Kevin Despain

Pregnancy modifies the large conductance Ca2+-activated K + channel and cGMP-dependent signaling pathway in uterine vascular smooth muscle

Regulation of uteroplacental blood flow (UPBF) during pregnancy remains unclear. Large conductance, Ca(2+)-activated K(+) channels (BK(Ca)), consisting of alpha- and regulatory beta-subunits, are expressed in uterine vascular smooth muscle (UVSM) and contribute to the maintenance of UPBF in the last third of ovine pregnancy, but their expression pattern and activation pathways are unclear. We examined BK(Ca) subunit expression, the cGMP-dependent signaling pathway, and the functional role of BK(Ca) in uterine arteries (UA) from nonpregnant (n = 7), pregnant (n = 38; 56-145 days gestation; term, approximately 150 days), and postpartum (n = 15; 2-56 days) sheep. The alpha-subunit protein switched from 83-87 and 105 kDa forms in nonpregnant UVSM to 100 kDa throughout pregnancy, reversal occurring >30 days postpartum. The 39-kDa beta(1)-subunit was the primary regulatory subunit. Levels of 100-kDa alpha-subunit rose approximately 70% during placentation (P < 0.05) and were unchanged in the last two-thirds of pregnancy; in contrast, beta(1)-protein rose throughout pregnancy (R(2) = 0.996; P < 0.001; n = 13), increasing 50% during placentation and approximately twofold in the remainder of gestation. Although UVSM soluble guanylyl cyclase was unchanged, cGMP and protein kinase G(1alpha) increased (P < 0.02), paralleling the rise and fall in beta(1)-protein during pregnancy and the puerperium. BK(Ca) inhibition not only decreased UA nitric oxide (NO)-induced relaxation but also enhanced alpha-agonist-induced vasoconstriction. UVSM BK(Ca) modify relaxation-contraction responses in the last two-thirds of ovine pregnancy, and this is associated with alterations in alpha-subunit composition, alpha:beta(1)-subunit stoichiometry, and upregulation of the cGMP-dependent pathway, suggesting that BK(Ca) activation via NO-cGMP and beta(1) augmentation may contribute to the regulation of UPBF.

Large-Conductance Ca2+-Dependent K+ Channels Regulate Basal Uteroplacental Blood Flow in Ovine Pregnancy

Objectives: The mechanisms regulating basal uteroplacental blood flow (UBF) and the greater than 30-fold increase observed in normal pregnancy remain unclear. Although vascular growth contributes in early gestation, vasodilation accounts for the exponential rise seen in the last third of pregnancy. Large conductance potassium channels (BKCa) are expressed in uterine vascular smooth muscle (VSM), but the extent of their role in regulating UBF in pregnancy is unclear. Therefore, we determined if BKCa regulate basal UBF during ovine pregnancy. Methods: Studies were performed at 113 to 127 days and 135 to 150 days of gestation in eight pregnant ewes instrumented with uterine artery flow probes and uterine arterial and venous catheters. Tetraethylammonium chloride (TEA), a BKCa-specific inhibitor at less than 1.0 mM, was infused intra-arterially into the pregnant uterine horn over 60 minutes to achieve levels of 0.001-0.35 mM while continuously monitoring UBF, arterial pressure (MAP), and heart rate (HR). uterine arterial and venous blood was collected simultaneously to measure uterine cyclic guanosine monophosphate (rGMP) synthesis. Results: Intra-arterial TEA dose-dependently decreased basal UBF in the early (R = 0.81, n = 36, P <.001) an late (R = 0.72, n = 31, P <.001) study periods without altering contralateral UBF, MAP, and HR. The IC50 was 0.2 mM and basal UBF decreased ≥80% at 0.35 mM in both periods. Although UBF fell greater than 40% at estimated plasma TEA levels of 0.3 mM, uterine arterial cGMP was unchanged, uterine venous cGMP rose, and uterine cGMP synthesis was unchanged; therefore, upstream events associated with BKCa activation were unaffected by blockade. Conclusions: These are the first data demonstrating that BKCa are essential in the maintenance of basal UBF in the last third of ovine pregnancy.

