Chronic hypoxia, pregnancy, and endothelium-mediated relaxation in guinea pig uterine and thoracic arteries

Vasodilation that occurs during normal pregnancy is associated with enhanced relaxation and decreased contractile response to agonists, which are in part due to increased stimulated and basal nitric oxide (NO). In preeclampsia and/or pregnancies carried at high altitude (HA), this normal vascular adjustment is reversed or diminished. We previously reported that HA exposure did not inhibit the pregnancy-associated decrease in contractile response to agonist or basal NO in guinea pig uterine arteries (UA). We therefore sought to determine whether altitude interfered with effects of pregnancy on endothelium-dependent relaxation through a reduction in stimulated NO. We examined the relaxation response to ACh in UA and bradykinin in thoracic arteries (TA) and effects of NO inhibition with 200 microM N(G)-nitro-L-arginine (L-NNA) in arterial rings isolated from nonpregnant and pregnant guinea pigs exposed throughout gestation to low altitude (LA, 1,600 m, n = 26) or HA (3,962 m, n = 22). In pregnant UA, relaxation to ACh was enhanced (P < 0.05) at both altitudes and NO inhibition diminished, but did not reverse, ACh relaxation. The effect of L-NNA on the relaxation response to ACh was less in HA than in LA animals (P = 0.0021). In nonpregnant UA, relaxation to ACh was similar in LA and HA animals. L-NNA reversed the relaxation response to ACh at HA but not at LA. In TA, relaxation to bradykinin was unaltered by pregnancy or altitude and was completely reversed by NO inhibition. These data suggest that effects of NO inhibition are diminished in UA during pregnancy at HA. Additional studies are needed to confirm whether these effects are mediated through inhibition of stimulated NO. HA exposure did not inhibit relaxation to ACh, perhaps because of stimulation of other vasodilators.     

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.     

Cortisol-mediated potentiation of uterine artery contractility: effect of pregnancy

During pregnancy, maternal plasma cortisol concentrations approximately double. Given that cortisol plays an important role in the regulation of vascular reactivity, the present study investigated the potential role of cortisol in potentiation of uterine artery (UA) contractility and tested the hypothesis that pregnancy downregulated the cortisol-mediated potentiation. In vitro cortisol treatment (3, 10, or 30 ng/ml for 24 h) produced a dose-dependent increase in norepinephrine (NE)-induced contractions in both nonpregnant and pregnant (138-143 days gestation) sheep UA. However, this cortisol-mediated response was significantly attenuated by approximately 50% in pregnant UA. The 11 beta-hydroxysteroid dehydrogenase (11-beta HSD) inhibitor carbenoxolone did not change the effect of cortisol in nonpregnant UA but abolished its effect in pregnant UA by increasing the NE pD(2) in control tissues from 6.20 +/- 0.05 to 6.59 +/- 0.11. The apparent dissociation constant value of NE alpha(1)-adrenoceptors was not changed by cortisol in pregnant UA but was decreased in nonpregnant UA. There was no difference in glucocorticoid receptor density between nonpregnant and pregnant UA. Cortisol significantly decreased endothelial nitric oxide (NO) synthase protein levels and NO release in both nonpregnant and pregnant UA, but the effect of cortisol was attenuated in pregnant UA by approximately 50%. Carbenoxolone alone had no effects on NO release in nonpregnant UA but was decreased in pregnant UA. These results suggest that cortisol potentiates NE-mediated contractions by decreasing NO release and increasing NE-binding affinity to alpha(1)-adrenoceptors in nonpregnant UA. Pregnancy attenuates UA sensitivity to cortisol, which may be mediated by increasing type-2 11-beta HSD activity in UA.     

Cellular mechanisms of bradykinin-induced hyperpolarization in renal epitheloid MDCK-cells.

Previous studies have demonstrated that bradykinin hyperpolarizes the cell membrane of subconfluent MDCK cells by increase of the potassium conductance. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of bradykinin on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate diester (TPA). In untreated cells, bradykinin leads to a transient increase of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, increase of Cai, activation of potassium channels and hyperpolarization of the cell membrane. The effects of bradykinin on PD and Cai are still present in the absence of extracellular calcium. In cells pretreated with pertussis toxin the effect of bradykinin on inositol trisphosphate formation is almost abolished but bradykinin still leads to a transient increase of Cai and PD in the presence and absence of extracellular calcium. In cells pretreated with TPA the bradykinin-induced increase of inositol trisphosphate formation is blunted, the bradykinin-induced increase of Cai abolished, but the bradykinin-induced hyperpolarization still present. The observations indicate that bradykinin increases Cai in part by phorbol ester and pertussis toxin sensitive activation of phospholipase C. In addition, bradykinin is capable of enhancing Cai by utilizing pertussis toxin insensitive mechanisms. Furthermore, bradykinin is able to transiently enhance the potassium conductance without a general increase of intracellular calcium.     

