Modulation of catecholamine release by endogenous adenosine in the rat adrenal medulla

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.     

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

Fasudil attenuates sympathetic nervous activity in the adrenal medulla of spontaneously hypertensive rats

We investigated the effects of fasudil, a Rho kinase inhibitor, on hypertension in spontaneously hypertensive rats and on the catecholamine synthetic pathway. Ten-week-old male SHR and Wistar-Kyoto rats were administered fasudil (10 mg/kg/day s.c.) for 4 days. Systolic blood pressure was measured using the tail-cuff method. Catecholamine levels were measured with high-performance liquid chromatography-ECD methods. Tyrosine hydroxylase protein levels were measured in Western blot analysis. The tyrosine hydroxylase mRNA level was measured using real-time PCR methods. Fasudil significantly decreased systolic blood pressure in spontaneously hypertensive rats, but not in Wistar-Kyoto rats. Fasudil also significantly decreased catecholamine, tyrosine hydroxylase protein, and tyrosine hydroxylase mRNA levels in the adrenal medulla of spontaneously hypertensive rats. These results suggest that the depressor effects of fasudil on hypertension in spontaneously hypertensive rats may be related to inhibition of the catecholamine synthetic pathway.     

Tissue culture of rat adult decapsulated adrenal glands

Summary A method of cultivation involving both repeated trypsinisations (at room temperature) and explantation of the tissue fragments on polythene discs has been shown to be apt to the growth in vitro of rat adult decapsulated adreno-cortical tissue. This is the first time that the successful cultivation of such a tissue is reported. The technique and its applications are discussed.The effects of _1–24 corticotrophin (ACTH_1–24) on the rat adult adrenal cultures have been examined by both electron microscopy and autoradiography. Zona fasciculata and reticularis cells grown in the absence of ACTH for long terms (15–16 days) survive and proliferate as dedifferentiated elements. If ACTH_1–24 is added to the cultures, adrenocortical cells will, within 2 days, simultaneously increase their proliferation rate and differentiate. After 7 days of treatment, cortical cells exhibit not only fully differentiated but even hypertrophic morphologic features. Significant stimulations of adrenal DNA, RNA and gross protein synthesis have been found to take place at different times after the starting of the ACTH_1–24 treatment. These data are discussed in relation to the findings previously reported in literature.Rat adult adrenal gland tissue cultures are proposed as a non-previously available tool for investigations into the physiopathology of the adrenal cells to be carried out in a carefully controlled environment.A Preliminary report on part of this material was given at the annual meeting of the Société Française de Microscopie Elécronique, Nantes, May 1972.Authors wish to thank Drs. P. G. Andreis and A. S. Belloni for their skilful assistance in the autoradiographic experiments. Thanks are also due to Mr. G. Gottardo for his excellent technical help in electron microscopic work. This work was partly supported by a contract with the CNR-Italy (No 69.0172/115.3439).     

Ultrastructural radioautography of the incorporation of tritiated leucine by the rat adrenal medulla in vivo

Summary Sections of tissues from the adrenal medullae of young rats were subjected to radioautography after a single intravenous injection of L-leucine 4,5 ³H to identify the sites of synthesis and follow the migration of newly-formed proteins in both adrenaline-storing (A) and noradrenaline-storing (N) cells. As early as 2 min after injection of leucine ³H, the label was highest in the rough endoplasmic reticulum (RER) of A and N cells, suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the RER into the Golgi complex of both cell types. Some label was already present over the secretory granule matrix (chromogranins) by 2 min but the peak was reached at 1 h in both A and N cells. By 4 h, the label over the secretory granules had diminished, indicating a release of newly-synthetized chromogranins outside the cells. The label over the hyaloplasm was relatively high at 2 min but it decreased rapidly to low levels. In contrast, the label over the cell surface continually increased to reach the highest levels among all organelles at 4 h in both cell types. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the hyaloplasm, before reaching the surface of A and N cells.Supported in part by the Quebec Heart Foundation, the Medical Research Council of Canada (Grant MT-1973), the J.-L. Levesque Foundation, the Ministry of Education of Quebec (Formation de Chercheurs et Action Concertée) and the Fond de l'Université de Montréal (Cafir)     

Direct connections between mitochondria and catecholamine-storage vesicles demonstrated by high voltage electron microscopy in rat adrenal medulla

Summary Tubular channels from mitochondria to catecholamine-storage vesicles have been demonstrated in thick sections of adrenal medullary tissue from hypoglycemia-stressed rats by the use of the high voltage electron microscope. The function of these connections is not presently known although they may serve as channels for the transport of materials such as high-energy nucleotides from one organelle to the other. The present study has examined only the adrenal medulla, but it should be considered that such connections may also exist in other neural cells and possibly other cells in which there is intracellular transport of ATP.This work was supported by NIH DRR 70-4136, WVU Medical Corporation Research Grants, Biomedical Research Support Grant 5 SOI RR05433, WVU Senate Research Grant, and West Virginia Heart Association Research Grant. We wish to thank Barbara Coalgate, Billie Pack, and the staffs of the HVEM laboratories at U.S. Steel and University of Colorado.     

