Biomedical subjects

M S Liu

Publications and source records attributed to M S Liu.

At least 19 recordsLinked to original sources

PubMed2001

Calcium uptake by sarcoplasmic reticulum is impaired during the hypodynamic phase of sepsis in the rat heart.

Alterations of the ATP-dependent Ca2+ uptake in the cardiac sarcoplasmic reticulum (SR) during the 2 hemodynamically distinct phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham-operated. The SR vesicles were isolated by sucrose gradient centrifugation. The results show that the rates of ATP-dependent Ca2+ uptake in the cardiac SR were unaffected during the early hyperdynamic phase, whereas they were decreased by 41-46% (P < 0.01) during the late hypodynamic phase of sepsis. Analysis of the kinetics of Ca2+ transport indicates that during the late phase of sepsis, the Vmax values of Ca2+ pump for ATP and Ca2+ were decreased, whereas the affinities of Ca2+ pump for ATP and Ca2+ were unaffected. Magnesium stimulated, whereas vanadate inhibited the ATP-dependent Ca2+ uptake, but the Mg2+-stimulated and the vanadate-inhibited Ca2+ uptake activities were significantly lower during the late sepsis. Phosphorylation of SR by the cAMP-dependent and the calmodulin-dependent protein kinases stimulated the ATP-dependent Ca2+ uptake in the control and the early septic experiments, whereas it failed to stimulate Ca2+ uptake in the late sepsis. The extent of the phosphorylation-stimulated Ca2+ uptake activities was reduced by 65-69% (P < 0.01) during the early sepsis, and they were completely abolished during the late sepsis. These data indicate that the ATP-dependent Ca2+ uptake in cardiac SR was impaired during the late hypodynamic phase of sepsis. The impaired Ca2+ uptake during late sepsis was associated with a defective phosphorylation of SR proteins. Because the ATP-dependent Ca2+ uptake by cardiac SR plays an important role in the regulation of contraction-relaxation coupling, our findings may contribute to the understanding of the pathogenesis of altered cardiac function during the progression of sepsis.

Adenosine Triphosphate↗

PubMed2001

Impairment of the ryanodine-sensitive calcium release channels in the cardiac sarcoplasmic reticulum and its underlying mechanism during the hypodynamic phase of sepsis.

Changes in Ca2+-induced Ca2+ release in cardiac sarcoplasmic reticulum (SR) during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The 45Ca2+ release studies show that the amount of Ca2+ released from the passively and the actively loaded SR vesicles was unaffected during the early sepsis (9 h after CLP), but it was significantly decreased during the late phase (18 h after CLP) of sepsis. The [3H]ryanodine binding assays reveal that the Bmax for ryanodine binding was unaffected during the early phase, but was decreased by 32.1% during the late phase of sepsis. The affinity of ryanodine receptor for Ca2+ remained unchanged during sepsis. ATP, AMP-PCP, and caffeine stimulated binding, while MgCl2 and ruthenium red inhibited [3H]ryanodine binding in control, early sepsis, and late sepsis groups. The EC50 and IC50 values for these regulators were unaffected during the progression of sepsis. Digestion of control SR with phospholipase A2 decreased [3H]ryanodine binding and the decrease was reversible by the addition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS). Addition of PC, PE, or PS to the SR isolated from septic rats stimulated [3H]ryanodine binding. These data demonstrate that Ca2+-induced Ca2+ release from cardiac SR remained relatively unaffected during the early phase, but was significantly impaired during the late phase of sepsis. The sepsis-induced impairment in SR Ca2+ release is a result of a quantitative reduction in the number of Ca2+ release channels. Furthermore, the reduction is associated with a mechanism involving a modification of membrane lipid profile in response to certain stimuli such as activation of phospholipase A2.

Adenosine Triphosphate↗

PubMed2001

Altered phosphorylation and calcium sensitivity of cardiac myofibrillar proteins during sepsis.

