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Maternal mortality rates are at an all-time high across the world and are set to increase in subsequent years. Cardiovascular disease is the leading cause of death during pregnancy and postpartum, especially in the United States. Therefore, understanding the physiological changes in the cardiovascular system during normal pregnancy is necessary to understand disease-related pathology. Significant systemic and cardiovascular physiological changes occur during pregnancy that are essential for supporting the maternal-fetal dyad. The physiological impact of pregnancy on the cardiovascular system has been examined in both experimental animal models and in humans. However, there is a continued need in this field of study to provide increased rigor and reproducibility. Therefore, these guidelines aim to provide information regarding best practices and recommendations to accurately and rigorously measure cardiovascular physiology during normal and cardiovascular disease-complicated pregnancies in human and animal models. [ABSTRACT FROM AUTHOR]

In light of evidence that immature arteries contain a higher proportion of noncontractile smooth muscle cells than found in fully differentiated mature arteries, the present study explored the hypothesis that age-related differences in the smooth muscle phenotype contribute to age-related differences in contractility. Because [Ca.sup.2+] handling differs markedly between contractile and noncontractile smooth muscle, the present study specifically tested the hypothesis that the relative contributions of [Ca.sup.2+] influx and myofilament sensitization to myogenic tone are upregulated, whereas [Ca.sup.2+] release is downregulated, in immature [14 days postnatal (P14)] compared with mature (6 mo old) rat middle cerebral arteries (MCAs). Myofilament [Ca.sup.2+] sensitivity measured in [beta]-escin-permeabilized arteries increased with pressure in P14 but not adult MCAs. Cyclopiazonic acid (an inhibitor of [Ca.sup.2+] release from the sarcoplasmic reticuhim) increased diameter and reduced [Ca.sup.2+] in adult MCAs but increased diameter with no apparent change in [Ca.sup.2+] in P14 MCAs. [La.sup.3+] ([Ca.sup.2+] influx inhibitor) increased diameter and decreased [Ca.sup.2+] in adult MCAs, but in P14 MCAs, [La.sup.3+] increased diameter with no apparent change in [Ca.sup.2+]. After treatment with both [La.sup.3+] and CPA, diameters were passive in both adult and P14 MCAs, but [Ca.sup.2+] was decreased only in adult MCAs. To quantify the fraction of smooth muscle cells in the fully differentiated contractile phenotype, extents of colocalization between smooth muscle [alpha]-actin and SM2 myosin heavy chain were determined and found to be at least twofold greater in adult than pup MCAs. These data suggest that compared with adult MCAs, pup MCAs contain a greater proportion of noncontractile smooth muscle and, as a consequence, rely more on myofilament [Ca.sup.2+] sensitization and [Ca.sup.2+] influx to maintain myogenic reactivity. The inability of [La.sup.3+] to reduce cytosolic [Ca.sup.2+] in the pup MCA appears due to [La.sup.3+]-insensitive noncontractile smooth muscle cells, which contribute to the spatially averaged measurements of [Ca.sup.2+] but not contraction. cerebral arteries; myogenic reactivity; myofilament calcium sensitivity; postnatal maturation; smooth muscle [alpha]-actin; smooth muscle phenotype; SM2 myosin heavy chain doi: 10.1152/ajpheart.00214.2010.

In the developing fetus, cerebral artery (CA) contractility demonstrates significant functional differences from that of the adult. This may be a consequence of differential activities of [[alpha].sub.1]-adrenergic receptor ([[alpha].sub.1]-AR) subtypes. Thus we tested the hypothesis that maturational differences in adrenergic-mediated CA contractility are, in part, a consequence of differential expression and/or activities of [[alpha].sub.1]-AR subtypes. In CA from fetal (~140 days) and nonpregnant adult sheep, we used wire myography and imaging, with simultaneous measurement of tension and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), radio-immunoassay, and Western immunoblots to examine phenylephrine (Phe)-induced contractile responses. [The [alpha].sub.1A]-AR antagonists (5-MU and WB-4101) completely inhibited Phe-induced contraction in adult but not fetal CA; however, [[[Ca.sup.2+]].sub.i] increase was reduced significantly in both age groups. The [alpha].sub.1D]-AR antagonist (BMY-7378) blocked both Phe-induced contractions and [Ca.sup.2+] responses to a significantly greater extent in adult compared with fetal CA. In both age groups, inhibition of [[alpha].sub.1A]-AR and [[alpha].sub.1B]-AR, but not [[alpha].sub.1D]-AR, significantly reduced inositol 1,4,5-trisphosphate responses to Phe. Western immunoblots demonstrated that the [[alpha].sub.1]-AR subtype expression was only ~20% in fetal CA compared with the adult. Moreover, in fetal CA, the [[alpha].sub.1D]-AR was expressed significantly greater than the other two subtypes. Also, in fetal but not adult CA, Phe induced a significant increase in activated ERK1/2; this increase in phosphorylated ERK was blocked by [[alpha].sub.1B]-AR (CEC) and [[alpha].sub.1D]-AR (BMY-7378) inhibitors, but not by [[alpha].sub.1A]-AR inhibitors (5-MU or WB-4101). In conclusion, in the fetal CA, [[alpha].sub.1B]-AR and [[alpha].sub.1D]-AR subtypes play a key role in contractile response as well as in ERK activation. We speculate that in fetal CA [[alpha].sub.1B]-AR and [[alpha].sub.1D]-AR subtypes may be a critical factor associated with cerebrovascular growth and function. vascular smooth muscle; development; maturation doi: 10.1152/ajpheart.00112.2010.

