Renal nerves affect rate of achieving sodium balance in spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) has an elevated efferent sympathetic nerve activity, suggesting that the renal handling of sodium and water may be altered. This study evaluated the renal neurogenic influence on the rate of achieving sodium balance in adult SHRs and Wistar-Kyoto (WKY) rats after either a step increase or step decrease in fixed sodium intake. Conscious, unrestrained rats with either innervated or denervated kidneys were initially placed on a low-sodium (0.3 mEq/d) or high-sodium (5.0 mEq/d) intake by intravenous infusion. Hourly urinary sodium excretion was determined 24 hours before and 72 hours after sodium intake had been increased from low to high or decreased from high to low. After either step change in fixed sodium intake, both innervated SHRs and innervated WKY rats achieved sodium balance within 24 hours. Similarly, the time course of achieving sodium balance was nearly identical between WKY rats with innervated and denervated kidneys after either switch in sodium intake. In SHRs receiving a step increase in sodium intake, both innervated and denervated kidneys increased urinary sodium excretion equally for 9 hours; however, at this time, innervated SHRs continued to increase sodium excretion rapidly, whereas denervated rats were delayed in a further response. Thus, innervated SHRs achieved sodium balance approximately 18 hours sooner than denervated SHRs. Differences in urinary sodium excretion did not result from concomitant changes in plasma renin activity or mean arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of furosemide and verapamil on the NaCl dependency of macula densa-mediated renin secretion.

The present studies in perfused specimens of the juxtaglomerular apparatus microdissected from rabbit kidneys were performed to quantitatively evaluate the relation between macula densa NaCl concentration and renin secretion and to study the effect of furosemide and verapamil on NaCl dependency of renin release. Renin secretion was found to decrease exponentially when macula densa NaCl concentration was increased from 26/7 mmol/L (Na/Cl) to 46/27, 66/47, and 86/67 mmol/L. Increasing Na/Cl concentrations from 86/67 to 106/87 mmol/L had no further effect on renin secretion. [Cl]1/2, the chloride concentration producing the half-maximal effect, was 30 mmol/L. Addition of 50 mumol/L furosemide to the luminal fluid caused renin secretion to become essentially independent of macula densa NaCl concentration. This effect was due to both an increase of renin secretion at high NaCl concentrations and a decrease of renin release at low NaCl concentrations. Verapamil added to the superfusate at a concentration of 1 mumol/L also abolished NaCl dependency of renin secretion; most of this effect was due to an increase of renin release at high luminal NaCl. These results suggest that Na-2Cl-K cotransport and calcium flux through voltage-gated channels are two mechanisms required for the expression of NaCl-dependent renin release. Identification of the cellular localizations of these two critical membrane proteins in the renin control pathway requires further study.

Effects of continuous infusion of endothelin-1 in pregnant sheep.

Plasma concentration of endothelin-1, a potent vasoconstrictor produced by the vascular endothelium, has been observed to be significantly increased in a number of pathophysiological states, including preeclampsia. In the present study we have evaluated the effects of elevated plasma endothelin-1 in pregnant sheep by continuous exogenous endothelin-1 administration. Nine pregnant ewes (110+/-5 days' gestation) were instrumented for measurements of maternal mean arterial pressure, renal blood flow, and uterine blood flow. After recovery, endothelin-1 was infused intravenously for 4 hours at a dose that was adjusted to raise mean arterial pressure by approximately 20 mm Hg by the end of the first hour (range 5 to 20 ng/kg per minute). Mean arterial pressure, renal blood flow, uterine blood flow, urinary protein excretion, hematocrit, and plasma endothelin-1 concentration were measured hourly, and renal and uterine vascular resistances were calculated. Endothelin-1 produced significant increases (% change from baseline at t=4 hours) in mean arterial pressure (45+/-8%), renal vascular resistance (353+/-66 %), and uterine vascular resistance (59+/-21%). Endothelin-1 also increased microvascular permeability both systemically and within the kidney, as suggested by marked increases in hematocrit (0.27+/-0.01 to 0.32+/-0.01) and urinary protein concentration (0.95+/-0.1 to 7.9+/-3.2 mg/mL per mg creatinine). There was a highly significant correlation (P<.0001) between plasma endothelin-1 and mean arterial pressure, renal vascular resistance, uterine vascular resistance, hematocrit, and urinary protein content in all sheep studied. In addition, plasma endothelin-1 corresponded well with the time course of the changes in cardiovascular parameters and urinary protein excretion observed. These results provide evidence to suggest that elevation of circulating endothelin-1 in pregnant sheep can produce cardiovascular and hemodynamic changes that in many ways resemble the human disease preeclampsia. This supports the hypothesis that endothelial cell damage and/or dysfunction that is associated with increased production of endothelin-1 could directly contribute to the progression of preeclampsia.

