Characteristics of intracellular Ca²⁺ signals consisting of two successive peaks in hepatocytes during liver regeneration after 70% partial hepatectomy in rats

Figures & data

Table 1 Primer list for RT-PCR

Gene name	Sequence
CYP1A2
Sense	5′-TGACCTCTTTGGAGCTGGGTT-3′
Antisense	5′-AACCCAGCTCCAAAGAGGTCA-3′
CYP2B1
Sense	5′-GGGCAATGCCTTCGCCAAGACA-3′
Antisense	5′-CAAATGCGCTTTCCTGTGGAGAA-3′
CYP2E1
Sense	5′-ACAATGACAAGAAGTGTCTGAGGCTCAT-3′
Antisense	5′-ATGACATATCCTTGGAACACAGTAT-3′
S100A4
Sense	5′-CTGGATGTAATAGTGTCCACCTTCCA-3′
Antisense	5′-TTGCTCAGCACTGTGCACATGTATGA-3′
Calpain
Sense	5′-ACGAGAGGATCTCGCAGAGA-3′
Antisense	5′-CCTAACATTGAGACTAGCATTA-3′
Calmodulin
Sense	5′-GAGGTTGATGAAATGATCAGGGAAGCA-3′
Antisense	5′-AGAACACCCGCTTGCAGTAGTGCAACT-3′
HSP70
Sense	5′-CGATGAAGCTGTTGCCTATGGTGCAGCTGT-3′
Antisense	5′-TTCTTGGACCATGCGCTCAATATCCTCCT-3′
HSP90
Sense	5′-GCCTGGAGCTACCAGAGGATGAGGAAGA-3′
Antisense	5′-CACTTCTTCCATGCGAGAGGCATCCT-3′
VEGF-C
Sense	5′-CCAGTGTGTCTGCAAGAGAACATGT-3′
Antisense	5′-AACCAGTCTCCTTAAAAGGCTTTATGC-3′
VEGF-D
Sense	5′-GACATGCTGTGGGATAACACCAAATGTA-3′
Antisense	5′-GTATTTCTTAGAGGCCAAACGTCTG-3′
β-Actin
Sense	5′-TGACGGTCAGGTCATCACTA-3′
Antisense	5′-CCACACAGAGTACTTGCGCT-3′

Figure 1 Time course of physiological profiles during liver regeneration after 70% PH in rats.

Notes: (A) Body weight (g). (B) Serum ALT activity (IU/L). (C) The R_r of liver regeneration after 70% PH was calculated by R_r = 100 × (total weight of regenerated liver/total weight of the liver lobe pieces excised/0.7). Each point presented as mean ± SD for six or more rats.
Abbreviations: ALT, alanine aminotransferase; PH, partial hepatectomy; SD, standard deviation.

Figure 2 Time course of physiological effects of drugs in rats during liver regeneration after 70% PH.

Notes: (A) Serum ALT activity (IU/L) at 24 hours after ip administration of 0.2 mL/kg CCl₄ to normal rats. (B) Serum ALT activity at 24 hours after ip administration of 0.2 mL/kg CCl₄ to rats during liver regeneration after 70% PH. (C) V_dss of SAM. After intravenous administration of SAM (68.5 mg/kg of body weight), serum SAM was measured several times for 90 minutes by the fluorometric method. V_dss and other parameters of SAM were estimated by pharmacokinetics. (D) V_dss of EB. After intravenous administration of EB (10 mg/kg of body weight), serum EB concentrations were measured several times for 120 minutes by the absorbance method. V_dss of EB was estimated by pharmacokinetics. Each point is presented as mean ± SD for three rats.
Abbreviations: ALT, alanine aminotransferase; EB, Evans blue; PH, partial hepatectomy; SAM, salicylamide; SD, standard deviation; V_dss, distribution of volume at a steady state after drug administration.

Table 2 Pharmacokinetic parameters of SAM in rats after intravenous administration of SAM 68.5 mg/kg of body weight

Days	AUC (µg/mL min)	CL (mL/min/kg)	MRT (min)	Vdss (mL/kg)
0	13.00±1.08	3.55±0.33	20.30±0.50	72.37±8.42
1	13.00±1.28	2.47±0.40	35.55±2.49	87.08±9.35
2	21.12±4.89	2.80±0.46	31.52±6.68	85.36±5.55
3	18.16±0.77	3.01±0.30	28.28±0.48	85.41±9.96
4	19.04±2.61	3.18±0.18	28.17±5.83	90.68±23.71
5	18.11±0.20	3.06±0.25	26.46±0.26	80.81±5.83
6	16.02±1.89	3.23±0.13	23.69±2.39	78.53±2.34
7	14.78±1.55	2.97±0.11	22.29±1.06	66.25±5.58
8	17.59±1.46	2.74±0.11	26.43±1.76	72.16±1.81
9	15.70±3.02	2.47±0.02	27.48±0.92	67.95±1.60
10	22.03±1.02	2.35±0.11	32.59±1.32	76.44±0.23

Note: Parameters show mean ± SD.

