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Effect of nitric oxide on responses of the human uterine arteries to vasopressin
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Vascular Pharmacology 48 (2008) 9 – 13 www.elsevier.com/locate/vph
Effect of nitric oxide on responses of the human uterine arteries to vasopressin Anna Kostrzewska a , Beata Modzelewska a,⁎, Tomasz Kleszczewski a , Satish Batra b a
Department of Biophysics, Medical University of Bialystok, ul. Mickiewicza 2A, Bialystok, Poland b Department of Obstetrics and Gynaecology, University Hospital, S-221 85 Lund, Sweden Received 17 January 2007; accepted 20 September 2007
Abstract Nitric oxide (NO) is known to be an important relaxant of contractile activity in various muscles including the human uterine arteries. It has been suggested that NO plays a role in modulation of vascular action of arginin vasopressin (AVP), a strong vasoconstrictor of the human uterine arteries. Therefore, the purposes of this study were to investigate an involvement of endogenous NO in regulation of responses of the human intrauterine arteries to AVP and examine the effect of exogenous NO on contractions of the human intrauterine arteries evoked by AVP. Pretreatment of the artery rings with L-NA, an inhibitor of NO synthase significantly increased the resting force and enhanced the artery responses to AVP. The opposite effect has been observed after administration of 10− 6 mol/L sodium nitroprusside (SNP). Pretreatment of the artery rings with 10− 7 M CTX, a blocker of Ca2+-sensitive potassium channels with large conductance, did not change significantly their responses to AVP. Glibenclamide (1.5·10− 6 mol/L), a blocker of ATP-dependent potassium channels and apamin (10− 8 M), a specific blocker of Ca2+-sensitive potassium channels with small conductance strongly enhanced the maximum responses of the artery rings to AVP. Pretreatment with CTX significantly decreased the relaxation induced by SNP while apamin attenuated the sensitivity to SNP resulted in rightward shift of the concentration–response curve to SNP. In conclusion, this study indicates that: NO plays a role in regulation of both the vascular tone of the human intramyometrial arteries and their response to AVP. Ca2+-sensitive K+ channels with small and large conductance are involved in the SNP-induced relaxation of these arteries. The pathways of this relaxation cannot be sufficiently explained at this moment and need further investigation. © 2007 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Potassium channel; Vasopressin
1. Introduction Nitric oxide (NO) is known to be an important relaxant of contractile activity in various muscles. It is generally accepted that NO activates soluble guanylate cyclase (Bracamonte et al., 1999) causing activation of several mechanisms leading to vasorelaxation. In some vessels, NO may activate K+ channels leading to hyperpolarization of the cell membrane. Nitric oxide may open K+ channels directly or by mechanisms mediated by cGMP (Bolotina et al., 1994; Fukami et al., 1998; Hennan and ⁎ Corresponding author. Department of Biophysics, Medical Academy of Bialystok, ul. Mickiewicza 2A, 15-230 Bialystok, Poland. Tel.: +48 85 748 56 67; fax: +48 85 748 54 16. E-mail address: [email protected] (B. Modzelewska). 1537-1891/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2007.09.003
Diamond, 1998). Several types of K+ channels may be involved in NO-induced relaxation, including ATP-dependent K + channels (Murphy and Brayden, 1995), Ca2+-sensitive K+ channels with small (Simonsen et al., 1997) and large (Bychkov et al., 1998) conductance and even voltage-dependent K+ channels (Zhao et al., 1997). It has been proven that like other smooth muscles, the human uterine arteries are able to produce NO (Jovanovic et al., 1994). Arginin vasopressin (AVP), a strong vasoconstrictor of the human uterine arteries has been implicated as one of the important agents in the pathophysiology of dysmenorrhoea. It has also been suggested that NO plays a role in modulation of vascular action of AVP (Gardiner et al., 1991). Therefore, the purposes of this study were to (1) investigate an involvement of endogenous nitric oxide in regulation of responses of the human
10
A. Kostrzewska et al. / Vascular Pharmacology 48 (2008) 9–13
intrauterine arteries to arginin vasopressin and (2) examine the effect of exogenous NO on contractions of the human intrauterine arteries evoked by arginin vasopressin. 2. Method 2.1. Tissue collection Specimens of the uterine artery were obtained from nonpregnant women, aged 41–51 years, undergoing hysterectomy for benign gynaecological disorders. The local ethical committee approved the study. Immediately after removing of the uterus, arteries with diameters 0.6–1.2 mm were excised from it, and placed in physiological salt solution (PSS) at 0 °C, aerated with carbogen (Kostrzewska et al., 1998). 2.2. Artery tissue bath experiments Under a dissecting microscope 3 mm wide rings of arteries were prepared. Experiments were performed on rings without endothelium. The endothelium was removed mechanically by gentle rubbing of the intimal surface with a stainless-steel wire. The strips were mounted in an organ bath containing PSS at pH 7.4 and a temperature 37 °C, and bubbled with carbogen. The preparations were allowed to equilibrate for 1–2 h. During the equilibration period the passive tension was adjusted to obtain an optimal value on their length-tension curve as determined by the tension developed in response to 80 mmol/L of potassium chloride (80 mmol/L K+). Responses of artery to AVP were recorded under isometric conditions. Before each experiment strips were (several times) activated by 80 mmol/L K + administration. The muscle responses to K+ depolarization were treated as a control. Strips showing unstable responses to the K+ depolarization were not used in the experiments. To confirm successful removal of endothelium acetylcholine (10− 5 mol/L) was administered to the rings precontracted with 10− 7 mol/L AVP. The preparation exhibiting acetylcholine-induced relaxation not higher than 10%
Fig. 2. Concentration–response curves of AVP-induced contractions of the human intrauterine arteries: Responses obtained in absence (○) and presence (●) of 10− 3 mol/L L-NA; (B) Responses obtained in the absence (○) and presence (▲) of 10− 6 mol/L SNP. Responses are expressed as percent of the response to 80 mmol/L K+ observed before the onset of the experiment. Data represent means ± SEM for 5 and 4 tissues, respectively.