Goyal R, Mittal A, Chu N, Shi L, Zhang L, Longo LD. Maturation and the role of PKC-mediated contractility in ovine cerebral arteries. Am J Physiol Heart Circ Physiol 297: H2242-H2252, 2009. First published September 11, 2009; doi: 10.1152/ajpheart.00681.2009.--[Ca.sup.2+]- independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the [Ca.sup.2+]- independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular [Ca.sup.2+] concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries. protein kinase C-potentiated inhibitory protein of 17 kDa; development; extracellular signal-regulated kinase-1/2; RhoA; myosin light chain 20; caldesmon doi: 10.1152/ajpheart.00681.2009.

Chronic hypoxia during pregnancy has profound effects on uterine artery (UA) contractility and attenuates uterine blood flow. The present study tested the hypothesis that chronic hypoxia inhibits the pregnancy-induced reduction in pressure-dependent myogenic tone of resistance-sized UAs. UAs were isolated from nonpregnant ewes (NPUAs) and near-term pregnant ewes (PUAs) that had been maintained at sea level (~300 m) or at high altitude (3,801 m) for 110 days. In normoxic animals, the pressure-dependent myogenic response was significantly attenuated in PUAs compared with NPUAs. Hypoxia significantly increased myogenic tone in PUAs and abolished its difference between PUAs and NPUAs. Consistently, there was a significant increase in PKC-mediated baseline [Ca.sup.2+] sensitivity of PUAs in hypoxic animals. Hypoxia significantly increased phorbol 12,13-dibutyrate (PDBu)-induced contractions in PUAs but not in NPUAs. Whereas the inhibition of ERK1/2 by PD-98059 potentiated PDBu-mediated contractions of PUAs in normoxic animals, it failed to do so in hypoxic animals. Hypoxia decreased ERK1/2 expression in PUAs. PDBu induced membrane translocation of PKC-[alpha] and PKC-[epsilon]. Whereas there were no significant differences in PKC-[alpha] translocation among all groups, the translocation of PKC-[epsilon] was significantly enhanced in NPUAs compared with PUAs in normoxic animals, and hypoxia significantly increased PKC-[epsilon] translocation in PUAs. In the presence of PD-98059, there were no significant differences in PDBu-induced PKC-e translocation among all groups. Treatment of PUAs isolated from normoxic animals with 10.5% [O.sub.2] for 48 h ex vivo significantly increased PDBu-induced contractions and eliminated its difference between PUAs and NPUAs. The results suggest that hypoxia upregulates pressure-dependent myogenic tone through its direct effect in suppressing ERK1/2 activity and increasing the PKC signal pathway, leading to an increase in the [Ca.sup.2+] sensitivity of the myogenic mechanism in the UA during pregnancy. hypoxia; protein kinase C

Pre-natal cocaine exposure in rats resulted in decreased PKC[epsilon] protein expression in the heart of adult male but not female offspring. The present study determined its functional consequence of inhibiting cardioprotection mediated by ischemic preconditioning. Pregnant Sprague-Dawley rats were administered intraperitoneally saline or cocaine (30 mg x [kg.sup.-1] x [day.sup.-1]) from day 15 to day 21 of gestational age. Hearts were isolated from 3-mo-old offspring and were subjected to ischemia and reperfusion injury in a Langendorff preparation, with or without prior ischemic preconditioning. Preischemic values of left ventricular function were the same between the saline control and cocaine-treated animals. Ischemic preconditioning of two episodes of 5-min ischemia significantly decreased infarct size and enhanced postischemic functional recovery of the left ventricle in the saline control animals. This ischemic preconditioning was associated with increased phospho-PKC[epsilon], but not phospho-PKC[delta], levels and was blocked by a PKC[epsilon] translocation inhibitor peptide. Prenatal cocaine treatment abolished the ischemic preconditioning-mediated increase in phospho-PKC[epsilon] and cardioprotection in the heart of male offspring. In contrast, the cardioprotective effect was fully maintained in female offspring that were exposed to cocaine before birth. The results suggest that prenatal cocaine exposure causes a sex-specific loss of cardioprotection by ischemic preconditioning in adult offspring, which is most likely due to fetal programming of PKC[epsilon] gene repression, resulting in a downregulation of PKC[epsilon] function in the heart of adult male offspring. fetal programming; protein kinase C; ischemia

Charles SM, Zhang L, Longo LD, Buchholz JN, Pearce WJ. Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in [Ca.sup.2+] in rat cerebral arteries. Am J Physiol Regul Integr Comp Physiol 293: R737-R744, 2007. First published June 6, 2007; doi:10.1152/ajpregu.00869.2006.--Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([[[Ca.sup.2+]].sub.i]) were measured simultaneously in endothelium-denuded, fura-2-1oaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-too-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [[[Ca.sup.2+]].sub.i] was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [[[Ca.sup.2+]].sub.i]. However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilameut calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [[[Ca.sup.2+]].sub.i], rather than enhanced augmentation of myofilament calcium sensitivity. rat cerebral arteries; myofilament calcium sensitivity

Protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility and its adaptation to pregnancy. The present study tested the hypothesis that PKC differentially regulates [[alpha].sub.1]-adrenoceptor-mediated contractions of uterine arteries isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Phenylephrine-induced contractions of NPUA and PUA sheep were determined in the absence or presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). In NPUA sheep, PDBu produced a concentration-dependent potentiation of phenylephfine-induced contractions and shifted the dose-response curve to the left. In contrast, in PUA sheep, PDBu significantly inhibited phenylephrine-induced contractions and decreased their maximum response. Simultaneous measurement of contractions and intracellular free Ca2+ concentrations ([[Ca.sup.2+]].sub.i]) in the same tissues revealed that PDBu inhibited phenylephrine-induced [[Ca.sup.2+]].sub.i] and contractions in PUA sheep. In NPUA sheep, PDBu increased phenylephrine-induced contractions without changing [[Ca.sup.2+]].sub.i]. Western blot analysis showed six PKC isozymes, [alpha], [[beta].sub.1], [[beta].sub.II], [delta], [member of], and [zeta], in uterine arteries, among which [[beta].sub.I], [[beta].sub.II], and isozymes were significantly increased in PUA sheep. In contrast, PKC-[member of] was decreased in PUA sheep. In addition, analysis of subcellular distribution revealed a significant decrease in the particulate-to-cytosolic ratio of PKC-[member of] in PUA compared with that in NPUA sheep. The results suggest that pregnancy induces a reversal of PKC regulatory role on [[alpha].sub.1]-adrenoceptor-mediated contractions from a potentiation in NPUA sheep to an inhibition in PUA sheep. The differential expression of PKC isozymes and their subcellular distribution in uterine arteries appears to play an important role in the regulation of [Ca.sup.2+] mobilization and [Ca.sup.2+] sensitivity in [[alpha].sub.1]-adrenoceptor-mediated contractions and their adaptation to pregnancy. calcium mobilization; calcium sensitivity; adaptation; sheep

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 [Ca.sup.2+] sensitivity. In [beta]-escin-permeabilized arterial preparations, [Ca.sup.2+]-induced concentration-dependent contractions were significantly decreased in uterine arteries from pregnant animals compared with those of nonpregnant animals. Time-course studies showed that [Ca.sup.2+] increased phosphorylation of 20-kDa myosin light chain (ML[C.sub.20]), 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 ML[C.sub.20] and an accordance increase in the level of [Ca.sup.2+]-induced phosphorylated ML[C.sub.20] (ML[C.sub.20]-P) in uterine arteries during pregnancy. Simultaneous measurements of MC[L.sub.20]-P levels and contractions stimulated with [Ca.sup.2+] in the same tissues demonstrated a significant attenuation in the tension-to-ML[C.sup.20]-P ratio in uterine arteries during pregnancy. Activation of PKC with phorbol 12,13-dibutyrate (PDBu) potentiated [Ca.sup.2+]-induced contractions in uterine arteries from nonpregnant but not pregnant animals. Accordingly, inhibition of PKC attenuated [Ca.sup.2+]-induced contractions in uterine arteries from nonpregnant but not pregnant animals. PDBu produced contractions in the presence or absence of [Ca.sup.2+] 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 ML[C.sub.20] phosphorylation but down-regulates the thin-filament regulatory pathway by decreasing the contractile sensitivity of ML[C.sub.20]-P, resulting in attenuated baseline [Ca.sup.2+] sensitivity in the uterine artery. In addition, PKC plays an important role in the regulation of basal [Ca.sup.2+] sensitivity, which is downregulated during pregnancy. protein kinase C; thick filament; thin filament; myosin light chain; phosphorylation

The mechanisms of adaptation of uterine artery vascular tone to pregnancy are not fully understood. The present study tested the hypothesis that pregnancy decreases the PKC-mediated [Ca.sup.2+] sensitivity of the contractile process and attenuates myogenic tone in resistance-sized uterine arteries. In pressurized uterine arteries from nonpregnant (NPUA) and near-term pregnant (PUA) sheep, we measured, simultaneously in the same tissue, vascular diameter and vessel wall intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) as a function of intraluminal pressure. In both NPUA and PUA, membrane depolarization with KCl caused a rapid increase in [[[Ca.sup.2+]].sub.i] and a decrease in diameter. A pressure increase from 20 to 100 mmHg resulted in a transient increase in diameter that was associated with an increase in [[[Ca.sup.2+]].sub.i], followed by myogenic contractions in the absence of further changes in [[[Ca.sup.2+]].sub.i]. In addition, activation of PKC by phorbol 12,13-dibutyrate induced a decrease in diameter in the absence of changes in [[[Ca.sup.2+]].sub.i]. Pressure-dependent myogenic responses were significantly decreased in PUA compared with NPUA. However, pressure-induced increases in [[[Ca.sup.2+]].sub.i] were not significantly different between PUA and NPUA. The ratio of changes in diameter to changes in [[[Ca.sup.2+]].sub.i] was significantly greater for pressure-induced contraction of NPUA than that of PUA. Inhibition of PKC by calphostin C significantly attenuated the pressure-induced vascular tone and eliminated the difference of myogenic responses between NPUA and PUA. In contrast, the MAPKK (MEK) inhibitor PD-098059 had no effect on NPUA but significantly enhanced myogenic responses of PUA. In the presence of PD-098059, there was no difference in pressure-induced myogenic responses between NPUA and PUA. The results suggest that pregnancy down-regulates pressure-dependent myogenic tone of the uterine artery, which is partly due to increased MEK/ERK activity and decreased PKC signal pathway leading to a decrease in [Ca.sup.2+] sensitivity of myogenic mechanism in the uterine artery during pregnancy. sheep; protein kinase C; extracellular signal-regulated kinase

