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1 Division of Neurosurgery, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0517; and 2 Department of Pharmacology, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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NG-nitro-L-arginine methyl ester (L-NAME; 250 µg/5 µl), an inhibitor of NO synthase, or the vehicle artificial cerebrospinal fluid (aCSF; 5 µl) was administered intracerebroventricularly to conscious rats hemorrhaged (0.7 ml/min) to a 20% volume depletion. Hypotension was maximal 5 min after hemorrhage ended, with compensatory recovery to basal levels 20 min later, regardless of drug treatment. L-NAME, however, elevated (P < 0.05) blood pressure (vs. aCSF controls) 40-45 min after intracerebroventricular administration. In normovolemic rats, L-NAME produced a significant pressor response and increased plasma levels of vasopressin (VP) and oxytocin (OT). After hemorrhage, both hormone levels increased, but only OT was further enhanced by L-NAME. Thus centrally produced NO tonically inhibits OT and VP secretion under basal normovolemic conditions and selectively inhibits OT release during hypovolemia. Hemorrhage increased the rates of glucose utilization in the neural lobe, indicative of enhanced efferent neural functional activity. L-NAME further enhanced the metabolic activity in the entire hypothalamoneurohypophysial system of hemorrhaged animals. Several other brain structures involved in the regulation of blood pressure and the stress response were also metabolically affected by the hemorrhage and L-NAME.
nitric oxide; 2-deoxy-D-[14C]glucose; glucose utilization; hypovolemia; neural lobe
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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NO SYNTHASE, an enzyme that catalyzes the synthesis of NO from L-arginine, has been identified in several brain areas (2, 23) implicated in a diversity of biological functions (for review, see Ref. 19), such as plasticity of the nervous system, regulation of blood pressure and blood flow, and immunoresponses. Of particular interest to our study is the role of this ubiquitous gas molecule on the regulation of body fluid and blood pressure homeostasis. Immunohistochemical and in situ hybridization studies have demonstrated the presence of the enzyme protein in several structures within the neural circuitry controlling blood pressure and water balance. For example, NO synthase and its mRNA are present in the hypothalamic magnocellular neurons in the supraoptic and paraventricular nuclei (11), where vasopressin (VP) and oxytocin (OT) are synthesized. The enzyme is also abundant in the terminals of the hypothalamoneurohypophysial system in the neural lobe (2). Moreover, NO synthase has been identified in the subfornical organ (SFO), organum vasculosum laminae terminalis (OVLT), and median preoptic nucleus (32), structures in the lamina terminalis that have anatomic connections with the supraoptic and paraventricular nuclei essential for the control of VP release, sympathetic nervous system activity, drinking behavior, and salt appetite. NO synthase is also present in the nucleus of the solitary tract (31), the primary relay synapse of baroreceptor and volume receptor inputs to the central nervous system, where inhibition of this enzyme increases activity of the sympathetic nervous system (7). The enzyme has been identified in the terminals in the nucleus ambiguus (5) and caudal and rostral ventrolateral medulla (12, 17), structures implicated in the maintenance of cardiovascular homeostasis. Several of these nuclei establish anatomic and functional connections within a larger network in the forebrain, which involves the lamina terminalis, to subserve body fluid and blood pressure homeostasis in response to inputs from volume and baroreceptors.
To further clarify the role of centrally produced NO on blood pressure and body fluid homeostasis, we investigated the effects of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, on cerebral metabolism, neurohypophysial hormone secretion, and blood pressure responses during hypovolemia induced by hemorrhage.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals
The studies were carried out on 85 adult male Sprague-Dawley rats (234-345 g). Of these, 45 were used for the 2-deoxy-D-[14C]glucose ([14C]DG) experiments and the remaining 40 for the measurement of VP and OT levels in the plasma. The animals were kept three per cage under laboratory conditions with a 12:12-h dark-light cycle (lights on at 0600), with food and water ad libitum for 5-7 days before surgery.Preparation of Animals
The animals were anesthetized with a combination of ketamine (60 mg/kg im) and Nembutal (40 mg/kg ip). A 20-gauge stainless steel guide cannula, sealed with silicone to prevent leakage of cerebrospinal fluid (CSF), was stereotactically implanted into the right cerebroventricle on a flat skull at the following coordinates: 0.8 mm posterior to bregma, 1.2 mm lateral to midline, and 3.5 mm below the dura. The cannula was secured to the skull with two jeweler's screws and dental cement. The animals were placed in individual cages and allowed to recover from surgery for a period of 7 days. During this time, they were handled daily to condition them to the investigators and minimize the effects of stress during the experiments.On the day of the experiment, the animals were anesthetized with halothane (1.5% in O2) and the left femoral artery was catheterized for withdrawal of blood. For the [14C]DG experiments, the right femoral artery and vein were also catheterized for measurement of arterial blood pressure and blood sampling and administration of the tracer, respectively. The catheters were filled with heparinized saline (100 U/ml), exteriorized, and secured at the nape of the neck. Animals were allowed to recover from surgery for at least 2 h before the experimental manipulation, during which time water and food were not available. Experiments were performed during the light cycle of the day, between 1100 and 1600.
