt solution (BSS) (Hanks & Wallace, 1949) sodium bicarbonate (7.5%) and amino acid stock solutions were from Grand Island Biologicals, Grand Island, NY. N6, 0”-d/butyryl Insulin, cycloheximide, adenosine 3’,5’-cyclic monophosphate (dbcAMP) and theophylline were from Sigma Chemical Company, St. Louis, MO. The amino acids L-[4,5-3HJleucine (1 Ci/mmol) and I-aminocyclopentane-l-[i4C]carboxylic acid (cycloleucine) (6OCi/ mol) were from Amersham, Arlington Heights, IL. The basic incubation medium was MEM (Eagle, 1959) with 20% more glucose and a three-fold increase in amino acids (Shingoethe et al., 1967). Bovine serum albumin at 1% (w/v) was included in the medium. BSS was used to wash acini. Acini were prepared from mammary tissue obtained from lactating Holstein cows at slaughter (Park er al., 1979). In both protein secretion and amino acid uptake studies, approx 50mg of acini were suspended in 4 ml of MEM. All incubations were at 37°C and were terminated by cooling on ice water. Acinar suspensions were subsequently centrifuged at 2000 R for 10 min to separate supernatant and particulate fractions. The trichloroacetic acid-insoluble radioactivity in supernatant fractions was considered to represent secreted proteins. Methods for recovery of proteins by trichloroacetic acid (TCA) precipitation and determination of radioactivities were as described (Park et a/., 1979). Protein was measured according to Lowry et al. (1951) with bovine serum albumin as standard. Effects of dbcAMP, insulin and cycloheximide .on protein secretion by acini were evaluated by inclusion of these compounds (at levels specified in Figs and Tables) in the basic incubation media. Incubation was for 90min except where specified. For these studies unlabelled leucine was deleted from the medium and was replaced with

889

890

C. S. PARK, J. J. SMITH,W. N. EIGELand T. W. KEENAN

L-[4,5-3H]leucine at 0.5 pCi/ml. Secreted proteins were recovered after incubation and the specific activity (CPM/mg protein) was determined. For ammo acid uptake studies acini were preincubated in MEM at 37°C for 20min. The medium was then replaced with desired treatment media containing dbcAMP. insulin or cycloheximide as specified. After incubation for up to 220min, this medium was removed and replaced with MEM containing 0.5 ml unlabeled- and 0.5 @/ml [ ‘%I]cycloleucine. Incubation was continued for 15 min. After recovery of acini and washing with BSS, specific activities of acini were determined. Uptake of cycloleucine was calculated as: Cycloleucine uptake (nmols/mg protein) = acini specific activity (CPM/mg protein)/medium specific activity (CPM/nmol) (Borle & Studer, 1978). Main effects of treatments were examined by analysis of variance. Treatments were compared by Student-Newmar-Keuls test. Relationships among variables were assessed by regression analysis (Snedecor & Cochran, 1967).

I

OO

I 60

I loo

I

I50

INCUBATION PERIOD (minutes)

RESULTS Protein secretion

Theophylline, an inhibitor of CAMP phosphodiesterase, and cycloheximide, an inhibitor of protein synthesis, decreased release of proteins from bovine mammary acini (Fig. 1). With all treatments, protein secretion increased linearly over 150 min. Cycloheximide was somewhat more inhibitory than was theophylline, reducing the amount of released protein to 71% of control levels after 150 min. The reduction caused by theophylline at 5 and 1OmM was to 80 and 77% of control levels, respectively. Analysis of variance revealed that, in response to cycloheximide. theophylline and varying incubation times, the overall rate of protein secretion was significantly (P < 0.05) decreased. Influence of increasing the concentration of insulin in media containing dbcAMP or cycloheximide on protein released is shown in Fig. 2. Insulin alone or in media with cycloheximide stimulated protein release to about 130% of control values at optimum insulin concentrations. In the presence of dbcAMP

