Enhanced Contraction of Arterial Smooth Muscle Cell in Skin Artery Is Sensitive to Hyperpolarization Mediated by BK_Ca Channel in Chronic Constriction Injury Model Rat

Abstract

Previous our study found that improvement of skin blood flow associated with neuropathic pain using vasodilators is useful for alleviation of neuropathic pain. In this study, we aimed to elucidate the mechanism underlying enhanced vasorelaxation induced by vasodilators, which increase cAMP and cyclic guanosine monophosphate (cGMP), in chronic constriction injury model rat. We assessed vasorelaxation effect of vasodilators by measurement of isometric contraction in isolated plantar artery from chronic constriction injury of sciatic nerve model rats. Nifedipine, a voltage-dependent Ca²⁺ channel inhibitor, NS1619, Ca²⁺-activated K⁺ (BK_Ca) channel opener, and diazoxide, an ATP-sensitive potassium channel opener, -induced vasorelaxation in ipsilateral plantar artery was enhanced compared to the these in contralateral plantar artery. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and substance P, a NK₁ receptor agonist, caused vasorelaxation in both ipsilateral and contralateral artery. The vasorelaxation induced by SNP and substance P in ipsilateral artery is enhanced compared to the these in contralateral artery. Isoprenaline, a β adrenoceptor agonist, and salbutamol, a β₂ adrenoceptor agonist, caused strong vasorelaxation in ipsilateral artery but not in contralateral artery. Iberiotoxin, a BK_Ca channel inhibitor, prominently suppressed the enhanced vasorelaxation induced by SNP, substance P, isoprenaline and salbutamol. In summary, the enhanced contraction of arterial smooth muscle cell in skin artery is sensitive to hyperpolarization in chronic constriction injury model rat. Furthermore, β adrenoceptor agonist would be a good drug to improve the decreased skin blood flow because it has selective vasorelaxation to ipsilateral plantar artery.

INTRODUCTION

Acute pain is one of the important alert to prevent additional injury.¹⁾ On the other hand, chronic pain is thought as unnecessary pain.²⁾ Population of neuropathic pain patient is estimated with around 7–8% of general population.³⁾ Clinical drugs are administrated for alleviation of neuropathic pain but the effect of these drugs are unsatisfactory.⁴⁾ In neuropathic pain patients and model animals, reduced skin blood flow was found.^5–8) Our previous study has shown that hydralazine, a peripheral vasodilator, and phentolamine, an α adrenoceptor antagonist, ameliorated decreased skin blood flow and alleviated neuropathic pain in chronic constriction injury (CCI) mouse, suggesting an inverse relationship between skin blood flow and neuropathic pain.⁵⁾ Additionally, elcatonin, a calcitonin receptor agonist, preferentially suppresses the enhancement of ipsilateral plantar artery contraction in CCI rats,⁹⁾ while alleviating pain thresholds that had been enhanced by nerve injury.¹⁰⁾ Therefore, methods to relieve pain by improving blood flow are considered attractive.

We have found that the inactivation of sodium calcium exchanger (NCX) caused by decrease of Na⁺ gradient due to enhanced Na⁺ influx mediated by amiloride-sensitive pathway contributes to the Ca²⁺ elevation, which leads to enhanced plantar arterial contraction in CCI model rats.¹¹⁾ Thus, normalizing the regulatory mechanisms of calcium concentration would be a good target. Based on our previous results, it is plausible to say that enhanced vasocontraction is caused by intracellular molecular signaling and cAMP would suppress the enhanced vasocontraction by inhibiting some proteins involving in the enhanced intracellular signaling.⁹⁾ The present study aimed to clarify the mechanism of plantar artery relaxation by vasodilators to identify therapeutic targets in CCI rats. Our results showed that vasodilators increasing cAMP or cyclic guanosine monophosphate (cGMP) in arterial smooth muscle cell have higher affinity to noradrenaline (NAd)-induced pre-contraction in ipsilateral plantar arteries compared to contralateral ones and caused enhanced vasorelaxation mediated by large-conductance calcium activated potassium (BK_Ca) channel. The present study suggests that vasodilators increasing cAMP or cGMP would be useful for alleviation of neuropathic pain targeting for reduced skin blood flow.

