Impulse activity differentially regulates [Leu]enkephalin and catecholamine characters in the adrenal medulla.

Regulation of the putative peptide neurohumour [Leu]enkephalin and the catecholaminergic enzymes tyrosine hydroxylase and phenylethanolamine-N-methyl-transferase was examined in the rat adrenal medulla in vivo and in vitro. Surgical denervation of the adrenal gland or pharmacologic blockade of synaptic transmission, treatments known to decrease catecholamine traits, increased [Leu]enkephalin content. Medullas explanted to culture exhibited a 50-fold rise in [Leu]enkephalin in 4 days, whereas tyrosine hydroxylase remained constant, and phenylethanolamine-N-methyltransferase decreased to a new baseline level. Veratridine-induced depolarization prevented the accumulation of [Leu]enkephalin, an effect that was blocked by tetrodotoxin, which antagonizes transmembrane Na+ influx. These studies suggest that enkephalinergic and catecholamine characters are differentially regulated by impulse activity and depolarization in the adrenal medulla.

Biochemistry of information storage in the nervous system.

The use of molecular biological approaches has defined new mechanisms that store information in the mammalian nervous system. Environmental stimuli alter steady-state levels of messenger RNA species encoding neurotransmitters, thereby altering synaptic, neuronal, and network function over time. External or internal stimuli alter impulse activity, which alters membrane depolarization and selectively changes the expression of specific transmitter genes. These processes occur in diverse peripheral and central neurons, suggesting that information storage is widespread in the neuraxis. The temporal profile of any particular molecular mnemonic process is determined by specific kinetics of turnover and by the geometry of the neuron resulting in axonal transport of molecules to different synaptic arrays at different times. Generally, transmitters, the agents of millisecond-to-millisecond communication, are subject to relatively long-lasting changes in expression, ensuring that ongoing physiological function is translated into information storage.

Neurotransmitter plasticity at the molecular level.

Contrary to long-held assumptions, recent work indicates that neurons may profoundly change transmitter status during development and maturity. For example, sympathetic neurons, classically regarded as exclusively noradrenergic or cholinergic, can also express putative peptide transmitters such as substance P. This neuronal plasticity is directly related to membrane depolarization and sodium ion influx. The same molecular mechanisms and plastic responses occur in mature as well as developing neurons. Further, contrary to traditional teaching, adult primary sensory neurons may express the catecholaminergic phenotype in vivo. Transmitter plasticity is not restricted to the peripheral nervous system: ongoing studies of the brain nucleus locus ceruleus in culture indicate that specific extracellular factors elicit marked transmitter changes. Consequently, neurotransmitter expression and metabolism are dynamic, changing processes, regulated by a variety of defined factors. Transmitter plasticity adds a newly recognized dimension of flexibility to nervous system function.

Effects of sodium bicarbonate correction of metabolic acidosis on regional tissue oxygenation in very low birth weight neonates.

OBJECTIVE: To determine the effects of sodium bicarbonate (NaHCO3) correction of metabolic acidosis on cardiopulmonary, laboratory, and cerebral, renal and splanchnic regional oxygen saturation (rSO2) and fractional tissue oxygen extraction (FTOE) in extremely premature neonates during the first postnatal week. STUDY DESIGN: Observational cohort data were collected from 500 to 1250 g neonates who received NaHCO3 'half' corrections (0.3 * Weight (kg) * Base Deficit (mmol l(-1))) for presumed renal losses. RESULT: Twelve subjects with normal blood pressure and heart rate received 17 NaHCO3 corrections. Mean (±s.d.) gestational age was 27±2 week and birth weight was 912±157 g. NaHCO3 corrections provided a mean (±s.d.) 4.5±1.0 ml kg(-1) fluid bolus, shifted mean (±s.d.) base deficit from 7.6±1.8 to 3.4±2.1 mmol l(-1) (P<0.05), and increased median (±s.d.) pH from 7.23±0.06 to 7.31±0.05 (P<0.05). No significant changes in blood pressure, pulse oximetry, PCO2, lactate, sodium, blood urea nitrogen, creatinine or hematocrit were observed. Cerebral, renal and splanchnic rSO2 (74%, 66% and 44%, respectively, at baseline) and FTOE (0.21, 0.29 and 0.52, respectively, at baseline) were unchanged following NaHCO3 correction. CONCLUSION: NaHCO3 infusions decreased base deficits and increased pH though produced no discernible effects or benefits on cardiopulmonary parameters including rSO2 and FTOE. These findings warrant further prospective evaluation in larger populations with more significant metabolic acidosis to determine the utility of tissue oxygenation monitoring in differentiating metabolic acidosis due to oxygen delivery/consumption imbalance versus renal bicarbonate losses.