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([[Ca.sup.2+]]i) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 X [10.sup.-6] M] in the absence or presence of ERK1/2 inhibition (U-0126, [10.sup.-5] M). After PDBu [+ or -] ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, [caldesmon.sup.Ser789], myosin light [chain.sub.20] (ML[C.sub.20]), and CPI-17. PDBu induced significant increase in tension in the absence of increased [[Ca.sup.2+]]i. PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated [caldesmon.sup.Ser789] and ML[C.sub.20] levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 x [10.sup.-7] M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases ML[C.sub.20] phosphorylation in the thick filament pathway and increases [Ca.sup.2+] sensitivity. In addition, ERK1/ 2-dependent phosphorylation of [caldesmon.sup.Ser789] was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase. vascular smooth muscle; U-0126; caldesmon; CPI-17; myosin light chain 20

We previously demonstrated that ERK/PKC signaling pathways play a key role in regulation of C[a.sup.2+] sensitivity and contractility of the uterine artery. The present study tested the hypothesis that ERK and PKC differentially regulated myosin light chain phosphatase activity by phosphorylation of myosin phosphatase target protein-1 (MYPT-1) and CPI-17. Agonist-induced contractions and phosphorylation of MYPT-1/[Thr.sup.696]. MYPT-1/[Thr.sup.850] and CPI-17/[Thr.sup.38] were measured simultaneously in the same tissues of isolated near-term pregnant ovine uterine arteries. Phenylephrine produced time-dependent concurrent increases in the phosphorylation of ER[K.sub.44/42] and MYPT-1/[Thr.sup.850] that preceded contractions. In addition, phenylephrine induced phosphorylation of CPI-17/ [Thr.sup.38] that was concurrent with the contractions. In contrast, phenylephrine did not induce phosphorylation of MYPT-1/[Thr.sup.696] in the uterine artery. PD-098059 inhibited phosphorylation of ER[K.sub.44/42] and the initial peak phosphorylation of MYPT-1/[Thr.sup.850] but did not affect CPI-17/[Thr.sup.38] phosphorylation. Activation of PKC by phorbol 12,13-dibutyrate induced a time-dependent phosphorylation of CPI-17/[Thr.sup.38] that preceded contractions of the uterine artery. In addition, phorbol 12,13-dibutyrate activated PKC-[alpha] and induced a coimmuno-precipitation of PKC-[alpha] with caldesmon. The results suggest that phosphorylation of MYPT-1/[Thr.sup.850] and CPI-17/[Thr.sup.38] play important roles in regulation of agonist-mediated C[a.sup.2+] sensitivity in the uterine artery, in part by ERK and PKC. respectively. In addition, phosphorylated CPI-17 may regulate C[a.sup.2+] sensitivity by interacting with caldesmon and reversing its inhibitory effect on myosin ATPase. myosin light chain phosphatase; phenylephrine; calcium sensitivity; caldesmon

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.sup.2+] concentration, and phosphorylation of 20-kDa myosin light chain (ML[C.sub.20]) were measured simultaneously in uterine arteries isolated from nonpregnanl and near-term (140 days gestation) pregnant sheep. Phenylephrine-mediated intracellula, free [Ca.sup.2+] concentration, ML[C.sub.20] phosphorylation, and contraction tension were significantly increased in uterine arteries of pregnant compared with nonpregnant animals. In contrast, phenylephrine-mediated [Ca.sup.2+] sensitivity of MLC2o phosphorylation was decreased in the uterine arteries of pregnant sheep. Simultaneous measurement of phenylephrine-stimulated tension and ML[C.sub.20]. phosphorylalion in the same tissue indicated a decrease in ML[C.sub.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[C.sub.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. ML[C.sub.20] phosphorylation-independent contractions. The results suggest that in uterine arteries, pregnancy upregulates [[alpha].sub.1]-adrenoceptor-mediated [Ca.sup.2+] mobilization and ML[C.sub.20] phosphorylation. In contrast, pregnancy downregulates the [Ca.sup.2+] sensitivity of myofilaments, which is mediated by both thick- and thin-filament pathways. [[alpha].sub.1]-adrenoceptor; protein kinase C; calcium; myosin light chain phosphorylation; extracellular signal regulaled kinase