MATERIALS AND METHODS

Animals

Male Wistar rats (SLC, Shizuoka, Japan) were housed in a light-dark regular cycle, with food and water available ad libitum as the animal use protocol was approved by the Institutional Animal Care and Use Committee and according to the Guidelines for Animal Experiments established by the Japanese Pharmacological Society.

CCI rats were made by sciatic nerve ligation method as described previously.⁹⁾ Briefly, the unilateral sciatic nerve of 7-week-old male Wistar rats was surgically exposed and loosely ligated with 4-0 braid silk, 4 times with 1 mm space under the anesthesia with sevoflurane or isoflurane. After 4 weeks from the operation, CCI rats were used for experiments.

Measurement of Isometric Contraction in Isolated Plantar Arteries

CCI rats were treated with sodium pentobarbital (50 mg/kg, intraperitoneally) and euthanized by decapitation. Plantar arteries were isolated in ice-cold Krebs–Henseleit (KH) solution (in mM: NaCl, 118; KCl, 4.7; CaCl₂, 2.55; MgSO₄, 1.18; KH₂PO₄, 1.18; NaHCO₃, 24.8; and glucose, 11.1) aerated with 95% O₂/5% CO₂. The isolated plantar arteries were kept in KH solution overnight at 4 °C to prevent spontaneous contractions. The arteries were cut into ring segments around 2 mm in width. Each ring segment was mounted on a myograph (Multi Myograph Model 610M; Danish Myo Technology A/S, Aarhus, Denmark) with two tungsten wires of 40 µm diameter in KH solution aerated with 95% O₂/5% CO₂ at 37 °C. Isometric tension was measured and recorded using a data analysis program (Myodaq 2.01; Danish Myo Technology A/S, Aarhus, Denmark). The arteries were loaded with a resting tension of 5–10 mN. After suitable stabilization, KH solution was replaced to 80 mM KCl solution (in mM: NaCl, 42.7; KCl, 80; CaCl₂, 2.55; MgSO₄, 1.18; KH₂PO₄, 1.18; NaHCO₃, 24.8; and glucose, 11.1). This procedure was repeated until stable contraction was attained. We used only specimens in which acetylcholine (1 µM), a muscarinic receptor agonist, induced a relaxation of less than 10% on contraction induced by noradrenaline. The noradrenaline concentration was adjusted so that the noradrenaline-induced contraction was approximately 80% of the magnitude of the 80 mM KCl-induced contraction. Vasodilator-induced relaxation was assessed taking this contraction as 100%. When applying iberiotoxin, the inhibitor was administered 15 min before application of noradrenaline. Each cumulatively applied data set was fitted by Hill’s equation using Prism (Prism-graph pad software, San Diego, CA, U.S.A.), to estimate the half effective concentration (EC₅₀) and maximum relaxation of the vasodilators with the following equation:

  

where X is drug concentration, Y is effect, A is a minimum value of the curve, B is maximum value of the curve, B−A is the maximum relaxation, and C is the slope of the curve (Hill coefficient). Using these parameters, the effect of the ipsilateral and contralateral vessels was compared and are summarized in tables.

Drugs

Iberiotoxin, NS1619, nifedipine, salbutamol, sodium nitroprusside and noradrenaline were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Substance P was purchased from PEPTIDE Institute (Osaka, Japan). Other drugs were purchased from Wako (Osaka, Japan).

Statistical Analysis

Data are expressed as mean  ±  standard error of the mean (S.E.M.). Statistical significance was tested by Student’s t-test or paired t-test. p-Value less than 0.05 was considered significant.

RESULTS

Hyperpolarization Is the Point at Which the Ipsilateral Artery Exhibits Greater Sensitivity to Vasodilators

Nifedipine, an L-type voltage dependent calcium channel inhibitor, -induced vasorelaxation in the ipsilateral plantar artery was enhanced compared that in the contralateral artery (Fig. 1A, Table 1).

