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Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats and compared with age-matched mother-fed (MF) pups. The plasma insulin and glucagon contents were increased sixfold and twofold, respectively, in HC rats compared with MF rats. There was a distinct leftward shift in the glucose-stimulated insulin secretory pattern for HC islets. HC islets secreted insulin in the absence of any added glucose and in the presence of Ca(2+) channel inhibitors. The activities of glucokinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase complex were significantly increased in HC islets compared with MF islets. The protein contents of GLUT-2 and hexokinase were significantly increased in HC islets. These findings indicate that a nutritional intervention in the form of a HC formula only during the suckling period has a profound influence on pancreatic function, causing the onset of hyperinsulinemia.

This study was designed to compare the effect of fluid percussion brain injury (FPI) on the hypotensive cerebrovascular response in newborn and juvenile pigs as a function of time postinsult and to determine the role of endothelin-1 (ET-1) in any age-dependent differences in hypotensive cerebrovascular regulation after injury. Ten minutes of hypotension (10-15 ml blood/kg) decreased mean arterial blood pressure uniformly in both groups ( approximately 45%). In the newborn, hypotensive pial artery dilation (PAD) was blunted within 1 h, remained diminished for at least 72 h, but was resolved within 168 h postinjury (66 +/- 4, 69 +/- 4, 71 +/- 4, and 64 +/- 4% inhibition at 1, 4, 8, and 72 h post-FPI). During normotension, regional cerebral blood flow (rCBF) was decreased by FPI, and hypotension further reduced the already decremented rCBF for at least 72 h. Cerebrospinal fluid (CSF) ET-1 was increased from 26 +/- 4 to 206 +/- 25 pg/ml within 72 h post-FPI, whereas an ET-1 antagonist partially restored impaired hypotensive PAD and altered hypotensive rCBF. In contrast, hypotensive PAD and altered CBF were only inhibited for 4 h post-FPI in the juvenile (56 +/- 3 and 34 +/- 4% inhibition at 1 and 4 h post-FPI). CSF ET-1 was only increased from 27 +/- 4 to 67 +/- 9 pg/ml at 4 h, whereas the concentration returned to preinjury value by 8 h post-FPI. ET-1 antagonism similarly partially restored impaired hypotensive PAD and altered hypotensive rCBF. These data show that FPI disturbs cerebral autoregulation during hypotension both to a greater magnitude and for a longer duration in the newborn than in the juvenile. These data suggest that the greater FPI-induced ET-1 release in the newborn could contribute to age-dependent differences in impaired hypotensive cerebral autoregulation after FPI.

To study the effect of fetal hypoxemia on perinatal norepinephrine and epinephrine total body kinetics, 13 near-term fetal lambs were instrumented with vascular catheters under general anesthesia. One week later, norepinephrine and epinephrine kinetics were measured in normoxemic (n = 7) or spontaneously hypoxemic fetuses (n = 6) with isotope dilution methodology. Hypoxemic fetuses had lower body (P < 0.02) and placental (P = 0.01) weights and a threefold elevation in plasma norepinephrine (P < 0.005) and epinephrine (P < 0.025) associated with correspondingly higher total body norepinephrine (P < 0.005) and epinephrine (P < 0.05) spillovers. After birth, total body norepinephrine and epinephrine spillover increased 45% and 3.2-fold, respectively, in normoxemic animals (both P < 0.001). However, in the hypoxemic group, norepinephrine total body spillover was unchanged between fetal and 1-h lambs and then fell in 4-h lambs (P < 0.005). In addition, total body epinephrine release rose postnatally (P < 0.05) but less than in the normoxemic group (P < 0.02). No differences in norepinephrine or epinephrine total body clearance occurred between normoxemic and hypoxemic groups in either fetal or newborn lambs. These findings indicate that in hypoxemic and growth-restricted fetuses 1) elevated circulating norepinephrine and epinephrine levels are related to increased sympathoadrenal activity and 2) birth is associated with an initial maintenance and subsequent decline in global sympathetic activity but a blunting of adrenal medullary activation.

Melatonin is an important component of the avian circadian system. This study investigates the effects of pinealectomy (Pin-X) and melatonin implantation (Mel) on electroretinogram (ERG) rhythms in chicks. Feeding rhythms were monitored to obtain a phase reference for ERG recordings. Pin-X and Mel had little or no effect on feeding rhythms. Sham-operated Pin-X and vehicle implantation had no effect on ERG rhythms in the light-dark (LD) cycle or constant darkness (DD). ERG a- and b-wave amplitudes were higher during the day than during the night. The a- and b-wave implicit times were shorter during the day than during the night. a-Wave sensitivity was higher during the night than during the day, whereas b-wave sensitivity was not rhythmic. Pin-X abolished the circadian rhythm of b-wave amplitude and implicit time in DD but had no effect on a-wave rhythmicity. Mel abolished the rhythm of b-wave amplitude and of a- and b-wave implicit time in DD. Neither treatment affected ERG in LD. These results suggest that the circadian system regulates rhythmic visual function in the retina at least partially through Mel. The role played by the pineal gland and Mel may be specific to some physiological modalities (e.g., vision) while not influencing others (e.g., feeding).

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.

In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O(2) mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ET(A) receptor -/-, +/-, and +/+ genotypes were used. Isolated DA from term ET(A) +/+ fetuses contracted to O(2) (5-95%) and a thromboxane A(2) analog (ONO-11113, 0.1 microM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1-100 nM). Indomethacin (2.8 microM) was also a constrictor. ET(A) -/- DA, unlike ET(A) +/+ DA, contracted marginally to O(2) and ET-1 but responded to ONO-11113. O(2) contraction was also reduced in ET(A) +/- DA. In vivo, DA constricted equally in tracheotomized ET(A) -/- and ET(A) +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ET(A) -/- fetuses in utero, although it occurred in ET(A) +/+ and +/- littermates. We conclude that ET-1 mediates the DA constrictor response to O(2). Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

The O2 sensitivity of carotid chemoreceptor type I cells is low just after birth and increases with postnatal age. Chronic hypoxia during postnatal maturation blunts ventilatory and carotid chemoreceptor neural responses to hypoxia, but the mechanism remains unknown. We tested the hypothesis that chronic hypoxia from birth impairs the postnatal increase in type I cell O2 sensitivity by comparing intracellular Ca2+ concentration ([Ca2+]i) responses to graded hypoxia in type I cell clusters from rats born and reared in room air or 12% O2. [Ca2+]i levels at 0, 1, 5, and 21% O2, as well as with 40 mM K+, were measured at 3, 11, and 18 days of age with use of fura 2 in freshly isolated cells. The [Ca2+]i response to elevated CO2/low pH was measured at 11 days. Chronic hypoxia from birth abolished the normal developmental increase in the type I cell [Ca2+]i response to hypoxia. Effects of chronic hypoxia on development of [Ca2)]i responses to elevated K+ were small, and [Ca2+]i responses to CO2 remained unaffected. Impairment of type I cell maturation was partially reversible on return to normoxic conditions. These results indicate that chronic hypoxia severely impairs the postnatal development of carotid chemoreceptor O2 sensitivity at the cellular level and leaves responses to other stimuli largely intact.

