D(1) dopamine receptor regulation of NHE3 during development in spontaneously hypertensive rats.

To determine if the defective interactions among D(1)-like receptors, G proteins, and Na(+)/H(+) exchanger 3 (NHE3) are consequences of hypertension, we studied these interactions in rats, before (2--3 wk) and after (12 wk) the establishment of hypertension. To eliminate the confounding influence of second messenger action on D(1) receptor-NHE3 interaction, studies were performed in renal brush-border membranes (BBM) devoid of cytoplasmic second messengers. NHE3 activity increased with age in Wistar-Kyoto (WKY) rats (3 wk = 1.48 +/- 0.39, n = 13; 12 wk = 2.83 +/- 0.15, n = 16, P < 0.05) but not in spontaneously hypertensive rats (SHRs; 3 wk = 2.52 +/- 0.37, n = 11; 12 wk = 2.81 +/- 0.20, n = 16). D(1) receptor protein tended to decrease, whereas NHE3 protein tended to increase with age in both WKY and SHRs. However, the inhibitory effect of a D(1)-like agonist, SKF-81297, on NHE3 activity increased with age in WKY rats (3 wk = -40.7 +/- 5.3%, n = 10, 12 wk = -58.7 +/- 4.6%, n = 12, P < 0.05) but not in SHRs (3 wk = -27.6 +/- 5.9%, n = 11, 12 wk = -25.1 +/- 3.2%, n = 11). The decreased inhibitory effect of another D(1)-like agonist, fenoldopam, on NHE3 activity in SHRs was not caused by increased activity and binding of G beta gamma to NHE3 as has been reported in young WKY rats. G(s)alpha mediates, in part, the inhibitory effect of D(1)-like agonists on NHE3 activity. In WKY rats, fenoldopam increased G(s)alpha/NHE3 binding to the same extent in 2-wk-old (1.5-fold, n = 4) and adult (1.5-fold, n = 4) rats. In contrast, in SHRs, fenoldopam decreased the amount of G(s)alpha bound to NHE3 in 2-wk-old SHRs and had no effect in 4-wk-old and adult SHRs. These studies indicate that the decreased inhibitory effect of D(1)-like agonists on NHE3 activity in SHRs (compared with WKY rats) precedes the development of hypertension. This may be caused, in part, by a decreased interaction between G(s)alpha and NHE3 in BBM secondary to impaired D(1)-like receptor function.

Autonomic adjustments to severe hypotension in fetal and neonatal sheep.

In fetal sheep, severe hypotension causes heart rate (HR) slowing. Studies during development have also shown that a reflex bradycardia and hypotension can be elicited after chemostimulation with veratridine and is dependent on the age of the animal. In adults, a vagally mediated depressor reflex characterized by bradycardia, hypotension, and withdrawal of efferent sympathetic activity can be observed after stimulation of chemosensitive or mechanosensitive cardiac receptors with veratridine or in circumstances of reduced cardiac filling. This reflex, known as the Bezold-Jarisch reflex, plays a role in disease states such as myocardial ischemia and hemorrhage. The objectives of our study were to determine whether a sympathoinhibitor depressor reflex, along with the bradycardia, is observed during pharmacologically induced hypotension in fetal and newborn lambs. In both fetal and newborn lambs, HR and renal sympathetic nerve activity (RSNA) initially increased (p < 0.05) in response to nitroprusside infusion to reach a maximum value. The range (or "plateau") of mean arterial blood pressure over which maximum RSNA was maintained constant before withdrawal of sympathetic tone started to be observed was significantly (p < 0.05) smaller in fetuses (0.3 +/- 0.3 mm Hg) than newborn (6 +/- 1 mm Hg) lambs. Similarly, the plateau over which maximum HR was maintained before onset of bradycardia was significantly smaller in fetuses (4 +/- 1 versus 11 +/- 2 mm Hg). The mean arterial blood pressure level ("threshold") at which a depressor reflex was triggered was significantly (p < 0.05) lower in fetal than newborn sheep (35 +/- 2 versus 53 +/- 3 mm Hg for HR and 35 +/- 2 versus 57 +/- 2 mm Hg for RSNA). The rates of fall (slopes) for both HR and RSNA were also significantly (p < 0.05) more pronounced in fetuses (1.85 +/- 0.27 and 6.08 +/- 2.45%/mm Hg) than in newborns (1.21 +/- 0.16 and 1.97 +/- 0.32%/mm Hg). Bilateral vagotomy significantly increased the plateau of mean arterial blood pressure over which maximum RSNA and HR were maintained constant. Vagotomy also decreased the threshold for both RSNA and HR and the slope of the RSNA response to the nitroprusside infusion in newborn lambs. Results from this study show that activation of the arterial baroreflex during nitroprusside-induced hypotension is followed by withdrawal of sympathetic tone and bradycardia and that this depressor reflex is more pronounced in late-gestation fetuses than newborn lambs and is significantly attenuated after bilateral vagotomy in newborn lambs.