Large Conductance Ca2+—Activated K+ Channels Contribute to Vascular Function in Nonpregnant Human Uterine Arteries

Large conductance K + channels (BKCa) are expressed in uterine artery (UA) smooth muscle from nonpregnant and pregnant sheep and contribute to the regulation of basal vascular tone and responses to estrogen and vasoconstrictors. To determine if BKCa are expressed in women and contribute to UA function, we collected UA from nonpregnant women (n = 31) at elective hysterectomy and analyzed for subunit protein, localization with immunohistochemistry, and function using endothelium-denuded rings. UA expresses BKCa α -, β1- and β2-subunit protein. KCl and phenylephrine (PE, an α 1-agonist) caused dose-dependent vasoconstriction (P < .001), and UA precontracted with PE dose-dependently relaxed with sodium nitroprusside (SNP; P < .001).Tetraethylammonium chloride (TEA, 0.2-1.0 mM), a BKCa inhibitor, dose-dependently increased resting tone (P = .004; 28% ± 5.3% with 1.0 mM), enhanced PE-induced (10 − 6 M) vasoconstriction (P < .04), and attenuated SNP-induced relaxation at 1.0 mM (P = .02). BK Ca are expressed in human UA and modulate vascular function by attenuating vasoconstrictor responses and contributing to nitric oxide-induced vasorelaxation.

Differential Responses to Systemic and Local Angiotensin II Infusions in Conscious Postnatal Sheep

Angiotensin II (ANG II) increases blood pressure (MAP) via specific ANG II receptors (AT) and is considered important in regulating MAP after birth. In adult animals, AT1 receptors predominate in vascular smooth muscle (VSM) and mediate vasoconstriction. In newborn sheep, AT2 receptors, which do not mediate vasoconstriction, predominate in vascular smooth muscle until 2 wk postnatal when they are replaced by AT1. Thus, the mechanisms whereby ANG II increases MAP after birth are unclear. We examined the effects of ANG II on femoral vascular resistance (FmVR) and blood flow (FmBF) in serial studies of newborn sheep (n = 7) at 7–14 d, 15–21 d, and 22–35 d. Animals had femoral catheters implanted for systemic ANG II infusions and cardiovascular monitoring, and a flow probe was implanted on the contralateral artery proximal to the superficial saphenous artery, which contained a catheter for intra-arterial ANG II infusions. Studies were performed using a range of systemic and intra-arterial ANG II doses. Systemic ANG II increased MAP dose-dependently at all ages (p < 0.001); however, responses were not age dependent. FmBF rose dose dependently at 7–14 d (p < 0.001) and was unchanged at older ages. FmVR was unaffected at 7–14 d, but values increased dose dependently at 15–21 d and 22–3 5d (p < 0.001), although never exceeded relative increases in MAP. Local ANG II did not alter MAP, FmBF, or FmVR at any age. Although systemic ANG II increases MAP and FmVR dose dependently after birth, ANG II-induced vasoconstriction is attenuated. Furthermore, intra-arterial ANG II does not alter FmVR in the absence of systemic responses, suggesting incomplete vascular smooth muscle AT1 expression, stimulation of local ANG II antagonists, or ANG II-mediated release of another vasoconstrictor.

Differential Sensitivity to Angiotensin II and Norepinephrine in Human Uterine Arteries

