Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man

Levels of norepinephrine (NE) in human plasma have been determined by a radioenzymatic technique sufficiently sensitive to measure 0.014 ng NE per ml plasma. Several procedures which raise plasma NE levels have been compared and a standard procedure developed to evaluate sympathetic neuronal function based on the increments in plasma NE produced by postural change and a standard amount of exertion. The mean basal level of NE in plasma of 74 resting, supine, normal subjects ranging in age from 10 to 70 (mean 32.7 years) was 0.292 ± 0.016 (± SEM) ng/ml and ranged from 0.112 to 0.738 ng/ml. There was a significant correlation between age and basal levels of NE (L.R. = 0.33, p < 0.01). In 44 subjects who stood for 5 minutes after the basal sample of blood was obtained, the mean plasma level of NE increased to 0.538 ± 0.044 ng/ml and further increased to 0.778 ± 0.080 ng/ml after a subsequent isometric hand grip for 5 minutes.     

Insulin sensitivity regulated by feeding in the conscious unrestrained rat

Hepatic insulin sensitizing substance (HISS), a putative hormone released from the liver in response to insulin in fed animals, accounts for 50-60% of insulin action. HISS release is regulated by permissive control of the hepatic parasympathetic nerves. The objectives were to develop the rapid insulin sensitivity test (RIST) in conscious rats, and to assess the effects of anesthesia, atropine, feeding, and fasting on insulin action. The RIST index, expressed as milligrams glucose per kilogram body weight required to maintain euglycemia after a 50 mU/kg bolus of insulin, was similar in conscious and anesthetized rats (238.6+/-42.5 vs. 225.3+/-30.4 mg/kg). Atropine produced a 56% inhibition of insulin action in fed rats. After a 24 h fast, full HISS-dependent insulin resistance had developed as shown by a low RIST index that was not reduced further by atropine. Fasting caused a 10.5% decrease in insulin action per hour over six hours. HISS-dependent insulin resistance in 24-h fasted rats was reversed 4 h after re-feeding (90.9+/-12.3 vs. 204.5+/-30.5 mg/kg). We conclude that HISS-dependent and HISS-independent insulin action, as assessed by the RIST, is similar in conscious and pentobarbital-anesthetized rats. Pharmacological blockade of HISS-dependent insulin action and physiological regulation of HISS action by feeding-fasting is confirmed. Re-feeding fasted rats reversed HISS-dependent insulin resistance. Merits of use of the RIST in conscious versus anesthetized rats are discussed.     

Dopamine accumulation after dopamine beta-hydroxylase inhibition in rat heart as an index of norepinephrine turnover

Dopamine concentration in rat heart is normally very low, only a few percent of the concentration of norepinephrine. After treatment of rats with a dopamine beta-hydroxylase inhibitor, 1-cyclohexyl-2-mercapto-imidazole (CHMI), there was a rapid increase in dopamine concentration even before norepinephrine concentration had decreased perceptibility. This accumulation of dopamine was readily measured by liquid chromatography with electrochemical detection. Since the percentage change in dopamine was much greater than the percentage change in norepinephrine, especially at early times, measurement of dopamine accumulation rather than norepinephrine decline was considered as a useful measure of norepinephrine turnover. Drugs that act on noradrenergic receptors and are known to alter norepinephrine turnover were found to alter the rate of dopamine accumulation. Clonidine and guanabenz decreased dopamine accumulation after CHMI, whereas piperoxan (but not prazosin) increased dopamine accumulation after CHMI. Pergolide, a dopamine agonist whose lowering of blood pressure and cardiac rate has been suggested to be due to suppression of neurogenic release or norepinephrine, also decreased dopamine accumulation after CHMI. The results suggest that measuring dopamine accumulation may have advantages over measuring norepinephrine disappearance after dopamine beta-hydroxylase inhibition as an indicator of norepinephrine turnover in heart.     

Evaluation of malondialdehyde as an index of lead damage in rat brain homogenates

Lipid peroxidation in vitro homogenates of brain was examined as sequela of lead toxicity. The levels of malondialdehyde (MDA) in homogenates of rat brain (1 ml, 5% w/v) treated with lead (50 g) alone or in combination with ascorbic acid (100 g), alphatocopherol (100 g) or hydroquinone (100 g) were evaluated. The levels of MDA were consistently evoked by lead in a dose-related manner. The toxicity of lead was further advanced by the action of the pro-oxidant drug ascorbic acid on the brain. However, the anti-oxidant drugs alphatocopherol and hydroquinone decreased the toxic effect of lead on the brain. These results clearly show that the enhanced lipid peroxidation may provide a basis of lead-induced neurotoxicity.     

