Study of the mechanisms regulating human umbilical artery contractility

doi:10.4236/health.2010.24049

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Vol.2, No.4, 321-331 (2010) Health

doi:10.4236/health.2010.24049

Study of the mechanisms regulating human umbilical

artery contractility

António José Santos-Silva1, Elisa Cairrão1,2, Ignacio Verde3

1Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Avenida Infante Dom Henrique, Covilhã, Portugal

2Centro Hospitalar da Cova da Beira, Quinta do Alvito, Covilhã, Portugal

3Centro de Investigação em Ciências da Saúde, Universidade daBeira Interior, Avenida Infante Dom Henrique, Covilhã, Portugal;

iverde@fcsaude.ubi.pt

Received 10 November 2009; revised 27 December 2009; accepted 5 January 2010.

ABSTRACT

We studied the involvement of different types of

Ca2+ channels, cyclic nucleotides and different

kinases in the regulation of human umbilical

artery (HUA) contractility. The elucidation of the

precise mechanisms regulating the contractility

of this artery could be very important to reveal

potential therapeutic targets to treat HUA dis-

orders such as preeclampsia. The relevancy of

different types of Ca2+ channels on the regula-

tion of HUA tonus was analyzed. Among the

different Ca2+ channel inhibitors used, only the

L-type calcium channels (LTCC) inhibition in-

duced relaxation of HUA in Ca2+ containing me-

dium. The inhibition of T-type calcium channels

(TTCC) or TRP channels did not significantly

affect HUA contractility. In presence of Ca2+, the

intracellular increase of a cyclic nucleotide

(cAMP or cGMP) induces relaxation of HUA,

which was almost complete in histamine-con-

tracted HUA, and lower effect was observed in

arteries contracted by KCl and serotonin (5-HT).

Inhibition of PKA and PKG weakly reduced the

relaxations induced by the increase of cAMP

and cGMP respectively, suggesting that the re-

laxation induced by these nucleotides is not

totally mediated by the activation of their re-

spective kinases and that oth er mechani sms are

involved. In calcium containing solution, PP2A

inhibition produces relaxation of contracted

HUA. In KCl contracted arteries, the OA and

nifedipine relaxant effects are similar and not

additive, suggesting that PP2A could activate

LTCC. Besides, the increase of cyclic nucleo-

tides significantly increased the OA effect,

suggesting that the effect of PP2A inhibition is

independent of the cyclic nucleotide pathways.

The contractions induced by KCl, histamine and

5-HT in presence of Ca2+ were not significantly

affected by ROCK, ERK1/2 or p38MAPK inhibi-

tors. In absence of extracellular Ca2+, histamine

and 5-HT elicited contractions of HUA charac-

terized by two components, a rapid phasic con-

tractile component followed by a decrease of

the contraction until a tonic component. How-

ever, KCl elicited sustained contractions of HUA

in absence of extracellular Ca2+. As in presence

of calcium, the ERK1/2 and p38MAPK inhibitors

did not influence the contractions induced by

KCl, histamine or 5-HT in absence of extracel-

lular Ca2+. However, in these conditions, ROCK

inhibition significantly relaxed the contractions

induced by KCl and reduced the phasic and

tonic components of the contraction elicited

either by histamine or 5-HT. Our results show

that calcium-dependent contractions of HUA

depend on Ca2+ entry by LTCC, and these chan-

nels seems to be positive regulated by PP2A.

Cyclic nucleotides mediate HUA vasodilatation

but their dependent kinases are not the unique

responsible of this effect. HUA is able to con-

tract independently of Ca2+ influx by activating

the ROCK pathway and/or due to intracellular

Ca2+ release.

Keywords: Umbilical Artery; Smooth Muscle;

L-type Ca2+ Channels; ROCK; Cyclic AMP;

Cyclic GMP

1. INTRODUCTION

The mechanisms regulating smooth muscle contractility

in human umbilical artery (HUA) are very important for

optimum gas and nutrient exchange between foetus and

placenta. Since the umbilical blood vessels are not in-

nervated, the con trol of umbilical blood flow depends of

vasoactive substances either released locally or existing

in the circulation [1,2]. In general, vascular smooth

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

322

muscle (VSM) contractions are initiated by receptor or

ion channel activation and involve Ca2+ dependent

and/or independent mechanisms [3]. The increase of

cytosolic free Ca2+ can be originated by a transient and

rapid increase due to the Ca2+ release from the sar-

coplasmic reticulum and/or by a sustained extracellular

Ca2+ influx through Ca2+ channels [4,5]. This Ca2+ in-

crease leads to myosin light chain kinase activation and

contraction due to interaction between the thick and the

thin myofilaments [6]. VSM relaxation can be mediated

by activation of myosin light chain phosphatase which

has opposite effects than myosin light kinase activation.