[Regulation of NHE3 by D1 dopamine receptor during development of spontaneously hypertensive rats].

OBJECTIVE: To determine if spontaneous hypertension is secondary to defective interaction among dopamine receptor, G protein, and Na(+)/H(+) exchanger 3 (NHE3). METHODS: The inhibitory effect of a D(1) dopamine agonist upon NHE3 activity and its impact upon G(s)alpha/NHE3 binding in renal brush border membrane (BBM) of spontaneously hypertensive rats (SHRs) 2 - 3 weeks before and 12 weeks after the establishment of hypertension were examined. In order to avoid the confounding influence of second messenger on D(1) receptor/NHE3 interaction, study was made in BBM devoid of cytoplasmic component. RESULTS: NHE3 activity increased with age in Wister-Kyoto (WKY) rats but not in SHRS. D1 receptor expression did change with age in both WKY rats and SHRs. The inhibitory effect of a D(1)-like agonist on NHE3 activity increased with age in WKY rats but not in SHRs. In WKY rats, another D(1)-like agonist, fedoldopam, increased G(s)alpha/NHE3 binding to the same extent in 2 week old and adult rats, but decreased the amount of G(s)(alpha)bound to NHE3 in 2 week old SHRs. CONCLUSION: The decrease of inhibitory effect of D(1)-like agonist upon NHE3 activity in SHRs precedes the development of hypertension. Spontaneous hypertension may be caused, in part, by a decreased interaction between G(s)alpha and NHE3 in BBM secondary to D(1)-like receptor function.

Successful bone marrow transplantation in a patient with Schimke immuno-osseous dysplasia.

Early death in Schimke immuno-osseous dysplasia often results from renal failure and/or cell-mediated immunodeficiency. Kidney transplants have improved renal function, but effective therapy for the immunodeficiency has not yet been reported. We describe markedly improved marrow function 2 years after bone marrow transplantation in a boy with Schimke immunoosseous dysplasia.

Gbeta regulation of Na/H exchanger-3 activity in rat renal proximal tubules during development.

The decreased natriuretic action of dopamine in the young has been attributed to decreased generation of cAMP by the activated renal D(1)-like receptor. However, sodium/hydrogen exchanger (NHE) 3 activity in renal brush-border membrane vesicles (BBMV) can be modulated independent of cytoplasmic second messengers. We therefore studied D(1)-like receptor regulation of NHE activity in BBMVs in 2-, 4-, and 12-wk-old (adult) rats. Basal NHE activity was least in 2-wk-old compared with 4- and 12-wk-old rats. D(1)-like agonist (SKF-81297) inhibition of NHE activity was also least in 2-wk-old (-1 +/- 9%, n = 3) compared with 4 (-15 +/- 5%, n = 6)- and 12 (-65 +/- 4%, n = 6)-wk-old rats. The decreased response to the D(1)-like agonist in BBMV was not caused by decreased D(1) receptors or NHE3 expression in the young. G(s)alpha, which inhibits NHE3 activity by itself, coimmunoprecipitated with NHE3 to the same extent in 2-wk-old and adult rats. G(s)alpha function was also not impaired in the young because guanosine 5'-O-(3-thiotriphosphate) decreased NHE activity to a similar extent in 4-wk-old and adult rats. Galpha(i-3) protein expression in BBMV also did not change with age. In contrast, Gbeta expression and the amount of Gbeta that coimmunoprecipitated with NHE3 in BBMV was greatest in 2-wk-old rats and decreased with age. Gbeta common antibodies did not affect D(1)-like agonist inhibition of NHE activity in adult rats (8%) but markedly increased it (48%)in 4-wk-old rats. We conclude that the decreased inhibitory effect of D(1)-like receptors on NHE activity in BBMV in young rats is caused, in part, by the increased expression and activity of the G protein subunit Gbeta/gamma. The direct regulation of NHE activity by G protein subunits may be an important step in the maturation of renal tubular ion transport.

