Influence of oestrous cycle and pregnancy on the reactivity of the rat mesenteric vascular bed

J.J.Dalle Lucca1, A.S.O. Adeagbo2 and N.L. Alsip1,3

1 Center for Applied Microcirculatory Research and 2 Department of Physiology, Health Sciences Center, A1115, University of Louisville School of Medicine, Louisville, KY 40292, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an {alpha}1-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 µmol/l), and concentration–response curves to ACh generated. Responsiveness to ACh was tested in the presence of N{omega}-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo-oxygenase and K+ channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats.

Key words: acetylcholine/mesenteric vascular bed/microcirculation/oestrous cycle/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The hormonal status of a female fluctuates with each reproductive cycle and during pregnancy. Although the vascular alterations during pregnancy are well documented (Gant et al., 1987Go; Jovanovic and Jovanovic, 1997Go, 1998Go), the impact of female hormone concentrations on vascular reactivity during the reproductive cycle has not been studied extensively. Blood pressure is lower and urinary cAMP excretion is increased in women during the luteal phase versus the follicular phase (Chapman et al., 1997Go). Thus, changes in hormone concentrations may be reflected by marked systemic vasodilatation and/or refractoriness to vasopressors such as those reported during pregnancy (Pascoal et al., 1995Go). Systemic vascular resistance is determined primarily by the contractile state (tone) of the microcirculation, in particular the arterioles, and small arteries (Christensen and Mulvany, 1993Go). Many aspects of the control mechanisms of the microcirculation and small arteries are poorly understood, especially how reproductive hormones regulate their functions. Since the mesenteric vascular bed is representative of the splanchnic vasculature and the reactivity of these resistance vessels is particularly crucial to the understanding of blood pressure homeostasis (Pascoal et al., 1995Go), the first aim of this study was to determine if acetylcholine-induced vasodilatation in the isolated, perfused mesenteric vascular bed was affected by the day of the oestrous cycle and pregnancy.

The regulation of vascular tone and reactivity involves many factors. Endothelial cells modulate underlying vascular smooth muscle tone by releasing endothelium-derived relaxing factors such as nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF), and contracting factors including superoxide anion, thromboxane A2/prostaglandin H2 and endothelin 1 (Furchgott and Vanhoutte, 1989Go; Lévesque et al., 1994Go). All endothelium-derived relaxing factors diffuse to the vascular smooth muscle after release by endothelial cells. In smooth muscle cells, NO and PGI2 initiate vascular relaxation by increasing cGMP and cAMP respectively. In contrast, EDHF activates K+ channels thereby causing K+ efflux, hyperpolarization of vascular smooth muscle cells and vascular relaxation. The K+ channel upon which this substance acts is tissue dependent, and the involvement of EDHF seems to be more significant in smaller resistance vessels than in larger vessels. This substance is still unidentified, but may be an epoxyeicosatetraenoic acid (Campbell et al., 1996Go; Van de Voorde and Vanheel, 1997Go; Suzuki et al., 1998Go).

The reproductive hormones, in particular oestrogen, alter the production of the relaxing factors from the endothelium (Meyer et al., 1997Go). Oestrogen stimulates the synthesis of NO in the uterus and other tissues (Geary et al., 1998Go; Kirsch et al., 1999Go) and stimulates PGI2 production in vascular endothelial cells (Farhat et al., 1996Go; Myers et al., 1996Go; Jun et al., 1998Go). The ability of oestrogen to stimulate the production of EDHF has not been studied, although EDHF production may be enhanced in pregnant rats (Bobadilla et al., 1997Go) and guinea pigs (Keyes et al., 1998Go). This suggests that basal, as well as stimulated, release of endothelium-derived relaxing factors may fluctuate during the reproductive cycle and pregnancy. In male and female rats, relaxation to acetylcholine or carbachol in the mesenteric vascular bed is mediated by endothelium-derived NO (Fortes et al., 1990Go) and EDHF (Kamata et al., 1996Go) with the relative contribution of EDHF greater in female than male rats (McCulloch and Randall, 1998Go). Whether the relative contribution of the endothelial factors released by acetylcholine in this vascular bed is affected by the reproductive cycle or pregnancy is unknown.

