James C. Rose

Epidural Clonidine Produces Antinociception, But Not Hypotension, In Sheep

Intrathecally administered clonidine produces analgesia, but also produces hypotension. To assess the effects of epidural administration, the authors inserted lumbar epidural catheters in seven non-pregnant ewes, and injected, on separate days, clonidine (50–750 mcg), morphine (5–10 mg), and a clonidine-morphine combination (clonidine 150 mcg + morphine 5 mg). Clonidine produced dose-dependent antinociception and sedation, with the lowest maximally effective antinociceptive dose being 300 mcg. Morphine produced less intense antinociception than clonidine, and did not potentiate clonidines effect. Antinociception, but not sedation, following clonidine injection was reversed by epidural injection of the α2-adrenergic antagonist, idazoxan. Epidurally administered naloxone and prazosin did not reverse clonidines antinociceptive effect, nor did intravenously administered idazoxan. Epidurally administered clonidine did not decrease blood pressure or heart rate or affect arterial blood gas tensions or spinal cord histology. These data suggest that epidurally administered clonidine produces analgesia by a local, α2-adrenergic mechanism. In sheep, epidurally administered clonidine does not produce hypotension.

Angiotensin-(1-7)-Angiotensin-Converting Enzyme 2 Attenuates Reactive Oxygen Species Formation to Angiotensin II Within the Cell Nucleus

The angiotensin (Ang) type 1 receptor (AT1R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT1R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (1.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT2R as the predominant receptor subtype (≈80%) in young sheep, with the Ang-(1-7) (AT7R; Mas protein) and AT1R antagonists competing for the remaining sites. Conversely, in older sheep, the AT1R accounted for ≈85% of nuclear sites, whereas the Ang type 2 receptor and AT7R subtypes comprise ≈20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (97±22%; n=6) versus young animals (7±2%; P=0.01; n=4), and this was abolished by an AT1R antagonist. The AT7R antagonist D-Ala7-Ang-(1-7) increased ROS formation to Ang II by ≈2-fold (174±5% versus 97±22%; P<0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT7R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(1-7). The ACE2 inhibitor MLN4760 also exacerbated the Ang II–dependent formation of ROS (156±15%) and abolished the generation of Ang-(1-7) from Ang II. We conclude that an ACE2-Ang-(1-7)-AT7R pathway modulates Ang II–dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.

Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production

Expression of nuclear angiotensin II type 1 (AT(1)) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist (125)I-[Sar(1),Thr(8)]-ANG II ((125)I-sarthran) with the AT(1) antagonist losartan (LOS) or the AT(2) antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC (> or =70%) and PM (> or =80%) sites while LOS competition predominated in medullary NUC (> or =75%) and PM (> or =70%). Immunodetection with an AT(2) antibody revealed a single approximately 42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT(2) in the tubulointerstitium, AT(1) in the medulla and vasa recta, and both AT(1) and AT(2) in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-l-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear AT(2) receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure.

Angiotensin-(1-7)–Angiotensin-Converting Enzyme 2 Attenuates Reactive Oxygen Species Formation to Angiotensin II Within the Cell Nucleus. Inter-American Society of Hypertension

The angiotensin (Ang) type 1 receptor (AT1R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT1R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (1.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT2R as the predominant receptor subtype (≈80%) in young sheep, with the Ang-(1-7) (AT7R; Mas protein) and AT1R antagonists competing for the remaining sites. Conversely, in older sheep, the AT1R accounted for ≈85% of nuclear sites, whereas the Ang type 2 receptor and AT7R subtypes comprise ≈20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (97±22%; n=6) versus young animals (7±2%; P=0.01; n=4), and this was abolished by an AT1R antagonist. The AT7R antagonist D-Ala7-Ang-(1-7) increased ROS formation to Ang II by ≈2-fold (174±5% versus 97±22%; P<0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT7R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(1-7). The ACE2 inhibitor MLN4760 also exacerbated the Ang II–dependent formation of ROS (156±15%) and abolished the generation of Ang-(1-7) from Ang II. We conclude that an ACE2-Ang-(1-7)-AT7R pathway modulates Ang II–dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.

Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97±3 versus 83±2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4±7.0 versus 36.0±2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8±1.2 versus 31.4±4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming–induced hypertension.

Alterations in fetal kidney development and elevations in arterial blood pressure in young adult sheep after clinical doses of antenatal glucocorticoids.