The distribution of Alz-50 immunoreactivity in the normal human brain.

Alz-50 is a monoclonal antibody that recognizes normal tau proteins as well as phosphorylated tau proteins that are associated with paired helical filaments in Alzheimer's disease. To establish an accurate baseline for future pathological studies, we examined the distribution of Alz-50 immunoreactivity in normal human brain from infancy to senescence. We found extensive staining patterns of somata and axonal profiles in the striatum, amygdala, hypothalamus, brainstem and spinal cord in all normals at all ages. Similar normal staining patterns were seen in the brains of patients who had suffered trauma, tumors, cerebral infarcts, grade 1 periventricular hemorrhages, and in those who had suffered from amyotrophic lateral sclerosis, Parkinson's disease, multi-systems atrophy and Shy-Drager syndrome. An absence of cell body staining and only minimal axonal staining was noted in the same brains with immunocytochemistry using PHF-1, a monoclonal antibody generated against paired helical filament proteins from Alzheimer brains. The characteristic staining pattern of Alz-50 in normal brains is substantially more extensive than has previously been recognized. This pattern, which presumably describes a specific class of tau proteins, must be distinguished from the pathological staining observed in neurodegenerative diseases.

The macula densa mechanism for control of renin secretion.

Many questions remain concerning the precise mechanism by which the luminal signal for renin secretion is transmitted from the MD to the JG granular cell. It appears likely that a number of pathways may participate in MD-mediated changes in renin release, some of the agents serving to modulate release, others participating directly in signal transmission. Collectively, the current information suggests a transmission pathway that involves several steps. First, changes occurring in MD NaCl transport, which result directly from alterations in luminal NaCl concentration, act to initiate the response. Second, conveyance of this signal to the extraglomerular mesangial cells via a change in the ionic environment of the EGM field and/or a paracrine factor produced by the MD (perhaps NO) appear likely to be the next step. The subsequent steps probably include changes in prostaglandin production by the EGM, and possible participation of other paracrine factors produced by both endothelial and extraglomerular mesangial cells.

Heat shock-like protein is transferred from glia to axon.

Glia-axon protein transfer was examined in the squid giant axon. Proteins synthesized by the glial sheath surrounding the axon were labeled with [3H]leucine. Raising the temperature of the incubation medium from 20 degrees C to 30 degrees C increased the synthesis of glial proteins that resembled heat-shock proteins. These proteins were among the group known to be transferred into the axon. Thus, glia provide the axon with proteins that may be involved in the reaction to trauma.

Neurofibrillary tangles in the cerebral cortex of sheep.

Neurofibrillary degeneration, including neurofibrillary tangles (NFTs) and neuritic plaques, is an important pathological hallmark of Alzheimer's disease (AD). Unfortunately, no practical animal model of neurofibrillary degeneration has been described. We report here the presence of structures in the cerebral cortex of sheep, Ovis aries, that resemble Alzheimer NFTs and neuritic plaques. NFT-like structures and clusters of degenerating neurites are stained by silver impregnation and thioflavin-S, and are immunoreactive with antibodies against tau microtubule-associated proteins. Viewed under the electron microscope, tau-immunoreactive tangles consist of paired helical filaments. Naturally occurring neurofibrillary structures in sheep cortex provide a model for studying the pathobiology of Alzheimer's disease.

Hemodynamic effects of platelet-activating factor in nonpregnant and pregnant sheep.