Experimental Design
The protocols used in these experiments were reviewed and approved by the Animal Care and Use Committee of The University of Texas Medical Branch, Galveston, TX.Two series of experiments were performed.
Series 1. HORMONE
MEASUREMENT. Four groups of rats
(n = 10 each), two normovolemic and
two hypovolemic, were studied. Hematocrits were determined from the
arterial blood under basal conditions and immediately after hemorrhage.
Hemorrhage was induced by withdrawal of blood from the femoral artery
via a withdrawal-infusion pump (Harvard Apparatus, South Natick, MA) at
a rate of 0.7 ml/min, until a depletion of 20% of total blood volume
(13 ml/kg of body weight) was achieved, 4-6 min later. A 30-gauge
stainless steel injector, which extended 0.5 mm below the tip of the
intracerebroventricular guide cannula, was then inserted into each
animal and left in place for 5 min before delivering the vehicle,
artificial CSF (aCSF; 5 µl), or
L-NAME (250 µg/5 µl) (Sigma
Chemical, St. Louis, MO). At corresponding times, normovolemic rats
also received aCSF or L-NAME
intracerebroventricularly. Two minutes later, animals were decapitated
and trunk blood was collected into chilled siliconized tubes containing
100 U of heparin. The blood samples were centrifuged immediately (3,000 g at 4°C for 30 min), and the
plasma was separated and kept frozen (
20°C) until assayed
for VP and OT.
To verify the position of the intracerebroventricular cannula, 5 µl of 2% Evans blue solution were injected intracerebroventricularly after the animals were decapitated. The brains were fixed in 10% buffered neutral Formalin solution for at least 2 days and then were cut frontally in a brain slicer (David Kopf Instruments, Tujunga, CA). The track of the internal cannula and the distribution of the dye in the ventricular system were analyzed under a dissecting microscope. Only data from animals with the dye distributed throughout the ventricular system were used.
RADIOIMMUNOASSAY. Plasma samples were
thawed in an ice bath and centrifuged at 1,500 g for 30 min before extraction of VP and OT. VP was extracted from 0.7-1.5 ml of plasma using
bentonite, acid acetone, and petroleum ether. Dried extracts of plasma
were stored at 20°C until assayed by RIA. Standard curves
for VP assay ranged from 0.5 to 16 pg/tube. The sensitivity of the
assay was 0.7 pg/tube, with <0.01% cross-reactivity with OT and
arginine8-vasotocin. For each
sample assayed, triplicates of two or three dilutions were run.
Recovery of hormone extracted from plasma was estimated by addition of
15 and 20 pg/tube of synthetic VP (Sigma Chemical) to 16 samples of 1.0 ml of pooled plasma. For each recovery, eight blank samples were run.
Recovery averaged 62.2 ± 2%, with an intra-assay coefficient of
variation of 12 ± 6% (n = 4). OT
was extracted from 0.5-1.0 ml of plasma using cold acetone-ether
and measured by RIA. Standard curves for OT assay ranged from 0.78 to
25 pg/tube. The sensitivity of the RIA for OT was 0.7 pg/tube, and the
cross-reactivity with VP and
arginine8-vasotocin were <0.01%
and <0.1%, respectively. Recovery of synthetic OT (Sigma Chemical),
10 and 20 pg/tube, calculated from 16 samples, averaged 88.7 ± 4%.
The interassay and intra-assay coefficients of variation were
18.4 ± 0.8% (n = 6) and
8 ± 1.54% (n = 8), respectively. Data from VP and OT were corrected for 100% recovery.