Therapies to prevent the onset or progression of pulmonary hypertension in newborns have received little study compared with those in adult models. We wanted to determine whether nifedipine treatment prevents the increased pulmonary vascular resistance, blunted pulmonary vascular responses to acetylcholine, and reduced lung endothelial nitric oxide synthase (eNOS) amounts that we have found in a newborn model of chronic hypoxia-induced pulmonary hypertension. Studies were performed with 1- to 3-day-old piglets raised in room air (control) or 10% O2 (hypoxia) for 10-12 days. Some piglets from each group were given nifedipine (3-5 mg/kg sublingually three times a day). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. Pulmonary vascular responses to acetylcholine and eNOS amounts were assessed in excised lungs. The calculated value of the pulmonary vascular resistance for nifedipine-treated hypoxic piglets (0.09 +/- 0.01 cmH(2)O. ml(-1). min. kg) was almost one-half of the value for untreated hypoxic piglets (0.16 +/- 0.01 cmH(2)O. ml(-1). min. kg) and did not differ from the value for untreated control piglets (0.05 +/- 0.01 cmH(2)O. ml(-1). min. kg). Pulmonary arterial pressure responses to acetylcholine and whole lung homogenate eNOS amounts were less for both nifedipine-treated and untreated hypoxic piglets than for untreated control piglets. Nifedipine treatment attenuated pulmonary hypertension in chronically hypoxic newborn piglets despite the persistence of blunted responses to acetylcholine and reduced lung eNOS amounts.

Afferent sensory fibers are the primary neuroanatomic link between nutrient-related events in the gastrointestinal tract and the central neural substrates that modulate ingestion. In this study, we evaluated the peripheral gastric effects of leptin (OB protein) on brain stem neuronal activities using an in vitro neonatal rat preparation. We also tested gastric leptin effects as a function of age in neonates. For approximately 33% of the nucleus tractus solitarius units observed, gastric leptin (10 nM) produced a significant activation of 188.2 +/- 8.6% (mean +/- SE) compared with the control level of 100% (P < 0.01). Concentration-dependent leptin effects have also been shown. The remaining neurons (67%) had no significant response to gastric leptin application. Next, we evaluated the peripheral gastric effects of leptin (10 nM) on brain stem unitary activity in three different age groups (1-2 days old, 3-5 days old, and 7-8 days old) of neonatal rats. In the 1- to 2-day-old and the 3- to 5-day-old groups, we observed that response ratios and activity levels were similar. However, there was a significant difference between the 7- to 8-day-old group and the two younger age groups in both the response ratios and the activation levels. The percentage of activation responses increased from approximately 26% in the 1- to 2-day-old and the 4- to 5-day-old age groups to 70% in the 7- to 8-day-old group (P < 0.05). The level of activation increased from 168.3 +/- 2.7% (compared with the control level) in the 1- to 2-day-old and the 4- to 5-day-old age groups to 231.4 +/- 11.9% in the 7- to 8-day-old group (P < 0.01). Our data demonstrate that peripheral gastric leptin modulates brain stem neuronal activity and suggest that gastric leptin has a significantly stronger effect in the 7- to 8-day-old animals than in the younger neonates.

This laboratory has previously reported that sustained reduction of blood flow in newborn intestine causes a triphasic increase in vascular resistance that occurs over 3-4 h and that these changes are mediated, in part, by loss of endothelial nitric oxide (NO) production. This study examines the effects of exposure to sustained low-flow perfusion on the subsequent response to three contractile agonists: ANG II, norepinephrine (NE), and endothelin-1 (ET-1). Gut loops from 3- and 35-day-old swine were exposed to low-flow conditions in vivo (i.e., reduction of flow to approximately 50% of baseline) for 30 min or 5 h. Thereafter, they were removed to an extracorporeal perfusion circuit for in vitro hemodynamic assessment; alternatively, the mesenteric artery perfusing the gut loop was removed and cut into rings for assessment of isometric tension development. Gut loops from 3-day-old subjects exposed to low-flow conditions demonstrated significantly increased contractile responses to ANG II, NE, and ET-1; also, mesenteric artery rings from these gut loops demonstrated a significant reduction of the ED50 for all three agonists. Similar changes were not observed in intestine or mesenteric artery rings from older subjects. Sustained blockade of endogenous NO synthesis with NG-monomethyl- L-arginine duplicated the effects of exposure to sustained low-flow perfusion. It appears that sustained reduction of blood flow in newborn intestine decreases constitutive NO production, which in turn causes a generalized enhancement of the contractile efficacy of ANG II, NE, and ET-1.

Bombesin (BN) suppresses food intake in rats whether given centrally or systemically. Although the brain BN-sensitive receptors are known to be essential for the anorexic effect of systemic BN, the mode of communication between the gut and the brain remains unclear. This study assessed whether the anorexic effect of systemic BN is mediated humorally or via neural circuits. Afferent neurons were lesioned using capsaicin (50 mg/kg sc) on postnatal day 2, and responses to BN were assessed during adulthood. Capsaicin treatment decreased body weight gain significantly from postnatal age 4-7 wk. Peripheral BN (4-16 micrograms/kg ip) dose dependently suppressed food intake in control animals. However, this effect was completely blocked in capsaicin-treated rats. In contrast to systemic effects, feeding-suppressant effects of centrally administered BN (0.01-0.5 microgram icv) were not affected by capsaicin treatment. This research suggests that peripheral BN communicates with the brain via a neuronal system(s) whose afferent arm is constituted of capsaicin-sensitive C and/or Adelta-fibers, whereas the efferent arm of this satiety- and/or anorexia-mediating circuitry is capsaicin resistant.

Neonatal pulmonary artery smooth muscle cells (PASMCs) exhibit enhanced growth capacity and increased growth responses to mitogenic stimuli compared with adult PASMCs. Because intracellular signals mediating enhanced growth responses in neonatal PASMCs are incompletely understood, we questioned whether 1) Gq agonists increase cAMP content and 2) increased cAMP is proproliferative. Endothelin-1 and angiotensin II increased both cAMP content and proliferation in neonatal but not in adult PASMCs. Inhibition of protein kinase C and protein kinase A activity nearly eliminated the endothelin-1- and angiotensin II-induced growth of neonatal PASMCs. Moreover, cAMP increased proliferation in neonatal but not in adult cells. Protein kinase C-stimulated adenylyl cyclase was expressed in both cell types, suggesting that insensitivity to stimulation of cAMP in adult cells was not due to decreased enzyme expression. Our data collectively indicate that protein kinase C stimulation of cAMP is a critical signal mediating proliferation of neonatal PASMCs that is absent in adult PASMCs and therefore may contribute to the unique proproliferative phenotype of these neonatal cells.

Glucocorticoid levels increase greatly at the time of birth in humans and sheep, coinciding with an increased ability of the kidney to reabsorb sodium. Cortisol induces proximal tubule apical membrane Na+/H+ exchanger maturation in near-term fetal sheep. Proximal tubule salt transport is ultimately dependent on Na+ pump activity, so we studied the effects of cortisol treatment on renal cortical Na+-K+-ATPase. We first looked at six 140 day gestation fetal sheep (term is 145) and compared their renal cortical Na+-K+-ATPase to that of six 1-day-old lambs. Na+-K+-ATPase activity increased 80% after birth. Then nine pairs of twin fetal sheep were chronically instrumented at 127 days gestation. After 72 h recovery, one twin was given a 48-h continuous intraperitoneal infusion of cortisol. Both twins were then killed, and their renal cortices were studied. Na+-K+-ATPase activity increased 122% with cortisol treatment; activity equaled that of 1-day-old lambs. Protein abundance of the alpha1-subunit of the Na+-K+-ATPase increased 19%; the beta1-subunit increased 39% with cortisol treatment. mRNA abundance of the alpha1-subunit increased 58%; the beta1-subunit increased 72%. These results indicate that cortisol matures Na+-K+-ATPase activity.

