This Article  • Abstract  • PDF  • Send to a friend  • Save in My Folder  • Save to citation manager  • Permissions  Citing Articles  • Citation map  • Citing articles on HighWire  • Citing articles on ISI (12)  • Contact me when this article is cited  Related Content  • Similar articles in this journal  Topic Collections  • Burns  • Infectious Diseases  • Bacterial Infections  • Critical Care/ Intensive Care Medicine  • Adult Critical Care  • Alert me on articles by topic

Role of Ca²⁺

Mohammed M. Sayeed, PhD

Arch Surg. 2000;135:1432-1442.

ABSTRACT
Alterations in cellular responses in various organ systems contribute to trauma-, burn-, and sepsis-related multiple organ dysfunction syndrome. Such alterations in muscle contractile, hepatic metabolic, and neutrophil and T-cell inflammatory-immune responses have been shown to result from cell-signaling modulations and/or impairments in the respective cell types. Altered Ca²⁺ signaling would seem to play an important role in the myocardial and vascular smooth muscle contractile dysfunction in the injury conditions; Ca²⁺-linked signaling derangement also plays a crucial role in sepsis-induced altered skeletal muscle protein catabolism and resistance to insulin-mediated glucose use. The injury-related increased hepatic gluconeogenesis and acute-phase protein response could also be caused by a pathophysiologic up-regulation of hepatocyte Ca²⁺-signal generation. The increased oxidant production by neutrophil, a potentially detrimental inflammatory response in early stages after burn or septic injuries, seems to result from an up-regulation of both the Ca²⁺-dependent as well as Ca²⁺-independent signaling pathways. The injury conditions would seem to cause an inappropriate up-regulation of Ca²⁺-signal generation in the skeletal myocyte, hepatocyte, and neutrophil, while they lead to a down-regulation of Ca²⁺ signaling in T cells. The crucial signaling derangement that causes T-cell proliferation suppression seems to be a decrease in the activation of protein tyrosine kinases, which subsequently down-regulates Ca²⁺ signaling. The delineation of cell-signaling derangements in trauma, burn, or sepsis conditions can lead to development of therapeutic interventions against the disturbed cellular responses in the vital organ systems.

INTRODUCTION

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

Alterations in cellular Ca²⁺ regulation can be caused by pathophysiologic stimuli that disrupt membrane functional/structural integrity and thereby impair transmembrane Ca²⁺ movements. This Ca²⁺ dysregulation can adversely affect the cytosolic Ca²⁺ concentration, [Ca²⁺]_i, in the resting cells as well as the intracellular signaling role of Ca²⁺ in the elicitation of cellular contractile, secretory, metabolic, proliferative, and/or cell-cell communicative or adhesive responses. The derangements in cellular responses can, in turn, initiate and/or exacerbate tissue and organ dysfunction. In acute-injury conditions, such as trauma, burn, and sepsis, the ensuing production of neuroendocrine and inflammatory stimuli can provoke cellular Ca²⁺ dysregulation, leading to inappropriate Ca²⁺_i signaling. Alternatively, tissue damage resulting from trauma-, burn-, and sepsis-induced hypoxia or ischemia could disrupt energy-linked membrane Ca²⁺ movements and disturb Ca²⁺_i homeostasis. The Ca²⁺_i-related disturbances could initially occur in certain tissues or organs, and then later affect remote tissues or organs. Thus, Ca²⁺_i dyshomeostasis and accompanying Ca²⁺_i-signaling derangements can become pathogenic mechanisms contributing to the development of multiple organ dysfunction and failure.

This article presents an overview of studies of cellular Ca²⁺ regulation and Ca²⁺-linked signaling in various organs and systems in burn and septic conditions. The overview relies heavily on studies in animal models of burn and septic injuries. To date, there are but few such studies in patients with trauma, burn, or sepsis. The article also provides a survey of mechanisms of Ca²⁺_i homeostasis and of Ca²⁺-linked signaling in muscle and nonmuscle cell types under physiological conditions. The survey of physiologic mechanisms is intended to serve as a frame of reference for the evaluation of signaling alterations in the trauma, burn, and septic injury conditions.

