Source of calcium for contractile responses of large and small human intramyometrial arteries

A. Kostrzewska1,3, B. Modzelewska1 and S. Batra2

1 Department of Biophysics, Medical Academy, ul. Mickiewicza 2A, 15–230 Bialystok, Poland and 2 Department of Obstetrics and Gynaecology, University Hospital, S-221 85 Lund, Sweden


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The role of calcium (Ca2+) released from intracellular stores and the entry of extracellular Ca2+ for vasopressin (AVP)-induced responses in large and small, human, intramyometrial arteries was investigated. There was no statistical difference as revealed by pD2 values (–log EC50), in the sensitivity of large and small vessels to AVP. Nimodipine caused an inhibition of contractions induced by low concentrations (10–10 mol/l) of AVP in both types of vessels but, at higher concentration (>10–10 mol/l), whereas responses in small arteries were diminished, in large arteries they remained unchanged. In Ca2+-free solution, responses of large and small arteries to potassium and to 10–10 mol/l AVP were abolished. With 10–6 mol/l AVP, response in small arteries was completely inhibited, whereas in large arteries it was reduced by ~50%. Additional experiments were done on large arteries. Thapsigargin (TSG), which causes depletion of internal Ca2+ stores, caused a significant reduction in responses. Following treatment with TSG, responses to AVP in Ca2+-free solution were almost completely inhibited but arteries responded again when incubated in normal physiological salt solution. The results indicate that in contrast to large arteries, small arteries are highly dependent on extracellular Ca2+. Response of large arteries showed considerable dependence on Ca2+ stored internally particularly, for maximum activation.

Key words: contraction/human uterine arteries/vasopressin


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well known that in common with other types of smooth muscle, contraction of vascular smooth muscle requires an increase in cytoplasmic calcium (Ca2+). The elevation of intracellular Ca2+ may result from either Ca2+ influx through specific membrane channels or as a release from internal store primarily in sarcoplasmic reticulum (SR). The relative contribution of intracellular and extracellular sources may depend on the tissue and mode of stimulation. Depolarization by high potassium (K+) opens voltage sensitive Ca2+ channels in the plasma membrane, causing contraction predominantly by the accelerated entry of extracellular Ca2+. However, contraction induced by interaction of certain agonists, such as vasopressin (AVP) in the case of uterine vessels, with their receptor results in both the release of intracellular Ca2+ and influx of extracellular Ca2+ (van Breemen and Saida, 1989Go).

The relative contribution of the two mechanisms depends on concentration of the agonist, on the type, and the most likely size of the blood vessel. In physiological conditions, both mechanisms are considered important in maintenance of vascular tone. Detailed information on the relative importance of these mechanisms in uterine arteries is needed to develop a rational approach to the pharmacological treatment of disorders such as dysmenorrhoea and possibly premature labour.

Based on the data from morphological studies that have revealed that conduit arteries contain a much more developed SR than resistance arteries, it has been suggested that Ca2+ release from the SR has a limited role in excitation–contraction coupling in resistance arteries (Somlyo, 1980Go; Ashida et al., 1988Go). Conversely, extracellular Ca2+ may only play a major role in the contractile activity of small resistance arteries.

The contribution of extracellular Ca2+ to contractile response can be assessed by application of selective organic Ca2+ channel blockers, whereas the assessment of the role of Ca2+ stored in SR is more difficult. With the availability of specific inhibitors of refilling of the internal Ca2+ store, it has become feasible to investigate the relative contribution of Ca2+ stored in SR. Thapsigargin (TSG), which inhibits Ca2+ uptake into the internal Ca2+ stores and thereby causes its depletion with continuous exposure, has been widely used as a tool for the assessment of the relative importance of intracellular Ca2+ in smooth muscle contraction.