Series 2. MEASUREMENT
OF GLUCOSE UTILIZATION. Four groups of rats
(n = 11 or 12 each), two normovolemic
and two hypovolemic, were studied. The procedures for inducing
hemorrhage and intracerebroventricular injections of aCSF and
L-NAME were the same as
described in the first series of experiments. The
[14C]DG experiment
started 2 min after intracerebroventricular injection of aCSF or
L-NAME in hypovolemic and
normovolemic rats. The period of measurement of glucose utilization was
initiated by an intravenous bolus injection of 125 µCi/kg of
[14C]DG (specific
activity 50-55 mCi/mmol; American Radiolabeled Chemicals, St.
Louis, MO). Fifteen to seventeen arterial blood samples of ~85 µl
each were collected at time intervals throughout the 45 min, and the
plasma was assayed for
[14C]DG and glucose
concentrations as described (27). At ~45 min after the administration
of [14C]DG, the
animals were killed by an intravenous injection of a lethal dose of
pentobarbital sodium. The brain was rapidly removed, frozen in
isopentane chilled to 45°C with dry ice, and covered with
embedding medium (M-1 embedding matrix; Shandon Lipshaw, Pittsburgh,
PA). The pituitary gland was also removed and mounted in frozen
embedding medium in a small aluminum foil container. The frozen tissues
were cut into 20-µm sections in a cryostat at 22°C and
autoradiographed together with calibrated
[14C]methyl
methacrylate standards as described (27). Selected sections were
stained with thionin for histological identification of areas of
interest. The local tissue concentrations of
14C were determined by
quantitative densitometric analysis of autoradiographs by means of a
charge-coupled device camera and an image-analysis system (Imaging
Research, St. Catherines, ON, Canada). An overlay program that allowed
superimposition of histological sections with the autoradiographs was
used to optimize the precision of the analysis of small nuclei. Glucose
utilization was calculated from the tissue concentration of
14C and the time courses of the
arterial plasma
[14C]DG and glucose
concentrations by means of the operational equation of the
[14C]DG method (27).
PHYSIOLOGICAL STATUS. The physiological status of each animal was assessed during the experiments by measuring the mean arterial blood pressure, hematocrit, plasma osmolality, and total protein concentration and arterial blood pH, PO2, and PCO2. Mean arterial blood pressure was measured intermittently throughout the experiment. Arterial blood pH, PO2, and PCO2 were measured immediately before the administration of [14C]DG. The other variables were measured under basal condition (sample 1), 4 min after the end of hemorrhage (sample 2), and 35 min after the administration of [14C]DG (sample 3). Arterial blood pressure was recorded from the femoral artery by means of a pressure transducer and Gould-Brush 2400 polygraph (Gould Electronics, Cleveland, OH). Arterial blood pH, PO2, and PCO2 were measured with a blood gas analyzer (Instrumentation Laboratory System BG3). Hematocrit was determined in microcapillary tubes. Plasma osmolality was quantified with a vapor pressure osmometer (model 5500; Wescor, Logan, UT), and the plasma protein concentration was measured with a hand-held refractometer (Schuco, Japan).
Statistics
VP and OT data were log transformed to reduce the variance differences among the treatment groups before analysis by a two-factor ANOVA. The two factors were defined as volemic state (normovolemic or hypovolemic) and drug treatment (aCSF or L-NAME). Homogeneity of variances among the treatment groups was evaluated using the Levine's test. Because the data in the control and hypovolemic groups did not have homogeneous variance even after log transformation, the untransformed raw data were analyzed using the Brown-Forsythe ANOVA approximation (3). Differences in group means were analyzed further by the Bonferroni t-test for multiple comparisons using separate group variance estimates. The cerebral glucose utilization data were analyzed by a two-factor ANOVA. When a significant interaction between the volemic state and drug treatment was found, the data were further analyzed by calculating the least-significant difference procedure for multiple comparisons. For the effects of hemorrhage on other physiological variables, the data were analyzed by a two-factor ANOVA (drug treatment and time) for repeated measures on one factor (time).| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Study 1: Neurohypophysial Hormones
Hemorrhage increased the plasma concentration of both VP and OT (P < 0.01), as expected (Fig. 1). The main effect of the drug treatment (aCSF vs. L-NAME) on VP secretion, however, was marginally significant (P = 0.07), without a measurable interaction between the volemic condition and drug treatment. A differential effect of L-NAME in normovolemic rats, however, could have been masked by the large differences in variance occurring in normovolemic and hemorrhaged animals. A further analysis by the Bonferroni t-test indicated that the increase in secretion of VP (means ± SE, n = 10) from 2.4 ± 0.4 pg/ml in the control group treated with aCSF to 4.6 ± 0.6 pg/ml after treatment with L-NAME in normovolemic rats was statistically significant (P < 0.05). The changes in hypovolemic rats [26.4 ± 5.0 pg/ml (aCSF) vs. 36.4 ± 5.0 pg/ml (L-NAME)] (Fig. 1), however, were not significant. This demonstrates that production of VP is tonically attenuated by NO in normovolemic condition, but this mechanism is removed (i.e., disinhibited) in the hypovolemic condition.