Different types of Ca2+ channels have been involved

in the control of VSM contractility, such as L-type Ca2+

channels (LTCC) [7,8], T-type Ca2+ channe ls (TTCC) [9],

store-operated Ca2+ channels (SOCC) [10] or stretch-

activated channels (SAC) [11,12]. Recently, the SOCC

and SAC have been identified as being transient receptor

potential (TRP) channels [13]. In VSM, different TRP

channels were linked with Ca2+ store depletion,

G-protein-coupled receptor activation, membrane stretch,

pho- spholipid signals and other factors. However, the

role of these channels was not clarified yet, probably due

to the lack of specific pharmacological tools, such as

specific activators and/or inhibitors [14]. The TTCC

were mainly involved in the regulation of cell prolifera-

tion [15]. Although these channels were involved in

smooth muscle contraction by some authors [16], the

information linking TTCC with VSM contractility is

scant and their relative importance in this process needs

further analysis. On the other hand, LTCC seems to be

dominant in the regulation of VSM contractility because

they have been appointed as the main pathway for Ca2+

entry. In HUA, some authors identified different LTCC

and TTCC [17] and the LTCC blockers have appeared to

be the most potent relaxants of this artery [18]. On the

other hand, in smooth muscle cells, LTCC could be

regulated by PP2A, although the number of studies is

very low and the direction of this modulation has re-

mained somewhat controversial. Some author have

shown that PP2A does not modulate LTCC in tracheal

smooth muscle [19], but in intestinal smooth muscle a

dual effect of phosphatase inhibitors on LTCC has been

reported [20]. In human umbilical vein, Groschner et al.

have suggested that inhibition of PP2A activates LTCC

[21].

Concerning the Ca2+ independent mechanism, it could

involve Ca2+-sensitization mediated by the activation of

the RhoA-kinase (ROCK) which causes inhibition of

myosin light ch ain phosph a tase, leading to an increase of

myosin light chain phosphorylation [5]. In this sense,

some authors have reported that rhoA/ROCK pathway

can contribute to agonist-induced contractions of HUA.

However, this effect seems to be limited to intracellular

Ca2+-induced contractions and may be more important in

sustaining contractions rather than the initial phase of

force development [22]. Other authors have suggested

the existence, in VSM cells, of a Ca2+-dependent Rho

stimulation mechanism linked to different receptor cou-

pled to G proteins [23]. Also, it has been described that

rabbit artery smooth muscle depolarization increases

Ca2+ sensitization due to ROCK activation [24]. On the

other hand, a role of some components of the MAPK

cascade and its substrates (ERK1 and ERK2) in modu-

lating the VSM contractility has been also suggested

[25]. Some roles of these kinases in the regulation of cell

proliferation and differentiation has been establish ed [26]

but their role in VSM contraction and relaxation is al-

most unknown. Some agonists inducing VSM contrac-

tion can also activate ERKs [27,28]. In cerebral arteries,

it was suggested that ERKs modulate Ca2+ sensitivity

and contractility [25]. Other authors have observed that

5-HT induces rat aorta contractions involving p38

MAPK or Erk MAPK pathways [29-31].

The cyclic nucleotides, cAMP or cGMP, are the main

second messenger involved in the regulation of vasodila-

tion [32]. Intracellular accumulation of cAMP and

cGMP can be achieved by stimulation of adenylate or

guanylate cyclase, respectively, or by inhibition of

phosphodiesterases (PDE) [33]. Concerning cAMP, in-

tracellular increase of this nucleotide induces relaxation

of different human arteries [34-36], including HUA [37].

Among the four families of PDE expressed in this

smooth muscle (PDE1, PDE3, PDE4 and PDE5), PDE4

has been shown as the key enzyme involved in the regu-

lation of HUA relaxation associated to cAMP [37].

Concerning cGMP, the increase of the intracellular level

of this nucleotide also ind uces artery vasodilatation [38 ],

including HUA vasodilatation [37,39]. PDE5 has been

shown as the key enzyme involved in the regulation of

HUA relaxation associated to cGMP [37]. Also, distinct

authors have described that in different arteries [40,41],

including HUA [39], the vasodilatation induced by

cGMP is mediated by activation of potassium channels.

Increases in cAMP and cGMP activate cAMP-dependent

protein kinase (PKA) and cGMP-dependent protein

kinase (PKG), respectively [42]. In VSM cells, the inhi-

bition of LTCC by PKG has been reported [43]. How-

ever, the cAMP pathway has been suggested to inhibit,

to enhance, or to have no effect on smooth muscle LTCC

[43]. Also, some authors have suggested that relaxation

induced by cyclic nucleotides is not totally mediated by

the activation of their respective kinases. In this sense,

other mechanisms were involved, such as cross-activa-

tion between cyclic nucleotide dependent kinases [44] or

the regulation of other proteins having Epac (exchange

protein directly activated by cAMP) which activates the

small GTPbinding protein Rap1 [45].

Despite of the great importance of HUA before and

during childbirth, the mechanisms involved in the regu-

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

323

lation of the contractility of this artery have been weakly

studied. Devoid of en ervation, the regulation of vascular

tone of the HUA depends only by the local release of

humoral factors, such as 5-HT and histamine [1,2]. In

this work we have evaluated the relevancy of some

mechanisms involved in the contraction and relaxation

of this artery, such as extracellular Ca2+, cyclic nucleo-

tides, Ca2+ channels and different kinase types.

2. METHODS

2.1. Tissue Preparation

Umbilical cord pieces of 3-7 cm were obtained from

normal term pregnancies with the consent of the donor

mothers. All procedures carried out with these samples

have been approved by the Ethics Committee of “Centro

Hospitalar da Cova da Beira EPE”. The umbilical cord

samples were collected in sterile physiological saline

solution (composition, mM: NaCl 110; CaCl2 0.15; KCl

5; MgCl2 2; HEPES 10; NaHCO3 10; KH2PO4 0.5;

NaH2PO4 0.5; Glucose 10; EDTA 0.49). In order to

avoid contaminatio n and tissue degradatio n, penicillin (5

U/ml), streptomycin (5 µg/ml), amphotericin B (12.5

ng/ml) and antiproteases (leupeptine, 0.45 mg/l; ben-

zamidine, 26 mg/l; and trypsin inhibitor, 10 mg/l) were

added to the physiological saline solution. Umbilical

artery rings of 3-5 mm were isolated from the surround-

ing connective tissue. Vascular endothelium was me-

chanically removed by gentle rubbing with a cotton bud

introduced through the arterial lumen. These denuded

HUA rings were used to perform contractility experi-

ments.

