Richard B. Marchase

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.

Efficient Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator Requires a Tyrosine-based Signal

We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192–5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2–78) and carboxyl-terminal tail regions (residues 1391–1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2–78) and CFTR-(1391–1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440–1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had ∼40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 microM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

Glucosamine improves cardiac function following trauma-hemorrhage by increased protein O-GlcNAcylation and attenuation of NF-κB signaling

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-alpha and IL-6 mRNA, IkappaB-alpha phosphorylation, NF-kappaB, NF-kappaB DNA binding activity, ICAM-1, and MPO activity. LPS (2 microg/ml) increased the levels of IkappaB-alpha phosphorylation, TNF-alpha, ICAM-1, and NF-kappaB in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in IkappaB-alpha phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GlcNAc levels and attenuated the LPS-induced activation of NF-kappaB. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-kappaB pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock.

Glucosamine administration during resuscitation improves organ function after trauma hemorrhage.

ABSTRACT Stress-induced hyperglycemia is necessary for maximal rates of survival after severe hemorrhage; however, the responsible mechanisms are not clear. One consequence of hyperglycemia is an increase in hexosamine biosynthesis, which leads to increases in levels of O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins. This modification has been shown to lead to improved survival of isolated cells after stress. In view of this, we hypothesized that glucosamine (GlcNH2), which more selectively increases the levels of O-GlcNAc administration after shock, will have salutary effects on organ function after trauma hemorrhage (TH). Fasted male rats that underwent midline laparotomy were bled to a mean arterial blood pressure of 40 mmHg for 90 min and then resuscitated with Ringer lactate (four times the shed blood volume). Administration of 2.5 mL of 150 mmol L−1 GlcNH2 midway during resuscitation improved cardiac output 2-fold compared with controls that received 2.5 mL of 150 mmol L−1 NaCl. GlcNH2 also improved perfusion of various organs systems, including kidney and brain, and attenuated the TH-induced increase in serum levels of IL-6 (902 ± 224 vs. 585 ± 103 pg mL−1) and TNF-&agr; (540 ± 81 vs. 345 ± 110 pg mL−1) (values are mean ± SD). GlcNH2 administration resulted in significant increase in protein-associated O-GlcNAc in the heart and brain after TH. Thus, GlcNH2 administered during resuscitation improves recovery from TH, as assessed by cardiac function, organ perfusion, and levels of circulating inflammatory cytokines. This protection correlates with enhanced levels of nucleocytoplasmic protein O-GlcNAcylation and suggests that increased O-GlcNAc could be the mechanism that links stress-induced hyperglycemia to improved outcomes.

Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia/reperfusion in adult mouse cardiomyocytes

An increase in cytosolic Ca2+ via a capacitative calcium entry (CCE)-mediated pathway, attributed to members of the transient receptor potential (TRP) superfamily, TRPC1 and TRPC3, has been reported to play an important role in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia-reperfusion (I/R). Therefore, we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Adult cardiomyocytes isolated from wild-type (WT) mice and from mice overexpressing TRPC3 in the heart were subjected to 90 min of ischemia and 3 h of reperfusion. After I/R, viability was 51 +/- 1% in WT mice and 42 +/- 5% in transgenic mice (P < 0.05). Apoptosis assessed by annexin V was significantly increased in the TRPC3 group compared with WT (32 +/- 1% vs. 21 +/- 3%; P < 0.05); however, there was no significant difference in necrosis between groups. Treatment of TRPC3 cells with the CCE inhibitor SKF-96365 (0.5 microM) significantly improved cellular viability (54 +/- 4%) and decreased apoptosis (15 +/- 4%); in contrast, the L-type Ca2+ channel inhibitor verapamil (10 microM) had no effect. Calpain-mediated cleavage of alpha-fodrin was increased approximately threefold in the transgenic group following I/R compared with WT (P < 0.05); this was significantly attenuated by SKF-96365. The calpain inhibitor PD-150606 (25 microM) attenuated the increase in both alpha-fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca2+ overload stress, but it did not affect the response to TNF-alpha-induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, to increased calpain activation.

