Adrenoceptors mediating contraction in the human uterine artery

Pharmacological characterization of adrenoceptors mediating smooth muscle contraction was performed in isolated preparations from the human uterine artery. The mixed alpha 1- and alpha 2-adrenergic agonist, noradrenaline (NA) and the selective alpha 1-agonists, phenylephrine and methoxamine, all contracted the smooth muscle preparations in a concentration-dependent manner. The responses were antagonized competitively by the selective alpha 1-antagonist, prazosin, yielding pA2 values for the three agonists (8.33-9.08) typical for an interaction with alpha 1-receptors. The alpha 2-selective receptor agonists, clonidine and BHT 920, did not exert any contractile effects in the isolated uterine arteries, and the alpha 2-adrenergic antagonist, yohimbine, counteracted the contractile effect of NA only at high concentrations. The concentration-response curve for NA was unaffected by the alpha 2-selective antagonists, rauwolscine and idazoxan. The results suggest that the postjunctional contractile receptors in the human uterine artery primarily are of the alpha 1 type, and give no evidence for any substantial involvement of alpha 2-receptors in this important tributary vessel of the human female reproductive tract.     

Neuropeptide Y (NPY) co-exists with tyrosine hydroxylase and potentiates the adrenergic contractile response of vascular smooth muscle in the human uterine artery.

The human uterine artery was studied by immunocytochemistry and in vitro pharmacology. Nerve fibres containing immunoreactivity for neuropeptide Y (NPY) were encountered in the media, adventitia, smooth muscle layers and surrounding the vasa vasorum. Approximately 50% of the nerve fibres containing NPY also stored immunoreactivity for tyrosine hydroxylase (TH) and TH activity was found in no other fibres. Noradrenaline (NA) contracted the isolated uterine artery in a concentration dependent manner. The presence of increasing doses of NPY shifted the concentration-response curve for NA to the left. Consequently the pD2-values were increased indicating a potentiation of the adrenergic effects induced by NPY. The results demonstrate the existence of NPY in adrenergic nerve fibres surrounding the human uterine artery. A close co-operation between NPY and NA in the neuronal control of smooth muscle is suggested.     

Dual repressive effect of angiotensin II-type 1 receptor blocker telmisartan on angiotensin II-induced and estradiol-induced uterine leiomyoma cell proliferation

BACKGROUND: Although uterine leiomyomas or fibroids are the most common gynecological benign tumor and greatly affect reproductive health and well-being, the pathophysiology and epidemiology of uterine leiomyomas are poorly understood. Elevated blood pressure has an independent, positive association with risk for clinically detected uterine leiomyoma. Angiotensin II (Ang II) is a key biological peptide in the renin-angiotensin system that regulates blood pressure. METHODS: In this study, we investigated the potential role of Ang II (1-1000 nM) in the proliferation of rat ELT-3 leiomyoma cells in vitro. RT-PCR and western blot analysis with cell proliferation and DNA transfection assays were performed to determine the mechanism of action of Ang II. RESULTS: Ang II induced ELT-3 leiomyoma cell proliferation (P < 0.01) and the expression of Ang II type 1 receptor (AT(1)R) and AT(2)R mRNA and protein was confirmed. Regarding the intracellular signaling pathway, the Ang II-induced cell proliferation was AT(1)R-, epidermal growth factor receptor-, extracellular-regulated kinase- and protein kinase C-dependent but was not dependent on the AT(2)R or phosphatidylinositol-3 kinase or JAK kinase. The AT(1)R blocker telmisartan, effectively repressed Ang II-induced and estradiol-induced cell proliferation (P < 0.01). AT(1)R, but not AT(2)R, plays a role in Ang II-induced ELT-3 cell proliferation. CONCLUSIONS: These experimental findings in vitro highlight the potential role of Ang II in the proliferation of leiomyoma cells.     

