Insulin dependence of the actions of growth hormone and somatostatin on splanchnic biogenic amines of the dog

It is known from studies previously conducted in this laboratory that an iv injection of ovine growth hormone (GH, 100 micrograms/kg BW) or an equimolar amount of somatostatin (SRIF, 7.5 micrograms/kg BW), given to normal conscious dogs into a saphenous vein, leads to a significant increase in hepatic portal plasma serotonin and a simultaneous decrease in the concentrations of dopamine, norepinephrine and epinephrine. The changes take place within 12 minutes after the injection and are observed only in the portal circulation. The purpose of the present experiment was to investigate whether or not similar results could be obtained in diabetic animals. Mongrel dogs were rendered diabetic by surgical pancreatectomy and fitted with an indwelling hepatic portal catheter. Radioenzymatic methods were employed for quantitative measurements of plasma free serotonin and catecholamines. No response was noted when the same type of experiments as those conducted in normal dogs were now carried out in trained, fully conscious totally pancreatectomized dogs deprived of exogenous insulin supply. When the same animals were given an injection into a peripheral vein of 50 mU/kg BW regular crystalline insulin (a small dose that affected neither plasma glucose nor biogenic amine levels) 10 minutes prior to the administration of the other hormones, the usual response to both GH and SRIF was restored, i.e. the data were comparable to those of normal dogs. It is concluded that the GH/SRIF effect on gut biogenic amines is insulin dependent.     

Somatostatin mediates the effect of growth hormone surges on splanchnic biogenic amines

Experiments were conducted in trained, conscious dogs fitted with an indwelling portal catheter. Radioenzymatic methods were employed for the quantitative measurement of plasma-free serotonin and catecholamines. An injection of ovine growth hormone (GH, 100 micrograms/kg) or an equimolar amount of somatostatin (somatotropin release inhibitory factor, SRIF, 7.5 micrograms/kg) into a saphenous vein led, within the first 15 min, to a transient but significant increase in plasma serotonin and a decrease in the concentrations of dopamine, norepinephrine, and epinephrine. The changes were frequently in excess of 40% of baseline values, and were found only in the portal and not in the peripheral circulation. When the animals were pretreated with an antiserum specifically directed against SRIF, GH surges no longer caused alterations in the portal levels of biogenic amines. Thus, the effects of spike concentrations of GH on plasma serotonin and catecholamines are apparently mediated by SRIF, a novel and unexpected function for a hormone that is known as an inhibitor of GH secretion.     

The effect of growth hormone on biogenic amines in the hepatic portal circulation of the dog

The effects of a spike concentration of growth hormone (GH) on hepatic portal and peripheral levels of free serotonin and catecholamines were studied by improved radioenzymatic methods in trained, conscious, normal, adult dogs fitted with an indwelling portal catheter. An injection of ovine GH (6 or 100 micrograms/kg) into a cephalic vein produced in the hepatic portal circulation a transient, statistically significant rise of serotonin and a concomitant significant reduction in the concentration of dopamine, norepinephrine, and epinephrine. No change was found in the peripheral circulation, partly because the amines were conjugated to sulfates and glucuronides and these derivatives are not detectable by our assays. Thus, a pulse of GH not only stimulates the release of pancreatic hormones and glucose turnover, but also affects the portal profile of glucoregulatory bioamines. The present investigation lends further support to our view that the splanchnic area represents an endocrine system whose preferential target is the liver.     

Catecholamine Release and Excretion in Rats with Immunologically Induced Preganglionic Sympathectomy