Early enteral feeding does not affect the incidence of necrotizing enterocolitis.

To begin to determine the optimal time for initiating enteral feedings, 34 sick, very low birth weight infants were prospectively selected from all neonates of less than 1,500 g (N = 116) and randomly divided into two groups. Infants were fed either on day 1 (early) or 7 (late) of life, according to a feeding protocol which included parenteral nutrition and a scheduled progression from sterile water to 2.5% dextrose, half-strength, and finally full-strength formula over seven days. The incidence of necrotizing enterocolitis and subsequent hospital course were compared. Initiating enteral feedings on day 1 did not significantly increase the incidence of necrotizing enterocolitis, produce a clustering of cases, or induce an earlier onset of necrotizing enterocolitis. The overall incidence of necrotizing enterocolitis in sick, very low birth weight neonates was 29% (5/17) and 35% (6/17) in the early and late groups, respectively, compared with 4.2% (2/47) in minimally sick, very low birth weight neonates. No significant differences between groups were seen in obstetrical complications, birth weight, gestational age, Apgar scores, presence of patent ductus arteriosus or intraventricular hemorrhage, use of umbilical catheterization, and respiratory or oxygen requirements. Infants fed enterally from day 1 did show a significantly higher energy and protein intake during the second week of life. These data show that providing dilute, early enteral calories does not adversely affect the incidence of necrotizing enterocolitis.

Noninvasive diagnosis of persistent fetal circulation versus congenital cardiovascular defects.

Congenital cardiovascular anomalies associated with right-left atrial or ductal shunts must be excluded before a diagnosis of persistent fetal circulation (PFC) can be made. Despite the advent of 2-dimensional echocardiography (2-D echo), this differentiation can be difficult and may require cardiac catheterization with selective angiography. Fifteen consecutive cases were analyzed in which difficulty was encountered with this differential diagnosis, and experience with the use of cardiac auscultation, the 12-lead electrocardiogram (ECG), arterial blood gas determinations and 2-D echo, both alone and with injection of venous contrast material, is reviewed. Electrocardiographic abnormalities of ventricular axis, hypertrophy or dominance (p = 0.002) and suspicion of cardiovascular disease on 2-D echo (p = 0.011) were the most useful findings in differentiating patients with PFC from those with congenital cardiovascular abnormalities. The ECG was the most sensitive test (100% sensitivity, 90% specificity), while 2-D echo was the most specific (100% specificity, 75% sensitivity). Evidence of right-left shunting at atrial or ductal levels or both did not differentiate between the groups; both groups had evidence of such shunts. A decision tree was developed to facilitate this differential diagnosis, which uses the ECG and 2-D echo. If the ECG reveals no abnormalities of ventricular axis, dominance or hypertrophy, the 2-D echo shows no structural abnormalities, and total anomalous pulmonary venous return and coarctation/interruption of the aorta are specifically excluded, a congenital cardiovascular anomaly is effectively eliminated. We suggest that this approach can optimize the management of the cyanotic newborn with suspected PFC by eliminating the risks of cardiac catheterization and angiography without missing the diagnosis of a major structural cardiovascular anomaly.

Cholinergic regulation of rat preproenkephalin RNA in the adrenal medulla.

Expression of the rat preproenkephalin (ppENK) gene involves transsynaptic cholinergic mechanisms. We evaluated the effects of cholinergic agonist treatments in vivo on the expression of adrenomedullary ppENK RNA. Cholinergic treatment with nicotinic + muscarinic receptor agonists resulted in a synergistic 100-fold rise in steady-state ppENK messenger RNA levels, but only a 30- to 35-fold rise in initiation of steady-state ppENK RNA transcripts. The levels of initiated ppENK steady-state RNA peaked at two days, whereas mature (1.45 kb) ppENK mRNA levels continued to rise, peaking at four days. This suggested that other transcriptional (attenuation or alternative splicing) or post-transcriptional (RNA stabilization) regulatory mechanisms must be operative. As multiple ppENK RNA start sites exist, we examined how usage of multiple sites was altered by cholinergic treatments. The predominant start site changed from E2 in the basal state, to E4 after primary cholinergic stimulation, to E3 after re-treatment. This represents novel example of differential usage of multiple RNA initiation start sites in vivo. Differences in initiated and mature transcripts are consistent with at least four mechanisms involved in control of cholinergic-induced ppENK RNA expression: (i) simply new initiation of RNA transcripts, (ii) differential usage of the multiple RNA start sites, (iii) stabilization of mRNA transcripts, and (iv) attenuation and/or alternative RNA splicing of RNA transcripts.