2.2. Artery Tension Recording

2.2.1. Relaxation Studies in Ca2+ Cont aining Medium

The HUA rings were placed in organ bath chambers

(LE01.004, Letica) containing Krebs-bicarbonate solu-

tion (composition, mM: NaCl 119, KCl 5.0, NaHCO3

25, KH2PO4 1.2, CaCl2 0.5, MgSO4 1.2, EDTA 0.03,

glucose 11) at 37ºC and continuously gassed with car-

bogen. The artery rings were suspended between two

parallel stain- less steel wires and tension measurement

was performed using isometric transducers (TRI201,

Panlab SA, Spain), amplifier (ML118/D Quad Bridge,

ADInstruments), interface PowerLab/4SP (ML750,

ADInstruments) and a computerized system with

Chart5 PowerLab software (ADInstruments). For

analysis, the isometric tension measured has been ex-

pressed in milligrams (mg) of force elicited by the ar-

tery in presence of drugs. To analyze the relaxation data,

we used the percentage of reduction on the maximal

contraction induced by the contractile agents. During

the resting periods, the organ bath solution was

changed every 15 min. Initially, the rings were equili-

brated for 60 min until a resting tension of 1000 mg

was achieved. After this, the rings were challenged

with 5-HT (1µM) to test their viability. Rings that in-

duced a maximal contraction lower than 1 g when

challenged with 5-HT were excluded from the study.

Afterwards, the rings were contracted using KCl (60

mM), histamine (10



M) and 5-HT (1



M). To deter-

mine the involvement of distinct types of Ca2+ channels,

the LTCC blocker nifedipine (10



M), the TTCC

blocker mibefradil (10



M) and the TRP blocker

2-aminoethoxydiphenyl borate (APB; 100



M) have

been used.

To analyze the involvement of the cAMP or cGMP

pathways the following drugs were used in some cases:

rolipram (1



M), a PDE4 selective inhibitor; forskolin

(10



M), an adenylate cyclase activator; KT-5720 (KTa;



M), a PKA inhibitor; sodium nitroprusside (SNP;



M) a guanylate cyclase stimulator; dipyridamol (3



M), a PDE5 inhibitor; and KT-5823 (KTg; 1



M), a

PKG inhibitor. To evaluate the possible involvement of

PP2A, okadaic acid (OA; 5 nM) has been used in some

experiments. Control experiments with ethanol, the vehi-

cle used to dissolve some drugs, were alway s perform ed.

2.2.2. Relaxation S tudies in Ca2+-free Medium

To analyze the HUA contractility in absence o f Ca2+, we

used a Krebs solution without Ca2+ (composition, mM:

NaCl 119, KCl 5.0, NaHCO3 25, KH2PO4 1.2, MgSO4

1.2, EDTA 0.03, EGTA 0.5, glucose 11). The rings were

also contracted using KCl (60 mM), histamine (10



or 5-HT (1



M).

To analyze the modulation of contractility by some

kinases in Ca2+-free contractions, the following drugs

were used in some experiments: Y-27632 (Y27; 10



M),

a ROCK inhibitor; PD-98059 (PD; 50



M) an ERK1/2

inhibitor; and SB-203580 (SB; 25



M), a p38MAPK

inhibitor. In some experiments, nifedipine (10



M) and

OA were also used.

2.3. Drugs and Chemicals

All drugs and chemicals have been purchased from

Sigma-Aldrich Quimica (Sintra, Portugal), except for-

skolin and rolipram, which were purchased from Biogen

Cientifica (Madrid, Spain). Forskolin, rolipram and

dipyridamol, were initially dissolved in ethanol and all

the other drugs were initially disso lved in distilled water.

Final solutions were obtained by dilution with Krebs

solution. The final concentration of ethanol in the organ

bath did never exceed 0.1%.

2.4. Statistical Analysis

Statistical analysis of the data has been performed using

the SigmaStat Statistical Analysis System, version 1.00

(1992). Results have been expressed as mean ± s.e.m. of

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

324

n experiments. Comparison among multiple groups was

analyzed by using a one-way ANOVA followed by

Tukey or Dunnet´s post hoc tests to determine signifi-

cant differences among the means. Comparison among

two groups was analyzed by using Student-t test. Prob-

ability levels lower than 5% were considered significant

(P < 0.05)

3. RESULTS

3.1. Effect of Ca2+ Channel Inhibitors in

Contracted HUA

The HUA rings without endothelium were contracted by

KCl (60 mM), histamine and 5-HT. The presence of KCl

(60 mM), 5-HT (1 µM) and histamine (10 µM) elicited

maximal contractile effects of 2041.8 ± 94.3 (n = 66),

1798.0 ± 103.9 (n = 53) and 1382.6 ± 89.4 mg (n = 44)

respectively. As already exposed in a previous work [46],

the contraction induced by histamine is lower than that

produced by KCl or 5-HT (P < 0.05; one-way ANOVA

with Tukey post hoc test). After contraction by these

agents, the relaxant effect of blockers of different Ca2+

channels was analysed (Figure 1). The relaxations in-

duced by nifedipine on KCl and histamine contracted

HUA were similar, however the effect on 5-HT con-

tracted arteries was lower (P < 0.05). In general, all the

relaxing drugs tested in this work have lower effects on

HUA contracted by 5-HT than on contractions induced

by KCl or histamine. On the other hand, relaxations in-

duced by mibefradil (TTCC blocker) and APB (TRP

channels blocker) were very small, independently of the

contractile agent.

