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Toxin resistance and reduced secretion in a mouse L-cell mutant defective in herpes virus propagation.

The mouse L-cell mutant gro29 was selected originally for its inability to propagate herpes simplex virus; it shows severe defects in virus egress and the transport and processing of viral glycoproteins after infection. In this report, we show that uninfected gro29 cells display pleiotropic changes in protein secretion, oligosaccharide processing, and sensitivity to the toxins ricin and modeccin. Specifically, the rate of secretion of a nonglycosylated protein, human growth hormone, was reduced 70% in gro29 cells compared with the parental L cells. A direct measurement of the transport capacity of Golgi membranes in a cell-free assay suggests that gro29 cells contain less functional Golgi than parental cells. Despite this deficiency, N-linked oligosaccharides were processed efficiently in mutant cells, although there were differences in the structure of the mature forms. Lectin intoxication assays revealed that gro29 cells were cross-resistant to killing by the cytotoxic lectins ricin and modeccin, but not to wheat germ agglutinin, Ricinus communis agglutinin RCA120, or leucoagglutinin. Fluorescence labeling using fluorescein-conjugated lectins showed that uninfected gro29 cells expressed relatively few ricin-binding molecules, suggesting a possible mechanism for toxin resistance. These studies provide evidence that the processes of protein secretion, lectin intoxication, and herpes virus maturation and egress may share a common cellular component.

Animals↗

Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component.

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

Carboxymethylcellulose Sodium↗

[Differential diagnosis of platelet disorders].

Platelet disorders frequently represent a cause of bleeding disorders with a late manifestation and spontaneous bleeding. Disturbances of cellular hemostasis can be of quantitative nature due to an altered production or destruction of platelets. Qualitative disturbances can be associated with defects of adhesion, secretion or degranulation. Drug induced reactions, inflammatory processes and autoimmune reactions are the most frequent underlying disorders. Even a late manifestation, however, does not exclude congenital disorders. In the differential diagnosis of thrombocyte disorders the anamnestic analysis of the clinical circumstances of manifestation, of a family background and potentially interfering drugs are of central importance. Template bleeding time, aggregometry and flow cytometry are complementary methods for the characterization of functional defects. First of all, a von Willebrand syndrome as the most frequent congenital form of a mucocutaneous bleeding pattern needs to be excluded. The clinical context is very important in the analysis of disturbances of platelet turnover. Reticulated platelets allow the quantitative assessment of reduced production or increased destruction. Platelet indices, morphological assessment of blood and bone marrow and immunological tests allow the pathogenetical classification of thrombocytopenia. Idiopathic thrombocytopenia (ITP) is a frequent diagnosis by exclusion. The analysis of glycoprotein expression and the genetic characterization of suspected congenital defects are only performed in selected cases. Clinical and laboratory assessment are complementary in the discrimination of secondary forms of thrombosis from clinically relevant clonal disturbances.

Blood Platelet Disorders↗

Maturity-onset diabetes of the young (MODY), MODY genes and non-insulin-dependent diabetes mellitus.

Maturity-onset diabetes of the young (MODY) is a genetically and clinically heterogeneous subtype of non-insulin-dependent diabetes mellitus (NIDDM) characterised by early onset, autosomal dominant inheritance and a primary defect in insulin secretion. To date, three MODY genes have been identified on chromosomes 20q [hepatocyte nuclear factor (HNF-4 alpha)/MODY1], 7p (glucokinase/MODY2) and 12q (HNF-1 alpha/MODY3). Mutations in glucokinase/MODY2 result in mild chronic hyperglycaemia due to reduced pancreatic beta-cell responsiveness to glucose as well as decreased net accumulation of hepatic glycogen and increased hepatic gluconeogenesis following meals. In contrast, MODY1 and MODY3 are characterised by severe insulin secretory defects and major hyperglycaemia associated with microvascular complications. The role of the three known MODY genes in susceptibility to the more common late-onset from of NIDDM remains uncertain. Genetic studies seem to exclude any function as major susceptibility genes, although they may play a minor role in a polygenic context or a major role in particular populations.

Age of Onset↗

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

Animals↗

Characterization of the hlyB gene and its role in the production of the El Tor haemolysin of Vibrio cholerae O1.

El Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood. In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site-directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid-exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane-associated protein.

Amino Acid Sequence↗

[Maturity-onset diabetes of the young--MODY. Molecular-genetic, pathophysiological and clinical characteristics].

Maturity-onset diabetes of the young (MODY) is a genetically and clinically heterogeneous subtype of type 2 diabetes characterised by an early onset, an autosomal dominant inheritance, and a primary defect in insulin secretion. MODY comprises 2-5% of cases of type 2 diabetes. So far, six MODY genes have been identified (MODY1-6): hepatocyte nuclear factor (HNF-4 alpha), glucokinase, HNF-1 alpha, HNF-1 beta, insulin promoter factor 1(IPF-1), and neurogenic differentiation factor 1 (NEUROD1). MODY2 and MODY3 are the most common forms of MODY. Mutations in glucokinase/MODY2 result in a mild form of diabetes. In contrast, MODY3 and some of the other MODY forms are characterised by major insulin secretory defects and severe hyperglycaemia associated with microvascular complications. About 25% of known MODY is caused by mutations in yet unknown genes and present results suggest that other monogenic forms of type 2 diabetes might exist. The diagnosis of MODY has implications for the clinical management of the patient's diabetes. The identification of MODY genes also opens new perspectives in the understanding of the molecular basis of diabetes and may probably contribute to the definition of novel targets for drug development and gene therapy.

Diabetes Mellitus, Type 2↗

Plasma beta-endorphin level in cases of luteal phase defect.

OBJECTIVE: To evaluate beta-endorphin secretion in euprolactinemic cases of luteal phase defect (LPD). DESIGN: Serial blood samples from the 18th to the 26th day of the menstrual cycle were assayed for beta-endorphin, progesterone (P), estradiol (E2), and prolactin (PRL) in cases of LPD and controls. Diagnosis of LPD was based on determinations of serum P and premenstrual endometrial biopsy. SETTING: From Cairo University Hospitals. PATIENT, PARTICIPANTS: Twenty-six women with LPD and 8 normal fertile women (controls) were chosen. INTERVENTIONS: None. MAIN OUTCOME MEASURES: beta-Endorphin, P, E2, and PRL concentrations were determined by the corresponding 125I radioimmunoassay. RESULTS: Plasma beta-endorphin level in cases of LPD varied from 2.58 to 9.14 pmol/L, whereas the level of controls varied from 2.41 to 5.57 pmol/L. The mean value of plasma beta-endorphin in cases of LPD was significantly higher than that of controls by 146% (P less than 0.0005). In spite of the significant decrease of serum P in cases of LPD, serum E2 level did not differ significantly from that of controls. CONCLUSION: The possible sources of beta-endorphin rise and its implication in the etiology of LPD are explained.

Adult↗

Growth hormone accelerates tubular acid secretion.

The effect of growth hormone on tubular H+ secretion by the hypophysectomized and intact rat was studied in the isolated functioning kidney. Net acid secretion was estimated as the sum of HCO3- absorption plus NH+4 excretion. Kidneys from either intact or hypophysectomized rats were isolated and perfused over a 90-min time course during which either recombinant human growth hormone (GH), insulin-like growth factor-1 (IGF-1), porcine insulin, or vehicle was added; hormone response was then compared with the time controls. Compared with kidneys from intact rats, hypophysectomized rat kidneys exhibited a marked acidification defect, net H+ secretion, 13,530 +/- 600 vs. 17,860 +/- 810 (SE) nmol/ml of glomerular filtrate (GF). Administering GH (50 nM) increased net H+ secretion within 15 min in both hypophysectomized and intact groups to a maximum of 17,950 +/- 910 and 20,960 +/- 1,100 nmol/ml GF, respectively; neither insulin nor IGF-1 (50 nM) was able to mimic GH's effect. Addition of 1 mM amiloride completely abolished the GH-accelerated acid secretion and greater than 70% of the basal net acid secretion rate. Furthermore, GH-enhanced volume absorption was also abolished by amiloride, although neither NaCl nor glutamine absorption was affected. GH-accelerated acid secretion and coupled volume absorption could be observed at concentrations as low as 3.5 nM with half-maximal effect at 12 nM, which is within the range of GH concentration achieved during episodic GH surges. Finally administering GH in vivo to hypophysectomized rats enhanced net acid secretion and urinary acidification, consistent with accelerated tubular H+ secretion as one physiological expression of GH action.

