Neuroendocrine regulation of salt and water metabolism

Neurons which release atrial natriuretic peptide (ANPergic neurons) have their cell bodies in the paraventricular nucleus and in a region extending rostrally and ventrally to the anteroventral third ventricular (AV3V) region with axons which project to the median eminence and neural lobe of the pituitary gland. These neurons act to inhibit water and salt intake by blocking the action of angiotensin II. They also act, after their release into hypophyseal portal vessels, to inhibit stress-induced ACTH release, to augment prolactin release, and to inhibit the release of LHRH and growth hormone-releasing hormone. Stimulation of neurons in the AV3V region causes natriuresis and an increase in circulating ANP, whereas lesions in the AV3V region and caudally in the median eminence or neural lobe decrease resting ANP release and the response to blood volume expansion. The ANP neurons play a crucial role in blood volume expansion-induced release of ANP and natriuresis since this response can be blocked by intraventricular (3V) injection of antisera directed against the peptide. Blood volume expansion activates baroreceptor input via the carotid, aortic and renal baroreceptors, which provides stimulation of noradrenergic neurons in the locus coeruleus and possibly also serotonergic neurons in the raphe nuclei. These project to the hypothalamus to activate cholinergic neurons which then stimulate the ANPergic neurons. The ANP neurons stimulate the oxytocinergic neurons in the paraventricular and supraoptic nuclei to release oxytocin from the neural lobe which circulates to the atria to stimulate the release of ANP. ANP causes a rapid reduction in effective circulating blood volume by releasing cyclic GMP which dilates peripheral vessels and also acts within the heart to slow its rate and atrial force of contraction. The released ANP circulates to the kidney where it acts through cyclic GMP to produce natriuresis and a return to normal blood volume

Release of plasma atrial natriuretic peptide after volume expansion is not related to pituitary-adrenal axis diurnal variation in normal subjects

The existence of a circadian rhythm of atrial natriuretic peptide (ANP) in humans is controversial. We studied the plasma ANP response to isotonic blood volume expansion in the morning and in the afternoon and its relationship with adrenocorticotropic hormone (ACTH)-cortisol diurnal variation in seven normal subjects. Basal plasma ANP level was similar in the morning (19.6 ± 2.4 pg/ml) and in the afternoon (21.8 ± 4.8 pg/ml). The ANP peak obtained with saline infusion (0.9% NaCl, 12 ml/kg) in the morning (49.4 ± 8 pg/ml) did not differ from that obtained in the afternoon (60.3 ± 10.1 pg/ml). There was no correlation between the individual mean cortisol and ACTH levels and the ANP peak obtained with saline infusion. These data indicate no diurnal variation in plasma ANP secretion induced by blood volume expansion and no relationship between plasma ANP peak and ACTH-cortisol diurnal variation

Rapid eye movement (REM) sleep deprivation reduces rat frontal cortex acetylcholinesterase (EC 3.1.1.7) activity

Rapid eye movement (REM) sleep deprivation induces several behavioral changes. Among these, a decrease in yawning behavior produced by low doses of cholinergic agonists is observed which indicates a change in brain cholinergic neurotransmission after REM sleep deprivation. Acetylcholinesterase (Achase) controls acetylcholine (Ach) availability in the synaptic cleft. Therefore, altered Achase activity may lead to a change in Ach availability at the receptor level which, in turn, may result in modification of cholinergic neurotransmission. To determine if REM sleep deprivation would change the activity of Achase, male Wistar rats, 3 months old, weighing 250-300 g, were deprived of REM sleep for 96 h by the flower-pot technique (N = 12). Two additional groups, a home-cage control (N = 6) and a large platform control (N = 6), were also used. Achase was measured in the frontal cortex using two different methods to obtain the enzyme activity. One method consisted of the obtention of total (900 g supernatant), membrane-bound (100,000 g pellet) and soluble (100,000 g supernatant) Achase, and the other method consisted of the obtention of a fraction (40,000 g pellet) enriched in synaptic membrane-bound enzyme. In both preparations, REM sleep deprivation induced a significant decrease in rat frontal cortex Achase activity when compared to both home-cage and large platform controls. REM sleep deprivation induced a significant decrease of 16% in the membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) in the 100,000 g pellet enzyme preparation (home-cage group 152.1 ± 5.7, large platform group 152.7 ± 24.9 and REM sleep-deprived group 127.9 ± 13.8). There was no difference in the soluble enzyme activity. REM sleep deprivation also induced a significant decrease of 20% in the enriched synaptic membrane-bound Achase activity (home-cage group 126.4 ± 21.5, large platform group 127.8 ± 20.4, REM sleep-deprived group 102.8 ± 14.2). Our results suggest that REM sleep deprivation changes Ach availability at the level of its receptors through a decrease in Achase activity