Fig. 1. Concentration–Response Curves of Relaxation by Nifedpine (A), NS1619 (B) or Diazoxide (C) to NAd-Induced Contraction in the Contralateral (Open Symbols) or Ipsilateral (Closed Symbols) Plantar Arteries Isolated from CCI Rats (n = 4–7)

After NAd-induced contraction reached steady state, each vasodilators were applied in cumulative manner. Data were expressed as means ± S.E.M.

Table 1. Parameters for the Concentration Response Curves of Vasodilators to NAd-Induced Contraction in the Contralateral or Ipsilateral Plantar Arteries Isolated from CCI Rats

	Contralateral		Ipsilateral
	pEC50	Max	pEC50	Max
Isoprenaline	7.39 ± 0.06	16.04 ± 2.00	7.32 ± 0.14	75.40 ± 10.22**
Salbutamol	6.78 ± 0.05	37.39 ± 4.26	8.04 ± 0.13**	88.15 ± 2.07**
Substance P	7.97 ± 0.04	88.58 ± 1.33	8.25 ± 0.06**	92.17 ± 1.34
SNP	6.83 ± 0.14	82.58 ± 4.38	8.12 ± 0.09**	97.21 ± 0.33*
Nifedipine	8.02 ± 0.05	65.99 ± 5.57	8.31 ± 0.04**	86.44 ± 2.95*

Isoprenaline, salbutamol, sodium nitroprusside, substance P and nifedipine-induced vasorelaxation was normalized by noradrenaline-induced pre-contraction in contralateral and ipsilateral plantar artery in chronic constriction injury rat. pEC₅₀ is the negative logarithm of the effective concentration required by 50% response of the maximum relaxation (Max). Data are presented as means ± S.E.M. *p < .05 and **p < .01 vs. contralateral side.

We then examined the effects of activators on each of two different potassium channels that could affect calcium channel activity. NS1619, a large conductance calcium-activated potassium channel opener, and diazoxide, an ATP-sensitive potassium channel opener, -induced vasorelaxation in the ipsilateral plantar arteries were both enhanced compared to those in the contralateral ones (Figs. 1B, C). Because the concentration-response relationships of NS1619 and diazoxide in the contralateral plantar arteries were not saturated and the fitting failed to converge, the parameters (pEC₅₀ and Max) could not be calculated.

Vasodilators That Mediate Adenylate Cyclase or Guanylate Cyclase Preferentially Inhibit Contraction of Ipsilateral Arteries

Maximum relaxation in concentration-response curve of isoprenaline, a β adrenoceptor agonist, in the ipsilateral plantar artery was larger than that in contralateral artery but significant difference of pEC₅₀ was not detected (Fig. 2A, Table 1). Maximum relaxation and pEC₅₀ in concentration-response curve of salbutamol, a β₂ adrenoceptor agonist, in the ipsilateral plantar artery was larger than that in contralateral artery (Fig. 2B, Table 1). pEC₅₀ in concentration-response curve of substance P, NK₁ receptor agonist, in the ipsilateral plantar artery was larger than that in contralateral artery but significant difference of maximum relaxation was not detected (Fig. 2C, Table 1). Maximum relaxation and pEC₅₀ in concentration-response curve of sodium nitroprusside (SNP), an NO donor, in the ipsilateral plantar artery was larger than the one in contralateral one (Fig. 2D, Table 1).

Fig. 2. Concentration Response Curves of Relaxation by Isoprenaline (A), Salbutamol (B), Substance P (C) or Sodium Nitroprusside (D) to NAd-Induced Contraction in the Contralateral (Open Symbols) or Ipsilateral (Closed Symbols) Plantar Arteries Isolated from CCI Rats (n = 4–7)

After NAd-induced contraction reached steady state, each vasodilators were applied in cumulative manner. Data were expressed as means ± S.E.M.