Epidemiologic studies have yielded controversial information regarding an association between antenatal steroid administration and elevations in arterial blood pressure (BP). The aim of the study was to determine whether antenatal administration of a clinically relevant dose of steroids at a time when fetal nephrogenesis is at its highest results in abnormal kidney development and adult hypertension. Pregnant sheep were treated with either vehicle or betamethasone. Maternal injections were given 24 h apart at 80 d of gestational age (dGA; 0.55 of gestation). Animals were studied either as fetuses or as immature adults. Fetuses were delivered by cesarean section at 135 dGA. Adults were studied at 6 mo of age. Betamethasone administration did not induce premature labor or intrauterine growth restriction. In the betamethasone-exposed group, we found at 135 dGA a 25.5% decrease in the number of glomeruli with no differences in fetal kidney weight. In adults, mean, systolic, and diastolic arterial BPs were significantly higher, whereas there were no significant differences in heart rate over the same study period. The major finding of this study is that a single course of antenatal steroids alters renal development and is associated with elevations in arterial BP in lambs at 6 mo of age. We conclude that antenatal glucocorticoid administration under the National Institutes of Health consensus guidelines may alter human fetal renal development.

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast.

Epidural clonidine analgesia in obstetrics: sheep studies

Epidural clonidine administration produces analgesia by a nonopiate, spinal mechanism, and offers advantages over other epidural agents for labor analgesia. To examine clonidines acute maternal and fetal effects, the authors injected clonidine, 300 micrograms, epidurally in seven chronically prepared, near term ewes. Unlike epidural saline injection, clonidine increased maternal and fetal serum glucose (by 178 +/- 30% and 190 +/- 30%, respectively; mean +/- SEM, P less than .01) 1 h following injection. Maternal and fetal serum cortisol and arterial blood gas tensions were unchanged following clonidine. Epidural clonidine injection produced minor decreases (10-15%) in heart rate in ewe and fetus, without altering maternal and fetal blood pressure, intra-uterine pressure, or uterine blood flow. Maternal and fetal serum clonidine concentrations peaked at 58 +/- 8 and 73 +/- 5 min following injection, respectively, and declined with similar half-lives. Heart rate correlated negatively with serum clonidine concentration in both ewe and fetus (P less than .05). Apart from hyperglycemia, which does not occur in humans, these results in sheep suggest that epidurally administered clonidine does not adversely affect the fetus and may be evaluated as an analgesic in obstetrics.

Effects of Nitroglycerin and Nitroprusside on the Uterine Vasculature of Gravid Ewes

The effects of nitroglycerin (TNG) and sodium nitroprusside (SNP) on mean aortic pressure (MAP), uterine blood flow (UBF), uterine vascular conductance (UVC), and pulse rate (PR) were compared when the two agents were infused to prevent and treat hypertension induced by norepinephrine (NE) in gravid ewes. When infused alone, TNG, 19 üg/kg/min, decreased MAP 19 per cent and increased PR 33 per cent from control values (P lt; 0.05), but did not significantly change UBF or UVC. In comparison, SNP, 3 üg/kg/min, decreased MAP 20 per cent and increased PR 43 per cent (P lt; 0.05), and did not significantly change UBF or UVC. When given alone, four successive 2-min infusions of NE produced dose-related increase in MAP and decreases in UBF, UVC, and PR; values were significantly different from control with the two higher doses of NE. Although MAP, UBF, and UVC were still significantly changed from control levels when NE was given in the presence of the above infusions of TNG or SNP, MAP was lower and UBF and UVC were higher compared with when NE was given alone (P lt; 0.05). When given to control hypertension induced by a continuous infusion of NE, TNG or SNP produced uterine vasodilatation and significantly increased UBF. Nitroglycerin and SNP were equally effective in counteracting the maternal hypertension and antagonizing the uterine vascular effect of NE. It is concluded that TNG and SNP counteract uterine vasoconstriction resulting from alpha-adrenergic stimulation and do not produce a shunt of blood flow away from the uterine vasculature when used to control hypertension in gravid ewes.

Vasopressin is important for restoring cardiovascular homeostasis in fetal lambs subjected to hemorrhage.

To determine if the posterior pituitary hormone vasopressin is important for maintaining fetal cardiovascular homeostasis during hypovolemic stress, in seven chronically catheterized fetal lambs we induced hemorrhage of 20% of estimated blood volume in the presence and in the absence of a potent antagonist to the pressor effects of vasopressin. The study was a paired crossover design with at least 48 hours separating experiments in the same animal. Injection of the vasopressin antagonist did not alter basal fetal heart rate or arterial blood pressure, but hemorrhage of 2% of estimated fetal blood volume per minute for 10 minutes produced a greater fall in blood pressure (13 +/- 2 versus 10 +/- 2 torr, p less than 0.05) when the blocker was present than when it was absent. Arterial blood pressure remained below control levels longer following hemorrhage when the fetuses were pretreated with the antagonist (49.7 +/- 6 versus 26.6 +/- 6 minutes, p less than 0.01), and the integrated fall in arterial blood pressure with hemorrhage was greatest (283 +/- 53 versus 169 +/- 57 mm Hg . min p less than 0.01) when the blocker was used. The fall in heart rate following hemorrhage was similar with and without blocker pretreatment. These results indicate that vasopressin plays a physiologic role in blood pressure regulation in fetal lambs during periods of hypovolemia.