Gastroduodenal resistance and neural mechanisms involved in saline flow decrease elicited by acute blood volume expansion in anesthetized rats

We have previously demonstrated that blood volume (BV) expansion decreases saline flow through the gastroduodenal (GD) segment in anesthetized rats (Xavier-Neto J, dos Santos AA & Rola FH (1990) Gut, 31: 1006-1010). The present study attempts to identify the site(s) of resistance and neural mechanisms involved in this phenomenon. Male Wistar rats (N = 97, 200-300 g) were surgically manipulated to create four gut circuits: GD, gastric, pyloric and duodenal. These circuits were perfused under barostatically controlled pressure (4 cmH2O). Steady-state changes in flow were taken to reflect modifications in circuit resistances during three periods of time: normovolemic control (20 min), expansion (10-15 min), and expanded (30 min). Perfusion flow rates did not change in normovolemic control animals over a period of 60 min. BV expansion (Ringer bicarbonate, 1 ml/min up to 5% body weight) significantly (P<0.05) reduced perfusion flow in the GD (10.3 ± 0.5 to 7.6 ± 0.6 ml/min), pyloric (9.0 ± 0.6 to 5.6 ± 1.2 ml/min) and duodenal (10.8 ± 0.4 to 9.0 ± 0.6 ml/min) circuits, but not in the gastric circuit (11.9 ± 0.4 to 10.4 ± 0.6 ml/min). Prazosin (1 mg/kg) and yohimbine (3 mg/kg) prevented the expansion effect on the duodenal but not on the pyloric circuit. Bilateral cervical vagotomy prevented the expansion effect on the pylorus during the expansion but not during the expanded period and had no effect on the duodenum. Atropine (0.5 mg/kg), hexamethonium (10 mg/kg) and propranolol (2 mg/kg) were ineffective on both circuits. These results indicate that 1) BV expansion increases the GD resistance to liquid flow, 2) pylorus and duodenum are important sites of resistance, and 3) yohimbine and prazosin prevented the increase in duodenal resistance and vagotomy prevented it partially in the pylorus

Effect of pyloroplasty and fundectomy on the delay of gastric emptying and gastrointestinal transit of liquid elicited by acute blood volume expansion in awake rats

We evaluated the effects of fundectomy and pyloroplasty on the delay of gastric emptying (GE) and gastrointestinal (GI) transit of liquid due to blood volume (BV) expansion in awake rats. Male Wistar rats (N = 76, 180-250 g) were first submitted to fundectomy (N = 26), Heinecke-Mikulicz pyloroplasty (N = 25) or SHAM laparotomy (N = 25). After 6 days, the left external jugular vein was cannulated and the animals were fasted for 24 h with water ad libitum. The test meal was administered intragastrically (1.5 ml of a phenol red solution, 0.5 mg/ml in 5% glucose) to normovolemic control animals and to animals submitted to BV expansion (Ringer-bicarbonate, iv infusion, 1 ml/min, volume up to 5% body weight). BV expansion decreased GE and GI transit rates in SHAM laparotomized animals by 52 and 35.9% (P<0.05). Fundectomy increased GE and GI transit rates by 61.1 and 67.7% (P<0.05) and prevented the effect of expansion on GE but not on GI transit (13.9% reduction, P<0.05). Pyloroplasty also increased GE and GI transit rates by 33.9 and 44.8% (P<0.05) but did not prevent the effect of expansion on GE or GI transit (50.7 and 21.1% reduction, P<0.05). Subdiaphragmatic vagotomy blocked the effect of expansion on GE and GI transit in both SHAM laparotomized animals and animals submitted to pyloroplasty. In conclusion 1) the proximal stomach is involved in the GE delay due to BV expansion but is not essential for the establishment of a delay in GI transit, which suggests the activation of intestinal resistances, 2) pyloric modulation was not apparent, and 3) vagal pathways are involved