Brain swelling after acute hyponatremia in prepubescent rats, in contrast to adults, has recently been associated with an increase in brain sodium and a high mortality that could be prevented by preadministration of testosterone. To reexamine the effect of acute hyponatremia in young brain, we measured brain water and solute content in prepubescent rats after induction of hyponatremia over 4 h with water and arginine vasopressin. An 18% decrease in plasma sodium was associated with a 13% increase in brain water and a decrease in brain sodium and glutamate contents. No animals died. To assess the effect of sex hormones on brain adaptation, prepubescent rats were pretreated with estrogen or testosterone before acute hyponatremia. Brain sodium and potassium contents were significantly reduced in comparison to normonatremia in testosterone-pretreated but not estrogen-pretreated animals. However, there was no difference between estrogen-pretreated and testosterone-pretreated groups in mortality or in brain contents of water, electrolytes, or major organic osmolytes. In conclusion, we found that brain adaptation to acute hyponatremia in prepubescent rats is similar to that observed in adults.

This study was designed to test the hypothesis that a reduced number of nephrons from birth leads to increased arterial pressure in adulthood. Newborn Sprague-Dawley rat pups were uninephrectomized during the first 24 h after birth. In chronically instrumented adult animals (approximately 22 wk), mean arterial pressure on a normal (0.20%)-Na+ diet was higher in uninephrectomized rats (133 +/- 2 mmHg vs. 121 +/- 2 mmHg in controls, P < 0.0001). Body weights were not significantly different, but the total kidney-to-body weight ratio was significantly reduced by 14% in adult uninephrectomized animals (P < 0.05). Glomerular filtration rate was reduced by approximately 30% in uninephrectomized rats (1.84 +/- 0.09 vs. 2.63 +/- 0.14 ml/min, P < 0.0002), and effective renal plasma flow was reduced to a lesser degree (6.37 +/- 0.38 vs. 7.87 +/- 0.51 ml/min, P < 0.03), such that the filtration fraction was also reduced (0.291 +/- 0.007 vs. 0.338 +/- 0.014, P < 0.01). After 7-10 days on a high (3.15%)-Na+ diet, arterial pressure increased more in uninephrectomized animals than in controls (20 +/- 3 vs. 1 +/- 1 mmHg, P < 0.003). Thus surgical removal of 50% of the nephrons, when done during development, caused reduced renal function and a salt-sensitive hypertension in adulthood. These data suggest that a reduced nephron endowment from birth, caused by genetic and/or perinatal environmental factors, could contribute to essential hypertension in adulthood.

Thioredoxin (TRX) is a potent protein disulfide oxidoreductase important in antioxidant defense and regulation of cell growth and signal transduction processes, among them the production of nitric oxide. We report that lung TRX and its reductase, TR, are specifically upregulated at birth by O2. Throughout the third trimester, mRNAs for TRX and TR were expressed constitutively at low levels in fetal baboon lungs. However, after premature birth (125 or 140 of 185 days gestation), lung TRX and TR mRNAs increased rapidly with the onset of O2 or air breathing. Lung TRX mRNA also increased in lungs of term newborns with air breathing. Premature animals (140 days) breathing 100% O2 develop chronic lung disease within 7-14 days. These animals had greater TRX and TR mRNAs after 1, 6, or 10 days of life than fetal control animals. In 140-day animals given lesser O2 concentrations (as needed) who do not develop chronic lung disease, lung TRX and TR mRNAs were also increased on days 1 and 6 but not significantly on day 10. In fetal distal lung explant culture, mRNAs for TRX and TR were elevated within 4 h in 95% O2 relative to 1% O2, and the response was similar at various gestations. In contrast, TRX protein did not increase in lung explants from premature animals (125 or 140 days) but did in those from near-term (175-day) fetal baboons after exposure to hyperoxia. However, lung TRX protein and activity, as well as TR activity, eventually did increase in vivo in response to hyperoxia (6 days). Increases in TRX and TR mRNAs in response to 95% O2 also were observed in adult baboon lung explants. When TRX redox status was determined, increased O2 tension shifted TRX to its oxidized form. Treatment of lung explants with actinomycin D inhibited TRX and TR mRNA increases in 95% O2, indicating transcriptional regulation by O2. The acute increase in gene expression for both TRX and TR in response to O2 suggests an important role for these proteins during the transition from relatively anaerobic fetal life to O2 breathing at birth.

Biliary lipids, composed of bile acids, cholesterol, and phosphatidylcholine, are a major source of luminal lipid in the small intestine. In the present study in a newborn swine intestinal epithelial cell line (IPEC-1), taurocholate and phosphatidylcholine were found to have no effect on apolipoprotein B (apo B) secretion but did significantly increase the basolateral secretion of apo A-I. This regulation of apo A-I secretion occurred at the pretranslational level for taurocholate and at the posttranslational level for phosphatidylcholine. The regulation of apo A-I secretion by phosphatidylcholine did not involve changes in apo A-I degradation and may involve mobilization of a preformed pool of apo A-I. Cholesterol, whether solubilized with taurocholate or phosphatidylcholine, had no effect on the secretion of either apo B or apo A-I. However, when taurocholate, phosphatidylcholine, and cholesterol were combined, apo B secretion was decreased, and the increase in apo A-I secretion noted with taurocholate and phosphatidylcholine alone was ablated. Another primary bile acid, taurochenodeoxycholate, was found to decrease apo B secretion but had no effect on apo A-I secretion. However, the significance of this effect is uncertain, since this bile acid caused significant cellular membrane injury, as evidenced by increased apical medium lactate dehydrogenase activity. Phosphatidylcholine, but not taurocholate, dramatically increased the basolateral secretion of radiolabeled phospholipid with a modest increase in cellular triglyceride radiolabeling. Furthermore, this effect of phosphatidylcholine on lipid synthesis did not require significant hydrolysis or uptake of the phosphatidylcholine molecule. Studies using radiolabeled taurocholate did not demonstrate active transport of taurocholate by these cells.

Carbonic anhydrase (CA) III is very abundant in type I skeletal muscle, but its function is still debated. Our aims were to examine CA III expression during growth and determine whether the effects of CA inhibition previously observed in adult muscles could be seen in younger rats in which CA III levels are lower. CA III content and activity were measured in soleus muscles from 10- to 100-day-old rats, and the influence of CA inhibitor on fatigue and hexosemonophosphate content was quantified in vitro. CA III activity and content increased fivefold between 10 and 100 days of age. Data analysis revealed that the influence of CA inhibitor on fatigue was to some extent positively and linearly related to the level of CA III activity. Hexosemonophosphate accumulation with CA inhibition also became more significant with age. In conclusion, CA III level in soleus muscle does not stabilize before 3 mo after birth; data also confirm that the effects of CA inhibitors are due to inhibition of the CA III isoform.

Prolonged maternal separation inhibits endogenous heat production in infant mammals exposed to cold. This inhibition of thermogenesis occurs many hours before energy stores have been fully depleted. The need to protect energy resources during separation-induced starvation may be signaled by declining levels of leptin, a hormone that acts as a "fat signal" and a regulator of energy utilization; in fact, starvation reduces leptin levels in adult mice and infant rats. It is not known, however, whether leptin has a functional role during starvation in infants. Such a role may be found in the regulation of nonshivering thermogenesis by brown adipose tissue (BAT), a specialized organ that provides heat to infant mammals, including humans, during cold exposure. Heat produced by BAT allows the cold-exposed infant to prevent the detrimental effects of hypothermia on physiology and behavior and, ultimately, growth. Here we show that leptin disinhibits BAT thermogenesis during cold exposure in infant rats after 18 h of maternal separation. This finding demonstrates that leptin is more than simply an adipostat for the regulation of body weight; specifically, leptin modulates thermogenesis and energy utilization in the early postnatal period.

