Cold-stored platelets: A product with function optimized for hemorrhage control.

Early administration of blood products following severe trauma is pivotal for establishing hemostasis and achieving successful outcomes. Platelet transfusions, in particular, provide rapid control of hemostasis and help to restore platelet dysfunction induced by trauma. In the U.S. platelets used for therapeutic purposes are stored at room temperature with a limited shelf life of 5-7 days. Issues with room temperature storage of platelets, including an increased risk of bacterial growth and a decline in platelet hemostatic function, have led to a resurgence in interest in cold-stored platelets for therapeutic transfusion. This review presents the current state of cold-stored platelets and cold-stored whole blood as treatment for actively bleeding patients. Usage of cold stored platelets in alternative areas, such as in the field of regenerative medicine, is also discussed.

Measuring plasminogen activator inhibitor activity in plasma by two enzymatic assays.

We compared two methods that measure plasminogen activator inhibitor (PAI) activity in plasma based on the ability of PAI to inhibit tissue plasminogen activator (tPA) or urokinase (uPA) in order to determine which method most accurately measures plasma PAI activity after stressors, like hemorrhage. Plasma PAI activity was significantly elevated after hemorrhage in both assays. Using standard curves derived from rhPAI-1, we found that the tPA-PAI assay was more sensitive than the uPA-PAI assay. However, we measured a 10-fold difference in PAI activity as measured between assays, suggesting that some endogenous plasma constituents (tPA, uPA, plasminogen or plasmin) may interfere with the accurate determination of PAI activity. Increasing the amount of plasma in each assay led to a progressive increase in PAI activity. However, removing either tPA or plasminogen from the tPA-PAI assay unmasked the presence of some endogenous tPA and plasminogen. Furthermore, increasing plasma volume in either assay increases measured plasma tPA, but not uPA. Finally, plasma tPA is elevated after hemorrhage, whereas plasma uPA is not. These results suggest that endogenous tPA and plasminogen may interfere with the measurement of plasma PAI activity in the tPA-PAI assay after hemorrhage or other stresses. The uPA-PAI assay does not have this confounding problem because endogenous uPA does not interfere with the assay, nor does it rise during hemorrhage.

The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase.

The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.

Kalirin, a multifunctional PAM COOH-terminal domain interactor protein, affects cytoskeletal organization and ACTH secretion from AtT-20 cells.

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

Expression of RESP18 in peptidergic and catecholaminergic neurons.

We examined the expression of regulated endocrine-specific protein of 18-kD (RESP18) in selected peptidergic and catecholaminergic neurons of adult rat brain. In the hypothalamic paraventricular, supraoptic, and accessory nuclei, RESP18 mRNA was highly expressed in neurons immunostained for oxytocin and vasopressin. RESP18 mRNA was also highly expressed in paraventricular nucleus neurons immunostained for corticotropin-releasing hormone, thyrotropin-releasing hormone, and somatostatin. RESP18 mRNA was expressed in POMC cells of the arcuate nucleus, in neuropeptide Y cells of the dorsal tegmental nucleus, lateral reticular nucleus, and hippocampus, and in brainstem catecholaminergic neurons. RESP18 mRNA expression was high in all paraventricular and arcuate neurons, but RESP18 protein was detectable in the perikarya of a subset of these neurons, suggesting an important post-transcriptional component to the regulation of RESP18 expression. RESP18 antisera immunostained perikarya but not axon fibers or terminals. Sub-cellular fractionation of homogenates of several hypothalamic nuclei identified RESP18 protein in fractions enriched in endoplasmic reticulum. The presence of 22- and 24-kD RESP18 isoforms in the neural lobe of the pituitary indicated that some RESP18 protein exited the endoplasmic reticulum. The post-transcriptional regulation of RESP18 expression and localization of RESP18 protein primarily to the endoplasmic reticulum suggests that RESP18 plays a regulatory role in peptidergic neurons.

Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme.

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways.

Blood flow, vascular resistance, and blood volume after hemorrhage in conscious adrenalectomized rat.

