Molecular forms of α-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of α-melanocyte-stimulating hormone (α-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of α-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-α-MSH, while monoacetyl-α-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-α-MSH was the major form of α-MSH. The profile of α-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-α-MSH and diacetyl-α-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of α-MSH predominate in PI tissue, while nonacetylated α-MSH is the major form in AL tissue. It appears, however, that acetylation of α-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.     

Immunoreactive dynorphin-A in bovine anterior pituitary and its in vitro release

The anterior lobe (AL) of the bovine pituitary contained and released, during in vitro culture, a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that occurring in neural tissue. Bovine AL tissue from intact females contained less ir-DYN-A than did AL tissue from castrated males. Enzymatically dispersed AL cells contained and released ir-DYN-A in vitro. Preincubation of dispersed AL cells for 18 hr, rather than 1.5 hr, increased the content and release of ir-DYN-A as well as LH. Addition of gonadotropin-releasing hormone (GnRH) to tissue slices or dispersed cells stimulated release of LH, but in contrast to published observations from rat AL, GnRH had no effect on release of ir-DYN-A. Addition of estradiol-17 beta, with or without progesterone, increased release of ir-DYN-A but not LH during 2-hr cultures. In summary, bovine AL contains and releases in vitro a large molecular weight form of ir-DYN-A. Although this ir-DYN-A was not coreleased with LH, a reproductive role was suggested by in vivo and in vitro effects of gonadal hormones on ir-DYN-A in the bovine anterior pituitary.     

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.     

Thyroidal ANF: a possible mediator of autocrine regulation in the porcine thyroid gland.

Atrial natriuretic factor immunoreactivity (ir-ANF) was examined in thyroid tissue sections and cultured thyroid cells using immunohistochemical staining with a monoclonal anti-ANF antibody. Localization of ir-ANF in perinuclear granules in cultured cells and in the basal region of follicular cells in sectioned tissue suggests that ir-ANF is a basally secreted product. Thyroidal ir-ANF was also characterized using reversed phase high performance liquid chromatography (RP-HPLC) of acidic thyroid extracts. An ir-ANF peak coeluting with synthetic rat ANF(99-126) suggests that thyroidal ir-ANF may be identical in form to circulating atrial ANF. However, the detection of ir-ANF in cultured thyroid cells confirms that the immunoreactivity is locally produced. Saturation analysis revealed high affinity ANF receptors (Kd = 0.1 nM; MBC = 17.2 fmol/mg protein) on these cultured cells, and a competition study demonstrated the ability of extracted thyroidal ir-ANF to inhibit 125I rat ANF binding to the membrane receptors. The evidence presented here suggests that ir-ANF in the thyroid may be secreted locally to exert an autocrine effect on neighboring follicular cells.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.     

Urokinase-type and tissue-type plasminogen activators have different distributions in cultured bovine capillary endothelial cells

The cell extracts and conditioned medium from cultured bovine capillary endothelial (BCE) cells were examined to determine the types of plasminogen activator (PA) present in each of these two fractions. The fractions were first analyzed by fibrin autography after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The cell extracts contained two species of PA of Mr 48,000 and 28,000. Multiple forms of PA were detected in the conditioned medium: variable amounts of the Mr 48,000 and 28,000 forms and a broad band of activity with Mr in the range of 67,000-93,000. The major fraction of the Mr 48,000 form was in the cell extract. Treatment of the cells with 12-0-tetradecanoyl phorbol-13-acetate or with a preparation containing angiogenic activity resulted in a proportionate increase in the levels of all forms. The Mr 48,000 form was demonstrated to be a urokinase-like PA, since it was immunoprecipitated with antibodies to urokinase. When conditioned medium or cell extracts from biosynthetically labelled BCE cells were incubated with antiserum to urokinase, the Mr 48,000 form was immunoprecipitated only from the cell extract. The Mr 67,000-93,000 forms were demonstrated to be tissue-type PAs, since they were immunoprecipitated with antibodies to tissue PA. When the same conditioned medium or cell extracts were incubated with antiserum to tissue-type PA, the Mr 67,000-93,000 forms were immunoprecipitated only from the conditioned medium. Therefore, BCE cells are able to produce both tissue-type PA, which is primarily secreted, and urokinase-type PA, which remains primarily cell associated.     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs)

We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and alpha-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1-39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18-39))-like immunoactivity. Acetylated alpha-MSHs were not detectable at Day 50. alpha-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl alpha-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1-39) levels were negligible when compared to increasing alpha-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of alpha-N- and alpha-N,O-acetylated forms of alpha-MSHs were detected during middle and late stage fetal development. Diacetyl alpha-MSH was the predominant form of alpha-MSH in adult NIL extracts. No acetylated alpha-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1-39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and alpha-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.     

Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: role of Rathke's pouch

The ontogenesis of immunoreactive (ir) ACTH cells and ir alpha-MSH cells in rat hypothalamus was studied in vivo and in vitro. Ir ACTH cells first appeared in the neuroepithelial cell layer lining the floor of the third ventricle on Day 13.5 of gestation, whereas ir alpha-MSH first appeared in the cytoplasm of several ir ACTH cells in the basal part of the arcuate nucleus of the hypothalamus on Day 19.5. When the medial-basal hypothalamus of 12.5-day embryos was cultured alone, a few ir ACTH cells were found after culture for 10 days, but not 3 days, and no ir alpha-MSH cells were observed in the cultures. When the hypothalamus was cultured with Rathke's pouch (intact or without the intermediate lobe anlage), ir ACTH cells appeared within 3 days. In these cultures on Days 6 and 10, long beaded fibers were seen projecting from cells in the neuronal tissue, and some cells showed immunolabeling for alpha-MSH. When the hypothalamus was cocultured with oral epithelium instead of Rathke's pouch, the appearance of neuronal ir ACTH cells was like that in cultures of hypothalamus alone. These in vitro findings suggest that stimulus from the anterior lobe anlage of the pituitary is necessary for normal development of ir ACTH/alpha-MSH cells in the hypothalamus.     

Immunological studies of canine ovarian antigens

Studies were conducted to determine the feasibility of producing canine ovary antiserum in the rabbit and to characterize the antigenic composition of the canine ovary. Ovaries from dogs were obtained and corpora lutea (CL) macroscopically removed. Following homogenization of ovaries, adult male rabbits were immunized by injecting the ovarian preparation. Unabsorbed antiserum cross-reacted with canine ovarian extract and with other reproductive and non-reproductive organ extracts to form precipitin bands. Species cross-reactivity was also observed by testing the unabsorbed antiserum with extracts from organs of the cat and rat. Absorption of antiserum with canine liver and lung extracts removed antibodies not specific to the canine ovary. One band was observed when such absorbed antiserum was allowed to react with the canine ovarian extract. The absorbed antiserum produced no bands with extracts from other canine reproductive and non-reproductive organs tested. These experiments suggested that the canine ovary contained at least 1 organ-specific antigen. This organ-specific antigen was located in the isolated canine ovarian cells by the immunofluorescence technique.     

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.     

An investigation of N-terminal pro-opiocortin peptides in the rat pituitary

Extracts of neurointermediate lobe (NIL) and anterior lobe (AL) of the rat pituitary, and material released from perfused rat pars distalis (PD) and pars intermedia (PI) cells were gel chromatographed and monitored using three antisera, each recognizing different regions of the non-corticotropin (ACTH)-lipotropin (LPH) portion of pro-opiocortin (POC). Two peaks (termed N-POC I) which emerged close to the elution position of rat beta-LPH were detected. The first peak was reduced significantly in the PI. Two smaller N-POC fragments which eluted near beta-endorphin were detected only in extracts and secretions of intermediate lobe tissue. One peak cross-reacted in the gamma 3-melanotropin (MSH) assay (N-POC III) whereas the other peak possessed amino (N)-terminal N-POC immunoreactivity (N-POC II). The results demonstrated differences in the distribution and nature of N-POC peptides released and extracted from the PD and PI of the rat pituitary, and suggest that the enzymic processing of N-POC is different in the two pituitary lobes.     

Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

Characterization of alpha-melanocyte-stimulating hormone in rat pancreas

Reverse-phase high performance liquid chromatography and radioimmunoassay were used to characterize alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in rat pancreas. Relative to synthetic alpha-MSH standards, serial dilutions of pancreas extracts showed parallel and concentration dependent displacement of (125I) alpha-MSH from alpha-MSH antibody. Chromatographic separation revealed immunoreactive material coeluting with synthetic N,O-diacetyl alpha-MSH, which accounted for 78% of total alpha-MSH materials in this tissue. The remainder of immunoreactive alpha-MSH coeluted with synthetic alpha-MSH, desacetyl alpha-MSH, or their methionine sulfoxides. In contrast with anterior pituitary, it appears that biosynthetic processing of alpha-MSH from pro-opiomelanocortin (POMC) may be similar in rat pancreas and pituitary intermediate lobe, since their relative alpha-MSH immunoreactive elution profiles were similar. These findings support the hypothesis of tissue specific regulation of biosynthetic processing of POMC.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

Changes in hypothalamic and pituitary content of immunoreactive alpha-melanocyte-stimulating hormone during the gestational and postpartum periods in the rat

alpha-Melanocyte-stimulating hormone (alpha-MSH) was measured in the mediobasal hypothalamus (MH), median eminence (ME), preoptic-suprachiasmatic area (POA-SCN), anterior (AL), and posterior lobes (PL) of the pituitary gland during the gestational and postpartum periods in the rat. The content of alpha-MSH in the MH and POA-SCN compared to estrous levels was lower during the later days of gestation and decreased further in the MH during lactation in association with the elevated plasma prolactin (Prl). Distinct increases in the ME content of alpha-MSH compared to estrous levels occurred on Days 8 and 12 of the gestational period and Day 14 of the postpartum period. A significant increase in PL content of alpha-MSH compared to Days 5-11 and 17-20 occurred on Day 4 of gestation, and no significant changes were detected in the AP concentration of alpha-MSH throughout the period studied. In vitro, PLs and ALs from females on Day 4 of gestation secreted more alpha-MSH into the incubation medium than tissues from animals on Day 20. These results suggest that alpha-MSH of both brain and pituitary origin may play a role in mediating some of the physiological changes which occur during pregnancy and lactation.     

Immunoreactive dynorphin-A in ovine anterior pituitary and effects of estradiol-17 beta administration

Anterior lobe (AL) tissue of the ovine pituitary gland contained a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that found in pituitary neurointermediate lobe. Administration of estradiol-17 beta or estradiol-17 beta plus progesterone to ovariectomized sheep decreased AL tissue concentrations of ir-DYN-A but did not affect any LH parameter. Enzymatically dispersed AL cells also contained ir-DYN-A, but specific release during in vitro incubation was too low to be detected even when cells were exposed to gonadotropin-releasing hormone.     

Plasma alpha-melanocyte-stimulating hormone during the menstrual cycle in women

alpha-Melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) immunoreactivity (IR) was measured in the blood of 22 healthy women with normal ovulatory process in the early and late follicular (near to ovulation) phases and in the early luteal phase of the menstrual cycle. Plasma alpha-MSH IR ranged from undetectable values to 81.3 pg/ml, the highest levels being found in the late follicular phase (15.52 +/- 4.16 pg/ml). In contrast, plasma ACTH IR was always detectable (range: 18.5-63.2 pg/ml), but its concentration did not differ significantly between the 3 phases of the menstrual cycle. High-pressure liquid chromatography fractionation of Sep pak C18-purified alpha-MSH IR revealed in all 3 phases the presence of 3 major peaks of alpha-MSH IR, coeluting with desacetyl-alpha-MSH, alpha-MSH and diacetyl-alpha-MSH, respectively. The most abundant peak always coeluted with authentic desacetyl-alpha-MSH, and the ratio between this deacetylated and the other 2 acetylated forms was similar in the 2 follicular phases (1:1.25 and 1:1.16 in the early and late phase, respectively), but significantly different in the luteal phase (1:0.48). The fluctuations in plasma concentration of the above MSH-related peptides suggest that different rates of alpha-MSH acetylation and release take place in the pituitary gland depending on the phase of the menstrual cycle.     

Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture.