Acute blood volume expansion delays the gastrointestinal transit of a charcoal meal in awake rats

The present study evaluates the effect of blood volume expansion on the gastrointestinal transit of a charchoal meal (2.5 ml of an aqueous suspension consisting of 5% charcoal and 5% gum arabic) in awake male Wistar rats (200-270 g). On the day before the experiments, the rats were anesthetized with ether, submitted to left jugular vein cannulation and fasted with water ad libitum until 2 h before the gastrointestinal transit measurement. Blood volume expansion by iv infusion of 1 ml/min Ringer bicarbonate in volumes of 3, 4 or 5% body weight delayed gastrointestinal transit at 10 min after test meal administration by 21.3-26.7% (P<0.05), but no effect was observed after 1 or 2% body weight expansion. The effect of blood volume expansion (up to 5% body weight) on gastrointestinal transit lasted for at least 60 min (P<0.05). Mean arterial pressure increased transiently and central venous pressure increased and hematocrit decreased (P<0.05). Subdiaphragmatic vagotomy and yohimbine (3 mg/kg) prevented the delay caused by expansion on gastrointestinal transit, while atropine (0.5 mg/kg), L-NAME (2 mg/kg), hexamethonium (10 mg/kg), prazosin (1 mg/kg) or propranolol (2 mg/kg) were ineffective. These data show that blood volume expansion delays the gastrointestinal transit of a charcoal meal and that vagal and yohimbine-sensitive pathways appear to be involved in this phenomenon. The delay in gastrointestinal transit observed here, taken together with the modifications of gastrointestinal permeability to salt and water reported by others, may be part of the mechanisms involved in liquid excess management.

Rapid eye movement sleep deprivation induces an increase in acetylcholinesterase activity in discrete rat brain regions

Some upper brainstem cholinergic neurons (pedunculopontine and laterodorsal tegmental nuclei) are involved in the generation of rapid eye movement (REM) sleep and project rostrally to the thalamus and caudally to the medulla oblongata. A previous report showed that 96 h of REM sleep deprivation in rats induced an increase in the activity of brainstem acetylcholinesterase (Achase), the enzyme which inactivates acetylcholine (Ach) in the synaptic cleft. There was no change in the enzyme's activity in the whole brain and cerebrum. The components of the cholinergic synaptic endings (for example, Achase) are not uniformly distributed throughout the discrete regions of the brain. In order to detect possible regional changes we measured Achase activity in several discrete rat brain regions (medulla oblongata, pons, thalamus, striatum, hippocampus and cerebral cortex) after 96 h of REM sleep deprivation. Naive adult male Wistar rats were deprived of REM sleep using the flower-pot technique, while control rats were left in their home cages. Total, membrane-bound and soluble Achase activities (nmol of thiocholine formed min-1 mg protein-1) were assayed photometrically. The results (mean ± SD) obtained showed a statistically significant (Student t-test) increase in total Achase activity in the pons (control: 147.8 ± 12.8, REM sleep-deprived: 169.3 ± 17.4, N = 6 for both groups, P<0.025) and thalamus (control: 167.4 ± 29.0, REM sleep-deprived: 191.9 ± 15.4, N = 6 for both groups, P<0.05). Increases in membrane-bound Achase activity in the pons (control: 171.0 ± 14.7, REM sleep-deprived: 189.5 ± 19.5, N = 6 for both groups, P<0.05) and soluble enzyme activity in the medulla oblongata (control: 147.6 ± 16.3, REM sleep-deprived: 163.8 ± 8.3, N = 6 for both groups, P<0.05) were also observed. There were no statistically significant differences in the enzyme's activity in the other brain regions assayed. The present findings show that the increase in Achase activity induced by REM sleep deprivation was specific to the pons, a brain region where cholinergic neurons involved in REM generation are located, and also to brain regions which receive cholinergic input from the pons (the thalamus and medulla oblongata). During REM sleep extracellular levels of Ach are higher in the pons, medulla oblongata and thalamus. The increase in Achase activity in these brain areas after REM sleep deprivation suggests a higher rate of Ach turnover.