Characterization of enkephalins in rat adrenal medullary explants.

In the rat, removal of depolarizing stimuli to the adrenal medulla by surgical denervation in vivo or by explanting adrenal medullae has been shown to dramatically increase preproenkephalin mRNA, and enkephalin-containing (EC) peptides. To further elucidate the cellular basis of these effects and the role of transsynaptic influences on post-translational processing, we have defined the time course, and characterized EC peptides in rat adrenal medullary explants in control and depolarized states. The rise in EC peptides begins after 1 day in culture and reaches a peak at 4-7 days. Although the onset of the increase in EC peptides in culture is delayed by 12-24 h compared to the changes seen in vivo, following surgical denervation, the time course of peak and duration is remarkably similar. Size exclusion chromatography (SEC) revealed that the major species of newly appearing EC peptides in explanted glands is a high molecular weight peptide of approximately 18,000 with a Met-/Leu-enkephalin ratio of approximately 6. These results suggest that proenkephalin, the initial precursor of the EC peptide family, is the major EC peptide that accumulates in rat adrenal medullary explants. A low-molecular weight EC peptide, found by high-performance liquid chromatography to be free Met-enkephalin, is a minor component of the culture induced increase in EC peptides. Culturing of medullae in the presence of depolarizing concentrations of K+ prevents the accumulation of the proenkephalin-like EC peptides and free enkephalins.(ABSTRACT TRUNCATED AT 250 WORDS)

Quiescent variability of cerebral, renal, and splanchnic regional tissue oxygenation in very low birth weight neonates.

OBJECTIVE: In extremely premature neonates, data concerning the normal baseline variability of near-infrared spectroscopy (NIRS)-derived regional oxygen saturation (rSO2) are lacking. We sought to determine: 1) the quiescent variability of cerebral, renal, and splanchnic rSO2 in clinically stable, undisturbed very low birth weight neonates and 2) the effects of different data averaging epochs on site-specific variability. STUDY DESIGN: In this prospective, observational study, neonates between 500 and 1250 g underwent seven days of continuous, real-time cerebral, renal, and splanchnic NIRS monitoring starting within the first seventy-two postnatal hours. Demographic, cardiopulmonary, bedside care, and rSO2 data were collected. rSO2 variability was analyzed utilizing data from quiescent periods identified using pre-specified stability criteria. Between- and within-monitoring site comparisons of data averaging methods were made utilizing ANOVA. RESULT: Twenty-four subjects (GA 27 ± 0.3 wk, birth weight 988 ± 34 g; mean ± SEM) were monitored. Coefficients of variation (CoVar = SD/mean) were calculated for each monitoring site using varied data averaging epochs. CoVar was lowest for cerebral, intermediate for renal, and highest for splanchnic rSO2 (P < 0.01). For renal and splanchnic sites, shorter epochs (5- and 15-min) resulted in significantly smaller CoVars [P < 0.01 and P < 0.05, respectively]. Splanchnic variability was highly dependent on epoch length, ranging from 16% over 5 min to 23% over 60 min. CONCLUSION: 1) rSO2 variability differs significantly between monitoring sites and 2) shorter data sampling epochs decrease rSO2 variability. These observations may assist clinicians in operationally defining minimally significant departures to enable medical decision making utilizing this monitoring technique.

Monitoring regional tissue oxygen extraction in neonates <1250 g helps identify transfusion thresholds independent of hematocrit.