KClHistamine 5-HT

(6)

(4)

(6)

(4)

(5)

(8)

% of relaxation

Nifedipine

Mibefradil

APB

Figure 1. Effects of different Ca2+ channel inhibitors on HUA.

Rela xati on ind uced by nife dipine (10



M), mibefradil (10



and APB (100



M) on contractions elicited by KCl (60 mM),

histamine (10



M) and 5-HT (1



M). The bars represent the

means and the lines the S.E.M. of the number of experiments

indicated near the bars. Bars with different letters indicate sig-

nificant differences in the effect of nifedipine (P < 0.05,

one-way ANOVA with Tukey post hoc test).

Thus, TTCC or TRP inhibitors did not relax HUA and

the relaxation induced by the inhibition of LTCC has

been bigger when arteries were contracted by depolari-

sation or by histamine than by 5-HT receptor activation.

3.2. Effect of Cyclic Nucleotides in Ctracted

HUA

The effect of cAMP increase on HUA contracted arteries

has been analysed by using forskolin (adenylate cyclase

stimulator) and rolipram (PDE4 inhibitor). The conjoint

application of rolipram (1 µM) and forskolin (10 µM)

have relaxed the contractions induced by KCl on 41.0%

(Figure 2). In contrast, the effect of these two drugs ap-

plied together on 5-HT contracted arteries was only 10%

(Figure 2). These drugs almost fully relaxed the HUA

contracted by histamine (10 μM). Thus, the increase of

cAMP have induced almost a full relaxation on hista-

mine contracted HUA, 41% when arteries are contracted

by depolarisation and a very small effect on 5-HT con-

tracted arteries.

The effect of PKA inhibition on these relaxations has

been analysed by using KTa (1 µM). The PKA inhibition

induced a significant reduction on the forskolin plus

rolipram effect in KCl and histamine contracted arteries

(P < 0.05, Figure 2), but was not efficient reducing this

effect in 5-HT contracted arteries (P > 0.05; Figure 2).

Besides, even in KCl and histamine contractions, the

PKA inhibition did reduce in a tiny way the relaxation

induced by the cAMP increase, suggesting that cAMP

relaxation is partially indep enden t of PKA an d ind icating

the existence of another pathway linked to cAMP.

FSK + ROLFSK + ROL + KTaFSK + ROLFSK + ROL + KTaFSK + ROLFSK + ROL + KTa

100

FSK

+ ROL

+ KTa

FSK

+ RO L

FSK

+ ROL

+ KTa

FSK

+ ROL

FSK

+ ROLFSK

+ ROL

+ KTa

(8)

KCl

Histamine

5-HT

(9)

(6)

(5)

(6)

% of relaxation

Figure 2. Relaxant effect of cAMP increase on HUA con-

tracted arteries. Relaxation of HUA induced by the combina-

tion of forskolin (FSK; 10



M) and rolipram (ROL; 1



M) on

contractions elicited by KCl (60 mM), histamine (10



M) and

5-HT (1



M). The effect of KTa (1



M) on this relaxation is

also shown. The bars represent the means and the lines the

S.E.M. of the numbers of experiments indicated near the bars.

Significant differences versus FSK+ROL effect are shown (* P <

0.05, Student-t test).

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

325

The effect of cGMP on HUA contracted arteries has

been also analysed by using SNP (guanylate cyclase

stimulator) and dipyridamol (PDE5 inhibitor). The big-

ger relaxant effect of conjoint application of dipyridamol

and SNP has been obtained in HUA contracted by hista-

mine (90.8%), being the effect on 5-HT- and KCl- con-

tracted HUA considerably lower, 33.0% and 30.8% re-

spectively (Figure 3). Thus, the increase of cGMP has

induced almost a full relaxation (90.8%) on histamine

contracted HUA, and around 30% of relaxation on arter-

ies contracted by KCl or 5-HT.

The effect of PKG inhibition on these relaxations has

been analysed by using KTg (1 µM). The PKG inhibi-

tion have induced a significant reduction on the SNP

plus dipyridamol effect on histamine and 5-HT con-

tracted arteries (P < 0.05; Figure 3), but the reduction

induced in KCl contracted arteries was not significant (P >

0.05; Figure 3). As for PKA, the PKG inhibition did not

completely reduce the relaxation induced by the cGMP

increase, also suggesting the contribution of another

pathway distinct than the PKG activation.

3.3. Effect of Phosphatase 2A Inhibition on

Contracted HUA

HUA rings were contracted by KCl (60 mM), histamine

and 5-HT, and the effect of OA (phosphatase 2A inhibi-

tor; 5 nM) on these contract i o ns was anal ys e d.