Altered phosphorylation and Ca(2+) sensitivity of cardiac myofibrillar proteins during different phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). The results show that phosphorylation of troponin I (TnI) was increased by 268% during the early phase (9 h after CLP) but decreased by 46% during the late phase (18 h after CLP) of sepsis. Phosphorylation of C protein was increased by 76% during the early phase but decreased by 41% during the late phase of sepsis. Phosphorylation of myosin light chain-2 (MLC-2) remained unaltered during the early phase but was decreased by 38% during the late phase of sepsis. Phosphorylation of TnT was unaffected during the progression of sepsis. The increases in the phosphorylation of TnI and C protein during early sepsis were associated with the decrease in the Ca(2+) sensitivity of myofilaments and the increases in myocardial changes in tension development (+dP/dt(max)) and cAMP level. The decreases in the phosphorylation of TnI and C protein during late sepsis coincided with the declines in the activities of myofibrillar ATPase, Ca(2+) sensitivity of myofilaments, myocardial +/-dP/dt(max), and cAMP content. The increases and the decreases in the phosphorylation of TnI and C protein, +/-dP/dt(max), and the tissue cAMP level were sensitive to isoproterenol stimulation and propranolol inhibition. These findings suggest that alterations in the phosphorylation of myofibrillar proteins, such as TnI, C protein, and MLC-2, and changes in the activities and the Ca(2+) sensitivity of myofibrillar ATPase may contribute to the altered cardiac function during the progression of sepsis. Furthermore, the sepsis-induced alterations in the phosphorylation and Ca(2+) sensitivity of cardiac myofibrillar proteins were mediated via a beta-adrenergic receptor pathway.

Adenosine Triphosphatases↗

PubMed2000

Biphasic redistribution of muscarinic receptor and the altered receptor phosphorylation and gene transcription are underlying mechanisms in the rat heart during sepsis.

OBJECTIVE: The purpose of this study was to investigate intracellular redistribution of muscarinic cholinergic receptor (m2AChR) and the roles of receptor phosphorylation and gene transcription as underlying mechanisms in the rat heart during different phases of sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). The density of m2AChR in the sarcolemmal and light vesicle fractions was studied using [3H]-quinuclidinyl benzilate ([3H]-QNB). Phosphorylation of m2AChR was studied by labeling of the myocardial ATP pool by perfusing isolated hearts with [32P]H3PO4 followed by identification of the phosphorylated m2AChR with SDS-PAGE. The steady-state level of m2AChR mRNA was determined by RT-PCR and Southern blot analysis. RESULTS: Septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP, early sepsis) and a subsequent hypocardiodynamic (18 h after CLP, late sepsis) state. During early sepsis, the Bmax for [3H]-QNB binding was increased in sarcolemma (+69%) but decreased in light vesicles (-22%), whereas during late sepsis, the Bmax was decreased in sarcolemma (-20%) but increased in light vesicles (+32%). The sum of Bmax for sarcolemmal and light vesicle fractions was increased during early sepsis (+43%) but decreased during late sepsis (-14%). The phosphorylation of m2AChR was decreased during early sepsis (-73%) but increased during late sepsis (+36% to +90%). The m2AChR mRNA abundance was increased during early sepsis (+52%) but decreased during late sepsis (-28%). CONCLUSIONS: The m2AChR in the rat heart was externalized from light vesicles to sarcolemma (overexpression) during early sepsis but internalized from surface membranes to intracellular sites (underexpression) during late sepsis. Furthermore, changes in the receptor phosphorylation and gene transcription are responsible for the biphasic redistribution and the altered expression of m2AChR in the rat heart during the progression of sepsis.

Analysis of Variance↗

PubMed2000

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection.

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.

Automation↗

PubMed2000

Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection.

DNA amplification technology has been applied to clinical diagnosis of infectious disease, genetic disorder, and cancer. After in vitro amplification of a particular DNA region, the methods of analysis for these amplified samples play a pivotal role in clinical diagnosis. Conventional gel electrophoresis has been routinely used in the lab for checking DNA. The whole procedure is time consuming and requires more than 1 ng of DNA for detection. To achieve greater performance in DNA diagnosis, we demonstrated capillary electrophoresis with laser induced fluorescence detection for analysis of amplified DNA. The analysis of DNA could be completed within 3 min and the data is directly entered into the computer. Considering the automatic and rapid process, we believe that this method could be routinely utilized for the clinical diagnosis of amplified DNA products.

