Adrenal vein catecholamines and neuropeptides during splanchnic nerve stimulation in cats

Splanchnic nerve stimulation in bursts at low (5 Hz) and high (50 Hz) frequency (30 V, 1 msec; train duration 1 sec; train rate 0.5/second) was employed in 10 cats under halothane anesthesia, during 10-minute periods, while blood samples were concurrently collected from the adrenal vein and femoral artery for the measurement of norepinephrine (NE), epinephrine (EPI), dopamine (DA), Met-enkephalin (ME), neuropeptide Y (NPY), peptide YY (PYY) and neurotensin (NT). In Group I (n = 5), splanchnic nerve stimulation was initially applied at 5 Hz followed after 20 min by a 50 Hz stimulus, while in Group II (n = 5) the stimulation sequence was reversed. Adrenal vein and femoral artery plasma levels of catecholamines and neuropeptides were not significantly affected by the stimulation sequence, while a significant decrease in blood pressure response was observed in Group II during the 5 Hz stimulation as compared to Group I, indicating desensitization. Splanchnic nerve stimulation at 5 Hz caused a preferential increase in adrenal vein NE (9-fold) versus EPI (7-fold) levels as compared to baseline, while 50 Hz stimulation led to further comparable increases in NE (5-fold) and EPI (6-fold) levels. Significant increases in adrenal vein DA and neuropeptide levels were only observed during 50 Hz stimulation, with DA showing a 5-fold, ME a 2.6-fold and NPY a 3-fold increase as compared to 5 Hz stimulation, and NT a 3.6-fold increase as compared to baseline. Present findings indicate different dynamics in the movement of catecholamines and neuropeptides from the adrenal.     

Chromatographic characterisation of the circulating neuropeptide Y immunoreactivity from patients with phaeochromocytoma

Samples of adrenal medullary phaeochromocytoma from two patients were extracted in acid. The tissue concentrations of neuropeptide Y (NPY)-like immunoreactivity were found to be 0.94 and 8.11 micrograms/g. Plasma from these patients subjected to NPY radioimmunoassay after Sephadex G-75 gel filtration chromatography revealed circulating plasma concentrations of 1889 and 2079 pg/ml, compared to levels in normal plasma of less than 50 pg/ml, treated in the same way. Further characterisation of this plasma NPY-like immunoreactivity by HPLC showed this peptide to possess identical elution behaviour to human NPY standard. It is suggested that circulating NPY may be a contributory factor in the symptoms exhibited by some patients with adrenal medullary phaeochromocytoma.     

Distinguishing splanchnic nerve and chromaffin cell stimulation in mouse adrenal slices with fast-scan cyclic voltammetry

Electrical stimulation is an indispensible tool in studying electrically excitable tissues in neurobiology and neuroendocrinology. In this work, the consequences of high-intensity electrical stimulation on the release of catecholamines from adrenal gland slices were examined with fast-scan cyclic voltammetry at carbon fiber microelectrodes. A biphasic signal, consisting of a fast and slow phase, was observed when electrical stimulations typically used in tissue slices (10 Hz, 350 μA biphasic, 2.0 ms/phase pulse width) were applied to bipolar tungsten-stimulating electrodes. This signal was found to be stimulation dependent, and the slow phase of the signal was abolished when smaller (≤250 μA) and shorter (1 ms/phase) stimulations were used. The slow phase of the biphasic signal was found to be tetrodotoxin and hexamethonium independent, while the fast phase was greatly reduced using these pharmacological agents. Two different types of calcium responses were observed, where the fast phase was abolished by perfusion with a low-calcium buffer while both the fast and slow phases could be modulated when Ca2(+) was completely excluded from the solution using EGTA. Perfusion with nifedipine resulted in the reduction of the slow catecholamine release to 29% of the original signal, while the fast phase was only decreased to 74% of predrug values. From these results, it was determined that high-intensity stimulations of the adrenal medulla result in depolarizing not only the splanchnic nerves, but also the chromaffin cells themselves resulting in a biphasic catecholamine release.     

Innervation of the mink pineal gland with neuropeptide Y (NPY)-containing nerve fibers

Summary An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY (32–36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this spegcies are a component of the central innervation or originnate from extracerebral parasympathetic ganglia.     