Okadaic acid (5 nM) have relaxed the contracted HUA

and this effect was bigger on contractions induced by

SNP+DIPSNP + DIP + KTgSNP+DIPSNP + DIP + KTgSNP+DIPSNP + DIP + KTg

100

(5)

KCl

H i s ta mine

5-HT

(9)

(6)

(8)

(9)

% of relaxation

SNP

+ DIPSNP

+ DIP

+ KTg

SNP

+ DIPSNP

+ DIP

+ KTg

SNP

+ DIPSNP

+ DIP

+ KTg

Figure 3. Relaxant effect of cGMP increase on HUA con-

tracted arteries. Relaxation of HUA induced by the combina-

tion of SNP (10



M; guanylate cyclase activator) and dipyri-

didamol (DIP; 3



M) on contractions elicited by KCl (60 mM),

histamine (10



M) and 5-HT (1



M). The effect of KTg (1



on this relaxation is also shown. The bars represent the means

and the lines the S.E.M. of the numbers of experiments indi-

cated near the bars. Significant differences versus SNP+DIP

effect are shown (*P < 0.05, Student-t test).

KCl than when contraction were induced by histamine

or 5-HT (P < 0.05; Figure 4(a)). Thus OA relaxes better

the contractions induced by depolarisation, when LTCC

are activated.

In KCl contracted arteries, the OA relaxation was

similar than the induced by nifedipine. When these two

drugs were applied together, the relaxant effect was not

bigger than the relaxation induced by nifedipine or OA

applied alone (P > 0.05; Figure 4(b)). However, the

presence of forsk olin and ro lipram, dr ugs which in crease

the cAMP levels, significantly enlarged the OA effect

(P<0.05; Figure 4(b)). Also, the presence of SNP and

dipyridamol, drugs increasing the cGMP levels, signifi-

cantly amplified the OA effect (P < 0.05; Figure 4(b)).

Thus, the relaxant effect of LTCC inhibition and phos-

phatase 2A inhibition are not additive or synergic.

However, the cyclic nucleotides have augmented the

relaxation induced by PP2A inhibition.

KClHistamine 5-HT

% of relaxation

(7)

(6) b

(5)

(a)

AONIFAO+NIFAO+FSK+ROL AO+SNP+DIP

100

(7)

(8)

% of relaxation on KCl contraction

(4)

(5) *

(4)

AO NIF AO

+ NIFAO

+FSK

+ ROL

+ SNP

+DIP

OA OA OA OA

(b)

Figure 4. Relaxant effect of PP2A inhibition in HUA con-

tracted arteries. a) Relaxation induced by OA (5 nM) on HUA

contractions elicited by KCl (60 mM), histamine (10



M) and

5-HT (1



M); b) Effect of nifedipine (NIF; 10



M), forskolin

(FSK; 10



M) plus rolipram (ROL; 1



M) and SNP (10



plus dipyridamol (DIP; 3



M) in the OA relaxation. Statistical

significant differences versus the OA effect are indicated (*P <

0.05, one-way ANOVA with Dunnet´s post hoc test).

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

326

3.4. Effect of Rock Inhibition in HUA Con-

tractility

The role of different kinases in HUA contractility has

been analysed by using the following inhibitors: Y27

(ROCK inhibitor; 10



M); PD (ERK1/2 inhibitor; 50



M); and SB (p38MAPK inhibitor; 25



M).

Firstly, the effect of these inhibitors on HUA contrac-

tility was analysed in Ca2+ containing medium. In these

conditions, ROCK inhibition did not induce significant

relaxation (P > 0.05) of HUA contracted by KCl (0.3±

0.2%, n=8), by histamine (0.1 ± 0.1%; n=5) or by 5-HT

(0.8±0.8%; n=6). Neither, in the same conditions,

ERK1/2 inhibition did not induce significant relaxation

(P>0.05) of HUA contracted by KCl (3.0 ± 1.5%; n=9),

by histamine (1.7 ± 1.7%; n=5) or by 5-HT (1.1±1.1%;

n=4). Also, in presence of extracellular Ca2+, p38MAPK

inhibition did no t induce significan t relax ation (P > 0.05)

of HUA contracted by KCl (1.2 ± 1.0%; n=8), by hista-

mine (1.1 ± 0.7%; n = 7) or by 5-HT (1.6 ± 1.0%; n=5).

Thus, ROCK, ERK1/2 or p38MAPK inhibition did not

affect the contraction induced either by KCl, histamine

or 5-HT in presence of extracellular Ca2+.

The effect of these inhibitors has been also analysed

in absence of extracellular Ca2+ (0Ca medium). In these

conditions KCl has induced sustained contractions

(1013.6 ± 79.2 mg; n = 18) that were significantly lower

than the induced in presence of Ca2+ (1859.8 ± 70.9 mg;

n = 95)(P < 0.05; Student-t test). The ERK1/2 or p38

MAPK inhibition did not significantly influence the

contractions induced by KCl in absence of Ca2+ (Figure

6(a)). Besides, the inhibition of PP2A or LTCC did not

affect the contractions induced by KCl in absence of

extracellular Ca2+ (Figure 6(a)). However, ROCK inhi-

bition by Y27 relaxed on 81.6% the contractio n s indu ced

by KCl in Ca2+ free medium (Figure 6(a)). Figure 5(a)

shows a recor d of an experiment in which KCl (60 mM)

induces contraction of HUA in Ca2+-free medium and

Y27 relaxes this contraction.

