Changes of adenosine triphosphate-dependent calcium uptake in microsomal fractions of rat liver during sepsis

Changes of adenosine triphosphatedependent calcium uptake in microsomal fractions of rat liver during sepsis Tsann-Long Maw-Shung

Hwang, MD, Ying-Tung Lau, PhD, Ming-Miig Liu, PhD, Taipei, Taiwan, and St. Louis, MO.

Tsai,

BS, and

Background. Intracellular calcium concentration is an important regulator of cellular metabolism. Endoplasmic reticulum membranes play an important role in the regulation of qtoplasmic calcium in the mammalian liver. The characterization of the changes of calcium uptake in endoplasmic reticulum may contribute to the potential intracellular mechanisms for cellular dysfunction during sepsis. Methods. The effects of sepsis on the calcium uptake in rough endoplasmic reticulum of rat liver were studied. Sepsis was induced by means of cecal ligation and puncture (CLP). The control rats underwent sham operation. Microsomal fractions were isolated from the liver with differential centrifugation. Results. The calcium uptake by liver endoplasmic reticulum was decreased by 30 % to 35 % (p c 0.05) during early sepsis (9 hours after CLP) and by 38% to 43 % (p < 0.05) during late sepsis (18 hours after CLP), respectively. The maximum velocity values for adenosine triphosphate (ATP) and for Ca” were also decreased by 25 % to 37% (p < 0.05) d uring early sepsis and by 35 % to 42 % (’ < 0.05) during late sepsis. The Michaelis-Menten constant for ATP and Ca” transport had no difference among three groups. The magnesium stimulation and vanadate inhibitgr activity were also decreased by 17% to 38 % (p < 0.05) during early sepsis and by 34 % to 50 % (‘ < 0.05) during late sepsis. Conclusions. These data demonstrate that ATP-dependent calcium uptake in rough endoplasmic reticulum of rat liver was impaired during early and late sepsis. Because the low intracellular calcium concentration plays an important role in the regulation of cellular function, an impairment in the ATP-dependent calcium uptake by endoplasmic reticulum during early and late sepsis may have a pathophysiologic significance in contributing to the development of altered hepatic metabolism during sepsis. (Surgery 1997;121:662-7.) From the Department of Surgq & Physiology, Chang Gung Memorial Medical College, Taipei, Taiwan, and Dqatiment of Pharmacological Louis University School of Medicine, St. Louis, MO

SEPSIS IS THE MOST COMMON cause Of XUIkipk organ failure (MOF) . The endotoxin mediates the pathophysiologic changes associated with sepsis that are known on the basis of extensive in vivo animal studies. Although the macrophage hypothesis, microcirculatory hypothesis, and gut hypothesis provide more understanding of the pathophysiology of MOF,l the cellular dysfunctions that may precede MOF are still mostly unclear. Recently, graded changes of cellular calcium activity (calcium sig-

Supported Gung 31664

by grants NSCSZ-0419-182-061 Memorial Hospital and National and HL30080.

Accepted

for publication

Jan.

and Institutes

CMRP 379 of Chang of Health grants GM-

7, 1997.

Reprint requests: Tsann-Long Hwang, MD, Department of Surgery, Chang Gung Memorial Hospital, 199, Tung-Hwa N. Rd., Taipei, Taiwan, R.O.C. Copyright

0 1997

0039-6060/97/$5.OOtO

662

SURGERY

by Mosby-Year 11/56/80395

Book,

Inc.

Hospital, Chang Gung & Physiological Science,

St.

