The distribution of opioid binding subtypes in the bovine adrenal medulla

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1-13). Specific [3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1-13). Delta opioid sites, labelled with [3H][D-Ala2,D-Leu5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [3H][D-Ala2, NMePhe4,Gly-ol5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.     

Localization and release of immunoreactive vasoactive intestinal polypeptide in bovine adrenal medulla

The concentration of immunoreactive (IR) vasoactive intestinal polypeptide (VIP) in extracts from bovine adrenal medulla was 29.9 +/- 7.2 pmol/g wet wt., which was about 100 times that of IR neurotensin and 30 times that of IR somatostatin. Chromatographic analysis showed that most of the IR-VIP was the same molecular size as synthetic VIP(1-28). On retrograde perfusion of isolated bovine adrenal gland, release of VIP with catecholamine (CA) was marked on stimulation with high K+, but slight on stimulation with acetylcholine, which induced marked release of CA. These results suggest that most of the VIP is localized not in CA storing granules in chromaffin cells, but in other intraadrenal neuronal components. In immunohistochemical studies, IR VIP fibers with large varicosities were observed around the vessels in the adrenal medulla.     

The effects of bradykinin, angiotensin and acetylcholine on the bovine adrenal medulla

1. The response of the adrenal medulla to the intra-arterial injection of angiotensin, bradykinin and acetylcholine and to splanchnic nerve stimulation has been investigated in day-old and 3- to 18-month-old calves and in adult cats.2. Bradykinin and angiotensin had no appreciable effect on catecholamine release in the day-old calf nor did these peptides potentiate the response of the gland to acetylcholine.3. In older calves the response of the gland to bradykinin and angiotensin was marginally above the control output, whereas acetylcholine and splanchnic nerve stimulation caused a 10-100 fold increase in the rate of secretion. The response to acetylcholine but not to the peptides was related to the dose administered.4. The adrenal medulla of the cat was stimulated by angiotensin and bradykinin but the maximum amounts released by the peptides were much smaller than the discharge elicited either by splanchnic nerve stimulation or by acetylcholine. No dose-response relationship could be found for either adrenaline or noradrenaline released by angiotensin, or for noradrenaline released by bradykinin. The slight increase in adrenaline output which followed the injection of bradykinin was dependent on the dose but the slope of the curve (b = 0.161) was much less than that for acetylcholine (b = 0.636).     

A novel substance P binding site in bovine adrenal medulla.

Radioligand binding techniques were used to characterize the substance P (SP) binding site on membranes prepared from bovine adrenal medullae. 125I-labelled Bolton-Hunter substance P (BHSP), which recognises the C-terminally directed, SP-preferring NK1 receptor, showed no specific binding. In contrast, binding of [3H]SP was saturable (at 6 nM) and reversible, with an equilibrium dissociation constant (Kd) 1.46 +/- 0.73 nM, Bmax 0.73 +/- 0.06 pmol/g wet weight and Hill coefficient 0.98 +/- 0.01. Specific binding of [3H]SP was displaced by SP greater than neurokinin A (NKA) greater than SP(3-11) approximately SP(1-9) greater than SP(1-7) approximately SP(1-4) approximately SP(1-6), with neurokinin B (NKB) and SP(1-3) very weak competitors and SP(5-11), SP(7-11) and SP(9-11) causing negligible inhibition (up to 10 microM). This potency order is quite distinct from that seen with binding to an NK1 site, a conclusion confirmed by the lack of BHSP binding. It appears that Lys3 and/or Pro4 are critical for binding, suggesting an anionic binding site. These data suggest the existence of an unusual binding site which may represent a novel SP receptor. This site appears to require the entire sequence of the SP molecule for full recognition.     

Vascular and adrenocortical responses to a specific antagonist of angiotensin II.

Angiotensin II receptors in vascular smooth muscle and adrenal cortex have been characterized in the dog. The evidence was derived chiefly from experiments that assessed the ability of a structural analog of angiotensin II, [Sar1, Ile8] AII, to antagonize the effects of exogenously administered angiotensin II on arterial pressure and aldosterone secretion. [Sar1, Ile8] AII is a potent and specific blocker of the pressor response to angiotensinII; in the adrenal cortex, it is a much less effective inhibitor of aldosterone biosynthesis. These results indicate differences in the receptor sites for angiotensin II in vascular smooth muscle and adrenal cortex. Further, they raise the possibility that angiotensin II stimulates aldosterone secretion by mechanisms other than have already been proposed.     

