Residual NO modulates contractile responses and membrane potential in isolated rat mesenteric arteries

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Nitric Oxide

journal homepage:www.elsevier.com/locate/yniox

Residual NO modulates contractile responses and membrane potential in

isolated rat mesenteric arteries

Olcay Ergürhan K

ıroğlu

a

, Özlem Yorulmaz Özü

a

, Mustafa Emre

b

,

İhsan Bayel

a

,

Eda Karabal Kumcu

a

, M. Ata Seçilmi

ş

a,∗

a_{Department of Pharmacology, Çukurova University, Medical School, Adana, 01130, Turkey} b_{Department of Biophysics, Çukurova University, Medical School, Adana, 01130, Turkey}

A R T I C L E I N F O

Keywords: Isometric contraction

NOS inhibitor-insensitive residual NO Mesenteric artery

Membrane potential

A B S T R A C T

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS in-hibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01–10 μM), an α1-adrenoceptor agonist, led to depolarisation and concentration-dependent contraction in mesenteric arteries. The depolarisation and contractile responses were augmented by NA or L-NAME. Hydroxocobalamine (HC) or carboxy-PTIO (c-PTIO) also caused to further increase the membrane de-polarization and contractions induced by phenylephrine in the presence of NOS inhibitors. Chemical removal of endothelium by saponin, tyrosin kinase inhibitor erbstatin A, but not calmodulin inhibitor calmidazolium in-hibited the additional membrane depolarisation and contractile responses induced by L-NA or L-NAME and L-NA or L-NAME plus HC. Thesefindings show that residual NO modulates the contractile responses in isolated rat mesenteric arteries by exerting a tonic inhibitor effect on the depolarization and vasoconstriction induced by phenylephrine.

1. Introduction

In the control of blood pressure, vascular endothelium has a key role via transducing chemical and mechanical signals into vasoactivity by the release at least three vasodilators such as prostacyclin[1], nitric oxide (NO) [2–4] and endothelium-derived hyperpolarizing factor (EDHF) [5–7]. Generation of endothelial NO is stimulated by some chemical agonists in a Ca2+_{/calmodulin dependent manner [8]}

whereas endothelial NO is also formed by mechanical stimuli such as isometric contraction, pressure and shear stress depending on the ac-tivation of tyrosine kinase inhibitor-sensitive pathway [9,10]. After activation of mechanical or chemical stimuli, vascular NO is generated from L-arginine by the endothelial nitric oxide synthase (eNOS) in the endothelial cells[11].

It is well known that endothelial NO and EDHF have tonic inhibitor role in vasoconstrictor process in mesenteric arteries because en-dothelium removal, NOS inhibition or blockade of K+ channels po-tentiates the sensitivity to vasoconstrictor agents[6,12–15]. It has been

also demonstrated the activity of isometric contractions-induced NO in small mesenteric arteries [10,16,17]. In addition to NO, myogenic contractions in mesenteric arteries[13]and pulsatile or cyclic stretch in coronary arteries stimulate EDHF expression and release which mod-ulate the vascular tonus[18,19]. However, EDHF is only evaluated in the absence of NO and prostanoids[20]. For this purpose nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors together are most commonly used pharmacological tools to assess EDHF activity in re-sistance arteries. In the previous studies, it was shown that non-NO and non-prostanoid vasorelaxant responses attributed to EDHF were ad-ditionally inhibited in the presence of NO scavengers such as he-moglobin, hydroxocobalamine (HC) and carboxy-PTIO (c-PTIO) in rabbit carotid artery[21], human and porcin coronary arteries[22,23] and rat mesenteric arteries[24], suggesting residual NO contributes EDHF response. Recently, we reported that shear stress-induced NO has NOS-inhibitors resistant component known as residual NO which plays modulator role in the perfusion pressure increased by phenylephrine in the isolated rat mesenteric bed[17]. Meanwhile, the possible existence

http://dx.doi.org/10.1016/j.niox.2017.10.003

Received 10 July 2017; Received in revised form 28 September 2017; Accepted 11 October 2017

∗_{Corresponding author.}

E-mail address:asecilmis@cu.edu.tr(M.A. Seçilmiş).