Systemic hypotension causes a greater degree of vasoconstriction in intestine from 3- than from 35-day-old postnatal swine. To determine the basis for this age-dependent difference, systemic hypotension (pressure reduction to approximately 50% of baseline) was induced by creating pericardial tamponade in postnatal swine instrumented to allow measurement of intestinal hemodynamics and oxygenation in vivo. Hypotension caused gut vascular resistance to increase 77 +/- 6% in 3-day-old subjects but only 18 +/- 3% in 35-day-old subjects. Prior blockade of alpha1-receptors with phentolamine, vasopressin receptors with [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP, or surgical denervation of the gut loop had no effect on hypotension-induced gut vasoconstriction. Losartan, which blocks angiotensin AT1 receptors, significantly attenuated hypotension-induced gut vasoconstriction in both age groups. BQ-610, which blocks endothelin ETA receptors, also limited the magnitude of vasoconstriction but only in younger subjects. This effect may have been consequent to an interaction between endothelin and angiotensin, inasmuch as a subpressor concentration of endothelin increased the contractile response to angiotensin in mesenteric artery rings. The substantial rise in 3-day-old gut vascular resistance was partly consequent to a locally mediated vasoconstriction that occurred in response to pressure and/or flow reduction during hypotension, as evidenced by the significant attenuation of this constriction when blood flow was held constant by controlled-flow perfusion to the gut loop during hypotension. Intestinal O2 uptake was compromised to a significantly greater degree in 3- than in 35-day-old subjects during hypotension. This difference was primarily due to the inability of younger intestine to increase O2 extraction in the face of reduced blood flow and may be mediated, in part, by an effect of angiotensin II on intestinal capillary perfusion.

Alkalosis-induced relaxation was measured in precontracted arterial rings from 1-wk-old piglets exposed to normoxia or to 3 days of chronic hypoxia. In normoxic piglet arteries, alkalosis-induced relaxation was blunted in arteries without functional endothelium and in arteries treated with nitric oxide synthase or guanylate cyclase inhibitors but not in arteries treated with cyclooxygenase inhibitors or Ca2+- and ATP-dependent K+-channel inhibitors. Inhibition of voltage-dependent K+ channels with 10(-3) M 4-aminopyridine also failed to block alkalosis-induced relaxation. 4-Aminopyridine at 10(-2) M did block the response, but this may have been due to sustained vascular smooth muscle depolarization. Arteries from hypoxic piglets exhibited greater contraction to the thromboxane mimetic U-46619, decreased endothelium-dependent relaxation, and blunted alkalosis-induced relaxation. The residual relaxation was eliminated by nitric oxide synthase but not by cyclooxygenase or voltage-dependent K+-channel inhibition. Alkalosis-induced relaxation of newborn piglet pulmonary arteries appears to be mediated by the nitric oxide-cGMP pathway and is attenuated after 3 days of hypoxia, likely because of decreased nitric oxide activity.

To characterize pulmonary and total body norepinephrine and epinephrine kinetics in the immediate newborn period, the effects of desipramine were studied in six fetal lambs chronically instrumented at 133-134 days gestation (term 147 days) and delivered 1 wk later by cesarean section under spinal anesthesia. Norepinephrine and epinephrine kinetics were determined with isotope dilution methodology 4 h after birth and repeated 30 min after desipramine (2 mg/kg iv). At baseline, the lungs accounted for 35 +/- 10 and 47 +/- 13% of whole body norepinephrine clearance (93 +/- 8 ml. min-1. kg-1) and spillover (188 +/- 29 ng. min-1. kg-1) and 15 +/- 2 and 19 +/- 7% of whole body epinephrine clearance (82 +/- 4 ml. min -1. kg -1) and release (22.7 +/- 2.7 ng. min-1. kg-1), respectively. Desipramine decreased pulmonary norepinephrine and epinephrine clearance and spillover to near-zero levels, whereas whole body norepinephrine clearance fell by 51 +/- 3% (P < 0.001), norepinephrine spillover by 54 +/- 6% (P < 0.005), epinephrine clearance by 30 +/- 6% (P < 0.01), and epinephrine spillover by 34 +/- 11% (P < 0.05). These results indicate that, in the immediate newborn period, pulmonary removal and release of norepinephrine and epinephrine is mediated by a desipramine-sensitive process that accounts for a major portion of associated reductions in whole body norepinephrine and epinephrine clearance and release.

Nitric oxide (NO) is an excitatory neurotransmitter in the hypoxic ventilatory response (HVR). Furthermore, neuronal NO synthase (nNOS) activity in the developing rat correlates with the magnitude of late hypoxic ventilatory depression. To test the hypothesis that repeated short exposures to hypoxia may modify late HVR characteristics in young rats, we conducted 30-min hypoxic challenges in 2- to 3-day-old rat pups, before (Pre) and 6 h after (Post) they completed a series of eight cycles consisting of 5 min of hypoxia and 10 min of normoxia (Hyp-Norm) or normoxia throughout (Norm-Norm). In an additional group, similar challenges were performed after administration of either intraperitoneal vehicle or 25 mg/kg 7-nitroindazole (7-NI). Ventilation (VE) was measured using whole body plethysmography. Although no changes in peak VE responses occurred with episodic hypoxia (Pre vs. Post, P = not significant), late VE reductions were markedly attenuated in Post (DeltaVE from early to late: 7.2 +/- 1.5 ml/min in Pre vs. 4.5 +/- 1.1 ml/min in Post; P < 0.002). Furthermore, 7-NI treatment of Post animals was associated with late VE reductions to Pre levels in Hyp-Norm-exposed animals. Western blots of protein equivalents from the caudal brain stem revealed increased nNOS expression in Hyp-Norm compared with Norm-Norm (P < 0.01). Current findings suggest that repeated short hypoxic exposures improve the ability to sustain VE, which appears to be mediated by increased nNOS expression and activity in brain stem respiratory regions. We postulate that changes in nNOS may play a role in respiratory control plasticity.

The role of tyrosine phosphorylation was investigated using protein tyrosine phosphatase inhibitors in newborn pigs equipped with a cranial window in vivo. We tested the hypothesis that cyclooxygenase and nitric oxide (NO) synthase are physiological targets for tyrosine phosphorylation in cerebral circulation. Phenylarsine oxide dilated pial arterioles and increased prostacyclin and prostaglandin E2 in cortical periarachnoid fluid; these responses were inhibited by indomethacin. Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NNA) inhibited the vasodilation to phenylarsine oxide; the effects of NO synthase inhibitors and indomethacin were additive. Cyclooxygenase-mediated vascular responses were assessed using topical application of arachidonic acid. Phenylarsine oxide and sodium orthovanadata potentiated vasodilation and prostanoid synthesis in response to arachidonic acid. Nomega-nitro-L-arginine methyl ester and Nomega-nitrol-arginine did not affect vasodilation or prostanoid production in response to arachidonic acid, indicating no cross talk between cyclooxygenase and NO synthase. These data indicate that cyclooxygenase and NO synthase are physiological targets for tyrosine phosphorylation in the cerebral circulation of newborn pigs.