was treated as denuded endothelium and used in experiments. After recording the control responses, responses to AVP were recorded in the presence of test substances. Only one concentration–response curve was made with each preparation. Quantification of the responses was done by calculation of area under the curve. The area was measured from the baseline over a 10 min period after each stimulus. The effects were evaluated by comparing experimental responses with the controls (set as 100%). 2.3. Drugs and solutions The composition of the solution used was (mmol/L): NaCl 136.9; KCl 2.70; MgCl2 1.05; NaH2PO4 0.4; CaCl2 1.80; Table 1 Effects of L-NNA, SNP, CTX, apamin and glibenclamide on parameters of the concentration–response curve to vasopressin
Fig. 1. The influence of sodium nitroprusside (SNP) and L-NA on the resting force developed by human intrauterine artery. The responses are expressed as percent of the resting force recorded before a compound administration. Data represent means ± SEM for 5 experiments.
Pre-treatment
Emax (%)
pD2
N
Control −3 L-NNA (10 mol/L) SNP (10− 6 mol/L) CTX (10− 7 mol/L) Apamin (10− 8 mol/L) Glibenclamide (1.5 · 10− 6 mol/L)
136.8 ± 4.6 223.8 ± 3.7 a 118.4 ± 5.6 114.5 ± 15.3 240.6 ± 7.0 a 295.3 ± 15.1 a
9.123 ± 0.113 8.682 ± 0.057 a 9.210 ± 0.219 8.945 ± 0.364 9.369 ± 0.111 9.430 ± 0.213
6 5 4 5 6 4
a
Significantly different from the control value.
A. Kostrzewska et al. / Vascular Pharmacology 48 (2008) 9–13
11
tion by a computer program (GraphPAD Software, San Diego, CA, USA). 2.4. Statistical analysis The data were analyzed with ANOVA or Student t-test where appropriate. The probability value of 0.05 was accepted as significant for differences between groups of data. Throughout the paper the results are expressed as mean ± S.E.M. and n denotes the number of tissues (n) obtained from
Fig. 3. Concentration–response curves of AVP-induced contractions of the human intrauterine arteries: (A) Responses obtained in the absence (○) and presence (■) of 10− 8 mol/L apamin; (B) Responses obtained in the absence (○) and presence (●) of 10− 7 mol/L CTX; Responses obtained in the absence (○) and presence (▲) of 1.5 · 10− 6 mol/L glibenclamide. Responses are expressed as percent of the response to 80 mmol/L K+ observed before the onset of the experiment. Data represent means ± SEM for 6 and 4 tissues, respectively.
NaHCO3 2.00; glucose 5.55. Depolarization was induced by elevating the KCl concentration to 80 mmol/L while removing an equimolar amount of NaCl. Vasopressin (AVP), NG-nitro-Larginine (L-NA), charybdotoxin (CTX), apamin and sodium nitroprusside (SNP) purchased from the Sigma Chemical Company were dissolved in distilled water, glibenclamide in DMSO. All substances were added directly to the muscle bath. The concentration of DMSO in the bath never exceeded 0.1% v/v. Where appropriate, control responses were recorded in the presence of 0.1% of DMSO (Kostrzewska et al., 1998). Concentration–response curve was fitted to the logistic equa-
Fig. 4. The effect of K+ channel blockers on the relaxation of the human intrauterine arteries caused by SNP: (A) Concentration–response curves to sodium nitroprusside (SNP) in the absence (○) and presence (■) of 10− 8 mol/L apamin; (B) Concentration–response curves to SNP in the absence (○) and presence (●) of 10− 7 mol/L CTX; (C) Concentration–response curves to SNP in the absence (○) and presence (▲) of 1.5 · 10− 6 mol/L glibenclamide. Responses are expressed as percent of the response to 10− 6 mol/L AVP. Data represent means ± SEM for 7 and 4 tissues, respectively.