Amiloride↗

Insulin secretion, insulin sensitivity, and hepatic insulin extraction in first-degree relatives of type 2 diabetic patients.

To identify early metabolic abnormalities in type 2 diabetes mellitus, we measured insulin secretion, sensitivity to insulin, and hepatic insulin extraction in 48 healthy normal glucose-tolerant Brazilians, first-degree relatives of type 2 diabetic patients (FH+). Each individual was matched for sex, age, weight, and body fat distribution with a person without history of type 2 diabetes (FH-). Both groups were submitted to a hyperglycemic clamp procedure (180 mg/dl). Insulin release was evaluated in its two phases. The first was calculated as the sum of plasma insulin at 2.5, 5.0, 7.5, and 10.0 min after the beginning of glucose infusion, and the second as the mean plasma insulin level in the third hour of the clamp procedure. Insulin sensitivity index (ISI) was the mean glucose infusion rate in the third hour of the clamp experiment divided by the mean plasma insulin concentration during the same period of time. Hepatic insulin extraction was determined under fasting conditions and in the third hour of the clamp procedure as the ratio between C-peptide and plasma insulin levels. FH+ individuals did not differ from FH- individuals in terms of the following parameters [median (range)]: a) first-phase insulin secretion, 174 (116-221) vs 207 (108-277) microU/ml, b) second-phase insulin secretion, 64 (41-86) vs 53 (37-83) microU/ml, and c) ISI, 14.8 (9.0-20.8) vs 16.8 (9.0-27.0) mg kg-1 min-1/ microU ml-1. Hepatic insulin extraction in FH+ subjects was similar to that of FH- ones at basal conditions (median, 0.27 vs 0.27 ng/microU) and during glucose infusion (0.15 vs 0.15 ng/ micro U). Normal glucose-tolerant Brazilian FH+ individuals well-matched with FH- ones did not show defects of insulin secretion, insulin sensitivity, or hepatic insulin extraction as tested by hyperglycemic clamp procedures.

Adult↗

Genetics of type 2 diabetes mellitus.

The clinical picture of type 2 diabetes mellitus (T2DM) is formed by impairment in insulin secretion and resistance to insulin action. As a result of intensive efforts of the scientists around the world mutations and polymorphisms in a number of genes were linked with monogenic and polygenic forms of T2DM. Two major strategies were used in this research: genome scanning and the candidate gene approach. Monogenic forms, despite their rarity, constitute a field where substantial progress has been made in the dissection of the molecular background of T2DM. Monogenic forms of T2DM with profound defect in insulin secretion include subtypes of maturity onset diabetes of the young (MODY), maternally inherited diabetes with deafness (MIDD) caused by mitochondrial mutations, and rare cases resulting from insulin gene mutations. The majority of proteins associated with MODY are transcription factors, such as hepatocyte nuclear factor 4alpha (HNF-4alpha), HNF-1alpha, insulin promoter factor-1 (IPF-1), HNF-1beta, and NEUROD1. They influence expression of the other genes through regulation of mRNA synthesis. Only MODY2 form is associated with glucokinase, a key regulatory enzyme of the beta cell. There are striking differences in the clinical picture of MODY associated with glucokinase and MODY associated with transcription factors. Three monogenic forms of T2DM characterized by severe insulin resistance are the consequence of mutations in the PPARgamma, ATK2, and insulin receptor genes. Patients with monogenic T2DM, particularly with MODY, sometimes, develop discrete extra-pancreatic phenotypes; for example, lipid abnormalities or a variety of cystic renal diseases. Efforts aiming to identify genes responsible for more common, polygenic forms of T2DM were less effective. These forms of T2DM have a middle/late age of onset and occur with both impaired insulin secretion and insulin resistance. Their clinical picture is created by the interaction of environmental and genetic factors, such as frequent polymorphisms of many genes, not just of one. These polymorphisms may be localized in the coding or regulatory parts of the genes and are present, although with different frequencies, in T2DM patients as well as in healthy populations. Sequence differences in a few genes have been associated, so far, with complex, polygenic forms of T2DM, for example, calpain 10, PPARgamma, KCJN11, and insulin. In addition, some evidence exists that genes, such as adiponectin, IRS-1, and some others may also influence the susceptibility to T2DM. It is expected that in the nearest future more T2DM susceptibility genes will be identified.