Ca²⁺_i HOMEOSTATIS AND Ca²⁺_i LINKED SIGNALING

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

In the resting (basal) state of cells, an unregulated passive movement of Ca²⁺ into the cell occurs across the plasma membrane because of the steep transmembrane gradient of Ca²⁺ concentration, [Ca²⁺], namely, a 10,000-fold higher [Ca²⁺] in the extracellular environment than in the cell interior. Such Ca²⁺ movement is, however, greatly hampered because of a low permeability of the plasma membrane to Ca²⁺. In physiologic states, the resting cells' steady state [Ca²⁺]_i (40-100 nmol/L) is maintained by an active Ca²⁺ extrusion out of cells, which is presumably of a magnitude equal to that of the passive movement into the cells. On physiologic activation of various cell types via neural and endocrine stimuli, [Ca²⁺]_i is transiently elevated to 150 to 1000 nmol/L. Such transient [Ca²⁺]_i elevations constitute the Ca²⁺ signals serving to elicit various cellular responses ascribed to be mediated by the neuroendocrine stimuli. The amplitude and the duration of the Ca²⁺ signals presumably vary depending on the type of stimulus-receptor system and the type of response generated in a given cell system.

The neurotransmitter- and hormone-mediated elevation of [Ca²⁺] above the steady state level results from an influx of Ca²⁺ into the cytosolic compartment from the extracellular and/or sarcoendoplasmic reticulum (SER) compartments. These Ca²⁺ fluxes also follow [Ca²⁺] gradients. However, unlike the unregulated passive movements, they are specifically triggered by the neuroendocrine stimuli and apparently result from regulated increases in permeability of the plasma and SER membranes to Ca²⁺. Such increases in membrane permeability are actually caused by "unplugging" or "gating" of Ca²⁺-specific membrane pores or channels. The gating of plasma-membrane Ca²⁺ channels is modulated either by an electrical stimulus modifying the potential difference across the membrane and thereby enhancing Ca²⁺ conductance through the channel, or by a hormone binding to its specific receptor, which itself serves as a Ca²⁺ channel. The hormone-bound channel exhibits enhanced Ca²⁺ conductance. The gating of the SER Ca²⁺ channel is in turn dependent either on the electrical or hormonal activation of the plasma membrane Ca²⁺ channel; it leads to the release of the SER Ca²⁺ into the cytosol. Recent studies in cardiac and vascular smooth muscle have shown that the SER Ca²⁺ channel, at its cytosolic end, is sensitive to localized Ca²⁺ "sparks" produced by the Ca²⁺ coming through the plasma membrane Ca²⁺ channels.^1-2 Such Ca²⁺ sparks occur over a narrow spatial range close to the SER membrane.

SIGNALING ROLE OF PHOSPHOLIPASES AND KINASES

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

In hormone-stimulated muscle cells, the hormone-receptor interactions in the plasma membrane lead to activation of 1 or more isoforms of the intramembrane enzyme, phospholipase C (PLC), which catalyzes the formation of inositol 1,4,5-trisphosphate (InsP₃) from the membrane-derived phosphoinositides.³ Inositol 1,4,5-trisphosphate serves as an intracellular ligand that binds to its specific receptor in the SER membrane, triggering the release of SER Ca²⁺ into the cytosolic compartment. The transient Ca²⁺ signals, generated under physiological conditions, are then followed by a return of [Ca²⁺] to the steady state level because of activation of an energy requiring reuptake of Ca²⁺ by the SER and active Ca²⁺ extrusion to the cell exterior.^4-5 The elevation in [Ca²⁺]_i in cardiomyocytes is primarily caused by SER Ca²⁺ release⁶ and by both Ca²⁺ entrance through the plasma membrane Ca²⁺ channel and release from SER in the vascular smooth myocytes.⁷ Whereas the generation of the Ca²⁺_i transients commonly occurs subsequent to electrical stimulation of the cardiac and smooth muscle cells, the hormonal stimulations causing InsP₃ formation serve to modulate ongoing contractile responses in both of these cell systems. The Ca²⁺ signaling in the muscle cells, whether it results from purely electrical stimuli or is modulated by the hormonal stimuli, plays a final response-generating role.

In a variety of nonmuscle cell types, the signaling to secretory, proliferative, and cell-cell communicative and adhesive responses are initiated not only with the activation of phospholipases, but also with kinases and/or phosphatases. In some of these cell types, the signaling process is initiated via activation of the guanine nucleotide-binding G proteins (G_q) (which activate phospholipases); in others, there is an initial activation of protein tyrosine kinases (PTK).⁸ The activation of PTK in some instances may lead to stimulation of a phospholipase and an elevation in [Ca²⁺]_i.⁹ Unlike its role as a distal signaling effector element in the various muscle cells, Ca²⁺ signal in the nonmuscle cells seems to serve as an intermediary signaling element. A further signaling by kinases and/or phosphatases, occurring distal to the Ca²⁺ signal, is known to have a direct role in the elicitation of the cellular response. That cellular responses can be elicited entirely independently of Ca²⁺ signal has also been shown in a variety of nonmuscle cell types.^10-11