The role of protein O-linked β-N-acetylglucosamine in mediating cardiac stress responses

The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of its adverse effects in mediating a range of chronic disease processes, including diabetes, cancer and neurodegenerative diseases. However, at the cellular level it has been shown that O-GlcNAc levels are increased in response to stress; augmentation of this response improved cell survival while attenuation decreased cell viability. Thus, it has become apparent that strategies that augment O-GlcNAc levels are pro-survival, whereas those that reduce O-GlcNAc levels decrease cell survival. There is a long history demonstrating the effectiveness of acute glucose-insulin-potassium (GIK) treatment and to a lesser extent glutamine in protecting against a range of stresses, including myocardial ischemia. A common feature of these approaches for metabolic cardioprotection is that they both have the potential to stimulate O-GlcNAc synthesis. Consequently, here we examine the links between metabolic cardioprotection with the ischemic cardioprotection associated with acute increases in O-GlcNAc levels. Some of the protective mechanisms associated with activation of O-GlcNAcylation appear to be transcriptionally mediated; however, there is also strong evidence to suggest that transcriptionally independent mechanisms also play a critical role. In this context we discuss the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis including the role of non-voltage gated, capacitative calcium entry as a potential mechanism contributing to this protection.

The protective effects of PUGNAc on cardiac function after trauma-hemorrhage are mediated via increased protein O-GlcNAc levels.

We have previously shown that administration of glucosamine after trauma-hemorrhage (TH) improved cardiac output and organ perfusion, and this was associated with increased levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins in the heart and brain. An alternative means of increasing O-GlcNAc levels is by inhibition of O-linked N-acetylglucosaminidase, which catalyzes the removal of N-acetylglucosamine from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc). The goal of this study, therefore, was to determine whether PUGNAc administration after TH also improves recovery of organ perfusion and function. Fasted male rats were bled to and maintained at a mean arterial blood pressure of 40 mmHg for 90 min, followed by fluid resuscitation. Intravenous administration of PUGNAc (200 &mgr;mol/kg body weight) 30 min after the onset of resuscitation significantly improved cardiac output compared with the vehicle controls (12.3 ± 1.3 mL/min per 100 g body weight vs. 25.5 ± 2.0 mL/min per 100 g body weight; P < 0.05), decreased total peripheral resistance (6.6 ± 0.8 mmHg/mL per minute per 100 g body weight vs. 3.7 ± 0.3 mmHg/mL per minute per 100 g body weight; P < 0.05), and increased perfusion of critical organ systems, including the kidney and liver, determined at 2 h after the end of resuscitation. Treatment with PUGNAc also attenuated the TH-induced increase in plasma IL-6 levels (864 ± 112 pg/mL vs. 392 ± 188 pg/mL; P < 0.05) and TNF-&agr; levels (216 ± 21 pg/mL vs. 94 ± 11 pg/mL; P < 0.05) and significantly increased O-GlcNAc levels in the heart, liver, and kidney. Thus, PUGNAc, like glucosamine, improves cardiac function and organ perfusion and reduced the level of circulating IL-6 and TNF-&agr; after TH. The similar effects of glucosamine and PUGNAc support the notion that the protection associated with both interventions is mediated via increased protein O-GlcNAc levels.

Aging leads to increased levels of protein O-linked N-acetylglucosamine in heart, aorta, brain and skeletal muscle in Brown-Norway rats

Changes in the levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer’s and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5- and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30–107%) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by ∼30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.

STIM1/Orai1-mediated SOCE: current perspectives and potential roles in cardiac function and pathology.

Store-operated Ca²⁺ entry (SOCE) is critical for Ca²⁺ signaling in nonexcitable cells; however, its role in the regulation of cardiomyocyte Ca²⁺ homeostasis has only recently been investigated. The increased understanding of the role of stromal interaction molecule 1 (STIM1) in regulating SOCE combined with recent studies demonstrating the presence of STIM1 in cardiomyocytes provides support that this pathway co-exists in the heart with the more widely recognized Ca²⁺ handling pathways associated with excitation-contraction coupling. There is now substantial evidence that STIM1-mediated SOCE plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo, and there is growing support for the contribution of SOCE to Ca²⁺ overload associated with ischemia/reperfusion injury. Here, we provide an overview of our current understanding of the molecular regulation of SOCE and discuss the evidence supporting the role of STIM1/Orai1-mediated SOCE in regulating cardiomyocyte function.