On the other hand, in Ca2+ free medium histamine and

5-HT have elicited two step contractions characterized

by a rapid phasic component, 2-3 min after the receptor

stimulation, followed by a decrease of the contraction

until a tonic component, 15-20 min after. The ERK1/2 or

p38MAPK inhibition did not affect the phasic or tonic

components of the contraction elicited by histamine or

5-HT (P > 0.05; Figures 6(b) and 6(c)). However,

ROCK inhibition significantly reduced the phasic and

tonic components of the contraction elicited either by

histamine or 5-HT (P < 0.05; Figures 6(b) and 6(c)).

Figure 5(b) shows a record of an experiment in which,

in Ca2+ free medium, 5-HT elicited two step contractions

characterized by a phasic and tonic components which

were reduced after ROCK inhibition.

Thus, ROCK inhibition decreases the tension induced

either by KCl, histamine or 5-HT in Ca2+ free medium.

0 1020304050607080

0.0

0.2

0.4

0.6

0.8

1.0

Y27 10 M

KCl 60mM

TENSION (g)

TIME (min)

(a)

0510 15 20 2590 95100105110

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9 5-HT 1 M

Y27 10 M

5-HT 1 M

TENSION (g)

TIME (min )

(b)

Figure 5. Original records of two tension experiments

with HUA rings in Ca2+ free medium. a) Ca2+-free me-

dium contration of HUA induced by KCl (60 mM) which

is relaxed by Y27 (10



M); b) Ca2+-free medium contrac-

tion of HUA induced by 5-HT (1



M) in presence and in

absence of Y27 (10



M).

4. DISCUSSION

The present study investigated the involvement of some

mechanism, such as different types of Ca2+ channels,

cyclic nucleotides and different kinases, in th e regulation

of the HUA contractility. The elucidation of the precise

mechanism regulating the contractility of this artery

could be very important to detect potential therapeutic

targets to treat HUA disorders such as preeclampsia.

We firstly investigated th e relevanc y of different types

of Ca2+ channels on the regulation of HUA tonus. Our

results show that the inhibition of LTCC relaxes con-

tracted HUA, even if this effect does not reach 100% of

relaxation. Other authors have previously reported that,

among different Ca2+ channel blockers, nifedipine is the

most potent umbilical vasodilator [18]. At the vascular

level, it has been shown that nifedipine does not fully

relax contractions induced by KCl. The existence of in-

tracellular Ca2+ release when the arteries are contracted

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

327

Y27 AOPDSBNIF

100

% of relaxation on KCl contraction

in 0Ca m edium

(a)

HISHIS + Y27HIS + PDHIS + SB

200

400

600

800

1000

1200

(6)

(8)

(18)

(6)

(8)

(18)

Contraction induced by HIS (mg)

in 0Ca medium

Phasic contraction

Tonic contraction

(4) #

(4)

(b)

5-H

5-HT + Y2

5-HT + PD5-HT + SB

200

400

600

800

1000

120

(5)

(14)

Contraction induced by 5-HT (mg)

in 0Ca medium

Phasic contraction

Tonic con traction

(4)

(c)

Figure 6. Rel a xan t e ffect of different inhibitors of kinases

on HUA cont racted in Ca2+-free medium. a) Effect of Y27

(10



M), OA (5 nM), PD (50



M), SB (25



M) and

nifedipine (NIF; 10



M) on KCl induced contraction in

Ca2+-free medium; b) Effect of Y27 (10



M), PD (50



and SB (25



M) on the phasic and tonic contraction in-

duced by histamine (10



M) in Ca2+-free medium; c) Ef-

fect of Y27 (10



M), PD (50



M) and SB (25



M) on the

phasic and tonic contraction induced by 5-HT (1



M) in

Ca2+-free medium. The bars represent the means and the

lines the S.E.M. of the numbers of experiments indicated

near the bars. Statistical significant differences versus the

effect in absence of inhibitor are indicated (*P < 0.05,

one-way ANOVA with Dunnet´s post hoc test).

by KCl has been suggested as responsible of the lack of

a full relaxant effect of LTCC inhibitors [7]. In this sense,

and as we will discuss later, the ability of KCl to in duce

HUA contractions in Ca2+-free medium could be due to

stimulation of intracellular Ca2+ release. The relaxant

effect of nifedipine was stronger when HUA were con-

tracted by depolarization or by histamine than in arteries

contracted by 5-HT. Mikkelsen et al. also have observed

that, in human pulmonary arteries, the contraction in-

duced by 5-HT is more resistant to nifedipine than the

contraction mediated by depolarization [8]. On the other

hand, several studies demonstrated that TTCC and TRP

channels are involved in VSM contraction in different

arteries [5,9,10,47]. However, our results show the ab-

sence of effect of mibefradil and APB, suggesting that

Ca2+ entry through TTCC and TRP channels does not

contribute to HUA contraction.

The precise regulation of the intracellular levels of

cAMP and cGMP plays an important role in many phy-

siological processes, including vascular smooth muscle

contractility [32]. However, the mechanisms by which

increases in cAMP and cGMP concentration lead to ar-

tery relaxation are still unclear. Some authors have re-

ported that forskolin, a direct stimulator of adenylate

cyclase, induced relaxation of different human vessels

such as dorsal artery [34], pulmonary artery [35], pla-

cental vessels [36] and umbilical artery [37]. On the

other hand, among the four families of PDE expressed in

this smooth muscle (PDE1, PDE3, PDE4 and PDE5),

PDE4 has been shown as the key enzyme involved in the

regulation of HUA relaxation associated to cAMP [37].