Enhanced Vasorelaxation Was Strongly Inhibited by Calcium-Activated Potassium Channel Inhibitor

One hundred nanomolar iberiotoxin, an inhibitor of BK_Ca channel, strongly inhibited salbutamol-, substance P- or SNP- induced vasorelaxation in the ipsilateral plantar arteries (Fig. 3, Table 2). Significant difference of pEC₅₀ in concentration response curve of substance P in the presence of iberiotoxin (100 nM) between ipsilateral and contralateral plantar arteries was not detected (Table 2). However, significant difference of pEC₅₀ and maximum relaxation in concentration-response curve of SNP still remained (Table 2). The inhibitory effect of salbutamol was weak so that parameters could not calculated.

Fig. 3. Concentration Response Curves of Relaxation by Salbutamol (A), Substance P (B) or Sodium Nitroprusside (C) to NAd-Induced Contraction in the Contralateral (Open Symbols) and Ipsilateral (Closed Symbols) Plantar Arteries Isolated from CCI Rats in the Presence of Iberiotoxin (n = 4–6)

Iberiotoxin (100 nM) was applied 15 min before NAd; each vasodilator was administered cumulatively after NAd-induced contraction reached steady state. Data are expressed as means ± S.E.M.

Table 2. Parameters for the Concentration Response Curves of Relaxation by Substance P or Sodium Nitroprusside to NAd-Induced Contraction in the Contralateral or Ipsilateral Plantar Arteries Isolated from CCI Rats in the Presence of Iberiotoxin

	Contralateral		Ipsilateral
	pEC50	Max	pEC50	Max
Substance P	8.83 ± 0.07	81.01 ± 0.61	8.98 ± 0.06	81.72 ± 3.42
SNP	6.84 ± 0.15	70.92 ± 3.31	7.25 ± 0.03*	94.78 ± 0.73**

Sodium nitroprusside and substance P-induced vasorelaxation in the presence of iberiotoxin (100 nM) was normalized by noradrenaline-induced pre-contraction in contralateral and ipsilateral plantar artery in chronic constriction injury rat. pEC₅₀ is the negative logarithm of the effective concentration required by 50% response of the maximum relaxation (Max). Data are presented as means ± S.E.M. *p < .05 and **p < .01 vs. contralateral side.

DISCUSSION

Previous our study showed that improvement of reduced skin blood flow by vasodilators is correlated with alleviation of neuropathic pain in CCI mice and enhanced cutaneous vasocontraction is caused by NCX inactivation following reduction of skin blood flow in CCI rats.^5,11)

The present study found that the effect of vasodilators, which increase cAMP mediated by G_s protein coupling receptors or cGMP to ipsilateral plantar arterial contraction is potent than the that to contralateral plantar arterial contraction in CCI rats. In addition to it, the enhanced vasorelaxation caused by increased cAMP or cGMP is followed by activation of BK_Ca channel.¹²⁾ Therefore, it is safe to say that vasorelaxation of the ipsilateral plantar artery due to hyperpolarization is enhanced compared to the contralateral plantar artery.

In smooth muscle cells, an increase in intracellular Ca²⁺ concentration leads to vasoconstriction, accompanied by depolarization due to activation of receptor coupling cation channels.¹²⁾ Potassium channels, on the other hand, play an important role in inhibiting smooth muscle cell tone.¹³⁾ In particular, activation of BK_Ca channel strongly contributes to smooth muscle cell relaxation.¹⁴⁾ BK_Ca channel is activated by elevation of intracellular Ca²⁺ concentration and phosphorylation mediated by protein kinases.¹²⁾ Isoprenaline and salbutamol is a non-specific β adrenoceptor and specific β₂ adrenoceptor agonist, respectively.¹⁵⁾ Activation of β adrenoceptors increases intracellular cAMP followed by activation of protein kinase A (PKA).¹⁶⁾ Furthermore, substance P is a NK₁ receptor agonist and NK₁ receptor conjugates G_q-proteins.¹⁷⁾ Activation of NK₁ receptor in endothelial cell causes elevation of intracellular Ca²⁺ concentration followed by nitric oxide synthase (NOS) activation.^18,19) NO produced by NOS activates guanylate cyclase to produce cGMP followed by activation of PKG.²⁰⁾ Activated PKA and PKG activate BK_Ca channel by phosphorylation of α and β-subunit.¹⁴⁾