Fetal programming has profound effects on cardiovascular function in later adult life. We tested the hypothesis that chronic hypoxic exposure during fetal development downregulates endogenous cardioprotective mechanisms in adult rats. Time-dated pregnant rats were divided between normoxic and hypuxic (10.5% [O.sub.2] from days 15 to 21 of gestation) groups. The male progeny were studied at 2 mo of age. Rats were subjected to heat stress (42[degrees]C for 15 min). After 24 h, hearts were excised and subjected to 30 min of global ischemia and 1 h of reperfusion. Prenatal hypoxia did not change adult rat body weight and heart weight, but significantly increased the cross-sectional area of a left ventricular (LV) myocyte. Heat stress significantly improved postischemic recovery of LV function in normoxic control rats, but not in prenatally hypoxic rats. The infarct size in the LV resulting from ischemia-reperfusion was reduced by the heat stress pretreatment in control rats, but not in prenatally hypoxic rats. In accordance, heat stress significantly increased LV myocardial content of heat shock protein 70 only in normoxic control rats. In addition, there was a significant decrease in the LV myocardial content of the PKC-[member of] isoform in prenatally hypoxic rats compared with control rats. We conclude that prenatal hypoxia causes in utero programming of hsp70 gene in the LV, leading to an inhibition of its response to heat stress and a loss of cardioprotection in later adult life. fetal programming; heat shock protein 70; protein kinase C-[member of]; ischemia-reperfusion injury

We have demonstrated that extracellular signal-regulated kinase (ERK) plays an important role in the regulation of uterine artery contraction. The present study tested the hypothesis that ERK regulates thick and thin filament regulatory pathways in the uterine artery. Isometric tension, intracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), and 20-kDa myosin light chain (L[C.sub.20]) phosphorylation were measured simultaneously in uterine arteries isolated from near-term (140 days gestation) pregnant sheep. Phenylephrine produced time-dependent increases in [[[Ca.sup.2+]].sub.i] and L[C.sub.20] phosphorylation that preceded the contraction, which were inhibited by the MEK (ERK) inhibitor PD-098059. In addition, PD-098059 decreased the intercept of the regression line of L[C.sub.20] phosphorylation vs. [[[Ca.sup.2+]].sub.i] but increased the rate of tension development vs. L[C.sub.20] phosphorylation. In contrast to phenylephrine, phorbol 12,13-bibutyrate (PDBu) produced contractions without changing [[[Ca.sup.2+]].sub.i] or L[C.sub.20] phosphorylation. PD-098059 potentiated PDBu-induced contractions without affecting [[[Ca.sup.2+]].sub.i] and L[C.sub.20] phosphorylation. PDBu produced time-dependent increases in phosphorylation of p42 and p44 ERK and ERK-dependent phosphorylation of caldesmon at [Ser.sup.789] in the uterine artery. PD-098059 blocked PDBu-mediated phosphorylation of p42 and p44 ERK and caldesmon. The results indicate that ERK may regulate force by a dual regulation of thick and thin filaments in uterine artery smooth muscle. ERK potentiates the thick filament regulatory pathway by enhancing L[C.sub.20] phosphorylation via increases in [[[Ca.sup.2+]].sub.i] and [Ca.sup.2+] sensitivity of L[C.sub.20] phosphorylation. In contrast, ERK attenuates the thin filament regulatory pathway and suppresses contractions independent of changes in L[C.sub.20] phosphorylation in the uterine artery. isometric tension; intracellular free calcium concentration; myosin light chain phosphorylation; PD-098059; phenylephrine; phorbol 12,13-dibutyrate

We previously demonstrated that cortisol regulated [[alpha].sub.1]-adrenoceptor-mediated contractions differentially in nonpregnant and pregnant uterine arteries. Given that chronic hypoxia during pregnancy has profound effects on maternal uterine artery reactivity, the present study investigated the effects of chronic hypoxia on cortisol-mediated regulation of uterine artery contractions. Pregnant (day 30) and nonpregnant ewes were divided between normoxic control and chronically hypoxic [maintained at high altitude (3,820 m), arterial P[O.sub.2]:60 mmHg for 110 days] groups. Uterine arteries were isolated and contractions measured. In hypoxic animals, cortisol (10 ng/ml for 24 h) increased norepinephrine-induced contractions in pregnant, but not in nonpregnant, uterine arteries. The 11[beta]-hydroxysteroid dehydrogenase inhibitor carbenoxolone did not change cortisol effects in nonpregnant uterine arteries, but abolished it in pregnant uterine arteries by increasing norepinephrine p[D.sub.2] (-log E[C.sub.50]) in control tissues. The dissociation constant of norepinephrine-[[alpha].sub.1]-adrenoceptors was not changed by cortisol in nonpregnant, but decreased in pregnant uterine arteries. There were no differences in the density of glucocorticoid receptors between normoxic and hypoxic tissues. Cortisol inhibited the norepinephrine-induced increase in [Ca.sup.2+] concentrations in nonpregnant arteries, but potentiated it in pregnant arteries. In addition, cortisol attenuated phorbol 12,13-dibutyrate-induced contractions in normoxic nonpregnant and pregnant uterine arteries, but had no effect on the contractions in hypoxic arteries. The results suggest that cortisol differentially regulates [[alpha].sub.1]-adrenoceptor-and PKC-mediated contractions in uterine arteries. Chronic hypoxia suppresses uterine artery sensitivity to cortisol, which may play an important rule in the adaptation of uterine vascular tone and blood flow in response to chronic stress of hypoxia during pregnancy. pregnancy; norepinephrine; protein kinase C; glucocorticoid receptors; calcium sensitivity

