Proteostasis, or cellular protein homeostasis, relies on the regulation of protein synthesis, folding, conformational maintenance, and degradation (1). Deviations from optimal proteostasis can result in serious pathologies and accelerate the aging of an organism. Proteostasis is maintained by several control systems, all of them of equal importance (1). Protein degradation is partly regulated by the ubiquitin-proteasome system (UPS), which ensures rapid and specific turnover of proteins. UPS modifies cellular and protein functions, including cell cycle, cell signalling, DNA repair, chromatin modifications, and protein trafficking (2). These are mediated by ATPase (ubiquitination enzymes encoded by the
In our opinion, testing of the impact of 1,4-dihydropyridine (1,4-DHP) derivatives, a vast group of compounds with different pharmacological activities, on the UPS could be a prospective research branch. A big group of these compounds has been synthesised in the Latvian Institute of Organic Synthesis over the last few years. Some of them manifest interesting effects besides antioxidant activity (9), as they can modify cell proliferation (10), bind DNA and proteins, or stimulate DNA repair by activating DNA repair enzymes (11, 12). These novel 1,4‑DHP derivatives have a weak Ca2+ channel blocker activity and are water-soluble unlike “classical” Ca2+ channel blockers, which are hydrophobic. Yet they can modify the expression of several genes and proteins (13, 14), including the proteasome gene
This study was approved by the Animal Ethics Committee of the Food and Veterinary Service (Riga, Latvia) and was carried out according to the guidelines of the 1986 European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (16). Male Wistar rats (215.0±5.6 g) were purchased from the Laboratory of Experimental Animals, Riga Stradins University, Riga, Latvia. Animals were kept at 22±0.5 °C with a 12 h light/dark cycle and fed standard laboratory diet.
All drugs used in the study – metcarbatone, etcarbatone, glutapyrone, styrylcarbatone (J-9-125), and AV-153 Na and Ca salts (Figure 1) were synthesised at the Latvian Institute of Organic Synthesis. Other chemicals were purchased from Sigma-Aldrich Chemie (Taufkirchen, Germany).
Rats were divided into control and treatment groups. The latter received 0.05 mg/kg or 0.5 mg/kg of metcarbatone, etcarbatone, glutapyrone, styrylcarbatone, AV-153-Na, or AV-153-Ca
Total RNA was isolated from the kidneys, blood, and liver with a TRI reagent (Sigma Aldrich, Taufkirchen, Germany). RNA was purified from DNA with a DNA-free kit (Ambion, Austin, TX, USA) and its quantity and purity determined with a NanoPhotometer ® NP 80 spectrophotometer (ImplenGMBH, Munich, Germany).
The quality of RNA was analysed with gel electrophoresis. cDNA was synthesised from the obtained RNA (5 μg from kidneys and liver, and 2 μg from blood) with random hexamer primers (RevertAid™ First Strand cDNA Synthesis Kit, Fermentas, Vilnius, Lithuania).
mRNA expression of
Primers were designed using Primer‑BLAST software ( 1 7 ) . Primer sequences were:
Oligonucleotides were supplied by Metabion International AG (Martinsried, Germany). qPCR reactions were performed using a StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Cycling conditions were as follows: one cycle at 95 ºC for 10 min, 40 cycles at 95 ºC for 15 sec, and one cycle at 60 ºC for 1 min (Applied Biosystems StepOne software, version 2.1). The specificity of amplification products was verified by dissociation curve: one cycle at 95 ºC for 15 sec, one at 60 ºC for 1 min, and one at 95 ºC for 15 sec. The cycle threshold (Ct) values are presented in Tables 1 and 2.