Sympathetic responses to cardiopulmonary vagal afferent stimulation during development.

Previous work in our laboratory has demonstrated impairment of cardiopulmonary reflex control of renal sympathetic nerve activity (RSNA) during the newborn period. The present study was designed to test the hypothesis that this delayed maturation is secondary to incomplete central integration of vagal afferent input. Term fetal (135-140 days; n = 6), newborn (3-7 days of age; n = 8), and young adult (6-8 wk old; n = 8) sheep anesthetized with alpha-chloralose underwent vagal afferent nerve stimulation. All animals had undergone prior sinoaortic denervation to eliminate influences from the arterial baroreceptors. After determination of optimal stimulation parameters, RSNA responses to gradual increases in stimulation frequency (1.0-16 Hz) were recorded and compared by one-way ANOVA. RSNA decreased progressively with increased frequency of stimulation in all three groups of animals. When comparing the three groups at any given frequency of stimulation, reflex withdrawal of RSNA tended to be more pronounced in newborn lambs (P < 0.05 for 1 and 4 Hz). Heart rate (HR) was also noted to decrease significantly with vagal afferent stimulation in each of the groups, but no significant differences in the reflex decreases in HR were noted among the three groups of animals. These results demonstrate that central integration of vagal afferent input is intact in fetal and newborn sheep. These results suggest that the delayed maturation of cardiopulmonary reflex-mediated changes in RSNA seen early in development appears to depend on intrinsic alterations in baroreceptor function rather than incomplete central integration.

Effect of antenatal glucocorticoids on sympathetic nerve activity at birth in preterm sheep.

Renal sympathetic nerve activity (RSNA) increases rapidly after delivery of term fetal sheep and parallels the rise in heart rate (HR) and arterial pressure. To examine the RSNA response at birth in immature lambs, experiments were performed in chronically instrumented preterm fetal sheep (118- to 125-day gestation, term 145 days) before and after delivery by cesarean section. HR remained unchanged from fetal values at 1 and 4 h after birth, whereas mean arterial blood pressure (MABP) decreased significantly (P < 0.05) by 4 h after delivery. RSNA significantly decreased after premature birth in all animals studied (n = 6), achieving only 39 +/- 17% of fetal RSNA (P < 0.05; all results are mean +/- SE). Because cardiovascular function after premature birth is improved by the use of antenatal corticosteroids, we also tested the hypothesis that corticosteroid administration would evoke a more pronounced sympathetic response in prematurely delivered lambs (n = 7, 118- to 125-day gestation). After maternal administration of dexamethasone (5 mg i.m., 48 and 24 h before delivery), RSNA increased after birth in six of seven fetuses to 166 +/- 32% of the fetal RSNA value. Dexamethasone treatment also decreased the sensitivity of baroreflex-mediated changes in HR in response to increases in MABP. Because the sympathetic response at birth is depressed in preterm compared with term lambs, we performed an additional study (n = 8) to determine if immature sheep are capable of mounting a sympathetic response to cold. In utero cooling produced rapid and sustained increases in MABP (20 +/- 4%), HR (26 +/- 6%), and RSNA (282 +/- 72%) (all P < 0.05), consistent with a generalized sympathoexcitation. These results suggest that sympathoexcitation is absent after premature delivery despite the presence of functional descending autonomic pathways. Furthermore, exogenous corticosteroids appear to have a maturational effect on the sympathetic response at birth, which may be one mechanism by which maternal steroid administration improves postnatal cardiovascular homeostasis.