The present study was designed to evaluate the dilatation to acetylcholine in the isolated, perfused mesenteric vascular bed from virgin female rats at various stages of the oestrous cycle (oestrus, dioestrus-1, dioestrus-2 and pro-oestrus), and in pregnant rats (gestation day 16). The contribution of the endothelial-derived products–NO, prostanoids and EDHF–as mediators of acetylcholine-induced vascular relaxation in each group was also explored. In pregnant females, there is reduced contraction to vasoconstrictors which is thought to be secondary to increased synthesis of NO and PGI2 (Magness et al., 1992Go; Kim et al., 1994Go) and possibly EDHF (Keyes et al., 1998Go). Therefore, in the mesenteric vascular beds from cycling and pregnant rats, (i) the ability of a selective {alpha}1-adrenergic agonist, cirazoline, to induce tone, and (ii) the influence of NO, prostanoids, and EDHF on this cirazoline-induced tone were also assessed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
N{omega}-nitro-L-arginine (L-NA), tetrabutylammonium iodide (TBA), acetylcholine (ACh) bromide and ibuprofen (IBU) were purchased from Sigma Chemical Co., St Louis, MO, USA. Cirazoline hydrochloride (CRZ) was purchased from RBI/Sigma, Natick, MA, USA. Ketamine was purchased from Ft Dodge (Columbus, OH, USA) and xylazine from Butler Co. (Columbus, OH, USA). All components of the modified Krebs solution (physiological salt solution; PSS) were purchased from Sigma Chemical Co. L-NA was dissolved in PSS; all other compounds were dissolved in distilled water.

Animals
Virgin female Sprague–Dawley rats weighing 200–225 g (Harlan, Indianapolis, IN, USA) were housed in a temperature- and humidity-controlled room of our Association for the Accreditation of Laboratory Animal Care (AALAC) -approved animal care facility with a 12 h light:12 h dark light cycle and access to distilled water and rat chow ad libitum. A vaginal smear was performed daily to determine the stage of oestrous cycle. Animals were monitored for at least two oestrous cycles and only those animals exhibiting regular 4 day oestrous cycles were used in this study. An animal was mated when her vaginal smear indicated that she was in pro-oestrous. The day that spermatozoa were present in the vaginal smear was considered day 0 of pregnancy.

Perfused mesenteric vascular bed
Female virgin rats on each day of the oestrous cycle (oestrus, dioestrus-1, diestrus-2, pro-oestrus) and pregnant rats on gestation day 16 were used in these studies. Following anaesthesia with an i.p. injection of ketamine (37.5 mg/kg) combined with xylazine (5.0 mg/kg), the abdominal cavity was opened and the superior mesenteric artery cannulated through an incision at its confluence with the dorsal aorta. The entire mesenteric vascular bed was then flushed with heparinized PSS, and the small intestinal borders were trimmed off. The bed was then transferred to a warmed (37°C) chamber where it was perfused with a gas mixture (95% O2:5% CO2)-saturated PSS maintained at 37°C. The perfusion rate was kept constant at 5ml/min using a peristaltic pump (Masterflex, Cole-Palmer Instruments Co., Vernon Hills, IL, USA). Changes in perfusion pressure were recorded via a Statham pressure transducer (model P23XL) coupled to a Grass polygraph recorder (model 7H).

The composition of PSS (in mmol/l) was: NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 12.5, glucose 11.1. The pH of the PSS was maintained at 7.4 after saturation with a 95% O2:5% CO2 gas mixture. Tissues were allowed to equilibrate for at least 30 min before the start of all experiments.

All experiments in this study involved measuring vasodilatation to ACh in the absence, and in the presence of inhibitors of nitric oxide synthase (NOS), cyclo-oxygenase, and K+ channels or combinations of these agents. Therefore, vascular tone in the isolated, perfused mesenteric vascular bed was generated by continuous infusion of CRZ, a synthetic imidazole derivative which is stable in aqueous medium at physiological pH and produces stable tone for many hours in this preparation (Adeagbo and Triggle, 1993Go).