Short- and long-term effects of vinblastine on the rat adrenal medulla

Summary The effects of a single high dose (10mg/kg) of vinblastine (vb) sulfate (Velbe, Lilly) on the ultrastructure, catecholamine (CA) content and activity of CA-synthesizing enzymes of the rat adrenal medulla were studied for up to 120h after intravenous injection of the drug.By 1 h, microtubules were virtually absent from chromaffin cells and preganglionic cholinergic axons, and typical paracrystals had appeared inside the nerve fibers. By 16h microtubules were completely reconstituted and paracrystals had disappeared. From 16h onwards, there was an increasing depletion of storage granules from adrenaline (A) — producing cells, which coincided with biochemical determinations showing a reduction of adrenal A to about 40 % of control levels by 48 h, with noradrenaline (NA) remaining in the range of controls. Both A- and NA-storing cells showed an extensive proliferation of the rough endoplasmic reticulum (ER). Vb caused a marked increase in tyrosine hydroxylase (TH; +113%) and dopamine -hydroxylase (DBH; +82%) activities after 48 h. Splanchnicotomy completely abolished the vb-mediated increase in TH and DBH activities. A smaller increase (+ 47 %) in enzyme activity was observed with phenylethanolamine N-methyltransferase (PNMT). Vb (10^–5M) had no apparent effect on granule content and the amount of rough ER in chromaffin cells, which were cultured for 48 h.The results demonstrate that a single high dose of vb has relatively little short-term effects on the rat adrenal medulla, but causes drastic long-term changes in CA-content and enzyme activities that are mediated by the preganglionic nerves. These changes could be interpreted as an effort to compensate for a loss of CA-stores in peripheral adrenergic nerves (cf. Cheney et al., 1973). The differential long-term effect of vb on adrenal NA and A might be due to the lower induction of PNMT as compared to TH and DBH activities and/or to a preferential release of A versus NA, which may occur at high frequencies of stimulation of the splanchnic nerves.Supported by grants from the Deutsche ForschungsgemeinschaftDedicated to Professor G. Petry in honor of his 65th birthday     

Rapid purification and properties of protein kinase C from bovine adrenal medulla

Protein kinase C was purified from bovine adrenal medulla using a rapid procedure which resulted in an approximate 1500-fold increase in specific activity. The characteristics of the enzyme are reported and for the first time it is possible to compare the effects of TPA on secretion from intact and permeabilized cells with the effect of TPA on protein kinase C purified from the same secretory tissue.     

Role of proto-oncogenes in the regulation of proenkephalin mRNA expression induced by repeated nicotine injections in rat adrenal medulla

We have studied the effect of repeated systemic administrations of nicotine (3 mg/kg) at 30 min intervals on proenkephalin (proENK) mRNA level in rat adrenal gland. Northern blot analysis has shown that proENK mRNA expression was enhanced by repeated nicotine administrations. Additionally, repeated administrations of nicotine transiently induced the c-fos and c-jun mRNA levels after the first-third nicotine administration, and the c-fos and c-jun mRNA levels were returned to the basal level after the seventh administration of nicotine. c-Fos, c-Jun and Fra-2 protein levels were persistently increased until the seventh administration. The repeated nicotine administrations also elevated phospho-CREB without altering total CREB level in all tested groups. Immunohistochemical analysis showed that the increase of c-Fos and c-Jun proteins by repeated nicotine administrations is mostly medulla specific, while Fra-2 immuno reactivity was shown both in medulla and cortex. The repeated nicotine administrations enhanced the AP-1 and ENKCRE-2 DNA binding activities. Furthermore, the cross-competition studies revealed that the AP-1 proteins, rather than CREB, actively bind to ENKCRE-2 DNA domain. These results suggest that proENK mRNA expression induced by repeated nicotine administrations may be mediated by AP-1 proteins, such as c-Fos, c-Jun and Fra-2 rather than CREB via interacting to the ENKCRE-2 DNA binding domain in rat adrenal medulla.     