Role of endogenous ANG II and AT1 receptors in regulating arterial baroreflex responses in newborn lambs.

The present study was designed to test the hypothesis that endogenous angiotensin II (ANG II) influences baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) early in life and to determine whether these actions are mediated by angiotensin AT1 or AT2 receptors. To test this hypothesis, we studied the effects of systemic and central administration of losartan, a selective AT1 receptor antagonist, and PD-123319, a selective AT2 antagonist, on baroreflex-mediated control of HR and RSNA in conscious newborn lambs. Systemic administration of losartan decreased resting mean arterial blood pressure (MABP) from 70 +/- 3 to 58 +/- 4 mmHg (P < 0.05) without producing reflex increases in HR or RSNA. The baroreflex response curves were shifted to the left as indicated by a decrease in the arterial pressure at the midpoint of the curve for HR (83 +/- 3 to 75 +/- 4 mmHg) and RSNA (74 +/- 2 to 69 +/- 3 mmHg; P < 0.05 for both). Losartan also reset HR and RSNA baroreflex curves when changes in baseline blood pressure were prevented by simultaneous infusion of phenylephrine. In contrast, a sustained decrease in arterial pressure of 10-12 mmHg with nitroprusside failed to shift the baroreflex function curves. PD-123319 had no effect on baseline HR, MABP, RSNA, or baroreflex responses. Lateral ventricle administration of losartan but not PD-123319 also produced a decrease in arterial pressure (81 +/- 4 to 73 +/- 3 mmHg, P < 0.05) and reset the baroreflex for HR and RSNA toward lower pressure. These results demonstrate that, early in life, endogenous ANG II exerts a tonic effect on baroreflex control of HR and RSNA to shift the curves toward higher pressure levels. The alterations in arterial baroreflex function appear independent of direct ANG II effects on arterial pressure and are mediated by AT1 receptors.

Angiotensin II and the maturation of renal cortical Na+/H+ exchanger activity during fetal life in sheep.

The postnatal rise in renal Na+ reabsorption is associated with an increase in proximal tubule apical membrane Na+/H+ exchanger (NHE) activity in sheep. Inasmuch as circulating angiotensin II (ANG II) levels increase immediately after birth and ANG II is known to upregulate NHE activity in the adult proximal tubule, we postulated that ANG II plays a role in mediating maturational changes in NHE activity. We therefore studied the effects of ANG II infusion (10 micrograms/h) for 24 h on renal cortical NHE activity in chronically instrumented, twin ovine fetuses (129 +/- 2 days gestation, term is 145 days, n = 10 pairs); one twin of each pair served as a control. After 24 h, the fetuses were killed and brush-border membrane vesicles (BBMV) were prepared from the renal cortices. Postinfusion plasma ANG II levels were significantly higher and plasma renin activities were significantly lower in treated fetuses compared with controls. Kinetic analysis revealed an increase in NHE activity after ANG II treatment; however, the difference was not statistically significant: maximal velocity (in nmol.s-1.mg protein-1) control 1.65 +/- 0.50, treated 2.31 +/- 0.66 (P = 0.11, n = 9 pairs); Michaelis constant control 8.29 +/- 1.17 mM, treated 9.84 +/- 1.26 mM (P = 0.11). Northern blots of total RNA from the cortices of these animals were hybridized to a D-[32P]UTP-labeled antisense RNA probe prepared from a 1.3-kb rat NHE3 cDNA fragment. There were no differences between the groups in NHE3 mRNA levels (32P counts were control 413 +/- 54, treated 340 +/- 46). ANG II does not appear to play an important role in the regulation of NHE activity in the proximal tubule of the near-term sheep fetus.