Renal adaptive responses during the 24 h after delivery in term newborn lambs include marked increases in both glomerular filtration rate (GFR) and sodium reabsorption. This study investigated the effects of ventilation style on cardiovascular, renal, and endocrine adaptations in preterm newborn lambs. Lambs (n = 62) were delivered by cesarean section at 131 days gestation (term = 150 days), treated with surfactant, and randomized to one of three ventilation strategies: high-frequency oscillation (12 Hz), high rate (50 breaths/min; tidal volume = 8 ml/kg), or low rate (15 breaths/min; tidal volume = 15 ml/kg). Lambs (5 or 6/group) were ventilated for 2, 5, 10, and 24 h to maintain arterial PCO2 between 45 and 50 mmHg. Plasma vasopressin levels decreased to <25 pg/ml by 10 h, and fractional sodium excretion decreased to <1% by 16 h in all groups. However, cardiac output, renal plasma flow, and GFR values did not change over time for any of the groups. The style of ventilation employed had no measurable effects on overall cardiovascular, renal, or endocrine function. We conclude in ventilated preterm lambs that 1) the ventilation style does not affect the time course for postnatal adaptation, 2) adaptive changes in renal tubular sodium reabsorption are evident by 16 h after birth, and 3) changes in preterm newborn renal sodium reabsorption occur in the absence of postnatal changes in renal plasma flow or GFR.

Studies were conducted in young postnatal swine to determine if substance P (SP) participates in the regulation of postnatal intestinal hemodynamics and oxygenation. SP was present in homogenates of whole intestine from postnatal swine in an age-dependent manner as follows: 1 day old and never fed, 126 +/- 35; 3 days old and fasted, 148 +/- 30; and 14 days old, 51 +/- 10 pg/mg protein (P < 0.01, 14- vs. 1- or 3-day-olds). Phenylephrine-precontracted rings of mesenteric artery from 3-day-old subjects mounted for tension recording within buffer-filled myographs demonstrated brisk relaxation in response to SP (EC50, 2 x 10(-10) M). This relaxation was eliminated by mechanical removal of the endothelium or blockade with the L-arginine analog NG-monomethyl-L-arginine (L-NMMA) and was significantly attenuated by pretreatment with N-acyl-L-Trp-3,5-bis-(trifluoromethyl) benzyl ester (NATB), a highly selective NK-1 receptor antagonist (pA2 5 x 10(-10) M). Infusion of exogenous SP into the mesenteric artery of innervated in vivo gut loops reduced intestinal vascular resistance 35% and increased tissue oxygen uptake 40% in both 3- and 14-day-old subjects. By contrast, blockade of the NK-1 receptor for SP with NATB increased intestinal vascular resistance 19% in 3-day-old subjects but only 5% in 14-day-old subjects (P < 0.01). SP-induced changes in gut vascular resistance were significantly attenuated by prior coinfusion of NATB or L-NMMA, indicating that the peptide exerted this vascular effect via theNK-1 receptor, which is linked to endothelial cell nitric oxide synthase. Both NATB and L-NMMA attenuated flow-induced dilation within pump-perfused in vitro gut loops from 3-day-old subjects. SP appears to participate in the regulation of the newborn intestinal circulation, especially during the first days after birth.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by > 90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability (Po). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating Po declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.

Swallowing represents a primary physiological function that provides for the ingestion of food and fluid. In precocial species, swallowing activity likely develops in utero to provide for a functional system during the neonatal period. The chronically instrumented ovine fetal preparation has provided the opportunity for recent advances in understanding the regulation of in utero swallowing activity. The near-term ovine fetus swallows fluid volumes (100-300 ml/kg) that are markedly greater, per body weight, than that of the adult (40-60 ml/kg). Spontaneous in utero swallowing and ingestive behavior contribute importantly to the regulation of amniotic fluid volume and composition, the acquisition and potential recirculation of solutes from the fetal environment, and the maturation of the fetal gastrointestinal tract. Fetal swallowing activity is influenced by fetal maturation, neurobehavioral state alterations, and the volume of amniotic fluid. Furthermore, intact dipsogenic mechanisms (osmolality, angiotensin II) have been demonstrated in the near-term ovine fetus. It remains unknown to what degree, if any, fetal swallowing may be influenced by nutrient appetite, salt appetite, or taste. Nevertheless, the development of dipsogenic and additional regulatory mechanisms for ingestive behavior occurs during fetal life and may be susceptible to changes in the pregnancy environment. This review describes what is currently known regarding the in utero development of ingestive behavior and the importance of this activity for fetal and perhaps ultimately adult fluid homeostasis.

Studies were conducted to determine if the response of in vitro mesenteric artery from 3- and 35-day-old swine to acute hypoxia was age dependent. Isometric tension developed by mesenteric artery rings was measured using a standard myograph apparatus. When the buffer aeration gas was changed from 95% O2-5% CO2 to 95% N2-5% CO2, phenylephrine-precontracted rings from both age groups consistently demonstrated a triphasic response, consisting of, in order, an initial, brief dilation, a sharp contraction, and a sustained loss of tone. The only portion of the triphasic response that was age dependent was the constrictor response, hypoxic vasoconstriction (HVC), which was significantly greater in rings from younger animals. HVC appeared to be mediated by a hypoxia-induced loss of constitutive nitric oxide production. Thus HVC was eliminated by endothelial removal, significantly attenuated by pretreatment with NG-monomethyl-L-arginine (L-NMMA), but not with NG-monomethyl-D-arginine, restored by coadministration of L-arginine, and accentuated by pretreatment with superoxide dismutase. Blockade of endothelin A receptors with BQ-610 or inhibition of cyclooxygenase or lipoxygenase activities with indomethacin or phenidone had no effect on HVC in either group. HVC appeared to be dependent on reduction in PO2, not on reduced ATP secondary to hypoxia, as it did not occur in rings administered 2,4-dinitrophenol, an agent that uncouples oxidative phosphorylation. The magnitude of HVC, which appears to be mediated by hypoxia-induced supression of NO production, is greater in mesenteric artery from 3-day-old swine than from 35-day-old swine.

In altricial species, such as humans and rats, much of the development of autonomic systems occurs postnatally. Consequently, vulnerabilities exist early in postnatal development when immature autonomic functions are challenged by external factors such as variations in ambient temperature (Ta). Ta profoundly influences sleep/wake state structure in adult animals and humans, and exposure to excessive warmth has been implicated as a risk factor in sudden infant death syndrome. To better understand the relationship between temperature and sleep during development, we investigated the effect of Ta variation on sleep/wake state structure and sleep intensity in developing rats. In this experiment, sleep intensity was measured by the intensity of slow-wave activity during slow-wave sleep. Neonatal Long-Evans hooded rat pups were surgically prepared for chronic sleep/wake state and brain temperature (Tbr) recording. Two-hour recordings of sleep/wake state and Tbr were obtained from rats on postnatal day 12 (P12), P14, P16, P18, and P20 at a Ta of either 28.0-30.0, 33.0-35.0, or 38.0-40.0 degrees C. Ta significantly influenced sleep/wake state structure but had little, if any, effect on sleep intensity in developing rats.

Nonciliated bronchiolar (Clara) cells metabolize environmental toxicants, are progenitor cells during development, and differentiate postnatally. Because differentiating Clara cells of neonatal rabbits are injured at lower doses by the cytochrome P-450-activated cytotoxicant 4-ipomeanol than are those of adults, the impact of early injury on the bronchiolar epithelial organization of adults was defined by treating neonates (3-21 days) and examining them at 4-6 wk. Bronchiolar epithelium of 6-wk-old animals treated on day 7 was most altered from that of control animals. Almost 100% of the bronchioles were lined by zones of squamous epithelial cells. Compared with control animals, the distal bronchiolar epithelium of 4-ipomeanol-treated animals had more squamous cells (70-90 vs. 0%) with a reduced overall epithelial thickness (25% of control value), fewer ciliated cells (0 vs. 10-20%), a reduced expression of Clara cell markers of differentiation (cytochrome P-4502B, NADPH reductase, and 10-kDa protein), and undifferentiated nonciliated cuboidal cell ultrastructure. We conclude that early injury to differentiating rabbit Clara cells by a cytochrome P-450-mediated toxicant inhibits bronchiolar epithelial differentiation and greatly affects repair.