Atrial natriuretic peptide attenuates pressor but enhances natriuretic responses to angiotensin II in pregnant conscious goats

This study was designed to examine the actions of ANP in acute, ANGII-mediated hypertension during pregnancy. Effects on blood pressure, blood volume, and renal Na and K excretion were evaluated in conscious goats (n= 6). ANP (2 μrg min^-1), ANGII (0.5 μg min^-1), or ANGII + ANP (doses the same as for each peptide alone) was infused intravenously for 60 min. The pressor response to ANGII was reduced in pregnant goats. This reduction was seen in systolic, but not in diastolic pressure. ANP decreased pressure by 5–10 mmHg both in pregnancy and in non-pregnancy. When ANGII + ANP was infused, blood pressure initially rose as with ANGII but then declined. ANP suppressed only the elevated systolic pressure. Plasma protein concentration and haematocrit was reduced by ANGII but increased by ANP alone or together with ANGII, thereby implying fluid shift into the vasculature by ANGII and opposite movement by ANP. ANGII increased renal Na excretion to 1500 μmol min^-1in non-pregnancy, but only to half of that in pregnancy. ANP alone caused small natriuresis, but enhanced ANGII-induced natriuresis to near 3000 μmol min^-1in both non-pregnant and pregnant goats. In summary, ANP further attenuated the blunted blood-pressure rise due to ANGII in pregnant goats, and reduced plasma volume, but enhanced renal Na excretion as in non-pregnant goats. This implies that with the present combination ANP and ANGII caused a near maximal natriuretic response that was not modified by the systemic cardiovascular changes occurring in pregnant goats.     

Angiotensin II in human seminal fluid

The renin-angiotensin system (RAS) and angiotensin II are important in sperm function and male fertility. Angiotensin II type I (AT1) receptors have been identified in developing and ejaculated human spermatozoa, and angiotensin can stimulate sperm motility, the acrosome reaction and binding to the zona pellucida. However, there is little information on the availability of the hormone to spermatozoa during the reproductive process. Seminal plasma and blood plasma obtained from normal and subfertile subjects was extracted, and angiotensin content was analysed by radioimmunoassay. Values obtained for blood angiotensin II were within the normal range at 16.0 +/- 3.1 pg/ml (mean +/- SEM). Values for seminal plasma were usually 3-5 fold higher, at 51.6 +/- 9.3 pg/ml (n = 34, P < 0.0001). High performance liquid chromatography analysis showed that approximately 80% of the immunoreactive angiotensin was attributable to angiotensin II itself. However, seminal plasma angiotensin II concentrations were not correlated with blood angiotensin II, sperm concentration or sperm motility. The results show that immunoreactive angiotensin from a source other than the circulation is available to spermatozoa in human ejaculates. The results are consistent with the concept that angiotensin II has an important role in male fertility.     

Predominant role for nitric oxide in the relaxation induced by acetylcholine in human uterine artery

The effect of acetylcholine on the isolated human uterine arteryrings was investigated. Acetylcholine (10^–10 M to 6x 10^–5M) induced concentration- and endothelium-dependent relaxation(pD2=7.4±0.02, maximal response was 77.5±6.3%of relaxation induced by papaverine at 3x10–4 M), diethylcarbamazine(104 M) and tetra-ethylammonium (3x10^–4 M) had no effectson acetylcholine-evoked relaxation. Methylene blue (10–5M) and NG-monomethyl-L-arginine (L-NMMA) (3x10^–6 to 3x10^–5 M) antagonized relaxation induced by acetylcholine.The inhibition of endothelium-dependent relaxation by L-NMMA(10–5 M) was reversed by L-arginine (10–5 M) butnot by D-arginine (10^–4 M). It is concluded that in uterineartery acetylcholine induces endothelium-dependent relaxation.It is suggested that the acetylcholine-induced relaxation ofisolated uterine artery is probably mediated via endothelialnitric oxide formation     

Vasoactive intestinal peptide regulates angiotensin II catabolism in the rabbit

Although vasoactive intestinal peptide (VIP) is natriuretic it stimulates renin and aldosterone secretion. Therefore, to effect a natriuresis, VIP may need to modulate the sodium conserving actions of the renin angiotensin system (RAS) by another means. One possibility is that it alters the rate of disappearance from the circulation of one or more components of the RAS. We sought to determine whether VIP regulates the rate of catabolism of angiotensin II (Ang II). Steady state metabolic clearance studies of Ang II were undertaken with and without simultaneous VIP infusion. These studies were performed in rabbits on low, normal and high sodium diets, as dietary sodium has been shown to affect the metabolism of both VIP and Ang 11. The effects of VIP on plasma Ang 11 concentration and secretion were also studied. VIP decreased Ang II catabolism in rabbits on low (P < 0.05) and normal sodium diets (P < 0.05). Plasma levels of Ang II increased significantly in response to VIP in rabbits on these diets (low, P < 0.04; normal, P < 0.05). In contrast, in rabbits on a high sodium diet VIP increased the rate of catabolism of Ang II (P < 0.001). Thus we conclude that the effect of VIP on sodium excretion may be modulated by its effects on Ang II metabolism. The decrease in Ang II catabolism seen in rabbit on low and normal sodium diets may prevent or ameliorate any natriuresis while the more rapid degradation of Ang II which occurs in dietary sodium excess may enhance the natriuretic effect of VIP.     