Arginine vasopressin modulation of arterial baroreflex responses in fetal and newborn sheep.

The present study was designed to test the hypothesis that the influence of circulating vasopressin (AVP) on the arterial baroreflex control of renal sympathetic nerve activity (RSNA) and heart rate (HR) changes during development. To test this hypothesis, we studied arterial baroreflex-mediated control of HR and RSNA in the presence of increasing plasma levels of AVP in conscious, chronically instrumented fetal, newborn, and adult sheep. In fetal and newborn sheep, increasing plasma AVP levels (from < 10 to > 200 microU/ml) increased resting levels of mean arterial blood pressure (MABP) and decreased HR and RSNA. HR and RSNA baroreflex responses to variations of MABP with nitroprusside and phenylephrine infusion were not modified by elevated AVP levels in either newborn or fetal sheep, except for a small decrease in maximal HR response to nitroprusside infusion in the newborn animals. In contrast, in adults, AVP caused bradycardia and a decrease in RSNA without change in MABP, accompanied by resetting of the arterial baroreflex (decrease in maximal HR and RSNA, decrease in RSNA gain, and shift of HR to lower pressure). To test the hypothesis that the inability of AVP to reset the arterial baroreflex early during development was not secondary to maximal stimulation of V1 receptors during baseline conditions, we investigated the effect of V1-receptor blockade on baseline cardiovascular and arterial baroreflex function in newborn lambs. Administration of a V1-receptor antagonist produced no significant changes in resting MABP, HR, and RSNA and did not influence arterial baroreflex-mediated changes in HR and RSNA. These results indicate that, contrary to adults, circulating AVP does not modulate the arterial baroreflex in fetal and newborn sheep.

Dietary sodium effects on renin and angiotensinogen gene expression in preweanling WKY and SHR.

The influence of altered dietary sodium on angiotensinogen and renin gene expression was examined in young normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Artificial rearing was used to increase or decrease dietary sodium intake during the preweanling period. In normally reared control animals, renal renin and liver angiotensinogen mRNA decreased between 6 and 30 postnatal days of age. In contrast, in the central nervous system, angiotensinogen mRNA increased between 6 and 30 days of age, and renin mRNA remained stable. Dietary sodium manipulation between postnatal days (PD) 6 and 18 significantly influenced renal renin gene expression, with low-sodium diet increasing renin mRNA on PD12 and PD18 and high-sodium diet decreasing renin mRNA on PD18. Liver angiotensinogen mRNA decreased for animals on either diet on PD12 and PD18. Brain angiotensinogen and renin mRNA were not affected by dietary sodium levels. There were no strain-related differences in the response to high and low dietary sodium. These results demonstrate that 1) the peripheral and central renin-angiotensin systems do not have a common ontogenetic pattern of development, 2) they are independently regulated in response to dietary sodium variations, and 3) young WKY and SHR share very similar ontogenetic patterns of angiotensinogen and renin gene expression.

Cardiovascular, renal, and endocrine responses to graded volume expansion in lambs during maturation.

To further investigate the maturation of the cardiopulmonary baroreflex, we measured the effects of a 45-min blood volume expansion to an increase in right atrial pressure of approximately 4 mmHg in chronically instrumented newborn (n = 17) and older lambs (n = 14). Measurements included various parameters of endocrine, cardiovascular, and renal function and concomitant recording of renal sympathetic nerve activity (RSNA). During blood volume expansion, RSNA was inhibited to a similar extent in newborns and older lambs when atrial pressures were increased by approximately 4 mmHg. A sympathoinhibition persisted in newborns but was only transient in older lambs. In newborn lambs, heart rate decreased in response to blood volume expansion, whereas heart rate remained constant after blood volume expansion in older lambs. The renal and endocrine responses to blood volume expansion were, however, similar in newborns and older lambs. These data provide evidence that when atrial pressures are matched, the renal and endocrine responses to blood volume expansion are similar, but there are differential cardiovascular and RSNA responses. Any reduced ability of the newborn kidney to excrete a volume load is therefore probably related to maturational differences in its distribution between the capacitance vessels and the heart.

Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.

Since dopamine produced by the kidney is an intrarenal regulator of sodium transport, an abnormality of the dopaminergic system may be important in the pathogenesis of hypertension. In the spontaneously hypertensive rat (SHR), in spite of normal renal production of dopamine and receptor density, there is defective transduction of the D1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na+/H+ exchanger [NHE] and Na+/K+ATPase activity) by dopamine. To determine if impaired D1 receptor regulation of NHE in proximal tubules is related to hypertension, studies were performed in a F2 generation from female Wistar Kyoto (WKY) and male SHR crosses. A D1 agonist, SKF 81297, inhibited (37.6 +/- 4.7%) NHE activity in brush border membranes of normotensive F2s (systolic blood pressure < 140 mm Hg, n = 7) but not in hypertensive F2s (n = 21). Furthermore, a D1 agonist, SKF 38393, when infused into the renal artery, dose dependently increased sodium excretion in normotensive F2s (n = 3) without altering renal blood flow but was inactive in hypertensive F2s (n = 21). Since the major D1 receptor gene expressed in renal proximal tubules is the D1A subtype, we determined the importance of this gene in the control of blood pressure in mice lacking functional D1A receptors. Systolic blood pressure was greater in homozygous (n = 6) and heterozygous (n = 5) mice compared to normal sex matched litter mate controls (n = 12); moreover, the mice lacking one or both D1A alleles developed diastolic hypertension. The cosegregation with hypertension of an impaired D1 receptor regulation of renal sodium transport and the development of elevated systolic and diastolic pressure in mice lacking one or both D1A alleles suggest a causal relationship of the D1A receptor gene with hypertension.

Chronic hypertension and altered baroreflex responses in transgenic mice containing the human renin and human angiotensinogen genes.

We have generated a transgenic model consisting of both the human renin and human angiotensinogen genes to study further the role played by the renin-angiotensin system in regulating arterial pressure. Transgenic mice containing either gene alone were normotensive, whereas mice containing both genes were chronically hypertensive. Plasma renin activity and plasma angiotensin II levels were both markedly elevated in the double transgenic mice compared with either single transgenic or nontransgenic controls. The elevation in blood pressure caused by the human transgenes was independent of the genotype at the endogenous renin locus and was equal in mice homozygous for the Ren-1c allele or in mice containing one copy each of Ren-1c, Ren-1d, or Ren-2. Chronic overproduction of angiotensin II in the double transgenic mice resulted in a resetting of the baroreflex control of heart rate to a higher pressure without significantly changing the gain or sensitivity of the reflex. Moreover, this change was not due to the effects of elevated pressure itself since angiotensin-converting enzyme inhibition had minimal effects on the baroreflex in spontaneously hypertensive BPH-2 control mice, which exhibit non-renin-dependent hypertension. This double transgenic model should provide an excellent tool for further studies on the mechanisms of hypertension initiated by the renin-angiotensin system.

Regulation of renal sympathetic nerve activity at birth.

The present studies were designed to assess the contribution of onset of respiration, separation from the placenta, and a decrease in environmental temperature on the increase in renal sympathetic nerve activity (RSNA) that occurs at birth. In the first series of experiments, heart rate (HR), mean arterial blood pressure (MABP), and RSNA were recorded in chronically instrumented near-term fetal sheep (n = 12) before and during in utero ventilation (V), V + oxygenation (V + O), and V + O + umbilical cord occlusion (V + O + CO). RSNA increased by 49 +/- 16% during V alone (P < 0.05), whereas no additional changes were seen with V + O or V + O + CO. HR and MABP did not change with any intervention. In a second series of experiments (n = 10), changes in fetal HR, MABP, and RSNA in response to in utero cooling were recorded. Cooling of the fetal core temperature by -3.1 +/- 0.2 degree C produced a rapid and sustained increase in RSNA (330 +/- 155%), HR (25 +/- 11%), and MABP (10 +/- 2%) consistent with generalized sympathoexcitation. In a third series of studies (n = 3), we found that brain stem transection between the rostral pons and posterior hypothalamus abolishes the increases in RSNA seen at birth. These results suggest that cooling is a major contributor to the postnatal rise in RSNA and that brain centers at the level of or above the hypothalamus are involved in mediating sympathoexcitation at birth.