Bradykinin stimulates GTP hydrolysis in NG108-15 membranes by a high-affinity, pertussis toxin-insensitive GTPase

In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells.     

TNF-alpha enhances contraction and inhibits endothelial NO-cGMP relaxation in systemic vessels of pregnant rats

Tumor necrosis factor-alpha (TNF-alpha) is elevated in the plasma of preeclamptic women and may have a role in pregnancy-induced hypertension. However, whether the hemodynamic effects of TNF-alpha reflect the direct effects on vascular reactivity is unclear. We tested the hypothesis that TNF-alpha impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. We measured isometric contraction in aortic strips isolated from virgin and pregnant Sprague-Dawley rats (nontreated vs. treated for 2 h with 10-1,000 pg/ml TNF-alpha). In endothelium-intact vascular strips, TNF-alpha caused greater enhancement of phenylephrine (Phe) contraction in pregnant than virgin rats. TNF-alpha caused significant inhibition of ACh- and bradykinin-induced vascular relaxation and nitrite/nitrate production that were more prominent in pregnant than virgin rats. N(G)-nitro-L-arginine methyl ester [L-NAME, 100 microM, an inhibitor of nitric oxide (NO) synthase] or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 1 microM, an inhibitor of cGMP production in smooth muscle) inhibited ACh relaxation and enhanced Phe contraction in nontreated but to a lesser extent in TNF-alpha-treated vessels, particularly those of pregnant rats. Endothelium removal enhanced Phe contraction in nontreated but not TNF-alpha-treated vessels, especially those of pregnant rats. Relaxation of Phe contraction with the NO donor sodium nitroprusside was not different between nontreated and TNF-alpha-treated vessels. Thus TNF-alpha enhances vascular contraction and inhibits endothelium-dependent NO-cGMP-mediated vascular relaxation in systemic vessels, particularly those of pregnant rats. The results support a direct role for TNF-alpha as a possible mediator of increased vascular resistance associated with pregnancy-induced hypertension.     

Pregnancy and estradiol decrease GTPase activity in the guinea pig uterine artery

The mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner are poorly understood. We propose that it is consequential to estrogen-mediated alterations in G protein-mediated signal transduction. Aortas and uterine (UAs) and mesenteric arteries (MAs) were obtained from late-pregnant, nonpregnant, or ovariectomized guinea pigs chronically treated with 17beta-estradiol. High-affinity GTPase activity was assayed enzymatically. The cGMP generated in response to the endothelium-dependent agonist ACh was measured in UAs incubated with or without cholera toxin (CTX, which inhibits G(s)alpha). Pregnancy significantly decreased UA but not aorta or MA GTPase activity. 17beta-Estradiol decreased UA GTPase activity compared with untreated ovariectomized animals. ACh increased cGMP in pregnant but not nonpregnant UAs. Pretreatment of nonpregnant UAs with CTX increased ACh-induced cGMP levels similar to pregnancy. Thus pregnancy and estradiol decrease the GTPase activity of a CTX-sensitive G protein in UAs, increasing receptor-dependent cGMP release. This alteration in receptor-mediated G protein coupling in UAs may contribute to the characteristic cardiovascular adaptation to pregnancy.     

Effect of cortisol on norepinephrine-mediated contractions in ovine uterine arteries