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For OT secretion, a significant interaction between hemorrhage and drug treatment was observed. This indicates that L-NAME potentiates the stimulatory effect of hemorrhage on OT secretion, as well as elevates basal levels of the hormone.
Study 2: [14C]DG Experiments
Physiological variables. Hemorrhage induced an immediate fall in the mean arterial blood pressure, which continued to decrease even after the bleeding stopped, reaching values of ~60 mmHg 5 min later, at time 0 (Fig. 2). Blood pressure then increased slowly in both hypovolemic groups that received either aCSF or L-NAME intracerebroventricularly. Although the values in both groups had returned to basal levels by 20 min, in animals treated with L-NAME blood pressure continued to increase, becoming statistically greater than controls receiving aCSF at 40 and 45 min after intracerebroventricular injection. In normovolemic rats, intracerebroventricular inhibition of NO synthase with L-NAME caused a gradual increase in the mean arterial blood pressure, beginning at 15 min and continuing for the duration of the experiment. In animals that received aCSF, blood pressure remained unchanged throughout the experiment.
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Hematocrit decreased in normovolemic rats, but by a similar magnitude in both groups receiving either aCSF or L-NAME (Table 1), probably due to the sampling of blood for the measurement of the various physiological variables, as well as for the [14C]DG experiments. In hemorrhaged rats, the magnitude of the fall in hematocrit was larger than in normovolemic rats, as expected. The total protein concentration in plasma also declined slightly in normovolemic rats, but the magnitude of the drop was larger in hypovolemic rats. Plasma osmolality was similar in all groups of rats and was affected by neither the hemorrhage nor the inhibitor of NO synthase. Arterial blood pH, PO2, and PCO2 were within normal physiological parameters in both normovolemic and hypovolemic rats.
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Rates of cerebral and neural lobe glucose utilization. The evoked metabolic responses of several structures within the forebrain and hindbrain were affected by the hemorrhage and by the central inhibition of NO synthase with L-NAME (Table 2). Regarding the magnocellular system, after hemorrhage a significant increase in glucose utilization was observed in the neural lobe, but not in the supraoptic and paraventricular nuclei. The enhanced activity originating in the perikarya of the magnocellular neurons in both nuclei was instead reflected by the higher rates of glucose metabolism in the neural lobe, because the [14C]DG method measures preferentially the metabolic activity of axonal terminals rather than cell bodies (10). Many forebrain structures anatomically connected with the hypothalamo-neurohypophysial system were also affected by the hemorrhage. These included the lateral hypothalamus, amygdala, suprachiasmatic nuclei, lateral and median preoptic nuclei, and medial and lateral septum (Table 2). Glucose utilization in all these structures decreased significantly in response to hemorrhage. In the median eminence, but not in the other circumventricular organs such as the SFO, OVLT, and area postrema, the rates of glucose utilization were enhanced (P < 0.05) by the hemorrhage, indicating increased efferent neural activity. Within the hindbrain, glucose utilization was reduced by hemorrhage (P < 0.05) in the nucleus ambiguus, rostral ventrolateral medulla, and inferior olivary nuclei, most certainly due to the removal of an inhibitory input (i.e., disinhibition) to these nuclei. When the central production of NO was inhibited with L-NAME, the evoked metabolic response increased in 14 out of 24 structures examined from the forebrain to the hindbrain, regardless of whether the animal was hemorrhaged or not. In response to L-NAME, glucose metabolism increased significantly in the entire hypothalamoneurohypophysial system, i.e., supraoptic and paraventricular nuclei and neural lobe, as well as in the OVLT (Table 2). The rates of glucose utilization were also enhanced by L-NAME in several other structures in the forebrain, such as the periventricular nucleus and medial preoptic area, including those affected by hemorrhage, i.e., lateral hypothalamus, amygdala, and lateral preoptic area. A significant interaction between hemorrhage and L-NAME was observed in the medial preoptic area (P = 0.04) and only marginally in the lateral hypothalamus (P = 0.06). In both structures, L-NAME enhanced glucose utilization in hypovolemic but not in normovolemic animals.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The results of these experiments show that hemorrhage and inhibition of the central production of NO affect blood pressure, secretion of neurohypophysial hormones, and cerebral evoked metabolic responses. In the following sections each of these variables will be discussed separately.