nals) have been recognized as major factors of the regulation of cellular functions that are involved in several pathophysiologic conditions including sepsis. Calcium signals are generated by transient or sustained changes in the concentration of calcium ions. The cytosolic calcium ion concentration in the resting liver is only a small fraction of the total cellular calcium that is the sum of this ion in different po01,~, 3 comprising the mitochondrial matrix space, the endoplasmic reticulum, the cytosol, calcium-binding proteins4 the inner and outer leaflets of the plasma membrane with the glycocalyx,‘-* the Golgi complex,8~g the lysosomes,lo and secretory granules. The maintenance of different calcium activity gradients among the various compartments and graded and coordinated changes are basic requirements for normal cellular function. The calcium activity gradient in the hepatocyte from the cytosol to the extracellular space is in the range of 1 to 10,000 in the resting state and can decrease to 1 to

suw? Volume 121, Number

Hwang

1000 in the stimulated state. The large gradients are maintained mainly by the very low calcium permeability of the plasma membrane. We have found that the adenosine triphosphate (ATP)-dependent Ca*+ transport, which plays an important role in the regulation of intracellular Ca2+ homeostasis in hepatocytes, by plasma membranes was decreased by 30% to 50% during late sepsis. I1 Such changes could exert a pathophysiologic significance via altering hepatic glucose metabolism during septic shock. Endoplasmic reticulum (ER) membranes are also postulated to play a role in the regulation of the levels of cytoplasmic calcium in the mammalian liver. Active calcium uptake into vesicles derived from the ER of liver, coupled with a Ca*+-ATPase, was first described in 1975.l* The Ca*+-ATPase has been identified by the demonstration of its phosphorylated intermediate in ratr515 and human liver microsomes.16 ATP-dependent Ca*+ sequestration was reported to be sensitive to oxidative damage.l’ Thle present study was designed to compare the properties of ATP-dependent calcium uptake from rat liver ER during early and late sepsis. The characterization of the changes of calcium uptake of the ER should shed more light on the potential intracellular mechanisms, for cellular dysfunction during early and late sepsis. MATERIAL

AND

et al.

663

6

METHODS

Animal model. Male Sprague-Dawley rats weighing 275 to 325 gm were used. They were fed and observed for at least 5 days before the experiment; the diseased rats with body weight loss were excluded. All animals were fasted overnight with free access to water before the study. They were randomly placed into three groups: early sepsis, late sepsis, and control group for experiments. Induction of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et all8 with minor modification.“~ lg After ether anesthesia was induced, a midline laparotomy was performed, and the cecum was ligated with 3-O silk ligation and punctured twice with an l&gauge needle. Then the cecum was returned to the peritoneal cavity, and the abdomen was closed in two layers. Control rats underwent sham operation. All animals were injected with 4 ml/100 gm body weight normal saline solution after operation and also 7 hours after operation. Animals were fasted but had free access to water after operative procedures. Nine or 18 hours after CLP or sham operation, the septic and control animals were given anesthetic again and their livers were removed. Early and late sepsis refers to those animals killed at 9 and 18 hours after CLP, respectively. All procedures involving rats were performed according to the guidelines of the Na-

tional Institutes of Health “Guide for the Care and use of Laboratory Animals. ” Preparation of rough and smooth ER of rat liver. Microsomal fractions were isolated from the liver. Three microsomal fractions, one mainly corresponding to the rough ER (roughs microsomes), another to intermediate part, and the other to the smooth I ER (smooth I microsomes), were prepared as described by Dallner. *‘s21 Briefly, the liver was removed and homogenized. Twenty-five percent (w/v) liver homogenates were prepared by five passes in a glass homogenizer tightly fitted with a motor-driven Teflon pestle. The homogenates were (diluted 1:lO (v/v) with homogenous buffer and then centrifuged at 1000 gfor 10 minutes. The supernatant was centrifuged at 8000 g for 20 minutes. The postmitochondrial supernatant was sedimented at 104,100 gfor 70 minutes, and the pellet was resuspended in 24 ml homogenous buffer. A 6 ml sample was layered on top of 5 ml 0.6 mol/L sucrose, 6 ml 0.75 mol/L sucrose, and 10 ml 1.3 mol/L sucrose (containing 15 mmol,/L CsCl and 5 mmol/L HEPES, pH 7.2). Smooth I microsomes (at the 0.75/1.3 mmol/L sucrose interface), intermediate microsomes (at 1.3 mol/L sucrose), and rough microsomes (pellet) were separated, recovered, diluted, or resuspended (10 ml, final volume) with 100 mmol/L KCl, 20 mmol/L NaCl, 10 mmol/L HEPES, pH 7.2, and centrifuged at 104,100 gfor 70 minutes. The resulting pellets were resuspended in 0.25 mol/L sucrose and 5 mmol/L HEPES, pH 7.2. Marker enzyme characterizations for rough and smooth ER subfractions. Purity of fraction was monitored by measuring glucose-6phosphatase and 5’-nucleotidase, which was determined by a calorimetric method.** The optical density at 660 nm in the enzyme activities from spectrophotometer was recorded (Table I). The ER subfractions with ideal marker enzyme activity were used for the following studies. Measurement of calcium uptake in rough ER. The rough ER subfractions were individually prepared in each of the three experimental groups. All subfractions were derived from a single rat, and each group consisted of six rats. The microsomal fractions were incubated at 37” C in a medium that has the following composition (mmol/L): KCl, 120; MgCls, 4; NaNs, 5; oxalate, 3; HEPES/KOH, 40; pH 7.2, in the presence of 5 mmol/L ATP and an ATP-regenerating system (5 mmol/L creatine phosphate and creatine phosphokinase, 5 pmol/L units/ml), 20 pmol/L CaCls, and 0.1 pCi/ml [45Ca] Cls. The incubation was started by the addition of a small volume (l/50 of the volume of the incubation mixture) of the microsomal suspensions to the prewarmed (5 minutes at 37” C) complete medium to achieve 0.08 to 0.1 mg microsomal protein/ml. Measurements of calcium uptake under different conditions. The dependence of calcium uptake on var-