Small branches of the uterine artery are considered to be resistance vessels and thereby particularly important in the local regulation of uterine blood flow. There is also some evidence of greater reactivity to vasoactive substances of small-sized branches of the uterine artery compared to large-sized arteries (Ekstrom et al., 1991Go). In order to distinguish the pathways of activation involved in excitation–contraction coupling of small and large intramyometrial arteries, we used nimodipine, a potent Ca2+ channel inhibitor, and TSG, a selective inhibitor of the SR Ca2+ pump, in addition to measurements of contractile responses in Ca2+-free medium.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specimens of the uterine artery were obtained from non-pregnant women, aged 41–51 years, undergoing hysterectomy for benign gynaecological disorders. The study was approved by the local ethical committee. Immediately after removal of the uterus, arteries with diameters 0.3–0.5 mm and 0.6–1.2 mm were excised from it, and placed in physiological salt solution (see below) at 0°C, aerated with 95% oxygen and 5% carbon dioxide. Under a dissecting microscope, ~3 mm wide rings of arteries were prepared. The strips were mounted in an organ bath containing physiological salt solution at pH 7.4 and temperature 37°C, and bubbled with 95% oxygen and 5% carbon dioxide. The preparations were allowed to equilibrate for 1–2 h. During the equilibration period the passive tension was adjusted several times until the resting tension became stable at 3 mN.

Responses of artery to AVP were recorded under isometric conditions. Before each experiment, strips were activated (several times) by 80 mmol/l K+ administration. The muscle responses to K+ depolarization were designated as control responses. Strips showing unstable responses to K+ depolarization were not used in the experiments. After recording a control response, responses to AVP were recorded in the presence of test substances.

Quantification of 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 at 100%).

Solutions
The composition of the physiological salt solution (PSS) used was (mmol/l): NaCl 136.9; KCl 2.68; MgCl2 1.05; NaH2PO4 1.33; CaCl2 1.80; NaHCO3 25.00; glucose 5.55 bubbled with 95% oxygen and 5% carbon dioxide. Depolarization was induced by elevating the KCl concentration to 80 mmol/l while removing an equimolar amount of NaCl. For Ca2+-free solution CaCl2 was replaced by 2 mmol/l EGTA. AVP was dissolved in distilled water, nimodipine and thapsigargin in dimethylsulphoxide (DMSO). The concentration of DMSO in the bath never exceeded 0.2% v/v and this concentration is known to have no influence on the response (Kostrzewska et al., 1996Go).

Concentration–response curves were fitted to experimental data by a computer program based on the Hill equation (Kenakin, 1984Go; GraphPAD Software, San Diego, CA, USA).

Statistical analysis
The Mann–Whitney test was used for comparison of EC50 (concentration for 50% of maximum response) values. The statistical parametric procedures were designed for determination of differences in potency and were based on normal distributions; log EC50 or –log EC50 (=pD2) values were calculated. Changes in reactions compared with controls were analysed with Student's t-test. 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 ± SEM and n denotes the number of strips obtained each from a different patient.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Application of AVP (10–11–10–6 mol/l) produced concentration-dependent contractions in small (0.3–0.5 mm) and large (0.6–1.2 mm) vessels with pD2 values equal to 9.59 ± 0.12 and 9.24 ± 0.23 respectively (Table IGo). There was no statistically significant difference in the pD2 values.


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Table I. The effect of nimodipine on the contractile response induced by vasopressin in the human intrauterine arteries
 
The extent of contribution of Ca2+ influx to responses of intrauterine arteries to AVP was investigated by using a Ca2+ channel blocker, nimodipine or incubating the tissues in Ca2+-free medium.

Effect of nimodipine
In arteries of both sizes, 10–5 mol/l nimodipine inhibited contractions induced by AVP at concentrations lower than 10–10 mol/l (Figure 1Go). For contractions induced by AVP at concentrations higher than 10–10 mol/l, the effect of incubation with nimodipine depended on size of the vessel. In small arteries, we observed a decrease of the maximum responses whereas in large diameter arteries the responses remained unchanged. The parameters of the concentration–response curves calculated for results obtained in the absence and presence of nimodipine are shown in Table IGo.



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Figure 1. Concentration–response curves of vasopressin (AVP)-induced contractions of human intrauterine arteries in absence ({square}, {circ}) and presence ({blacksquare}, •) of 10–5 mol/l nimodipine: (A) small vessels (0.3–0.5 mm); (B) large-sized vessels (0.6–1.2 mm). Responses are expressed as percentage of the control response to 80 mmol/l K+ observed before administration of nimodipine. Data represent means ± SEM for four experiments.

 
Effect of Ca2+-free medium
A 6 min incubation in the Ca2+-free solution completely abolished the responses of arteries of both sizes to 80 mmol/l K+ and 10–10 mol/l AVP. Responses of artery rings to 10–6 mol/l AVP, however, depended on the artery diameter. In Ca2+-free medium there was no response to 10–6 mol/l AVP in rings with diameter 0.3–0.5 mm, whereas the contractions induced in large vessels were reduced by ~50% (Figure 2Go).