Influence of Hemorrhage and NO on Arterial Blood Pressure
Hemorrhage induced a rapid fall in blood pressure, which continued to decrease even after the bleeding stopped. When L-NAME was administered intracerebroventricularly to hypovolemic and normovolemic rats, the mean arterial blood pressure increased gradually and became significantly different from the respective controls receiving aCSF at times varying between 15 and 40 min. Although the mechanism involved in this delayed pressor response is not known, it apparently does not involve catecholamines from the adrenal glands (14) nor the
1-adrenergic receptors (15). These results indicate that centrally produced NO maintains resting arterial blood pressure as well as contributes to the delayed hypotensive response after hemorrhage. Although it seems paradoxical, this mechanism is probably important to maintain vasodilation and,
therefore, blood flow to vital organs following hemorrhage.
Influence of Hemorrhage and NO on the Hypothalamoneurohypophysial System
These studies confirm our previous report (8) that under basal normovolemic condition, NO tonically inhibits secretion of OT and VP from the magnocellular system, as circulating levels of both hormones became elevated after inhibiting NO synthase activity with L-NAME. Increased plasma VP in normovolemic animals was also described by Chiu and Reid (4) after intravenous infusion of L-NAME to conscious rabbits. They did not, however, measure plasma levels of OT. Similarly to our earlier finding (8), basal secretion of OT was shown to be under a stronger inhibitory control by NO mechanisms than VP, judging by the larger increase in circulating levels of OT (4.8 times) than VP (2.3 times) in the same animals after central injection of L-NAME. The effects seen in our study were immediate, as they were detected within 2 min following injection of the NO synthase inhibitor. This time point was chosen because the peak OT and VP response occurs between 2 and 5 min after intracerebroventricular injection of L-NAME in euvolemic animals (unpublished results). A rapid effect with a short onset was also expected because of the transient half-life of NO and the short half-life (1.5-5 min) of the neurohypophysial hormones in plasma. The short time course for affecting hormone release in the presence of a more sustained inhibition of NO synthase (24) indicates that compensatory mechanisms regulating secretion of magnocellular neurons become operant and effective in lowering plasma hormone levels by 15 min after intracerebroventricular L-NAME. The response to L-NAME is unlikely to result from a nonspecific factor, such as stress, because control rats treated with aCSF had low levels of VP and OT. Chiu and Reid (4) observed that when the inhibitor of NO synthase is injected intravenously, the increase in plasma levels of VP is delayed, being observed within 15 min. Inhibition of NO synthase at an active site(s), in proximity to the cerebroventricular system, is achieved very rapidly after intracerebroventricular injection of L-NAME, as contrasted with a longer time to onset when the enzyme inhibitor is injected systemically. Unpublished data from our laboratory demonstrate that the activity of NO synthase measured in homogenates of hypothalamic tissue containing the paraventricular nuclei is inhibited by 95% as early as 2 min after intracerebroventricular administration of 250 µg of L-NAME, but not D-NAME. The duration of the inhibitory response is prolonged. Salter et al. (24) demonstrated that the activity of NO synthase in several brain structures remains inhibited up to 6 h after intracerebroventricular injection of 30 µg of L-NAME, a dose about eight times lower than the one used in the present study.The prompt hormonal response to L-NAME after its intracerebroventricular administration could result from enzyme inhibition in circumventricular organs of the lamina terminalis or in the paraventricular nuclei themselves, which lie in close proximity to the third ventricle. Recently, Sancesario et al. (25) described processes of neurons expressing NADPH-diaphorase (and, therefore, potentially NO synthase) contacting CSF in the lateral and third ventricles of the rat brain that could also mediate the observed central effects of L-NAME on the magnocellular system. These CSF-contacting neurons arise from the periventricular hypothalamic nucleus, and their morphology indicates they could subserve a sensory function to signal changes in the composition of CSF to other brain regions within the central nervous system.