Cortisol potentiated norepinephrine (NE)-mediated contractions in ovine uterine arteries (UA). We tested the hypothesis that cortisol regulated alpha(1)-adrenoceptor-mediated pharmacomechanical coupling differentially in nonpregnant UA (NUA) and pregnant UA (PUA). Cortisol (10 ng/ml for 24 h) significantly increased contractile coupling efficiency of alpha(1)-adrenoceptors in NUA, but increased alpha(1)-adrenoceptor density in PUA. Cortisol potentiated NE-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] synthesis in both NUA and PUA, but increased coupling efficiency of alpha(1)-adrenoceptors to Ins(1,4,5)P(3) synthesis only in NUA. Carbenoxolone alone did not affect NE-mediated Ins(1,4,5)P(3) production, but significantly enhanced cortisol-mediated potentiation of NE-stimulated Ins(1,4,5)P(3) synthesis in PUA. In addition, cortisol potentiated the NE-induced increase in Ca(2+) concentration in PUA, but increased NE-mediated contraction for a given amount of Ca(2+) concentration in NUA. Collectively, the results indicate that cortisol potentiates NE-mediated contractions differentially in NUA and PUA, i.e., by upregulating alpha(1)-adrenoceptor density leading to increased Ca(2+) mobilization in PUA while increasing alpha(1)-adrenoceptor coupling efficiency and myofilament Ca(2+) sensitivity in NUA. In addition, the results suggest that pregnancy increases type 2 11 beta-hydroxysteroid dehydrogenase activity in the UA.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Evidence for microvascular dysfunction after prenatal dexamethasone at 0.7, 0.75, and 0.8 gestation in sheep

Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM (n = 7) or saline (n = 7) was given starting at 103 days of gestation (dGA; term approximately 149 days). Fetal femoral arteries (approximately 300-microm internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health.     

Chronic hypoxia augments uterine artery distensibility and alters the circumferential wall stress-strain relationship during pregnancy.

Pregnancy-associated increases in uterine artery (UA) blood flow are due, in part, to vasoactive and growth-related changes that enlarge UA diameter. Although active and passive mechanical factors can contribute to this enlargement, their role is less well understood. We hypothesized that pregnancy increased UA distensibility and/or decreased myogenic tone. Given the fetal growth restriction and lower UA flow seen under chronic hypoxia, we further hypothesized that chronic hypoxia opposed these normal active and passive mechanical changes. UA were isolated from 12 nonpregnant and 12 pregnant (0.7 gestation) guinea pigs housed under normoxia or chronic hypoxia (3,960 m) and studied by pressure myography. Pregnancy increased UA diameter similarly under normoxia and hypoxia. Although chronic hypoxia raised resting tone in UA from nonpregnant guinea pigs to approximately 20% and tone was greater in preconstricted pregnant chronically hypoxic vs. normoxic UA (both P<0.01), there was an absence of myogenic response (i.e., an increase in tone with rising pressure) in all groups. Pregnancy increased UA distensibility 1.5-fold but did not change stiffness or the stress-strain relationship. Compared with vessels from normoxic pregnant animals, hypoxic pregnancy raised UA distensibility fourfold, decreased stiffness (rate constant b=3.80+/-1.06 vs. 8.92+/-1.25, respectively, P<0.01), lowered elastin by 50%, and shifted the stress-strain relationship upward such that four times as much strain was present at a given stress. We concluded that increased distensibility and low myogenic tone contribute to enlarging UA diameter and raising UA blood flow during pregnancy. Chronic hypoxia exaggerates the rise in distensibility and alters the stress-strain relationship in ways that may provoke vascular injury.     

Effects of pregnancy and chronic hypoxia on contractile responsiveness to alpha 1-adrenergic stimulation

Decreasedcontractile response to vasoconstrictors in uterine and nonuterinevessels contributes to increased blood flow to the uterine circulationduring normal pregnancy. Pregnancies complicated by preeclampsiaand/or chronic hypoxia show a reversal or diminution of thesepregnancy-associated changes. We sought to determine whether chronichypoxia opposes the reduction in contractile response in uterine andnonuterine vessels during normal pregnancy and, if so, whetherdecreased basal nitric oxide (NO) activity was involved. We examinedthe contractile response to phenylephrine (PE) in guinea pig uterineartery (UA), mesenteric artery (MA), and thoracic aorta (TA) ringsisolated from nonpregnant or pregnant guinea pigs that had been exposedthroughout gestation to either low (1,600 m,n = 47) or high (3,962 m,n = 43) altitude. In the UA, pregnancyreduced contractile sensitivity to PE and did so similarly at low andhigh altitude (EC₅₀: 4.0 × 10⁸ nonpregnant, 9.3 × 10⁸ pregnant at lowaltitude; 4.8 × 10⁸nonpregnant, 1.0 ×10⁸pregnant at high altitude; both P < 0.05). Addition of the NO synthase inhibitornitro-L-arginine (NLA; 200 mM)to the vessel bath increased contractile sensitivity in the pregnant UA(P < 0.05) and eliminated the effectof pregnancy at both altitutes. NLA also raised contractile sensitivityin the nonpregnant high-altitude UA, but contractile response withoutNLA did not differ in the high- and low-altitude animals. In the MA,pregnancy decreased contractile sensitivity to PE at high altitudeonly, and this shift was reversed by NO inhibition. In the TA, neitherpregnancy nor altitude affected contractile response, but NO inhibition raised contractile response in nonpregnant and pregnant TA at bothaltitudes. We concluded that pregnancy diminished contractile responseto PE in the UA, likely as a result of increased NO activity, and thatthese changes were similar at low and high altitude. Counter to ourhypothesis, chronic hypoxia did not diminish the pregnancy-associatedreduction in contractile sensitivity to PE or inhibit basal NO activityin the UA; rather it enhanced, not diminished, basal NO activity in thenonpregnant UA and the pregnant MA.