DNA↗

PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and is involved in atherosclerosis. ACAT-1 protein is located mainly in the ER. The hydropathy plot suggests that ACAT-1 protein contains multiple transmembrane segments. We inserted either the hemagglutinin tag or the HisT7 tag at various hydrophilic regions within the human ACAT-1 protein and used immunofluorescence microscopy to determine the topography of the tagged proteins expressed in mutant Chinese hamster ovary cells lacking endogenous ACAT. All of the tagged proteins are located mainly in the ER and retain full or partial enzyme activities. None of the tagged proteins produces detectable intracellular degradation intermediates. Treating cells with digitonin at 5 micrograms/ml permeabilizes the plasma membranes while leaving the ER membranes sealed; in contrast, treating cells with 0.25% Triton X-100 or with cold methanol permeabilizes both the plasma membranes and the ER membranes. After appropriate permeabilization, double immunostaining using antibodies against the N-terminal region and against the inserted tag were used to visualize various regions of the tagged protein. The results show that human ACAT-1 in the ER contains seven transmembrane domains.

Algorithms↗

PubMed1999

Transcriptional regulation of alpha1-adrenoceptor gene in the rat liver during different phases of sepsis.

Changes in alpha1-adrenoceptor (alpha1AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic phase (9 h after CLP, early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [3H]prazosin binding studies show that the density of alpha1AR was increased by 30% during the early phase while it was decreased by 24% during the late phase of sepsis. Western blot analyses reveal that alpha1AR protein level was elevated by 48% during early sepsis but was decreased by 55% during late sepsis. Northern blot analyses depict that the steady-state level of alpha1bAR mRNA was enhanced by 21% during the early phase but was declined by 29% during the late phase of sepsis. Nuclear run-off assays show that the transcription rate of alpha1bAR gene transcript was increased by 76% during early sepsis while it was decreased by 29% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of alpha1bAR mRNA remained unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the increase in the rate of transcription of alpha1bAR gene paralleled with the elevations in the alpha1bAR mRNA abundance and alpha1AR protein level, while during the late phase of sepsis, the decrease in the rate of transcription of alpha1bAR gene coincided with the declines in the alpha1bAR mRNA abundance and the alpha1AR protein level in the rat liver. These observations indicate that the altered expression of alpha1AR genes in the rat liver during the progression of sepsis was regulated transcriptionally.

Adrenergic alpha-Antagonists↗

PubMed1999

Alterations of G-protein and adenylate cyclase signaling in rat liver during the progression of sepsis.

Changes in the protein level of various subunits of G-protein and the activity of adenylate cyclase in rat liver plasma membranes during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that the protein levels of Galphai-2 and Galphai-3 were unchanged during the early hypermetabolic (hyperglycemic) phase (9 h after CLP), whereas Galphaai-2 and Galphaai-3 were increased by 32.4 and 59.1%, respectively, during the late hypometabolic (hypoglycemic) phase (18 h after CLP) of sepsis. The protein levels of Galphas and Gbeta remained unaltered during both the early and the late phases of sepsis. The activity of adenylate cyclase remained unchanged during the early phase, whereas it was decreased by 26% (p < .05) during the late phase of sepsis. Since the G-protein/adenylate cyclase signaling system mediates hormonal control of hepatic glucose metabolism, the observed increases in the Galphai-2 and Galphai-3 protein levels coupled with a decrease in the activity of adenylate cyclase may contribute to the development of the hypoglycemia during the late stage of sepsis.

Adenylyl Cyclases↗

PubMed1999

Liver protein kinase A activity is decreased during the late hypoglycemic phase of sepsis.