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A. Kostrzewska et al. / Vascular Pharmacology 48 (2008) 9–13
different patients. In the case when the same protocol was run on two strips from the same uterus, the data were averaged. 3. Results To investigate whether endogenous NO may influence the AVP-induced contractions of the human intramyometrial arteries, the effects of L-NA (4 · 10− 3 mol/L), an inhibitor of NO synthase, on the artery response to AVP was tested. When added to the bathing solution, L-NA caused a small increase of the resting force developed by the artery rings. The opposite effects have been observed after administration of 10− 6 mol/L SNP (Fig. 1). Application of AVP (10− 11–10− 7 mol/L) produced concentration-dependent contractions, with − logEC50 (pD2) = 9.123 ± 0.113 (n = 6). The magnitude of the maximum response calculated as the percent of the response to 80 mmol/L K+ was 136.8 ± 4.6% (n = 6). Pretreatment of the tissues with L-NA increased significantly the maximum response of the tissue to AVP. In contrast, the incubation with SNP did not change the artery response to AVP (Fig. 2; Table 1). Pretreatment of the artery rings with 10− 7 mol/L CTX, a blocker of Ca2+ -sensitive potassium channels with large conductance, for 15 min did not change significantly their responses to AVP (Fig. 3B). Glibenclamide (1.5 · 10− 6 mol/L), a blocker of ATP-dependent potassium channels and apamin (10− 8 mol/L), a specific blocker of Ca2+-sensitive potassium channels with small conductance strongly enhanced the maximum responses of the artery rings to AVP (Fig. 3A and C). The Emax ratios were 1.9 and 1.8 for glibenclamide and apamin, respectively (Table 1). There were no statistically significant changes of the pD2 values of the concentration– response curves obtained in the absence and after incubation with glibenclamide or apamin (Table 1). Fig. 4 shows the effects of apamin, CTX and glibenclamide on relaxation induced by SNP in the artery rings precontracted with AVP( 10− 6 mol/L). Preincubation with apamin (10− 8 mol/L) and 10− 7 mol/L CTX resulted in rightward shift of the concentration - response curves to SNP. The effect was statistically significant (p b 0.05). The influence of 1.5 · 10− 6 mol/L glibenclamide on the SNP-induced relaxation was complex. The pretreatment with glibenclamide decreased the relaxation caused by SNP at concentrations b10− 7 mol/L but did not change the relaxation induced by higher concentrations of SNP (Fig. 4C). The effect was statistically insignificant (twoway ANOVA; p N 0.5). 4. Discussion In this study, pretreatment of the artery rings with L-NA, an inhibitor of NO synthase significantly increased the resting force. This result is consistent with the observation that there is nonendothelial nitric oxide production in the human intrauterine artery (Jovanovic et al., 1994). In addition, the inhibition of NO synthase by L-NNA significantly enhanced the artery responses to AVP. A similar effect has been observed in collateral vascular bed of rats (Chan et al., 1999). These observations suggest that NO, apart from a role in regulation of the vascular tone of the
human intramyometrial arteries, is involved in the modulation of their response to AVP. The observation that SNP decreases the basal force of the artery strips supports the idea that NO is involved in the control of vascular tone. On the other hand, SNP administered prior to AVP did not change the artery response to this compound. The last finding suggests that the pathways by which endogenous and exogenous NO modulate the artery response to AVP are different. 4.1. Effect of blockers of K+ channels on the artery response to AVP Pretreatment of the artery rings with CTX did not change their response to AVP. However, it has been reported that in some arteries the large conductance, Ca2+-sensitive K+ channels are inhibited by protein kinase C (PKC) (Schubert and Nelson, 2000). It is known that activation of V1a receptors results in diacylglycerol (DAG) production and subsequent activation of PKC (Birnbaumer, 2000). Recently, it has been reported that in small arteries from brain of newborn pigs vasopressin impaired vasodilatation caused by activation of K+ channels (Salvucci and Armstead, 2000). Thus, the lack of the CTX effect on the artery response to AVP may indicate that, the activity of the CTX-sensitive Ca2+-dependent K+ channels is impaired during the artery response to AVP. In our experiments, preincubation with apamin, a specific blocker of Ca2+-dependent K+ channels with small conductance resulted in the enhancement of AVP-induced contractions. This finding suggests that apamin-sensitive K+ channels exist in the human intramyometrial arteries and they are involved in regulation of AVP-induced force development in these muscles. Glibenclamide at the concentration 10− 6 mol/L is relatively selective as an ATP-sensitive K+ channel blocker (Kessler et al., 