Available online 12 October 2017

1089-8603/ © 2017 Elsevier Inc. All rights reserved.

of residual NO may inhibit activity of EDHF. In this case, data obtained from the experiments performed in these arteries may be mis-interpreted. Indeed, the presence of NO prevents EDHF up-regulation as a back-up mechanism[25]and it has been also reported that the pro-duction of EDHF is damped by NO in the pig coronary artery [26]. However, in addition to vasoconstriction, residual NO also may mod-ulate membrane potential increased by vasoconstrictor agents in me-senteric arteries.

Therefore, we tested the effect of NO scavengers on the pheny-lephrine-induced increase in vessel tone and membrane potential in the presence of NOS inhibitor L-NA or L-NAME to evaluate the possible existence of residual NO in response to vasoconstriction in the isolated rat mesenteric arteries. Our results demonstrated the modulator role of residual NO on the contraction and membran potential in the isolated rat mesenteric arteries under conditions of NOS/COX inhibition. 2. Materials and methods

2.1. Preparation of the isolated rat mesenteric arteries

The protocol of the study was approved by the ethics committee of Medical Faculty at Çukurova University. Male Wistar Albino rats weighing 200–250 g were used in our experiments. Rats were main-tained on a 12:12 h light/dark cycle and the room temperature was constant during housing. Animals were decapitated after halothane (60 mg/kg) anesthesia. The abdomen was immediately opened and superior mesenteric artery was removed by cleaning from connective tissues. Superior mesenteric artery segments, length in ± 2 mm, were mounted under 300 mg tension between two stainless hook, one end attached to an isometric force transducer (MAY FDT 10-A) and tissues were placed in 20 ml organ bath containing Krebs - Henseleit solution (composition in mM: NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2,

NaHCO3, 25, KH2PO41.2, glucose 11, Na2EDTA 0.01) to measure the

tissue's contractile responses isometrically. During the experiments, organ bath temparature was constant at 37 °C and medium was aereted with 95% O2and 5% CO2. Changes in isometric force were recorded

and stored using data acquisition system (BIOPAC MP30 Systems, Inc). All experiments were performed in non-prostanoid medium-induced by indomethacine (1μM).

2.2. Organ bath experiments

In the beginning of every experiment, after 1h_{equilibration period,}

each ring was precontracted with 40 mM KCl. After the tonus reached to a steady-state, the tissues were washed with fresh Krebs solution to return to basal tone. Then, a single dose of acethylcholine was applied to the tissues pre-contracted with phenylephrine (2–5 μM) to test the functional endothelium. The rings relaxed with acethycholine less than 50% were discarded. Then the rest of the tissues were contracted with phenylephrine (0,001–30 μM) cumulatively. After 30 min of resting period of fresh Krebs solution, phenylephrine (0,001–30 μM) added to the organ bath to constrict the tissues again. This protocol was accepted as control group. In the rest of the groups, experimental design was performed through the control group protocol. In the other experi-ments, test agents such as L-NA, L-NAME, c-PTIO, hydroxocobalamine, erbstatin A or calmidazolium was added to the organ bath medium after thefirst series of phenylephrine responses were obtained.