Experimental protocols
Protocol 1
The purpose of this series of experiments was two-fold. The first was to determine the pressor effect of CRZ infusion and the relative modulation of this by nitric oxide synthesis inhibition with L-NA (100 µmol/l), cyclo-oxygenase inhibition with IBU (10 µmol/l) and K+ channel blockade with TBA (1 mmol/l). The second purpose of these experiments was to determine the influence of the oestrous cycle and pregnancy on ACh-induced dilatation and the relative contribution of NO, prostanoids and EDHF in mediating this response. The experiments were carried out in mesenteric vascular beds isolated from female rats at different stages of the oestrous cycle and late pregnancy.

Following the equilibration period, CRZ (1 µmol/l) was added to the perfusion medium and remained for the entire protocol. Once the developed tone plateaued, a concentration–response relationship to ACh was established by giving bolus injections (0.1 ml) of ACh into the perfusion tube (10–4 to 10 nmol). After the tissue had recovered from the highest concentration of ACh, L-NA was added to the perfusion solution (PSS) and co-infused with CRZ. After 20 min, the bolus injections of ACh were repeated. IBU was then added to the perfusion solution already containing CRZ and L-NA. After the tone reached a plateau (usually 20 min), ACh concentrations were given for a third time. Finally, TBA was added to the perfusion solution along with the CRZ, L-NA and IBU and again, after tone had levelled off (usually 40 min), the ACh injections were repeated. At the end of each experiment, sodium nitroprusside (SNP, 10 µmol/l) was administered to assess the ability of the vessel to dilate. The tissue was always allowed to return to its pre-injection pressure before another concentration of ACh or an inhibitor was given. One group of virgin rats on dioestrous day 1 and one group of pregnant rats were subjected to the protocol without the addition of L-NA, IBU or TBA to the PSS. These groups served as time controls.

The increase in perfusion pressure (tone) generated by CRZ and any change in tone produced by L-NA, IBU and TBA were compared for the different stages of oestrous cycle and the pregnant group. Concentration–response curves for the response to ACh were constructed in presence of CRZ (control), CRZ and L-NA (L-NA present), CRZ, L-NA and IBU (L-NA + IBU present) and CRZ, L-NA, IBU and TBA (L-NA, IBU and TBA present).

Protocol 2
The purpose of this protocol was to determine if the order in which inhibitors were added to the perfusion solution affected the vascular response to ACh. In these experiments, TBA was added to the perfusion solution prior to L-NA. For this protocol, pregnant rats and virgin rats during dioestrous-1 were used since they represent the two groups with the largest difference in reproductive hormone concentrations. In isolated, perfused mesenteries from pregnant and dioestrous-1 virgin animals, CRZ (1 µmol/l) was added to the perfusion solution to induce vascular tone. Bolus injections of ACh were given at increasing concentrations (10–4 to 10 nmol) into the perfusion tube to determine the control concentration–response relationship. The K+-channel blocker TBA (1 mmol/l) was then introduced into the perfusion solution and, after a 40 min equilibration period, ACh responsiveness was re-determined. Next, the NOS inhibitor L-NA (100 µmol/l) was introduced into the suffusion solution, in addition to CRZ and TBA. After at least a 20 min equilibration period, the vascular responsiveness to ACh was tested again. SNP (10 µmol/l) was injected at the end of each experiment to assess the ability of the vessel to dilate. The tissue was always allowed to return to its pre-injection pressure before another concentration of ACh or an inhibitor was given.

Concentration–response curves for ACh were constructed in presence of CRZ (control), CRZ and TBA (TBA present) and CRZ, TBA and L-NA (TBA + L-NA present).

Statistical analysis
Change in tone due to the addition of CRZ, L-NA, IBU or TBA to the perfusion solution was expressed in mmHg. Concentration–response curves were obtained for ACh alone (control), in the presence of CRZ and L-NA (L-NA present), CRZ, L-NA and IBU (L-NA+IBU present), and CRZ, L-NA, IBU and TBA (L-NA+IBU+TBA present). Relaxation to ACh at each concentration was expressed as a percentage of the total developed tone. Each concentration–response curve was used to determine the concentration of ACh that gave 50% of the maximal response (EC50) and the maximal response. Change in perfusion pressure, EC50s, and responses at each concentration of ACh were separately compared by one-way analysis of variance (ANOVA) and Newman–Keul's multiple range tests when a significant difference existed between groups. A P value <0.05 was taken as significant. Group data are reported as mean ± SEM.