During hemorrhage, inhibition of OT release by NO mechanisms became more intense, resulting in an even greater attenuation of OT secretion, whereas the inhibitory effect of NO on VP release into plasma was removed (i.e., disinhibited). This was evidenced by the potentiated rise in plasma levels of OT, but not VP, in hemorrhaged animals treated with L-NAME. In accordance with our results, Chiu and Reid (4) demonstrated that intravenous inhibition of NO synthase with L-NAME did not further increase plasma VP levels during hemorrhage in conscious rabbits. Thus at least a part of the mechanism modulating VP secretion during hemorrhage involves the selective removal of an inhibitory effect of NO on vasopressinergic neurons.
Contrasting with the neuroendocrine changes described here, we have demonstrated previously that consumption of water elicited by hemorrhage is significantly reduced by intracerebroventricular L-NAME (13). Therefore, NO, presumably derived from within the central nervous system, facilitates drinking behavior while it inhibits selectively OT, but not VP, secretion from the magnocellular neuroendocrine system. Collectively, during hypovolemia, NO promotes the preferential release of VP into plasma, which, in conjunction with facilitating drinking, contributes to the restoration of body fluid homeostasis.
Local Rates of Cerebral Glucose Utilization
Influence of hemorrhage and NO on the hypothalamo-neurohypophysial system. The local rates of cerebral glucose metabolism in several brain regions were affected by the hemorrhage and inhibition of NO synthase. Although there was no complete temporal correspondence between the hormonal and metabolic measurements in our studies, an overlap of physiological events occurred in the beginning of the compensatory response to hypovolemia. Although the duration of the [14C]DG method is 45 min, the significant events depicted by the method are those occurring during the first 20 min after injection of the tracer (see Ref. 9). Therefore, it can be considered that the elevated rates of glucose utilization in the neural lobe are partially due to increased firing rates in the magnocellular neurons that resulted in increased plasma levels of VP and OT. Metabolic activity, however, was not increased in the supraoptic and paraventricular nuclei, where the perikarya of the magnocellular neurons are located. This differential increase reflects the higher metabolic activity at the axon terminals (10) of the neural lobe, where the surface-to-volume ratio is larger than at the perikarya in the magnocellular nuclei. Regardless of the hemorrhage, however, L-NAME increased glucose utilization in the supraoptic and paraventricular nuclei, as well as the neural lobe in association with elevated plasma levels of neurohypophysial hormones.Influence of hemorrhage and NO on circumventricular organs and other forebrain structures. Hemorrhage increased significantly glucose utilization in the median eminence, a circumventricular organ. This probably reflects the increased activity of the axon terminals in the external zone that influences release of adenohypophysial hormones from the anterior pituitary. Part of this enhanced metabolic activity may relate to stimulation of adrenocorticotrophs because secretion of ACTH increases during hemorrhage to enhance the activity of the sympathetic nervous system (6). In other circumventricular organs, however, such as the SFO and OVLT, which contain angiotensin II binding sites (18), glucose utilization remained unchanged. It is likely that the amount of angiotensin II formed in response to hypovolemia was lower than the threshold and, therefore, insufficient to increase the metabolism in these structures to detectable levels. In this regard, it has been shown that the threshold of plasma angiotensin II levels to stimulate drinking behavior is reached after 48 h of water deprivation (30).
Surprisingly, glucose metabolism was reduced by hemorrhage in the median preoptic nucleus. Although this nucleus contains neurons that are responsive to angiotensin II (18), plasma osmolality, and changes in blood pressure (1), its electrical stimulation can lead to inhibition of magnocellular neurons in the supraoptic and paraventricular nuclei (20). Moreover, neurons in the median preoptic nucleus are inhibited or excited by decreases in blood pressure, although a majority of them seem to be stimulated by hypotension. In several other forebrain structures, such as the lateral hypothalamus, amygdala, lateral preoptic area, medial and lateral septum, and suprachiasmatic nuclei, the rates of glucose utilization were decreased significantly by the hemorrhage. With the exception of the suprachiasmatic nuclei, these structures participate in the regulation of neurohypophysial hormones secretion, drinking behavior, and blood pressure. The medial septum, for example, exerts an inhibitory influence on the magnocellular VP and OT neurons of the supraoptic nuclei (22). Hemorrhage, therefore, seems to reduce the synaptic influences on the activity of septal neurons, presumably facilitating release of neurohypophysial hormones. The lateral hypothalamus and lateral preoptic area participate in mechanisms regulating drinking behavior (21) as well as blood pressure responses (16).