The present study was designed to assess the dose-related effects of platelet-activating factor (PAF) on systemic, renal, and uterine hemodynamics in nonpregnant sheep and to evaluate how pregnancy might alter these responses. Nonpregnant and pregnant (110 +/- 5 days gestation) ewes were instrumented for conscious measurements of maternal mean arterial pressure (MAP), renal blood flow (RBF), uterine blood flow (UBF), hematocrit, and urinary protein concentration. After recovery, dose-response curves to PAF were generated by systemic infusion at 10, 30, and 100 ng. kg(-1). min(-1) (15 min/dose) into the maternal femoral vein. The above parameters were measured, and renal and uterine vascular resistances (RVR and UVR, respectively) were calculated. In pregnant sheep, PAF increased MAP, RVR, UVR, and urinary protein concentration. We also observed increases in hematocrit, indicative of reduced blood volume secondary to increased systemic microvascular protein permeability. These responses were similar in nonpregnant sheep, with the exception of UVR in nonpregnant ewes being decreased (and thus UBF was increased), whereas in pregnant sheep, UVR was increased, which resulted in decreased UBF. This suggests that pregnancy alters the mechanism of action of PAF within the uterine vasculature in a way that can reduce UBF and thereby potentially compromise placental perfusion.

Comparison of labeled heat shock proteins in neuronal and non-neuronal cells of Aplysia californica.

Aplysia californica has been used to study the protein synthetic response of nervous tissue to stress induced by elevated temperatures. The abdominal and pleural ganglia as well as associated connectives were exposed to various temperatures for 30 min, labeled with [33S]methionine at room temperature, and then analyzed by sodium dodecyl sulfate gel electrophoresis. All cells examined responded to temperatures of greater than 31 degrees C by a reduction in levels of labeled actin, as well as by the enhanced labeling of proteins with apparent Mr of 70,000 and 110,000. Two-dimensional electrophoresis indicated that the molecular weight and isoelectric focusing properties are similar to the heat shock proteins (HSPs) observed in other systems. In addition to these major HSPs, heat-induced proteins with molecular weights ranging from 70,000 to 90,000 were highly labeled in the neurosecretory bag cells. Further cell type-specific differences in the protein synthetic response to elevated temperatures were revealed by quantitation of the major HSPs. Levels of labeled HSPs were significantly lower in ganglion cells as compared to the non-neuronal connective cells. In addition, the decrease in actin levels appeared to be less dramatic in the ganglion cells. Analysis of the cellular compartmentalization of HSPs suggests that both neurons and glia are capable of HSP synthesis. Studies in the squid have demonstrated that HSPs are transferred from adaxonal glia into the axoplasm (Tytell, M., S. G. Greenberg, and R. J. Lasek, unpublished observation).(ABSTRACT TRUNCATED AT 250 WORDS)

Neurofilament protein synthesis in DRG neurons decreases more after peripheral axotomy than after central axotomy.

Cytoskeletal protein synthesis was studied in DRG neurons after transecting either their peripheral or their central branch axons. Specifically, the axons were transected 5-10 mm from the lumbar-5 ganglion on one side of the animal; the DRGs from the transected side and contralateral control side were labeled with radiolabeled amino acids in vitro; radiolabeled proteins were separated by 2-dimensional (2D) PAGE; and the amounts of radiolabel in certain proteins of the experimental and control ganglia were quantified and compared. We focused on the neurofilament proteins because they are neuron-specific. If either the peripheral or central axons were cut, the amounts of radiolabeled neurofilament protein synthesized by the DRG neurons decreased between 1 and 10 d after transection. Neurofilament protein labeling decreased more after transection of the peripheral axons than after transection of the central axons. In contrast to axonal transections, sham operations or heat shock did not decrease the radiolabeling of the neurofilament proteins, and these procedures also affected the labeling of actin, tubulin, and the heat-shock proteins differently from transection. These results and others indicate that axonal transection leads to specific changes in the synthesis of cytoskeletal proteins of DRG neurons, and that these changes differ from those produced by stress to the animal or ganglia. Studies of the changes in neurofilament protein synthesis from 1 to 40 d after axonal transection indicate that the amounts of radiolabeled neurofilament protein synthesis were decreased during axonal elongation, but that they returned toward control levels when the axons reached cells that stopped elongation.(ABSTRACT TRUNCATED AT 250 WORDS)

A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis.

Paired helical filaments (PHFs) are prominent components of Alzheimer disease (AD) neurofibrillary tangles (NFTs). Rather than isolating NFTs, we selected for PHF populations that can be extracted from AD brain homogenates. About 50% of PHF immunoreactivity can be obtained in 27,200 x g supernatants following homogenization in buffers containing 0.8 M NaCl. We further enriched for PHFs by taking advantage of their insolubility in the presence of zwitterionic detergents and 2-mercaptoethanol, removal of aggregates by filtration through 0.45-microns filters, and sucrose density centrifugation. PHF-enriched fractions contained two to five proteins of 57-68 kDa that displayed the same antigenic properties as PHFs. Since the 57- to 68-kDa PHF proteins are antigenically related to tau proteins, they are similar to the tau proteins previously observed in NFTs. However, further analysis revealed that PHF-associated tau can be distinguished from normal, soluble tau by PHF antibodies that do not recognize human adult tau and by one- and two-dimensional PAGE.

