Comp. Biochem. Physiol. Vol. 102B, No. 4, pp. 701-706, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992 Pergamon Press Ltd
CONSTITUTIVE PROTEIN SECRETION BY GUINEA-PIG SEMINAL VESICLE EPITHELIAL CELLS D. A. HUDSON Cell and Developmental Biology Research Unit, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. Tel: (644) 721-000; Fax: (644) 712-070 (Received 20 January 1992) Abstract--1. Secretion of pulse-labelled protein by the isolated epithelium of guinea-pig seminal vesicle epithelium was rapid, unaffected by cholinergic and adrenergic drugs, cyclic nucleotides or changes in the sodium, potassium and calcium concentrations of the "chase" medium. 2. Low temperature, NH4C1, hyper- and hypo-osmolarity and membrane-stabilizing agents inhibited secretion which was also dependent on aerobic metabolism. 3. Monensin reduced secretion of the six labelled, relatively low molecular weight proteins recovered from the medium in a concentration-dependent, apparently non-specific manner.
INTRODUCTION Seminal vesicles synthesize a n d secrete a variety of different molecules ( M a n n a n d L u t w a k - M a n n , 1981) including tissue-specific proteins (Aumuller a n d Seitz, 1990). F o u r secretory proteins have been isolated from the guinea-pig seminal vesicles, a n d considerable progress has been m a d e in determining the m e c h a n i s m o f p r o d u c t i o n of these by the a n d r o g e n - d e p e n d e n t epithelial cells (Veneziale et al., 1977; N o r v i t c h et al., 1989). However, there is virtually no i n f o r m a t i o n o n the process o f secretion o f these proteins per se. The object o f the present study, therefore, was to survey some of the characteristics o f secretion in vitro employing "pulse-chase" experiments with the isolated epithelium of guinea-pig seminal vesicles. MATERIALS AND METHODS Animals Either Dunkin-Hartley or various outbred strains of adult, non-breeding male guinea-pigs were used. They were maintained under standard conditions, allowed free access to food (adequate in vitamin C) and water and killed by intraperitoneal injection of sodium pentobarbitone. Isolation of the seminal vesicle epithelium Seminal vesicles were removed from the anaesthetized animal and placed in pre-cooled 0.9% NaC1. Following removal of blood vessels and adipose tissue, the vesicles were opened along their length and the bulk of the contained secretion carefully removed by means of a spatula. The epithelium was scraped from the underlying muscle with a scalpel blade, placed in pre-warmed 0.9% NaCI and vigorously shaken to remove secretory material attached to the tissue. Pieces of epithelium of roughly equal weight (approx. 30 rag) were suspended in pre-warmed Krebs-Ringer bicarbonate buffer (pH7.3) containing glucose (10raM) and incubated for about 20 min with occasional shaking and one change of the medium. Usually, this treatment was sufficient to remove remaining secretory material and damaged cells from the epithelium. Protein labelling For the pulse-chase experiments, washed pieces of tissue were routinely incubated for 20min at 37°C in 701
pre-warmed Krebs-Ringer bicarbonate buffer equilibrated with 95%O2:5%CO 2. The buffer contained L-[4,53H]leucine (0.5-1.0/1Ci/ml) although in a few experiments L-[U-'4C]lysine (0.1-0.2/~Ci/ml) was used to label the proteins. At the end of the incubation period the epithelium was rapidly washed in 2 or 3 changes of large volumes of pre-warmed Krebs-Ringer buffer, blotted on a moist paper towel and dispensed into 4 ml of fresh buffer containing l mM non-radioactive leucine (or lysine) to which other additions were made, as indicated in the text. The discharge period was usually 60rain at 37°C under aerobic conditions with constant shaking of the medium. At the end of the discharge period the tissues were removed, washed in approx. I0 ml of fresh buffer, again blotted on a moistened paper towel and weighed. Recovery of labelled protein Collection of the labelled protein involved homogenizing the tissue pieces in cold saline with a motor-driven homogenizer. Ice-cold trichloracetic acid (TCA) was then added both to the homogenates and to the media to a final concentration of 10% and kept in an ice-water mixture overnight. A zero-minute control incubation was performed for each discharge experiment. The protein precipitates were collected by centrifugation at 1000g for 10min and washed twice in 5% TCA. The final sediment was usually washed with ethanol/ether (l:l,v/v) or acetone and dissolved in 0.5 M NaOH at 50°C. The contained radioactivity was measured in a Beckman LS3801 liquid scintillation counter after neutralization with HCI and addition of Triton X-100/toluene-based scintillation fluid. Where appropriate, incubations were carried out to check on the possible effects of solvents (ethanol and dimethylsulphoxide, DMSO) used for some of the chemicals tested (monensin, cytochalasin B, A23187, etc). Typically, pieces of epithelium pooled from two animals were used in each experiment. Every experimental variable was tested in at least two experiments so that each result would be based on measurements from randomly dispersed tissue from at least four different animals. Secretion was calculated as the amount of labeUed protein in the incubation medium as a percentage of labelled proteins in both the tissue and medium. Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard.