Effects of lead and/or zinc exposure during the second stage of rapid postnatal brain growth on delta-aminolevulinate dehydratase and negative geotaxis of suckling rats

Lead has been shown to produce cognitive and motor deficits in young rats that could be mediated, at least in part, by inhibition of the zinc-containing heme biosynthetic enzyme delta-aminolevulinate dehydratase (ALA-D). In the present study we investigated the effects of lead and/or zinc treatment during the second stage of rapid postnatal brain development on brain, kidney and blood ALA-D specific activity, as well as the negative geotaxis behavior of rats. Eight-day-old Wistar rats were injected intraperitoneally with saline, lead acetate (8 mg/kg) and/or zinc chloride (2 mg/kg) daily for five consecutive days. Twenty-four hours after treatment, ALA-D activity was determined in the absence and presence of DL-dithiothreitol (DTT). The negative geotaxis behavior was assessed in 9- to 13-day-old rats. Treatment with lead and/or zinc did not affect body, brain or kidney weights or brain- or kidney-to-body weight ratios of the animals. In spite of the absence of effect of any treatment on ALA-D specific activity in brain, kidney and blood, the reactivation index with DTT was higher in the groups treated with lead or lead + zinc than in the control group, in brain, kidney and blood (mean ± SEM; brain: 33.33 ± 4.34, 38.90 ± 8.24, 13.67 ± 3.41; kidney: 33.50 ± 2.97, 37.60 ± 2.67, 15.80 ± 2.66; blood: 63.95 ± 3.73, 56.43 ± 5.93, 31.07 ± 4.61, respectively, N = 9-11). The negative geotaxis response behavior was not affected by lead and/or zinc treatment. The results indicate that lead and/or zinc treatment during the second stage of rapid postnatal brain growth affected ALA-D, but zinc was not sufficient to protect the enzyme from the effects of lead in brain, kidney and blood.

Nitrergic modulation of vasopressin, oxytocin and atrial natriuretic peptide secretion in response to sodium intake and hypertonic blood volume expansion

The central nervous system plays an important role in the control of renal sodium excretion. We present here a brief review of physiologic regulation of hydromineral balance and discuss recent results from our laboratory that focus on the participation of nitrergic, vasopressinergic, and oxytocinergic systems in the regulation of water and sodium excretion under different salt intake and hypertonic blood volume expansion (BVE) conditions. High sodium intake induced a significant increase in nitric oxide synthase (NOS) activity in the medial basal hypothalamus and neural lobe, while a low sodium diet decreased NOS activity in the neural lobe, suggesting that central NOS is involved in the control of sodium balance. An increase in plasma concentrations in vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP), and nitrate after hypertonic BVE was also demonstrated. The central inhibition of NOS by L-NAME caused a decrease in plasma AVP and no change in plasma OT or ANP levels after BVE. These data indicate that the increase in AVP release after hypertonic BVE depends on nitric oxide production. In contrast, the pattern of OT secretion was similar to that of ANP secretion, supporting the view that OT is a neuromodulator of ANP secretion during hypertonic BVE. Thus, neurohypophyseal hormones and ANP are secreted under hypertonic BVE in order to correct the changes induced in blood volume and osmolality, and the secretion of AVP in this particular situation depends on NOS activity.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the mycobacteria growth indicator tube (MGIT) system