664

Hwang

SUFY

et al. June

Table.

Marker

enzymes of subfractions 5’-Nucleotidase

6-

5

0

Control

0 A

Early Sepsis Late Sepsis

10

Time

15

(min)

Fig. 1. Effect of sepsis on time course ofATPdependent uptake in rat liver ER. Incubation on abscissa. Vertical bars represent

Ca2+ time was varied as indicated SEM. *p < 0.01. #p < 0.05.

Control Smooth I Intermediate Rough Homogenate Early sepsis Smooth I Intermediate Rough Homogenate Late sepsis Smooth I Intermediate Rough Homogenate

Glucose-6-bhosfihate

part

0.90 0.41 0.43 0.09

11.89 12.49 14.95 4.15

part

16.20 ? 1.87 5.54 I 0.87

13.34 11.33

part

5.88 t 1.20 4.42 t 0.65

part part

Data are mean 2 SEM. Enzyme activities are expressed each group.

13.82 5.16 4.61 3.30

2 -+ t ?

of ER

15.66 4.82 5.82 5.01

part

? L c t

in pmol/mg/hr.

1997

0.81 0.65 0.40 0.20 Number

f ? ? ‘-’

0.59 0.59 0.75 0.14

i t 13.72 c 4.53 ?

0.54 0.52 1.18 0.40

11.85 13.45 14.84 4.38

0.28 0.69 1.32 0.39

+ + 2 t-

of experiments

is 6 for

RFSULTS 5

4-

1 #

*

f

&l

$g&

*

*

* I-

WI/

i/ * o!

-

I

.

1

0

I

.

2

I

3

.

0

Control

0

Earlysspsis

A

Late sepsis 1

.

4

Mg*Coneentration(mM) Fig. 2. Effect liver

of Mg’+

ER of septic

on ATP-dependent

and control

groups.

Ca2’ uptake

I

5

in rat

*p < 0.01. #p < 0.05.

ious uptake times and different concentrations of magnesium or ATP in the three groups were measured. The inhibition effect of vanadate on the uptake of calcium was also measured with different concentrations of vanadate. The calcium uptake with or without the presence of glucose&-phosphate in the smooth I and rough ER at 2 and 20 minutes were also compared among the three groups. Statistics. All data were expressed as mean -C-SEM. One-way ANOVA followed by multiple comparison procedures were used for statistical analysis. A p value less than 0.05 was considered statistically significant.