Diabetes Mellitus, Type 2↗

Metolazone pharmacokinetics and pharmacodynamics in renal transplantation.

Metolazone pharmacokinetics and pharmacodynamics were investigated in five renal transplant patients and five creatinine clearance matched controls. Whereas the time to peak metolazone excretion was similar in both groups (with the exception of one renal transplant patient who subsequently was found to have chronic rejection), the percent of the administered dose excreted over 48 h was significantly less in renal transplant patients (8% in renal transplant patients vs 24% in controls). Diminished bioavailability of metolazone or tubular secretion likely accounted for this disparity in metolazone excretion. Both groups developed diuretic tolerance as indicated by hysteresis in the dose response relationship. Cumulative sodium excretion over three successive 12-h time intervals did not differ between groups. Despite this comparable natriuresis, potassium excretion was significantly less in the renal transplant patients during the first day of the study. Accordingly, metolazone administration may provide a means to "unmask" subclinical, tubular secretory dysfunction in the transplanted kidney, as exemplified by defects in metolazone secretion and potassium excretion.

Adult↗

Decreased adrenal medullary catecholamine release in spontaneously diabetic BB-Wistar rats. Role of hypoglycemia.

We have demonstrated previously that spontaneously diabetic BB-Wistar rats exhibit decreased adrenal medullary catecholamine secretion in response to splanchnic nerve terminal stimulation. We hypothesized that this abnormality is caused by changes in the sensitivity of the adrenomedullary chromaffin cells to acetylcholine (ACh). To study this hypothesis, we isolated adrenal glands from control and spontaneously diabetic BB-Wistar rats, perfused them with ACh, and measured catecholamine secretion. Adrenal catecholamine release in response to ACh was significantly decreased at 2, 8, and 16 weeks after the onset of diabetes compared with age-matched, nondiabetic control rats. Catecholamine release in response to perfusion with 20 mM K+ was the same in adrenals from diabetic and control rats. The decreased responsiveness of diabetic rat adrenals to perfusion with ACh was significantly correlated with a decrease in the release of catecholamines in response to splanchnic nerve stimulation. A similar defect in catecholamine secretion was also seen in adrenals harvested from nondiabetic BB-Wistar rats following a 3-h period of acute hypoglycemia; however, the adrenal response to potassium was also decreased as was the catecholamine content of the adrenal. Conversely, nondiabetic BB-Wistar rats made diabetic with streptozocin (STZ) and maintained in a hyperglycemic state did not exhibit catecholamine hyposecretion 2 weeks after STZ administration. Collectively, the data describe decreased adrenomedullary response to cholinergic stimulation in spontaneously diabetic rats as early as 2 weeks after the onset of diabetes and that a similar, although more severe, hyposecretion occurs after acute, severe hypoglycemia.

Acetylcholine↗

Glucose refractoriness of pancreatic beta-cells in rat models of non-insulin dependent diabetes.

A decreased insulin response, preferentially to glucose, has been considered a hallmark of non-insulin dependent diabetes mellitus (Type 2) in humans. Syndromes resembling human diabetes occur spontaneously in many animal species and can also be induced by treating animals with drugs or viruses, excising their pancreases or manipulating their diet. Among these models, rat diabetes induced by neonatal streptozotocin administration (n-STZ models) has been first recognized as an adequate tool to study the long-term consequences of a gradually reduced beta-cell mass. More recently, the GK (Goto Kakisaki) Wistar rat has become available and is now considered as a promising spontaneous rat model of non-insulin dependent diabetes. We and others have found that defects in insulin secretion and action develop in the n-STZ and the GK models, which in many ways resemble those described in human non-insulin dependent diabetes. This review is aimed to sum up with a comparative approach, the informations so far collected in the n-STZ and GK models concerning the cellular mechanisms leading to the desensitization of their beta-cells to glucose. Taken together, the data reinforce the view that the impairment of glucose-induced insulin release in n-STZ and GK rats is clearly related to a defect in oxidative glycolysis. This leads to a severe decrease in the mitochondrial oxidative catabolism of glucose-derived pyruvate. Its coincides with a lower ATP/ADP ratio in glucose-stimulated islets and a subsequent alteration of ionic events tightly coupled to the fuel function of the hexose in islet cells, i.e. the decrease in K+ conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

In vivo insulin secretion and action in hyperglycemic rat.