In this sense, we used forskolin (adenylate cyclase

stimulator) and rolipram (PDE4 inhibitor) as drugs that

can elicit the maximal increase in cAMP levels in HUA

smooth muscle cells. The conjoint application of both

drugs elicited different degree of relaxation depending

on the contractile agent used. When applied together,

these drugs almost fully relaxed histamine contracted

HUA, but only 41% and 10% of relaxation was obtained

on HUA contracted by KCl and 5-HT, respectively. We

have previously shown that 5-HT2A, 5-HT1B/1D and

5-HT7 receptors are present in HUA smooth muscle cells.

The HUA contraction induced by 5-HT are mainly me-

diated by the activation of 5-HT2A, which activation in-

creases IP3 levels, and 5-HT1B/1D receptors, which acti-

vation inhibits adenylate cyclase [48]. Concerning the

histamine receptors, H1 receptor activation induces con-

traction and H2 and H3 receptors activation mediates

HUA relaxation through the increase of cAMP intracel-

lular levels [48]. According with th ese previo u s findings,

the activation of different 5-HT receptors in HUA inh ib-

its adenylate cyclase and the activation of different his-

tamine receptors induces contraction, but also induces a

small increase of cAMP levels due to H2 and H3. Thus,

histamine contraction s are more susceptible to activato rs

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

328

of adenylate cyclase or to PDE4 inhibitors than 5-HT

contractions. On the other hand, apparently KCl does not

affect adenylate cyclase activity and the effect of forskolin

and rolipram has been lower than in histamine-contracted

arteries and bigger than HUA contracted by 5-HT.

Is well known that NO activates soluble guanylate cy-

clase increasing cGMP levels, which induces vasodilata-

tion [38], including HUA vasodilatation [37,39]. On the

other hand, PDE5 has been shown as being the key en-

zyme involved in the regu lation of HUA relaxatio n asso-

ciated to cGMP [37]. In this sense, we used SNP

(guanylate cyclase stimulator) and rolipram (PDE5 in-

hibitor) as drugs that can elicit the maximal increase in

cGMP levels in HUA smooth muscle cells. As for cAMP,

the conjoint application of these drugs has elicited dif-

ferent degree of relaxation depending on the contractile

agent used. The biggest relaxant effect was observed in

contraction induced by histamine, followed by contrac-

tions produced by 5-HT and by KCl depolarization.

Numerous authors have described that cGMP induced

vasodilatation is mediated by activation of potassium

channels in different arteries [40,41], including HUA

[39]. The regulation of voltage-dependent potassium

channels (KV) by KCl and 5-HT could be responsible of

these differences. It has been described that vascular

contraction induced by KCl is mainly due to the influx

of extracellular Ca2+ via voltage-dependent Ca2+ chan-

nels [1], but Kv channel inactivation at high potassium

concentrations (60 mM) was also demonstrated in some

blood vessels [49]. Recently, using patch clamp tech-

niques, some authors have demonstrated that 5-HT de-

creases Kv channel activity in cells of rat pulmonary [50]

and mesenteric [51] arteries. Thus, as the cGMP relaxant

effect seems to be mediated by potassium channels acti-

vation, this effect is lower when HUA are contracted by

KCl and 5-HT because these agents inhibit these chan-

nels.

Our results have shown that the PKA inhibition in-

duced a significant reduction on the forskolin plus rolip-

ram effect in KCl and histamine contracted arteries, even

if this effect is small. The relaxant effect of cAMP in-

crease in 5-HT contracted arteries was very low (around

10%) and KTa have failed to significantly decrease the

relaxations induced by forskolin plus rolipram. Also, the

PKG inhibition induced a significant reduction on the

SNP plus dipyridamol effect in histamine and 5-HT con-

tracted arteries. The analysis of these results suggests

that both PKA and PKG are involved in HUA relaxa tion

mediated by cAMP and cGMP respectively. However,

the contribution of these kinases is very small and in

some cases was not significant. These results suggest

that the relaxation induced by cyclic nucleotides is not

totally mediated by the activation of their respective

kinases and other mechanisms can be involved as de-

scribed by other authors [44,45]

As we mentioned before, LTCC are critically impor-

tant for regulating HUA contraction. It has been de-

scribed that LTCC can be dephosphoralyted by PP2A,

and inhibition of PP2A was found to result in ch anges in

functional properties of the LTCC [21]. However, there

is small number of studies on this matter in smooth mus-

cle and the direction of this modulation has remained

somewhat controversial. Some authors have shown that

in tracheal smooth muscle PP2A does not modulate

LTCC [19]. Also, it has been shown a dual effect of

phosphatase inhibitors on Ca2+ channels from intestinal

smooth muscle cells [20]. At the vascular level, Gro-

schner et al. have demonstrated that, in human umbilical

vein, when PP2A is inhibited by OA there is increase of

LTCC activity [21]. Our results show that PP2A inhibi-

tion produces relaxation of HUA, namely when this ar-

tery is contracted by KCl and histamine. This relaxant

effect is bigger in arteries contracted by KCl. Our results

also show that in KCl contracted arteries, the OA effect

is similar to the nifedipine effect. When applied together,

the relaxant effect is not bigger than the relaxation in-

duced by nifedipine or OA applied alone. These results

suggest that PP2A could activate LTCC in HUA. On the

other hand, the increase of cAMP or cGMP induced by

the conjoint application of cyclase activators and PDE

inhibitors significantly increased the OA effect. These

results suggest that the relaxation induced by PP2A inhi-

bition is independent of the cyclic nucleotide pathway.