Abstract: Plasma and urinary catecholamines were quantified to assess global sympathoadrenal function in rats with preganglionic lesions caused by antibodies to acetyl-cholinesterase (AChE). Rats were given intravenous injections of normal mouse IgG or murine monoclonal anti-acetylcholinesterase IgG (1.5 mg). Five or 16 days afterward, basal blood samples were taken through indwelling arterial cannulae. A few hours later, the rats were immobilized for 10 min in padded restrainers, and another blood sample was drawn. HPLC determinations showed low basal levels of norepinephrine and epinephrine (<0.2 ng/ml in all rat plasma samples). In control rats, immobilization stress increased levels of plasma catecholamines up to 35-fold. In rats tested 5 days after injection of antibody, the norepinephrine response was much smaller (15% of control), and (he epinephrine response was nearly abolished (5% of control). There was some recovery at 16 days after antibody treatment, but stress-induced catecholamine release was still markedly impaired. Reduced stress-induced release: was not accompanied by major changes in tissue epinephrine or norepinephrine (heart, spleen, adrenal glands, and brain), although adrenal dopamine content dropped by 60%. Urinary excretion was studied in parallel experiments to gain insight into the effects of AChE anti-bodies on basal sympathoadrenal activity. Epinephrine, norepinephrine, dopamine, and selected metabolites were quantified in 24-h urine samples collected at frequent intervals for 30 days after antibody injection. No statistically gnificant changes were detected in the urinary output of dopamine, 3-methoxytyramine, normetanephrine, or 3-methoixy-4-hydroxyphenylglycol. On the other hand, epinephrine and norepinephrine output increased sharply at the time of antibody injection and then fell significantly below control levels. Norepinephrine output returned to normal after 2 weeks, but epinephrine output remained depressed. These results are consistent with previous evidence of widespread and persistent antibody-mediated βmade to the preganglionic sympathetic system.     

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.     

Effect of bupropion on hippocampal neurotransmitters and on peripheral hormonal concentrations in the rat.

The purpose of the present study was to administer an acute dose of the dual dopamine norepinephrine reuptake blocker bupropion in freely moving rats and to monitor the extracellular neurotransmitter concentrations in the hippocampus via in vivo microdialysis and the peripheral hormonal concentrations via catheterization. A microdialysis probe was inserted in the hippocampus, and samples for serotonin, dopamine, and norepinephrine were collected every 20 min before and after the injection of 17 mg/kg of bupropion, for a total sampling time of 180 min. A catheter was placed in the vena femoralis of the second group of rats, and blood samples were collected before and after bupropion injection for quantification of growth hormone, prolactin, corticosterone, adrenocorticotropin hormone, and beta-endorphins. All neurotransmitter levels (dopamine, norepinephrine, and serotonin) significantly increased after bupropion injection. This was accompanied by a significant decrease in prolactin concentrations, whereas the other hormones showed no statistically significant variation. It can, therefore, be concluded that, although bupropion has dual reuptake proprieties, the observed effects both at the central and at the peripheral level seem to be ruled by the dopaminergic system.     

The effect of cold on adrenergic neurotransmission in canine saphenous arteries and veins

The effect of severe cold (5 to 10 degrees C) on adrenergic neurotransmission was compared in the isolated cutaneous (saphenous) artery and vein of the dog. The vein contracted to sympathetic nerve stimulation at temperatures as low as 10 degrees C; higher temperatures were needed for the artery to contract. Both blood vessels contracted to exogenous norepinephrine at temperatures as low as 5 degrees C. However, the contractile response to exogenous norepinephrine was less in the saphenous artery, and contractions to high K+ solution were depressed by cooling more in the artery than in the vein. During electrical stimulation of the sympathetic nerves in saphenous arteries and veins previously incubated with labeled norepinephrine, progressive cooling from 37 to 5 degrees C caused a sharp decline in overflow of [3H]norepinephrine and its metabolites. However, overflow of labeled norepinephrine in both blood vessels continued at very cold temperatures. Thus the inability of the saphenous artery to contract to sympathetic nerve stimulation at 10 degrees C can be explained by a greater sensitivity of the arterial smooth muscle to the direct depressant effect of cold, rather than to a differential release or metabolism or norepinephrine in the arterial wall or a loss of responsiveness to norepinephrine at very cold temperatures.     