2.3. Electrophysiological studies

Mesenteric artery rings were longitudinally opened and placed on an organ chamber with the endothelial side upward. Tissues were su-perfused with Krebs-Henseleit solution and gassed with 95% O2, 5%

CO2at 37 °C at constant flow rate (4 ml/min) for 1 h. The smooth

muscle cells from the intimal initial inner side were implanted with borosilicate 3M KCl capillary microelectrodes (tip resistances:

40–60 mΩ) coupled by an Ag/AgCl junction to a high impedance ca-pacitance neutralizing amplifier (Nihon Kohden, Model MEZ-7200, Tokyo, Japan). As a reference electrode, an agar-bridge containing 3 M KCl was used. The resting membrane potential was monitored via a storage oscilloscope (Hitachi Model VC-6045, Tokyo, Japan). Three minutes after recording the stable resting membrane potential, acet-ylcholine was added to the medium to examine the integrity of en-dothelium and then the effect of phenylephrine on the membrane po-tential was recorded. All agents tested in in vitro studies were also used for recording the changes on the increased membrane potential by phenylephrine of the tissues in accordance with the experimental pro-tocol.

2.4. Drugs and solutions

Acetylcholine chloride, phenylephrine hydrochloride, Nω -nitro-L-arginine (nitro-L--nitro-L-arginine), Nω-nitro- L--nitro-L-arginine methyl ester, in-domethacin, hydroxocobalamine, and carboxy-PTIO were all purchased from Sigma Co. (St. Lois, MO, USA). Erbstatin A was obtained from Tocris and saponin was purchased from Fluka Chemie AG Deisenhofen, Germany. Indomethacin, calmidazolium were dissolved in dimethyl sulfoxide (DMSO). Erbstatin A was dissolved in ethanol. The final concentration of DMSO or alcohol in the 1 L of Krebs solution, 0,01%, did not affect phenylephrine-induced contractions. Nitro-L-arginine and saponin were directly dissolved in Krebs solution on the day of use. The other agents were dissolved in distilled water.

2.5. Statistical analysis

All data are expressed as mean ± S.E.M in the membrane potential and tension. The increase in the tension induced by each phenylephrine concentration was expressed as a percentage of 40 mM KCl contraction. One–way analysis of variance (ANOVA) followed by the Bonferonni post-hoc test was used for statistical analysis. P values less than 0.05 were considered significant.

3. Results

3.1. Concentration-response curves for phenylephrine in the presence of L-NA, L-NA + HC, L-NA + c-PTIO

In the control experiments, cumulative addition of phenylephrine (0,001–30 μM), an alpha-1 adrenergic receptor agonist, to the organ bath caused to concentration-dependent contractions in the isolated rat mesenteric arteries. NOS inhibition with Nω-nitro-L-arginin (100μM; L-NA) augmented the contractions induced by phenlyephrine at all con-centrations from 0.01μM. In the presence of L-NA, an NO scavenger hydroxocobalamine (HC; 100μM) or carboxy- PTIO (c-PTIO; 300 μM) caused to further increase especially significant in the lower con-centrations of phenylephrine (0.01–0.1 μM) (Figs. 1 and 2). Similar results were obtained with L-NAME (300μM) and L-NAME(300 μM) plus HC(100μM) or c-PTIO (300 μM) in the isolated mesenteric arteries contracted with phenlyephrine (0,001–30 μM) (Figs. 3 and 4). 3.2. Concentration-response curves for phenylephrine in the presence of Erbstatin A, Erbstatin A + L-NA, Erbstatin A + L-NA + HC

In the presence of Erbstatin A (300μM), a tyrosine kinase inhibitor, neither L-NA (100μM) nor L-NA (100 μM) plus HC (100 μM) affected the contractions induced by phenylephrine (0,001–30 μM) (Fig. 5). 3.3. Changes in the membrane potential in the presence of phenylephrine, phenylephrine + L-NA or phenylephrine + L-NA + HC in the isolated rat mesenteric arteries

resting membrane potential of the isolated rat mesenteric arteries. Addition of L-NA (100μM) caused further significant depolarization in the membrane potential compared with phenylephrine alone. Treatment with HC (100μM) produced further significant increase in the membrane potential compared with phenylephrine + L-NA (Fig. 6). Similar results were also observed in the presence of L-NAME (300μM) or L-NAME (300μM) + HC (100 μM) on the depolarisation induced by phenylephrine in the rat mesenteric arteries (Fig. 7).