Pregnancy: Oxygen promotes contraction by endothelin-1 in human umbilical artery

The influence of oxygen on the contractile response to endothelin-1in the human umbilical artery was investigated in vitro. Segmentsof human umbilical artery were suspended in organ baths to recordthe circular motor activity induced by endothelin-1 at a pO₂of 12 kPascal (kPa) or 45 kPa. Endothelin-1 induced a concentration-dependentcontraction which was significantly larger at 45 kPa O₂ comparedwith the contractile response at 12 kPa O₂.     

Pregnancy: Ultrasonographic features of uterine blood flow during the first 16 weeks of pregnancy

Uterine blood flow volume has been thought to increase in alinear fashion throughout pregnancy, but previous studies inearly pregnancy may have not been performed often enough orin sufficient numbers of patients. We measured uterine arteryblood flow volume, average velocity, vessel cross-sectionalarea, resistance index, and spiral artery resistance index withDoppler ultrasound at 1–3 week intervals from gestational(post-menstrual) weeks 5–6 to week 16 in 44 normal, spontaneous,single pregnancies. Uterine artery blood flow volume and velocityincreased gradually until the end of week 9, and then rapidlyfrom weeks 10–16. Uterine artery vessel size increasedlinearly. The uterine artery resistance index was the inverseof volume and velocity, in contrast to the spiral artery resistanceindex, which decreased linearly. These findings indicate thatearly pregnancy changes in uterine and spiral artery blood flowoccur by different mechanisms, and that when investigating uterineblood flow in early pregnancy, studies need to begin by week6 and need to be performed at least biweekly.     

Temporary uterine artery occlusion for treatment of menorrhagia and uterine fibroids using an incisionless Doppler-guided transvaginal clamp: case report

We report the successful treatment of a 43-year-old woman with menorrhagia and multiple uterine fibroids by temporary uterine artery occlusion. Using a Doppler-guided transvaginal clamp, her uterine arteries were non-invasively identified and occluded by mechanical compression against the cervix for 6 h. Following removal of the clamp, blood flow in the uterine arteries returned immediately. Menorrhagia symptoms were tracked with the Ruta Menorrhagia Severity Scale. Uterine and fibroid volumes were measured by analysis of magnetic resonance images. The patient's self-reported menorrhagia symptoms were significantly reduced at 6 months (70% reduction in Ruta score) and both uterine volume and fibroid volume had decreased by more than 44% at 6 months. This case report illustrates the potential applicability of a simple-to-use, non-surgical device for the treatment of menorrhagia and uterine fibroids by temporary uterine artery occlusion.     

Pregnancy protects against antiangiogenic and fibrogenic effects of angiotensin II in rat hearts

Aim: To investigate the difference between physiological and pathological cardiac remodelling induced, respectively, by pregnancy and angiotensin (Ang) II, and to test the hypothesis that pregnancy protects against Ang II effects. Methods: Female Wistar rats, pregnant (n = 12) and non‐pregnant (n = 12), were implanted with mini‐pumps containing saline (sham) or 150 ng kg^?1 min^?1 Ang II. Ten days later echocardiography and blood pressure measurement were performed. Expression of 22 genes was assessed using RT‐PCR. Microscopic sections of LV were prepared to determine collagen content (Sirius Red staining), vessel density (β‐actin immunolabelling) and myocytes diameter (Toluidine Blue). Results: Heart weight (HW) was increased in Ang II treated groups compared with their controls. Furthermore, HW of Ang II treated pregnant rats (1.0 ± 0.03 g) was higher than that in non‐pregnant sham (0.7 ± 0.02 g), pregnant (0.8 ± 0.01 g) and Ang II treated non‐pregnant (0.8 ± 0.02 g) rats. Relative LV wall thickness showed similar pattern. Aortic pressure was significantly increased in Ang II groups. Collagen content was increased in Ang II (4.0 ± 0.5%) compared with sham (1.5 ± 0.1%) but reduced again when treated rats were pregnant (2.8 ± 0.4%). Vessel density was reduced by 47.8% after Ang II treatment in non‐pregnant and by only 13.9% in pregnant rats. Gene expression analysis showed increased expression of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), anykrin repeat domain‐containing protein 1 (Ankrd‐1), protein kinase C‐α and ‐δ and tumour suppressor gene TP53 (p53) in Ang II treated groups and upregulation of ANF, BNP and Ankrd‐1 remained when pregnancy was combined with Ang II. Pregnancy reduced expression of: α‐myosin heavy chain, tumour necrosis factor‐α, p53, endothelial nitric oxide synthase and inducible nitric oxide synthase. Conclusion: Pregnancy seems to counteract the detrimental effects of Ang II on fibrosis and angiogenesis in heart. In addition, pregnancy and Ang II lead to partly opposite changes in the expression of some genes important for heart function.     