1997). In the present study we used glibenclamide at concentration 1.5 · 10− 6 mol/L. At this concentration glibenclamide substantially inhibits the KATP current in smooth muscle cells (Quayle et al., 1995). In our experiments, glibenclamide did not change the resting force developed by the artery rings. Therefore, the augmentation of the responses to vasopressin in the presence of glibenclamide suggests that, in the case of AVP-evoked contractions, the ATP-sensitive K+ channels are involved in regulation of the force development. In 1992, Wakatsuki et al. have found that ATP-sensitive K+ channels, active in nonstimulated patches of cultured smooth muscle cells from porcine coronary artery, were blocked by vasopressin (Wakatsuki et al., 1992). The present results imply that paths activated by AVP to contract the human intrauterine arteries differ from those described for porcine coronary artery. 4.2. Effect of blockers of K+ channels on the SNP-induced relaxation In AVP contracted arteries, preincubation of the artery ring with glibenclamide resulted in slight, statistically nonsignificant decrease of the artery sensitivity to SNP. This suggests that ATP-dependent K+ channels are not involved in relaxation
A. Kostrzewska et al. / Vascular Pharmacology 48 (2008) 9–13
induced by NO donated by SNP. The presence of apamin attenuated the sensitivity to SNP as indicated by rightward shift of the concentration–response curve for SNP. These data suggest an existence of NO-mediated regulation of the apamin sensitive, Ca2+-dependent K+ channel. Also CTX added prior to AVP administration attenuated the SNP-induced relaxation of the human intrauterine artery. This finding seems to contradict the assumed impairment of the CTX-sensitive Ca2+-dependent K+ channels by PKC stimulated by the activation of V1a receptors. However, CTX and PKC influence the channel activity via different mechanisms. PKC attenuates the activity of the large conductance, CTXsensitive K+ channels decreasing their open probability (Minami et al., 1993; Schubert et al., 1999). Thus, the appropriate stimuli may still activate the channels. In contrast, CTX plugs the channel pore preventing ions movement across the cell membrane (Kaczorowski and Garcia, 1999; MacKinnon and Miller, 1988). Therefore, the present results imply that CTXsensitive K+ channels are involved in the modulation of the SNP-induced relaxation of the arteries precontracted with AVP. 5. Conclusions In conclusion, this study indicates that: (1) NO plays a role in regulation of both the vascular tone of the human intramyometrial arteries and their response to AVP and (2) Ca2+-sensitive K+ channels with small and large conductance are involved in the SNP-induced relaxation of these arteries. The pathways of this relaxation cannot be sufficiently explained at this moment and need further investigation. References Birnbaumer, M., 2000. Vasopressin receptors Trends Endocrinol. Metab. 11, 406–410. Bolotina, V.M., Najibi, S., Palacino, J.J., Pagano, P.J., Cohen, R.A., 1994. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368, 850–853. Bracamonte, M.P., Burnett, J., J.C., Miller, V.M., 1999. Activation of soluble guanylate cyclase and potassium channels contribute to relaxation to nitric oxide in smooth muscle derived from canine femoral veins. J. Cardiovasc. Pharmacol. 34, 407–413. Bychkov, R., Gollasch, M., Steinke, T., Ried, C., Luft, F.C., Haller, H., 1998. Calcium-activated potassium channels and nitrate-induced vasodilation in human coronary arteries. J. Pharmacol. Exp.Ther. 285, 293–298. Chan, C.C., Lee, F.Y., Wang, S.S., Chang, F.Y., Lin, H.C., Chu, C.J., Tai, C.C., Lai, I.N., Lee, S.D., 1999. Effects of vasopressin on portal-systemic
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collaterals in portal hypertensive rats: role of nitric oxide and prostaglandins. Hepatology. 30, 630–635. Fukami, Y., Toki, Y., Numaguchi, Y., Nakashima, Y., Mukawa, H., Matsui, H., Okumara, K., Ito, T., 1998. Nitrolycerin-induced aortic relaxation mediated by calcium-activated potassium channel is markedly diminished in hypertensive rats. Life Sci. 63, 1047–1055. Gardiner, S.M., Compton, A.M., Kemp, P.A., Bennett, T., 1991. Effect of NGnitro-L-arginine methyl ester or indomethacin on differential regional and cardiac hemodynamic action of arginine vasopressin and lysine vasopressin in conscious rats. Br. J. Pharmacol. 102, 65–72. Hennan, J.K., Diamond, J., 1998. Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP. Br. J. Pharmacol. 123, 959–967. Jovanovic, A., Grbovic, L., Tulic, I., 1994. L-arginine induces relaxation of human uterine artery with both intact and denuded endothelium. Eur. J. Pharmacol. 256, 103–107. Kaczorowski, G.J., Garcia, M.L., 1999. Pharmacology of voltage-gated and calcium-activated potassium channels. Curr. Opin. Cheml. Biol. 3, 448–458. Kessler, C., Loeffler, C., Linde, C., Baumlin, Y., Quast, U., 1997. Activators of protein kinase A induce a glibenclamide-sensitive 86Rb+ efflux in rat isolated aorta. Naunyn-Schmideberg's Arch. Pharmacol. 