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Establishment of tissue viability over time
The ability of the isolated, perfused mesenteric vascular bed to respond to repeated applications of ACh was tested in a group of virgin (n = 4) and a group of pregnant rats (n = 4). In both groups, the response to ACh was identical in all four curves obtained. A representative tracing is shown in Figure 1Go.



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Figure 1. Representative tracing from a time control experiment. The responsiveness to acetylcholine (ACh) was tested repeatedly in an isolated, perfused mesenteric vascular bed taken from a rat in dioestrus-1. Tone was induced with cirazoline (1 µmol/l) during the entire protocol. Segment A is analogous to the control response in Figures 3 and 4GoGo; segment B was obtained 20 min after the last dose of ACh in segment A; segment C was obtained 20 min after the last dose of ACh in segment B; segment D was obtained 40 min after the last dose of ACh in segment C. Sodium nitroprusside (SNP) was given as a bolus injection after the last dose of ACh. Similar results were obtained in tissues taken from pregnant animals. Scale of tracing is in mmHg.

 
Cirazoline-induced tone and its modulation by L-NA, IBU and TBA
The basal perfusion pressure of mesenteric vascular beds perfused with normal PSS (i.e. 4.7 mmol/l KCl) was 22.6 ± 0.3 mmHg (n = 58). Continuous infusion of CRZ (1 µmol/l) produced a significant increase in perfusion pressure in all experimental groups; however, the increase was significantly smaller in pregnant and pro-oestrous groups compared with the oestrous, dioestrous day 1 and dioestrous day 2 groups (Figure 2Go; open bars). Addition of L-NA (100 µmol/l) to the perfusion solution in addition to CRZ caused a further increase in tone in all groups (Figure 2Go; filled bars). The pro-oestrous group exhibited less total tone than all other groups, and the pregnant group developed less overall tone than the oestrous and dioestrous day 1 group. When the increase in tone to L-NA was expressed as a percentage increase above the CRZ-induced tone for each individual animal, that in the pregnant group was significantly larger than in the oestrous, dioestrous day 1 and dioestrous day 2 groups (413 ± 101% for pregnant, 284 ± 78% for pro-oestrous, 95 ± 29% for dioestrous day 2, 146 ± 28% for dioestrous day 1, and 111 ± 33% for oestrous).



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Figure 2. Bar graph showing the increase in perfusion pressure (mean ± SEM) in the isolated, perfused mesenteric vascular bed after adding cirazoline alone (1 µmol/l, open bars) or with N{omega}-nitro-L-arginine (L-NA) (100 µmol/l, filled bars) into the perfusion solution. The cirazoline-induced constriction was smaller in the pro-oestrous (Proest) and pregnant (Preg) groups. However, after addition of L-NA, the total perfusion pressure remained smaller only in the pro-oestrous group. Groups with same letter designation were not different, P <0.05. When the increase in tone to L-NA was expressed as the percentage of tone established by cirazoline, the percentage increase seen in the pregnant group (413 ± 101%) was significantly greater than the oestrous, dioestrous-1 (D-1) and dioestrus-2 (D-2) groups (111 ± 33%, 146 ± 28% and 95 ± 29% respectively). The pro-oestrous group had an intermediate value of 284 ± 78% that was not significantly different from any other group.

 
The addition of IBU (10 µmol/l) to the perfusion solution containing CRZ and L-NA did not cause a significant change in tone. Although adding TBA (1 mmol/l) to the perfusion medium containing CRZ, L-NA and IBU caused a transient reduction in perfusion pressure, vessel tone recovered to pre-application level without adjusting the concentration of CRZ.

Effect of L-NA and/or IBU on ACh-induced relaxation
Bolus injections of ACh (0.0001–1 nmol) initiated concentration-dependent decreases in perfusion pressure of CRZ pre-constricted mesenteric vascular beds of all experimental groups. The response to ACh was not affected by the day of the oestrous cycle or by pregnancy (Table IGo).


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Table I. Control responses to acetylcholine
 
In the presence of L-NA, there was a significant attenuation of the response to ACh in the oestrous, dioestrous day 1, dioestrous day 2 and pro-oestrous groups, but maximal responses were not altered (Figure 3Go). L-NA did not alter the response to ACh in isolated, perfused mesenteric vascular beds removed from pregnant rats (Figure 3Go, bottom graph). The addition of IBU to the perfusion medium did not modify ACh-induced vasodilatation in any experimental group (Figure 3Go).