Localization of vasopressin binding sites in rat brain by in vitro autoradiography using a radioiodinated V1 receptor antagonist

Vasopressin may act in the brain as a neurotransmitter or neuromodulator to influence blood pressure, memory, body temperature and brain development. In order to localize probable central nervous system sites for these actions, we have used 125I-labelled 1-d(CH2)5, 7-sarcosine-8-arginine vasopressin, a specific V1-receptor antagonist, and in vitro autoradiography to map brain vasopressin binding sites. High levels of binding were found in the choroid plexus, blood vessels, lateral septum, bed nucleus of stria terminalis, accumbens nucleus, central nucleus of amygdala, stigmoid hypothalamic nucleus, suprachiasmatic nucleus, arcuate nucleus, nucleus of the solitary tract, area postrema and parts of the hippocampus, thalamus, superior colliculus, and inferior olivary nuclei. Many of these regions are known to be vasopressin-sensitive and to contain vasopressin fibres. Significantly there was no binding to the paraventricular nor the supraoptic nuclei. Displacement of the radioligand from the lateral septum with unlabelled vasopressin analogues gave a rank order of potencies: d(CH2)5-D-Tyr2(Et)Val4-desGly9-arginine-vasopressin approximately equal to d(CH2)5-Tyr2-(Me)arginine-vasopressin approximately equal to arginine-vasopressin approximately equal to d(CH2)5-Sar7-arginine-vasopressin greater than [1-deamino, 8-D-arginine]-vasopressin approximately equal to oxytocin much greater than vasopressin4-9, consistent with binding to V1 receptor subtype. These studies confirm and extend previous findings of V1 receptors in the rat brain. In particular, several new regions of vasopressin receptor binding have been identified, possibly due to the advantages of a radioiodinated ligand with high receptor affinity without binding to neurophysins. Future study of these regions may prove fruitful in elucidating the central actions of vasopressin.     

Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain

The non-peptide angiotensin II receptor compounds DuP 753 and WL 19 were utilized to detect subtypes of [125I]Sar1-Ile8-angiotensin II binding to angiotensin II receptors in the rat brain. In rat forebrain homogenates, DuP 753 and WL 19 produced a partial displacement of [125I]Sar1-Ile8-angiotensin II binding with DuP 753 displacing approximately 65% of the binding and WL 19 displacing approximately 35% of the binding. Using the techniques of quantitative receptor autoradiography, a distinct regional distribution of the subtypes of angiotensin II antagonist bind was detected. The angiotensin II-1 binding site (the receptor subtype preferentially displaced by DuP 753) appeared to predominate in the dipsogenic, cardiovascular and endocrine areas, including the subfornical organ, paraventricular and periventricular nuclei of the hypothalamus, anterior pituitary, dorsal motor nucleus of the vagus, nucleus of the solitary tract and the area postrema. Additional areas that contained predominantly the angiotensin II-1 receptor subtype were the ventral hippocampus, substantia gelatinosa of the trigeminal nucleus, nucleus of the lateral olfactory tract, piriform cortex and median preoptic nucleus. The angiotensin II-2 binding site (displaced by WL 19) was the predominant subtype in the thalamus, inferior olive, lateral septum, subthalamic nucleus, locus coeruleus, medial geniculate and medial amygdala. Several areas of the brain appeared to contain both receptor subtypes, including the superior and inferior colliculi, and the olfactory bulb. The angiotensin II-1 binding site was concentrated in areas of the brain involved in mediating angiotensin II effects on drinking, endocrine status and blood pressure. Localization of angiotensin II-2 sites in the thalamus and areas of the brain which process sensory information suggests a novel modulatory role for angiotensin II at this receptor subtype. These results indicate that DuP 753 and WL 19 are highly selective for angiotensin II binding site subtypes in the brain and that, in general these subtypes are compartmentalized in distinct brain regions. The non-peptide compounds used in these studies should provide excellent tools to discern the functional role of angiotensin II receptor subtypes in the brain.     