Hemorrhage leads to cardiovascular collapse and death in adrenal-insufficient animals. To determine whether the cardiovascular collapse is due to vasodilation and/or failure to restore blood volume, we used radiolabeled microspheres and 125I-labeled albumin to measure blood flow and blood volume in conscious adrenalectomized (ADX) rats after 15 ml.kg-1.3 min-1 hemorrhage. In ADX rats, hemorrhage led to a greater fall than in sham rats in blood flow in the stomach, small intestines, cecum, colon, spleen, hepatic portal vein, kidney, testis, lung, thymus, bone, fat, forebrain, cerebellum, and brainstem. The greater fall in blood flow was caused by an increase in vascular resistance in these organs except brain and hepatic artery. Sham rats maintained or increased brain and hepatic artery blood flow after hemorrhage whereas flow decreased and remained depressed in ADX rats. ADX rats failed to restore blood volume, whereas sham rats completely restored blood flow by 2 h. We conclude that cardiovascular collapse in ADX rats does not result from vasodilatation but may result from a failure to restore blood volume. The failure to restore blood volume and the low blood flow to organs, especially brain and liver, may contribute to mortality in ADX rats after hemorrhage.

The expression of regulated endocrine-specific protein of 18 kDa in peptidergic cells of rat peripheral endocrine tissues and in blood.

We examined the cellular localization of regulated endocrine-specific protein of 18 kDa (RESP18) and mRNA in peripheral endocrine tissues. In situ hybridization and immunocytochemistry identified RESP18 mRNA in most cells of the anterior and intermediate pituitary, with RESP18 protein apparent in many anterior pituitary cells but very few intermediate pituitary cells. In the adrenal medulla and superior cervical ganglion, RESP18 mRNA co-localized with dopamine beta-mono-oxygenase and neuropeptide Y. In the thyroid, RESP18 mRNA was localized to C-cells. RESP18 mRNA was expressed in most of the cells of the pancreatic islets, co-localizing with insulin, glucagon, and somatostatin. No RESP18 mRNA or protein was detected in the adrenal cortex, ovary, neural lobe of the pituitary, parathyroid, exocrine pancreas, thyroid follicular cells, placenta, mammary tissue, liver, lung, or atria. As in the intermediate lobe of the pituitary, high levels of RESP18 mRNA in the pancreatic islets and adrenal medulla did not always correlate with immunodetectable RESP protein, suggesting that post-transcriptional mechanisms are important in controlling RESP18 expression. Western blot analyses identified 18 kDa RESP and higher molecular weight isoforms of RESP in most tissues and in plasma. Subcellular fractionation of the anterior pituitary identified 18 kDa RESP18 in fractions enriched in endoplasmic reticulum and secretory granules, with the higher molecular weight isoforms of RESP18 concentrated in fractions enriched in secretory granules. The broad neuroendocrine distribution of RESP18 suggests that it subserves an important function in the secretory pathway that is common to the production of many secreted peptides.

Stage-specific expression of RESP18 in the testes.

RESP18 (regulated endocrine-specific protein of 18 KD) is an endoplasmic reticulum (ER) protein that was identified by coordinate dopaminergic regulation with pro-opiomelanocortin in the rat neurointermediate pituitary. Many attributes of RESP18 suggest an important function in neuroendocrine cells. Several neuropeptides, growth factors, and enzymes involved in biosynthesis of classical chemical neurotransmitters, have been identified in germ cells, Sertoli cells, and spermatozoa. In this study, screening of reproductive tissues revealed high levels of RESP18 protein and mRNA in the testes but not in ovaries or epididymis. The testes and sperm expressed 18-KD RESP18 and a unique 19-KD isoform. To better understand RESP18 expression in the testes, we have examined the stages of the cycle of the seminiferous epithelium by immunohistochemistry and Western blot analyses. Immunohistochemical analysis showed that RESP18 protein was expressed exclusively in spermatocytes and maturing spermatids. RESP18 protein was expressed at high levels in Step 1-8 round spermatids, in which the PC4 prohormone convertase, nerve growth factor, and proenkephalin are also expressed. Western blots, Northern blots, and indirect immunofluorescence staining demonstrated RESP18 expression in sperm.

Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme.

The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.