The fine structure of the newborn rat adrenal cortex

Summary The structural changes of the zona juxtamedullaris of the rat adrenal cortex at birth, have been examined by the light and the electron microscope. In this zone clusters of medullary cells lying among the strands of cortical tissue were observed. In the inner portion of the zona juxtamedullaris two types of adrenocortical cells were found: light and very-dark cells. The latter are smaller than the light cells and are always in close connection with the medullary tissue. The ultrastructural features of the very-dark cells suggest that these elements are in degeneration. This finding supports the hypothesis that at birth there is a partial degeneration of the rat zona juxtamedullaris, i.e. the zone corresponding to the fetal zone of some mammalian species.It is proposed that in all mammalian species at birth there is a partial regression of the zona juxtamedullaris and that the regression of the fetal zone is only the quantitative increase of this phenomenon. This hypothesis is discussed in relation to numerous data demonstrating that there are enzymatic conditions in the rat during fetal life, which permit a discrete hypertrophy of the adrenal cortex.The author wishes to express his appreciation to Dr. A. Gambino and to Mr. G. Gottardo for technical assistance.     

Different types of small granule-containing cells and neurons in the guinea-pig adrenal medulla

Summary An electron microscopic, histoand biochemical study was carried out on the adrenal medulla of newborn and adult guinea-pigs giving special emphasis to small granule-containing (SGC) cells. Adrenaline (A) was the predominating catecholamine (CA) both in newborn (70–90 % of total CA) and adult (85–90%) guinea-pig adrenals. In analogy to the biochemical findings electron microscopy revealed a high predominance of A cells, which contained large granular vesicles with an average diameter of 180 nm. Most noradrenaline (NA) storing cells showed granular vesicles of a considerably smaller average diameter (80 nm) and had a higher nuclear-cytoplasmic ratio. These cells were termed SGC-NA cells. NA cells with large granular vesicles (average diameter 170 nm) were extremely rare. Another type of SGC cells contained granular vesicles with cores of low to medium electron-density (SGC-NA-negative cells). Biochemical determinations made it unlikely that these cells contained predominantly dopamine (DA). SGC cells were scarcely innervated by cholinergic nerves. They formed processes, which were found both in the adrenal cortex and medulla contacting blood vessels including sinusoid capillaries, steroid producing cells of the reticularis and fasciculata zone and processes, which were interpreted to belong to medullary nerve cells.Two types of neurons were present in the guinea-pig adrenal medulla, one resembling the principal neurons in sympathetic ganglia, the other, which, according to its morphology, occupied an intermediate position between principal neurons and SGC cells.In adrenomedullary grafts under the kidney capsule, which were studied three weeks after transplantation, ordinary A cells resembled SGC-NA negative cells with respect to their ultramorphology. Processes of transplanted principal neurons showed uptake of 5-hydroxydopamine and, hence, were considered to be adrenergic. Despite the lack of extrinsic nerves to the transplants, few principal neurons received cholinergic synapses, the origin of which is uncertain to date.Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/4)Dedicated to Professor H. Leonhardt in honor of his 60th birthday.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Prostaglandins E₁ and E₂ are specifically bound by particulate fractions from bovine adrenal medulla. The subcellular localization of these binding sites has been investigated by comparing their distribution in subcellular fractions obtained by differential and gradient centrifugation to those of marker enzymes for various organelles. Prostaglandin E₂ binding sites were purified about 16-fold with respect to the homogenate in a fraction which was highly enriched in plasma membranes on the basis of the activities of the marker enzymes acetylcholinesterase and calcium-dependent ATPase, which were both purified by about 12-fold in this fraction. The plasma membrane fraction contained relatively low activities of marker enzymes for mitochondria (monoamine oxidase), lysosomes (acid phosphatase), endoplasmic reticulum (glucose-6-phosphatase), Golgi (galactosyl transferase) and chromaffin granule membranes (dopamine β-hydroxylase). The only other fractions enriched in prostaglandin E₂ binding sites were those for the endoplasmic reticulum and the Golgi, in which the binding sites were purified about 4-fold and 7-fold, respectively. This is probably due mainly to contamination with plasma membranes, since calcium-dependent ATPase and acetylcholinesterase were each purified to a similar extent in these two fractions. These data suggest that the high-affinity prostaglandin E₂ binding sites of the adrenal medulla are localized primarily on the plasma membranes of the medullary cells.     

Central angiotensin II increases biosynthesis of tyrosine hydroxylase in the rat adrenal medulla

Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.     

Calmodulin binding and protein phosphorylation in adrenal medulla coated vesicles

Coated vesicles from bovine adrenal medulla contained clathrin and major detergent-insoluble polypeptides of 120-100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 A cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120-100, 50 or 49 kDa polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.     