     

Bradykinin –induced vasodilatation: Role of age,ACE1-inhibitory peptide,mas- and bradykinin receptors

Bradykinin exerts its vascular actions via two types of receptors, the non-constitutively expressed bradykinin receptor type 1 (BR1) and the constitutive type 2 receptor (BR2). Bradykinin-induced vasorelaxation is age-dependent, a phenomenon related to the varying amounts of BR1 and BR2 in the vasculature.Isoleucine-proline-proline (Ile-Pro-Pro), a bioactive tripeptide, lowers elevated blood pressure and improves impaired endothelium-dependent vasorelaxation in hypertensive rats. It inhibits angiotensin converting enzyme 1 (ACE1). Other mechanisms of action have also been postulated.The aims of the study were to clarify the underlying mechanisms of the age-dependency of bradykinin-induced vasodilatation such as the roles of the two bradykinin receptors, the mas-receptor and synergism with Ile-Pro-Pro.The vascular response studies were conducted using mesenteric artery and aorta rings from normotensive 6 wk. (young) and 22 wk. (old) Wistar rats. Cumulative dosing of acetylcholine, bradykinin and angiotensin(1–7) (Ang(1–7))were tested in phenylephrine-induced vasoconstriction with or without 10 min pre-incubation with antagonists against BR1-, BR2- or mas-receptors, Ang(1–7) or ACE1-inhibitors captopril and Ile-Pro-Pro.The bradykinin-induced vasorelaxation in vitro was age-dependent and it was improved by pre-incubation with Ile-Pro-Pro, especially in old rats with endothelial dysfunction. The mas-receptor antagonist, D-Pro₇-Ang(1–7) abolished bradykinin-induced relaxation totally. Interestingly, BR1 and BR2 antagonists only slightly reduced bradykinin-induced vasorelaxation, as an evidence for the involvement of other mechanisms in addition to receptor activation.In conclusion, bradykinin-induced vasorelaxation was age-dependent and Ile-Pro-Pro improved it. Mas receptor antagonist abolished relaxation while bradykinin receptor antagonist only slightly reduced it, suggesting that bradykinin-induced vasorelaxation is regulated also by other mechanisms than the classical BR1/BR2 pathway.     

Regulation of baseline Ca2+ sensitivity in permeabilized uterine arteries: effect of pregnancy

The adaptation of contractile mechanisms of the uterine artery to pregnancy is not fully understood. The present study examined the effect of pregnancy on the uterine artery baseline Ca2+ sensitivity. In beta-escin-permeabilized arterial preparations, Ca2+ -induced concentration-dependent contractions were significantly decreased in uterine arteries from pregnant animals compared with those of nonpregnant animals. Time-course studies showed that Ca2+ increased phosphorylation of 20-kDa myosin light chain (MLC20), which preceded the tension development in vessels from both pregnant and nonpregnant animals. When compared with vessels from nonpregnant animals, there was a significant increase in the protein level of MLC20 and an accordance increase in the level of Ca2+ -induced phosphorylated MLC20 (MLC20-P) in uterine arteries during pregnancy. Simultaneous measurements of MCL20-P levels and contractions stimulated with Ca2+ in the same tissues demonstrated a significant attenuation in the tension-to-MLC20-P ratio in uterine arteries during pregnancy. Activation of PKC with phorbol 12,13-dibutyrate (PDBu) potentiated Ca2+ -induced contractions in uterine arteries from nonpregnant but not pregnant animals. Accordingly, inhibition of PKC attenuated Ca2+ -induced contractions in uterine arteries from nonpregnant but not pregnant animals. PDBu produced contractions in the presence or absence of Ca2+ in the beta-escin-permeabilized arteries, which were significantly decreased in uterine arteries from pregnant compared with nonpregnant animals. The results suggest that pregnancy upregulates the thick-filament regulatory pathway by increasing MLC20 phosphorylation but downregulates the thin-filament regulatory pathway by decreasing the contractile sensitivity of MLC20-P, resulting in attenuated baseline Ca2+ sensitivity in the uterine artery. In addition, PKC plays an important role in the regulation of basal Ca2+ sensitivity, which is downregulated during pregnancy.     