Location of neurons that express regulated endocrine-specific protein-18 in the rat diencephalon.

In situ hybridization for regulated endocrine-specific protein-18 messenger RNA showed a distinct and limited pattern of expression in the hypothalamus, midline thalamus, amygdala and hippocampus of the rat. High levels of regulated endocrine-specific protein-18 messenger RNA were found in the magnocellular neurons of the hypothalamic paraventricular, supraoptic and accessory nuclei, in the neurons of the periventricular, medial tuberal, arcuate, lateral and perifornical nuclei, infundibular stalk, and in the ventrolateral division of the ventromedial nucleus and compact division of the dorsomedial nucleus. Lower levels of regulated endocrine-specific protein-18 messenger RNA were found in the parvocellular divisions of the paraventricular nucleus as well as in the bed nucleus of the stria terminalis, median preoptic nucleus, medial preoptic nucleus, medial and lateral preoptic areas, subfornical organ, suprachiasmatic nucleus, anterior hypothalamic area, zona incerta, ventromedial nucleus, dorsomedial nucleus and tuber cinereum. Regulated endocrine-specific protein-18 messenger RNA was also found in thalamic structures including the paraventricular, central medial, intermediodorsal, anterodorsal, rhomboid and reticular nuclei. Signal was also identified in the medial and lateral habenula, in the central, medial, basomedial and anterior cortical nuclei of the amygdala, and in the CA1-CA3 and dentate gyrus of the hippocampus. Dopamine may regulate regulated endocrine-specific protein-18 expression in the CNS because (i) regulated endocrine-specific protein-18 was originally identified in melanotropes based on its regulation by dopaminergic agents and (ii) many of the nuclei that contain regulated endocrine-specific protein-18 also receive dopaminergic input. The localization of regulated endocrine-specific protein-18 in the diencephalon suggests that regulated endocrine-specific protein-18 is involved in regulation of limbic and autonomic function, neuroendocrine control of salt and water balance, reproductive function and feeding behavior.

Plasminogen activator inhibitor-1 mRNA is induced by hemorrhage in endothelial and mesothelial cells of the rat liver.

We have recently shown that plasminogen activator inhibitor-1 (PAI-1) mRNA is elevated after hemorrhage in various tissues including liver. In this study, we set out to identify the cell types in the liver that are responsible for the increase in PAI-1 mRNA after hemorrhage using in situ reverse transcription-polymerase chain reaction (in situ RT-PCR). Male Sprague-Dawley rats were cannulated and subjected to a 20 ml/kg hemorrhage within 3 min or 300 micrograms/kg of endotoxin. Four hours later, the livers were harvested, fixed, frozen, and sectioned. RT-PCR showed an increase of PAI-1 mRNA in liver 4 h after hemorrhage or endotoxin-treatment. Standard in situ hybridization could not detect PAI-1 mRNA in the livers of either the hemorrhage, endotoxin, or control groups. However, in situ RT-PCR detected PAI-1 mRNA in vascular endothelial cells and capsular mesothelial cells, but not in hepatocytes, in both the hemorrhage and endotoxin groups. No signal was found in the control rats, or when the experimental protocol was modified to 1) omit the RT step, 2) precede the RT step with RNA digestion, or 3) use an irrelevant probe. These results demonstrate that hemorrhage induces PAI-1 mRNA in endothelial and mesothelial cells of liver.

Plasminogen activator inhibitor-1 rises after hemorrhage in rats.

Large hemorrhage leads to hypercoagulability, a phenomenon that has never been well explained. Because an elevation of plasminogen activator inhibitor (PAI)-1 increases procoagulant activity, we have determined whether plasma PAI activity and tissue PAI-1 mRNA are elevated after hemorrhage. Sprague-Dawley rats were bled (20 or 15 ml/kg) 4 days after cannulation. Plasma PAI activity was determined by the capacity of plasma to inhibit tissue-type plasminogen activator activity. Changes of PAI-1 mRNA in various tissues were detected by high-performance liquid chromatography after reverse transcription and polymerase chain reaction. Hemorrhage (20 ml/kg) significantly elevated plasma PAI activity at 0.5, 1, 2, 4, 6, and 8 h after hemorrhage and PAI-1 mRNA in liver at 1, 2, 4, and 6 h after hemorrhage. The PAI-1 message was also significantly elevated in lung, heart, and kidney at 4 h after hemorrhage. The increases of PAI-1 mRNA after 20 ml/kg hemorrhage were significantly greater than those after 15 ml/kg hemorrhage. These findings indicate that large hemorrhage can induce the increases in PAI activity and PAI-1 message and suggest that induction of PAI-1 may be involved in the thrombogenic responses observed after large hemorrhage.