Chronic hypoxia during pregnancy is one of the most common insults to fetal development. We tested the hypothesis that maternal hypoxia induced apoptosis in the hearts of near-term fetal rats. Pregnant rats were divided into two groups, normoxic control and continuous hypoxic exposure (10.5% [O.sub.2]) from day 15 to 21 of gestation. Hearts were isolated from fetal rats of 21-day gestational age. Maternal hypoxia increased hypoxia-inducible factor-l[alpha] protein in fetal hearts. Chronic hypoxia significantly increased the percentage and size of binucleated myocytes and increased apoptotic cells from 1.4 [+ or -] 0.14% to 2.7 [+ or -] 0.3% in the fetal heart. In addition, the active cleaved form of caspase 3 was significantly increased in the hypoxic heart, which was associated with an increase in caspase 3 activity. There was a significant increase in Fas protein levels in the hypoxic heart. Chronic hypoxia did not change Bax protein levels but significantly decreased Bcl-2 proteins. In addition, chronic hypoxia significantly suppressed expression of heat shock protein 70. However, chronic hypoxia significantly increased expression of the anti-apoptotic protein 14-3-3 [theta], among other 14--3-3 isoforms. Chronic hypoxia differentially regulated [beta]-adrenoreceptor ([beta]-AR) subtypes with an increase in [[beta].sub.1]-AR levels but no changes in [[beta].sub.2]-AR. The results demonstrate that maternal hypoxia increases apoptosis in fetal rat heart, which may be mediated by an increase in Fas and a decrease in Bcl-2 proteins. Chronic hypoxia-mediated increase in [[beta].sub.1]-AR and decrease in heat shock proteins may also play an important role in apoptosis in the fetal heart. fetus

Cortisol potentiated norepinephrine (NE)mediated contractions in ovine uterine arteries (UA). We tested the hypothesis that cortisol regulated [[alpha].sub.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].sub.1]-adrenoceptors in NUA, but increased [[alpha].sub.1]-adrenoceptor density in PUA. Cortisol potentiated NE-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)[P.sub.3]] synthesis in both NUA and PUA, but increased coupling efficiency of [[alpha].sub.1]-adrenoceptors to Ins(1,4,5)P3 synthesis only in NUA. Carbenoxolone alone did not affect NE-mediated Ins(].,4,5)[P.sub.3] production, but significantly enhanced cortisol-mediated potentiation of NE-stimulated Ins(1,4,5)[P.sub.3] synthesis in PUA. In addition, cortisol potentiated the NE-induced increase in [Ca.sup.2+] concentration in PUA, but increased NE-mediated contraction for a given amount of [Ca.sup.2+] concentration ill NUA. Collectively, the results indicate that cortisol potentiates NE-mediated contractions differentially in NUA and PUA, i.e., by upregulating [[alpha].sub.1]-adrenoceptor density leading to increased [Ca.sup.2+] mobilization in PUA while increasing [[alpha].sub.1]-adrenoceptor coupling efficiency and myofilament [Ca.sup.2+] sensitivity in NUA. In addition, the results suggest that pregnancy increases type 2 11[beta]-hydroxysteroid dehydrogenase activity in the UA. [[alpha].sub.1]-adrenoceptor; 11[beta]-hydroxysteroid dehydrogenase; inositol 1,4,5-trisphosphate; calcium; pregnancy

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or pre-eclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am d Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PG[I.sub.2]) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PG[I.sub.2] biosynthetic pathway (cPL[A.sub.2], COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant. nitric oxide; prostacyclin; calcium; kinases; programming

Cortisol-mediated potentiation of uterine artery contractility: effect of pregnancy. Am J Physiol Heart Circ Physiol 283: H238-H246, 2002. First published February 28, 2002; 10.1152/ajpheart.00842.2001.--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 ~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 p[D.sub.2] in control tissues from 6.20 [+ or -] 0.05 to 6.59 [+ or -] 0.11. The apparent dissociation constant value of NE [[alpha].sub.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 ~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].sub.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. nitric oxide; [[alpha].sub.1]-adrenoceptor; 11-[beta]HSD; glucocorticoid receptor

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered [[alpha].sub.2]-adrenergic receptor ([[alpha].sub.2]-AR) density and/or affinity. In fetal (~140 days) and adult sheep, we measured [[alpha].sub.2]-AR density and affinity with the antagonist [[sup.3]H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the [[alpha].sub.2]-AR antagonists yohimbine and idazoxan and contractile responses to the [[alpha].sub.2]-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, [[alpha].sub.2]-AR density was 201 [+ or -] 18 and 52 [+ or -] 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, [10.sup.-7] M yohimbine significantly decreased the p[D.sub.2] for NE-induced tension in the presence of 3 x [10.sup.-5] M cocaine, [10.sup.-5] M deoxycorticosterone, and [10.sup.-6] M tetrodotoxin. In fetal, but not adult,. MCA, UK-14304 induced a significant decrease in p[D.sub.2] for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of [10.sup.-6] M idazoxan to block [[alpha].sub.2]-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, [[alpha].sub.2]-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional [[alpha].sub.2]-AR. cerebrovascular circulation; vascular smooth muscle; norepinephrine; clonidine; UK-14304; yohimbine; idazoxan; tetrodotoxin; fetus