OBJECTIVE: We sought to characterize the effects of "booster" packed red blood cell transfusions on multisite regional oxygen saturation in very low birth weight neonates during the first postnatal week and to examine the utility of fractional tissue oxygen extraction as an estimate of tissue oxygenation adequacy. STUDY DESIGN: Data were collected in an observational near-infrared spectroscopy (NIRS) pilot survey of 500-1250 g neonates during the first postnatal week. A before-after analysis of "booster" transfusions, defined as empiric 15 mL/kg transfusion following 10 mL/kg cumulative phlebotomy losses, was conducted upon cardiopulmonary, laboratory, and spectroscopy data. RESULT: Ten neonates (gestational age 26 ± 0 wk; birth weight 879 ± 49 g) received 14 transfusions at 3 ± 0 postnatal days. Mean hematocrit increased from 35.2 ± 1.2 to 38.5 ± 1.2 % (P < 0.05) following transfusion; pH, base deficit, lactate, creatinine, and cardiopulmonary parameters were unchanged. Cerebral, renal, and splanchnic tissue oxygenation increased 10, 18, and 16%, with concomitant decreases in calculated oxygen extraction of 27, 30, and 9% (all P < 0.05), consistent with enhanced tissue oxygenation. These findings were not observed in a non-transfused comparison group of nine patients. CONCLUSION: "Booster" transfusions improved indices of regional tissue oxygenation while no departures were observed in conventional cardiovascular assessments. We speculate that NIRS-derived oxygenation parameters can provide an objective, graded, and continuous estimate of oxygen delivery-consumption balance not evident using standard monitoring techniques.

Nicotinic receptor partial agonists alter catecholamine homeostasis and response to nicotine in PC12 cells.

Repeated stress is a major public health concern where many stress responses are mediated by neuronal nicotinic acetylcholine receptors. In the present study we evaluated the effects of the nicotinic receptor partial agonists, cytisine and its derivative 3-(pyridin-3'-yl)-cytisine (3-pyr-Cyt) on two main biological outputs associated with activation of nAChR-release of neurotransmitters and increase in catecholamine biosynthesis to replenish the releasable pool. We compared these substances to the maximal response triggered by nicotine (full agonist) in PC12 cells. Cytisine, 3-pyr-Cyt or nicotine induced time-, dose- and Ca(2+)-dependent significant release of norepinephrine (NE) into the culture media. These effects were completely inhibited by mecamylamine but not by α-bungarotoxin, and only partially affected by α-conotoxin AulB, consistent with the involvement of α3ß4 receptors. Co-application of cytisine (or 3-pyr-Cyt) and nicotine resulted in attenuated nicotine-induced NE release. Cytisine or 3-pyr-Cyt alone induced a modest rise in tyrosine hydroxylase (TH) mRNA levels (index of the cell's catecholamine biosynthetic capacity). We conclude that both, cytisine and 3-pyr-Cyt (i) display typical partial agonist properties at naturally existing ganglionic nAChR (α3ß4 and α7 nAChR) with regard to catecholamine homeostasis (i.e. NE release and re-synthesis) and (ii) modulated the effect of nicotine during combined treatment.

Postnatal betamethasone vs dexamethasone in premature infants with bronchopulmonary dysplasia: a pilot study.

OBJECTIVE: As effects of glucocorticoids differ with respect to preparation, dose and duration, we hypothesized that a postnatal regimen of a low-dose, short-course betamethasone treatment had comparable efficacy and a better safety profile compared to the conventional high-dose, dexamethasone. STUDY DESIGN: To test our hypothesis, we selected premature neonates with a birth weight 10 postnatal days with an FiO(2)>0.4 and no ability to wean mechanical support for >or=3 consecutive days. These neonates either received twice daily dexamethasone 0.25 mg kg(-1) per dose intravenously for 3 days tapered to 0.125 mg kg(-1) per dose for 4 days (June 1999 to December 2000) or betamethasone 0.125 mg kg(-1) per day intramuscularly once per day for 3 days (January 2001 to December 2002). RESULT: We found a significant reduction in FiO(2) after 3 days in both glucocorticoid treatment groups. There were no significant differences between the two treatment groups in the clinical parameters including decrease in FiO(2), oxygenation index, mean airway pressure and percent extubation. Duration of ventilation, number of oxygen days and length of hospital stay were comparable in the two groups. Of particular interest, the betamethasone group showed fewer adverse effects, such as poor weight gain and high blood glucose, than the dexamethasone group. CONCLUSION: A short course of low-dose betamethasone has comparable efficacy and seemingly a better short-term safety profile compared to conventional dexamethasone treatment.