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Figure 2. Effects of Ca2+-free medium on responses of small (A) and large (B) diameter human intrauterine arteries to vasopressin (AVP). Responses are expressed as percentage of the control response to 80 mmol/l K+. Data represent means ± SEM for four experiments. *Indicates significantly different from control (P < 0.05).

 
Effect of thapsigargin
To assess the role of Ca2+ released from internal stores in responses of large arteries to 10–6 mol/l AVP, rings of the arteries were incubated with 10–6 mol/l TSG for 30–60 min to inhibit Ca2+ uptake into the SR (Low et al., 1993Go). These experiments were done using only 10–6 mol/l AVP, as responses to low concentration of AVP appeared to be dependent on the influx of extracellular Ca2+.

There was no significant change of the basal tension following incubation with TSG. After 30 or 60 min incubation in PSS solution containing TSG, the artery responses to 10–6 mol/l AVP decreased significantly. A second application of AVP did not evoke a contractile response. The difference between responses observed after 30 and 60 min pretreatment with TSG was not statistically significant. A prior incubation in Ca2+-free medium following pretreatment of arteries with TSG (60 min) did not significantly change their responses to AVP (Figure 3Go).



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Figure 3. Effects of incubation with thapsigargin (TSG) (10–6 mol/l) on responses of large-diameter (0.6–1.2 mm) intrauterine arteries to 10–6 mol/l vasopressin (AVP): in normal solution before incubation with TSG (A), after incubation with TSG for 30 min (B) and 60 min (C), after incubation with TSG for 60 min in Ca2+-free medium (D). Data represent means ± SEM for four experiments. *Indicates values significantly different from control (A; P < 0.05).

 
In order to accelerate emptying of Ca2+ stores and prevent TSG-induced Ca2+ influx, in another set of experiments, artery rings were incubated with TSG for 30 min in Ca2+-free medium (Xuan et al., 1992Go). In each experiment, after pretreatment with TSG, one ring was stimulated with AVP in Ca2+-free medium while a parallel ring was incubated in Ca2+-containing PSS (40–60 min), which allowed refilling of internal stores before administration of AVP. After refilling of Ca2+ stores, all tissues responded to AVP although the responses were significantly decreased. In Ca2+-free medium, three of five rings did not respond at all to AVP whereas the remaining two responded with small contractions (Figure 4Go). The mean response in this group (2.8 ± 2.0%) did not differ significantly from zero. A second application of AVP did not evoke a contractile response.



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Figure 4. Effect of depletion of Ca2+ stores and their restoration on responses of large-diameter (0.6–1.2 mm) intrauterine arteries to 10–6 mol/l vasopressin (AVP) in the presence of thapsigargin (TSG): response in normal physiological salt solution (PSS) (A), response in Ca-free medium (B), response after replacing Ca-free medium with PSS (C). Data represent means ± SEM for five experiments. *Indicates values significantly different from control (A; P < 0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vasopressin has previously been shown to contract both human non-pregnant myometrium and uterine arteries (Kostrzewska et al., 1993Go, 1997Go).

The present data show that, in Ca2+-free medium, the human intrauterine arteries do not contract when stimulated with low concentrations of AVP. This effect does not depend on the size of the artery. These results indicate that responses to Ca2+ entering the cytoplasm from extracellular space plays a key role in contractile responses of both small and large arteries to low concentrations of AVP. This is supported by the data showing a complete inhibition of the responses in the presence of a Ca2+ channel blocker, nimodipine. Together these two sets of data suggest that, in arteries of both sizes, calcium entering through dihydropyridine-sensitive channels is essential for activation of contractions caused by low concentrations of AVP (not higher than 10–10 mol/l). Recently, a tight and direct coupling between the V1a receptor and dihydropyridine calcium channel has been found in rat glomerulosa cells (Grazzini et al., 1996Go) and preglomerular arterioles (Iversen and Arendshorst, 1998Go). The extreme sensitivity to nimodipine suggests that in the human intrauterine arteries, a coupling may exist between vasopressin receptor and dihydropyridine sensitive calcium channel.