Kidney and blood real-time PCR cycle threshold (Ct) values
Kidneys | Blood | |||||||
---|---|---|---|---|---|---|---|---|
Control | 20,6336 | 21,2849 | 20,2966 | 18,5916 | 19,3183 | 24,7317 | 23,9346 | 23,4343 |
19,9632 | 21,0402 | 20,1468 | 18,5301 | 19,1140 | 24,9544 | 23,9330 | 23,6485 | |
20,5162 | 20,6843 | 19,9793 | 18,4140 | 19,5568 | 23,5918 | 23,6477 | 22,6171 | |
19,7661 | 20,8542 | 20,0899 | 18,4168 | 19,5473 | 25,3724 | 23,5371 | 23,2719 | |
20,4493 | 21,1992 | 20,3281 | 18,7329 | 19,2752 | 24,2432 | 23,6730 | 22,2758 | |
20,0575 | 20,7519 | 19,8123 | 18,2283 | 18,0247 | 24,1311 | 23,7417 | 21,8996 | |
19,8994 | 20,8551 | 20,1670 | 18,5733 | 19,6721 | 24,1230 | 22,6278 | 22,9333 | |
19,9792 | 21,2709 | 20,5719 | 18,8297 | 18,5737 | 23,8446 | 23,0603 | 22,4729 | |
20,3273 | 21,4559 | 20,4872 | 18,9060 | 18,9473 | 24,8475 | 24,5772 | 23,0626 | |
20,7163 | 21,1733 | 20,4132 | 18,8730 | 19,7215 | 24,5549 | 23,4031 | 23,6338 | |
20,1879 | 21,1945 | 20,4594 | 18,7461 | 18,7025 | 24,7850 | 24,1693 | ||
18,7703 | 24,3940 | 24,0506 | ||||||
Metcarbatone 0.05 mg/kg | 21,1421 | 21,1700 | 20,2690 | 18,7102 | 19,1992 | 24,9793 | 24,9819 | 23,4209 |
20,6063 | 20,9449 | 20,0149 | 18,6103 | 18,2122 | 25,0053 | 24,6967 | 22,8411 | |
20,9352 | 21,4263 | 20,5656 | 18,9078 | 19,6951 | 24,3028 | 23,8090 | 22,9598 | |
20,5415 | 20,9494 | 20,0938 | 18,4946 | 18,7135 | 24,5634 | 24,1763 | 22,6806 | |
Metcarbatone 0.5 mg/kg | 20,8324 | 20,9915 | 20,1140 | 18,8390 | 18,9062 | 25,2567 | 23,8337 | 23,6736 |
21,1187 | 21,0831 | 20,1682 | 18,7917 | 19,2715 | 25,2724 | 24,5450 | 23,6550 | |
21,0912 | 21,1248 | 20,3896 | 18,8515 | 18,5728 | 24,6956 | 23,9355 | 22,8742 | |
20,6640 | 21,1151 | 20,0875 | 18,7441 | 18,6701 | 25,2925 | 24,5882 | 23,2121 | |
Etcarbatone 0.05 mg/kg | 20,6521 | 21,3114 | 20,2236 | 18,6247 | 18,8542 | 24,8812 | 24,1604 | 22,9606 |
21,2283 | 21,3560 | 20,3069 | 18,6993 | 19,1319 | 24,8758 | 24,7965 | 22,9433 | |
21,3387 | 21,4437 | 20,1647 | 18,7293 | 18,1798 | 24,6700 | 24,4486 | 22,5183 | |
Etcarbatone 0.5 mg/kg | 20,6957 | 21,2241 | 20,1316 | 18,4727 | 19,2888 | 24,9479 | 24,4943 | 23,1595 |
21,4283 | 21,6113 | 20,5876 | 18,8926 | 19,2577 | 24,8016 | 24,0260 | 23,0911 | |
21,0995 | 21,5415 | 20,3180 | 18,8051 | 18,9519 | 24,5479 | 24,2079 | 22,4058 | |
20,9716 | 21,4134 | 20,4994 | 18,8108 | 19,0836 | 24,6532 | 24,6596 | 22,5140 | |
Styrylcarbatone 0.05 mg/kg | 21,4476 | 21,4452 | 20,5627 | 18,6984 | 19,5796 | 23,9558 | 23,0535 | 22,6217 |
20,9010 | 21,0415 | 20,1628 | 18,3986 | 19,0160 | 24,8889 | 24,1739 | 23,2842 | |
21,3434 | 21,2509 | 20,2865 | 18,6662 | 18,1215 | 23,5157 | 23,3238 | 21,4245 | |
21,6191 | 22,0462 | 21,5846 | 18,9777 | 19,5722 | 25,4267 | 24,9225 | 23,4448 | |
Styrylcarbatone 0.5 mg/kg | 21,5660 | 21,5113 | 21,2236 | 18,9375 | 19,1745 | 23,8688 | 23,2048 | 22,2596 |
21,2131 | 21,5601 | 20,7720 | 18,7713 | 19,5076 | 24,7026 | 23,6100 | 23,0168 | |