Product inhibition of carboxypeptidase H.

Carboxypeptidase H is one of several enzymes required for the processing of peptide hormone precursors. In this study, inhibition of carboxypeptidase H by its peptide products was investigated. Carboxypeptidase H activity in bovine adrenal medulla chromaffin granules and rat adrenal medulla homogenate was inhibited by the peptides Met- and Leu-enkephalin, vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone, with oxytocin and ACTH 1-14 having the least effect, at concentrations of 2-20 mM. Inhibition by amidated peptide products (vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone) show that the final products of the precursor processing pathway can regulate carboxypeptidase H. These levels of peptides are similar to known intragranular peptide concentrations indicating that product and feedback inhibition of carboxypeptidase H may play a role in the control of neuropeptide synthesis. The proenkephalin-derived peptides Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, and Met-enkephalin-Arg6-Phe7 competitively inhibited bovine and rat carboxypeptidase H with Ki values of 12.0, 6.5, 7.0, and 5.5 mM, respectively. The significantly greater Ki for Met-enkephalin may reflect the effects of higher intragranular concentration of Met-enkephalin, since one proenkephalin molecule contains four copies of Met-enkephalin and only one copy of each of the other enkephalin peptides. Thus, the products from one multivalent precursor molecule may equivalently inhibit carboxypeptidase H activity. Product inhibition of carboxypeptidase H and perhaps other processing enzymes may serve to limit the maximum peptide concentration within the secretory vesicle.

Neonatal stress: effects of hypoglycemia and hypoxia on adrenal tyrosine hydroxylase gene expression.

Catecholamines (CA) are released from and resynthesized in the adrenal medulla in response to stress. In the mature animal, stimulus-secretion-synthesis coupling occurs through transsynaptic (neuronal) activity. In contrast, in the immature animal, before functional adrenal innervation, certain stressors (hypoglycemia and glycopenia) do not result in CA release. Additionally, it is not known whether release and biosynthesis remain coupled in the neonate as they are in the adult. Therefore, to evaluate whether neonatal stressors can induce CA biosynthesis at the genomic level "directly" before function adrenal innervation, we studied the expression of the tyrosine hydroxylase (TH) gene, the rate-limiting enzyme in CA biosynthesis. Newborn rat pups were made either hypoxic, hypoglycemic, or cellularly glycopenic (2-deoxyglucose). Neither hypoxic stress nor insulin-induced hypoglycemic stress altered steady state levels of TH mRNA in the neonate. However, cellular glycopenia resulted in a significant 2-fold rise in TH mRNA levels (p < 0.05). As expected, each of these stressors increased TH mRNA levels in the mature adult rat. Thus, neonatal hypoxia and hypoglycemia appear to require intact neurogenic impulse activity, whereas cellular glycopenia may "directly" induce TH RNA, perhaps through hormonal mechanisms. This developmental model allows for the analysis of mechanisms governing adrenal CA release separate from those governing biosynthesis at the level of TH RNA. Acute neonatal hypoxic stress results in adrenal CA release without increasing TH RNA. Intrauterine growth retardation from chronic prenatal hypoxemia results in neonatal CA depletion and decreased CA responsiveness. We speculate that chronic hypoxia alters CA pathways, increasing the susceptibility of these infants to later stressors.

Heart rate and blood pressure responses to umbilical cord compression in fetal lambs with special reference to the mechanism of variable deceleration.

We examined heart rate and blood pressure responses to umbilical cord compression in fetal lambs. Fetal heart rate (FHR) responses resembling variable deceleration occurred only after umbilical blood flow was reduced by at least 50%. These changes during partial cord occlusion varied directly with the reduced umbilical blood flow and were abolished by atropine; no significant changes in arterial pressure were observed. Complete cord occlusion caused severe bradycardia, a progressive increase in arterial pressure, and delayed recovery of FHR. With partial cord occlusion, the bradycardia was of chemoreceptor origin and was vagally mediated; with complete occlusion the bradycardia may have resulted from both chemoreceptor and baroreceptor stimulation. During prolonged partial cord occlusion, FHR decreased initially, then recovered to above control value; this occurred in the face of a significant acidosis. Thus, FHR responses to cord compression are dependent on the actual percentage of reduction in umbilical blood flow and on its duration.