Co-storage of enkephalins and adrenaline in the bovine adrenal medulla

Fluorescence histochemical and immunohistochemical techniques have been used to examine the distribution of catecholamine-containing and enkephalin-containing cells in sections of adult bovine adrenal medulla. Noradrenaline-containing cells were identified by fluorescence microscopy following perfusion fixation with 4% paraformaldehyde (formaldehyde-induced fluorescence, technique of Era¨nko¨⁸). Adrenaline-containing cells did not fluoresce under these conditions. Adrenaline-synthesizing cells were identified by immunofluorescence with an antiserum to bovinephenyl-N-methyl transferase. An antiserum to bovine dopamine-β-hydroxylase was used to identify noradrenaline plus adrenaline cells in the same section. Leu- and met-enkephalin-containing cells were identified immunohistochemically with their respective antisera. To determine whether there was a preferential association of leu- or met-enkephalin with adrenaline or noradrenaline cells, these various antisera were used singly or sequentially on sections treated with formaldehyde in which the localization of endogenous noradrenaline fluorescence had been recorded and then the fluorescence removed by washing overnight.Immunoreactive leu- and met-enkephalin were found to be associated exclusively with adrenaline-synthesizing cells. The finding that both enkephalins are localized in the one cell type (adrenaline cells) in the bovine adrenal medulla is consistent with the proposed common precursor model for synthesis of the two opioid pentapeptides. These findings on co-storage of enkephalins with adrenaline in the adrenal medulla may have implications for other areas of the peripheral and central nervous system where co-storage of catecholamines and enkephalins is known to occur.     

Localisation and pharmacological characterisation of [3H]bremazocine binding in the bovine adrenal medulla.

[3H]Bremazocine (5 nM), in the presence of excess unlabelled mu and delta opioid ligands labelled two anatomically distinct populations of binding sites in the bovine adrenal medulla; a high density over the peripheral adrenaline-containing region of the medulla and a lower density over the central noradrenaline-containing region. This non-mu, non-delta opioid binding was specific (diprenorphine sensitive) but did not appear to involve classical kappa (kappa 1), sigma or PCP binding sites being insensitive to high concentrations of dynorphin (1-13), 3-PPP or MK-801. A significant proportion of the binding at both locations was however sensitive to competition by U50,488H or metorphamide. These data provide further evidence to support the existence of multiple opioid binding sites in the bovine adrenal medulla.     

Enkephalins are associated with adrenergic granules in bovine adrenal medulla

The subcellular localization of enkephalins was studied in the bovine adrenal medulla. In the adrenal medulla enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg⁶-Phe⁷ and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸) are found free and in the form of cryptic peptides included in larger precursors. Total Met-enkephalin immunoreactivity, which includes free and cryptic peptides, was determined after a sequential enzymatic treatment with trypsin and carboxypeptidase B. Total Met-enkephalin immunoreactivity, dopamine beta-hydroxylase and catecholamines were found to have a parallel distribution in the various subcellular fractions. The bulk of the total Met-enkephalin immu noreactivity (42%) was recovered in the large granule fraction. The large granule fraction also contained 38% of the total dopamine beta-hydroxylase activity, and 42% of the total catecholamines. Enkephalins are thus concentrated in the chromaffin granules.Chromaffin granules were also separated according to the method of Terland & coworkers¹⁷ into two fractions: one containing the dense noradrenergic vesicles and the other containing lighter adrenergic vesicles. Total Met-enkephalin immunoreactivity was restricted to the fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to total Met-enkephalin immu noreactivity was 97.This study is in accord with immunocytochemical observations which have indicated that enkephalins are located in adrenergic and not in the noradrenergic cells in the bovine adrenal medulla.     

Microinjection of angiotensin II in the caudal ventrolateral medulla induces hyperalgesia

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT₁) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT₁-immunoreactive neurons occurred in the CVLM, only 3% of AT₁-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT₁ antagonist losartan. The present study demonstrates that Ang II can act on AT₁ receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT₁-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.     