21,0897 | 21,4685 | 20,4881 | 18,7617 | 18,9420 | 24,6156 | 23,8653 | 23,2664 | |
21,4455 | 21,6779 | 20,9215 | 18,9282 | |||||
Glutapyrone 0.05 mg/kg | 22,0678 | 22,8544 | 21,6248 | 19,8827 | 18,9078 | 23,7505 | 22,4539 | 22,0790 |
21,0281 | 22,1696 | 20,8820 | 19,3440 | 19,1537 | 23,5877 | 22,7654 | 22,3149 | |
21,1787 | 21,9149 | 20,8096 | 19,0287 | 20,1436 | 25,5499 | 23,8626 | 23,8251 | |
21,3287 | 21,5062 | 20,4549 | 18,7512 | 19,6614 | 24,1258 | 23,0498 | 23,1506 | |
Glutapyrone 0.5 mg/kg | 21,0105 | 21,8076 | 20,5749 | 18,8806 | 19,3639 | 24,6586 | 23,1130 | 22,9958 |
20,6910 | 21,6908 | 20,2656 | 18,6775 | 19,6876 | 25,6195 | 23,7417 | 24,0393 | |
21,1142 | 21,4909 | 20,2270 | 18,7062 | 19,4588 | 24,3839 | 23,1421 | 22,8727 | |
21,3715 | 21,4707 | 20,0467 | 18,8292 | 19,4118 | 24,7691 | 23,3703 | 23,2199 |
Liver real-time PCR cycle threshold (Ct) values
Kidneys | Liver | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Control | 20,5970 | 22,5839 | 19,2551 | 19,1623 | 19,9986 | 19,8560 | 23,0557 | 21,1670 | 18,6385 | 19,8489 |
20,4384 | 22,5746 | 19,0794 | 18,8645 | 19,5620 | 19,5794 | 22,9181 | 21,1984 | 18,6208 | 19,6455 | |
20,4358 | 22,5837 | 19,1140 | 18,8865 | 19,7310 | 19,9334 | 23,3011 | 21,1796 | 19,6168 | 20,4226 | |
20,5461 | 22,7381 | 19,0685 | 19,1585 | 19,7124 | 19,7203 | 23,1264 | 21,1636 | 19,4941 | 20,2836 | |
20,3772 | 22,3517 | 19,0689 | 19,2026 | 20,2427 | 19,1888 | 22,9224 | 21,0717 | 19,1761 | 19,8373 | |
20,3640 | 22,2591 | 18,9613 | 18,9177 | 19,7695 | 19,7114 | 23,1850 | 21,1812 | 19,1708 | 20,0905 | |
20,4210 | 22,1444 | 19,0667 | 18,8171 | 19,3981 | 19,8140 | 23,5071 | 21,3909 | 19,3937 | 20,2497 | |
20,5429 | 22,3808 | 19,2618 | 20,5558 | 19,7631 | 23,3798 | 21,3486 | 19,0927 | 20,2443 | ||
20,4095 | 22,3303 | 18,9823 | 20,3727 | 19,6435 | 23,5475 | 21,2771 | 19,3322 | 20,4155 | ||
19,9648 | 22,0525 | 18,7116 | 19,2846 | |||||||
AV-153-Na 0.05 mg/kg | 21,1756 | 22,9314 | 19,3199 | 19,5803 | 20,0066 | 19,3193 | 22,6985 | 21,0440 | 18,3758 | 18,9400 |
22,0513 | 23,4752 | 19,6423 | 19,9317 | 20,6172 | 18,8415 | 22,8245 | 20,9705 | 18,3795 | 19,0355 | |
21,4856 | 23,2240 | 19,4647 | 19,6623 | 19,8643 | 19,5196 | 22,7070 | 21,0585 | 18,3857 | 19,0187 | |
21,6234 | 23,2381 | 19,5993 | 19,5928 | 19,6469 | 18,8631 | 22,7883 | 20,9608 | 18,3031 | 18,9503 | |
AV-153-Na 0.5 mg/kg | 21,9367 | 23,2775 | 19,4898 | 19,8077 | 20,0014 | 19,6059 | 22,7776 | 20,9764 | 18,4171 | 19,0567 |
21,6564 | 23,2385 | 19,5634 | 19,4900 | 19,8338 | 19,6035 | 22,8215 | 21,0093 | 18,4078 | 19,0701 | |
21,2077 | 23,2004 | 19,4074 | 19,5859 | 19,9741 | 19,5527 | 22,8589 | 20,9877 | 18,3254 | 19,1177 | |
AV-153-Ca 0.05 mg/kg | 19,7019 | 22,0370 | 19,1800 | 18,7166 | 19,3175 | 19,3393 | 22,9415 | 20,8086 | 19,1408 | 19,8715 |
19,8564 | 22,2813 | 19,3695 | 18,7706 | 19,2143 | 19,6280 | 23,3113 | 21,0154 | 19,1477 | 20,3142 | |