Changes in protein kinase A (PKA, or cAMP-dependent protein kinase) activity in the rat liver during different metabolic phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into 3 groups: control, early sepsis, and late sepsis. Early and late sepsis refer to those animals killed at 9 and 18 h, respectively, after CLP. Hepatic PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and diethylaminoethyl (DEAE)-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two peaks of PKA, type I (eluted at low ionic strength) and type II (eluted at high ionic strength), were collected and their activities were determined on the basis of the rate of incorporation of [gamma-32-P]ATP into histone. The results show that during early sepsis, both type I and type II PKA activities remained unchanged. During late sepsis, type I PKA activity was decreased by 40.7-53.6%, whereas type II PKA activity was unaffected. Kinetic analysis of the data on type I PKA during the late phase of sepsis reveals that the Vmax (maximal velocity) values for ATP, cAMP, and histone were decreased by 40.7, 53.6, and 47.3%, respectively whereas the Km (substrate concentration required for half-maximal enzymatic activity) values for ATP, cAMP, and histone were unaltered. These data indicate that type I PKA was inactivated during the late hypoglycemic phase of sepsis in the rat liver. Because PKA-mediated phosphorylation plays an important role in the regulation of hepatic glucose metabolism, an inactivation of PKA may contribute to the development of hypoglycemia during the late phase of sepsis.

Animals↗

PubMed1999

Transcriptional and posttranscriptional regulation of beta(2)-adrenergic receptor gene in rat liver during sepsis.

Changes in beta(2)-adrenergic receptor (beta(2)-AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic (9 h after CLP; early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [(3)H]dihydroalprenolol binding studies show that the density of beta(2)-AR was decreased by 12 and 35% during the early and late phases of sepsis, respectively. Western blot analyses depict that the beta(2)-AR protein level was reduced by 37 and 72% during early and late sepsis, respectively. The reverse transcription polymerase chain reaction and Southern blot analyses reveal that the steady-state level of beta(2)-AR mRNA was decreased by 37% during early phase and 77% during late phase of sepsis. Nuclear run-off assays show that the rate of transcription of beta(2)-AR mRNA was reduced by 36% during early sepsis and 64% during late sepsis. The stability assays indicate that the half-life of beta(2)-AR mRNA was shortened by 21 and 50% during the early and late phases of sepsis, respectively, indicating that the rate of degradation of beta(2)-AR mRNA was progressively enhanced during sepsis. These findings demonstrate that the beta(2)-AR gene was underexpressed in the liver during the progression of sepsis, and, furthermore, the underexpression of the beta(2)-AR gene was the result of a reduction in the rate of transcription coupled with an enhancement in the rate of degradation of beta(2)-AR gene transcripts. Thus our findings that the transcriptional and posttranscriptional regulation of beta(2)-AR gene associated with decreases in beta(2)-AR number and its protein expression may provide a molecular mechanistic explanation for the development of hypoglycemia during the late stage of sepsis.

Animals↗

PubMed1998

Progressive internalization of beta-adrenoceptors in the rat liver during different phases of sepsis.

Changes in the distribution of beta-adrenoceptors (beta ARs) in the plasma membrane and the light vesicle fractions of rat liver during different phases of sepsis were studied using [3H]dihydroalprenolol binding and photoaffinity labeling with [125I]iodocyanopindolol diazirine. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit an initial hypermetabolic (hyperglycemic) phase (9 h after CLP; early sepsis) followed by a hypometabolic (hypoglycemic) phase (18 h after CLP; late sepsis). The radioligand studies show that in the plasma membranes, the density of beta ARs was decreased by 28-32% and 46-69% during the early and the late phases, respectively, of sepsis. In the light vesicles, the density of beta ARs was increased by 25-30% and 30-35% during the early and the late phases, respectively, of sepsis. The total number of the receptor binding sites (the sum of that in plasma membrane plus light vesicle) was decreased by 11-12% and 21-35% during the early and the late phases, respectively, of sepsis. These results indicate that beta ARs were progressively internalized from surface membranes to the intracellular sites and, furthermore, they were underexpressed in the rat liver during the progression of sepsis. Since hepatic glucose metabolism is known to be regulated by catecholamines, in part, through beta AR mediation, an internalization/underexpression of hepatic beta ARs may play a role in the altered glucose homeostasis during sepsis, particularly in the late hypometabolic phase of sepsis.

Affinity Labels↗

PubMed1998

Phospholipase A2 activities are decreased during early but increased during late phases of sepsis in rat heart.

