1 Department of Biophysics, Medical Academy, ul. Mickiewicza 2A, 15230 Bialystok, Poland and 2 Department of Obstetrics and Gynaecology, University Hospital, S-221 85 Lund, Sweden
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Abstract |
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Key words: contraction/human uterine arteries/vasopressin
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Introduction |
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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 excitationcontraction coupling in resistance arteries (Somlyo, 1980; Ashida et al., 1988
). 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., 1991). In order to distinguish the pathways of activation involved in excitationcontraction 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.
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Materials and methods |
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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., 1996).
Concentrationresponse curves were fitted to experimental data by a computer program based on the Hill equation (Kenakin, 1984; GraphPAD Software, San Diego, CA, USA).
Statistical analysis
The MannWhitney 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.
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Results |
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Effect of nimodipine
In arteries of both sizes, 105 mol/l nimodipine inhibited contractions induced by AVP at concentrations lower than 1010 mol/l (Figure 1). For contractions induced by AVP at concentrations higher than 1010 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 concentrationresponse curves calculated for results obtained in the absence and presence of nimodipine are shown in Table I
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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 106 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 3).
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Discussion |
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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 1010 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., 1996) and preglomerular arterioles (Iversen and Arendshorst, 1998
). 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 (>1010 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., 1991; Rüegg et al., 1991
). 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, 1994
; Nakajima et al., 1996
). 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 106mol/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., 1996). 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., 1994; Tribe et al., 1994
). 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.
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Notes |
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References |
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Submitted on April 13, 2000; accepted on May 31, 2000.