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has increased the need for rapid drug susceptibility tests, which are needed for adequate patient treatment. The objective of the present study was to evaluate the mycobacteria growth indicator tube (MGIT) system to detect multidrug-resistant M. tuberculosis strains. The MGIT system was compared with two standard methods (proportion and resistance ratio methods). One hundred clinical M. tuberculosis isolates [25 susceptible to isoniazid (INH) and rifampicin (RIF), 20 resistant to INH, 30 resistant to INH-RIF, and 25 resistant to INH-RIF and other drugs] obtained in the State of São Paulo were tested for INH and RIF susceptibility. Full agreement among the tests was found for all sensitive and all INH-resistant strains. For RIF-resistant strains results among the tests agreed for 53 (96.4%) of 55 isolates. Results were obtained within 6 days (range, 5 to 8 days), 28 days and 12 days when using MGIT, the proportion method and the resistance ratio methods, respectively. The MGIT system presented an overall agreement of 96% when compared with two standard methods. These data show that the MGIT system is rapid, sensitive and efficient for the early detection of multidrug-resistant M. tuberculosis.

Neuroendocrine control of body fluid homeostasis

Angiotensin II and atrial natriuretic peptide (ANP) play important and opposite roles in the control of water and salt intake, with angiotensin II promoting the intake of both and ANP inhibiting the intake of both. Following blood volume expansion, baroreceptor input to the brainstem induces the release of ANP within the hypothalamus that releases oxytocin (OT) that acts on its receptors in the heart to cause the release of ANP. ANP activates guanylyl cyclase that converts guanosine triphosphate into cyclic guanosine monophosphate (cGMP). cGMP activates protein kinase G that reduces heart rate and force of contraction, decreasing cardiac output. ANP acts similarly to induce vasodilation. The intrinsic OT system in the heart and vascular system augments the effects of circulating OT to cause a rapid reduction in effective circulating blood volume. Furthermore, natriuresis is rapidly induced by the action of ANP on its tubular guanylyl cyclase receptors, resulting in the production of cGMP that closes Na+ channels. The OT released by volume expansion also acts on its tubular receptors to activate nitric oxide synthase. The nitric oxide released activates guanylyl cyclase leading to the production of cGMP that also closes Na+ channels, thereby augmenting the natriuretic effect of ANP. The natriuresis induced by cGMP finally causes blood volume to return to normal. At the same time, the ANP released acts centrally to decrease water and salt intake.

Usefulness of a rapid immunometric assay for intraoperative parathyroid hormone measurements

Intraoperative parathyroid hormone (IO-PTH) measurements have been proposed to improve operative success rates in primary, secondary and tertiary hyperparathyroidism (PHP, SHP and THP). Thirty-one patients requiring parathyroidectomy were evaluated retrospectively from June 2000 to January 2002. Sixteen had PHP, 7 SHP and 8 THP. Serum samples were taken at times 0 (before resection), 10, 20 and 30 min after resection of each abnormal parathyroid gland. Samples from 28 patients were frozen at -70ºC for subsequent tests, whereas samples from three patients were tested while surgery was being performed. IO-PTH was measured using the Elecsys immunochemiluminometric assay (Roche, Mannheim, Germany). The time necessary to perform the assay was 9 min. All samples had a second measurement taken by a conventional immunofluorimetric method. We considered as cured patients who presented normocalcemia in PHP and THP, and normal levels of PTH in SHP one month after surgery and who remained in this condition throughout the follow-up of 1 to 20 months. When rapid PTH assay was compared with a routine immunofluorimetric assay, excellent correlation was observed (r = 0.959, P < 0.0001). IO-PTH measurement showed a rapid average decline of 78.8% in PTH 10 min after adenoma resection in PHP and all patients were cured. SHP patients had an average IO-PTH decrease of 89% 30 min after total parathyroidectomy and cure was observed in 85.7%. THP showed an average IO-PTH decrease of 91.9%, and cure was obtained in 87.5% of patients. IO-PTH can be a useful tool that might improve the rate of successful treatment of PHP, SHP and THP.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 µM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.