3.4. Changes in the membrane potential in the presence of phenylephrine, phenylephrine + L-NA, phenylephrine + L-NA + HC in the endothelium-denuded isolated rat mesenteric ring preparations

In the endothelium-denuded arteries, phenylephrine caused sig-nificant depolarization in the resting membrane potential while L-NA (100μM) or L-NA (100 μM) + HC (100 μM) did not produce any fur-ther effect on the increase in the membrane potential induced by phenylephrine (Fig. 8).

3.5. Effects of calmidazolium, calmidazolium + L-NA,

calmidazolium + L-NA + HC in the isolated rat mesenteric vascular strips depolarized by phenylephrine

In the presence of calmidazolium, phenylephrine (0.1μM) caused to an increase in the membrane potential. The increase observed on the membrane potential with L-NA or L-NA plus HC was not affected by Ca-calmodulin inhibition (Fig. 9). The results obtained with L-NAME were similar with the ones induced by L-NA (Fig. 10).

3.6. Changes in the membrane potential in the presence of Erbstatin A, Erbstatin A + L-NA, Erbstatin A + L-NA + HC in the isolated rat mesenteric vascular strips depolarized by phenylephrine

In the presence of Erbstatin A, a protein kinase inhibitor, pheny-lephrine (0.1 μM) caused to an increase in the membrane potential while addition of L-NA (100μM) or L-NA + HC (100 μM) did not cause any further change in the membrane potential increased by

-9 -8 -7 -6 -5 -4 0 100 200 300

Control

L-NA

L-NA+HC

*

*

*

+

*

*

* *

*

+

+

+

Log[Phenylephrine]M

noi

tc

art

no

c

de

cu

dni

-l

C

K

fo

%

Fig. 1. The effects of L-NA (100 μM) and L-NA (100 μM) + HC (100 μM) on the con-tractions induced by phenylephrine (0,001–30 μM) in the isolated rat mesenteric arteries. The contractile responses are expressed as a percentage of 40 mM KCl-induced vaso-contraction.∗(p < 0.05) indicates statistical significance compared to the control and+

(p < 0.05) indicates statistical significance compared to L-NA groups.

-9 -8 -7 -6 -5 -4 0 100 200 300 Control L-NA L-NA+c-PTIO

*

+

*

*

*

+

*

*

* *

+ Log[Phenylephrine]M noi tc art no c de cu dni -l C K fo %

Fig. 2. The effects of L-NA (100 μM) and L-NA (100 μM) + c-PTIO (300 μM) on the contractions induced by phenylephrine (0,001–30 μM) in the isolated rat mesenteric arteries. The contractile responses are expressed as a percentage of 40 mM KCl-induced vasocontraction.∗(p < 0.05) indicates statistical significance compared to the control and+_{(p < 0.05) indicates statistical significance compared to L-NA groups.}

-9 -8 -7 -6 -5 -4 0 100 200 300 400

Control

L-NAME

L-NAME+HC

*

+

*

*

*

+

*

*

*

*

+

Log[Phenylephrine]M

noi

tc

art

n

oc

d

ec

u

d

ni-l

C

K

f

o

%

Fig. 3. The effects of L-NAME (300 μM) and L-NAME (300 μM) + HC (100 μM) on the contractions induced by phenylephrine (0,001–30 μM) in the isolated rat mesenteric arteries. The contractile responses are expressed as a percentage of 40 mM KCl-induced vasoconstriction.*_{(p < 0.05) indicates statistical significance compared to the control}

and+_{(p < 0.05) indicates statistical significance compared to L-NAME groups.}

-9 -8 -7 -6 -5 -4 0 100 200 300

Control

L-NAME

L-NAME+c-PTIO

*

+

*

*

*

+

*

*

*

*

+

Log[Phenylephrine]M

noi

tc

art

n

oc

d

ec

u

d

ni-l

C

K

f

o

%

Fig. 4. The effects of L-NAME (300 μM) and L-NAME (300 μM) + c-PTIO (300 μM) on the contractions induced by phenylephrine (0,001–30 μM) in the isolated rat mesenteric arteries. The contractile responses are expressed as a percentage of 40 mM KCl-induced vasoconstriction.*_{(p < 0.05) indicates statistical significance compared to the control}

phenylephrine in the isolated rat mesenteric vascular strip (Fig. 11). 4. Discussion