Adaptation of uterine artery thick- and thin-filament regulatory pathways to pregnancy

Little is known about the adaptation of uterine artery smooth muscle contractile mechanisms to pregnancy. The present study tested the hypothesis that pregnancy differentially regulates thick- and thin-filament regulatory pathways in uterine arteries. Isometric tension, intracellular free Ca(2+) concentration, and phosphorylation of 20-kDa myosin light chain (MLC(20)) were measured simultaneously in uterine arteries isolated from nonpregnant and near-term (140 days gestation) pregnant sheep. Phenylephrine-mediated intracellular free Ca(2+) concentration, MLC(20) phosphorylation, and contraction tension were significantly increased in uterine arteries of pregnant compared with nonpregnant animals. In contrast, phenylephrine-mediated Ca(2+) sensitivity of MLC(20) phosphorylation was decreased in the uterine arteries of pregnant sheep. Simultaneous measurement of phenylephrine-stimulated tension and MLC(20) phosphorylation in the same tissue indicated a decrease in MLC(20) phosphorylation-independent contractions in the uterine arteries of pregnant sheep. In addition, activation of PKC produced significantly lower sustained contractions in uterine arteries of pregnant compared with nonpregnant animals in the absence of changes in MLC(20) phosphorylation levels in either vessels. In uterine arteries of nonpregnant sheep, the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitor PD-098059 significantly increased phenylephrine-mediated, MLC(20) phosphorylation-independent contractions. The results suggest that in uterine arteries, pregnancy upregulates alpha(1)-adrenoceptor-mediated Ca(2+) mobilization and MLC(20) phosphorylation. In contrast, pregnancy downregulates the Ca(2+) sensitivity of myofilaments, which is mediated by both thick- and thin-filament pathways.     

Bradykinin stimulates a rise in cytosolic calcium in renal glomerular mesangial cells via a pertussis toxin insensitive pathway

Bradykinin elicits a complex response in the renal glomerulus which includes a reduction in the glomerular capillary ultrafiltration coefficient. To elucidate the biochemical mechanism of this response, we investigated calcium signalling in rat renal glomerular mesangial cells in culture using the calcium-sensitive fluorescent dye, Indo-1. Bradykinin was found to cause a concentration-dependent transient rise in cytosolic free calcium followed by a sustained slower secondary rise. The bradykinin response persisted with acute removal of extracellular calcium using EGTA, indicating that calcium entry from outside the cell did not mediate this primary response. Prolonged exposure to EGTA, which reduced intracellular stores, eliminated the calcium response to bradykinin but not to vasopressin, indicating differential sensitivity to intracellular calcium stores of these two hormonal responses. In agreement, prior stimulation with vasopressin significantly attenuated the response to bradykinin, but the converse did not occur. Aluminum fluoride and pertussis toxin were used to investigate the possible involvement of a guanyl nucleotide regulatory protein in signal transduction. Aluminum fluoride induced a transient rise in cytosolic calcium that was abrogated by prior exposure of the cells to pertussis toxin. This demonstrates the effectiveness of pertussis toxin and the presence of a calcium-signalling pathway susceptible to pertussis toxin in these cells. In contrast, the responses to bradykinin and vasopressin were unaffected by pertussis toxin. We conclude that bradykinin stimulates release of calcium from intracellular stores in glomerular mesangial cells via a pertussis toxin insensitive pathway. This mesangial response provides a direct biochemical basis for the bradykinin-induced fall in glomerular capillary ultrafiltration coefficient which has been observed in vivo.     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms involved in calcitonin gene-related Peptide-induced relaxation in pregnant rat uterine artery

