Processing of Proenkephalin in Adrenal Chromaffin Cells

The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.     

Regulation of Proenkephalin Synthesis in Adrenal Medullary Chromaffin Cells

The synthesis of proenkephalin was assessed in primary cultures of bovine adrenal medullary chromaffin cells by incubation of the cells with [35S]methionine, digestion of proenkephalin-derived peptides with trypsin and carboxy-peptidase B, and quantitation of radioactivity incorporated into Met-enkephalin following reversed-phase HPLC. Nicotine, histamine, and vasoactive intestinal peptide each enhanced the rate of proenkephalin synthesis approximately 10-fold when examined between 16 and 32 h after the drug or hormone addition. Inclusion of nifedipine (1 microM) partially blocked the stimulatory effect of nicotine, but not that of vasoactive intestinal peptide or histamine, or proenkephalin synthesis. Theophylline, tetrabenazine, and angiotensin II also increased the rate of proenkephalin synthesis (three- to eight-fold). These increases in the apparent rate of proenkephalin synthesis were not attributable to altered [35S]methionine specific radioactivity or rates of turnover and did not reflect similar increases in total protein synthesis. The half-life for turnover of Met-enkephalin sequences was 3-4 days in the cultured chromaffin cell. These studies directly show that proenkephalin synthesis is the primary regulatory step in control of chromaffin cell opioid peptide content.     

α1-Antichymotrypsin-Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in Chromaffin Granules and Inhibit the Proenkephalin Processing Enzyme "Prohormone Thiol Protease"

Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.     

The biosynthesis of sulfoquinovosyldiacylglycerol: studies with groundnut (Arachis hypogaea) leaves

The biosynthetic pathway of sulfoquinovosyldiacylglycerol (SQDG) was investigated using groundnut (Arachis hypogaea) leaf discs and 35S-labeled precursors. [35S]SO4(2-) was actively taken up by the leaf discs and rapidly incorporated into SQDG. After 2 h, 1.5% of the [35S]SO4(2-) added to the incubation medium was taken up, of which 28% was incorporated into SQDG. The methanol-water phases of the lipid extracts of the leaf discs were analyzed for the 35S-labeled intermediates. Up to 2 h of incubation, cysteic acid, 3-sulfopyruvate, 3-sulfolactate, 3-sulfolactaldehyde, and sulfoquinovose (SQ) which have been proposed as intermediates [Davies et al. (1966) Biochem. J. 98, 369-373] were not labeled. Only a negligible amount of radioactivity was observed in these compounds after incubation for 4 h and more. Addition of sodium molybdate inhibited the uptake of [35S]SO4(2-) as well as its incorporation into SQDG by the leaf discs, suggesting that 3'-phosphoadenosine-5'-phosphosulfate may be involved in the biosynthesis of SQDG. Addition of unlabeled cysteic acid to the incubation medium enhanced the uptake of [35S]SO4(2-) but did not affect its incorporation into SQDG. 35S-labeled cysteic acid was taken up by the leaf discs and metabolized to sulfoacetic acid but not incorporated into SQ or SQDG. These results show that cysteic acid is not an intermediate in SQDG biosynthesis. [35S]SQ was taken up by the leaf discs and incorporated into SQDG in a time-dependent manner. [35S]Sulfoquinovosylglycerol was also taken up by the leaf discs but not incorporated into SQDG. It is concluded that SQDG is not biosynthesized by the proposed sulfoglycolytic pathway in higher plants. Though [35S]SQ was converted to SQDG, the rates are much lower compared to [35S]SO4(2-) incorporation, which suggests that a more direct pathway involving sulfonation of a lipid precursor may exist in higher plants.     

Insulin-like growth factor I enhances proenkephalin synthesis and dopamine beta-hydroxylase activity in adrenal chromaffin cells

Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin-derived enkephalin-containing peptides and the activity of dopamine beta-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine beta-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was approximately 1 nM, with maximal effects observed at 10-30 nM. In contrast, insulin was 1000-fold less potent. Pretreatment of chromaffin cells with IGF-I increased the rate of [35S]proenkephalin synthesis 4-fold compared to untreated cells. Total protein synthesis increased only 1.5-fold under these conditions. These results suggest that IGF-I may be a normal regulator of chromaffin cell function.     

Characterization of Proenkephalin-Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [35S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.     

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.     

Sulfate incorporation from ascorbate 2-sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses.

Radioactivity was significantly incorporated from ascorbate 2-[35S]sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses. The extent of incorporation was comparable with that from inorganic [35S]sulfate. The radioactive chondroitin sulfate formed from ascorbate 2-[35S]sulfate gave two radioactive disaccharides on chondroitinase-ABC [EC 4.2.2.4] digestion. The incorporation was markedly decreased by inorganic sulfate. The time course of incorporation from ascorbate 2-[35S]sulfate and inorganic [35S]sulfate into chondroitin sulfate and the constituent disaccharides suggest that the incorporation rates from the two radioactive substances are different.     

An efficient and prolonged in vitro translational system from isolated cucumber etioplasts

Etioplasts were isolated from dark grown cucumber cotyledons pretreated with kinetin and gibberellic acid. When incubated in a cofactor enriched medium these etioplasts incorporated [35S] methionine into a hot trichloroacetic acid-insoluble fraction; this incorporation was linear for 8 h of incubation and was inhibited by chloramphenicol but not by cycloheximide. Over the same time period, the etioplasts showed continued linear synthesis of the chlorophyll precursors protochlorophyllide, Mg-protoporphyrin and protoporphyrin IX. Analysis of products of in vitro protein synthesis by etioplasts and cotyledons showed the thylakoid membrane polypeptide profiles to be identical. Continued incorporation of [35S] methionine into the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) for 8 h has been confirmed further by immunoprecipitation with anti-spinach RuBisCO. This competent in vitro translation system should be useful for future studies of chloroplast protein synthesis and gene expression.     

Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

Permeabilized cells treated with the adenosine triphosphate analog, [35S]adenosine-5'-0-(3-thiotriphosphate) ([gamma-35S]ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35S from the cells over a 20 min period. Treatment of permeabilized cells with ATP gamma S inhibited secretion and 35S incorporation into the cells. Pretreatment with ATP gamma S resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35S from [gamma-35S]ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells.     

Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.     

The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.     

Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan

The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein, much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000-100,000; pI 4.3-5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.     

The effect of Ca2+ ionophores upon the synthesis of proteins in cultured skeletal muscle

The influence of the Ca2+ ionophores, ionomycin and A23187 upon the incorporation of [35S]methionine into proteins of cultured chicken pectoralis muscle was studied during differentiation of myoblasts into multinucleated myotubes. Fusion was reversibly arrested by growing cells in low-calcium media from the time of plating. Exposure of normal and fusion blocked cultures to 10-6-10-5 M ionomycin or A23187 for 2-6 h on the second to fourth day of growth, resulted in a selective increase in the incorporation of [35S]methionine into two proteins of about 100 000 and 80 000 dalton. When 10-5 M ionomycin or A23187 were added to older cultures, all large myotubes contracted and detached from the plate. Only the adhering myoblasts and small myotubes incorporated [35s[methionine into the muscle proteins and showed increased incorporation of label into 100 000 and 80 000 proteins. After ionophore pulse, the adhering cells retained the ability to differentiate and accumulate myosin. The effect of Ca2+ ionophores upon the rate of protein synthesis is presumably related to increased influx of extracellular Ca2+ with a rise in the Ca2+ concentration of the cytoplasm. We conclude that Ca2+ sensitive mechanisms may regulate the synthesis of a select group of muscle proteins.     

Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx

Vascular endothelial cells are shielded from direct exposure to flowing blood by the endothelial glycocalyx, a highly hydrated mesh of glycoproteins, sulfated proteoglycans, and associated glycosaminoglycans (GAGs). Recent data indicate that the incorporation of the unsulfated GAG hyaluronan into the endothelial glycocalyx is essential to maintain its permeability barrier properties, and we hypothesized that fluid shear stress is an important stimulus for endothelial hyaluronan synthesis. To evaluate the effect of shear stress on glycocalyx synthesis and the shedding of its GAGs into the supernatant, cultured human umbilical vein endothelial cells (i.e., the stable cell line EC-RF24) were exposed to 10 dyn/cm2 nonpulsatile shear stress for 24 h, and the incorporation of [3H]glucosamine and Na2[35S]O4 into GAGs was determined. Furthermore, the amount of hyaluronan in the glycocalyx and in the supernatant was determined by ELISA. Shear stress did not affect the incorporation of 35S but significantly increased the amount of glucosamine-containing GAGs incorporated in the endothelial glycocalyx [168 (SD 17)% of static levels, P < 0.01] and shedded into the supernatant [231 (SD 41)% of static levels, P < 0.01]. Correspondingly with this finding, shear stress increased the amount of hyaluronan in the glycocalyx [from 26 (SD 24) x 10(-4) to 46 (SD 29) x 10(-4) ng/cell, static vs. shear stress, P < 0.05] and in the supernatant [from 28 (SD 11) x 10(-4) to 55 (SD 16) x 10(-4) ng x cell(-1) x h(-1), static vs. shear stress, P < 0.05]. The increase in the amount of hyaluronan incorporated in the glycocalyx was confirmed by a threefold higher level of hyaluronan binding protein within the glycocalyx of shear stress-stimulated endothelial cells. In conclusion, fluid shear stress stimulates incorporation of hyaluronan in the glycocalyx, which may contribute to its vasculoprotective effects against proinflammatory and pro-atherosclerotic stimuli.     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid and selective effects of thyrotropin on [35S]methionine incorporation into cytoplasmic and nuclear proteins in calf thyroid tissue slices

Previous studies have indicated that thyrotropin may induce general increases in RNA and protein synthesis in calf thyroid tissue slices. In this report, we show that thyrotropin selectively stimulates [35S]methionine incorporation into small numbers of specific cytoplasmic and nuclear proteins. We provide data on the time course of stimulation and on relative molecular masses and isoelectric points of hormone-response proteins. Calf thyroid tissue slices were incubated for 3 h, 6 h or 9 h in the presence or absence of thyrotropin (50 mU/ml); [35S]methionine (50-75 microCi/ml) was added for the final 3 h of incubation. Cytoplasmic and nuclear fractions were then prepared, and analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Thyrotropin increased [35S]methionine incorporation into two cytoplasmic and four nuclear proteins within 3 h; hormonal effects on the labeling of five of these six proteins were transient, and no longer evident after 6-9 h. In contrast, a second group of two cytoplasmic and four nuclear proteins exhibited increased labeling after a delay of 6-9 h. Our results suggest that thyrotropin selectively stimulates the synthesis of specific cytoplasmic and nuclear proteins in calf thyroid tissue slices, and that stimulation involves at least two mechanisms (one rapid, the other delayed).     

Effect of endothelial-cell-conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.