In the present study, it was investigated the residual NO activity in the presence of NOS inhibitors in response to vasoconstriction induced

by phenylephrine and we demonstrated that residual NO has a mod-ulator role in phenylephrine-induced contractions and membrane de-polarisation in the isolated rat mesenteric arteries.

In the blood vessels, stimulation of endothelial cells by endogenous or exogenous agonist leads to generation of the endothelial NO syn-thesized from L-arginine by eNOS Ca2+_{-calmodulin dependently,}

-9 -8 -7 -6 -5 -4 0 50 100 150 200

Erbstatin A

Erbstatin A+L-NA

Erbstatin A+L-NA+HC

Log[Phenylephrine]M

noi

tc

art

no

c

de

cu

dni

-l

C

K

fo

%

Fig. 5. The effects of Erbstatin A (300 μM), Erbstatin A (300 μM) + L-NA (100μM) or Erbstatin A (300 μM) + L-NA (100 μM) + HC (100 μM) on the contractions induced by phenylephrine (0,001–30 μM) in the isolated rat mesenteric arteries. The con-tractile responses are expressed as a percentage of 40 mM KCl-in-duced vasoconstriction. -50 -30 -10

Control

Phe

Phe+L-NA

Phe+L-NA+HC

*

*

*

+

+

#

mV

Fig. 6. Effects of phenylephrine (Phe; 0,1 μM), Phe (0,1 μM) +L-NA (100 μM), Phe (0,1μM) + L-NA (100 μM) + HC (100 μM) on the membrane potential of the rat me-senteric arteries.*_{(p < 0.05) indicates statistical significance compared to the control,}+

(p < 0.05) indicates statistical significance compared to Phe group, and#_{(p < 0.05)}

indicates statistical significance compared to Phe+L-NA group.

-50 -30 -10

*

*

*

+

+

#

Control

Phe

Phe+L-NAME

Phe+L-NAME+HC

mV

Fig. 7. Effects of phenylephrine (Phe; 0,1 μM), Phe (0,1 μM) +L-NAME (300 μM), Phe (0,1μM) + L-NAME (300 μM)+ HC (100 μM) on the membrane potential of the rat mesenteric arteries.*_{(p < 0.05) indicates statistical significance compared to the}

con-trol,+_{(p < 0.05) indicates statistical significance compared to Phe group, and}#

(p < 0.05) indicates statistical significance compared to Phe+L-NAME group.

-50 -30 -10

*

*

*

Control

Phe

Phe+L-NA

Phe+L-NA+HC

Endothel (-)

mV

Fig. 8. Effects of phenylephrine (Phe; 0,1 μM), Phe (0,1 μM) + L-NA (100 μM), Phe (0,1μM) + L-NA (100 μM) + HC (100 μM) on the membrane potential of the en-dothelium-denuded mesenteric arteries in rats.*_{(p < 0.05) indicates statistical}

sig-nificance compared to the control group.