702
D. A. HUDSON
Polyacrylamide gel electrophoresis and fluorography Incubation media were either dialyzed against distilled water or desalted on Sephadex GI0 (Pharmacia, Uppsala, Sweden), and lyophilized. Samples of the secretion proper were extracted with 0.9% NaC1, centrifuged at 1000 g for 10 rain and then treated in the same manner. Phenylmethylsulfonylfluoride (0.1 mM), iodoacetate (0.1 mM) or 200 K IU Aprotinin were added to the extraction solutions. SDS/polyacrylamide gel electrophoresis (SDS-PAGE) of the secreted proteins was carried out with slab gels (1.5 mm thick) using 5% acrylamide as described by Fairbanks et al. (1971), stained with Coomassie Blue, destained and dried. Apparent molecular weights were determined electrophoretically using bovine serum and egg albumin, fl-lactoglobulin, lysozyme, trypsinogen and pepsin as standards. The gels were treated for fluorography as described by Skinner and Griswold (1983) and exposed to Fuji RX X-ray film at - 80°C. Statistical significance was based on the Student t-test; values of P
0305-0491/92 $5.00+ 0.00 © 1992 Pergamon Press Ltd
CONSTITUTIVE PROTEIN SECRETION BY GUINEA-PIG SEMINAL VESICLE EPITHELIAL CELLS D. A. HUDSON Cell and Developmental Biology Research Unit, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. Tel: (644) 721-000; Fax: (644) 712-070 (Received 20 January 1992) Abstract--1. Secretion of pulse-labelled protein by the isolated epithelium of guinea-pig seminal vesicle epithelium was rapid, unaffected by cholinergic and adrenergic drugs, cyclic nucleotides or changes in the sodium, potassium and calcium concentrations of the "chase" medium. 2. Low temperature, NH4C1, hyper- and hypo-osmolarity and membrane-stabilizing agents inhibited secretion which was also dependent on aerobic metabolism. 3. Monensin reduced secretion of the six labelled, relatively low molecular weight proteins recovered from the medium in a concentration-dependent, apparently non-specific manner.