355, 483–490. Kostrzewska, A., Laudanski, T., Steinwall, M., Bossmar, T., Serradeil-Le Gal, C., Akerlund, M., 1998. Effects of the vasopressin V1a receptor antagonist SR 49059, on the response of human uterine arteries to vasopressin and other vasoactive substances. Acta Obstet. Gynecol. Scand. 77, 3–7. MacKinnon, R., Miller, C., 1988. Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel. J. Gen. Physiol. 91, 335–349. Minami, K., Fukuzawa, K., Nakaya, Y., 1993. Protein kinase C inhibits the Ca(2+)activated K+ channel of cultured porcine coronary artery smooth muscle cells. Biochem. Biophys. Res. Commun. 190, 263–269. Murphy, M.E., Brayden, J.E., 1995. Nitric oxide hyperpolarizes rabbit mesenteric arteries via ATP-sensitive potassium channels. J. Physiol. 486 (Pt 1), 47–58. Quayle, J.M., Bonev, A.D., Brayden, J.E., Nelson, M.T., 1995. Pharmacology of ATP-sensitive K+currents in smooth muscle cells from rabbit mesenteric artery. Am. J. Physiol. 269, C1112–C1118. Salvucci, A., Armstead, W.M., 2000. Vasopressin impairs K(ATP) and K(ca) channel function after brain injury. Brain Res. 887, 406–412. Schubert, R., Nelson, M.T., 2000. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol. Sci. 22, 505–512. Schubert, R., Noack, T., Serebryakov, V.N., 1999. Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells. Am. J. Physiol. 276, C648–C658. Simonsen, U., Prieto, D., Delgado, J.A., Hernández, M., Resel, L., Saenz de Tejada, I., García-Sacristán, A., 1997. Nitric oxide is involved in the inhibitory neurotransmission and endothelium-dependent relaxations of human small penile arteries. Clin. Sci. (Colch). 92, 269–275. Wakatsuki, T., Nakaya, Y., Inoue, I., 1992. Vasopressin modulates K-channel activities of cultured smooth muscle cells from porcine coronary artery. Am. J. Physiol. 263, H491–H496. Zhao, Y.J., Wang, J., Rubin, L.J., Yuan, X.J., 1997. Inhibition of K(V) and K(Ca) channels antagonizes NO-induced relaxation in pulmonary artery. Am. J. Physiol. 272, H904–H912.
Vascular Pharmacology 48 (2008) 9 – 13 www.elsevier.com/locate/vph
Effect of nitric oxide on responses of the human uterine arteries to vasopressin Anna Kostrzewska a , Beata Modzelewska a,⁎, Tomasz Kleszczewski a , Satish Batra b a
Department of Biophysics, Medical University of Bialystok, ul. Mickiewicza 2A, Bialystok, Poland b Department of Obstetrics and Gynaecology, University Hospital, S-221 85 Lund, Sweden Received 17 January 2007; accepted 20 September 2007
Abstract Nitric oxide (NO) is known to be an important relaxant of contractile activity in various muscles including the human uterine arteries. It has been suggested that NO plays a role in modulation of vascular action of arginin vasopressin (AVP), a strong vasoconstrictor of the human uterine arteries. Therefore, the purposes of this study were to investigate an involvement of endogenous NO in regulation of responses of the human intrauterine arteries to AVP and examine the effect of exogenous NO on contractions of the human intrauterine arteries evoked by AVP. Pretreatment of the artery rings with L-NA, an inhibitor of NO synthase significantly increased the resting force and enhanced the artery responses to AVP. The opposite effect has been observed after administration of 10− 6 mol/L sodium nitroprusside (SNP). Pretreatment of the artery rings with 10− 7 M CTX, a blocker of Ca2+-sensitive potassium channels with large conductance, did not change significantly their responses to AVP. Glibenclamide (1.5·10− 6 mol/L), a blocker of ATP-dependent potassium channels and apamin (10− 8 M), a specific blocker of Ca2+-sensitive potassium channels with small conductance strongly enhanced the maximum responses of the artery rings to AVP. Pretreatment with CTX significantly decreased the relaxation induced by SNP while apamin attenuated the sensitivity to SNP resulted in rightward shift of the concentration–response curve to SNP. In conclusion, this study indicates that: NO plays a role in regulation of both the vascular tone of the human intramyometrial arteries and their response to AVP. Ca2+-sensitive K+ channels with small and large conductance are involved in the SNP-induced relaxation of these arteries. The pathways of this relaxation cannot be sufficiently explained at this moment and need further investigation. © 2007 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Potassium channel; Vasopressin
1. Introduction Nitric oxide (NO) is known to be an important relaxant of contractile activity in various muscles. It is generally accepted that NO activates soluble guanylate cyclase (Bracamonte et al., 1999) causing activation of several mechanisms leading to vasorelaxation. In some vessels, NO may activate K+ channels leading to hyperpolarization of the cell membrane. Nitric oxide may open K+ channels directly or by mechanisms mediated by cGMP (Bolotina et al., 1994; Fukami et al., 1998; Hennan and ⁎ Corresponding author. Department of Biophysics, Medical Academy of Bialystok, ul. Mickiewicza 2A, 15-230 Bialystok, Poland. Tel.: +48 85 748 56 67; fax: +48 85 748 54 16. E-mail address: [email protected] (B. Modzelewska). 1537-1891/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2007.09.003