Human and rodent studies have demonstrated that calcitonin gene-related peptide (CGRP), a potent vasodilator, relaxes uterine tissue during pregnancy but not during labor. The vascular sensitivity to CGRP is enhanced during pregnancy, compared to nonpregnant human uterine arteries. In the present study, we hypothesized that uterine artery relaxation effects of CGRP are enhanced in pregnant rats compared to nonpregnant diestrus rats (NP-DE) and that several secondary messenger systems are involved in this process. We also hypothesized that the expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP1), and CGRP-B receptors are greater in pregnant rats. For vascular relaxation studies, uterine arteries from either NP-DE or Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP-A and CGRP-B receptor expressions were assessed by RT-PCR and Western immunoblotting, respectively. CGRP (10(-10)--10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in both NP-DE and Day 18 pregnant rat uterine arteries. Pregnancy increased the vasodilator sensitivity to CGRP significantly (P < 0.05) compared to NP-DE rats. CGRP receptor antagonist, CGRP8-37, inhibited CGRP-induced relaxation of pregnant uterine arteries. The CGRP-induced relaxation was not affected by NG-nitro-l-arginine methyl ester (L-NAME) (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by inhibitors of guanylate cyclase (ODQ, 10(-5) M) and adenylate cyclase (SQ 22536, 10(-5) M). CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by potassium channel blockers KATP (glybenclamide, 10(-5) M) and K(CA) (tetraethylammonium, 10(-3) M). The expression of CRLR and RAMP1 was significantly (P < 0.05) elevated during pregnancy compared to nonpregnant diestrus state (NP-DE). However, CGRP-B receptor proteins in uterine arteries were not altered with pregnancy compared to those of NP-DE. These studies suggest that CGRP-induced increases in uterine artery relaxation may play a role in regulating blood flow to the uterus during pregnancy and, therefore, in fetal growth and survival.     

Pregnancy alters hemodynamic responses to hemorrhage in conscious rabbits

Pregnant animals are less able to maintain mean arterial pressure (MAP) during hemorrhage compared with nonpregnant animals, but the hemodynamic basis of this difference is unknown. The hypothesis that pregnancy attenuates responses of cardiac output, as well as total peripheral resistance (TPR) and femoral conductance, to hemorrhage was tested in conscious rabbits in both the pregnant and nonpregnant state (n = 10). During continuous slow blood loss (2% of the initial blood volume per minute), MAP was maintained initially in both groups. However, MAP then abruptly decreased to <45 mmHg in all animals after a smaller percentage of the initial blood volume was removed in pregnant compared with nonpregnant rabbits (43.6 +/- 1.7%, nonpregnant; 29.6 +/- 2.2%, pregnant; P < 0.005). The more rapid transition to hypotension exhibited by pregnant rabbits was associated with greater initial falls in cardiac output (-56 +/- 10 ml/min, nonpregnant; -216 +/- 33 ml/min, pregnant; P < 0.005) and stroke volume (0.8 +/- 0.1 ml/beat, nonpregnant; -1.3 +/- 0.1 ml/beat, pregnant; P < 0.05). In addition, the increase in TPR as a function of the decrease in cardiac output was markedly attenuated (P < 0.0001) during pregnancy. Whereas femoral conductance decreased in nonpregnant rabbits, it did not change significantly in pregnant animals. In conclusion, the lesser ability of conscious pregnant rabbits to maintain MAP during hemorrhage is due largely to a greater decrease in cardiac output but also to inadequate reflex increases in TPR, possibly in part in the femoral vascular bed.     

Relations between contractile responses and beta-adrenoceptors in gastric fundus of diabetic rats.

Contractile responses to norepinephrine (NE), and the population of beta-adrenoceptors, were determined in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin (STZ), and age-matched controls. Relaxation and/or contraction of fundus strips of controls and diabetics were induced by 10(-5)M NE. Responses to NE were mainly relaxation in gastric fundus isolated from controls, and contraction in fundus isolated from diabetics. Contraction was blocked by 10(-8) M prazosin and relaxation was blocked by 10(-6) M propranolol. Relaxation by isoproterenol of contraction induced by 10(-6) M acetylcholine was significantly less in fundus from diabetics than in that from controls. The number of beta-adrenoceptors, measured with [125I] iodocyanopindolol as a ligand, was significantly less in gastric fundus membrane isolated from diabetics than in that from controls, but affinity was no different. The level of plasma catecholamine was higher in diabetics than in controls. Results suggest that depression of gastric fundus relaxation and increase of contraction by NE in diabetics could be due to fewer beta-adrenoceptor binding sites caused by down-regulation by higher catecholamine level in diabetic rats.