This work was designed to study the effects of insulin secretion and action in vivo of moderate hyperglycemia induced by glucose infusion during 4 days in unrestrained rats. The maintenance of a glycemia around 170 mg/dl throughout the infusion time necessitated a gradual increase of glucose infusion rate from 11.5 to 19 g/day. Throughout the infusion period, plasma insulin-to-glucose ratio remained much higher in hyperglycemic rats (HG) than in controls. Glucose tolerance and insulin secretion tests were performed 2 h after the end of the infusion, when glycemia and insulinemia were back to basal values. Incremental plasma glucose values were significantly lower in HG than in control rats without significant changes in incremental plasma insulin concentrations, suggesting an increased insulin efficiency. At the same insulin level, glucose utilization was higher in HG than in control rats during euglycemic-hyperinsulinemic clamps. These data show that short-term hyperglycemia and hyperinsulinemia do not induce a defect in insulin secretion in vivo and do increase tissue sensitivity to insulin.

Animals↗

Beta-cell function across the spectrum of glucose tolerance in obese youth.

The profile of insulin secretion and the role of proinsulin processing across the spectrum of glucose tolerance in obese youth have not been studied. The aims of this study were to define the role of insulin secretion and proinsulin processing in glucose regulation in obese youth. We performed hyperglycemic clamps to assess insulin secretion, applying a model of glucose-stimulated insulin secretion to the glucose and C-peptide concentration data. Thirty obese youth with normal glucose tolerance (NGT), 22 with impaired glucose tolerance (IGT), and 10 with type 2 diabetes were studied. The three groups had comparable anthropometric measures and insulin sensitivity. The glucose sensitivity of first-phase secretion showed a significant stepwise decline from NGT to IGT and from IGT to type 2 diabetes. The glucose sensitivity of second-phase secretion was similar in NGT and IGT subjects yet was significantly lower in subjects with type 2 diabetes. Proinsulin-to-insulin ratios were comparable during first- and second-phase secretion between subjects with NGT and IGT and were significantly increased in type 2 diabetes. Obese youth with IGT have a significant defect in first-phase insulin secretion, while a defect in second-phase secretion and proinsulin processing is specific for type 2 diabetes in this age-group.

Adolescent↗

An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant.

We describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre-pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid-cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post-1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.

Adult↗

Apolipoprotein[a] secretion from hepatoma cells is regulated in a size-dependent manner by alterations in disulfide bond formation.

Apolipoprotein[a] (apo[a]) is a large disulfide linked glycoprotein synthesized by hepatocytes. We have examined the role of disulfide bond formation in the processing of apo[a] using human and rat hepatoma cells expressing apo[a] isoforms containing varying numbers of kringle 4 (K4) domains, following treatment with DTT. Hepatoma cells expressing 6- or 9-K4 isoforms revealed approximately 90% inhibition of apo[a] secretion following DTT treatment, although larger isoforms containing 13- or 17-K4 domains demonstrated continued secretion (up to 30% of control values), suggesting that a fraction of the larger isoforms is at least partially DTT resistant. Wash-out experiments demonstrated that these effects were completely reversible for all isoforms studied, with no enhanced degradation associated with prolonged intracellular retention. DTT treatment was associated with enhanced binding of apo[a] with the endoplasmic reticulum-associated chaperone proteins calnexin, calreticulin, and BiP, which was reversible upon DTT removal. The chemical chaperone 6-aminohexanoic acid, previously demonstrated by others to rescue defective apo[a] secretion associated with alterations in glycosylation, failed to alter the secretion of apo[a] following DTT treatment. The demonstration that DTT modulates apo[a] secretion in a manner influenced by both the type and number of K4 repeats extends understanding of the mechanisms that regulate its exit from the endoplasmic reticulum.

Aminocaproic Acid↗