Regulation Of Protein Secretion Research Articles

The importance of proline on long-term hepatocyte function in a collagen gel sandwich configuration: Regulation of protein secretion

A serum-free culture system for primary hepatocytes which maintains stabel high-level hepatocyte function for prolonged periods in culture has been developed. Isolated rat primary hepatocytes were cultured in serum-free media between two layer of gelled collagen in a sandwich configuration which reinstates the cellular polarity necessary for long-term function in vitro. Theese serum-free hepatocyte cultures maintained near physiological rates of albumin and transferrin secretion for a minimum of 26 days in culture. L-Proline was shown to be critical for both the approach to steady state and maximal level of protein secretion. Analysis of does-response data gave K(m) values of 2.9 and 1.7 microg/mL for albumin and transferrin secretion, respectively.

Use of regulated secretion in protein production from animal cells: an overview.

Traditional industrial cell culture processes require extensive downstream product refining due to low product titer and purity in the spent growth medium. A controlled secretion process incorporating cells derived from endocrine or exocrine organs could potentially alleviate this processing burden by dynamically decoupling product recovery from cell growth and product biosynthesis. In addition, such specialized secretory cells may be uniquely capable of performing desirable post-translational processing of the secretory product. We briefly review the biology of regulated protein secretion as well as the biology and biochemistry of the signal transduction mechanisms by which regulated systems respond to environmental stimuli. Drawing on these and other basic principles from cell biology and bioengineering, we describe the important features of a controlled secretion process. Among other issues we discuss the choice of cell lines, expression systems, cell culture methods, and bioreactor configurations. We extensively analyze the kinetics of regulated secretion in the context of a controlled secretion process. This discussion is illustrated with experimental results from two model cell lines, recombinant AtT-20 and beta TC3, expressing recombinant human endocrine hormones or native murine insulin respectively.

Regulation of ACTH secretory pathways in cultured pituitary cells.

Although chloroquine, an agent that disrupts regulated protein secretion, has previously been shown to decrease the adrenocorticotropic hormone (ACTH) secretory response to adenosine 3',5'-cyclic monophosphate or corticotropin-releasing factor (CRF) in AtT-20 and rat anterior pituitary cells, respectively, it has no effect on the response to vasopressin. The present study extended experiments with chloroquine to cultured sheep anterior pituitary cells, which have a greater maximum response to vasopressin. Chloroquine (200 microM) had no effect on basal ACTH secretion or on stimulation by vasopressin. In contrast to the rat, the net response to CRF was tripled by chloroquine in ovine cells. The effect of chloroquine on the response to CRF was more effective by coexposure of cells to CRF and chloroquine than by pretreatment with chloroquine. Monensin or vinblastine did not increase the ACTH response to CRF. The results indicate ACTH release in response to vasopressin is chloroquine insensitive in this way, can be dissociated from the mechanism that responds to CRF, and would be consistent with the CRF response mechanism involving pathways that can alter the secretory pool of ACTH. When chloroquine acts to increase the response to CRF, it is likely not to act by stabilizing the CRF-receptor complex.

Constitutive and regulated secretion of proteins.

SORTING 257 CYTOSKELETON INVOLVEMENT WITH PROTEIN SECRETION 266 SECRETION IN EPITHELIAL CELLS .... 277 RECYCLING OF THE SECRETORY VESICLE MEMBRANE ..... . . 281

Pathways of Protein Secretion in Eukaryotes

Protein secretion from cells can take several forms. Secretion is constitutive if proteins are secreted as fast as they are synthesized. In regulated secretion newly synthesized proteins destined for secretion are stored at high concentration in secretory vesicles until the cell receives an appropriate stimulus. When both constitutive and regulated protein secretion can take place in the same cell a mechanism must exist for sorting the correct secretory protein into the correct secretory vesicle. The secretory vesicle must then be delivered to the appropriate region of plasma membrane. Transfection of DNA encoding foreign secretory proteins into regulated secretory cells has provided insight into the specificity of sorting into secretory vesicles.

VIP as a neurotransmitter or a neuromodulator in the regulation of protein secretion in rat lacrimal gland

VIP as a neurotransmitter or a neuromodulator in the regulation of protein secretion in rat lacrimal gland

Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density: Plasminogen activator, procollagen and fibronectin

To study the relationship between cell growth control, cell contact, and protein secretion, we examined the production of plasminogen activator, procollagen, and fibronectin by Chinese hamster ovary (CHO) fibroblasts, both as a function of position in the cell cycle and as a function of cell density. CHO fibroblasts that were synchronized at hourly intervals throughout the cell cycle by mitotic selection in an automated roller bottle apparatus secreted plasminogen activator only during the G2 and M phases of the cell cycle (10–14 h after mitotic selection). Cell-associated plasminogen activator activity was variable during G1 and S, but was greatly reduced during G2 and M. In contrast, secretion of the connective tissue matrix proteins, procollagen and fibronectin, was controlled by cell density rather than by cell cycle position. Type III procollagen and fibronectin were secreted throughout the cell cycle with no pronounced variations. Type I procollagen was not secreted by cycling cells and was observed in confluent cultures only after 24–48 h. To correlate these changes in protein secretion patterns with cell shape and contact, we used scanning electron microscopy (SEM) to study the appearance of CHO cells after mitotic selection. Actively dividing cells retained a high proportion of rounded, ruffled, and blebbed cells during all phases of the cell cycle. Only with increased cell density in contact-inhibited confluent cultures did most cells begin to flatten and spread. Thus, secretion of and attachment to extracellular matrix did not occur in rapidly dividing cells, but appeared to require the increased cell-cell contact and spreading that accompanies contact inhibition of growth. On the other hand, increased secretion of plasminogen activator was directly related to cell division and may be part of a sequence of events that allows cells growing in culture to loosen extracellular attachments in preparation for rounding and cytokinesis.

Regulation of protein secretion and metabolism in rat salivary glands. Effects of norepinephrine and carbachol on the glycogenolysis in submaxillary glands.

Carbachol and norepinephrine induce glycogen breakdown in rat submaxillary glands incubated in vitro. These effects are suppressed by specific inhibitors of cholinergic receptors (atropine, a muscarinic blocker) and of adrenergic receptors (propranolol, a beta-blocker and phentolamine, an alpha-blocker). The beta-adrenergic effect seems to be mediated through the adenosine 3':5'-monophosphate system, whereas the alpha-adrenergic and cholinergic effects are dependent of Ca2+ influx into cell. In the two latter systems, it is suggested that a direct activation of phosphorylase b kinase by calcium takes place. These data clearly indicate that glycogenolysis in rat submaxillary glands in vitro can be independently affected by sympathetic and parasympathetic agents via specific receptors.