Plasma free and sulfoconjugated catecholamines during sustained exercise

Previous research established a relationship between circulating sulfoconjugated norepinephrine (NE-SO4) and oxygen consumption at various exercise intensities. In this study, the stability of the NE-SO4 response was examined during sustained exercise at a constant relative intensity. Seven trained men bicycled at 78 +/- 3% of their maximal O2 consumption for 28 min and then rested on the ergometer for a comparable duration. After a 30-min rest, plasma samples were collected through an indwelling catheter at 7-min intervals during the exercise and recovery periods. Free NE and epinephrine increased sixfold during exercise. These changes were accompanied by increases in sulfoconjugated catecholamines, but only NE-SO4 achieved statistical significance (rest, 712 +/- 602; exercise, 1,329 +/- 1,163 pg/ml). This occurred at three collection periods (14, 21, and 28 min). Approximately 35, 52, and 95% of NE, epinephrine, and dopamine, respectively, existed as sulfoconjugated during exercise. Subject variation was present in the sulfoconjugated catecholamine response that could not be attributed to corresponding differences in circulating free catecholamine release. These findings implicate blood flow as a factor in the sulfoconjugation of NE, but not epinephrine or dopamine.     

Direct evidence that an increase in aortic norepinephrine level in response to insulin-induced hypoglycemia is due to increased adrenal norepinephrine output

This study reports on the major source of circulating norepinephrine that is known to increase, progressively, during sustained hypoglycemia induced by intravenous insulin administration. Plasma concentrations of epinephrine, norepinephrine, and dopamine were simultaneously determined for adrenal venous and aortic blood in dogs anesthetized with sodium pentobarbital. The model used allowed us to perform a functional adrenalectomy (ADRX), while continuously monitoring the adrenal medullary secretory function. Under basal conditions, the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine were 0.169 +/- 0.074, 0.067 +/- 0.023, and 0.011 +/- 0.003, respectively. Plasma concentrations (ng/mL) of aortic epinephrine, norepinephrine, and dopamine were 0.132 +/- 0.047, 0.268 +/- 0.034, and 0.034 +/- 0.009. Following insulin injection (0.15 IU/kg, i.v.), the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine increased gradually (p less than 0.05), reaching the values of 0.918 +/- 0.200, 0.365 +/- 0.058, and 0.034 +/- 0.007 30 min after insulin administration. Similarly, aortic epinephrine, norepinephrine, and dopamine concentrations (ng/mL) increased significantly (p less than 0.05) to 0.702 +/- 0.144, 0.526 +/- 0.093, and 0.066 +/- 0.024. The aortic glucose concentration (mg/dL) was diminished from 81.8 +/- 4.1 to 36.9 +/- 3.4 (p less than 0.01). After taking the blood sample at 30 min following insulin administration, ADRX was immediately performed. Five minutes after the onset of ADRX, the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine increased further to 1.707 +/- 0.374 (p less than 0.05), 0.668 +/- 0.139 (p less than 0.05), and 0.052 +/- 0.017.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epinephrine-induced hepatic potassium movements before and after adrenergic blockade.

The epinephrine-induced loss and subsequent uptake of K+ by the liver was studied by measuring hepatic arterio-venous K+ differences and splanchnic blood flows in anesthetized dogs with chronically implanted portal vein catheters and celiac and superior mesenteric artery flow probes. When epinephrine was administered intraportally, neither alpha- nor beta-adrenergic blockade, singly or in combination, had significant effects upon the hyperkalemic or the hypokalemic phases in either hepatic venous or systemic arterial blood. It was concluded that the movements of K+ into and out of the liver caused by epinephrine are not mediated by the classical adrenergic receptors as defined by inhibition by specific blocking agents.     

Plasma catecholamines in the aorta and the phrenicoabdominal vein in exercising dogs

Plasma epinephrine and norepinephrine concentrations were measured in the aorta and phrenicoabdominal vein in five dogs at rest and during short-duration mild- and moderate-intensity exercise and during prolonged mild-intensity exercise. Plasma epinephrine and norepinephrine concentrations increased with exercise in both the aorta and the phrenicoabdominal vein. Plasma epinephrine concentration was much higher in the phrenicoabdominal vein than in the aorta (24-43 times). Plasma epinephrine concentrations in the aorta and phrenicoabdominal vein were significantly correlated (r = 0.88). This confirms that peripheral epinephrine concentration is a reliable index of the activity of the adrenal medulla during exercise. The epinephrine-to-norepinephrine ratio in the phrenicoabdominal vein was stable (4:1) throughout the experimental protocol, suggesting that the proportion of the two amines released by the adrenal medulla did not vary through this range of adrenal activity in dogs.     