The activities of marker enzymes in subfractions of ER among the three groups are shown in Table I. The activity of 5’-nucleotidase in smooth I subfraction was enriched twofold to threefold as compared with the other subfractions. The activity of glucose-Gphosphatase in smooth I, intermediate, and rough subfractions was enriched about threefold as compared with the homogenate. The calcium uptake of rough ERwith a different time course in the three groups was shown in Fig. 1. The calcium uptake in the early and late septic groups was significantlylower than that in the control group (p < 0.05). The calcium uptake of rough ER with different concentrations of magnesium was revealed in Fig. 2. Most points of the early septic group (p < 0.05) and all points of the late septic group (p < 0.05) were significantly lower than those of the control group. Fig. 3, A shows the calcium uptake of rough ER with different concentrations of ATP. Those of the early and late septic groups were significantly lower than those of the control group (p < 0.05). Analysis of the data with Eadie-Hofstee plots indicates that the maximum velocity (Vmax) for ATP was also decreased in the early and late sepsis groups (4.90 2 0.47, 3.78 + 0.61, and 3.33 + 0.43 nmol/mg/min for control and early and late sepsis groups, respectively). The Michaelis-Menten constant (Km) for ATP for Ca*’ transport remained a nonsignificant difference during early and late sepsis (2.31 ? 0.51, 2.54 ? 0.89, and 2.29 ? 0.42 mmol/L for control and early and late sepsis groups, respectively) (Fig. 3, B). These data showed that the inhibition of ATP-dependent Ca*’ transport in rat liver ER during early and late sepsis is noncompetitive in nature, which

SurgeTy

Hwang

Volum,e 121, Number

o!

.

0

et al.

665

6

,

.

,

.

2

1

,

.

3

ATP

,

0

Control

0

Early sepsis

A

Late Sepsis

.

4

,

.

5

A

Free Ca?ihce.tmtion

(M)

, 6

(mM)

0 cmtml . EsrtYsepir . Late se* 1.5-

B 0.5v.8

0.1

0.4

0.6

0.8

1.0

1.2

1.4

I.6

I.R

2.0

1.2

Fig. 4. Effect of sepsis on ATP-dependent Ca2+ uptake in rat liver ER as function of different Ca’+ concentrations. A, Sub strate-velocity relationship; B, Eadie-Hofstee plots. Vertical bars represent SEM. *p < 0.01. #p < 0.05.

Fig. 3. Effect of sepsis on ATP-dependent Caz’ uptake in rat liver ER as function of different ATP concentrations. A, Sub strate-velocity relationship; B, Eadie-Hofstee plots. *p < 0.01. #p < 0.05.

is associated with a mechanism not affecting the affinity toward ATP. Fig. 4, A shows the effect of sepsis on ATP-dependent Ca2+ transport in rat ER as a function of different calcium concentrations. Analysis of the data with EadieHofstee plots indicated that the Vmax for Ca2+ was significantly depressed in the early and late sepsis groups (3.48 + 0.35, 2.59 + 0.24, and 2.20 t 0.2 nmol/mg/ min for control and early and late sepsis groups, respectively). The Km for Ca*+ had no difference among the three groups (1.25 +- 0.17,1.63 1. 0.19, and 1.70 + 0.17 pmol/L for control and early and late sepsis gro~xps, respectively) (Fig. 4, B). The results indicate that the inhibition of the ATP-dependent Ca2+ transport in rat

liver ER during early and late sepsis is associated with a mechanism not affecting the affinity toward Ca2+. The inhibition effect of vanadate on the uptake of calcium in rough ER was studied and is shown in Fig. 5. The inhibition of calcium uptake in the early and late groups was significantly lower than that in the control group (p < 0.05). The effects of early and late sepsis on calcium uptake with or without the presence of glucose-&phosphate in the smooth I and rough ER are shown in Figs. 6 and 7. The increase of Ca2+ uptake during the 20-minute period was more significant in the presence of glucose-6phosphate. Comparing the three groups, the calcium uptake during the 20-minute period significantly decreased during early and late sepsis in the smooth I and rough ER, especially in the smooth I parts (p < 0.05).

666 Hwang

et al.

swe?Y June

1997

5

4

I

0 comol T

0

Early sepsis

A

Late sa@eis

* * “I

. 0.0

012

.