Using infant piglets, we studied the effects of nonspecific inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg) on vascular pressures, regional blood flow, and cerebral metabolism before 8 min of cardiac arrest, during 6 min of cardiopulmonary resuscitation (CPR), and at 10 and 60 min of reperfusion. We tested the hypotheses that nonspecific NO synthase inhibition 1) will attenuate early postreperfusion hyperemia while still allowing for successful resuscitation after cardiac arrest, 2) will allow for normalization of blood flow to the kidneys and intestines after cardiac arrest, and 3) will maintain cerebral metabolism in the face of altered cerebral blood flow after reperfusion. Before cardiac arrest, L-NAME increased vascular pressures and cardiac output and decreased blood flow to brain (by 18%), heart (by 36%), kidney (by 46%), and intestine (by 52%) compared with placebo. During CPR, myocardial flow was maintained in all groups to successfully resuscitate 24 of 28 animals [P value not significant (NS)]. Significantly, L-NAME attenuated postresuscitation hyperemia in cerebellum, diencephalon, anterior cerebral, and anterior-middle watershed cortical brain regions and to the heart. Likewise, cerebral metabolic rates of glucose (CMRGluc) and of lactate production (CMRLac) were not elevated at 10 min of reperfusion. These cerebral blood flow and metabolic effects were reversed by L-arginine. Flows returned to baseline levels by 60 min of reperfusion. Kidney and intestinal flow, however, remained depressed throughout reperfusion in all three groups. Thus nonspecific inhibition of NO synthase did not adversely affect the rate of resuscitation from cardiac arrest while attenuating cerebral and myocardial hyperemia. Even though CMRGluc and CMRLac early after resuscitation were decreased, they were maintained at baseline levels. This may be clinically advantageous in protecting the brain and heart from the damaging effects of hyperemia, such as blood-brain barrier disruption.

Abnormalities of premature newborn adaptation after preterm birth result in significant perinatal mortality and morbidity. We assessed the effects of short-term (24 h) fetal betamethasone exposure on preterm newborn baboon pulmonary and cardiovascular regulation and renal sodium handling during the first 24 h after birth. Male fetal baboons (Papio) (124-day gestation, term 185 days) received ultrasound-guided intramuscular injections of saline (n = 5) or betamethasone (0.5 mg/kg; n = 5). Fetuses were cesarean delivered 24 h later, treated with 100 mg/kg surfactant, and ventilated by adjusting peak inspiratory pressures to maintain PCO2 values of 35-50 mmHg for 24 h. Betamethasone- vs. saline-treated mean +/- SE newborn body weights (0.45 +/- 0.02 vs. 0.41 +/- 0.01 kg) were similar. Although prenatal betamethasone did not affect postnatal lung function (PCO2, arterial/alveolar O2 gradient, or dynamic compliance), plasma hormone (cortisol or thyroxine), or catecholamine levels, mean arterial pressure (25 +/- 1 vs. 32 +/- 1 mmHg), plasma sodium concentration (132 +/- 2 vs. 138 +/- 1 meq/l), glomerular filtration rate (0.07 +/- 0.02 vs. 0.16 +/- 0.02 ml.min-1.kg-1), and renal total sodium reabsorption (1.5 +/- 0.5 vs. 16.0 +/- 3.0 mu eq.min-1.kg-1) values were significantly lower in saline-treated than in betamethasone-treated newborns at 24 h. We conclude that despite the fact that there are no pulmonary and endocrine effects, antenatal glucocorticoid exposure alters premature newborn baboon vascular and renal glomerular function and improves sodium reabsorption after preterm delivery.

Neonatal treatment of rats with angiotensin-converting enzyme inhibitors or the angiotensin II type 1 receptor antagonist losartan induces irreversible renal histological abnormalities, mainly papillary atrophy, in association with an impairment in urinary concentrating ability. In the present study, sodium and potassium balance were assessed during high and low sodium intake and dietary potassium restriction in adult Wistar rats treated neonatally with enalapril (10 mg x kg(-1) x day(-1)) from 3 to 24 days of age. During balance studies, neonatally enalapril-treated rats showed 1) normal adaptation to dietary sodium restriction, 2) sodium retention during dietary sodium loading, and 3) a transient, modest, renal potassium wastage during dietary potassium restriction. Renal clearance determinations under pentobarbital anesthesia showed elevated fractional excretions of sodium and potassium and osmolar clearance without changes in glomerular filtration rate or effective renal plasma flow in enalapril-treated compared with vehicle-treated rats. Thus, in addition to the impaired urinary concentrating ability, adult rats treated neonatally with enalapril demonstrated alterations in renal sodium and potassium handling, which may be related to the prevailing papillary atrophy.

Ibuprofen, a cyclooxygenase (COX) inhibitor nonselective for either COX-1 or COX-2 isoform, upregulates cerebrovascular prostaglandin E2 (PGE2) and PGF2alpha receptors in newborn pigs. COX-2 was shown to be the predominant form of COX and the main catalyst of prostaglandin synthesis in the newborn brain. We proceeded to establish direct evidence that COX-2-generated prostaglandins govern PGE2 and PGF2alpha receptor density and function in the cerebral vasculature of the newborn. Hence, we determined PGE2 and PGF2alpha receptor density and functions in brain vasculature by using newborn pigs treated with saline, ibuprofen, COX-1 inhibitor (valerylsalicylate), or COX-2 inhibitors (DUP-697 and NS-398). Newborn brain PGE2 and PGF2alpha concentrations were significantly reduced by ibuprofen, DUP-697, and NS-398 but not by valerylsalicylate. In newborn pigs treated with DUP-697, NS-398, and ibuprofen, PGE2 and PGF2alpha receptor densities in brain microvessels were increased to adult levels; there was also a significant increase in inositol 1,4,5-trisphosphate (IP3) production and cerebral vasoconstrictor effects of 17-phenyl trinor PGE2 (EP1 receptor agonist), M&B-28767 (EP3 receptor agonist), PGF2alpha, and fenprostalene (PGF2alpha analog). Treatment with ibuprofen or DUP-697 also increased the upper blood pressure limit of cerebral cortex and periventricular blood flow autoregulation from 85 to > or = 125 mmHg (uppermost blood pressure studied). However, valerylsalicylate treatment did not affect cerebrovascular PGE2 and PGF2alpha receptors, IP3 production, or vasoconstrictor effects in newborn animals. These in vivo and in vitro observations indicate that COX-2 is mainly responsible for the regulation of PGE2 and PGF2alpha receptors and their functions in the newborn cerebral vasculature.

In the rat heart there is a postnatal switch in the expression of isoforms of both Na-K-ATPase and myosin heavy chain (MHC). Here we investigated factors controlling isoform changes in cultures of neonatal cardiomyocytes. In serum-free medium, the compositions of either Na-K-ATPase or MHC isoforms resembled the neonatal phenotype. Thyroid hormone induced the MHC isoform switch but increased expression of all Na-K-ATPase isoforms to various extents. Dexamethasone failed to induce the MHC switch and inhibited Na-K-ATPase alpha 1 isoform expression without inducing the other isoforms. With both hormones, the adult phenotype for both MHC and Na-K-ATPase was seen but with low Na-K-ATPase alpha 2. The paucity of alpha 2 protein was not predicted by studies of mRNA levels. In serum, there was a gradual decline of Na-K-ATPase alpha 3 and the appearance of alpha 2, but again at a relatively low level. Expression of Na-K-ATPase alpha 2 was significantly upregulated when cardiomyocytes were cocultured with sympathetic neurons from superior cervical ganglia, without changes in the MHC isoforms. Thus innervation is postulated to play a specific role in modulating Na-K-ATPase gene expression.