Differential gene expression and regulation of renal angiotensin II receptor subtypes (AT1 and AT2) during fetal life in sheep.

Previous studies have shown that angiotensin II subtype 2 (AT2) receptors appear early during renal embryonic development. Factors involved in the regulation of AT2 receptors during renal development, however, have not been investigated. The present study was designed 1) to characterize the ontogeny of renal AT2 gene expression during the last half of gestation in fetal sheep and newborn lambs, 2) to compare changes in AT1 and AT2 gene expression during renal development, 3) to determine the influence of AII in modulating renal AT1 and AT2 gene expression during fetal life, and 4) to characterize the role of cortisol in modulating renal AT2 gene expression during the last trimester of gestation in fetal sheep. To perform these studies, we first isolated and cloned a polymerase chain reaction product that has 92 and 90% homology with the cDNA encoding the human and rat AT2 receptors, respectively. Using this sheep AT2 cDNA probe, we demonstrated that the sheep AT2 gene was encoded in a single locus. In addition, we showed that renal AT2 mRNA expression was high early during fetal life (60-90-d gestation) and decreased rapidly thereafter. In contrast, the expression of renal AT1 receptor gene was low at 60-d gestation and increased during the last trimester of gestation. We found that a continuous i.v. infusion (1 mL/h) of AII (9.5 mM/n) for 24 h, which raised plasma AII levels from 84 +/- 9 pg/mL to 210 +/- 21 pg/mL, decreased the expression of both renal AT1 and AT2 genes in third trimester fetal sheep. On the other hand, we observed that cortisol, known to decrease AT1 gene expression in the fetus, had no effect on AT2 gene expression. In summary, this study demonstrates that AII, but not glucocorticoids, contributes to the regulation of renal AT2 gene expression during development and that there is differential regulation of AT1 and AT2 receptors.

Alpha 1B-adrenergic receptors in rat renal microvessels.

Although several alpha-adrenergic receptor genes are expressed in the rat kidney, their expression in the renal vasculature has not been studied. Since pharmacological studies have suggested that an alpha 1B-adrenergic receptor may mediate renal vasoconstriction, we studied the expression of alpha 1B-adrenergic receptors in renal microvessels, from 10- to 14-week-old male spontaneously hypertensive rats (SHR) and their normotensive control, the Wistar-Kyoto rat (WKY). In these microvessels, isolated by perfusion with iron, alpha 1B-adrenergic receptor mRNA levels (by ribonuclease protection assay) were similar in SHR and WKY rats. Photo-affinity labeling with [125I]-arylazidoprazosin demonstrated the presence of alpha 1B-adrenergic receptor protein. Maximum receptor density (determined by 3H-prazosin binding: Bmax 59.8 +/- 4.1 and 58.7 +/- 4.3; Kd 0.48 +/- 0.05 nM and 0.31 +/- 0.06 nM in SHR and WKY, respectively) and chloroethylclonidine (CEC)-sensitive binding sites (determined by [125I]-(2-beta(4-hydroxyphenyl)-ethylaminomethyl)-tetralone binding) (125I-HEAT) were similar in SHR and WKY rats. There are two novel findings in these studies: (1) the alpha 1B-adrenergic receptor gene is expressed in renal microvessels of WKY and SHR; (2) alpha 1B-adrenergic receptor gene expression in renal microvessels is not altered in adult SHR. The failure to down-regulate expression of the alpha 1B-adrenergic receptor at the mRNA and protein level in the SHR could result in persistence of alpha 1B-adrenergic receptor effects and contribute to the increased vascular resistance in hypertension.

Transgenic animals in the study of blood pressure regulation and hypertension.