Fig. 1. Secretion of protein by bovine mammary acini in response to addition of theophylline and cycloheximide to culture medium with varying incubation times. Each line was fitted by regression analysis based on triplicate determinations for each data point. stimulation was much more pronounced, amounting to 180% of control levels. Although control (no dbcAMP or cycloheximide) acini responded to insulin 15% more than groups treated with cycloheximide, the difference between control and dbcAMP or cycloheximide groups was not significani (P > 0.05). Maximum response in all treatments was at 60-120 PU insulin/ml (Fig. 2). Secretion of proteins from all treatment groups exhibited quadratic response with increasing amounts of insulin up to 360 pU/ml. Combined analysis of variance indicated that effects of dbcAMP, cycloheximide and insulin on protein secretion were significant (P < 0.05). To determine optimum levels of theophylline and dbcAMP for insulin responsiveness, a dose-response study was conducted (Table 1). In the absence of insuthis

ximida (0.5

INSULIN

Fig. 2. Effect of varying concentrations

I 30

mg/ml)

(p.lJ/mM)

of insulin, dbcAMP and cycloheximide on secretion of protein by bovine mammary acini during 90min incubations. Curves were derived from quadratic equations with each data point representing the mean of four determinations,

891

Protein secretion and amino acid uptake Table 1. Effect of insulin, theophylline and dbcAMP on secretion of proteins by bovine mammary gland acini” Added insulin (200 @/ml) Theophylline (mM) 0 IO 5

No insulin dcbAMP (mM)

0

5

0

1c!O.Od 118.4 f 21.4 138.6 + 25.9

76.1 k 9.6 84.3 + 10.6 98.2 f 13.1

0.1

1.0 Insulin effect’ CV’ W

(%’ + SEM’) 100.0’ 122.8 + 19.6 143.0 + 20.8 127.3

63.1 + 6.9 69.1 & 7.7 74.5 + 8.6

21.3 3

10

70.2 + 8.1 63.6 + 5.1 77.7 + 10.3 69.5 k 9.9 91.4 f 13.7 80.2 + 11.1 128.2 114.9 18.8 3

Insulin effect

121.4 123.4 126.0 [123.6]*

” Acini were incubated with varing concentrations of theophylline and dbcAMP in the absence and presence of insulin for 90min. Abbreviations: SEM = Standard error of the mean; CV = Coefficient of variation; N = Number of observations/mean. bConcentration (2OO~U/ml) based on a dose responseastudy (Fig. 2). At this concentration, maximum milk protein secretion occurred. c.dPer cent of mean control value for which specific activity (CPM/mg protein) was 738. c*ePer cent of mean control value for which specific activity (CPM/mg protein) was 916. f Per cent increase in secretion of proteins due to addition of insulin at each level of dbcAMP (in column) and theophylline (in row). gOvera increase due to insulin.

lin, depressed secretion of proteins by theophylline concentrations of 5 and 1OmM were significant (P < 0.05) at each dbcAMP concentration. The greatest inhibition was recorded when the treatment included 1OmM theophylline and no dbcAMP. In contrast, dbcAMP had a stimulating effect (P < 0.05), independent of theophylline concentration, with maximum protein secretion occurring at 1 mM dbcAMP. Overall response patterns in the presence of insulin (2OO~U/ml) were similar, except that more protein was released into medium (Table I). Amino acid uptake

Results of studies on uptake of cycloleucine, a nonmetabolizable amino acid, over an incubation period of 220min are given in Table 2. Maximum uptake occurred after preincubation for from 40 to 90 min in both control and cycloheximide treated groups With dbcAMP treated acini, maximum cycloleucine transport occurred after 40 min’ preincubation. Theoretical preincubation time for maximum cycloleucine accumulation was calculated to. between 53 and 58 min under all treatments conditions. Cycloheximide, dbcAMP and preincubation times

significantly (P < 0.05) influenced cycloleucine accumulation. Cycloheximide inhibited uptake by 35% over control levels, whereas dbcAMP treatment resulted in an 18% increase in cycloleucine uptake at 40min preincubation. No difference (P < 0.05) in uptake between 0.1 and 1 mM dbcAMP was observed. Relatively small coefficients of variation (about 1619%) from treatment groups indicated that experimental variances originated primarily from treatment effects (Table 2). Dose response curves with insulin concentrations ranging from 0 to 36O$J/ml are in Fig. 3. Increasing insulin concentrations to about 60 pU/ml markedly stimulated cycloleucine uptake by control acini as well as by acini exposed to dbcAMP or cycloheximide. This insulin stimulation was most pronounced in dbcAMP and control groups. Increasing amounts of insulin produced a cubic response for cycloleucine uptake which was significant (P < 0.05) with all three treatments. DISCUSSION