The release of catecholamines from the adrenal medulla and its modulation by alpha 2-adrenoceptors in the anaesthetized dog

The adrenal nerve of anaesthetized and vagotomized dogs was electrically stimulated (10 V pulses of 2 ms duration for 10 min) at frequencies of 1, 3, 10, and 25 Hz. There was a correlation between the frequency of stimulation and the plasma concentrations of epinephrine, norepinephrine, and dopamine in the adrenal vein, mainly after the 1st min of stimulation and the maximal concentration was reached sooner with higher frequencies of stimulation. Moreover, the relative percentage of catecholamines released in response to the electrical stimulation was not changed by the frequency of stimulation. To test the hypothesis that a local negative feedback mechanism mediated by alpha 2-adrenoceptors exists in the adrenal medulla, the effects of the systemic administration of clonidine (alpha 2-antagonist) on the concentrations of catecholamines in the adrenal vein were evaluated during the electrical stimulation of the adrenal nerve (5 V pulses of 2 ms duration for 3 min) at 3 Hz. Moreover, the effects of the systemic injections of more specific alpha 2-agonist and antagonist (oxymetazoline and idazoxan) were tested on the release of catecholamines in the adrenal vein in response to electrical stimulation of the splanchnic nerve at 1 and 3 Hz frequencies. The injection of 0.5 mg/kg of yohimbine caused a significant increase in the concentrations of epinephrine and norepinephrine in the adrenal vein induced by the electrical stimulation of the adrenal nerve and the injection of 15 micrograms/kg of clonidine had no effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic potassium movements induced by sympathomimetic amines before and after adrenergic blockade.

The hepatic K+-mobilizing effects of phenylephrine and isoproterenol were studied in dogs equipped with chronic indwelling portal vein catheters. Animals anesthetized with sodium pentobarbital, received intraportal injections of these sympathomimetic amines, alone or in combination, before and after alpha, or beta, or combined adrenergic blockade. Hepatic K+ movements were assessed by measuring systemic arterial and hepatic venous K+ levels. It was concluded that adrenergic blockade exerted no significant influence on the ability of these agents to provoke the initial release and subsequent uptake of K+ by the liver.     

Cyclic AMP, the hyperresponsiveness factor from hog kidney

The isolation and identification of a material present in the plasma of hypertensive dogs and hypertensive human patients has been under study since 1972. The earliest experiments in relation to this work, noted that plasma from hypertensive dogs cause a hyperresponse to norepinephrine when both were administered by way of the vein. Employing a rat assay system that consisted of an anesthetized rat with polyethylene catheters in the vein for giving norepinephrine and the test fractions and a catheter in the artery for blood pressure monitoring, fractions from hog kidney were tested for hyperresponsiveness activity. The active material is very comparable to cyclic AMP in molecular weight, ultraviolet spectrum, paper chromatography, Enzyme hydrolysis and activity in the anesthetized rat system. This evidence indicates that the hyperresponsiveness factor of renal origin is cyclic AMP.     

Plasma concentrations of 5-HT, 5-HIAA,norepinephrine, epinephrine and dopamine in ecstasy users