SIGNALING MECHANISMS OF CELLULAR RESPONSES

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

The organ/system derangements that occur with trauma and burn injuries or with subsequent septic complications invariably result from functional deficits at the cellular level. Such cellular deficits reflect alterations in cellular responses to hormones and inflammatory mediators released in the injury conditions. The altered cellular responses, in turn, could be related to disturbances in hormone-, cytokine-, complement-, protein-, and eicosanoid-mediated cell-signaling pathways. To date, a number of studies have evaluated signaling alterations in the various cellular components of organs and systems that exhibit functional derangements in trauma, burn, and sepsis conditions.^12-15 These studies have allowed for the elucidation of the mechanisms of the dysfunctional state of the organs/systems.

A number of studies have assessed signaling aberrations that could cause cardiovascular dysfunction in the injury states. While some studies have failed to observe changes in membrane Ca²⁺-transport mechanisms in the myocardial cells after endotoxic-septic injury,¹⁶ several others have shown septic injury–related impairments in the active Ca²⁺ transport mechanisms in the myocardium.^17-22 Investigators have also supported Ca²⁺ signaling alterations because of elevated [Ca²⁺]_i in the cardiac myocytes in experimental burn and sepsis.^22-26 The aberrant myocardial Ca²⁺_i homeostasis could be caused by either hypoxic conditions or inflammatory mediators, such as tumor necrosis factor (TNF-) and nitric oxide.^27-31

The loss of vascular smooth muscle responsiveness to vasopressor agents, which is known to occur in the injured hosts, adversely affects the ability of the vasculature to provide for compensatory adjustments of arterial blood pressure. Such a loss of responsiveness by vasculature has been shown to be related to disturbances in Ca²⁺-related signaling events.³² The injury-related microvascular permeability changes, which contribute to disturbed fluid-protein flux across capillaries, have been traced to alterations in cyclic adenosine monophosphate (cAMP) generation and indirectly to alterations in Ca²⁺-dependent signaling pathways in the endothelial cells.³³ Thus, signaling alterations in the vascular smooth muscle and endothelial cells can cause blood pressure and flow dysregulation, as well as endothelial permeability defects in any organ or tissue, and thereby contribute to organ and tissue damage.

Although, to date, there is little known about signaling alterations in the paranchymal cells in kidneys, lungs, and the gastrointestinal tract in injured hosts, a number of studies from my laboratory have investigated the role of Ca²⁺-linked signaling pathways and related cellular responses in some of the vital nonimmune (skeletal myocytes and hepatocyte) and immune (neutrophils and T lymphocytes) cell systems in injured animals.^34-72

SKELETAL MYOCYTE SIGNALING TO PROTEIN-METABOLIC AND GLUCOSE-UPTAKE RESPONSES

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

Physiologic Mechanisms

The hormonal and neural stimuli collectively serve to enhance skeletal muscle's contractile and metabolic responses in a coordinated manner. Figure 1A shows signaling pathways that lead to generation of these responses. The Ca²⁺ sparks generated by the neural stimuli (via activation of the plasma membrane voltage-operated Ca²⁺ channel) lead to release of Ca²⁺ from SER into the cytosol to cause actin-myosin interactions.⁶ An -adrenergic receptor activation via epinephrine and norepinephrine can also lead to release of SER Ca²⁺ by activating the phospholipase C–InsP₃ pathway.³ The resulting Ca²⁺ signal produces not only the contractile, but also the glycogenolytic response (via activation of the glycogenolytic enzyme phosphorylase kinase) to provide for cellular energy for contraction. The hormonal activation of the -adrenergic receptor and the linked membrane enzyme adenylate cyclase, resulting in the generation of cAMP, supports muscle contraction in an indirect manner. Cyclic AMP causes activation of protein kinase A that phosphorylates an SER membrane protein, namely phospholamban.⁷³ The phosphorylation of phospholamban allows for the stimulation of SER Ca²⁺-adenosine triphosphatase, which is responsible for the active reuptake of Ca²⁺ into SER. Thus, the hormonal stimulation is responsible for "recharging" the SER Ca²⁺ reservoir and maintaining Ca²⁺ availability for contractile force development in the muscle. Cyclic AMP is also required for an optimal activation of phosphorylase kinase.⁷⁴ Whereas [Ca²⁺]_i elevation serves as the primary signal for the skeletal muscle responses, cAMP plays a secondary role. The skeletal muscle proteolysis is also modulated by Ca²⁺_i signaling via activation of the Ca²⁺-dependent protease, calpain.⁷⁵ Although Ca²⁺ signal is not directly involved in the muscle's insulin-mediated glucose uptake, augmented Ca²⁺ signaling has been shown to down-regulate the insulin-dependent glucose transporter (GLUT4).⁷⁶ The insulin-mediated signaling pathway involves activation of the membrane receptor–bound PTK responsible for the activation of the downstream signaling intermediates, insulin receptor substrate-1 and phosphatidyl-inositol-3-kinase (PI-3K).⁷⁷ The latter pathway is implicated in the transcription of GLUT4g (glucose transporter gene) as well as its translation into GLUT4 protein (GLUT4p) and GLUT4p incorporation into the plasma membrane.