Fos expression in rat celiac ganglion: an index of the activation of postganglionic sympathetic nerves

To develop an index of the activation of abdominal sympathetic nerves, we used Fos immunostaining of the celiac ganglion (CG) taken from rats receiving nicotine, preganglionic nerve stimulation, or glucopenic agents. Subcutaneous nicotine injection moderately increased Fos expression in the principal ganglionic cells of the CG (17 +/- 4 Fos+ per mm(2), approximately 12% of all principal CG cells), whereas subcutaneous saline had no effect (0 +/- 0 Fos+ per mm(2); n = 7; P < 0.01). Greater Fos expression was obtained by applying nicotine topically to the CG (71 +/- 8 Fos+ per mm(2); 52% of all principal CG cells, n = 5; P < 0.01 vs. topical saline, n = 4) and by preganglionic nerve stimulation (126 +/- 9 Fos+ per mm(2); 94% of all principal CG cells, n = 11; P < 0.01 vs. nerve isolation, n = 7). Moderate Fos expression was also observed in the CG after intraperitoneal 2-deoxy-D-glucose (2DG) injection (21 +/- 2 Fos+ per mm(2); 16% of all principal CG cells, n = 5; P < 0.01 vs. saline ip) or insulin injection (16 +/- 2 Fos+ per mm(2); 12% of all principal CG cells, n = 6; P < 0.01 vs. saline ip). Furthermore, Fos expression induced by 2DG was dose and time dependent. These data demonstrate significant Fos expression in the CG in response to chemical, electrical, and reflexive stimulation. Thus Fos expression in the CG may be a useful index to describe various levels of activation of its postganglionic sympathetic neurons.     

Evaluation of SQ 28,603, an inhibitor of neutral endopeptidase, in conscious monkeys.

The potent neutral endopeptidase inhibitor SQ 28,603 (N-(2-(mercaptomethyl)-1-oxo-3-phenylpropyl)-beta-alanine) significantly increased excretion of sodium from 4.9 +/- 2.3 to 14.3 +/- 2.1 muequiv./min and cyclic 3',5'-guanosine monophosphate from 118 +/- 13 to 179 +/- 18 pmol/min after intravenous administration of 300 mumol/kg (approximately 80 mg/kg) in conscious female cynomolgus monkeys. SQ 28,603 did not change blood pressure or plasma atrial natriuretic peptide concentrations in the normal monkeys. In contrast, 1-h infusions of 3, 10, or 30 pmol.kg-1.min-1 of human atrial natriuretic peptide lowered blood pressure by -3 +/- 4, -9 +/- 4, and -27 +/- 3 mmHg (1 mmHg = 133.322 Pa), increased cyclic guanosine monophosphate excretion from 78 +/- 11 to 90 +/- 6, 216 +/- 33, and 531 +/- 41 pmol/min, and raised plasma atrial natriuretic peptide from 7.2 +/- 0.7 to 21 +/- 4, 62 +/- 12, and 192 +/- 35 fmol/mL without affecting sodium excretion. In monkeys receiving 10 pmol.kg-1.min-1 of atrial natriuretic peptide, 300 mumol/kg of SQ 28,603 reduced mean arterial pressure by -13 +/- 5 mmHg and increased sodium excretion from 6.6 +/- 3.2 to 31.3 +/- 6.0 muequiv./min, cyclic guanosine monophosphate excretion from 342 +/- 68 to 1144 +/- 418 pmol/min, and plasma atrial natriuretic peptide from 124 +/- 8 to 262 +/- 52 fmol/mL. In conclusion, SQ 28,603 stimulated renal excretory function in conscious monkeys, presumably by preventing the degradation of atrial natriuretic peptide by neutral endopeptidase.     

Impact of portal glucose delivery on glucose metabolism in conscious, unrestrained mice

Previous studies in mice suggest that portal venous infusion of glucose at a low rate paradoxically causes hypoglycemia; this does not occur in dogs, rats, and humans. A possible explanation is that fasting status in the mouse studies may have altered the response. We sought to determine whether the response to portal glucose delivery in the mouse was similar to that seen in other species and whether it was dependent on fasting status. Studies were performed on chronically catheterized conscious mice. Catheters were placed into the portal and jugular veins and carotid artery 5 days before study. After a 5- or 16-h fast, glucose was infused into either the portal (PO) or the jugular vein (JU) for 6 h at 25 microg.g(-1).min(-1). [3-(3)H]glucose was infused into the JU to measure glucose turnover. In 5-h-fasted mice, PO and JU exhibited similar increases in arterial blood glucose from 155 +/- 11 to 173 +/- 19 and 147 +/- 8 to 173 +/- 10 mg/dl, respectively. Endogenous glucose production decreased and arterial insulin increased to the same extent in both PO and JU. A similar response was observed in 16-h-fasted mice; however, the proportion of hepatic glycogen synthesis occurring by the indirect pathway was increased by fasting. In summary, portal glucose delivery in the mouse did not cause hypoglycemia even when the duration of the fast was extended. The explanation of the differing response from previous reports in the mouse is unclear.     

