Effects of bradykinin on voltage-gated KV 4 channels in muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease

Abstract

Key points: During exercise, bradykinin (BK), a muscle metabolite in ischaemic muscles, exaggerates autonomic responses to activation of muscle afferent nerves in peripheral artery disease (PAD). We examined whether BK inhibits activity of K_V 4 channels in muscle afferent neurons of PAD rats induced by femoral artery occlusion. We demonstrated that: 1) femoral occlusion attenuates K_V 4 currents in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles and decreases the threshold of action potential firing; 2) BK has a greater inhibitory effect on K_V 4 currents in muscle DRG neurons of PAD rats; and 3) expression of K_V 4.3 is downregulated in DRGs of PAD rats and inhibition of K_V 4.3 significantly decreases activity of K_V 4 currents in muscle DRG neurons. Femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit activity of K_V 4 channels in muscle afferent neurons and this is likely involved in the exaggerated exercise pressor reflex in PAD.

Abstract: Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in patients with peripheral artery diseases (PAD) and in PAD rats induced by femoral artery occlusion. However, the precise signalling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. A-type voltage-gated K⁺ (K_V ) channels are quintessential regulators of cellular excitability in the various tissues. Among K_V channels, K_V 4 (i.e. K_V 4.1 and K_V 4.3) in primary sensory neurons mainly participate in physiological functions in regulation of mechanical and chemical sensation. However, little is known about the role of K_V 4 in regulating neuronal activity in muscle afferent neurons of PAD. In addition, bradykinin (BK) is considered as a muscle metabolite contributing to the exaggerated exercise pressor reflex in PAD rats with femoral artery occlusion. Our data demonstrated that: 1) K_V 4 currents are attenuated in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles of PAD rats, along with a decreasing threshold of action potential firing; 2) K_V 4 currents are inhibited by application of BK onto muscle DRG neurons of PAD rats to a greater degree; and 3) expression of K_V 4.3 is downregulated in the DRGs of PAD rats and K_V 4.3 channel is a major contributor to the activity of K_V 4 currents in muscle DRG neurons. In conclusion, data suggest that femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit the activity of K_V 4 channels in muscle afferent neurons likely leading to the exaggerated exercise pressor reflex observed in PAD.

Keywords: A-type voltage-gated K+ channels; bradykinin; dorsal root ganglion; peripheral artery disease.

Figure 1.. Kv4 channels are involved in AP firing in rat muscle DRG neurons (DiI-labeled).

(A): Kv4 currents in muscle neurons. Protocol 1 was used to record the total TEA-resistant K⁺ currents (IK_total), and protocol 2 to record the delayed rectifying current (IK_DR), TEA-R-IKA = IK_total− IK_DR. Traces 1 & 2 were TEA-R-IKA of a muscle DRG neuron before and after AmmTX3 (inhibitor to Kv4 channels). Application of 2 μM AmmTX3 for 5 min blocked ~80% of the density of TEA-R-IKA in DRG neurons. *P < 0.05, comparing to untreated neurons (n=8 in each group). (B): In muscle DRG neurons (silent), AP firing was seen when currents were injected. After its application for 5 min, 2 μM of AmmTX3 enhanced the firing frequency of DRG neurons compared with controls using the same current when step currents were injected from −50 pA to 500 pA in 10 pA increments per step, and the protocol was shown with 50 pA increments per step from 150 pA to 200 pA. Averaged data showing that the frequency of AP firing was increased by AmmTx3 with 200 pA injected current. *P < 0.05, comparing to untreated neurons (n=8 in each group).

Figure 2.. Decreases in Kv4 currents and threshold of AP firing in muscle DRG neurons of occluded rats.

(A): The representatives of TEA resistant Kv4 currents in muscle afferent neurons of both control rats and PAD rats. Protocol 1 was for TEA-resistant IK_total, and protocol 2 for IK_DR. (B): I-V curve of Kv4 current in rat DRG neurons. After 3 days of femoral artery occlusion, the density of Kv4 currents in DRG neurons was decreased when depolarizing voltage stepped over −30mV from −100mV. *P<0.05 between control and occlusion (n=22 in each group) at the same depolarizing voltage. (C): The representatives of AP firing in rat muscle DRG neurons when step currents were injected from −50pA to 500pA in 10pA increments per step. (D): Averaged threshold of AP was decreased in muscle DRG neurons of occluded rats compared with control rats. *P<0.05 between control (n=45) and occlusion (n=27). (E): The density of TEA-R-IKA in muscle DRG neurons of both control rats and occluded rats when 2 μM AmmTX3 was applied. *P<0.05 between 2 μM AmmTX3 treatment and vehicle control in each group; #P <0.05 between control rats (n=8) and occluded rats (n=7). (F): A histogram showing % inhibitory efficiency of 2 μM AmmTX3 on Kv4 in muscle DRG neurons of both the control rats and the occluded rats. *P <0.05, comparing to the control animals.

Figure 3.. The effect of BK on Kv4 currents and AP firing in rat muscle afferent neurons.

After 1μM BK of application for 5 min, Kv4 currents were reduced in DRG neurons of control rats when depolarizing voltage stepped over 10mV from −100mV (A), and in DRG neurons of occluded rats when depolarizing voltage stepped over −30mV (B) *P<0.05, BK (n=8) vs. control without BK (n=9). (C) & (D): Showing AP firing frequency in rat DRG neurons after current injections. BK enhanced AP firing frequency in DRG neurons of both control and PAD *P<0.05, BK vs. control without BK. # P<0.05 vs. control rats.

Figure 4.. BK receptors involved in the effects of BK on Kv4 currents in rat muscle afferent neurons.

All DRG neurons were pre-incubated with 1 μM R-715 or 200 nM HOE 140 for 20 min before recording. (A): The inhibitory efficiency of 1 μM R-715 on TEA-R-IKA in muscle DRG neurons. *P <0.05, respectively comparing with 1 μM BK alone in the control or occluded groups. No significant differences were seen in % inhibitory efficiency between 1 μM R-715 and 1 μM R-715 plus 1 μM BK (P >0.05 for control and occluded groups). (B): The inhibitory efficiency of 200 nM HOE 140 on TEA-R-IKA in muscle DRG neurons. *P <0.05, respectively comparing with 1 μM BK alone and 200 nM HOE 140 plus 1 μM BK in the control or occluded groups.

Figure 5.. Expression of K_V4.1 and K_V4.3 channels of DRGs and the effects of blocking individual K_V4.1 and K_V4.3 channel on the activities of K_V currents.

(A): Representative bands and (B): averaged data, showing that the total protein levels of K_V4.3 in DRGs were less in occluded rats (n=6) than in control rats (n=6). *P<0.05 vs control. K_V4.1 expression tended to be lower in DRGs of occluded rats. Dots indicate individual data. Represented lanes on the bands indicate DRGs protein sample from individual rats. β-actin was used as the internal protein control. (C): representative traces and (D): averaged data showing that blocking K_V4.3 subunits (500 nM of PaTx1) had a less inhibitory effect on K_V4 currents in DRG neurons of occluded rats than its effect in DRG neurons of control rats. The representative traces of TEA-R-IKA in the rat muscle DRG neurons: the black traces and red traces indicate before and after Kv4.1 and Kv4.3 inhibitors were applied for 5 min. *P < 0.05 vs. control rats (n=14 in each group). There were no significant differences observed in inhibitory effects of blocking K_V4.1 subunit (2 μM of JZX-XII) on K_V4 currents in DRG neurons of control rats (n=8) and occluded rats (n=10; P > 0.05 between control rats and occluded rats).