-50 -30 -10

*

*

*

Control CAL+Phe CAL+Phe+L-NA CAL+Phe+L-NA+HC

+

+

# mV

Fig. 9. Effects of calmidazolium (CAL; 1 μM), calmidazolium (1 μM) + L-NA (100 μM) or calmidazolium (1μM) + L-NA (100 μM) + HC (100 μM) on the membrane potential increased by phenylephrine (0.1μM) in the rat mesenteric arteries.*_{(p < 0.05) indicates}

statistical significance compared to the control and+_{(p < 0.05) indicates statistical}

significance compared to control and CAL + Phe groups,#_{(p < 0.05) indicates}

resulting inhibition of vasoconstriction[4,11]. Conversely, endothelial damage or NOS inhibition increases the sensitivity to contractile agents in the various arteries[2,15,27–29]. NO-induced relaxation of vascular smooth muscle is accompanied by the hyperpolarisation involved in directly activation of Ca2+_{-dependent and/or voltage-gated K}+

-chan-nels in rabbit aorta[30], pulmonary artery smooth muscle cells[31] and indirectly throuhg cGMP-dependent mechanism in rat mesenteric arteries[32]. In the present study, firstly, tone of mesenteric arteries were increased submaximally by phenylephrine. After phenylephrine-induced contraction reached to plateau, vasoconstrictor responses were augmented by NOS inhibitors, L-NA or L-NAME, suggesting modulator role of endothelial NO-induced by vasoconstriction due to pheny-lephrine application in the vascular tone. These results are similar with those observed on the isolated perfused rabbit heart[33]and rat me-senteric bed[10,16,17]in the presence of NOS inhibitors. It was shown that endothelial factors, NO or EDHF led to membrane hyperpolarisa-tion in vascular tissues such as rabbit saphaneous artery[34]and rat aorta [35,36]. It has been reported that activation ofα1 adrenergic

receptors by noradrenaline lead to depolarisation of smooth muscle cells in the rat mesenteric arteries due to release of intracellular Ca2+

induced by inositol triphosphate (IP3) which was inhibited by nitric

oxide donors [32]. In the present study, we also observed that NOS inhibition caused to further increase in the membrane potential induced

by phenylephrine in the mesenteric arteries. These findings are in agreement with the previous study showing that SNAP, an NO donor, caused to both relaxation and hyperpolarisation of noradrenaline-sti-mulated rat superior mesenteric artery[32].

In the presence of NOS and COX inhibitors, endothelium dependent-vasodilator responses may be attributed to an unidentified factor EDHF [5–7,20]. However, previous studies showed that even at concentra-tions thougt to be enough to inhibit NO synthesis, NOS inhibitors failed to completely abolish NO activity[23,37]. Because EDHF is observed only in non-NO and non-prastonoid medium, vasodilator and hy-perpolarizing effects of residual NO may be thought as EDHF actions in the experimental studies on the vascular endothelium. However in the presence of NOS and COX inhibitors, observed actions of NO scavengers (hydroxocobalamine, carboxy-PTIO and hemoglobin) through the ad-ditional inhibition on NO activity in the vascular smooth muscle can point out the residual NO activity. Chen et al. demonstrated that acetylcholine caused EDRF and EDHF release in rat blood vessels[5]. In addition, it was shown that acethylcholine- or histamine-induced en-dothelium-dependent relaxant responses in rat mesenteric vascular bed [38]and A-23187- or bradykinin-induced EDHF generation in canine coronary arteries were inhibited by calmodulin antagonist calmidazo-lium[39]. These results suggest that NO or EDHF activity induced by chemical agonist is dependent on intracellular calcium-calmodulin coupling. Meanwhile it has been showed that generation of NO through Ca2+_{-independent activation of eNOS is also induced by isometric}