View larger version (62K): [in this window] [in a new window] Figure 1. Diagrammatic sketches showing signaling pathways in skeletal myocyte (A), hepatocyte (B), neutrophil (C), and T lymphocyte (D). Abbreviations, in alphabetical order: AC indicates adenylate cyclase; AGP, -1 acid glycoprotein; ANG-II, angiotensin II; APP, acute-phase protein; APPg, APP gene; C5a, complement protein 5a; cAMP, cyclic adenosine monophosphate; CER, ceramide; CORT, glucocorticoid; CyclinD, E, C, cell-cycle genes; DAG, diacylglycerol; EPI, epinephrine; ER, endoplasmic reticulum; FBR, fibrinogen; Fyn, an Src protein kinase; Gi, inhibitory G protein subunit; Gq, G protein implicated in the activation of phospholipase C ; Gs, G protein that stimulates AC; GCG, glucagon; GLUT4g, GLUT4m, and GLUT4p, glucose transporter gene, messenger RNA, and protein; GM-CSF, granulocyte-macrophage colony stimulating factor; HGB, haptoglobin; IL, interleukin; IL-2R, interleukin 2 receptor; InsP3, inositol trisphosphate; IRS-1, insulin-receptor substrate; Jak, Janus kinases; Lck, an Src protein kinase; Lyn, an Src kinase; MAPK, mitogen activating protein kinase; NEPI, norepinephrine; NFATc, nuclear factor AT cytosolic; NF, nuclear factor kappa B; p47/p67, phosphoproteins responsible for NADPH oxidase activation; PAF, platelet activating factor; PEPCK, phosphoenol pyruvate carboxy kinase; PEPCKg, PEPCK gene; PI-3K, phosphotidylinositol-3-kinase; PKC, protein kinase C; PLC, phospholipase C; PLC, phospholipase C ; PLC, phospholipase C; PrK, phosphorylase kinase; PTK, protein tyrosine kinase; Rac, phosphoprotein responsible for NADPH oxidase activation; Ras, signaling protein critical in MAPK activation; Raf, signaling protein critical in MAPK activation; ROC, receptor-operated Ca2+ channel; SER, sarcoplasmic endoplasmic reticulum; Shc, adapter signaling protein; STAT 3/5, signal transduction activator protein 3 and 5; TcR, T-cell antigen receptor; TNF-, tumor necrosis factor ; TST, thiostatin; VOC, voltage-operated Ca2+ channel; VP, vasopressin; and Zap, an Src kinase.

Pathophysiologic Alterations

In burn, trauma, and septic conditions, the breakdown of protein into amino acids in skeletal muscle plays a major role in the heightened body-protein turnover. The amino acids released from the muscle are used for gluconeogenesis and for the acute phase protein (APP) synthesis in the liver. A net breakdown of protein in skeletal muscle under the injury conditions is presumably driven by glucocorticoids; it could also be driven by decreased insulin-mediated amino acid uptake into the muscle.^51-53 An appropriate level of gluconeogensis and APP response in inflammatory conditions are presumably adaptive processes. However, if allowed to proceed in an uncontrolled manner, these altered metabolic responses lead to muscle wasting and an excessive inflammatory response. Studies from our laboratory have shown a Ca²⁺-signaling up-regulation, which can lead to increased Ca²⁺_i availability in the skeletal muscle during sepsis (Figure 2A). The sepsis-related Ca²⁺-signaling up-regulation was correlated with the activation of a Ca²⁺-dependent neutral protease, calpain, in the skeletal myocytes.^54-57 Studies by other investigators have also supported a causal role of Ca²⁺ in skeletal muscle protein breakdown during sepsis.^78-79