Diamond, 1998). Several types of K+ channels may be involved in NO-induced relaxation, including ATP-dependent K + channels (Murphy and Brayden, 1995), Ca2+-sensitive K+ channels with small (Simonsen et al., 1997) and large (Bychkov et al., 1998) conductance and even voltage-dependent K+ channels (Zhao et al., 1997). It has been proven that like other smooth muscles, the human uterine arteries are able to produce NO (Jovanovic et al., 1994). Arginin vasopressin (AVP), a strong vasoconstrictor of the human uterine arteries has been implicated as one of the important agents in the pathophysiology of dysmenorrhoea. It has also been suggested that NO plays a role in modulation of vascular action of AVP (Gardiner et al., 1991). Therefore, the purposes of this study were to (1) investigate an involvement of endogenous nitric oxide in regulation of responses of the human
10
A. Kostrzewska et al. / Vascular Pharmacology 48 (2008) 9–13
intrauterine arteries to arginin vasopressin and (2) examine the effect of exogenous NO on contractions of the human intrauterine arteries evoked by arginin vasopressin. 2. Method 2.1. Tissue collection Specimens of the uterine artery were obtained from nonpregnant women, aged 41–51 years, undergoing hysterectomy for benign gynaecological disorders. The local ethical committee approved the study. Immediately after removing of the uterus, arteries with diameters 0.6–1.2 mm were excised from it, and placed in physiological salt solution (PSS) at 0 °C, aerated with carbogen (Kostrzewska et al., 1998). 2.2. Artery tissue bath experiments Under a dissecting microscope 3 mm wide rings of arteries were prepared. Experiments were performed on rings without endothelium. The endothelium was removed mechanically by gentle rubbing of the intimal surface with a stainless-steel wire. The strips were mounted in an organ bath containing PSS at pH 7.4 and a temperature 37 °C, and bubbled with carbogen. The preparations were allowed to equilibrate for 1–2 h. During the equilibration period the passive tension was adjusted to obtain an optimal value on their length-tension curve as determined by the tension developed in response to 80 mmol/L of potassium chloride (80 mmol/L K+). Responses of artery to AVP were recorded under isometric conditions. Before each experiment strips were (several times) activated by 80 mmol/L K + administration. The muscle responses to K+ depolarization were treated as a control. Strips showing unstable responses to the K+ depolarization were not used in the experiments. To confirm successful removal of endothelium acetylcholine (10− 5 mol/L) was administered to the rings precontracted with 10− 7 mol/L AVP. The preparation exhibiting acetylcholine-induced relaxation not higher than 10%
Fig. 2. Concentration–response curves of AVP-induced contractions of the human intrauterine arteries: Responses obtained in absence (○) and presence (●) of 10− 3 mol/L L-NA; (B) Responses obtained in the absence (○) and presence (▲) of 10− 6 mol/L SNP. Responses are expressed as percent of the response to 80 mmol/L K+ observed before the onset of the experiment. Data represent means ± SEM for 5 and 4 tissues, respectively.
was treated as denuded endothelium and used in experiments. After recording the control responses, responses to AVP were recorded in the presence of test substances. Only one concentration–response curve was made with each preparation. Quantification of the responses was done by calculation of area under the curve. The area was measured from the baseline over a 10 min period after each stimulus. The effects were evaluated by comparing experimental responses with the controls (set as 100%). 2.3. Drugs and solutions The composition of the solution used was (mmol/L): NaCl 136.9; KCl 2.70; MgCl2 1.05; NaH2PO4 0.4; CaCl2 1.80; Table 1 Effects of L-NNA, SNP, CTX, apamin and glibenclamide on parameters of the concentration–response curve to vasopressin
Fig. 1. The influence of sodium nitroprusside (SNP) and L-NA on the resting force developed by human intrauterine artery. The responses are expressed as percent of the resting force recorded before a compound administration. Data represent means ± SEM for 5 experiments.
Pre-treatment
Emax (%)
pD2
N
Control −3 L-NNA (10 mol/L) SNP (10− 6 mol/L) CTX (10− 7 mol/L) Apamin (10− 8 mol/L) Glibenclamide (1.5 · 10− 6 mol/L)
136.8 ± 4.6 223.8 ± 3.7 a 118.4 ± 5.6 114.5 ± 15.3 240.6 ± 7.0 a 295.3 ± 15.1 a
9.123 ± 0.113 8.682 ± 0.057 a 9.210 ± 0.219 8.945 ± 0.364 9.369 ± 0.111 9.430 ± 0.213
6 5 4 5 6 4
a
Significantly different from the control value.