contraction in the rabbit aorta [10] and mesenteric artery [16]. Therefore residual NO generation may be associated with the activation of kinases, because the responses were sensitive to protein kinase hibitor staurosporin and tyrosine kinase inhibitor erbstatin A, but in-sensitive to calmodulin inhibitor. Recently we showed that the activity of NOS inhibitor-insensitive residual NO in response to pressure in-creased by phenylephrine in mesenteric arteries[17]. In this study to evaluate the residual NO activity, in the presence of NOS inhibition, hydroxocobalamine an NO scavenger was applied to mesenteric strips, resulting further increase in membrane voltage and contraction. These responses were not affected by the presence of calmodulin inhibitor calmidazolium. These results suggest that residual NO generation-duced by vasoconstriction is independent on intracellular calcium crease and modulates membrane depolarisation and contraction in-duced by phenylephrine in rat mesenteric arteries. We also observed that neither NOS inhibition nor NO-scavenging affected the contraction and depolarisation of mesenteric arteries in the presence of tyrosine kinase inhibitor Erbstatin A. Thesefindings suggest that activity of NOS by isometric vasoconstriction depends on tyrosine kinase but not cal-cium-calmodulin coupling. Because endothelial eNOS is expressed only in vascular endothelium, we treated mesenteric arteries with saponin to examine whether the selectivity of hydroxocobalamine is dependent on endothelial NO. In the endothelium-denuded mesenteric arteries, phe-nylephrine caused significant depolarisation in the resting membrane potential. However application of NOS inhibitor or NOS inhibitor plus HC failed to produce any further effect on the contraction and the de-polarisation induced by phenylephrine, showing activity of residual NO is dependent on existence of endothelium rather than smooth muscle. Zhao et al. observed that phenylephrine-induced contractions were smaller in endothelium intact rat thorasic aorta than those elicited in the endothelium removed ones of the spontaneous hypertensive rats and these contractions were also augmented by NO scavengers, c-PTIO or haemoglobin in the presence of COX and NOS inhibitors [40]. However, increased EDHF activity were observed by using small con-ductance calcium activated potassium channel blocker UCL 1684 and intermediate conductance calcium activated potassium channel blocker TRAM 34 while decreased eNOS expression and reduced NO bioavail-ability were determined in the subcutaneous arteries obtained from type-2 diabetic patients[41]. These results suggest that residual NO or EDHF may have a compensator role for the pathological conditions such as endothelial dysfunction or diminished eNOS expression. As the -50 -30 -10 * * * + +# Control CAL+Phe CAL+Phe+L-NAME CAL+Phe+L-NAME+HC mV

Fig. 10. Effects of calmidazolium (CAL; 1 μM), calmidazolium (1 μM) + L-NAME (300μM) or calmidazolium (1 μM) + L-NAME (300 μM) + HC (100 μM) on the mem-brane potential increased by phenylephrine (0.1μM) in the rat mesenteric arteries.*

(p < 0.05) indicates statistical significance compared to the control and+_{(p < 0.05)}

indicates statistical significance compared to control and CAL + Phe groups, #

(p < 0.05) indicates statistical significance compared to Phe + CAL and Phe + CAL + L-NAME groups. -50 -30 -10

*

*

*

Control Erbstatin A+Phe Erbstatin A+Phe+L-NA Erbstatin A+Phe+L-NA+HC mV

Fig. 11. Effects of Erbstatin A (300 μM), Erbstatin A (300 μM) + L-NA (100 μM) or Erbstatin A (300μM) + L-NA (100 μM) + HC (100 μM) on the membrane potential increased by phenylephrine (0.1μM) in the rat mesenteric arteries.*_{(p < 0.05) indicates}

contribution of endothelial factors may show differences according to species and/or tissues, it is needed further experimental studies using selective inhibitors to determine the contribution of residual NO or EDHF to the compensating mechanisms.

5. Conclusion

In conclusion, our data indicates that contractions induced by phenylephrine cause to endothelium-dependent, Ca2+_-calmodulin

in-dependent and tyrosine kinase inhibitor-sensitive residual NO release playing a modulatory role in both membrane potential and smooth muscle contraction in the rat in mesenteric arteries.

Acknowledgements

This study was supported by the Çukurova University Research Foundation (TF 2009BAP49). The authors wish to thank Ahmet Kantur and Zeynep Akıllı for their technical assistance. We are also indebted to Çukurova University Experimental Research Center (DETAUM) for supply of the rats.

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