20,1111 | 21,8349 | 19,0583 | 18,6593 | 19,3757 | 19,3697 | 22,8512 | 20,9235 | 18,9642 | 20,2187 | |
19,6506 | 23,1935 | 21,0658 | 19,3068 | 20,0824 | ||||||
AV-153-Ca 0.5 mg/kg | 20,3086 | 22,0432 | 19,5138 | 18,8374 | 19,3591 | 19,4528 | 22,9892 | 20,7577 | 19,0212 | 19,3202 |
20,5201 | 22,7760 | 18,8733 | 19,2125 | 20,2666 | 19,5880 | 23,2170 | 21,2606 | 19,0527 | 19,6300 | |
19,5801 | 21,8729 | 18,8382 | 18,7051 | 19,3437 | 18,8662 | 23,0295 | 21,3512 | 18,8270 | 19,3601 | |
20,1326 | 22,0602 | 18,9902 | 18,8147 | 19,6876 | 19,5368 | 23,2560 | 21,5740 | 19,0952 | 19,4394 | |
20,1226 | 21,8402 | 18,8235 | 18,8101 | 19,4492 | 19,5666 | 23,0126 | 21,1141 | 18,7163 | 19,2355 |
Reference gene stability was analysed with BestKeeper provided in RefFinder (
In the kidney, metcarbatone at both doses significantly increased the expression of the
The effect of metcarbatone on
Metcarbatone (mg/kg) | Kidney fold difference (SEM range) | Blood fold difference (SEM range) | |
---|---|---|---|
Control | 1.00 (0.93–1.07) | 1.00 (0.87–1.14) | |
0.05 | 1.44 (1.34–1.54)* | 0.76 (0.56–1.04) | |
0.50 | 1.61 (1.50–1.74)** | 0.53 (0.48–0.58) | |
Control | 1.00 (0.93–1.08) | 1.00 (0.84–1.18) | |
0.05 | 1.51 (1.40–1.63)** | 0.55 (0.39–0.77) | |
0.50 | 1.69 (1.60–1.79)** | 0.58 (0.51–0.67) | |
Control | 1.00 (0.94–1.07) | 1.00 (0.90–1.12) | |
0.05 | 1.43 (1.34–1.54)* | 0.83 (0.68–1.01) | |
0.50 | 1.43 (1.34–1.53)* | 0.60 (0.55–0.64)* |
*P<0.05 and **P<0.01 compared to control
The
The effect of etcarbatone on
Etcarbatone (mg/kg) | Kidney fold difference (SEM range) | Blood fold difference (SEM range) | |
---|---|---|---|
Control | 1.00 (0.93–1.07) | 1.00 (0.87–1.14) | |
0.05 | 1.46 (1.29–1.65)* | 0.61 (0.52–0.70) | |
0.50 | 1.36 (1.29–1.43)* | 0.85 (0.84–0.87) | |
Control | 1.00 (0.93–1.08) | 1.00 (0.84–1.18) | |
0.05 | 1.82 (1.56–2.12)** | 0.45 (0.37–0.55) | |
0.50 | 1.61 (1.52–1.71)** | 0.66 (0.59–0.74) | |
Control | 1.00 (0.94–1.07) | 1.00 (0.90–1.12) | |
0.05 | 1.72 (1.52–1.95)** | 0.79 (0.71–0.88) | |
0.50 | 1.62 (1.53–1.72)** | 1.07 (0.99–1.17) |
*P<0.05 and **P<0.01 compared to control
Styrylcarbatone significantly increased the expression of the
The effect of styrylcarbatone on
Styrylcarbatone (mg/kg) | Kidney fold difference (SEM range) | Blood fold difference (SEM range) | |
---|---|---|---|
Control | 1.00 (0.93–1.07) | 1.00 (0.87–1.14) | |
0.05 | 1.65 (1.53–1.79)** | 0.99 (0.78–1.26) | |
0.50 | 1.53 (1.43–1.64)** | 1.13 (0.93–1.37) | |
Control | 1.00 (0.93–1.08) | 1.00 (0.84–1.18) | |
0.05 | 1.63 (1.39–1.90)** | 0.87 (0.64–1.18) | |
0.50 | 1.41 (1.36–1.47)* | 1.18 (0.97–1.44) | |
Control | 1.00 (0.94–1.07) | 1.00 (0.90–1.12) | |
0.05 | 2.05 (1.98–2.13)*** | 1.09 (0.90–1.32) | |
0.50 | 1.83 (1.75–1.91)*** | 1.08 (0.84–1.39) |
*P<0.05, **P<0.01, and ***P<0.0001 compared to control
In the kidney, the higher dose of glutapyrone significantly increased