Several structures affected by hemorrhage were metabolically enhanced by the inhibition of NO synthase. These structures included the lateral hypothalamus, amygdala, and the lateral preoptic area. Three structures, however, whose evoked metabolic activity was not modified by hemorrhage, i.e., the OVLT, periventricular nucleus, and the medial preoptic area, had enhanced glucose utilization after L-NAME. These data indicate that afferent fibers, perhaps projecting from the SFO to the OVLT and median preoptic nucleus, are tonically attenuated by NO synthesized and released locally to influence the magnocellular system or blood pressure regulation.
Influence of hemorrhage and NO on the hindbrain structures. Hemorrhage reduced significantly the rates of glucose utilization in several hindbrain structures, including the rostral ventrolateral medulla, nucleus ambiguus, and inferior olivary nuclei. Participation of these structures in the regulation of cardiovascular function and the baroreflex has been demonstrated by others (26, 29). No significant decrease in glucose utilization was seen in the nucleus of the solitary tract or the caudal ventrolateral medulla (which receives direct input from the nucleus of the solitary tract). Metabolic activity, however, decreased significantly in its projection site in the rostral ventrolateral medulla. Neurons in this area connect directly with preganglionic neurons of the sympathetic nervous system in the intermediolateral column of the spinal cord, and their activity is tonically inhibited by the caudal ventrolateral medulla via a GABAergic mechanism (29). During hypovolemia, decreased input from volume receptors and baroreceptors removes this inhibition of the rostral ventrolateral medulla, ultimately resulting in an increased activity of the sympathetic system. The nucleus ambiguus, on the other hand, contains preganglionic neurons of the parasympathetic nervous system that innervate the heart and receive inputs from the nucleus of the solitary tract (5). Its stimulation causes a decrease in heart rate. Glucose utilization in the nucleus ambiguus was significantly reduced by hemorrhage and enhanced by L-NAME. The present studies, however, do not allow the establishment of a relationship between the heart rate and the evoked metabolic response in this brain region, because this parameter was not monitored during the experiment.
The rates of glucose utilization in the inferior olivary nucleus, whose role in cardiovascular regulation was described by Smith and Nathan (26), were significantly decreased by hemorrhage. Stimulation of this nucleus inhibits the depressor and cardioinhibitory responses of the carotid sinus reflex. It is possible that removal of this inhibitory effect during hemorrhage leaves the baroreflex unopposed to counteract the hypotension. Similarly to the effects seen in the nucleus ambiguus, L-NAME increased metabolic activity in the inferior olivary nucleus.
Perspectives
It is becoming increasingly evident that NO produced in the central nervous system has an important physiological role in the regulation of fluid balance and blood pressure. NO exerts a tonic inhibitory influence on secretion of VP and OT during normovolemic isosmotic conditions (present data). When arterial blood pressure decreases during hemorrhage, the inhibitory influence of NO on vasopressinergic neurons is removed, while that on oxytocinergic neurons is increased. Thus, in conjunction with facilitating drinking behavior in hemorrhaged rats (13), NO enables a preferential release of VP relative to OT to promote a positive water balance during hypovolemia. The mechanism by which NO promotes the preferential release of VP appears to involve formation of a prostaglandin, as indomethacin, a blocker of cyclooxygenase, prevents the facilitated hormone response after L-NAME (28). On the other hand, centrally produced NO is important to maintain the resting arterial blood pressure and the delayed, but not the acute, hypotension after hemorrhage.| |
ACKNOWLEDGEMENTS |
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The authors thank Dr. Karen Pettigrew for advice on statistical analysis of hormonal data and Cherie Barker, Royce Couch, and Lynn Burke for their editorial assistance.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grant 2-RO1-NS-23055 (to M. Kadekaro and J. Y. Summy-Long).
Address for reprint requests: M. Kadekaro, Dept. of Surgery, Division of Neurosurgery, The Univ. of Texas Medical Branch, Galveston, TX 77555-0517.
Received 14 July 1997; accepted in final form 7 January 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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