View larger version (24K): [in this window] [in a new window] Figure 2. Signaling assessments in skeletal myocytes (A), hepatocytes (B), neutrophils (C), and T lymphocytes (D) obtained from injured rats with and without diltiazem (DZ) or indomethacin treatments. The skeletal myocytes were obtained from septic rats given live Escherchia coli, and labeled with calcium 45 (45Ca) to assess Ca2+ fluxes (A). The hepatocytes were isolated from rats made septic via endotoxin administration (B). Hepatocyte cytosolic [Ca2+]i concentration was quantified using indomethacin-1 fluorometry. Blood neutrophils were isolated from burn-injured rats (25% total burn surface area skin scald), and [Ca2+]i quantified using fura-2 fluorometry (C). Neutrophil protein kinase C (PKC) activity was measured in the membrane fractions after its translocation from the cytosolic fraction. The T lymphocytes were obtained from rats that became septic after intra-abdominal implantation of pellets laden with E coli and Bacteroides fragilis. The T lymphocyte [Ca2+]i was measured using fura-2 fluorometry. The T-lymphocyte Fyn activation was assessed using immunoblot and in vitro kinase measurements. Diltiazem treatment, at 2 mg/kg, was given at the time of inoculation of bacteria or endotoxin. Indomethacin treatment, at 2 mg/kg, was given at the time of bacterial implantation and repeated at 24 hours.44-45,61-63,67

The skeletal myocyte protein catabolic response in injury conditions could be exacerbated by a concomitant development of glucose-uptake resistance to insulin. Evidently, a decrease in glucose availability in the skeletal muscle because of insulin resistance could promote use of the catabolism-derived amino acids as alternative fuel for cellular energy production. Insulin resistance was demonstrated in skeletal muscle in septic injury via measurements of insulin-dependent transport of a nonmetabolizable glucose analogue, 3-methyl glucose (3MG) (Figure 3A).^{43, 60-61} The 3MG flux was substantially suppressed in septic rats compared with sham rats. A role of Ca²⁺ signaling in the suppression of insulin-mediated 3MG transport is indicated from the observed effects of treatment of septic rats with the Ca²⁺ entry blocker diltiazem (DZ) at the therapeutic dose, 1-2 mg/kg.^60-61 Diltiazem serving as a blocker of voltage-operated Ca²⁺ channel can suppress Ca²⁺-induced Ca²⁺ release from the intracellular stores and thus down-regulate Ca²⁺ signaling. This action of DZ in septic rats was evident from the observed decrease in Ca²⁺ flux in the skeletal muscle of DZ-treated septic rats (Figure 2A). Because DZ simultaneously prevented the decrease in insulin-mediated 3MG flux in septic rat skeletal muscle (Figure 3A), we postulated that sepsis-related up-regulation of Ca²⁺ signaling causes the decrease in insulin-mediated glucose uptake, which leads to development of insulin resistance. Studies by other investigators have supported such a role of enhanced [Ca²⁺]_i in insulin resistance in the adipose tissue.⁷⁶ Insulin's action on skeletal myocytes is mediated through insulin-receptor PTK activation, followed by the formation of insulin-receptor substrate-1 and the subsequent activation of PI-3K (Figure 1A). The PI-3K pathway has been shown to be sensitive to Ca²⁺, such that an enhanced [Ca²⁺]_i can suppress PI-3K activation, which can effectively decrease GLUT4 protein expression and its incorporation into the sarcolemma. Thus a Ca²⁺ signal caused down-regulation of PI-3K pathway could lead to the skeletal myocyte's resistance to insulin. It should be pointed out that skeletal muscle stimulation via -adrenergic signal leads to activation of protein kinase C (PKC) signaling, which might up-regulate the mitogen-activated protein kinase (MAPK) pathway to augment GLUT4 expression (Figure 1A). This action, via -adrenergic signal, would seem to be counter to the augmented Ca²⁺ signaling–mediated inhibition of the insulin receptor substrate-1/PI-3K pathway. At present, one can only speculate that Ca²⁺-mediated inhibition of the PI-3K pathway has a more potent effect in down-regulating GLUT4 expression than the -adrenergic receptor–linked PKC-mediated GLUT4 up-regulation. A further support of the inhibitory effect of enhanced Ca²⁺ signaling on insulin-mediated skeletal muscle glucose uptake came from experiments in sham rats. Sham rat skeletal muscle 3MG transport was substantially decreased when the transport measurements were carried out in the presence of the compound BAYK8644.⁶² This compound is known to augment Ca²⁺ influx via sarcolemmal voltage-operated Ca²⁺ channel. The compound BAYK8644-caused downregulation of 3MG transport was prevented when sham rat skeletal muscle was pretreated with DZ. Diltiazem's effectiveness in preventing both the up-regulation of Ca²⁺ flux and the downregulation of insulin-mediated 3MG flux, during sepsis (in vivo) or in the presence of BAYK (in vitro), lends support to a linkage between enhanced Ca²⁺ signaling and insulin resistance in skeletal myocyte in the injury conditions.