I

.

0:s

0.4

0:6

.

I

1.0

I

Time (min)

1.2

Vanadate(mM) Fig. 5. Effect of sepsis on inhibition of ATP-dependent Ca2’ uptake in rat liver ER with different vanadate concentrations. *p < 0.01.#p < 0.05.

Time (min)

Fig. 6. Effect of sepsis on calcium uptake with presence of glucose-&phosphate in smooth I and rough ER. Vertical bars represent SEM. Con., Control; ES., early sepsis; LX, late sepsis. *p < 0.01. *jJ < 0.05. DISCUSSION Calcium levels in the cytoplasm are regulated by at least three processes. One is the modulation of the calcium flux across the plasma membrane, the second is the uptake or release of calcium by or from the mitochondria, and the third is the exchange of calcium between cytosol and ER.23 Previous studies have shown an alternation in Ca2+specific membrane transport systems during exposure to endotoxin.24a25 We have also demonstrated that the ATP-dependent calcium transport in rat liver plasma membranes was impaired during late sepsisI Johns et

Fig. ‘7. Effect of sepsis on calcium uptake without presence of glucose-&phosphate in smooth I and rough ER. Vertical bars represent SEM. Con., Control; ES., early sepsis; LS., late sepsis. *p< 0.01.

alI7 have also proved that ATP-dependent calcium sequestration was uniquely sensitive to oxidative damage. The sepsis-induced changes of calcium homeostasis impaired the calcium transport either in plasma membrane or in the ER. The data presented in this study demonstrate that ATP-dependent calcium uptake in rat liver ER was also impaired during early and late sepsis. The increased cytosolic calcium may contribute to the altered hepatic glucose metabolism during sepsis. Because of its role in uptake of intracellular calcium from the cell, an impairment in the ER calcium uptake during sepsis results in increased cytosolic free calcium concentration. An increase in the cytosolic free calcium concentration in hepatocytes has been shown to activate phosphorylase bkinase and inactivate glycogen synthase through calciurn-calmodulin-dependent protein kinase mediation.2628 Activation of phosphorylase b kinase and inactivation of glycogen synthase would in turn increase the rate of glycogen breakdown and decrease the rate of glycogen synthesis. An increase in the cytosolic calcium concentration resulting from the impairment of the ATP-dependent calcium uptake in the ERcontributes to the acceleration of gluconeogenesis as seen during sepsis. The enzymatic basis of microsomal calcium transport is a Ca2+-ATPase. The ATPdependent uptake of calcium by rat liver ER is dependent on Mg2’. The microsomal Ca2+-ATPase exhibits the same stimulation by Mg2+ as calcium transport. The effects of ATP and Mg2+ on hepatic microsomal calcium uptake were impaired in early and late sepsis. The vanadate may induce calcium release and cause inhibitory effect of calcium uptake of hepatic microsomes. Both early and late

SU%@Y Volume

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et al.

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121, Number 6

sepsis resulted in impairment of the inhibitory effect by the vanadate. Because of its enzymatic activity, smooth I ER exhibits a much higher calcium accumulating capacity than the rough ER.2g In the presence of glucose-6-phosphate, it markedly stimulates MgATP-dependent calcium accumulation in both fractions. The effects of sepsis on the calcium uptake with or without glucose-Gphosphate, shown in Figs. 6 and 7, revealed the decreased effects in both fractions especially in the presence of glucose&-phosphate and in the smooth I ER. Different portions of ER behave differently with calcium uptake, and they depressed most sensitively in the smooth I subfraction. Ca*’ transport in liver ER involves formation of a phosphorylated intermediate of Ca2t-ATPase.‘3‘16 The inhibition of Ca2+ transport affects only the Vmax but not the Km for ATP and Ca*+ during early and late sepsis as shown in Figs. 3 and 4, which represents a mechanism not affecting the affinity toward Ca2+. The calcium transport of ER included calcium uptake and sequestration. Further study of the changes of calcium release of ER in sepsis will provide more information about calcium transport. REFERENCES 1. &itch further 2. Murphy

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