Secretion of milk proteins and lactose has been shown to occur by exocytotic discharge from tpithe-

Table 2. E&ct of cycloheximide and dbcAMP on uptake of cycloleucine” Incubation time (min)

Cotitrol

Cycloheximide (0.5 pp/ml)

dbcAMP (0.1 mM)

dbcAMP (l.OmM)

(nmols/mg protein)b 0 40 90 150 220 CVb

9.7 55.4 46.1 35.9 36.7

* + + f f

0.7 6.4 2.5 2.8 2.9

16.9

9.7 f 30.1 f 33.7 f 26.4 f 22.0 f

0.5 2.9 3.0 2.4 1.8

16.4

10.2 * 0.4 65.2 + 7.7 48.8 f 6.3 45.3 _+ 7.3 40.0 f 3.2 17.3

9.8 f 64.3 * 53.8 f 46.8 f 40.5 f

1.8 3.9 7.0 9.2 3.1

18.8

a Bovine mammary acini were preincubated with cycloheximide and dbcAMP for the indicated time prior to addition of a mixture with [‘4C]cycloleucine and 0.5 mM cycloleucine. Uptake was then measured over 15min (See Methods). b Each value represent mean (n = 3) f SEM, CV = coefficient of variation.

892

C. S. PARK. J.

J.

SMITH.

A 0 o

W. N.

EIGEL and

T. W. KEENAN

dbcAMP (I mM) Control Cycloheximide (0.5 mg/ml)

INSULIN

(+/ml)

Fig. 3. Dose response curves for insulin stimulation of cycloleucine uptake. Bovine mammary acini were preincubated with varying concentrations of insulin, dbcAMP and cycloheximide for 90 min prior to addition of cycloleucine. Subsequent incubation was for 15 min. Uptake was measured as described in the text. All curves were fitted by cubic equations with n = 3.

dial cells (for review see Keenan et al., 1978). Introduction of theophylline, an inhibitor of CAMP phosphodiesterase, to media containing bovine mammary acini resulted in inhibition of milk protein secretion (Table 1, Fig. 1). Loizzi et al. (1975) found theophylline at 1OmM to inhibit lactose synthesis. They suggested that this was due to an increase in CAMP levels as a result of phosphodiesterase inhibition; CAMP could then interact with microtubules producing a decline in lactose release (for a review of the functional relationships between CAMP and microtubules, see Rasmussen, 1970). In the present study inhibitory effects of theophylline on protein secretion were modest, amounting 20-30% inhibition at 10 mM. Previous studies have shown that addition of CAMP to acini prepared from bovine mammary tissue (Park et al., 1979) and to mammary tissue slices from rabbit and ewe (Ollivier-Bousquet & Denamur, 1975) result in accelerated intracellular transit and release of milk proteins. These results would tend to suggest that the observed inhibitory effects of theophylline on milk protein secretion occur through mechanisms other than increased levels of intracellular CAMP. Cycloheximide has previously been demonstrated to inhibit protein synthesis by bovine mammary acini (Park et al., 1979) and protein synthesis in diced mouse mammary tissue (Lobitz & Neville, 1971). Our studies on transport of cycloleucine indicated that inhibition occurred through decreased amounts of amino acids available to mammary epithelial cells (Fig. 3). Similar results have been obtained with other tissues (Elsas et al. 1968; Le Cam & Freychet, 1976; Lobitz & Neville, 1977; Rillema, 1976; Tews et al., 1970). Further, inhibition by cyclohexmide was maintained in the presence of insulin. Cycloheximide has been reported to inhibit insulin stimulation of amino acid uptake in diaphragm (Elsas et al., 1968). Stimulatory effects of insulin on protein secretion through interaction with DNA and RNA synthesis