We also have analyzed the effect of some kinases on

HUA contractility. Our results show that ROCK,

ERK1/2 or p38MAPK inhibition does not affect the

contraction induced by KCl, histamine or 5-HT in Ca2+-

containing extracellular solution. Other authors have

obtained significant relaxation of HUA contracted by

5-HT after ROCK inhibition, but using higher concen-

trations of Y27 (100 µM) which can inhibit also myosin

light chain kinase directly [22]. Also, Tasaki et al. ob-

served that SB significantly inhibits 5-HT-induced con-

tractions in rat aorta [30]. Some authors indicated that

5-HT induces contractions of rat aortic smooth muscle

by activating the MAPK pathway [29]. Also, activation

of p38 MAPK [30] and Erk MAPK [31] by 5-HT have

been shown in rat aorta.

Our results suggest that in the contractions induced by

KCl, histamine and 5-HT in presence of Ca2+ there is not

involvement of pathways concerning ROCK, ERK1/2 or

p38MAPK. Sakurada et al. have shown the existence of

a Ca2+-dependent Rho stimulation mechanism in VSM

from rabbit aorta which is activated b y excitatory recep-

tor agonists [23]. However, in HUA our results exclude

this possibility, because in presence of extracellular Ca2+

ROCK does not seem to be activated by KCl, histamine

or 5-HT.

On the other hand, in absence of extracellular Ca2+,

histamine and 5-HT elicited contractions of HUA char-

acterized by two components, a rapid phasic contractile

A. J. Santos-Silva et al. / Health 2 (2010) 321-331

329

component, 2-3 min after stimulation by the agonist,

followed by a decrease of the contraction until a tonic

component, 15-20 min after. The initial transient com-

ponent has been associated with Ca2+ release from the

sarcoplasmic reticulum, whereas the tonic component

seems to be dependent on the increase on extracellular

Ca2+. Both contractile responses are dependent on the

Ca2+ sensitization and two main pathways have been

implicated in this phenomenon: the inhibition of myosin

light chain phosphatase (MLCP) by ROCK; and the

phosphorylation of the thin filament proteins by

p38MAPK and ERK1/2 [5 ,52]. In contrasts, KCl elicited

sustained contractions of HUA in absence of extracellu-

lar Ca2+. This effect could be induced by a progressive

Ca2+ release from intracellular Ca2+ stores and/or an in-

crease of Ca2+ sensitization. These data agree with the

obtained by Tufan et al. in HUA, which have suggested

that Ca2+ independent isozymes of protein kinase C may

also be involved in the contractions produced by KCl in

absence of extracellular Ca2+ [1]. Some authors have

also suggested that depolarization by KCl induces intra-

cellular Ca2+ release in human arteries [7]. Besides, it

has been described that KCl depolarization increases

Ca2+ sensitization by ROCK activation in smooth muscle

cells from rabbit arteries [24]. As expected, in absence of

extracellular Ca2+, nifedipine did not relax the contrac-

tions elicited by KCl. Also in these conditions, OA did

not relax the contractions elicited by KCl. Once more,

these data suggest a functional relationship between the

PP2A and LTCC in the regulation of HUA contractility.

Concerning the kinases, the ERK1/2 and p38MAPK

inhibitors did not reduced or relax the contractions in-

duced by KCl, histamine or 5-HT in absence of ex-

tracellular Ca2+. Thus, these results demonstrate that

KCl-induced contractions are not linked to the activation

of ERK1/2 and p38MAPK. However, in Ca2+-free me-

dium, the ROCK inhibition significantly relaxed the

contractions induced by KCl and reduced the phasic and

tonic components of the contraction elicited either by

histamine or 5-HT. Similar results were obtained by Ark

et al. when HUA were contracted by 5-HT in Ca2+ free

medium [22]. Our data demonstrate that ROCK is capa-

ble of mediating the contractile response after stimula-

tion of a receptor agonist or by depolarization. Thus, in

absence of Ca2+, the sustained contractions elicited by

KCl and the phasic and tonic components of the contrac-

tion induced by histamine or 5-HT depend on ROCK

activation. Consequently, these contractions depend of

Ca2+ sensitization by the inhibition of MLCP by ROCK.

Our data show that the relevancy of the Ca2+ dependent

or independent mechanism in HUA varies in function of

the extracellular Ca2+ level, and further experiments are

necessary to deeply study this dependence. On the other

hand, our results suggest that HUA is a good sample to

study the Ca2+ sensitization mechanism induced by

ROCK.

In conclusion, our results demonstrate that the LTCC

are the main way for Ca2+ entry and is involved in HUA

contractions induced by depolarization or by agonists. A

positive regulation of LTCC by PP2A seems to occur in

HUA smooth muscle cells. Cyclic nucleotides are in-

volved in HUA vasodilatation. The relaxant effect of

cyclic nucleotides is partially due to the activation of

their respective kinases, but other pathways are also in-

volved. HUA is able to contract independently of Ca2+

influx by activating the ROCK pathway and/or due to

intracellular Ca2+ release.

5. ACKNOWLEDGEMENTS

We thank the donor mothers and the Gynaecoogy-Obste -trics Depart-

ment staff of “Centro Hospitalar da Cova da Beira” (Covilhã, Portugal)

for their disinteested collabration and the “Fundação para a Ciência e a

Tecnologia” (Portugal) for supporting the.grant SFRH/BDE/15532/

2004.

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