Characterization of the portal signal during 24-h glucose delivery in unrestrained, conscious rats

To characterize the "portal signal" during physiological glucose delivery, liver glycogen was measured in unrestrained rats during portal (Po) and peripheral (Pe) constant-rate infusion, with minimal differences in hepatic glucose load (HGL) and portal insulin between the delivery routes. Hepatic blood flows were measured by Doppler flowmetry during open surgery. Changes in hepatic glucose, portal insulin, glucagon, lactate, and free fatty acid concentrations were generally similar in either delivery except for glucagon at 4 h. Hepatic glycogen, however, increased continuously in Po and was higher than Pe at 8 and 24 h, although it decreased to the level of Pe upon the removal of Po at 8 h. There was a near-linear relationship between hepatic glycogen and HGL in either delivery, with the slope being twice as high in Po and the intercepts converging to basal HGL. The hepatic response to Po did not alter upon 80% replacement by Pe. These results suggest that negative arterial-portal glucose gradients increase the rate of hepatic glycogen synthesis against the incremental HGL in an all-or-nothing mode.     

Altered baroreflex function after tail suspension in the conscious rat

Experiments were performed on conscious chronically instrumented rats to determine the contribution of peripheral V2-vasopressinergic receptors in any alteration of baroreceptor reflex (BRR) sensitivity on release from 1 wk of 30 degrees head-down tilt resulting from tail suspension. Initial experiments determined changes in plasma volume (PV) occurring over this period by use of the Evans Blue dye dilution technique. PV was determined immediately before tail suspension and on day 7 of the stimulus. PV, erythrocyte volume, and total blood volume were all significantly diminished on day 7, whereas hematocrit was unchanged. Other rats were instrumented with pulsed Doppler flow probes on the ascending aorta for determination of cardiac output and with arterial and venous catheters 7-10 days before study. Immediately before tail suspension, control cardiac output, mean arterial blood pressure, and heart rate values were determined. In addition, BRR sensitivity was estimated both before and after intravenous administration of a V2-receptor antagonist by assessing the slope of the pulse interval-mean arterial blood pressure relationship in response to a series of pressor doses of phenylephrine. BRR sensitivity was determined on the last day of head-down tilt, 1 min after release from tail suspension, and 10 min after administration of a specific V2-vasopressinergic antagonist. BRR sensitivity tended to fall on day 7 of tail suspension compared with control and was significantly increased after release. However, BRR sensitivity was not altered by intravenous V2 antagonist administration either before tail suspension or after release.(ABSTRACT TRUNCATED AT 250 WORDS)     

I. Determination of the endogenous and evoked release of catecholamines from the hypothalamus and caudate nucleus of the conscious and unrestrained rat

The action of cathecholamines within the CNS is important for the expression of numerous vegetative and behavioral functions. To understand the role these amines play, it is necessary to measure changes in the levels of these transmitter substances by utilizing new developments and methodology in the behaving animal. Utilizing new developments in methodology, it is possible to measure the release of amines into perfusates obtained from specific sites in the brain of the rat under basal and evoked conditions without prior purification or concentration.Using the push-pull perfusion technique, perfusates were obtained from the hypothalamus and caudate nucleus and analyzed by liquid chromatography with electrochemical detection. It is possible to readily determine basal release of dopamine from the caudate nucleus. Detection of both dopamine and noradrenaline is possible under ephedrine stimulated conditions from both the caudate nucleus and the hypothalamus. Although levels of serotonin (5-HT) were detected in brain perfusates, it may not be of neuronal origin. It may be possible to use these techniques to delineate the roles these amines play in various physiological functions.     