Lack of effect of opioid compounds on angiotensin II responses of bovine adrenal medullary cells

Angiotensin II (10 nM) increased basal adrenaline and noradrenaline secretion from cultured bovine adrenal chromaffin cells by 2.5- to 3-fold and 4- to 6-fold, respectively, and stimulated basal accumulation of inositol phosphates more than 2-fold. Etorphine and diprenorphine in the range 10⁻⁹ to 10⁻⁵ M had no effect on the catecholamine secretion induced by angiotensin II, and, at 10⁻⁸ and 10⁻⁵ M, had no effect on angiotensin II-induced inositol phosphate accumulation. The functions of adrenal medullary opioid receptors remain to be determined.     

Distribution of [125I]angiotensin II binding sites in the rat brain: a quantitative autoradiographic study

Angiotensin II receptors have been localized by quantitative autoradiography in the rat central nervous system after labeling with [125I]angiotensin II. A highly discrete distribution of these receptors was found throughout the rat brain. The highest density was seen in regions of the medulla, hypothalamus and circumventricular organs where angiotensin II could potentially produce cardiovascular, dipsogenic and neuroendocrine responses. The distribution of angiotensin II receptors correlates relatively well with the previously reported distribution of angiotensin immunoreactive nerve terminals as well as areas determined by various physiological techniques to be sensitive to angiotensin II. Finally, the anatomical localization of angiotensin II receptor populations has revealed several areas of the brain where the effects of this peptide have not been investigated. Many of these nuclei are involved in the transmission and processing of somatic and visceral sensory information. These results suggest a broader role for the central renin-angiotensin system in modulating several types of sensory input.     

Potassium ion as a regulator of adrenal angiotensin II receptors

The importance of potassium as a regulator of angiotensin II receptors of two target tissues has been investigated by combining high-K+ diet in rats with a converting enzyme inhibitor (Captopril; SQ 14,225) or angiotensin II. K+ loading alone produced the characteristic increase in Ka and decrease in number of smooth muscle receptors and decrease in Ka and increase in number of adrenal receptors. The combination of Captopril and high-K+ diet blocked the development of most of these effects. In smooth muscle, the number of angiotensin II receptors was 40% higher (n = 5) and Ka was 94% lower (n = 5); in the adrenal the Ka was 29% higher (n = 4) than with K+ loading alone. However, development of the increment in number of adrenal receptors occurred in spite of Captopril treatment. In addition, angiotensin II infusion concomitantly with high-K+ diet resulted in a further increment in the number of adrenal receptors. These studies represent the first example of an ion as a regulator of a peptide hormone receptor and emphasize that different mechanisms of regulation are involved in adrenal and smooth muscle.     

The topography of gangliosides in the membrane of the chromaffin granule of bovine adrenal medulla

We have studied the topography of the gangliosides of the adrenal chromaffin granules by using neuraminidase to remove sialic acid from membrane gangliosides of intact and ruptured chromaffin granules. Residual sialic acid was then measured to compare the availability of gangliosides on the outer and inner surfaces of the membrane. Measurement of protein sialic acid served as a control since these residues are known to be on the inner surface of the membrane. Prolonged digestion of broken membranes showed that maximally 75% of both lipid and protein-bound sialic acid residues are available to neuraminidase. Prolonged digestion of intact granules produced no measureable loss of sialic acid from either protein or lipid fractions. Comparison of the thin-layer chromatograms of gangliosides extracted from digested and undigested membranes showed no preferential digestion of any component.We conclude that at least 75% of the gangliosides are on the inner leaflet of the membrane and suggest that all of the gangliosides are so located.     

Opossum adrenal medulla: II. Differentiation of the chromaffin cell

The ultrastructure of the opossum adrenal medulla was examined in its postnatal development. Maturation of chromaffin cells and genesis of chromaffin vesicles were of particular interest. The primitive sympathetic cell was seen to contain few organelles with no apparent polarity. Initial pheochro-moblasts contained more organelles with some polarity. Endoplasmic reticulum and the Golgi complex increased as the pheochromoblasts matured, which suggested increased synthetic activity. Structures resembling Golgi/endoplasmic reticulum/lysosome (GERL) systems were seen in the pheochromoblasts. It is suggested that some of the components of the chromaffin vesicle may be processed by the GERL while others come directly through the Golgi complex. It is stressed that the developing pheochromoblast in the opossum presents an interesting model in which to study the genesis of the chromaffin vesicle.