The effect of glutapyrone on
Glutapyrone (mg/kg) | Kidney fold difference (SEM range) | Blood fold difference (SEM range) | |
---|---|---|---|
Control | 1.00 (0.93–1.07) | 1.00 (0.87–1.14) | |
0.05 | 1.10 (0.95–1.26) | 1.49 (1.27–1.74) | |
0.50 | 1.21 (1.05–1.39) | 0.99 (0.86–1.14) | |
Control | 1.00 (0.93–1.08) | 1.00 (0.84–1.18) | |
0.05 | 1.40 (1.26–1.55) | 2.04 (1.94–2.14)* | |
0.50 | 1.73 (1.49–2.01)** | 1.66 (1.56–1.77) | |
Control | 1.00 (0.94–1.07) | 1.00 (0.90–1.12) | |
0.05 | 1.46 (1.28–1.66)* | 1.30 (1.19–1.42) | |
0.50 | 1.59 (1.46–1.73)** | 0.96 (0.84–1.11) |
*P<0.05 and **P<0.01 compared to control
AV-153-Na significantly increased the expression of
The effect of AV-153-Na on
AV-153-Na (mg/kg) | Kidneys fold difference (SEM range) | Liver fold difference (SEM range) | |
---|---|---|---|
Control | 1.00 (0.97–1.03) | 1.00 (0.95–1.05) | |
0.05 | 1.28 (1.21–1.35)* | 0.94 (0.82–1.08) | |
0.50 | 1.28 (1.12–1.46)* | 1.23 (1.19–1.26) | |
Control | 1.00 (0.99–1.02) | 1.00 (0.94–1.04) | |
0.05 | 1.64 (1.51–1.78)*** | 0.79 (0.71–0.87)* | |
0.50 | 1.68 (1.47–1.91)*** | 1.09 (1.08–1.11) | |
Control | 1.00 (0.97–1.03) | 1.00 (0.92–1.09) | |
0.05 | 1.39 (1.29–1.51)** | 1.19 (1.07–1.33) | |
0.50 | 1.47 (1.30–1.66)** | 1.61 (1.59–1.62)* | |
Control | 1.00 (0.92–1.08) | 1.00 (0.94–1.06) | |
0.05 | 2.00 (1.78–2.25)*** | 1.49 (1.32–1.68)** | |
0.50 | 2.17 (1.87–2.52)*** | 1.91 (1.86–1.95)*** |
*P<0.05, **P<0.01, and ***P<0.001 compared to control
In the kidney, AV-153-Ca significantly affected only the
The effect of AV-153-Ca on
AV-153-Ca (mg/kg) | Kidney fold difference (SEM range) | Liver fold difference (SEM range) | |
---|---|---|---|
Control | 1.00 (0.97–1.03) | 1.00 (0.95–1.05) | |
0.05 | 0.89 (0.76–1.03) | 0.96 (0.94–0.99) | |
0.50 | 1.00 (0.92–1.09) | 0.89 (0.81–0.97) | |
Control | 1.00 (0.99–1.02) | 1.00 (0.94–1.04) | |
0.05 | 0.62 (0.55–0.71)** | 1.05 (1.03–1.08) | |
0.50 | 0.85 (0.75–0.95) | 0.82 (0.71–0.95) | |
Control | 1.00 (0.97–1.03) | 1.00 (0.92–1.09) | |
0.05 | 0.85 (0.77–0.93) | 0.89 (0.86–0.93) | |
0.50 | 0.90 (0.84–0.96) | 0.96 (0.88–1.05) | |
Control | 1.00 (0.92–1.08) | 1.00 (0.94–1.06) | |
0.05 | 1.03 (0.96–1.11) | 0.87 (0.82–0.93) | |
0.50 | 0.98 (0.89–1.07) | 1.35 (1.22–1.48)* |
*P<0.05 and **P<0.01 compared to control
Most of the tested 1,4-DHP derivatives increased gene expression levels in the kidney but were mainly without a significant effect in the blood and liver. The general sensitivity of the kidney cells to 1,4‑DHP could simply be explained by accumulation of the compounds in the kidney before excretion, but there are no data to support it.
Comparing the effects of 1,4‑DHP derivatives on different proteasome subunit genes, we noticed that subunit mRNA expression did not follow a uniform pattern. Other authors have also reported divergent effects of drugs on different proteasome subunit gene expression. For example, cocaine mainly upregulated the