Recreational use of the synthetic methamphetamine derivative MDMA (3,4-methylenedioxymethamphetamine), the main constituent of the illegal drug "ecstasy", has increased dramatically in recent years. The reasons for ecstasy-associated cardiovascular complications like tachycardia, arrhythmias and hypertensive crises and psychiatric symptoms like psychotic episodes are not well understood. We have measured the plasma concentrations of 5-HIAA, 5-HT, norepinephrine, epinephrine and dopamine in 159 ecstasy users and controls. Ecstasy users showed elevated resting sympathetic activity, reflected in increased norepinephrine, epinephrine and dopamine levels. The levels of these catecholamines correlated positively with the cumulative dose and also with consumption during the last 30 days and 12 months. Although it is known that significant changes in 5-HT and 5-HIAA appear in the cerebrospinal fluid in ecstasy users, we could not detect alterations in serotonergic neurotransmitters in plasma in this large sample of subjects. Thus, in the drug-free interval, ecstasy users show lowered central serotonergic activity (lowered 5-HT and 5-HIAA concentrations in CSF) along with unchanged central noradrenergic and dopaminergic activity (HVA and MHPG unchanged in CSF) and elevated peripheral noradrenergic, dopaminergic and adrenergic activity along with unchanged peripheral serotonergic activity (plasma levels). We conclude, that the data presented here could argue for a noradrenergic hyperreactivity in the drug-free interval in ecstasy users resulting from previous ecstasy consumption. Also for an association with psychotic episodes and cardiovascular complications like tachycardia, arrhythmias.     

Alpha2 adrenergic and high affinity serotonergic receptor changes in the brain stem of streptozotocin-induced diabetic rats.

The brain stems (BS) of streptozotocin (STZ)-diabetic rats were studied to see the changes in neurotransmitter content and their receptor regulation. The norepinephrine (NE) content determined in the diabetic brain stems did not show an increase, while epinephrine (EPI) content increased significantly compared with control. The NE to EPI turnover showed a significant increase. The alpha2 adrenergic receptor kinetics revealed that the receptor affinity was significantly reduced during diabetes. In insulin treated rats the NE content decreased while EPI content remained increased as in the diabetic state. Insulin treatment increased the Bmax for alpha2 adrenergic receptors significantly while the increase in Kd reversed to normal. Unlabelled clonidine inhibited [3H]NE binding in BS of control diabetic and insulin treated diabetic rats showed that alpha2 adrenergic receptors consisted of two populations of binding sites with Hill slopes significantly away from unity. In diabetic animals the ligand bound weaker to the low affinity site than in controls. Insulin treatment reversed this alteration to control levels. The displacement analysis using (-)-epinephrine against [3H]yohimbine in control and diabetic animals revealed two populations of receptor affinity states. In control animals, when GTP analogue added with epinephrine, the curve fitted for a single affinity model; but in the diabetic BS this effect was not observed. In both the diabetic and control BS the effects of monovalent cations on affinity alterations were intact. Our data thus show that alpha2 adrenergic receptors have a reduced affinity due to an altered post receptor affinity regulation The serotonin (5-HT) content in the brain stem increased. Its precursor (5-hydroxy) tryptophan (5-HTP) showed an increase and its breakdown metabolite (5-hydroxy) indoleacetic acid (5-HIAA) showed a significant decrease. This showed that in serotonergic nerves there is a disturbance in both synthetic and breakdown pathways which lead to an increased 5-HT. The high affinity serotonin receptor numbers remained unaltered with a decrease in the receptor affinity. The insulin treatment reversed these altered serotonergic receptor kinetic parameters to control level. Thus our study shows a decreased serotonergic receptor function. These changes in adrenergic and serotonergic receptor function were suggested to be important in insulin function during STZ diabetes.     

Monoamine Neurotransmitters and Their Metabolites in the Mature Rabbit Brain Following Induction of Hydrocephalus

Functional and behavioral disturbances associated with hydrocephalus may be due in part to altered neurotransmitter function in the brain. Hydrocephalus was induced in adult rabbits by injection of silicone oil into the cisterna magna. These and controls were killed 3 days, 1 and 4 weeks post-injection. Tissue concentrations of norepinephrine, epinephrine, serotonin, dopamine, and the metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) levels were determined in fifteen brain regions using HPLC. There were decreases in hypothalamic and medullary dopamine, transient decreases in basal ganglia serotonin, increases in thalamic noradrenaline, and increases in hypothalamic and thalamic epinephrine. Changes in the primary neurotransmitters may be attributable to damage of their axonal projection systems. Metabolite concentrations increased in the cerebrum. Reduced clearance of extracellular fluid which accompanies cerebrospinal fluid stasis may explain the accumulation of metabolites.     