View larger version (32K): [in this window] [in a new window] Figure 3. Cellular response assessments in sepsis or burn rats. The skeletal myocyte responses to Escherichia coli sepsis were evaluated via measurements of insulin-sensitive transport of nonmetabolizable sugar, 3-methyl glucose (3MG) (A). In hepatocytes, -1 acid glycoprotein (AGP) messenger RNA expression evaluated the acute phase protein (APP) response (B), shown as a percentage of measurements in unoperated-on, untreated control rats. Activation of neutrophil with burn injury was assessed by measuring superoxide free radical (O2-) production (C). The proliferation of T lymphocytes was measured after stimulation of isolated T cells with concanavalin A (D). The treatment of the sepsis/burn rats was carried out as described in the legend to Figure 2.44-45,61, 66

HEPATOCYTE SIGNALING TO GLUCOREGULATORY AND ACUTE-PHASE PROTEIN RESPONSES

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

Physiologic Mechanism

Hepatocytes are influenced by a variety of stimuli of origin in the endocrine and immune systems.⁸⁰ The hormones epinephrine, norepinephrine, vasopressin, angiotensin II, and glucagon modulate hepatocyte glycogenolysis and gluconeogenesis.⁷⁴ The signaling pathways for the glycogenolytic or gluconeogenic responses are mediated by Ca²⁺-linked as well as cAMP-linked pathways.^{74, 81} The -receptor activation via epinephrine and norepinephrine is coupled to a stimulatory G protein–linked activation of adenylate cyclase resulting in the formation of cAMP (Figure 1B). The subsequent activation of cAMP-dependent protein kinase A is implicated in the stimulation of the hepatocyte glycogenolytic enzyme, phosphorylase kinase. Epinephrine and norepinephrine, acting through the -adrenergic receptor, lead to the activation of G_q protein and PLC1, causing rapid generation of InsP₃ and diacylglycerol (DAG).⁸² Like cAMP, the InsP₃ that caused [Ca²⁺]_i elevation also activates phosphorylase kinase. Both cAMP and Ca²⁺ are responsible for a maximum glycogenolytic response. Vasopressin and angiotension II can also act through the G_q-coupled PLC1 stimulation and trigger the Ca²⁺ signal to effect glycogenolysis. Glucagon receptor activation in the liver also causes the generation of cAMP and produces the glycogenolytic response.

Glucagon and glucocorticoids stimulate phosphoenol pyruvate carboxy kinase (PEPCK) transcription, which is the first committed step in the up-regulation of hepatic gluconeogensis.⁸¹ While glucagon stimulates PEPCK through cAMP generation, glucocorticoid-mediated stimulation follows interactions with its cytosolic receptor. Insulin, on the other hand, has a dominant inhibitory effect on PEPCK transcription. The stimulation of gluconeogenesis is also dependent on the Ca²⁺ signal generated via vasopressin or angiotension II.⁸² Although recent studies have suggested that an activation of an MAPK could prevent cAMP-mediated stimulation of PEPCK transcription, there is no support for the concept that insulin-mediated inhibition of PEPCK transcription involves the MAPK pathway.⁸³ Involvement of a PI-3K pathway, however, seems essential in the inhibitory regulation of PEPCK transcription by insulin.

During inflammatory states, hepatocytes elicit the APP response, resulting in the formation of proteins that influence host defense. The APPs include fibrinogen (coagulant), -2 macroglobulin (antiprotease), C-reactive protein (opsonin), haptoglobin (hemoglobin carrier), -1 acid glycoprotein (immunosuppressant), and thiostatin (antiprotease). Although the APP response is generally taken to be adaptive to inflammation, the precise roles played by APPs are not known.⁸⁴ The hepatic APP response is stimulated by cytokines TNF-, interleukin 1 (IL-1), and IL-6, and by glucocorticoids.⁸⁵ Interleukin 6 can act through the transmembrane protein receptor to activate PTK of the Janus kinase (JAK) family.⁸⁶