High-affinity angiotensin receptors in rat adrenal medulla

Angiotensin II receptors have been quantitated in single rat adrenal medullas by incubation of tissue sections with 125I-[Sar1]-AII, autoradiography with exposure to 3H-sensitive Ultrofilm, computerized densitometry and comparison with 125I-labelled standards. Rat adrenal medulla contains a single class of high affinity AII receptors with a Ka of 0.84 +/- 0.02 X 10(9) M-1 and a Bmax of 3259 +/- 502 fmol/mg protein, one of the highest densities in AII receptors found in rat tissues. These observations provide evidence for a local site of action of AII in the release of adrenal medullary catecholamines.     

Autoradiographic localization of strychnine-sensitive glycine receptor in bovine adrenal medulla

The presence of an uptake system and a functional glycine receptor in adrenal medulla chromaffin cells was investigated using an autoradiographic technique in adrenal gland slices. Specific³[H]-glycine binding was observed in both adrenal cortex and medulla slices, while only specific binding of [³H]strychnine was seen only in chromaffin cells and was not associated with cortical cells. [³H]Glycine binding sites in the cortex are apparently different from those of [³H]strychnine binding sites in the medulla since excess strychnine does not displace [³H]glycine from adrenal cortex but does so from medulla. This difference supports biochemical evidence for glycine transport into medulla cells and glycine receptor sites on the chromaffin cell membrane.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules ($\text{S}{}_{\mathrm{<mtext>H</mtext>}}\text{(}\text{average sedimentation coefficient}\text{) = 12 400}\text{S}$ in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin ($\text{?95\%}$ of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium.The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat

Summary The pre- and postnatal development of the adrenal medulla was examined in the rat by immunohistochemistry and by assay of catecholamines. Immunohistochemistry involved the use of antibodies to noradrenaline (NA), adrenaline (A) and the biosynthesizing enzymes dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Adrenal glands were obtained from animals from the 16th day of gestation to the 7th postnatal day at daily intervals, and at the 14th postnatal day, and from adult rats. Tissues were fixed in ice-cold, 4% paraformaldehyde, buffered at pH 7.3. Cryostat sections (7 m) were stained with the indirect immunofluorescence technique. Adrenals from the same developmental stages were assayed for the presence of DA (dopamine), NA and A by ion-pair reversed-phase liquid chromatography with electrochemical detection.In adult adrenals the majority of the medullary cells (approximately 80%) were highly immunoreactive to A and moderately immunoreactive to NA. They also showed immunoreactivity to both DBH and PNMT, i.e., they are synthesizing and storing A. The remaining cell clusters were only stained by antibodies to DBH and NA (NA-synthesizing and -storing cells). These findings correlate well with the relative concentrations of A and NA as determined by assay.Three developmental phases could be distinguished. In the first phase, the 16th and 17th prenatal day, medullary cells were only immunoreactive to DBH and NA, and only very small amounts of A as compared to NA were found. During the second period, from the 18th prenatal day to 2 or 3 days after birth, all medullary cells were immunoreactive to DBH, NA, PNMT and A, and during this phase the adrenaline concentration increased daily and became the predominant amine on the 20th day of gestation. Adrenaline represented 75% of total catecholamine on the 1st to 3rd day after birth. The third phase started at the 2nd or 3rd postnatal day and was characterized by the presence of an increasing number of medullary cells solely immunoreactive to DBH and NA, hence synthesizing and storing NA. The remaining cells were immunoreactive to DBH, NA, PNMT and A. Postnatally, the relative concentration of A continued to rise reaching 79% by the 4th postnatal day. These results indicate that initially the adrenal medullary cells are synthesizing and storing almost exclusively NA. Probably, adrenaline synthesis begins at the 16th–17th day of gestation and the cells are then capable of synthesizing and storing both NA and A (mixed cell type) with A synthesis and storage rapidly becoming predominant. Finally, after birth, separate NA-synthesizing and -storing cell types are formed and the so-called A cells stored predominantly (probably >90%) adrenaline with a small proportion of noradrenaline.In the medullary blastema and in the sympathetic ganglia of prenatal animals two cell types, only immunoreactive to DBH and NA, were observed. Presumably, these cells represent developing sympathetic neurons and extra-adrenal chromaffin cells; the latter cell type occasionally invades the adrenal gland. Thus, prospective medullary cells are able to synthesize and store NA before they have made contact with the cortical blastema but A-synthesizing cells are found only within the adrenal gland.Low but significant amounts of DA were found in the adrenal before birth and during the first two postnatal weeks but in the adult animal this accounted for less than 0.1% of total catecholamine.Preliminary reports of this study were made to the American Association of Anatomists (Anat. Rec. 196; 196A, 1980), the Dutch Anatomical Society (Acta Morphol. Neerl. Scand. 19; 330, 1981, and the XIIIth Acta Endocrinologica Congress (Acta Endocrinol. 97: Suppl. 243, 285, 1981)     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.