Objective:

2

To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca

2+

channel (LTCC) and related signaling pathway.

Methods:

2

Isometric tension recording was performed in HUV rings pre-contracted with K

+

. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping

in A7r5 cells

a rat vascular smooth muscle cell line

was performed to study the effect of hesperetin on LTCC current.

Results:

2

After depolarizing precontraction

hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7%±4.3% compared to matching vehicle

osmolality

and time controls (

P

<

0.0001). Importantly

hesperetin competitively inhibited BayK8644-induced contraction

shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49–2.40

]

in vehicle control to 11.30 nmol/L (95% CI 5.45–23.41) in hesperetin (

P

=0.0001). Moreover

hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase

adenylyl cyclase

PDE3

PDE4

and PDE5 (

P

<

0.01)

while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (

P

>

0.05). However

simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin's effect (

P

>

0.05). In whole-cell patch-clamping

hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7%±5.8% (

P

=0.0104).

Conclusions:

2

Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC

and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.