Stress-induced changes of circulating neuropeptide Y in the rat: comparison with catecholamines

Circulating concentrations of neuropeptide Y-like immunoreactivity (NPY), noradrenaline (NA) and adrenaline (AD) were measured in conscious, chronically catheterized rats submitted to various stress protocols. Basal plasma levels of NPY, NA and AD (194 +/- 52 fmol/ml, 0.90 +/- 0.11 pmol/ml and 0.52 +/- 0.07 pmol/ml) were increased by handling (+132%, +76% and +629%, respectively) and rose further during electric shock treatment. Adrenalectomy resulted in the complete disappearance of circulating adrenaline but did not alter either control or stress values of noradrenaline. In comparison circulating levels of NPY were reduced, but not significantly in adrenalectomized animals. Insulin stress induced a large increase in plasma AD levels and cold stress induced an increase in plasma NA levels, without any parallel change in NPY concentrations. These results demonstrate that NPY, which is colocalized with catecholamines in the peripheral nervous systems, is also released during stress responses and that its release parallels more closely changes in circulating NA than AD. Furthermore, stress-induced changes in circulating NPY-like immunoreactivity do not originate from the adrenal gland but mainly from the peripheral nervous system, and the release of NPY is dependent upon the nature of the stimulus.     

Distribution of neuropeptide Y immunoreactivity in the forebrain of the rat

The distribution of neuropeptide Y (NPY) like immunoreactivity was investigated in the forebrain of the rat with immunohistochemical methods. Specificity of the antisera was established by the absence of all immunoreactive staining in tissue incubated in antisera which had been preabsorbed with the pure NPY antigen. NPY containing cells were distributed widely in the forebrain. These included neocortex, basal forebrain, septum, bed nucleus of the stria terminalis, neostratium, hypothalamic arcuate nucleus, and intergeniculate leaflet. This study also demonstrated an extensive network of NPY fibers in various areas of the forebrain such as the prepotic area, the hypothalamus and the paraventricular nucleus of the thalamus. The distribution of avian pancreatic polypeptide and NPY was compared, and the possible importance of NPY is briefly discussed.     

Correlation between endogenous noradrenaline and glucose released from the liver upon hepatic sympathetic nerve stimulation in anesthetized dogs

The metabolic role of neurally released noradrenaline (NA) was studied in the liver of anesthetized dogs. Sustained stimulation with various frequencies was directly applied on the anterior plexus of hepatic nerves. Stimulation-induced changes in plasma concentrations of endogenous catecholamines in hepatic venous blood were determined in correlation with concomitant changes in those of glucose (GL). Mean basal values for hepatic venous NA, adrenaline, dopamine, and GL were 0.062, 0.022, 0.032 ng/mL, and 97.9 mg%, respectively. Among these catecholamines, NA was the only one being released significantly during stimulation. While hepatic venous NA increased rapidly during stimulation, being maximum within 3 min, hepatic venous GL increased gradually, reaching a maximum value 5 min after the onset of stimulation. A highly significant correlation (r = 0.90, P less than 0.001) was found between changes in hepatic venous NA and GL concentrations observed during stimulation at various frequencies (2-16 Hz). However, hepatic vasoconstricting responses to stimulation were not correlated with increased hepatic venous GL. An alpha-blockade with phentolamine (2 mg/kg, iv) resulted in diminished release of GL by approximately 50% (P less than 0.05) and reduced hepatic arterial vasoconstriction by approximately 47% (P less than 0.01) upon stimulation (8 Hz, 5 min), even though NA release was markedly enhanced. We conclude that in the dog, NA is the sole catecholamine released within the liver in response to direct hepatic nerve stimulation, and NA thus released mediates the hepatic glycogenolysis via alpha-adrenoceptors.     

Plasma concentration of neuropeptide Y in patients with adrenal hypertension.