A. Kostrzewska et al. / Vascular Pharmacology 48 (2008) 9–13
11
tion by a computer program (GraphPAD Software, San Diego, CA, USA). 2.4. Statistical analysis The data were analyzed with ANOVA or Student t-test where appropriate. The probability value of 0.05 was accepted as significant for differences between groups of data. Throughout the paper the results are expressed as mean ± S.E.M. and n denotes the number of tissues (n) obtained from
Fig. 3. Concentration–response curves of AVP-induced contractions of the human intrauterine arteries: (A) Responses obtained in the absence (○) and presence (■) of 10− 8 mol/L apamin; (B) Responses obtained in the absence (○) and presence (●) of 10− 7 mol/L CTX; Responses obtained in the absence (○) and presence (▲) of 1.5 · 10− 6 mol/L glibenclamide. Responses are expressed as percent of the response to 80 mmol/L K+ observed before the onset of the experiment. Data represent means ± SEM for 6 and 4 tissues, respectively.
NaHCO3 2.00; glucose 5.55. Depolarization was induced by elevating the KCl concentration to 80 mmol/L while removing an equimolar amount of NaCl. Vasopressin (AVP), NG-nitro-Larginine (L-NA), charybdotoxin (CTX), apamin and sodium nitroprusside (SNP) purchased from the Sigma Chemical Company were dissolved in distilled water, glibenclamide in DMSO. All substances were added directly to the muscle bath. The concentration of DMSO in the bath never exceeded 0.1% v/v. Where appropriate, control responses were recorded in the presence of 0.1% of DMSO (Kostrzewska et al., 1998). Concentration–response curve was fitted to the logistic equa-
Fig. 4. The effect of K+ channel blockers on the relaxation of the human intrauterine arteries caused by SNP: (A) Concentration–response curves to sodium nitroprusside (SNP) in the absence (○) and presence (■) of 10− 8 mol/L apamin; (B) Concentration–response curves to SNP in the absence (○) and presence (●) of 10− 7 mol/L CTX; (C) Concentration–response curves to SNP in the absence (○) and presence (▲) of 1.5 · 10− 6 mol/L glibenclamide. Responses are expressed as percent of the response to 10− 6 mol/L AVP. Data represent means ± SEM for 7 and 4 tissues, respectively.
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different patients. In the case when the same protocol was run on two strips from the same uterus, the data were averaged. 3. Results To investigate whether endogenous NO may influence the AVP-induced contractions of the human intramyometrial arteries, the effects of L-NA (4 · 10− 3 mol/L), an inhibitor of NO synthase, on the artery response to AVP was tested. When added to the bathing solution, L-NA caused a small increase of the resting force developed by the artery rings. The opposite effects have been observed after administration of 10− 6 mol/L SNP (Fig. 1). Application of AVP (10− 11–10− 7 mol/L) produced concentration-dependent contractions, with − logEC50 (pD2) = 9.123 ± 0.113 (n = 6). The magnitude of the maximum response calculated as the percent of the response to 80 mmol/L K+ was 136.8 ± 4.6% (n = 6). Pretreatment of the tissues with L-NA increased significantly the maximum response of the tissue to AVP. In contrast, the incubation with SNP did not change the artery response to AVP (Fig. 2; Table 1). Pretreatment of the artery rings with 10− 7 mol/L CTX, a blocker of Ca2+ -sensitive potassium channels with large conductance, for 15 min did not change significantly their responses to AVP (Fig. 3B). Glibenclamide (1.5 · 10− 6 mol/L), a blocker of ATP-dependent potassium channels and apamin (10− 8 mol/L), a specific blocker of Ca2+-sensitive potassium channels with small conductance strongly enhanced the maximum responses of the artery rings to AVP (Fig. 3A and C). The Emax ratios were 1.9 and 1.8 for glibenclamide and apamin, respectively (Table 1). There were no statistically significant changes of the pD2 values of the concentration– response curves obtained in the absence and after incubation with glibenclamide or apamin (Table 1). Fig. 4 shows the effects of apamin, CTX and glibenclamide on relaxation induced by SNP in the artery rings precontracted with AVP( 10− 6 mol/L). Preincubation with apamin (10− 8 mol/L) and 10− 7 mol/L CTX resulted in rightward shift of the concentration - response curves to SNP. The effect was statistically significant (p b 0.05). The influence of 1.5 · 10− 6 mol/L glibenclamide on the SNP-induced relaxation was complex. The pretreatment with glibenclamide decreased the relaxation caused by SNP at concentrations b10− 7 mol/L but did not change the relaxation induced by higher concentrations of SNP (Fig. 4C). The effect was statistically insignificant (twoway ANOVA; p N 0.5). 4. Discussion In this study, pretreatment of the artery rings with L-NA, an inhibitor of NO synthase significantly increased the resting force. This result is consistent with the observation that there is nonendothelial nitric oxide production in the human intrauterine artery (Jovanovic et al., 1994). In addition, the inhibition of NO synthase by L-NNA significantly enhanced the artery responses to AVP. A similar effect has been observed in collateral vascular bed of rats (Chan et al., 1999). These observations suggest that NO, apart from a role in regulation of the vascular tone of the