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Figure 3. Vasodilator responses of isolated, perfused mesenteric vascular beds to increasing concentrations of acetylcholine in virgin rats on each day of the oestrous cycle and pregnant rats. Data are plotted as percentage decrease (mean ± SEM) in perfusion pressure, with 100% being total relaxation. Control curves (•) were obtained in the presence of cirazoline alone (1 µmol/l). L-NA curves ({blacksquare}) were obtained in the presence of cirazoline and L-NA (100 µmol/l), and ibuprofen (IBU, {blacktriangleup}) curves in the presence of cirazoline, L-NA and IBU (10 µmol/l). Note the marked attenuation of the response to acetylcholine in all groups after addition of 1 mmol/l tetrabutylammonium iodide ({blacklozenge}) into the perfusion solution containing cirazoline, L-NA and IBU. *Significant difference from control value; #, significant difference from L-NA value, P <0.05.

 
Effect of TBA on NO/prostanoid-independent vasodilatation to ACh
In all experimental groups, treatment with L-NA alone or in combination with IBU, to inhibit NOS and cyclo-oxygenase respectively (Figure 3Go), gave no or only partial blockade of the ACh-induced vasodilatation. Thus, the effect of a K+ channel antagonist TBA on vasodilator responses to ACh was tested in the presence of L-NA and IBU. TBA in the presence of L-NA and IBU significantly reduced the vasodilator response of the mesenteric vascular bed to ACh in all groups (Figure 3Go).

When TBA was introduced into the perfusion solution containing CRZ prior to L-NA (protocol 2), it caused a significant reduction in ACh-induced dilatation of the mesenteric vascular beds from both pregnant and virgin dioestrous-1 rats (Figure 4Go). The addition of L-NA to the TBA- and CRZ-containing perfusion medium did not cause any further attenuation of the response to ACh (Figure 4Go).



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Figure 4. Vasodilator response of the isolated, perfused mesenteric vascular bed to increasing concentrations of acetylcholine in pregnant and virgin dioestrous-1 rats. Data are plotted as percentage decrease in perfusion pressure (mean ± SEM), with 100% being total relaxation. The control curves ({blacklozenge}) were obtained in the presence of cirazoline alone (1 µmol/l). Tetrabutylammonium iodide (TBA) curves ({blacklozenge}) were obtained in the presence of cirazoline and TBA (1 mmol/l). LNA curves ({blacksquare}) were obtained in the presence of cirazoline, TBA and LNA (100 µmol/l). The response to acetylcholine in the presence of TBA ({blacklozenge}) was markedly attenuated in all groups. *Significant difference from control value, P <0.05.

 
To test the ability of the mesenteric vascular beds to relax at the end of each experiment, a bolus injection of the endothelium-independent relaxing factor, SNP (1 µmol/l) was injected. In all experiments SNP produced maximal relaxation (100% of total-induced tone).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three important results emerged from the present study. First, mesenteric vascular beds from pregnant rats and rats in the pro-oestrous stage are hyporesponsive to the {alpha}1-adrenergic agonist CRZ compared with mesenteric beds from virgin rats on the other days of the oestrous cycle. Second, when NOS was inhibited with L-NA, mesenteric vascular beds from pregnant rats showed a higher percentage increase in perfusion pressure than those from virgin animals. Third, vasodilatation initiated by ACh is mediated predominantly through activation of K+ channels, thus suggesting an important role for EDHF in mesenteric vessels of female rats.

Normal human gestation consistently leads to hyporesponsiveness to the pressor effects of angiotensin II, and perhaps also other pressure stimuli (Pan et al., 1990Go; Lindheimer et al., 1992). The refractoriness of the mesentery from pregnant rats to cirazoline observed in the present study is consistent with those findings. Since this effect was also seen in animals in pro-oestrus, this could be related to the increased hormone concentrations, presumably oestrogen, during pregnancy and pro-oestrus. It was reported recently (Chapman et al., 1997Go) that women during the luteal phase of the menstrual cycle exhibited a lower systemic arterial pressure compared with women in the follicular phase. Aortic rings from rats in pro-oestrus showed a reduction in the maximal contraction to noradrenalin and increased synthesis of dilator prostaglandins compared with aortas from rats in oestrus, dioestrus or met oestrus (Zamorano et al., 1994Go). Both these studies were performed when oestrogen concentrations were elevated, suggesting that oestrogen could be responsible for these vascular alterations.