The autonomic nervous system is intimately involved in regulating cardiovascular function. Sensing mechanisms dispersed throughout the circulation, including arterial baroreceptors, low pressure receptors, and chemosensitive receptors, continually evoke reflexes designed to maintain cardiovascular homeostasis. Although there is a growing body of knowledge regarding neural regulation of the adult cardiovascular system, characterization and understanding of these physiological systems during development is limited. This review highlights developmental changes in the arterial and cardiopulmonary baroreflex during fetal and postnatal life and contrasts the function of these responses with those seen in the adult. Baroreceptors are functional in the immature animal and reset toward higher pressure levels with maturation. In our ovine model, the sensitivity of the efferent limb of the baroreflex is greatest during fetal life and decreases with postnatal development. As in the adult, angiotensin II and arginine vasopressin interact with the sympathetic nervous system early during development to alter baroreflex control of the cardiovascular system. However, the extent to which these hormonal systems influence autonomic reflexes during the fetal and newborn period appears vastly different than in the adult. Endogenous angiotensin II significantly contributes to resetting of the arterial baroreflex early in life, whereas even high circulating levels of vasopressin have little effect on baroreflex function until adulthood. Finally, the ability of cardiopulmonary mechanoreceptors to regulate cardiovascular function is impaired early in development, in sharp contrast to the heightened sensitivity of the arterial baroreflex at this stage of maturation. The potential importance of these autonomic reflexes on cardiovascular function during the perinatal period is highlighted.

During late gestation, the lungs of rats contain retinyl esters, but their concentration decreases considerably at the time of birth. The regulation of the acquisition and utilization of these stored retinoids remains poorly understood, although it has been hypothesized that they are involved in surfactant production and alveolar septal formation. Previous investigations demonstrated that exogenous retinoic acid increases elastin production in cultured neonatal lung fibroblasts and increases the number of alveoli when it is administered to neonatal rats. It has been hypothesized that these pulmonary stores of retinyl esters may regulate the perinatal expression of various genes in the lung, including elastin. To test this hypothesis, inhibitors of retinoid metabolism were used to reduce the flux of retinyl esters to retinoic acid, and the effects of this maneuver on elastin gene expression were analyzed. Inhibitors of alcohol and aldehyde dehydrogenases and of retinyl ester hydrolases decreased the steady-state level of tropoelastin mRNA without reducing alpha 1(I) procollagen mRNA. The magnitude of the effects of the inhibitors was retinol dependent and was significantly reduced in lung tissue that was obtained from vitamin A-deficient fetuses. These findings suggest that the late gestational pulmonary stores of retinoids may increase elastin gene expression during the fetal and early postnatal life in the rat.

Chronic unilateral ureteral obstruction (UUO) in early development activates the intrarenal renin-angiotensin system and leads to profound renal vasoconstriction, renal growth arrest, and interstitial fibrosis. To investigate the response of the AT1 and AT2 subtypes of the angiotensin II (ANG II) receptors to UUO, Sprague-Dawley rats underwent UUO or control sham operation in the first 48 h of life and were studied 1-28 days later. Renal mRNA for renin, AT1 and AT2 receptor, and receptor binding and distribution were determined. In contrast to controls, renin mRNA increased from 14 to 28 days in the obstructed kidney. After ipsilateral UUO, AT1 mRNA was suppressed at 1 day, but had increased compared with controls at 28 days. AT2 receptor mRNA fell rapidly in all kidneys from 1 to 3 days of age, after which it remained undetectable. Compared with the intact opposite kidney, AT2 mRNA was suppressed in the obstructed kidney 1 day after UUO. Compared with controls, AT1 and AT2 receptor binding was decreased by ipsilateral UUO at 1 day, whereas AT1 binding was increased at 28 days. Renal ANG II content was increased in the obstructed compared with the intact opposite kidney 28 days after UUO. In view of the increase in renal renin and angiotensin II production resulting from UUO, increased renal AT1 mRNA and receptor binding are likely to contribute to the vasoconstriction and interstitial fibrosis of the neonatal kidney after prolonged UUO.

Transferrin (TF), the major iron-transporting protein in vertebrates, is mainly synthesized in the liver. Although its source in lung is unknown, TF is a major inhibitor for lipid peroxidation and microbial propagation in lung fluid, and iron-free TF has been shown in rabbits to decrease the severity of respiratory failure and to improve surfactant activity. This study shows that TF is produced and secreted by the lung. In baboons and humans. TF gene expression displays distinct temporal patterns in different lung cells as revealed by in situ hybridization. Although expression of TF mRNA in submucosal glands remains active during development and throughout adulthood, its level in airway epithelial increases with advancing gestational age, reaches its peak before birth, declines 6-12 mo after birth, and diminishes in the older adult. In premature baboons maintained on ventilatory support, expression of TF mRNA is suppressed in both airway epithelium and glands. TF production by airway epithelia before birth most likely prevents oxidative damage in the newborn period, and its loss during injury may allow further lung damage.

To determine if mechanical signals or alterations in intracellular Ca2+ concentration ([Ca2+]i) affect myosin heavy chain (MHC) gene expression in spontaneously beating, neonatal rat ventricular myocytes, contractile activity was inhibited with verapamil, KCl, or 2,3-butanedione monoxime (BDM), and their acute and chronic effects on myocyte shortening, [Ca2+]i, and MHC gene expression were examined. Despite their differing effects on [Ca2+]i, verapamil, KCl, and BDM all inhibited contractile activity and markedly downregulated beta-MHC mRNA levels to 24 +/- 5, 21 +/- 7, and 6 +/- 2% of contracting cells, respectively. In contrast, these inhibitors of contraction upregulated alpha-MHC mRNA levels to 163 +/- 19, 156 +/- 7, and 198 +/- 20% of contracting cells, respectively. Transient transfection with a rat beta-MHC promoter-luciferase expression plasmid demonstrated that all inhibitors of contraction significantly decreased beta-MHC promoter activity. Paradoxically, contractile arrest also inhibited alpha-MHC promoter activity, suggesting that increased alpha-MHC mRNA levels resulted from posttranscriptional mechanisms. Actinomycin D mRNA stability assays indicated that alpha-MHC mRNA half-life was prolonged in noncontracting cells (33 h) compared with contracting myocytes (14 h). Contraction-dependent alterations in MHC gene expression were not dependent on release of angiotensin II or other growth factors into the culture medium. Thus intrinsic mechanical signals rather than alterations in [Ca2+]i regulate alpha-MHC and beta-MHC gene expression by both transcriptional and posttranscriptional mechanisms.

Near-infrared spectrophotometry (NIRS) potentially provides a tool for noninvasive tissue oxygenation and blood volume monitoring. Cerebral monitoring could be useful in the prevention of hypoxic ischemic brain injury in newborns. This study sought to validate such NIRS measurements in normoventilated, hypocapnic, and hypoxemic states in the brain of newborn piglets vs. arterial (SaO2) and sagittal sinus blood hemoglobin saturation (SssO2) and blood volume measurements with 99mTc-labeled erythrocytes. NIRS measurements of cerebral blood volume (CBV) were performed with both oxyhemoglobin and indocyanine green as tracers, and changes in CBV were monitored by following the change in the concentration of total hemoglobin (i.e., oxyhemoglobin + deoxyhemoglobin). NIRS CBV measurements did not correlate well with the radioactive measurements. NIRS measurements of oxygenation, however, correlated well with a weighted mean value of SaO2 and SssO2 (r = 0.90; P < 0.0001). Multiple linear regression of the oxygenation index (i.e., oxyhemoglobin - deoxyhemoglobin) on SaO2 and SssO2 suggested that NIRS sees hemoglobin in tissue in a venous-to-arterial ratio of 2:1. Therefore, in this study, NIRS reliably monitored changes in cerebral tissue oxygenation but not in CBV.