Physiology: Description of circadian rhythm in uterine artery blood flow during the peri-ovulatory period

Uterine artery blood flow was assessed by transvaginal colourand pulsed Doppler ultrasound prospectively in six women duringthe peri-ovulatory period. All patients had regular ovulatorymenstrual cycles and a mid-luteal serum progesterone consistentwith spontaneous ovulation in the preceding cycle. Each patientunderwent serial transvaginal ultrasound examination and Dopplerassessment of blood flow in the uterine arteries. When the meanfollicular diameter was >16 mm or day –2 from the estimatedday of ovulation was reached, patients were scanned at 6 hourlyintervals at 0600, 1200, 1800 and 2400 h until follicular rupture.The pulsatility index (PI) and time averaged maximum velocity(TAMX) were calculated as Doppler indices of impedance to bloodflow and velocity respectively. A venous blood sample was takenat each visit for subsequent hormonal analysis. The mean uterineartery PI showed a marked daily fluctuation with a nadir occurringmost commonly at 0600 h. A comparison between the mean PI valuesat 0600 and 1800 h showed significantly lower results at 0600h in both dominant (P < 0.05) and non-dominant (P < 0.02)uterine arteries. Furthermore, mean uterine artery TAMX showeddaily fluctuations with peak values most commonly occurringat 0600 h with the nadir occurring during the afternoon andlate evening. There was no temporal relationship between thefluctuations in PI and changes in luteinizing hormone, folliclestimulating hormone, oestradiol or progesterone concentrations.These observations suggest that there is a circadian rhythmin uterine artery blood flow during the peri-ovulatory periodwhich appears to be independent from the hormonal changes.     

Pulsatility index of uterine artery in pregnant and non-pregnant women

In 60 infertile women, 73 uterine arterial pulsatility indices(PI) were measured by transvaginal colour Doppler sonography.The aims were to assess uterine perfusion response in infertilewomen during spontaneous ovarian cycles, and to analyse thechange of uterine perfusion in pregnant cases. The mean PI values(± SD) of 67 non-pregnant cycles were 2.30 (±0.78) in the follicular phase, 2.51 (± 1.05) in the ovulatoryphase and 2.50 (± 0.97) in the mid-luteal phase. Themean PI values (± SD) of six pregnant cycles were 1.67(± 0.22), 1.89 (± 0.41) and 2.23 (± 0.69)in the corresponding phases respectively. The difference betweenthe PI values in the follicular phase for the pregnant and non-pregnantgroups was significant (P < 0.05), as well as that betweenthe follicular and mid-luteal phase for the pregnant group (P< 0.05). There was no significant difference between thePI values in the ovulatory or mid-luteal phase of the two groups.     

Diagnostic techniques: Intra-observer, interobserver, interultrasound transducer and intercycle variation in colour Doppler assessment of uterine artery impedance

In this study we assessed the variability of measuring uterineartery pulsatility index by colour Doppler ultrasound usingdifferent ultrasound transducers (abdominal 3 MHz, abdominal5 MHz, endovaginal 5 MHz with and without a full bladder). Wethen assessed the intra-observer coefficient of variation forthe different transducers and interobserver variability fortwo observers using the endovaginal probe. The intercycle variabilityand effects of a full bladder on uterine artery blood flow werealso assessed. The uterine vessels were most easily identifiedusing the endovaginal transducer. The most reproducible resultswere obtained using the endovaginal probe. The coefficientsof variation obtained for the vaginal route were intra-observer4.1%, interobserver 11.8% and intercycle 16.5%. The blood flowindices were significantly different between the endovaginaland both transabdominal 5 and 3 MHz probes (P = 0.0027 and 0.0005respectively). There was no significant effect of a full bladderon pulsatility index. This study suggests that transvaginalcolour Doppler ultrasound is reproducible and the results obtainedfrom clinical use would be reliable.     