Neurogenic regulation of rate of achieving sodium balance after increasing sodium intake.

Evidence that the renal sympathetic nerves have direct effects on renal tubular function suggests that neurogenic mechanisms may play an important role in the daily regulation of sodium balance. We evaluated the influence of the renal nerves on the rate of elevating urinary sodium excretion (UNaV) after a step increase in fixed sodium intake. Conscious rats with innervated (INN) or denervated (DNX) kidneys were placed on low-sodium intake (LNa = 0.3 meq/day) or a normal sodium intake (NNa = 1.0 meq/day) by intravenous infusion. Hourly changes in UNaV were determined 24 h before and 72 h after increasing sodium intake to either NNa or high-sodium intake (HNa = 5.0 meq/day). Switching from LNa to NNa, INN rats increased UNaV within 24 h; however, DNX rats did not begin to increase UNaV until hour 60. Cumulative sodium balance over 72 h was more positive in DNX rats (INN = 1.29 +/- 0.29 meq; DNX = 2.06 +/- 0.21 meq, P less than 0.05). During the LNa-to-HNa switch, both INN and DNX rats increased UNaV equally for 12 h; however, at this time INN rats continued to increase UNaV, whereas DNX rats did not. DNX rats had a net accumulation of 2.54 meq more sodium than INN rats over 72 h. Significant inhibition of plasma renin activity within the first 24 h occurred only in rats receiving the LNa-to-HNa switch in sodium intake, and this response was not different between rats with innervated and denervated kidneys. These data suggest that the renal nerves provide a rapid sodium excretory response to step increases in sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional role of angiotensin II type 1 and 2 receptors in regulation of uterine blood flow in nonpregnant sheep.

The objective was to determine the receptor subtype of angiotensin II (ANG II) that is responsible for vasoconstriction in the nonpregnant ovine uterine and systemic vasculatures. Seven nonpregnant estrogenized ewes with indwelling uterine artery catheters and flow probes received bolus injections (0.1, 0.3 and 1 microg) of ANG II locally into the uterine artery followed by a systemic infusion of ANG II at 100 ng x kg(-1) x min(-1) for 10 min to determine uterine vasoconstrictor responses. Uterine ANG II dose-response curves were repeated following administration of the ANG II type 2 receptor (AT(2)) antagonist PD-123319 and then repeated again in the presence of an ANG II type 1 receptor (AT(1)) antagonist L-158809. In a second experiment, designed to investigate the mechanism of ANG II potentiation that occurred in the presence of AT(2) blockade, nonestrogenized sheep received a uterine artery infusion of L-158809 (3 mg/min for 5 min) prior to the infusion of 0.03 microg/min of ANG II for 10 min. ANG II produced dose-dependent decreases in uterine blood flow (P < 0.03), which were potentiated in the presence of the AT(2) antagonist (P < 0.02). Addition of the AT(1) antagonist abolished the uterine vascular responses and blocked ANG II-induced increases in systemic arterial pressure (P < 0.01). Significant uterine vasodilation (P < 0.01) was noted with AT(1) blockade in the second experiment, which was reversed by administration of the AT(2) antagonist or by the nitric oxide synthetase inhibitor N(omega)-nitro-L-arginine methyl ester. We conclude that the AT(1)-receptors mediate the systemic and uterine vasoconstrictor responses to ANG II in the nonpregnant ewe. AT(2)-receptor blockade resulted in a potentiation of the uterine vasoconstrictor response to ANG II, suggesting that the AT(2)-receptor subtype may modulate uterine vascular responses to ANG II potentially by release of nitric oxide.

Effect of nitric oxide on renin secretion. I. Studies in isolated juxtaglomerular granular cells.