Changes in regional vascular resistance and blood volume after hemorrhage in fed and fasted awake rats.

To determine whether fasting alters the response of blood flow to hemorrhage, blood flow was measured by radiolabeled microspheres before and after a 20 ml.kg-1.3 min-1 hemorrhage in fed and fasted chronically cannulated male Sprague-Dawley rats. Restitution of blood volume, as determined by dilution of hematocrit, was attenuated in fasted rats, although the responses of arterial blood pressure, heart rate, cardiac output, and total peripheral resistance were not significantly different. Fasting only affected resting blood flow in the bronchial artery and fat and had no effect on resting vascular resistance in any organ studied. In both fed and fasted rats, hemorrhage led to a significant fall in blood flow to the stomach, small intestine, cecum, colon, spleen, pancreas, kidney, bronchial artery, thymus, and muscle and a rise in blood flow to the adrenals. However, fasting did not significantly alter the response of flow or vascular resistance to these organs. Fasting did alter the blood flow response to hemorrhage in bone, fat, and the hepatic artery. These results demonstrate that 24 h of fasting does not affect the responses of blood flow and vascular resistance to hemorrhage in most organs, even though restitution of blood volume is attenuated.

Ventromedial hypothalamic lesions inhibit corticosteroid feedback regulation of basal ACTH during the trough of the circadian rhythm.

We have determined the effects of bilateral electrolytic lesions of the ventromedial hypothalamus (VMH) on activity in the hypothalamo-pituitary-adrenal (HPA) system. Acutely, during the first 5 days, lesions of the anterior-medial VMH caused loss of the diurnal rhythms in food intake and plasma corticosterone (B) levels. Plasma B concentrations were elevated during the time of the normal trough of the basal diurnal rhythm in HPA axis activity and the diurnal rhythm in food intake was abolished, in agreement with the results of others. Consistent with hyperactivity in the HPA axis, lesioned rats had increased adrenal weight, decreased thymus and body weights and decreased plasma transcortin concentrations. To determine how lesions of the VMH provoke these increases in activity of the HPA system, the sensitivity of ACTH in adrenalectomized, lesioned rats to replacement with exogenous B was determined under basal conditions during the trough (morning-AM) and peak (evening-PM) of the diurnal rhythm in HPA axis activity. ACTH in lesioned rats in the AM was insensitive to feedback over the very low range of plasma B of 1-4 micrograms/dl, whereas sham-lesioned controls exhibited the normal, high sensitivity of ACTH to B at this time of day. There was no difference between the sensitivity of ACTH to this low range of B in the PM in VMH- and sham-lesioned rats. Two to 5 weeks after VMH lesions, as found by others, mean daily plasma B levels did not differ from sham-lesioned controls; however, plasma B during the AM was still mildly elevated in these rats. Inhibition of plasma B in the PM by dexamethasone was less effective in lesioned rats. Although HPA system responses to hypoglycemia, corticotropin-releasing factor and ACTH were normal, the lesioned rats exhibited obesity, hyperinsulinemia, hyperglycemia, hypertension and tachycardia, all signs consistent with mild hyperactivity of the PHA axis. Occupancy of type I, high-affinity corticosteroid receptors is known to control basal activity of the HPA system during the trough of the diurnal rhythm and to interact with glucocorticoid receptors to affect basal activity during the peak of the diurnal rhythm and during AM stress. We conclude that VMH lesions disrupt transmission of inhibitory signals, mediated by occupancy of type I corticosteroid receptors, that are initiated by a B feed-back site.

Role of intestinal fluid in restitution of blood volume and plasma protein after hemorrhage in awake rats.