The cytokine stimulation of hepatocytes may result from the activation of PTK, which in turn may or may not lead to generation of a Ca²⁺ signal through PLC and InsP₃ (Figure 1B).⁸⁷ Tumor necrosis factor and IL-1 share an ability to cause a translocation of the complex nuclear factor from cytosol to nucleus, where nuclear factor can bind to regulatory elements in the promoter regions of the APP genes.⁸⁸ Nuclear factor translocation could also be promoted by DAG resulting subsequent to PLC activation. In addition to its role of activating PKC, DAG activates the enzyme sphingomyelinase to cause release of ceramide, which is known to promote dissociation of nuclear factor from its inhibitor protein, I-, and its translocation to the nucleus. Interleukin 6 mediation of APP response involves phosphorylation of signal transduction activator protein (STAT) 3 protein by a JAK tyrosine kinase and translocation of STAT3 to the nucleus. Signal transduction activator protein 3 serves as the transcription factor for the transcription of APP genes.⁸⁹ Recent studies have suggested that insulin down-regulates STAT3 binding to APP gene-promoter region and thereby negatively modulates the hepatic APP response.⁹⁰

Pathophysiologic Alterations

Both observations in patients with critical injuries and studies in animal models of sepsis injury have indicated a pronounced up-regulation of gluconeogensis, which causes sustained hyperglycemia.^91-93 The runaway gluconeogensis is likely sustained by amino acids derived from excessive breakdown of muscle protein. Several studies support the concept that the altered gluconeogenic process is related to a parallel pronounced up-regulation in Ca²⁺ signaling in the septic injury conditions.^{36, 46-50} As shown in Figure 2B, compared with sham-rat hepatocytes, septic-rat hepatocytes exhibit substantially higher basal [Ca²⁺]_i. A role of the hormone-mediated Ca²⁺ mobilization into hepatocyte cytosolic compartment, through both receptor-operated Ca²⁺ channel and endoplasmic reticulum release of Ca²⁺, has been implicated in the up-regulation of gluconeogensis. Studies from our laboratory show that Ca²⁺ influx through the receptor-operated Ca²⁺ channel in hepatocytes could be blocked by DZ.^{44, 47-48} Although DZ at nanomolar concentrations is a specific blocker of the voltage-operated Ca²⁺ channel, it has been known to block receptor-operated Ca²⁺ channel at micromolar concentrations.^47-48 Our studies have supported DZ treatment of hepatocytes, in vitro, or DZ treatment of rats resulting in micromolar concentrations in circulation blocked Ca²⁺ influx into hepatocytes. We treated septic rats with DZ at 1-2 mg/kg dose, and found that such a treatment prevented the sepsis-related elevation of hepatocyte basal [Ca²⁺]_i (Figure 2B).

Figure 3B shows data taken from our studies on the hepatic APP response in the septic rats.⁵¹ The expressions of the APP -1 acid glycoprotein was up-regulated in the septic rats. Our studies showed sepsis caused up-regulation of several of the other APPs as well. Like its effect on basal [Ca²⁺]_i signaling, DZ treatment effectively prevented -1 acid glycoprotein up-regulation. The APP expressions are up-regulated by inflammatory mediators TNF-, IL-1, IL-6, and glucocorticoids. These mediators are expressed both within the liver and released systemically during sepsis and plausibly act on hepatocytes triggering Ca²⁺-independent activation of PTK. Moreover, catecholamines, vasopressin, and angiotension II, all of which are known to be released during sepsis, can act through the Ca²⁺-PKC pathway, and can potentiate the inflammatory mediator-induced APP response via the Ras-MAPK pathway. Our studies have also provided evidence that the pronounced up-regulation of the hepatic APP response in septic rats was accompanied by severe lactic acidosis and a significant liver tissue damage compared with that in the sham group of rats.⁵¹ The prevention of both [Ca²⁺]_i elevation and the pronounced APP response after treatment of septic rats with DZ provided evidence for a linkage between the heightened Ca²⁺ signaling and hepatic APP response during sepsis.

NEUTROPHIL SIGNALING TO O₂^- PRODUCTION RESPONSE

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  • Neutrophil signaling to o2-...  • T-lymphocyte signaling to...  • Summary  • Author information  • References

Physiologic Mechanisms

Neutrophils isolated from blood of healthy humans and animals respond to a variety of chemotactic agents (eg, formylated dipeptides and tripeptides, complement protein 5a [C5a], leukotriene B4 [LTB4], and IL-8) by generating superoxide anion (O₂^-).¹⁰ However, the response is of a much smaller magnitude than the potentiated response produced by the same neutrophils previously "primed" with either the same chemotactive agents at lower concentrations or other agonists, such as TNF- or granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumor necrosis factor and GM-CSF themselves do not cause a demonstrable production of O₂^-; their action seems to be a prerequisite for the generation of the potentiated response. The mechanisms by which neutrophils are first primed and then activated must reside in the signaling pathways that are turned on by the neutrophil priming and activating factors.