It is generally accepted that the etiology of essential hypertension is due to a complex interplay of genetic and environmental factors. A great deal of research effort over the past ten years has been focused on the identification of genes the variants of which predispose individuals to high blood pressure. Consequently, transgenic and knockout animals have become important research tools, providing experimental systems in which defined genetic manipulations can be introduced on uniform genetic backgrounds while minimizing environmental variation. These animal models have provided the means by which candidate genes thought to be involved in blood pressure regulation have been studied. Furthermore, these models can be used to test the significance of genes and gene variants identified via genome-wide searches as potential causes of hypertension. The purpose of this review is to provide a brief discussion of transgenic and knockout methodology and its application to study the genetic basis of hypertension.

Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo.

Vector-mediated gene transfer offers a direct method of correcting genetic pulmonary diseases and might also be used to correct temporary abnormalities associated with acquired, nongenetic disorders. Because the fetus or newborn may be a more immune tolerant host for gene transfer using viral vectors, we used replication defective recombinant adenoviral vectors to test the feasibility of gene transfer to the fetal pulmonary epithelium in vitro and in vivo. Both proximal and distal epithelial cells in cultured fetal lung tissues from rodents and humans diffusely expressed the lacZ transgene 3 d after viral infection. In vivo gene delivery experiments were performed in fetal mice and lambs. Delivery of Ad2/CMV-beta Gal to the amniotic fluid in mice produced intense transgene expression in the fetal epidermis and amniotic membranes, some gastrointestinal expression, but no significant airway epithelial expression. When we introduced the adenoviral vector directly into the trachea of fetal lambs, the lacZ gene was expressed in the tracheal, bronchial, and distal pulmonary epithelial cells 3 d after viral infection. Unexpectedly, reactive hyperplasia and squamous metaplasia were noted in epithelia expressing lacZ in the trachea, but not in the distal lung of fetal lambs. 1 wk after infection, adenovirus-treated fetuses developed inflammatory cell infiltrates in the lung tissue with CD4, CD8, IgM, and granulocyte/macrophage positive immune effector cells. Transgene expression faded coincident with inflammation and serologic evidence of antiadenoviral antibody production. While these studies document the feasibility of viral-mediated gene transfer in the prenatal lung, they indicate that immunologic responses to E1-deleted recombinant adenoviruses limit the duration of transgene expression.

Effect of cortisol on gene expression of the renin-angiotensin system in fetal sheep.

Components of the renin-angiotensin system have been found in a variety of tissues during fetal and postnatal life and appear to be developmentally regulated. We postulated that hormonal changes associated with parturition participate in the regulation of renin, angiotensinogen (Ao) and angiotensin type 1 receptor (AT1) gene expression. Cortisol, which increases rapidly in fetal blood before delivery, has been shown to influence the maturation of various systems in the developing fetus. To test the hypothesis that an increase in cortisol regulates fetal renin. Ao, and AT1 mRNA gene expression, we used Northern blot analysis to study the effects of an intraperitoneal infusion of cortisol (3 mg/h, 1 mL/h) for 48 h on the expression of these genes in twin ovine fetuses (n = 10 pairs) at 130-d gestation (term 145 d); one twin in each pair served as a saline-treated control (0.9% NaCl, 1 mL/h). Plasma cortisol levels were significantly higher in cortisol-treated fetuses (113 +/- 23 nmol/dL) than in twin controls (4.6 +/- 0.8 nmol/dL). Cortisol infusion significantly decreased AT1 receptor mRNA levels in kidney and liver by 24 +/- 7% and 27 +/- 8%, respectively, when compared with controls (p < 0.05), whereas in contrast, increased mRNA levels (p < 0.05) in heart right atrium (91 +/- 23%) and ventricle (59 +/- 20%). Renin mRNA levels decreased in renal cortex by 77 +/- 13% (p < 0.05) in cortisol-treated animals compared with controls. Hepatic Ao mRNA levels decreased by 15 +/- 5% in response to cortisol (p < 0.05), whereas no significant effect was seen on renal Ao gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)