L-type [Ca.sup.2+] channels in fetal and adult ovine cerebral arteries. Am J Physiol Regulatory Integrative Comp Physiol 282: R131-R138, 2002; 10.1152/ajpregu.00318.2001.--Recently, we reported that, whereas in cerebral arteries of the adult a majority of norepinephrine (NE)-induced increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) comes from release of the sarcoplasmic reticulum (SR) [Ca.sup.2+] stores, in the fetus the SR [Ca.sup.2+] stores are relatively small, and NE-induced increase in [[[Ca.sup.2+]].sub.i] results mainly from activation of plasma membrane L-type [Ca.sup.2+] channels (20). In an effort to establish further the role of L-type [Ca.sup.2+] channels in the developing cerebral arteries, we tested the hypothesis that, in the fetus, increased reliance on plasmalemmal L-type [Ca.sup.2+] channels is mediated, in part, by increased L-type [Ca.sup.2+] channel density. We used [sup.3]H-labeled (+)isopropyl-4-(2,1,3-benzoxadiazol-4-y1)-1, 4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) to measure L-type [Ca.sup.2+] channel density ([B.sub.max]) in the cerebral arteries, common carotid artery (CCA), and descending aortae of fetal (~140 gestation days), newborn (7-10 days), and adult sheep. In the cerebral and common carotid arteries, [B.sub.max] values (fmol/mg protein) of fetuses and newborns were significantly greater than those of adults. Western immunoblotting assay also revealed that the density of L-type [Ca.sup.2+] channel protein in the cerebral arteries and CCA was about twofold greater in the fetus than the adult. Finally, compared with the adult, fetal cerebral arteries demonstrated a significantly greater maximum tension and [[[Ca.sup.2+]].sub.i] in response to stimulation with the L-type [Ca.sup.2+] channel agonist Bay K 8644. In addition, Bay K 8644-stimulated fetal vessels demonstrated a maximal tension and [[[Ca.sup.2+]].sub.i] similar to that observed in response to stimulation with [10.sup.-4] NE. These results support the idea that fetal cerebrovascular smooth muscle relies more on extracellular [Ca.sup.2+] and L-type [Ca.sup.2+] channels for contraction than does the adult and that this increased reliance is mediated, in part, by greater L-type [Ca.sup.2+] channel density. This may have important implications in the regulation of cerebral blood flow in the developing organism. cerebral circulation; norepinephrine; vascular smooth muscle; intracellular calcium; PN200-110; Bay K 8644; fetus; newborn

ERK MAP kinases regulate smooth muscle contraction in ovine uterine artery: effect of pregnancy. Am J Physiol Heart Circ Physiol 282: H292-H300, 2002.--The present study investigated the potential role of extracellular signal-regulated kinase (ERK) in uterine artery contraction and tested the hypothesis that pregnancy upregulated ERK-mediated function in the uterine artery. Isometric tension in response to phenylephrine (PE), serotonin (5-HT), phorbol 12,13-dibutyrate (PDBu), and KCl was measured in the ring preparation of uterine arteries obtained from nonpregnant and near-term (140 days gestation) pregnant sheep. Inhibiting ERK activation with PD-98059 did not change the KCl-evoked contraction but significantly inhibited the contraction to 5-HT in both nonpregnant and pregnant uterine arteries. PD-98059 did not affect PE-induced contraction in the uterine arteries of nonpregnant sheep but significantly decreased it in the uterine arteries of pregnant sheep. In accordance, PE stimulated activation of ERK in uterine arteries of pregnant sheep, which was blocked by PD-98059. PD-98059-mediated inhibition of the PE-induced contraction was associated with a decrease in both intracellular [Ca.sup.2+] concentration and [Ca.sup.2+] sensitivity of contractile proteins in the uterine arteries of pregnant sheep. PDBu-mediated contraction was significantly less in pregnant than in nonpregnant uterine arteries. PD-98059 had no effect on PDBu-induced contraction in nonpregnant but significantly increased it in pregnant uterine arteries. In addition, PD-98059 significantly enhanced PDBu-stimulated protein kinase C activity. The results indicate that ERK plays an important role in the regulation of uterine artery contractility, and its effect is agonist dependent. More importantly, pregnancy selectively enhances the role of ERK in [[alpha].sub.1]-adrenoceptor-mediated contractions and its effect in suppressing protein kinase C-mediated contraction in the uterine artery. mitogen-activated protein kinase; PD-98059; calcium; protein kinase C; [[alpha].sub.1]-adrenoceptor; extracellular signal-regulated kinase

The hypothesis that plasma membrane L-type and receptor-operated Ca2+ channels play a major role in norepinephrine-induced vasoconstriction through changes in plasma membrane Ca2+ flux and that this may change with developmental age was tested. Findings indicate that especially in fetal, but also in adult, ovine middle cerebral arteries, CA2+ flux via L-type calcium channels has a primary function in norepinephrine-induced contraction.