in mammary epithelial cells in vitro has been reviewed (Forsyth, 1971; see also Tucker, 1974). We observed a conspicuous increase in protein secretion from acini incubated with insulin (Fig. 2). While the reason for this observation is not readily apparent, it is possible that insulin and dbcAMP could have synergistic effects on protein secretion by promoting amino acid uptake and/or transport. Tews et al. (1970) reported that insulin stimulated protein synthesis in rat liver slices by increasing uptake of amino acids and this same effect was also produced by addition of CAMP (cf. also (Elsas et al., 1968, Friedberg et al., 1970; Oka et al., 1974; Tews et al.. 1970)). With rat liver, tissue content of CAMP was increased in the presence of glucagon (Jost et al., 1970). In the present study we observed no difference in secretion between control and cycloheximide treatments in the presence of insulin, possibly suggesting that inhibitory effects of cycloheximide were counteracted by insulin. Recently there have been several studies on uptake of the nonmetabolizable amino acid analogs a-aminoisobutyric acid and cycloleucine (Le Cam & Fraychet, 1976, 1977). We observed that increasing preincubation times beyond 90min did not improve cycloleucine uptake (Table 2). In fact, theoretical maximum uptake occurred at preincubation times between 53 and 58 min. However, Lobitz & Neville (1977) observed that a-aminoisobutyric acid uptake remained constant for 6 hr in diced mouse mammary tissue. A linear increase in a-aminoisobutyric acid uptake occurred over periods of 120 min (Le Cam & Freychet, 1976) and 180 min (Le. Cam & Freychet, 1978) in rat hepatocytes. The contrast between our results with cycloleucine and those above for a-aminoisobutyrate may represent species or culture differences as opposed to increased availability of carrier proteins or involvement of repression-derepression mechanisms (Le Cam & Freychet, 1976). Rapid uptake of cycloleucine by acini may also reflect higher

893

Protein secretion and amino acid uptake metabolic activity and greater retention of cell polarity than with other systems (Katz et al., 1974). Our observation on cycloheximide inhibition of cycloleutine uptake is supported by investigations with other systems (Chambrut, 1969; Le Cam & Freychet, 1976; Lobitz & Neville, 1977; Tews et al., 1970). Lobitz & Neville (1977) observed that insulin stimulated amino acid uptake in mouse mammary tissue. These workers, as well as others, observed that insulin neither influenced synthesis of transport proteins (Reynolds et al., 1974; Riggs & Pan, 1972) nor prevented degradation of amino acid carrier proteins (Guidotti, 1974). Lobitz & Neville (1977) indicated that uptake and transport were controlled by negative feedback regulation from intracellular amino acid pools as has also been suggested for other tissues (Franchi-Gazzola et a[., 1973). Our’ studies on trans-’ port of cycloleucine indicated that cycloheximide decreased uptake of this amino acid; similar results have been obtained with other tissues (Elsas, 1968; Le Cam & Freychet, 1976; Lobitz & Neville, 1977; Riggs & Pan, 1972; Tews et al., 1970). Further, inhibition by cycloheximide was maintained in the presence of insulin. Cycloleucine uptake by insulin stimulated bovine mammary acini was enhanced by dbcAMP (Fig. 3). Our results indicate that uptake of cycloleucine by bovine acini was increased by insulin and dbcAMP, either singly or in combination. It is possible that these agents influence amino acid transport by lactating bovine mammary gland in vivo. Acknowledgements-This research was supported by mants PCM75-11908 and PCM77-27144 from the National Science Foundation and by grant GM 23889 from the National Institute of General Medical Science. Purdue University Agricultural Experiment Station Journal Paper No. 7565. REFERENCES BAR H. P. (1973) Epinephrine- and postaglandin-sensitive adenyl cyclase in rat mammary gland. Biochem. biophys. Acta

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