The chemotactic stimuli, formyl-methionyl-leucyl-phenylalanine, LTB4, C5a, and IL-8 act on neutrophils through interactions with their 7 transmembrane-domain receptors that are coupled to the pertussis toxin–sensitive inhibitory G protein (G_i). The activation of G_i causes its dissociation into the subunits, G_i and (Figure 1C). The subunit is implicated in the activation of the enzyme PLC, leading to formation of InsP₃ and DAG.⁹⁴ The InsP₃-mediated release of Ca²⁺ from an endoplasmic reticulum compartment gives rise to the Ca²⁺ signal. The DAG-mediated activation of the Ca²⁺-dependent PKC isoform plays a role in the phosphorylation and activation of the cytosolic proteins, p47phox and p67phox, which translocate to plasma membrane and complex with the O₂^--producing membrane enzyme NADPH oxidase.^95-96 The G_i subunit, resulting from the activation of G_i, is implicated in the activation of the PTK, Lyn, and the Shc protein, which in turn can activate the Ca²⁺-signal independent PI-3K and Ras-MAPK pathway⁹⁷ that leads to activation of the cytosolic protein Rac. In its activated form, Rac, like p47phox and p67phox, translocates to plasma membrane and complexes with the NADPH oxidase. The actions of TNF- and GM-CSF are also known to depend on the Ca²⁺-independent PTK activation coupling to both the PI-3K and Ras-MAPK pathways.⁹⁸

Pathophysiologic Alterations

Neutrophils play an important role in nonspecific host defense against pathogens invading the injured hosts. Such an adaptive role involves neutrophil activation and their chemotaxis from circulation to the tissue sites of pathogen invasion. At the site of invasion, the activated neutrophils phagocytize the pathogens and lyse the phagocytized pathogens by means of superoxide and related oxidants and proteases produced by the activated neutrophils. However, there is also evidence that neutrophils can become activated in circulation to such an extent that they can begin to release O₂^- into the extracellular spaces during their adhesion to and migration through the endothelium and extravascular interstitium.⁴² Such release of O₂^- can potentially cause oxidative damage to endothelial and interstitial cells, as well as to proteins within the interendothelial pores and interstitial extracellular matrix.⁴² The oxidative damage entails peroxidation of the membrane lipids and matrix proteins.

We have studied O₂^- production by neutrophils harvested from the blood of septic or burn rats.^{41, 45, 63-64} Both conditions led to increased levels of O₂^- production in early periods after the injury. There was a sizable increase in O₂^- production in the burn group compared with the sham (Figure 3C). The neutrophil basal [Ca²⁺]_i and PKC activity were also found to be up-regulated in the burn group, compared with the sham group (Figure 2A). The activation of neutrophil Ca²⁺-PKC pathway is implicated in the phosphorylation of cytosolic proteins, p47phox and p67phox, which are required for the assembly and activation of the O₂^--generating NADPH oxidase complex in the plasma membrane.⁴² In our studies, we have shown that up-regulation of Ca²⁺-PKC signaling and O₂^- production occurs along with increased expressions of p47phox and p67phox protein in early periods after burn injury.⁶² Our studies have shown that the presence of the burn injury–activated neutrophils in the proximity of endothelium causes microvascular injury.⁹⁹ There is also indication that migration of the neutrophils into target organs, such as lungs and intestine, causes oxidative tissue damage.⁶⁴

The elevated basal [Ca²⁺] and PKC activation in the burn injured rat neutrophils in all probability reflect that there was a sustained priming and stimulation of neutrophils in vivo. This could be because of endogenous mediators, such as TNF-, GM-CSF, C5a, platelet-activating factor, and IL-8.⁴² Whereas TNF- and GM-CSF prime neutrophils without involving Ca²⁺ signaling, the activation by C5a, platelet-activating factor, and IL-8 are dependent on both Ca²⁺-independent and Ca²⁺-PKC signaling. We noted that the treatment of burn rats with the Ca²⁺ entry blocker DZ was effective in preventing both the burn-induced elevations of Ca²⁺-PKC signaling (Figure 2C) and the heightened O₂^- production response (Figure 3C).⁴⁵ These observations suggested that Ca²⁺ blocker abrogated signaling alterations to prevent the heightened neutrophil.

T-LYMPHOCYTE SIGNALING TO PROLIFERATIVE RESPONSE

Jump to Section  • Top  • Introduction  • Ca2+i homeostatis and ca2+i...  • Signaling role of phospholipases...  • Signaling mechanisms of cellular...  • Skeletal myocyte signaling to...  • Hepatocyte signaling to...  •