Research was conducted to study the effects of chronic hypoxia on free intracellular Ca2+ concentration and Ca2+ sensitivity of myofilaments during agonist stimulation in sheep uterine arteries. Smooth muscle Ca2+ was determined with muscle contraction while the ring arterial ring was connected to an isometric force transducer. Results indicated that chronic hypoxia does not influence the electromagnetic coupling induced by KCl but reduces pharmaco-mechanical coupling induced by the activation of GTP-binding protein-coupled receptors in smooth muscle.

The effects of long-term, hypoxemia-induced reductions in cerebrovascular contractility on the density and affinity of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] were analyzed in the cerebral arteries of sheep. Vascular Ins(1,4,5)P3-R density increased as a function of developmental age in the common carotid artery and aorta of sheep. High-altitude, long-term hypoxia was also associated with a significant decrease in Ins(1,4,5)P3-R density in the common carotid artery and main bran cerebral blood vessels of fetal and adult sheep.

The effects of G protein activation on Ca2+ sensitivity were analyzed in ovine common carotid (Com), basilar artery (Bas), main branch middle cerebral arteries (MCA) and second-branch middle cerebral arteries (MCA-B). The sensitivity of ovine Com, Bas, MCA and MCA-B to Ca2+ was enhanced by adrenergic agonists via a G protein-dependent mechanism. Furthermore, cerebrovascular reactivity involved the modulation of contractile responses to all stimuli that increase intracellular Ca2+ concentration.

The cerebral arteries of fetal and adult sheep were analyzed to determine the effects of high-altitude acclimatization and long-term hypoxemic cerebrovascular responses to norepinephrine (NE) and other agonists. Analysis of normoxic fetal and adult sheep cerebral arteries indicated the role of high-altitude acclimatization in reducing the density of alpha1-adrenergic receptors. Furthermore, high-altitude acclimatization also reduced NE-induced inositol 1,4,5-triphosphate responses in adult common carotid artery and fetal posterior main branch cerebral arteries.

The role of alpha1-adrenergic receptor (alpha1-AR) density and norepinephrine (NE)-induced inositol 1,4,5-triphosphate (IP3) in NE-mediated contraction changes were analyzed in sheep cerebral arteries. Development differences in the contractility of cerebral artery to NE is mediated by changes in aplha1-AR density which affects vascular agonist sensitivity. Furthermore, the common carotid arteries of fetus and newborn infants exhibited little IP3 response to NE compared to the anterior and middle cerebral arteries.

The effect of long-term hypoxia on cardiovascular function was investigated by examining changes in alpha1-adrenergic receptors and their coupling functions in the ovine uterine artery. Administration of norepinephrine induced contractions in the main and fourth-branch uterine arteries but elevated altitudes depressed this response. It was concluded that chronic hypoxia in high-altitude animals depresses alpha1-adrenergic-induced contractions of uterine arteries.

The components of the alpha1-adrenergic receptor contractile pathway in the cerebral arteries were investigated based on their responses to norepinephrine (NE). Measurements of NE-induced contractions and NE-induced Ins(1,4,5)P3 synthesis in adult sheep indicated higher NE-induced maximum response in the middle cerebral arteries than in the common carotid (Com). Higher Ins(1,4,5)P3 release was observed in both Com and in the anterior, middle and posterior arteries (AMP). Contractile response of AMP was higher to NE as compared to potassium. These results suggest significant differences in the components of the cerebral vasculature.

Pregnancy enhances endothelium-dependent relaxation of ovine uterine artery: role of NO and intracellular [Ca.sup.2+]. Am J Physiol Heart Circ Physiol 281: H183-H190, 2001.--The present study tested the hypothesis that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine artery was mediated primarily by an increase in nitric oxide (NO) release, resulting in a reduction in smooth muscle intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). Uterine arteries obtained from nonpregnant and near-term (140 days gestation) pregnant sheep were used. The [Ca.sup.2+] ionophore A23187 induced endothelium-dependent relaxations in both nonpregnant and pregnant uterine arteries, with an increased relaxation in the pregnant tissue. In contrast, endothelium-independent relaxations induced by sodium nitroprusside were the same in nonpregnant and pregnant arteries. In addition, removal of the endothelium significantly increased noradrenaline-induced contractions in pregnant, but not nonpregnant, uterine arteries. In accordance, pregnancy increased both basal and A23187-stimulated NO releases in the uterine artery. Simultaneous measurement of tension and [[[Ca.sup.2+]].sub.i] in the smooth muscle demonstrated a linear correlation with the slope of unity between A23187-induced relaxation and the reduction of [[[Ca.sup.2+]].sub.i] in both nonpregnant and pregnant uterine arteries. The A23187-induced reduction of [[[Ca.sup.2+]].sub.i] was significantly enhanced in pregnant, compared with nonpregnant, uterine arteries. The results indicate that pregnancy increases NO release, which, through decreasing [[[Ca.sup.2+]].sub.i] in the smooth muscle, accounts for the increased endothelium-dependent relaxation of the uterine artery. Signal transduction pathways distal to NO production are not changed by pregnancy. calcium