The mechanisms of hypertension during primary hyperaldosteronism and Cushing's syndrome are not completely understood. An enhanced vascular sensitivity to noradrenaline has been described in both situations. Neuropeptide Y (NPY) induces direct vasoconstriction and potentiates the action of noradrenaline. Sodium retention and dexamethasone have been shown to increase circulating NPY levels in animals and the expression of NPY in neuroendocrine cells. In order to determine if NPY could be involved in the enhanced vascular sensitivity to noradrenaline associated with adrenocortical hyperactivity, we measured plasma NPY in patients with Cushing's syndrome (n = 26) and primary hyperaldosteronism (n = 15) and compared it with that of hypertensive patients with pheochromocytomas (n = 13) or essential hypertension (n = 51) and with normotensive controls (n = 47). The concentration of NPY-Like immunoreactivity (NPY-Li) (mean +/- S.E.) in controls was 39.6 +/- 3.0 pg/ml. Elevated concentrations were found in 77% of the samples collected from pheochromocytoma patients (1180.4 +/- 394.0 pg/ml). NPY-Li levels in patients with essential hypertension (35.0 +/- 2.6 pg/ml), primary hyperaldosteronism (31.3 +/- 3.9 pg/ml) and Cushing's syndrome (33.1 +/- 4.8 pg/ml) were not different from that of controls. NPY-Li levels in hypertensive and normotensive patients with Cushing's syndrome were similar (38.5 +/- 7.5 vs 24.2 +/- 3.7 pg/ml). No correlation was found between the NPY-Li level and the mean blood pressure at the time of sampling. Our results suggest that NPY is unlikely to be involved in the pathogenesis of hypertension associated with primary hyperaldosteronism and Cushing's syndrome.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Functional significance of alpha-stimulation and alpha-blockade on responses to cardiac nerve stimulation in anesthetized dogs

Experiments were conducted to determine if α-stimulants could inhibit responses to sympathetic nerve stimulation $\text{via}$ a feedback inhibition loop mediated by prejunctional α-receptors. Responses to cardiac nerve stimulation in anesthetized dogs were compared before, during the peak effect of a drug infusion, and during a second drug infusion subsequent to the administration of phentolamine (5 mg/kg i.v.). The drugs infused were norepinephrine, phenylephrine, clonidine, naphazoline - all α-stimulants - and guanethidine. All drugs caused marked elevations of blood pressure, an indication of α-stimulation, but only guanethidine caused significant blockade of responses to sympathetic nerve stimulation. In addition, phentolamine, an α-receptor blocker, and desipramine, an inhibitor of amine uptake, did not potentiate responses to sympathetic nerve stimulation. These results do not support the hypothesis that sympathetic nerves are under a functionally significant feedback loop mediated by α-receptors.     

Cholinergic stimulation of neuropeptide Y secretion from the isolated perfused rat stomach.

Neuropeptide Y (NPY) is present in both extrinsic sympathetic adrenergic nerve terminals and intrinsic nerves of the gastrointestinal (GI) tract. Based on this localization a number of functions have been attributed to GI NPY including regulation of blood flow, intestinal fluid and electrolyte transport, and motility. There is nothing currently known, however, about the regulation of its secretion from GI nerves. The effect of cholinergic agonists and antagonists on secretion of NPY immunoreactivity (NPY-IR) from the isolated perfused rat stomach was investigated in the present study. Perfusate samples were extracted and concentrated on SepPak cartridges. Basal levels of NPY-IR varied between 98 and 147 fmol/min. Release was stimulated by high potassium concentrations (50 mM) and acetylcholine (ACh; 1 microM). ACh-induced secretion was unaffected by atropine, but inhibited by hexamethonium. Further evidence for a nicotinic component in the regulation of NPY-IR secretion was obtained by the finding of hexamethonium-induced reduction in basal secretion and stimulation of secretion by 1,1-dimethyl-4-phenyl-piperazinium (DMPP). In conclusion, cholinergic agonists and antagonists can modulate gastric NPY-IR secretion, and the cholinergic stimulatory effects are probably mediated via nicotinic receptor stimulation at the level of the intrinsic ganglia.     

Changes in blood level of catecholamines during arterial blood pressure rise induced with sciatic nerve stimulation

In experiments on rabbits and dogs it was demonstrated that electrical stimulation of the centripetal fibres of the cut sciatic nerve causing a rise of the arterial blood pressure produced a significant increase in the plasma levels of adrenaline and noradrenaline. This effect was not observed in animals with sympathetic system blockade caused by administration of reserpine. These observations indicate that pressure increase after sciatic nerve stimulation is due to stimulation of the adrenergic system.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.