human intramyometrial arteries, is involved in the modulation of their response to AVP. The observation that SNP decreases the basal force of the artery strips supports the idea that NO is involved in the control of vascular tone. On the other hand, SNP administered prior to AVP did not change the artery response to this compound. The last finding suggests that the pathways by which endogenous and exogenous NO modulate the artery response to AVP are different. 4.1. Effect of blockers of K+ channels on the artery response to AVP Pretreatment of the artery rings with CTX did not change their response to AVP. However, it has been reported that in some arteries the large conductance, Ca2+-sensitive K+ channels are inhibited by protein kinase C (PKC) (Schubert and Nelson, 2000). It is known that activation of V1a receptors results in diacylglycerol (DAG) production and subsequent activation of PKC (Birnbaumer, 2000). Recently, it has been reported that in small arteries from brain of newborn pigs vasopressin impaired vasodilatation caused by activation of K+ channels (Salvucci and Armstead, 2000). Thus, the lack of the CTX effect on the artery response to AVP may indicate that, the activity of the CTX-sensitive Ca2+-dependent K+ channels is impaired during the artery response to AVP. In our experiments, preincubation with apamin, a specific blocker of Ca2+-dependent K+ channels with small conductance resulted in the enhancement of AVP-induced contractions. This finding suggests that apamin-sensitive K+ channels exist in the human intramyometrial arteries and they are involved in regulation of AVP-induced force development in these muscles. Glibenclamide at the concentration 10− 6 mol/L is relatively selective as an ATP-sensitive K+ channel blocker (Kessler et al., 1997). In the present study we used glibenclamide at concentration 1.5 · 10− 6 mol/L. At this concentration glibenclamide substantially inhibits the KATP current in smooth muscle cells (Quayle et al., 1995). In our experiments, glibenclamide did not change the resting force developed by the artery rings. Therefore, the augmentation of the responses to vasopressin in the presence of glibenclamide suggests that, in the case of AVP-evoked contractions, the ATP-sensitive K+ channels are involved in regulation of the force development. In 1992, Wakatsuki et al. have found that ATP-sensitive K+ channels, active in nonstimulated patches of cultured smooth muscle cells from porcine coronary artery, were blocked by vasopressin (Wakatsuki et al., 1992). The present results imply that paths activated by AVP to contract the human intrauterine arteries differ from those described for porcine coronary artery. 4.2. Effect of blockers of K+ channels on the SNP-induced relaxation In AVP contracted arteries, preincubation of the artery ring with glibenclamide resulted in slight, statistically nonsignificant decrease of the artery sensitivity to SNP. This suggests that ATP-dependent K+ channels are not involved in relaxation
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induced by NO donated by SNP. The presence of apamin attenuated the sensitivity to SNP as indicated by rightward shift of the concentration–response curve for SNP. These data suggest an existence of NO-mediated regulation of the apamin sensitive, Ca2+-dependent K+ channel. Also CTX added prior to AVP administration attenuated the SNP-induced relaxation of the human intrauterine artery. This finding seems to contradict the assumed impairment of the CTX-sensitive Ca2+-dependent K+ channels by PKC stimulated by the activation of V1a receptors. However, CTX and PKC influence the channel activity via different mechanisms. PKC attenuates the activity of the large conductance, CTXsensitive K+ channels decreasing their open probability (Minami et al., 1993; Schubert et al., 1999). Thus, the appropriate stimuli may still activate the channels. In contrast, CTX plugs the channel pore preventing ions movement across the cell membrane (Kaczorowski and Garcia, 1999; MacKinnon and Miller, 1988). Therefore, the present results imply that CTXsensitive K+ channels are involved in the modulation of the SNP-induced relaxation of the arteries precontracted with AVP. 5. Conclusions In conclusion, this study indicates that: (1) NO plays a role in regulation of both the vascular tone of the human intramyometrial arteries and their response to AVP and (2) Ca2+-sensitive K+ channels with small and large conductance are involved in the SNP-induced relaxation of these arteries. The pathways of this relaxation cannot be sufficiently explained at this moment and need further investigation. References Birnbaumer, M., 2000. Vasopressin receptors Trends Endocrinol. Metab. 11, 406–410. Bolotina, V.M., Najibi, S., Palacino, J.J., Pagano, P.J., Cohen, R.A., 1994. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368, 850–853. Bracamonte, M.P., Burnett, J., J.C., Miller, V.M., 1999. Activation of soluble guanylate cyclase and potassium channels contribute to relaxation to nitric oxide in smooth muscle derived from canine femoral veins. J. Cardiovasc. Pharmacol. 34, 407–413. Bychkov, R., Gollasch, M., Steinke, T., Ried, C., Luft, F.C., Haller, H., 1998. Calcium-activated potassium channels and nitrate-induced vasodilation in human coronary arteries. J. Pharmacol. Exp.Ther. 285, 293–298. Chan, C.C., Lee, F.Y., Wang, S.S., Chang, F.Y., Lin, H.C., Chu, C.J., Tai, C.C., Lai, I.N., Lee, S.D., 1999. Effects of vasopressin on portal-systemic
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