In the kidney, metcarbatone, etcarbatone, styrylcarbatone, and AV-153-Na increased the expression of all analysed genes, but glutapyrone and AV-153-Ca showed varying effects. Glutapyrone did not affect the expression of
In the blood, glutapyrone increased only
In the liver, AV-153-Na upregulated the expression of
1,4‑DHP derivatives as prospective drugs have already shown antioxidant activities and a wide range of antiaging, antibacterial, anticancer, and neuroprotective actions (10). Glutapyrone, a representative of the novel group of 1,4‑DHP derivatives with weak Ca2+ channel blocker activity has very low toxicity and multiple pharmacological properties, including concomitant effects on multiple neurotransmitter systems and antioxidant activities (22). Carbatone, another compound of this group, administered orally, showed fast absorption in the gastrointestinal tract and 62 % bioavailability. It quickly spreads across tissues and is excreted mostly through urine and faeces. This group of 1,4-DHP derivatives seems to have a very low cytotoxicity at the tested doses (unpublished data).
It also seems that the protective antioxidant activities of 1,4-DHP derivatives are achieved by targeting the mitochondria. They might be working through direct scavenging of reactive oxygen species and decomposition of hydrogen peroxide. Furthermore, they stimulate cell growth and differentiation (23). An
The downregulation of UPS genes in the liver seems to correspond with age. In old mice, this downregulation was reported to lead to the accumulation of IκBα in the cytoplasm, which prevented the activation of the NF-κB protein, which is important for hepatocyte survival and liver health (25). Older age also seems to be associated with lower mRNA levels of both proteasome beta subunits, which are directly involved in the proteolytic function of the proteasome and antioxidant activity (26), but some healthy centenarians were reported to have proteasome subunit mRNA levels close to young donors. Furthermore, one study showed that a stable transfection of either
Downregulated proteasomal gene expression is also associated with several pathologies. For instance, in patients with schizophrenia, dentate granule neurons showed decreased expression of several proteasome subunit and other genes involved in protein processing by proteasomes and ubiquitin, resulting in a deficient ubiquitin-proteasome function that can lead to reduced neuron responsiveness (28). In patients with Parkinson’s disease, both catalytic and regulatory subunits of the UPS, including the
To sum up, our research has confirmed the ability of several 1,4-DHP derivatives to increase the expression of proteasome subunit genes. This might be a promising property for the development of drugs for conditions associated with impaired proteasomal functions and low mRNA levels of proteasome subunits.