Substance P causes an indirect relaxation response via NO, one of the endothelium-derived relaxing factors (EDRFs).²¹⁾ Enhanced vasorelaxation induced by NO donor in the ipsilateral side was much stronger than the one induced by substance P. Although the effect of iberiotoxin showed that the relaxation response induced by NO is mediated by BK_Ca channel, the relaxation response to substance P is less inhibited by iberiotoxin. This result suggests that the latter, unlike the former, may involve a non-BK_Ca channel-mediated relaxation mechanism. Another member of the EDRFs, prostaglandin I₂ (PGI₂), produces cAMP and relaxes smooth muscle, as do β-adrenergic receptor agonists. Thus, substance P may cause relaxation via factors of endothelial origin other than EDRFs, e.g., endothelial-derived hyperpolarization factors (EDHFs). Otherwise, the effect of NO on contraction by NAd was enhanced in the ipsilateral plantar artery compared to the contralateral plantar artery, but conversely NO production in endothelial cells was reduced. It is plausible to say that these two may have been offset in relaxation by substance P. As a result, the contribution of the BK_Ca channel-mediated relaxation mechanism is reduced, so that substance P relaxation in the ipsilateral becomes less sensitive to iberiotoxin. Further research is needed on this point. Importantly, however, the small difference between the ipsilateral and contralateral plantar arteries in the relaxation response, which is less sensitive to iberiotoxin as in the action of substance P, indicates the superiority of BK_Ca channel as a therapeutic target for hypersensitive contractions.

Isoprenaline or Salbutamol causes large vasorelaxation in mesenteric, pulmonary artery and aorta.^22,23) On the other hands, in contralateral plantar artery, these β adrenoceptor agonists did not cause large vasorelaxation to NAd-induced contraction. β adrenoceptor agonists-induced vasorelaxation to NAd-induced contraction in the ipsilateral plantar artery was very large. The enhanced β adrenoceptor agonists-induced vasorelaxation was strongly suppressed by BK_Ca channel inhibitor. It is unclear how BK_Ca channel is involved in the selective mechanism where ipsilateral β adrenergic receptor stimulation elicits a stronger relaxation response than the contralateral side. It should be noted, however, that the adrenergic concentration required to induce a similar degree of contraction is lower in the ipsilateral vessels than in the contralateral vessels. The stronger effect of nifedipine on ipsilateral contraction also supports the possibility that ipsilateral arterial excitation was less than contralateral contraction.

Previous our study found that enhanced Ca²⁺ elevation caused by decreased NCX activity is a key player of enhanced vasocontraction in CCI model: NCX inhibitor scarcely affected enhanced contraction in ipsilateral plantar artery while contralateral plantar arterial condition was mimicked to the ipsilateral condition by the NCX inhibitor.¹¹⁾ In other words, even weak Ca²⁺ influx can cause sufficient Ca²⁺ elevation. In this study result, we found that contribution of voltage-dependent Ca²⁺ channel to NAd-induced contraction in ipsilateral side is larger than the that in contralateral side. Therefore, the ipsilateral side may be more sensitive to hyperpolarization than the contralateral side. This hypothesis also explains why the ipsilateral plantar artery is more sensitive than the contralateral plantar artery to hyperpolarization-mediated relaxation, which occurs not only with BK_Ca but also with K_ATP channel activation.

We showed in this paper that BK_Ca channel activation is an important component of the ipsilateral plantar artery preferential relaxation response, and that vasodilators that induce a BK_Ca channel-mediated relaxation response by second messengers such as cAMP and cGMP may be a potential option for future pain relief treatment. Especially, β adrenoceptor mediated-vasorelaxation would be a good therapeutic target.

Acknowledgments

This work was supported by JSPS KAKENHI Grant No. JP25670041.

Conflict of Interest

The authors declare no conflict of interest.

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