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. 2024 Mar 29;17(4):440.
doi: 10.3390/ph17040440.

The Xanthine Derivative KMUP-1 Inhibits Hypoxia-Induced TRPC1 Expression and Store-Operated Ca2+ Entry in Pulmonary Arterial Smooth Muscle Cells

Zen-Kong Dai  1   2 , Yi-Chen Chen  3 , Su-Ling Hsieh  4 , Jwu-Lai Yeh  3   5 , Jong-Hau Hsu  1   2 , Bin-Nan Wu  3   5
Affiliations

The Xanthine Derivative KMUP-1 Inhibits Hypoxia-Induced TRPC1 Expression and Store-Operated Ca2+ Entry in Pulmonary Arterial Smooth Muscle Cells

Zen-Kong Dai et al. Pharmaceuticals (Basel). .

Abstract

Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca2+]i through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O2/5% CO2 for 24 h to elucidate TRPC1 overexpression and observe the Ca2+ release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca2+ entry upon SR Ca2+ depletion in hypoxic PASMCs.

Keywords: KMUP-1; canonical transient receptor potential channel 1; hypoxia; protein kinases; pulmonary artery smooth muscle cells; store-operated calcium entry.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effects of KMUP-1 in normoxic and hypoxic pulmonary arterial smooth muscle cells (PASMCs). (A) Structure of KMUP-1. (B) TRPC1 protein showed no significant differences in KMUP-1 (1, 10, 100 μM)-treated PASMCs under normoxic conditions. (C) Various concentrations of KMUP-1 (1, 10, 100 μM) inhibited hypoxia-induced TRPC1 protein overexpression. The quantitation of this protein is shown in the lower panel. Results were presented as the mean ± SE, n = 7. # p < 0.05 compared with control (normoxia), r = 0.66; ** p < 0.01, *** p <0.001 compared with hypoxia, r = 0.81. C: control represents normoxia. r: correlation coefficient.
Figure 2
Figure 2
KMUP-1 inhibited hypoxia-enhanced TRPC1 expression via the cGMP/PKG pathway. PASMCs pretreated with KMUP-1 (1 μM), KT5823 (1 μM), 8-Br-cGMP (100 μM), KT5823+KMUP-1, and 8-Br-cGMP+KMUP-1 under hypoxic states. The quantitation of these proteins is shown in the lower panel. Results are presented as the mean ± SE, n = 6. # p < 0.05 compared with normoxia, r = 0.47; ** p < 0.01, *** p < 0.001 compared with hypoxia, r = 0.72; † p < 0.05 compared with KMUP-1 group, r = 0.56. 8-Br-cGMP: membrane-permeable analog of cGMP. r: correlation coefficient.
Figure 3
Figure 3
KMUP-1 inhibited hypoxia-enhanced TRPC1 expression via the cAMP/PKA pathway. PASMCs pretreated with KMUP-1 (1 μM), KT5720 (1 μM), 8-Br-cAMP (100 μM), KT5720+KMUP-1, and 8-Br-cAMP+KMUP-1 under hypoxic states. The quantitation of these proteins is shown in the lower panel. Results are presented as the mean ± SE, n = 6. # p < 0.05 compared with normoxia, r = 0.59; ** p < 0.01, *** p < 0.001 compared with hypoxia, r = 0.68; † p < 0.05 compared with KMUP-1 group, r = 0.61. 8-Br-cAMP: membrane-permeable analog of cAMP. r: correlation coefficient.
Figure 4
Figure 4
KMUP-1 inhibited hypoxia-enhanced TRPC1 expression via PKC pathway. PASMCs pretreated with KMUP-1 (1 μM), PMA (1 μM), chelerythrine (1 μM), PMA+KMUP-1, and chelerythrine+KMUP-1 under hypoxic states. The quantitation of these proteins is shown in the lower panel. Results are presented as the mean ± SE, n = 6. # p < 0.05 compared with normoxia, r = 0.48; ** p < 0.01, *** p < 0.001 compared with hypoxia, r = 0.70; † p < 0.05 compared with the PMA group, r = 0.52. PMA: phorbol 12-myristate 13-acetate. r: correlation coefficient.
Figure 5
Figure 5
Effects of exposure to hypoxia on capacitative calcium entry in PASMCs. (A) Change in [Ca2+]i in pulmonary arterial smooth muscle cells (PASMCs) from control or hypoxia and subjected to the restoration of extracellular Ca2+ following store depletion with TG (1 μM). (B) Bar graph illustrates mean ± SE change in [Ca2+]i (∆[Ca2+]i) in response to TG and Ca2+ restoration. All experiments were performed in the presence of nifedipine, n = 12 for control and n = 13 for hypoxia. *** p < 0.001 compared with control (normoxia). TG: thapsigargin.
Figure 6
Figure 6
Effects of KMUP-1 on capacitative calcium entry in PASMCs. (A) Change in [Ca2+]i in pulmonary arterial smooth muscle cells from hypoxic or pretreated KMUP-1 groups and subjected to the restoration of extracellular Ca2+ following store depletion with TG (1 μM). (B) Bar graph illustrates mean ± SEM change in [Ca2+]i (∆[Ca2+]i) in response to TG and Ca2+ restoration. All experiments were performed in the presence of nifedipine, n = 7–13 of independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with hypoxic group. TG: thapsigargin.
Figure 7
Figure 7
Effects of KT5823, KT5720, and PMA on KMUP-1-inhibited capacitative calcium entry in hypoxic PASMCs. (A): Change in [Ca2+]i in pulmonary arterial smooth muscle cells (PASMCs) from pretreated KMUP-1 (1 μM), KT5823 (1 μM) with KMUP-1, KT5720 (1 μM) with KMUP-1, PMA (1 μM) with KMUP-1 and then subjected to the restoration of extracellular Ca2+ following store depletion with TG (1 μM). (B): Bar graph illustrates mean ± SE change in [Ca2+]i (∆[Ca2+]i) in response to TG and Ca2+ restoration. All experiments were performed in the presence of nifedipine, n = 6 of independent experiments. # p < 0.05 compared with hypoxic group; * p < 0.05 compared with KMUP-1 group in hypoxic PASMCs. PMA: phorbol 12-myristate 13-acetate; TG: thapsigargin.
Figure 8
Figure 8
Diagram summarizing the actions of KMUP-1 on the store-operated calcium channel (SOCs) in hypoxic PASMCs. Data suggest that KMUP-1 inhibits TRPC1 encodes for SOCs, activates the PKA/PKG pathway, and inhibits the PKC pathway. SOCs: store-operated calcium channels; SR: sarcoplasmic reticulum; SERCA: sarco/endoplasmic reticulum Ca2+-ATPase; TG: thapsigargin; PMA: phorbol 12-myristate 13-acetate; 8-Br-cAMP: membrane-permeable analog of cAMP; 8-Br-cGMP: membrane-permeable analog of cGMP; VOCC: voltage-operated calcium channels.
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