To determine whether food and/or water in the gastrointestinal tract affects restitution of blood volume and plasma protein after hemorrhage, fed and 24-h-fasted awake rats received a 20 ml.kg-1 x 3 min-1 hemorrhage, and restitution of blood volume was measured by Evans blue dye and dilution of hematocrit. Restitution of blood volume and plasma protein in fed rats was complete by 2-4 h. In contrast, restitution was severely attenuated in fasted rats and was not complete by 24 h. Because initial blood volume was significantly lower in the fasted rats (55.4 +/- 1.7 vs. 64.9 +/- 2.5 ml/kg in fed), the percent blood lost during hemorrhage was significantly greater (36 vs. 31%). However, the attenuated restitution was not the result of the larger hemorrhage, as fed rats receiving a 36% hemorrhage also restored blood volume completely by 4 h. In fasted rats, complete restitution of blood volume did occur when either water or food and water were given 4 h after hemorrhage. Gastrointestinal water content fell (from 65.5 +/- 4.8 to 47.9 +/- 1.6 ml/kg) 2h after hemorrhage in fed but not in fasted rats (33.5 +/- 2.4 to 30.6 +/- 2.5 ml/kg). These data suggest that gastrointestinal fluid is essential for complete restoration of blood volume in the awake rat.

Regulated endocrine-specific protein-18: a short-lived novel glucocorticoid-regulated endocrine protein.

Regulated endocrine-specific protein-18 (RESP18) is an 18-kilodalton endocrine-specific transcript whose expression is regulated by a number of different physiological and pharmacological stimuli in different tissues. RESP18 messenger RNA was identified in all cell types in the anterior pituitary, at levels that varied 2-fold from the lowest (corticotropes and thyrotropes) to the highest (gonadotropes, somatotropes, and mammotropes); the melanotropes of the intermediate pituitary have levels of RESP18 messenger RNA comparable to the highest levels in cells in the anterior pituitary. Mouse RESP18 was cloned and used as the basis for biosynthetic studies on RESP18 in AtT-20 cells, which express RESP18 endogenously; mouse RESP18 was highly homologous to rat RESP18. Pulse-chase biosynthetic labeling studies showed that AtT-20 cells expressed much less RESP18 than the endogenous prohormone, POMC, but that glucocorticoid treatment lowered POMC and raised RESP18 biosynthetic rates so that they were nearly equimolar. Surprisingly, RESP18 was not processed to smaller peptides to any significant extent, nor was RESP18 or any smaller peptide secreted. Newly synthesized RESP18 normally disappeared from AtT-20 cell extracts with a half-life of less than 15 min; the intracellular half-life of RESP18 was increased strikingly after glucocorticoid treatment of the cells. Upon subcellular fractionation, RESP18 was found to be entirely particulate and to cofractionate with markers for the endoplasmic reticulum, rather than with markers for secretory granules, such as POMC and prohormone-processing enzymes. Therefore, RESP18 is a major glucocorticoid-responsive protein in the secretory pathway of corticotropes, but its function may be entirely within the neuroendocrine cell.

PACE4: a subtilisin-like endoprotease prevalent in the anterior pituitary and regulated by thyroid status.

Low stringency screening of a rat hypothalamic complementary DNA library for additional members of the subtilisin-like prohormone convertase (PC) family identified rat PACE4, which is 90% identical to human PACE4 in amino acid sequence, with much lower similarity to rat PC1, PC2, furin, PC4, or PC6. The rat PACE4 sequence has the Asp-His-Ser catalytic site triad, an Arg-Gly-Asp potential integrin binding site, and three potential sites for N-linked glycosylation. Rat PACE4 has a long COOH-terminal region, which is very rich in Cys residues (15%). The unique signal sequence of rat PACE4 mediates translocation across microsomal membranes during in vitro translation and secretion of PACE4 from stably transfected fibroblast cells. Rat PACE4 has a tissue and cell line distribution unlike any reported PC, including human PACE4, with high expression in the anterior pituitary and readily detectable expression in several brain regions, the atrium, and the ventricle; negligible PACE4 messenger RNA (mRNA) is detected in neurointermediate pituitary and many nonneuroendocrine tissues. PACE4 mRNA is prevalent in Buffalo rat liver and GH3 cells and present at low levels in AtT-20 cells, whereas it is undetectable in several other cell lines. In situ hybridization coupled with immunocytochemistry revealed that PACE4 is produced by somatotropes, mammotropes, and corticotropes, whereas less PACE4 mRNA was detected in thyrotropes. PACE4 mRNA levels in anterior pituitary are strikingly regulated by thyroid status, with more than a 10-fold increase seen from hypothyroid to hyperthyroid animals. The prevalence of PACE4 in anterior pituitary and the striking effect of thyroid status on PACE4 expression suggest a specific role for PACE4 in processing neuroendocrine peptides.