BACKGROUND: Changes in the activities of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in the rat heart during early hyperdynamic and late hypodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. PLA2 activity was measured based on the rate of hydrolysis of 1-palmitoyl-2-[1-(14)C]-oleoyl phosphatidylcholine. RESULTS: The results show that under physiological conditions, sPLA2 and cPLA2 activities were time and protein dependent. The optimal Ca2+ concentrations for sPLA2 and cPLA2 activities were 3 mM and 40 microM, respectively. During sepsis, sPLA2 activity was decreased by 25% (P < 0.01) during early phase while it was increased by 49% (P < 0.01) during late phase of sepsis. Similarly, cPLA2 activity was decreased by 23% (P < 0.01) during early sepsis while it was increased by 60% (P < 0.01) during late sepsis. CONCLUSIONS: Since PLA2 functions to maintain cell membrane integrity and function, a biphasic change in sPLA2 and cPLA2 activities may contribute to the development of the two cardiodynamically distinct phases during the progression of sepsis.

Animals↗

PubMed1998

GTP-binding protein mediated phospholipase A2 activation in rat liver during the progression of sepsis.

Effects of GTP-binding proteins on the activation of secretory phospholipaseA2 (sPLA2) and cytosolic phospholipaseA2 (cPLA2) in rat liver during two different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The results show that in the absence of G-protein modulator, hepatic sPLA2 and cPLA2 activities were activated by 40.8-46 and 91.6-105.8%, respectively, during early and late phases of sepsis. GTPgammaS and fluoroaluminate (AlF4-) stimulated sPLA2 and cPLA2 activities within each experimental group, i.e., control, early sepsis, and late sepsis. The GTPgammaS and AlF4(-)-stimulated sPLA2 and cPLA2 activities remained significantly elevated during early phase (22.3-65.6% increase) and late phase (32.5-109.1% increase) of sepsis. Further analyses demonstrate that cholera toxin significantly stimulated sPLA2 and cPLA2 activities within each experimental group, and that the cholera toxin stimulated sPLA2 and cPLA2 activities remained significantly higher during early phase (23.5-37% increase) and late phase (56.7-70% increase) of sepsis. In contrast, pertussis toxin significantly inhibited sPLA2 and cPLA2 activities within each experimental group, and that the pertussis toxin-inhibited sPLA2 and cPLA2 activities remained significantly higher in early septic (57-68.5% increase) and late septic (34.6-45.5% increase) experiments. These data demonstrate that cholera toxin-sensitive G alpha s and pertussis toxin-sensitive G alpha i were both involved in the activation of sPLA2 and cPLA2 activities in rat liver during the progression of sepsis.

Animals↗

PubMed1998

Inactivation of protein kinase C in rat liver during late hypoglycemic phase of sepsis.

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat liver during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. Hepatic PKC was extracted and partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. PKC activity was assayed based on the rate of incorporation of 32p from [gamma-32P]ATP into histone. The results show that during early sepsis, both membrane-associated and cytosolic PKC activities remained relatively unaltered. During late sepsis, membrane-associated PKC was unaffected while cytosolic PKC activity was decreased by 19.5-34.4%. Kinetic analysis of the data on cytosolic PKC during late phase of sepsis reveals that the Vmax values for ATP, histone, Ca2+, phosphatidylserine, and diacylglycerol were decreased by 23.4, 22.1, 19.5, 25, and 34.4%, respectively, with no changes in their Km values. These data indicate that cytosolic PKC activity was inactivated in rat liver during late hypoglycemic phase of sepsis. Since PKC-mediated phosphorylation plays an important role in regulating hepatic glucose metabolism, an inactivation of cytosolic PKC may contribute to the development of hypoglycemia during late phase of sepsis.

Animals↗

PubMed1998

Protein kinase C activity is increased in rat heart during the early hyperdynamic phase of sepsis.