INTRODUCTION Seminal vesicles synthesize a n d secrete a variety of different molecules ( M a n n a n d L u t w a k - M a n n , 1981) including tissue-specific proteins (Aumuller a n d Seitz, 1990). F o u r secretory proteins have been isolated from the guinea-pig seminal vesicles, a n d considerable progress has been m a d e in determining the m e c h a n i s m o f p r o d u c t i o n of these by the a n d r o g e n - d e p e n d e n t epithelial cells (Veneziale et al., 1977; N o r v i t c h et al., 1989). However, there is virtually no i n f o r m a t i o n o n the process o f secretion o f these proteins per se. The object o f the present study, therefore, was to survey some of the characteristics o f secretion in vitro employing "pulse-chase" experiments with the isolated epithelium of guinea-pig seminal vesicles. MATERIALS AND METHODS Animals Either Dunkin-Hartley or various outbred strains of adult, non-breeding male guinea-pigs were used. They were maintained under standard conditions, allowed free access to food (adequate in vitamin C) and water and killed by intraperitoneal injection of sodium pentobarbitone. Isolation of the seminal vesicle epithelium Seminal vesicles were removed from the anaesthetized animal and placed in pre-cooled 0.9% NaC1. Following removal of blood vessels and adipose tissue, the vesicles were opened along their length and the bulk of the contained secretion carefully removed by means of a spatula. The epithelium was scraped from the underlying muscle with a scalpel blade, placed in pre-warmed 0.9% NaCI and vigorously shaken to remove secretory material attached to the tissue. Pieces of epithelium of roughly equal weight (approx. 30 rag) were suspended in pre-warmed Krebs-Ringer bicarbonate buffer (pH7.3) containing glucose (10raM) and incubated for about 20 min with occasional shaking and one change of the medium. Usually, this treatment was sufficient to remove remaining secretory material and damaged cells from the epithelium. Protein labelling For the pulse-chase experiments, washed pieces of tissue were routinely incubated for 20min at 37°C in 701
pre-warmed Krebs-Ringer bicarbonate buffer equilibrated with 95%O2:5%CO 2. The buffer contained L-[4,53H]leucine (0.5-1.0/1Ci/ml) although in a few experiments L-[U-'4C]lysine (0.1-0.2/~Ci/ml) was used to label the proteins. At the end of the incubation period the epithelium was rapidly washed in 2 or 3 changes of large volumes of pre-warmed Krebs-Ringer buffer, blotted on a moist paper towel and dispensed into 4 ml of fresh buffer containing l mM non-radioactive leucine (or lysine) to which other additions were made, as indicated in the text. The discharge period was usually 60rain at 37°C under aerobic conditions with constant shaking of the medium. At the end of the discharge period the tissues were removed, washed in approx. I0 ml of fresh buffer, again blotted on a moistened paper towel and weighed. Recovery of labelled protein Collection of the labelled protein involved homogenizing the tissue pieces in cold saline with a motor-driven homogenizer. Ice-cold trichloracetic acid (TCA) was then added both to the homogenates and to the media to a final concentration of 10% and kept in an ice-water mixture overnight. A zero-minute control incubation was performed for each discharge experiment. The protein precipitates were collected by centrifugation at 1000g for 10min and washed twice in 5% TCA. The final sediment was usually washed with ethanol/ether (l:l,v/v) or acetone and dissolved in 0.5 M NaOH at 50°C. The contained radioactivity was measured in a Beckman LS3801 liquid scintillation counter after neutralization with HCI and addition of Triton X-100/toluene-based scintillation fluid. Where appropriate, incubations were carried out to check on the possible effects of solvents (ethanol and dimethylsulphoxide, DMSO) used for some of the chemicals tested (monensin, cytochalasin B, A23187, etc). Typically, pieces of epithelium pooled from two animals were used in each experiment. Every experimental variable was tested in at least two experiments so that each result would be based on measurements from randomly dispersed tissue from at least four different animals. Secretion was calculated as the amount of labeUed protein in the incubation medium as a percentage of labelled proteins in both the tissue and medium. Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard.
702
D. A. HUDSON
Polyacrylamide gel electrophoresis and fluorography Incubation media were either dialyzed against distilled water or desalted on Sephadex GI0 (Pharmacia, Uppsala, Sweden), and lyophilized. Samples of the secretion proper were extracted with 0.9% NaC1, centrifuged at 1000 g for 10 rain and then treated in the same manner. Phenylmethylsulfonylfluoride (0.1 mM), iodoacetate (0.1 mM) or 200 K IU Aprotinin were added to the extraction solutions. SDS/polyacrylamide gel electrophoresis (SDS-PAGE) of the secreted proteins was carried out with slab gels (1.5 mm thick) using 5% acrylamide as described by Fairbanks et al. (1971), stained with Coomassie Blue, destained and dried. Apparent molecular weights were determined electrophoretically using bovine serum and egg albumin, fl-lactoglobulin, lysozyme, trypsinogen and pepsin as standards. The gels were treated for fluorography as described by Skinner and Griswold (1983) and exposed to Fuji RX X-ray film at - 80°C. Statistical significance was based on the Student t-test; values of P