Effect of steroid hormones on serotonin,norepinephrine and epinephrine contents in the pineal-paraphyseal complex of the soft-shelled turtle (Lissemys punctata punctata)

Summary Steroids (testosterone, oestrogen, progesterone, corticosterone, dexamethasone and deoxycorticosterone) were administered intramuscularly (0.1 mg · 100 g bw^-1) on seven consecutive days to juvenile male soft-shelled turtles. Serotonin, norepinephrine and epinephrine contents of the pineal-paraphyseal complex were measured spectrofluorometrically 24 h after the last injection. Testosterone and oestrogen decreased serotonin, norepinephrine and epinephrine levels. Progesterone treatment resulted in an increase of serotonin level and a fall in norepinephrine and epinephrine levels. Corticosterone treatment caused an increase of serotonin level and a decrease of norepinephrine and epinephrine levels. Dexamethasone failed to alter serotonin content, increased norepinephrine and decreased epinephrine levels. Deoxycorticosterone decreased serotonin and elevated epinephrine content.Abbreviations 5-HIAA 5-hydroxyindole-acetic acid - 5-MTOH 5-methoxytryptophol - ANOVA analysis of variance; bw body weight - COMT catecholamine-o-methyl transferase - E epinephrine - HIOMT hydroxyindole-o-methyl transferase - MAO monoamine oxidase - MS mean sum of squares - NAT N-acetyltransferase - NE norepinephrine - SR synaptic ribbon - SS sum of squares - SV source of variation     

Rat serum stimulates serotonin N-acetyltransferase activity in pineal monolayer cultures

The effects of rat serum on serotonin N-acetyltransferase (NAT) activity and indole synthesis in monolayer cultures of neonatal rat pineal glands was examined. The addition of 5% rat serum to these cultures resulted in stimulation of NAT activity equal to that obtained with optimal concentrations of the adrenergic agonist norepinephrine (NE). Rat serum also increased the synthesis of both N-acetylserotonin and melatonin from tryptophan. Stimulation of NAT activity by rat serum was partially blocked by metoprolol and propranolol, but not by phentolamine or butoxamine. The serum factor responsible for the stimulation was stable to both freezing and boiling. No significant amounts of epinephrine, norepinephrine, or dopamine were detected in the serum.     

Interaction of propranolol with central serotonergic neurons

Central monoaminergic mechanisms are believed to be involved in cardiovascular regulation. The present study was designed to evaluate the involvement of central serotonergic pathways in the antihypertensive action of propranolol in pentobarbital anesthetized mongrel dogs. Ventriculocisternal perfusion of propranolol (25 ug/kg/min for 30 min) decreased serotonin turnover as indicated by a significant decrease in cerebrospinal fluid levels of 5-hydroxyindoleacetic acid (5-HIAA). This effect was accompanied by a significant reduction in mean arterial pressure and heart rate. These results indicate that propranolol decreases central serotonergic activity and suggests a possible role for central serotonergic pathways in the antihypertensive action of propranolol. Several studies have indicated that central serotonergic pathways participate in the regulation of blood pressure. Brainstem regions including the nucleus tractus solitarius, the raphe nucleus and the anterior hypothalamic preoptic region are involved in cardiovascular control and contain a dense population of serotonergic neurons. A centrally-mediated hypotensive effect of propranolol has been demonstrated. Centrally administered propranolol increases cerebrospinal fluid (CSF) levels of norepinephrine and reduces blood pressure possibly due to decreased peripheral sympathetic nerve activity. Central serotonergic pathways may also be involved in the antihypertensive action of some beta-adrenoceptor antagonists. Destruction of central serotonergic neurons with 5,7-dihydroxytryptamine and desipramine abolished the antihypertensive effect of intracisternal propranolol in sinoaortic denervated dogs. Acute administrations of (-)-propranolol and (-)-pindolol decreased the synthesis rate of serotonin, while acute administration of salbutamol, a beta 2-adrenoceptor agonist, increased 5-HIAA levels in rat brain structures. The present study was designed to evaluate the involvement of central serotonergic pathways in the antihypertensive action of propranolol.