Although the cardiovascular benefits of oestrogen therapy have been well documented (Mikkola et al., 1998Go), the mechanism by which oestrogen exerts its effects on the vasculature is not completely understood. Chronic administration of oestrogen has been associated with an enhanced release of NO and prostaglandins from the endothelium in both humans and experimental animals (Farhat et al., 1996Go). In addition, oestrogen therapy has been associated with coronary blood flow regulation in part by up-regulating NOS activity (Gorodeski et al., 1995Go). Furthermore, resistance vessels from the mesentery of ovariectomized female rats receiving oestrogen replacement, exhibited an increase in basal, but not ACh-stimulated, release of endothelium-derived relaxing factors (Meyer et al., 1997Go).

The present study also reported a relatively higher increase in perfusion pressure in the mesenteric vascular bed from pregnant rats after addition of L-NA into the perfusion solution. This agrees with previous studies (Weiner et al., 1994Go; Nathan et al., 1995Go) reporting an increase in basal vascular NO synthesis during pregnancy. If basal release of NO is increased in pregnancy, this could explain the refractoriness to CRZ observed in the pregnant group (Molnár and Hertelendy, 1992Go).

The data presented here also revealed that in the isolated perfused mesenteric vascular bed pre-constricted with CRZ, ACh-induced relaxation did not differ among pregnant and non-pregnant cycling rats. Similarly, it was reported (Yamasaki et al., 1996Go) that neither ACh-induced relaxation nor basal NO biosynthesis is increased in the late pregnant rat femoral resistance vessels under isometric conditions. However, others report that ACh-induced relaxation is enhanced in mesenteric arteries from pregnant rats (Kim et al., 1994Go; Gerber et al., 1998Go), and in carotid arteries from the pregnant guinea pig (Weiner et al., 1989Go). Taken together, these results suggest a divergent responsiveness to ACh between resistance and conductance vessels during pregnancy.

In this study, L-NA did not change the vascular reactivity of the mesenteric vascular bed to ACh in the pregnant group although it caused a small but statistically significant inhibition in the cycling virgin groups. This suggests two possibilities: (i) that during late pregnancy in rats, there is a shift to some mediator other than NO for the ACh vasodilator response in the mesentery; or (ii) that there is an up-regulation of NOS in the mesenteric vessels from the pregnant rat. If the latter were true, the concentration of L-NA used in the present study may not have been sufficient to inhibit the up-regulated NOS.

Thus, in the isolated, perfused mesenteric vascular bed, NO activity appeared to be up-regulated in pregnancy and possibly on pro-oestrus. This raises the question of why we did not observe an increase in the relaxation to ACh in these preparations. The lack of effect of pregnancy on ACh-induced dilation has been observed in other vessels such as the renal and uterine arteries (Jovanovic et al., 1994aGo; Kim et al., 1994Go). One possible reason for this seeming dichotomy is that the apparent increase in NO activity in the pregnant and pro-oestrous groups is caused by an increase in vascular smooth muscle NO activity rather than endothelium-released NO. In the human uterine artery, L-arginine-induced relaxation is mediated by non-endothelial NO production (Jovanovic et al., 1994bGo). Another possible reason is that since the portion of the ACh-induced dilation in this vascular bed attributable to NO is so small, it may not have been possible to detect any increase in the relaxation to ACh with the number of preparations reported here. In fact, although not statistically significant, at two points of the ACh concentration–response curve (0.001 and 0.01 nmol), the response of the pregnant group does appear larger than the virgin groups (data not shown).