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradation of the ECM during the different stages of mammary development. Mammary tissue extracts from virgin and pregnant CD-1 mice resolved by zymography contained three major proteinases of 60K (K = 10(3) Mr), 68K and 70K that degraded denatured collagen. These three gelatinases were completely inhibited by the tissue inhibitor of metalloproteinases. Proteolytic activity was lowest during lactation especially for the 60K gelatinase which was shown to be the activated form of the 68K gelatinase. The activated 60K form decreased prior to parturition but increased markedly after the first two days of involution. An additional gelatin-degrading proteinase of 130K was expressed during the first three days of involution and differed from the other gelatinases by its lack of inhibition by the tissue inhibitor of metalloproteinases. The activity of the casein-degrading proteinases was lowest during lactation. Three caseinolytic activities were detected in mammary tissue extracts. A novel 26K cell-associated caseinase--a serine arginine-esterase--was modulated at different stages of mammary development. The other caseinases, at 92K and a larger than 100K, were not developmentally regulated. To find out which cell type produced the proteinases in the mammary gland, we isolated and cultured mouse mammary epithelial cells. Cells cultured on different substrata produced the full spectrum of gelatinases and caseinases seen in the whole gland thus implicating the epithelial cells as a major source of these enzymes. Analysis of proteinases secreted by cells grown on a reconstituted basement membrane showed that gelatinases were secreted preferentially in the direction of the basement membrane. The temporal pattern of expression of these proteinases and the basal secretion of gelatinases by epithelial cells suggest their involvement in the remodelling of the extracellular matrix during the different stages of mammary development and thus modulation of mammary cell function.     

A new in vitro model for studies of pancreatic polypeptide secretion and biochemistry

A new model tissue (pseudoislet) is described for studies of pancreatic polypeptide (PP) secretion and biochemistry. It consists of islet-like aggregates of canine pancreatic endocrine cells which are formed and maintained on tissue culture. Immunocytochemical staining revealed that pseudoislets prepared from the duodenal end of the pancreas contained a predominance (40-60%) of F cells (the PP secreting cell). Also present were 10-25% exocrine cells and an equal proportion of A, B and D cells. Several studies were conducted to characterize the pseudoislets' capacity to secrete PP. Basal rates of PP release and the concentration of PP per pseudoislet remained constant during four weeks of culture. Stimulation at weekly intervals by carbachol (0.1 mM) resulted in a stable secretory rate for 2 weeks, that declined progressively at weeks 3 and 4. When studied in a perfusion system, carbachol-stimulated PP release occurred in a biphasic pattern, similar to the well-recognized biphasic release of insulin from perifused rat islets. Dose-response curves of four cholinergic agonists revealed clear differences in secretagogue activity. Acetylcholine and methacholine were found to be equipotent, followed in order of potency by carbachol and bethanechol. These histologic and secretory data show that canine pseudoislets are healthy tissues composed of a high proportion of F cells which secrete PP in response to cholinergic stimulation. The data suggest that the cultured canine pseudoislet model provides an excellent system useful in studies of PP secretion and biosynthesis.     

Establishment of transfected cell lines producing testicular angiotensin-converting enzyme. Structural relationship between its secreted and cellular forms.

Angiotensin-converting enzyme (ACE) is present in endothelial and epithelial cells of various tissues as well as in the circulating plasma. The structural relationship between the cellular and the secreted forms of ACE and the pathways to their biosynthesis have not been determined as yet mainly because of the unavailability of a natural cell line expressing ACE in tissue culture. To circumvent this problem we have permanently transfected a mouse epithelial line with an expression vector containing the recently cloned rabbit testicular ACE cDNA. Clonal derivatives of this line secreted large quantities of enzymatically active ACE. When these cells were cultured in serum-free medium, the only detectable protein in the culture medium was ACE. It has been suggested that a hydrophobic domain near the carboxyl terminus of the enzyme anchors it to the plasma membrane. To test this hypothesis we established cell lines expressing a truncated form of the active enzyme which is missing the putative anchoring domain. Pulse-chase experiments showed that the truncated ACE was secreted from the cells much faster than the native enzyme. Moreover, the secreted form of the native enzyme had a lower molecular weight than the corresponding cellular form. These results are consistent with the hypothesis that the hydrophobic domain is instrumental in keeping the enzyme cell-bound, and secretion is achieved physiologically by removal of this domain from the enzyme by a specific proteolytic cleavage.