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat heart during different cardiodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of altered myocardial function during sepsis. Sepsis was induced by cecal ligation and puncture. Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after cecal ligation and puncture. Cardiac PKC was extracted and partially purified by ammonium sulfate fractionation and diethylaminoethyl-cellulose chromatography. PKC activity was assayed on the basis of the rate of incorporation of 32P from [gamma-32P]adenosine triphosphate into histone. The results show that during early sepsis, cytosolic PKC activity was increased by 42-73%, whereas membrane associated PKC activity was unchanged. During late sepsis, both cytosolic and membrane associated PKC activities remained unchanged. Kinetic analysis of the data on cytosolic PKC during the early phase of sepsis reveals that the Vmax (maximal velocity) values for Ca2+, phosphatidylserine, and diacylglycerol were increased by 58, 42, and 50%, respectively, with no changes in their Km (substrate concentration required for half-maximal enzyme activity) values. These data indicate that cytosolic PKC activity was activated in rat heart during the early hyperdynamic phase of sepsis. Because PKC mediated phosphorylation plays an important role in regulating myocardial contractility, an activation in cytosolic PKC may contribute to the development of a hypercardiodynamic state during the early phase of sepsis.

Adenosine Triphosphate↗

PubMed1998

Phosphorylation of beta-adrenergic receptor leads to its redistribution in rat heart during sepsis.

The role of receptor phosphorylation on the redistribution of beta-adrenergic receptors (beta-ARs) in rat hearts during different phases of sepsis was investigated. Sepsis was induced by cecal ligation and puncture (CLP). Changes in the distribution of beta-ARs in the sarcolemmal and light vesicle fractions were studied using (-)-[4,6-propyl-3H]dihydroalprenolol ([3H]DHA). Phosphorylation of beta-ARs was studied by perfusing hearts with [32P]H3PO4 followed by identification of the phosphorylated beta-ARs with immunoprecipitation using anti-beta 1-AR antibody. The results show that septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP; early sepsis) and a subsequent hypocardiodynamic (18 h after CLP; late sepsis) state. [3H]DHA binding studies show that, during early sepsis, the maximum binding capacity (Bmax) was increased by 26% in sarcolemma but was decreased by 30% in light vesicles, whereas, during late sepsis, the Bmax was decreased by 39% in sarcolemma but increased by 31% in light vesicles. These data indicate that beta-ARs in the rat heart were externalized from light vesicles to sarcolemma during early sepsis but were internalized from surface membranes to intracellular sites during late sepsis. The immunoprecipitation studies reveal that the externalization of beta-ARs during early sepsis was coupled with a concomitant decrease (-28.5 to -30.6%, P < 0.01) in the receptor phosphorylation, whereas the internalization of beta-ARs during late sepsis was accompanied by a simultaneous increase (30.3 to 33.8%, P < 0.01) in the receptor phosphorylation. Because the phosphorylation/dephosphorylation of beta 1-ARs regulate their functional coupling and may reflect their subcellular distribution, it is suggested that the increase in receptor phosphorylation seen in late sepsis leads to the receptor internalization observed in late sepsis; similarly, externalization of (dephosphorylated) receptors in early sepsis may give rise to the apparent decrease in sarcolemmal receptor phosphorylation observed during this interval.

Adenosine Triphosphate↗

PubMed1997

Differential activation of protein kinase A in various regions of myocardium during sepsis.

Changes in the activities of protein kinase A (PKA) (cAMP-dependent protein kinase) in various regions of rat myocardium during different cardiodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 hr, respectively, after CLP. Cardiac PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and DEAE-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two types of PKA, Type I (eluted at low ionic strength) and Type II (eluted at high ionic strength), were collected, and their activities were determined based on the rate of incorporation of [gamma-32P]ATP into histone. Under physiological conditions, Type I PKA activities were unevenly distributed (left atrium > right atrium > pacemaker region > left ventricle > right ventricle > ventricular septum) while Type II PKA activities were evenly distributed among different regions of myocardium. During early sepsis, Type I PKA activities remained unchanged while Type II PKA activities were activated by 32 and 70% in right atrium and pacemaker regions, respectively. During late sepsis, Type I PKA activities were stimulated by 228% in ventricular septum while Type II PKA activities were not affected. These data demonstrate that different PKA activities exist in various regions of the myocardium and that PKA activities were preferentially activated in certain areas during the progression of sepsis. Since PKA plays an important role in the regulation of myocardial function and metabolism, the activation of PKA in different regions of myocardial during different stages of sepsis may contribute to the altered cardiac function during the progression of sepsis.

Animals↗