In an earlier study (Gant et al., 1987Go), it was found that inhibition of cyclo-oxygenase restored the vascular refractoriness to angiotensin II in pregnant women, suggesting that prostanoids are a pivotal mediator of vascular reactivity during pregnancy. In our study, ibuprofen did not alter ACh-induced relaxation in the presence of L-NA, demonstrating that cyclo-oxygenase products are not major contributors to ACh-induced dilatation in the female mesenteric vascular bed. This agrees with previous findings (Meyer et al., 1997Go) in which ACh-induced dilatation was not affected by cyclo-oxygenase inhibition in isolated mesenteric arterioles from ovariectomized or oestrogen-treated rats. Similarly, in recent studies using a double-perfused mesentery preparation from Wistar Kyoto and spontaneously hypertensive male rats, it was shown that the cyclo-oxygenase pathway was not implicated in the vasodilatation induced by ACh (Le Marquer-Domagala and Finet, 1997Go). These studies all indicate that, at least in the mesenteric vascular bed, prostanoids are not involved in the vascular response to ACh.

In all virgin and pregnant groups the majority of the ACh-induced dilatation was intact in spite of the presence of L-NA and IBU, suggesting that as in males, NO is not the major mediator of ACh-induced dilatation in the female mesenteric vascular bed (Adeagbo and Triggle, 1993Go; McCulloch and Randall, 1998Go). In contrast to L-NA and IBU, the addition of the potassium channel blocker TBA profoundly attenuated the relaxation to ACh, although TBA did not significantly alter basal tone. Thus, the majority of the dilatation to ACh in this vascular bed appears to be mediated by the release of a non-NO, non-prostanoid factor, which dilates by opening K+ channels. Although the EDHF has yet to be identified, it is known to promote vasodilatation by activating K+-channels, resulting in smooth muscle cell membrane hyperpolarization. Therefore, the use of K+-channel blockers or varying extracellular K+ has been used consistently to demonstrate EDHF-mediated relaxation (Adeagbo and Triggle, 1993Go; McCulloch and Randall, 1998Go). TBA has been shown to block EDHF-mediated responses in many vessels, including coronary artery (Nakashima et al., 1997Go; Popp et al., 1998Go; Kaw and Hecker, 1999Go), omental arteries (Ohlmann et al., 1997Go) and carotid artery (Dong et al., 1997Go).

This last finding agrees with that from a recent study (McCulloch and Randall, 1998Go) in which data from female rats on different oestrous cycle days were pooled and the responsiveness to carbachol in the isolated, perfused mesenteric vascular bed investigated. As in the present study, these workers found that EDHF is an important mediator of muscarinic receptor-induced dilatation in this vascular bed in the female. They and others (Kilpatrick and Cocks, 1994Go; Hatake et al., 1995Go) suggested that under normal conditions NO is the main mediator of ACh-induced relaxation in the female mesenteric vascular bed, but during NOS inhibition, there is an up-regulation of EDHF production that compensated for the lack of NO. This hypothesis was ruled out by the results from the second protocol of the present study (Figure 4Go). It was found that perfusion of the female mesenteric vascular bed with TBA, a non-selective K+ channel blocker, in the presence of an intact NO system, dramatically inhibited relaxation to ACh. When L-NA was perfused along with TBA, there was no further inhibition of the ACh-induced relaxation of the mesenteric vascular bed of virgin rats or pregnant rats.

In conclusion, it was found that in cycling virgin and late-pregnant female Sprague–Dawley rats, the major portion of ACh-induced dilatation of the mesenteric vascular bed was probably mediated by EDHF. In addition, the present study suggested that NOS may be up-regulated in the mesenteric vascular bed from pregnant and possibly pro-oestrous virgin rats compared with virgin rats on other oestrous cycle days.


    Acknowledgments
 
The authors would like to thank Julie Hornung for her technical expertise. This work was supported by grants from the American Heart Association, Ohio Valley Affiliate and the University of Louisville School of Medicine. Dr Dalle Lucca is the recipient of a Postdoctoral Fellowship from FAPESP-Sao Paulo Brazil.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adeagbo, A.S.O. and Triggle, C.R. (1993) Varying extracellular [K+]: a functional approach to separating EDHF- and EDNO-related mechanisms in perfused rat mesenteric arterial bed. J. Cardiovasc. Pharmacol., 21, 423–429.[ISI][Medline]

Bobadilla, R.A., Henkel, C.C., Henkel, E.C. et al. (1997) Possible involvement of endothelium-derived hyperpolarizing factor in vascular responses of abdominal aorta from pregnant rats. Hypertension, 30, 596–602.[Abstract/Free Full Text]

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Submitted on September 13, 1999; accepted on December 15, 1999.