In contrast to effects caused by low AVP concentrations, AVP at high concentrations (>10–10 mol/l) appears to induce contractile response independently of calcium influx through the dihydropyridine sensitive channels (as it was not blocked by nimodipine). The data suggest a significant involvement of the path of excitation linked to inositol 1,4,5-trisphosphate production resulting in Ca2+ release from intracellular stores in SR (Knot et al., 1991Go; Rüegg et al., 1991Go). However, using contraction as the only indicator of Ca2+ movement through the membrane, one cannot exclude the possibility that high concentrations of vasopressin could activate a population of AVP receptor operated channels which are not sensitive to dihydropyridine (Van Renterghem and Lazdunski, 1994Go; Nakajima et al., 1996Go). Assuming that this was the case, the concentration of these extra AVP receptors or receptor-operated channels would tend to be higher in large than in small-diameter arteries.

Interestingly, the effect of nimodipine on responses elicited by high concentrations of AVP depended on the artery diameter. In small arteries, the statistically significant decrease of responses to high concentrations of AVP observed in the presence of nimodipine indicates that extracellular Ca2+ entering cytoplasm through dihydropyridine sensitive channels plays a major role in the activation of maximal response to the agonist. The importance of Ca2+ influx for activation of contractions of the small arteries is confirmed by data showing fading of responses to 10–6mol/l AVP in Ca2+-free medium.

In the large-sized arteries, exposure to nimodipine at a concentration completely inhibiting responses to K+-induced depolarization, caused no change in response to high concentration of AVP. These data indicate that in these vessels, the amount of Ca2+ released from intracellular stores is sufficient to achieve complete activation of the response. The above finding supports the view that the role of SR diminishes, as the arteries become smaller (Low et al., 1996Go). However, absence of Ca2+ from extracellular space significantly decreased the response of large vessels even to a high concentration of AVP, which leads to the suggestion that a substantial amount of activating Ca2+ could be entering from outside, through a dihydropyridine insensitive path. Another possible explanation is that the amount of Ca2+ stored internally decreases during incubation in Ca2+-free medium.

The results obtained in tissue incubated with TSG in Ca2+-containing solution suggest that either emptying of Ca2+ stores in SR is a slow process, or that a part of intracellularly stored Ca2+ is not influenced by TSG treatment (Borin et al., 1994Go; Tribe et al., 1994Go). The lack of difference between effects obtained after 30 or 60 min incubation with TSG seems to support the latter possibility.

The incubation of the large arteries for 30 min in the Ca2+-free solution containing TSG caused almost complete inhibition of their response to high concentration of AVP. When tissues were reincubated in Ca2+-containing PSS and then stimulated with AVP, there was a substantial response to the first dose of AVP.

The lack of response to successive administrations of AVP in arteries pretreated with TSG indicates that a signal from the TSG-sensitive intracellular Ca2+ store is necessary to evoke a response to AVP.

In conclusion, the data of the current study indicate that there is a considerable difference in large and small diameter arteries with respect to the source of Ca2+ for full activation. The relative contribution of extracellular Ca2+ depends on the concentration of the agonist and more importantly on the size of the intramyometrial arteries. Small branches of uterine arteries depended more heavily on the influx of extracellular Ca2+ than the large-sized arteries. Since small branches of the uterine artery are considered important in the local regulation of blood flow, one would expect Ca2+ channel blockers to be good candidates for a possible use in treatment of dysmenorrhoea.


    Notes
 
3 To whom correspondence should be addressed at: Department of Biophysics, Medical Academy, ul. Mickiewicza 2A, 15–230 Bialystok, Poland. E-mail: akost{at}ckst.ac.bialystok.pl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ashida, T., Schaeffer, J., Goldman, W. et al. (1988) Role of sarcoplasmic reticulum in arterial contraction: comparison of ryanodine's effect in a conduit and a muscular artery. Circ. Res., 62, 854–863.[Abstract]

Borin, M.L., Tribe, R.M. and Blaustein, M.P. (1994) Increased intracellular Na+ augments mobilization of Ca2+ from SR in vascular smooth muscle cells. Am. J. Physiol., 266, C311–C317.[Abstract/Free Full Text]

Ekstrom, P., Alm, P. and Akerlund, M. (1991) Differences in vasomotor responses between main stem and smaller branches of the human uterine artery. Acta Obstet. Gynecol. Scand., 70, 429 –433.[Medline]