Effect of pancreastatin on pancreatic endocrine function in the conscious rat

We have studied the effects of pancreastatin on insulin and glucagon secretions in vivo in the conscious rat. Rats were prepared with a gastric fistula and with both external jugular veins cannulated. We found that an i.v. infusion of pancreastatin (1 and 10 nmol/kg/h) inhibited the plasma insulin response and increased the plasma glucose response to the intragastric infusion of glucose in a dose-dependent manner. Furthermore, the infusion of pancreastatin increased the plasma glucagon response to the i.v. infusion of arginine in a dose-dependent manner, and it inhibited the plasma insulin response. However, such an infusion of pancreastatin had no effect on the basal plasma glucose level, nor did it have any effect on plasma insulin and glucagon concentrations. Thus, it is suggested that in the rat, the newly discovered pancreastatin is a regulator of islet cell function.     

Subcutaneous PO2 as an index of the physiological limits for hemodilution in the rat.

This study was designed to test the hypothesis that changes in subcutaneous PO2 (PscO2) during progressive hemodilution will reliably predict a "critical point" at which tissue O2 consumption (VO2) becomes dependent on O2 delivery (QO2). Twelve pentobarbital-anesthetized male Sprague-Dawley rats (315-375 g) underwent stepwise exchange of plasma for blood (1.5 ml of plasma for each 1 ml of blood lost). The initial exchange was equal to 25% of the estimated circulatory blood volume, and each subsequent exchange was equal to 10% of the estimated circulatory blood volume. After nine exchanges, the hematocrit (Hct) fell from 42 +/- 1 to 6 +/- 1%. Cardiac output and O2 extraction rose significantly. PscO2 became significantly reduced (P < 0.05) after exchange of 45% of the blood volume (Hct = 16 +/- 1%). VO2 became delivery dependent when QO2 fell below 21 ml x min(-1) x kg body wt(-1) (mean Hct = 13 +/- 1%). Eight control rats undergoing 1:1 blood-blood exchange showed no change in PscO2, pH, HCO3(-), or hemodynamics. Measurement of PscO2 may be a useful guide to monitor the adequacy of QO2 during hemodilution.     

A model of ventilatory instability induced in the unrestrained rat.

A classic conditioning paradigm was used to examine the hypothesis that perturbations during sleep in the neonate rat can have a lasting impact on breathing. During the first 4 wk of life, stimuli were presented to rats during behaviorally defined sleep. In a conditioned hypoxic (CH) group, brief periods of hypoxic gas were used as the unconditioned stimulus. Tactile and auditory stimuli were used as the conditioned stimuli. In a conditioned control (CC) group, air was used as the unconditioned stimulus. A third group of unconditioned control (UC) rats was not exposed to the conditioning paradigm. Animals were provided routine care for 3.5 mo; ventilation was then assessed using plethysmography. Conditioning during neonatal life produced increased ventilatory irregularities and apnea during behaviorally defined sleep in adult rats. Both CH and CC rats showed a significantly greater number of apneic events compared with UC rats. Over a 2-h sleep period, CH rats exhibited a total of 105.1 +/- 9.4 (SE) apneic events, CC rats 69.4 +/- 4.2 events, and UC rats 42.1 +/- 3.1 events [F(2,18) = 25.568; P < 0.0001]. These findings suggest that experiences in the first few weeks of life will alter ventilatory patterning in the adult animal.     

Determination of simultaneous turnover of serotonin,dopamine and norepinephrine in the telencephalon of unrestrained behaving rats

Rats received an injection of tritiated tryptophan and tyrosine via a chronic indwelling jugular catheter, and at 60 and 90 minutes post-injection, were killed by near-freezing. The three biogenic monoamines were separated by ion exchange and thin layer chromatographyand were quantitated with respect to radioactivity and content. The decline in specific activities can be used to calculate rate constants and turnover of serotonin, dopamine and norepinephrine concurrently in unrestrained behaving animals.     

Statistical criteria for using short-term measurements as an index of 24-hour mean arterial pressure in unanesthetized unrestrained dogs

This study assessed the statistical validity of short time-interval measurements as estimators of true 24 hour mean arterial pressure in unanesthetized, unrestrained dogs. 24 hour intra-arterial pressure recordings were obtained using a stable FM telemetry system. The 24 hour pressure measurements approximated a normal distribution whose variance was inversely related to the selected averaging interval. Given the variance of a normal distribution one can calculate the 95% confidence interval for any single random measurement. Conversely the number of random samples necessary to be within a prescribed confidence interval can be determined. In this study, the 95% confidence interval for a single, random 30 minute arterial pressure average was calculated to be 11.2 mmHg. Only 4.8 +/- 1.4% of 480 individual 30 minute arterial pressure measurements fell beyond this confidence interval. These outlying values were distributed throughout the 24 hour period. The data suggest that randomly chosen short time-interval measurements may be a valid index of true 24 hour mean pressure if the average variance of a population is known and confidence intervals are defined.