BACKGROUND During pregnancy, uteroplacental responses to norepinephrine (NE) exceed systemic responses. In contrast, uteroplacental responses to angiotensin II (ANG II) are less than systemic. The explanation for these differences in uteroplacental sensitivity remain unclear but may reflect type 2 ANG II receptor (AT(2)R) predominance in uterine artery (UA) vascular smooth muscle (VSM). OBJECTIVE The objective of the study was to examine VSM sensitivity to KCl, NE, and ANG II in UA from nonpregnant (NP) and pregnant (P) women and determine VSM ANG II receptor subtype expression. METHODS Responses to KCl, NE, and ANG II were examined in endothelium-denuded UA rings from NP (n = 28) and P (n = 13; 34-40 wk gestation) women, and ANG II receptor subtype, α(1)-receptor and contractile proteins were measured. RESULTS KCl and NE dose dependently contracted UA (P < 0.001), P exceeding NP 2-fold or greater; but α(1)-receptor expression was unchanged. ANG II did not elicit dose effects in NP or P UA; however, P responses exceeded NP approximately 2-fold (P < 0.001) and were approximately 2.5-fold less than NE (P < 0.001). AT(2)R and AT(1)R expression were similar (P > 0.1) in VSM from NP and term P women. AT(1)R blockade abolished ANG II contractions (P < 0.001); AT(2)R blockade did not enhance ANG II sensitivity in UA with or without endothelium. Actin contents increased approximately 2-fold in term UA. CONCLUSIONS Sensitivity to α-stimulation exceeds ANG II in NP and P UA, explaining the differential uteroplacental sensitivity in pregnancy. Because AT(2)R predominate in UA VSM throughout reproduction, this contributes to the inherent refractoriness to ANG II in the uterine vasculature. The increase in UA contractile proteins at term P suggests remodeling, explaining the enhanced contractility seen.

The renin-angiotensin system in conscious newborn sheep: Metabolic clearance rate and activity

The role of the renin-angiotensin system (RAS) in regulating newborn mean arterial blood pressure (MAP) and tissue blood flow remains unclear. Although postnatal MAP increases, vascular responsiveness to infused angiotensin II (ANG II) is unchanged, possibly reflecting increased metabolic clearance rate of ANG II (MCRANG II). To address this, we examined MAP, heart rate, plasma ANG II and renin activity (PRA), and MCRANG II in conscious postnatal sheep (n = 9, 5–35 d old) before and during continuous systemic ANG II infusions to measure MCRANG II. Postnatal MAP increased (p < 0.02), whereas plasma ANG II decreased from 942 ± 230 (SEM) to 471 ± 152 and 240 ± 70 pg/mL at <10 d, 10–20 d, and 21–35 d postnatally (p = 0.05), respectively. Despite high plasma ANG II, PRA remained elevated, averaging 6.70 ± 1.1 ng/mL·h throughout the postnatal period, but decreased 35% (p = 0.01) during ANG II infusions. MCRANG II decreased approximately sixfold after birth and averaged 115 mL/min·kg during the first month. Circulating ANG II is markedly increased after birth, reflecting placental removal, high fetal MCRANG II, and enhanced RAS activity. Although circulating ANG II decreases as MAP increases, MCRANG II is unchanged, suggesting decreased ANG II production. Persistent vascular smooth muscle (VSM) AT2 receptor subtype (AT2R) expression after birth may modify the hypertensive effects of ANG II postnatally.

Effects of exercise on soleus in severe burn and muscle disuse atrophy.

BACKGROUND Muscle loss is a sequela of severe burn and critical illness with bed rest contributing significantly to atrophy. We hypothesize that exercise will mitigate muscle loss after burn with bed rest. MATERIALS AND METHODS Male rats were assigned to sham ambulatory (S/A), burn ambulatory (B/A), sham hindlimb unloading (S/H), or burn hindlimb unloading (B/H). Rats received a 40% scald burn or sham and were ambulatory or placed in hindlimb unloading, a model of bed rest. Half from each group performed twice daily resistance climbing. Hindlimb isometric forces were measured on day 14. RESULTS Soleus mass and muscle function were not affected by burn alone. Mass was significantly lower in hindlimb unloading (79 versus 139 mg, P < 0.001) and no exercise (103 versus 115 mg, P < 0.01). Exercise significantly increased soleus mass in B/H (86 versus 77 mg, P < 0.01). Hindlimb unloading significantly decreased muscle force in the twitch (12 versus 31 g, P < 0.001), tetanic (55 versus 148 g, P < 0.001), and specific tetanic measurements (12 versus 22 N/cm(2), P < 0.001). Effects of exercise on force depended on other factors. In B/H, exercise significantly increased twitch (14 versus 8 g, P < 0.05) and specific tetanic force (14 versus 7 N/cm(2), P < 0.01). Fatigue index was lower in ambulatory (55%) and exercise (52%) versus hindlimb (69%, P = 0.03) and no exercise (73%, P = 0.002). CONCLUSIONS Hindlimb unloading is a significant factor in muscle atrophy. Exercise increased the soleus muscle mass, twitch, and specific force in this model. However, the fatigue index decreased with exercise in all groups. This suggests exercise contributes to functional muscle change in this model of disuse and critical illness.

Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT(2) receptors (AT(2)R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant (n = 16) and term pregnant (n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner (P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM (P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content (P ≤ 0.01). UVSM AT(1)R and AT(2)R expression was similar throughout the UVB and unchanged during pregnancy (P > 0.1). AT(1)R inhibition blocked ANG II-mediated contractions; AT(2)R blockade, however, did not enhance contractions. AT(2)R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.

Effects of Systemic and Local Phenylephrine and Arginine Vasopressin Infusions in Conscious Postnatal Sheep

Mean arterial pressure (MAP) increases after birth, however, the mechanisms remain unclear. Systemic angiotensin II (ANG II) infusions increase MAP in newborn sheep, but the direct effects of ANG II on peripheral vascular resistance (PVR) are minimal. Thus, its systemic pressor effects may reflect release of other pressor agents, e.g. α-agonists and/or AVP, suggesting they contribute to postnatal regulation of MAP and PVR. To address this, we performed studies in conscious sheep at 7–14, 15–21, and 22–35 d postnatal, infusing phenylephrine (PE) or AVP systemically or intra-arterially into the hindlimb while measuring MAP, heart rate (HR), and femoral blood flow (FmBF). Basal MAP and FmBF rose, whereas HR and femoral vascular resistance (FmVR) fell (p ≤ 0.03) during the first month postnatal. Although systemic PE and AVP dose dependently increased MAP and FmVR and decreased FmBF and HR (p < 0.001, ANOVA) at all ages, responses were not age dependent. Notably, increases in FmVR exceeded increases in MAP, and responses to PE appeared to exceed AVP (p < 0.05). Hindlimb infusions of both agents decreased FmBF and increased FmVR dose dependently (p < 0.001, ANOVA) at all ages without altering MAP or HR. These responses also were not age dependent. Unlike ANG II, PE and AVP directly increase PVR in newborn sheep. Moreover, FmVR increases more than MAP at all doses, suggesting these agonists may contribute to postnatal MAP regulation and could mediate the effects of systemic ANG II on postnatal MAP.

Metabolism and synthesis of arginine vasopressin in conscious newborn sheep

Arginine vasopressin (AVP) is an important regulator of cardiovascular homeostasis in the fetus, but its role after birth is unclear. Although infused AVP increases mean arterial pressure (MAP) during the 1st mo after birth, pressor responses are unchanged, suggesting that vascular responsiveness is also unchanged. Alternatively, this could reflect increases in AVP metabolic clearance rate (MCR(AVP)). However, newborn AVP metabolism and synthesis are poorly studied. Therefore, we examined the pressor responses to infused AVP and the pattern of circulating AVP, AVP production rate (PR(AVP)), and MCR(AVP) in conscious newborn sheep (n = 5) at 9-38 days after birth. Basal MAP rose and heart rate (HR) fell during the study period (P < or = 0.02), while circulating AVP was unchanged (P > 0.1), averaging 3.01 +/- 0.86 pg/ml. Infused AVP elicited steady-state responses at 10-40 min, increasing plasma AVP and MAP and decreasing HR (P < 0.001). Although pressor responses were unchanged between 9 and 38 days, the rise in MAP correlated with increases in plasma AVP (R = 0.47, P = 0.02, n = 24). MCR(AVP) was unchanged throughout the 1st mo (P > 0.2), averaging 205 +/- 17 ml.kg(-1).min(-1), and was associated with an elevated PR(AVP), 973 +/- 267 pg.kg(-1).min(-1), which also was unchanged (P > 0.1). After birth, MCR(AVP) and PR(AVP) are elevated, probably accounting for the stable plasma AVP levels. The former is also likely to account for the stable pressor responses to infused AVP during the 1st mo. The reason for the elevated PR(AVP) is unclear but may relate to increases in vascular volume associated with postnatal growth.