Experiments were performed on juxtaglomerular granular cells (JGC) in short-term primary culture to determine the direct immediate effect of NO on renin secretion and to test whether JGC are able to generate NO. Renin secretion was measured repeatedly over short time intervals in a cell superfusion system. Renin release did not significantly decrease over a 40-min observation period in untreated JGC. Addition of sodium nitroprusside (SNP) caused a reduction in renin release (measured in nano-Goldblatt hog units vs. time, i.e., nGU/min) from 479 +/- 25, 423 +/- 70, and 388 +/- 54 nGU/min to 295 +/- 19 (n = 5), 102 +/- 21 (n = 7), and 71 +/- 9 nGU/min (n = 6) with 10(-5), 10(-4), and 10(-3) M SNP, respectively. In the presence of the guanylate cyclase inhibitor methylene blue at 10(-4) M, SNP at 10(-4) M had no significant effect on renin secretion. 8-Bromoguanosine 3',5'-cyclic monophosphate at 10(-4) M in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-3) M) caused a reduction of renin secretion to 50.1 +/- 3.6% of control. To examine the possibility that renin secretion is affected by NO release from JGC, we assessed the effect of the NO synthase (NOS) substrate L-arginine (10(-3) M) and the NOS blocker N omega-nitro-L-arginine (10(-4) M) on renin secretion. Renin release was not significantly altered by either stimulation or inhibition of NOS activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nitric oxide on renin secretion. II. Studies in the perfused juxtaglomerular apparatus.

To examine the possible role of NO in macula densa control of renin secretion, we examined the effects of varying NO availability on renin release in the isolated perfused rabbit juxtaglomerular apparatus (JGA). Gradual increments of luminal Na/Cl concentration ratio (mM/mM) from 26/7 over 46/27, 66/47, to 86/67 caused a progressive decrease in renin secretion from (as log of nano-Goldblatt hog units vs. time, i.e., log nGU/min) 1.09 +/- 0.34 to 0.46 +/- 0.24 log nGU/min, with the greatest change occurring at the first concentration step. The presence of 0.7 mM N omega-nitro-L-arginine (NNA), an NO synthase inhibitor, in the luminal fluid significantly reduced renin secretion at the lowest Na/Cl concentration ratio to 0.65 +/- 0.32 log nGU/min (P < 0.01 compared with control). Renin secretion at the higher Na/Cl concentration ratios was not significantly affected by NNA compared with control. In contrast to these results, the addition of the NO donor nitroprusside (1 mM) to the bath caused a reduction in renin secretion from 1.0 +/- 0.39 to 0.47 +/- 0.46 log nGU/min (P < 0.05), an effect that was reversed by bath addition of 0.01 mM methylene blue. Similarly, addition of L-arginine (0.7 mM) to the bath reduced renin secretion from 0.99 +/- 0.37 to 0.81 +/- 0.38 log nGU/min (P < 0.01), whereas addition of L-arginine to the luminal fluid increased renin secretion from 0.85 +/- 0.43 to 1.94 +/- 0.46 log nGU/min (P < 0.05). The stimulatory effect of luminal L-arginine was reversed by the luminal addition of NNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevation of nitrate levels in pregnant ewes and their fetuses.

OBJECTIVE: Nitric oxide is a potent vasodilator released by endothelial cells that may play an important role in modulating maternal and fetal vascular tone in normal pregnancy. The current study was designed to evaluate whether plasma or urine nitrite and nitrate (the metabolites of nitric oxide) concentrations are elevated in pregnant compared with those of nonpregnant sheep and whether the nitrate concentrations in the fetal circulation were increased in comparison with the maternal circulation. STUDY DESIGN: Eleven pregnant sheep and seven nonpregnant oophorectomized sheep were instrumented with catheters in the maternal and fetal femoral arteries and veins, uterine and umbilical veins, and amniotic cavity. Blood, urine, and amniotic fluid samples were collected for nitrate determination at least 5 days after surgery. After extraction nitrate was reduced to nitrite and quantitated with the Greiss reagent. RESULTS: Arterial plasma nitrate concentrations in the pregnant sheep were significantly elevated compared with those of nonpregnant sheet (5.0 +/- 0.9 vs 2.5 +/- 0.6 micromol/L, p < 0.05). The urinary nitrate concentrations were also significantly increased in the pregnant sheep compared with those of nonpregnant sheet (89.9 +/- 16.3 vs 23.1 +/- 4.5 nmol/mg creatinine, p < 0.01). Fetal plasma nitrate concentrations were ninefold higher than the maternal nitrate concentrations (43.9 +/- 7 vs 5.0 +/- 0.9 micromol/L, p < 0.01), whereas amniotic fluid concentrations were extremely high (133.8 +/- 13.8 micromol/L, n = 3). No venous-arterial differences were measurable across either the maternal or fetal sides of the placenta. CONCLUSION: Nitrate concentrations in pregnant sheet and their fetuses are increased. The increased nitrate concentrations in the maternal and fetal circulations may reflect the increased nitric oxide synthesis, which may in part mediate the cardiovascular adaptations to normal pregnancy and the low systemic and umbilical vascular resistance in the fetus.

Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.

Tau (tau) is a major constituent of paired helical filaments (PHF) found in Alzheimer's disease. The current study examines the possibility that the distinct properties of PHF-associated tau proteins (tau PHF) result from post-translational modifications of normal soluble tau (tau s). Following hydrofluoric acid (HF) treatment, tau PHF proteins are heat- and acid-stable, soluble in 2-(N-morpholino)ethanesulfonic acid buffers and display the same molecular weight, pI, and immunochemical properties as normal tau s. Alkaline phosphatase treatment of dissociated PHF results in similar, although less extensive, electrophoretic changes and a reduction in PHF-1 immunoreactivity. Therefore, phosphorylation of normal tau s appears to be responsible for the distinct properties of tau PHF. Although our results suggest that all of the normal tau isoforms are in PHF, the relative abundance of individual tau species differs in HF-treated PHF and tau s samples. Moreover, the loss of PHF following HF treatment suggests that post-translational modifications contribute to the structural stability of PHF.

Effect of prostaglandin synthesis inhibition on macula densa-stimulated renin secretion.

The purpose of the present studies was to evaluate directly the role of prostaglandins in macula densa-mediated renin release. Individual juxtaglomerular apparatus specimens were microdissected from rabbit kidney and perfused with a solution containing either high NaCl (Na+ = 141 meq/l; Cl- = 122 meq/l) or low NaCl (Na+ = 26 meq/l; Cl- = 7 meq/l) concentration. With a step decrease in perfusate NaCl (high to low), renin secretion rate was markedly stimulated (from 15.06 to 63.1 nGU/min, P < 0.01), and the response was almost fully reversible. When specimens were bathed with cyclooxygenase inhibitors flurbiprofen (10(-5) M) or flufenamic acid (10(-4) M), this macula densa-activated increase in renin release was largely or completely abolished (flurbiprofen, 3.5-10.5 nGU/min, not significant; flufenamic acid, 9.0-12.3 nGU/min, not significant). Exposing the macula densa to a step increase in perfusate NaCl concentration (low to high) resulted in a significant and reversible suppression of renin secretion in control specimens, but no significant suppression was seen in specimens treated with flufenamic acid. These data provide direct evidence to support the hypothesis that locally produced prostaglandins may act as a primary mediator of the renin response to macula densa activation.

Mechanism of uterine vascular refractoriness to endothelin-1 in pregnant sheep.

Endothelin-1 (ET-1) is a potent vasoconstrictor and produces marked pressor responses when given systemically. Studies in sheep have demonstrated that during pregnancy the uterine vasculature is refractory to exogenously administered ET-1. We hypothesize that this pregnancy-dependent refractoriness is due to an upregulation of local uterine metabolism of ET-1 and/or ET(B) receptors and/or downregulation of local uterine ET(A) receptors. To investigate these possibilities, 21 nonpregnant and 17 pregnant sheep were used. Dose-response curves to intravenous infusion of ET-1 and phenylephrine were generated for pregnant and nonpregnant sheep. ET-1 infused systemically demonstrated vasoconstriction in the systemic and renal vasculature of pregnant and nonpregnant animals and vasoconstriction in the uterine vasculature of nonpregnant animals. The pregnant animals showed no uterine vascular response to ET-1. In contrast, phenylephrine showed vasoconstriction in the systemic, renal, and uterine circulations in both pregnant and nonpregnant sheep. After experimentation, the animals were euthanized, and tissues were harvested for Western blot and activity analysis of neutral endopeptidase (NEP) or RT-PCR analysis of endothelin-converting enzyme (ECE) and ET(A) and ET(B) receptors. The content and activity of NEP in the uterine and renal vasculature of pregnant and nonpregnant animals were similar. RT-PCR demonstrated the presence of ECE in the uterine vasculature of pregnant and nonpregnant sheep. ET(A) receptor mRNA was significantly reduced in pregnant compared with nonpregnant sheep, whereas ET(B) receptor mRNA remained unchanged. We conclude that the uterine vascular refractoriness seen in the pregnant sheep is due to a downregulation of ET(A) receptors.