Grazzini, E., Durroux, T., Payet, M.D. et al. (1996) Membrane-delimited G protein-mediated coupling between V-1a vasopressin receptor and dihydropyridine binding sites in rat glomerulosa cells. Mol. Pharmacol., 50, 1273–1283.[Abstract]

Iversen, B. and Arendshorst, W. (1998) ANG II and vasopressin stimulates calcium entry in dispersed smooth muscle cells of preglomerular arterioles. Am. J. Physiol., 274, F498–F508.[Abstract/Free Full Text]

Kenakin, T.P. (1984) The classification of drugs and drug receptors in isolated tissues. Pharmacol. Rev., 36, 165–222.[ISI][Medline]

Knot, H.J., de Ree, M.M., Gahwiler, B.H. et al. (1991) Modulation of electrical activity and of intracellular calcium oscillations of smooth muscle cells by calcium antagonists, agonists and vasopressin. J. Cardiovasc. Pharmacol., 18, S7–S14.

Kostrzewska, A., Laudanski, T. and Batra, S. (1993) Effect of ovarian steroids and diethystilbestrol on the contractile responses of the human myometrium and intramyometrial arteries. Eur. J. Pharmacol., 233, 127–134.[ISI][Medline]

Kostrzewska, A., Laudanski, T. and Batra, S. (1996) Inhibition of contractile responses of human myometrium and intramyometrial arteries by potassium channels openers. Acta Obstet. Gynecol. Scand., 75, 886–891.[ISI][Medline]

Kostrzewska, A., Laudadski T. and Batra, S. (1997) Potent inhibition by tamoxifen of spontaneous and agonist induced contractions of the human myometrium and intramyometrial arteries. Am. J. Obstet. Gynecol., 176, 381–386.[ISI][Medline]

Low, A., Darby, P., Kwan, C.-Y. et al. (1993) Effects of thapsigargin and ryanodine on vascular contractility: cross-talk between sarcoplasmic reticulum and plasmalemma. Eur. J. Pharmacol., 230, 53–62.[ISI][Medline]

Low, A.M., Kotecha, N., Neild, T.O. et al. (1996) Relative contributions of extracellular Ca2+ and Ca2+ stores to smooth muscle contraction in arteries and arterioles of rat, guinea-pig, dog and rabbit. Clin. Exp. Pharmacol. Physiol., 23, 310–316.[ISI][Medline]

Nakajima, T., Hazama, H., Hamada, E. et al. (1996) Endothelin-1 and vasopressin active Ca2+-permeable non-selective cation channels in aortic smooth muscle cells: mechanism of receptor-mediated Ca2+ influx. J. Mol. Cell. Cardiol., 28, 707–722.[ISI][Medline]

Rüegg, U.T., Wallnoffer, A., Knot, H. et al. (1991) Vasopressin and calcium: studies in vascular smooth muscle cells and mesenteric resistance vessels. In Jard, S. and Jamison, R. (eds), Vasopressin. Colloque INSERM/John Libbey Eurotext Ltd, pp. 385–392.

Somlyo, A. (1980) Ultrastructure of vascular smooth muscle. In Bohr, D., Somlyo, A. and Sparks, A. (eds), Handbook of Physiology, Section 2: The Cardiovascular System. American Physiological Society, Bethesda, pp. 33–67.

Tribe, R.M., Borin, M.L. and Blaustein, M.P. (1994) Functionally and spatially distinct Ca2+ stores are revealed in cultured vascular smooth muscle cells. Proc. Natl Acad. Sci. USA, 91, 5908–5912.[Abstract]

van Breemen, C. and Saida, K. (1989) Cellular mechanisms regulating [Ca2+]i smooth muscle. Annu. Rev. Physiol., 51, 315–329.[ISI][Medline]

Van Renterghem, C. and Lazdunski, M. (1994) Identification of the Ca2+ current activated by vasoconstrictors in vascular smooth muscle cells. Eur. J. Physiol., 429, 1–6.[ISI][Medline]

Xuan, Y.-T., Wang, O.-L. and Whorton, A. (1992) Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and [-insensitive pathways. Am. J. Physiol., 262, C1258–C1265.[Abstract/Free Full Text]

Submitted on April 13, 2000; accepted on May 31, 2000.