Prostanoid receptors in human uterine myocytes: the effect of reproductive state and stretch

In the human, prostanoids are known to be important mediators of uterine relaxation and contraction during pregnancy and parturition. We have previously shown that stretch of uterine smooth muscle cells increased prostaglandin H synthase 2 (PGHS-2) mRNA expression, PGHS-2 peptide synthesis and activity, however, the net effect on uterine contractility of this increase in prostaglandin synthesis would be determined by the expression of the different prostanoid receptors. Therefore, the aims of this study were to establish the expression of prostanoid receptor mRNA in uterine myocytes obtained from women in different reproductive states and to test the hypothesis that stretch of uterine myocytes alters prostanoid receptor mRNA expression to promote uterine contractility. Myocytes were isolated from women undergoing hysterectomy (NP) and pregnant women undergoing LSCS either before (NL) or after the onset of labour (L) and were subjected to 11% stretch for 1 h (n = 6 in all cases). Copy numbers of the individual receptors varied widely with reproductive state but followed the pattern: FP > IP = DP = EP-4 > TP = EP-3 = EP-2 > EP-1. FP mRNA expression was significantly lower in the NL group compared to the NP group and EP-3, EP-4 and TP mRNA expression was significantly lower in both NL and L groups compared to NP group levels. The level of mRNA expression of EP-1, EP-2, DP and IP did not differ between NP, NL and L groups. Stretch of cells derived from the NP group resulted in a significant decrease in EP-4 mRNA expression alone and of the NL group a significant decrease in EP-2 mRNA expression alone. Stretch had no effect on cells derived from the L group. These data show that pregnancy is associated with a significant reduction in 3 of 4 pro-contraction associated prostanoid receptor mRNA expression and 1 of 4 pro-relaxant. Stretch elicited no consistent change in prostanoid receptor mRNA expression.     

Different action of angiotensin II and noradrenaline on cytosolic calcium concentration in isolated and perfused afferent arterioles

Compensatory, net fluid transfer across the capillaries was studied in the arm of man with plethysmographic technique during experimental hypovolaemia induced by lower body negative pressure (LBNP). Thirty, 60, and 110 cmH₂O LBNP evoked rapid transfer of fluid from tissue to blood at average rates of 0.053, 0.088 and 0.147 ml min^-1 100 ml^-1 soft tissue, i.e. graded responses typical for a true homeostatic regulation. Other experiments demonstrated a net fluid absorption not only from the arm but also from a wide range of skeletal muscle and skin regions in the body during experimental hypovolaemia, i.e. the more or less generalized response required if the absorption process is to contribute importantly to plasma volume regulation. In a third series of experiments it was shown that gradually applied LBNP was a much less efficient stimulus for fluid gain into the circulation than rapidly instituted LBNP, tentatively explaining the fairly slow plasma volume refill in man in previous literature after experimental, true and necessarily slow blood loss. Taken together, the findings described warrant the conclusion that the described process of fluid gain into the circulation may be a very important component in the overall homeostatic circulatory regulation in states of hypovolaemia. The data in fact suggest that the process might be capable of increasing plasma volume by as much as 600 ml within only 10 min, suggesting that such plasma volume control might be much more potent than previously believed.     

The importance of fibroblasts in remodelling of the human uterine cervix during pregnancy and parturition

It is well established that fibroblasts play a crucial role in pathophysiological extracellular matrix remodelling. The aim of this project is to elucidate their role in normal physiological remodelling. Specifically, the remodelling of the human cervix during pregnancy, resulting in an enabled passage of the child, is used as the model system. Fibroblast cultures were established from cervices of non-pregnant women, women after 36 weeks of pregnancy and women directly after partus. The cells were immunostained and quantified by western blots for differentiation markers. The cultures were screened for cytokine and metalloproteinase production and characterized by global proteome analysis. The cell cultures established from partal donors differ significantly from those from non-pregnant donors, which is in accordance with in vivo findings. A decrease in alpha-smooth actin and prolyl-4-hydroxylase and an increase in interleukin (IL)-6, IL-8 and matrix metalloproteinases (MMP)-1 and MMP-3 were observed in cultures from partal donors. 2D-gel electrophoresis followed by mass spectrometry showed that the expression of 59 proteins was changed significantly in cultures of partal donors. The regulated proteins are involved in protein kinase C signalling, Ca2+ binding, cytoskeletal organization, angiogenesis and degradation. Our data suggest that remodelling of the human cervix is orchestrated by fibroblasts, which are activated or recruited by the inflammatory processes occurring during the ripening cascade.