RESP18, a novel endocrine secretory protein transcript, and four other transcripts are regulated in parallel with pro-opiomelanocortin in melanotropes.

The homogeneous nature of the rat intermediate pituitary makes it a powerful model system in which to study peptide hormone secretion. Adult male rats were treated with bromocriptine, a dopamine agonist, or haloperidol, a dopamine antagonist, for 3 weeks. In cDNA libraries prepared from the neurointermediate pituitaries of these rats, pro-opiomelanocortin (POMC) expression exhibited the expected decrease in response to bromocriptine, and increase in response to haloperidol. We report the identification of six transcripts that are coregulated with POMC in the intermediate pituitary by these dopaminergic agents. In addition to demonstrating parallel dopamine-regulated expression of carboxy-peptidase E, chromogranin B, binding protein/glucose-regulated protein, and tenascin, two novel regulated transcripts are described. The expression of one of these novel transcripts, RESP18, is limited to neural and endocrine tissue. The RESP18 transcript is approximately 800 nucleotides in length; its cognate translation product is 20 +/- 1 kDa, contains a putative signal sequence, and has many characteristics of a secreted protein. Cell-free translation experiments in the presence of microsomal membranes demonstrate that the 20 +/- 1-kDa RESP18 protein is cleaved to an 18 +/- 1-kDa protein and sequestered within the lumen of the rough endoplasmic reticulum. Tissue in situ hybridization analysis shows that RESP18 mRNA is highly expressed in both the intermediate and anterior pituitary, as well as in the paraventricular and supraoptic nuclei of the hypothalamus.

A circulating shock protein depolarizes cells in hemorrhage and sepsis.

OBJECTIVE: A study to determine if both septic and hemorrhagic shock lead to the appearance of a substance that depolarizes cells in plasma was performed. SUMMARY BACKGROUND DATA: Transmembrane potential decreases in skeletal muscle, hepatocytes, and red blood cells early in the development of both hemorrhagic and septic shock. The associated movement of ions and water into cells leads to extracellular fluid loss and exacerbates shock. METHODS: Adult male Sprague-Dawley rats with indwelling arterial and venous cannulae were bled 20 mL/kg or received intravenously 2 x 10(10) Escherichia coli suspended in 400 mL of 0.9% saline. Blood samples were taken after hemorrhage and induction of sepsis to determine the presence of a plasma factor that depolarized red blood cells. Control rats were not injected with E. coli or bled. Plasma from bled and septic rats was processed by sequential precipitation with ammonium sulfate and subjected to gel filtration. RESULTS: Depolarizing activity was highest 20 minutes after hemorrhage and 60 minutes after E. coli injection, decreasing to control levels by 2 (hemorrhage) and 4 (sepsis) hours. Control rats showed no significant change in depolarizing activity. Tryptic and chymotryptic digestion eliminated the depolarizing activity, indicating that the active substance is, at least in part, a protein. Depolarizing activity from bled and septic processed plasma was confined essentially to the 70% ammonium sulfate fraction and the activity migrated with an apparent molecular mass of 200 kD after gel filtration. Separation of the complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) produced an identical pattern of bands in both bled and septic animals. CONCLUSIONS: A circulating plasma protein complex of high molecular weight causes cellular depolarization in both hemorrhage and sepsis and may be responsible for the associated increases in cell sodium and water seen in both hemorrhagic and septic shock.