Sixty-two bulbospinal neurons were recorded in the rostral ventrolateral medulla (RVLM) of neonatal rats using patch electrodes. Sixty-one percent of the recorded neurons identified by histology contained tyrosine-hydroxylase (C1 cells). Substance P increased the spontaneous firing rate of all recorded cells but had no effect on spike configuration. The peptide depolarized neurons that were silenced by injection of hyperpolarizing current and increased their input resistance. All cells (n = 12) were activated by a neurokinin (NK)1 receptor agonist but most were unaffected by an NK2- or an NK2-selective compound. In voltage clamp, substance P produced a current that was linearly related to the membrane voltage. This current reversed polarity close to the potassium equilibrium potential in 11 of 23 cells. It reversed at more hyperpolarized potentials or not at all in the rest of the cells. In conclusion, substance P upregulates the intrinsic discharge rate of C1 and other putative sympathoexcitatory cells in neonatal rats. This effect is mediated via NK1 receptors. The depolarization is mediated by a reduction in resting potassium conductance and possibly by an additional cationic conductance. These results support the possibility that substance P could play a role "in vivo" in setting the basal level of discharge of the vasomotor cells of RVLM and therefore in regulating sympathetic vasomotor tone.

To measure the source and rate of mucosal glutathione (GSH) synthesis, fed piglets (28 days old; 7.7 kg) received a 6-h infusion of intragastric [U-13C]glutamate (n = 11) either with (n = 5) or without (n = 6) an intragastric infusion of [1-13C]glycine (0-6 h) and [1,2-13C2(U-13C)]glycine (3-6 h). Eighty-four percent of the labeled mucosal GSH-glutamate and 86% of the luminal GSH-glutamate was 13C5. The tracer-to-tracee ratio of GSH-[U-13C]glutamate was 75% of that of mucosal glutamate. Sixty percent of the labeled mucosal glutamate was 13C1, 13C2, or 13C3, but the tracer-to-tracee ratios of these isotopomers in GSH-glutamate were not significantly different from zero. After 3 h of infusion, the tracer-to-tracee ratio of GSH-[U-13C]glycine was 46%, and after 6 h of infusion GSH-[13C1]glycine was 82% of that of mucosal glycine. This suggested that the half-life of mucosal GSH was 2.7 +/- 0.1 h. We concluded that, in fed piglets, mucosal GSH-glutamate derived largely from the direct metabolism of enteral glutamate rather than from glutamate that was metabolized within the mucosa.

Recent evidence suggests that, in adult animals with hypoxia-induced pulmonary hypertension, atrial natriuretic factor (ANF) may modulate pulmonary vascular tone and may have a protective effect. However, its role in the pathogenesis of pulmonary hypertension of the newborn is unknown. We hypothesized that, in the newborn, hypoxia-induced pulmonary hypertension would result in ANF receptor downregulation, resulting in decreased dilator response, favoring pulmonary vasoconstriction and vascular remodeling. Therefore, we studied, in 1-day-old piglets exposed to hypoxia (fraction of inspired O2 0.10) for 3 or 14 days to induce pulmonary hypertension, 1) ANF release by measuring circulating levels of ANF by radioimmunoassay in pulmonary artery and veins, 2) pulmonary vascular reactivity to ANF using isolated perfused lungs, and 3) binding characteristics by examining the concentration dependence of ANF binding and competitive binding of 125I-labeled ANF with ANF, brain natriuretic peptide, C-type natriuretic peptide, and the specific ligand for ANF clearance receptor on microsomes from pulmonary arteries (down to 100 microns). ANF circulating levels are increased after exposure to hypoxia compared with normoxia, reaching significance at 14 days (P < 0.005). The magnitude of ANF dilator response is diminished after exposure to hypoxia (P < 0.05). Saturation studies reveal that the number of ANF receptors is diminished in hypoxia after 3 days but reaches significance after 14 days (P < 0.01) compared with their respective normoxic control. At either condition, the majority of these receptors are of the functional type, whereas clearance receptors are virtually undectable. These results suggest that hypoxia increases circulating ANF and causes a decreased responsiveness of the pulmonary vasculature to ANF. Receptor down-regulation may explain part of the reduced dilator response, although the involvement of other mechanisms is not excluded.

This study characterizes the development of diurnal patterns of slow-wave sleep (SWS) distribution and SWS electroencephalographic (EEG) delta-power (DP) density in 12- to 24-day-old rats (P12-P24). Diurnal organization in sleep-wake distribution was established by P20. A decline in SWS DP across the light phase did not appear until P24. Before P20, SWS DP increased across the light phase in a pattern inverse to that typically seen in adult rats. At P20, SWS DP was evenly distributed across the light phase, and at P24, SWS DP declined across the light phase. The transient dissociation between diurnal organization in sleep-wake cycles and SWS DP suggests that circadian and homeostatic sleep regulatory mechanisms develop at different rates in the postnatal period.

The optimal dose, route of administration, and treatment-to-delivery interval necessary to induce beneficial extrapulmonary effects of glucocorticoids are not known. Pregnant ewes (127 days gestation) were randomized to receive maternal or fetal intramuscular injections of betamethasone (0.2 or 0.5 mg/kg body wt) or saline 24 h before cesarean delivery of their lambs. Three hours after delivery, low-dose maternal vs. control lamb mean arterial pressure [64 +/- 4 vs. 47 +/- 2 (SE) mmHg], glomerular filtration rate (1.7 +/- 0.2 vs. 0.7 +/- 0.1 ml.min-1.kg-1), and total renal sodium reabsorption (219 +/- 31 vs. 85 +/- 12 mueq.min-1.kg-1) were increased. Comparable increases were observed in the high-dose maternal and fetal groups without effects in the low-dose fetal group. This study provides the first quantitative data demonstrating that even short-term (24-h) antenatal betamethasone exposure alters preterm newborn cardiovascular and renal functions. These responses are route and dose dependent and are comparable to glucocorticoid-induced maturational effects after longer-term antenatal exposure.

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.

A cardiovascular reflex evoked by esophageal distension (ECR) in urethan-anesthetized male Sprague-Dawley rats was studied to 1) determine whether the relevant sensory input from the esophagus is conveyed by vagal and/or spinal afferents and 2) evaluate the effects and sites of action of antinociceptive agents. Esophageal distension evoked a rise in arterial blood pressure and heart rate that increased linearly with the log of inflation pressure (25-150 mmHg). Distension (100 mmHg for 20 s) of the lower esophagus was a more effective stimulus than distension of the upper esophagus. The ECR was attenuated by unilateral and abolished by bilateral cervical vagotomy and dose dependently inhibited by morphine (1.0-4.0 mg/kg iv) or by intrathecal (T4-T5) administration of dexmedetomidine (DX, 0.05-0.5 microgram), but not by intrathecal (T4-T5) morphine (4-16 micrograms) or intrathecal (L1-L2) or intravenous DX (0.05-0.5 microgram). The ECR was also inhibited by capsaicin and by the topical administration of DX or morphine to the solitary complex. The pressor response persisted after intravenous pancuronium, scopolamine, and methscopolamine. The ECR circuit appears to consist of vagal afferents, efferent sympathetic preganglionic pathways originating in the thoracic spinal cord, and bulbospinal neurons yet to be identified. This reflex fulfills some criteria of a nociceptive event, but this interpretation requires further investigation.