Abbreviations: AUC, area under the plasma concentration curve; CL, clearance; MRT, mean residence time; SAM, salicylamide; V_dss, distribution of volume at a steady state after drug administration.

Figure 3 [Ca²⁺]_i in hepatocytes during liver regeneration after 70% PH.

Notes: The [Ca²⁺]_i concentration was measured using the fluorescent Ca²⁺ indicator fura-2. Hepatocytes were loaded with 5 µM fura-2-AM under continuous shaking for 30 minutes. After two washes with PBS (pH 7.4), the cells were incubated at 37°C for 5 minutes. Hepatocytes (1×10⁶ cells) were resupended in 1 mL Ca²⁺- and Mg²⁺-free HBSS in a 10 mm quartz cuvette, and the fluorescence was measured at an emission wavelength of 500 nm and excitation of 340 or 380 nm using spectrofluorometer. Each point is presented as mean ± SD for three rats.
Abbreviations: AM, acetoxymethyl ester; PBS, phosphate-buffered saline; PH, partial hepatectomy; SD, standard deviation; HBSS, Hanks’ balanced salt solution.

Figure 4 Cell cycle distribution during liver regeneration after 70% PH.

Notes: G₁ (A), S (B), and G₂ (C) phases. Hepatocytes were isolated from 70% PH rat livers, and 0.5×10⁶ cells were fixed with 70% ethanol at 4°C. The cells were stained with PI, and their fluorescence was measured by a flow cytometer using a 575-nm band-pass filter. Cells were estimated on graphs of cell counts versus PI staining. Each point is presented as mean ± SD for six rats.
Abbreviations: PH, partial hepatectomy; PI, propidium iodide; SD, standard deviation.

Figure 5 Protein contents (A) and cytochrome P450 level (B) in microsomes per 1 g of wet liver in rats during liver regeneration after 70% PH.

Notes: (A) Protein contents in microsomes were measured using Folin phenol reagent.⁴² (B) Microsomal cytochrome P450 levels were estimated using the method of Omura and Sato.³⁰ Each point presents as the mean ± SD for three rats.
Abbreviations: PH, partial hepatectomy; SD, standard deviation.

Figure 6 RNA content (A) and expression levels of VEGF and β-actin (B) in the liver.

Notes: (A) Total RNA in 2 g of wet liver was isolated with acid phenol, and calculated by the optical density at 260 nm. Each point presents as the mean ± SD for four rats. (B) VEGF gene expression was estimated by RT-PCR. Day 4–6 and 4+3 indicate 6 hours before day 4 and 3 hours after day 4, respectively.
Abbreviation: SD, standard deviation.

Figure 7 Expression levels per total RNA unit content of several genes after 70% PH.

Notes: (A) CYP1A2, (B) CYP2E1, (C) CYP2B1, (D) S100A4, (E) calpain, (F) calmodulin, (G) HSP70, (H) HSP90, (I) VEGF-C, and (J) VEGF-D. Total RNA in wet livers was isolated with acid phenol, and the expression levels of the indicated genes were estimated by RT-PCR. The vertical axis is the ratio (%) of the expression level at each day compared with that at day 0. The expression levels were normalized against β-actin expression, shown in upper row. Results are presented as the mean ± SD for double analyses.
Abbreviations: PH, partial hepatectomy; SD, standard deviation.

Figure 8 Light microphotographs of H&E-stained liver sections of rats during liver regeneration after 70% PH (×10).

Notes: Rats were sacrificed at (A) day 0, (B) day 1, (C) day 2, (D) day 3, (E) day 4, (F) day 5, and (G) day 6 after 70% PH, the livers were immediately removed, and weighed. Tissue samples were routinely fixed in 4% neutral buffered formalin for several days, dehydrated, paraffin-embedded, and sectioned at 5 µm thickness. Following deparaffinization in xylene, sections were dehydrated, stained with H&E, and mounted on glass slides for pathological assessment. Scale bars show in 100 µm.
Abbreviations: H&E, hematoxylin and eosin; PH, partial hepatectomy.

LowryOHRosebroughNJFarrALRandallRJProtein measurement with the Folin phenol reagentJ Biol Chem195119326527514907713

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OmuraTSatoRThe carbon monoxide-binding pigment of liver microsomes: I. Evidence for its hemoprotein natureJ Biol Chem19642392370237814209971

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