Biochemical characterization of various populations of isolated bovine adrenal chromaffin cells

Various populations of bovine adrenal chromaffin cells were isolated first by successive digestions with collagenase (original cell preparation) followed by sedimentation through a stepwise bovine serum albumin gradient (cell layers I, II and III). At the fine structural level, the ratios between the number of adrenaline-cells and noradrenaline-cells were 1.9 in the original cell preparation and 0.9, 2.0 and 4.6 in cell layers I, II and III, respectively. The catecholamine content of each cell population was also measured by spectrofluorometry. The original cell preparation contained 20.1 and 12.2 nmol per 10⁶ cells of adrenaline and noradrenaline, respectively. Each cell layer had similar total amount of catecholamines (from 38.3 to 40 nmol per 10⁶ cells) but their adrenaline/noradrenaline content ratios varied from 0.6 in cell layer 1 to 1.3 and 3.3 in cell layers II and III, respectively. Incubation of the cells in the presence of acetylcholine (50 μM) induced a release of catecholamines which was proportional to the cell content of each amine. However, the percentage of total cell content released was much higher in cell layer I (20%) than in cell layers II (8%) and III (5%). Finally, each cell population was also analyzed for its ability to respond to a muscarinic stimulation of cyclic GMP level and to bind [³H]etorphine, a highly potent opiate agonist. Acetylcholine induced 3.15-, 2.15- and 4.21-fold increases in the levels of cyclic GMP in the original cell preparation, cell layers II and III, respectively, but not in cell layer I. Conversely, the high affinity opiate binding site for [³H]etorphine was almost exclusively confined to cell layer III (B_max of 28.4 fmol per 10⁶ cells as compared with 2.8–7.5 fmol in the other cell preparations). These results indicate that bovine adrenal chromaffin cells can be separated according to their content in adrenaline and noradrenaline and their response to nicotinic, muscarinic and opiate stimuli.     

Comparison of leucine enkephalin and adrenocorticotrophin effects on adrenal function in fetal and adult sheep

At Day 120-125 of gestation equimolar amounts of ACTH and leu-enkephalin injected in vivo provoked similar rises in plasma cortisol concentrations in chronically catheterized fetuses. There was no concomitant change in plasma DHEA concentrations, or in maternal cortisol concentrations. At term (Days 135-140) 2 out of 5 animals responded similarly to both leu-enkephalin and ACTH injections with a rise in plasma cortisol concentrations, but the other 3 animals, in which basal cortisol concentrations had already risen, showed no response to either agonist. In adult sheep, ACTH provoked a significant increase in the plasma cortisol concentrations, but equimolar amounts of leu-enkephalin were without effect. There was a significant output of cortisol in response to ACTH administration by collagenase-dispersed adrenal cells from term sheep fetuses in vitro. Leu-enkephalin had no effect on cortisol output from dispersed adrenal cells when added by itself, or with ACTH. We conclude that leu-enkephalin is able to function as a stimulator of pituitary-adrenal function during fetal life. The lack of effect of leu-enkephalin on adrenal cells implies that its action is exerted not directly at the adrenal gland, but indirectly at the level of the hypothalamus or pituitary through stimulation of the release of other corticotrophic substances.     

Location of immunoreactive leucine enkephalin in the digestive gland of the Mollusca Mizuhopecten yessoensis (Jay)

Distribution of leu-enkephalin-like peptides in the digestive gland of the Japanese scallop has been investigated by means of the indirect immunohistochemical method. The immunoreactive leu-enkephalin is revealed in supranuclear vacuoles of separate large secretory cells. These cells are situated in epithelium of the digestive tubules along the gland periphery. Their number is from 1-3 up to 5-6 per tubue section. The basal part of the cell and small apical vacuoles do not contain any products of the reaction. It is possible that in the bivalve molluscs, as in vertebrates, the leu-enkephalin-immunoreactive elements participate in regulation of digestion.     

Mass spectrometric identification of various molecular forms of dynorphin in bovine adrenal medulla

Deliberate miscompartmentalization of liver outer mitochondrial membrane (OMM) proteins and liver mitochondrial proteins has been achieved by polyethylene-glycol mediated OMM vesicle-hepatocyte or mitochondrial-hepatocyte fusion. Reductively methylated OMM and mitochondrial proteins (³H) are destroyed at rates remarkably similar to those for OMM ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 60–70 h) or mitochondrial proteins ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 84–104 h) in liver $\text{in vivo}$ when studied over 4–5 days in hepatocyte monolayers cultured in conditions giving stabilized endogenous protein catabolic rates mimicking endogenous $\text{in vivo}$ rates. Destruction of transplanted OMM proteins is partially sensitive to chloroquine, supporting some lysosomally mediated autophagic destruction of long-lived transplanted OMM proteins in hepatocyte monolayers.     

Presence of immunoreactive dynorphin in human cerebrospinal fluid

Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.     

Multiple forms of immunoreactive dynorphin in human pituitary and pheochromocytoma

Multiple forms of immunoreactive dynorphin (I-Dy) in human pituitary and pheochromocytoma were examined utilizing gel filtration and high performance liquid chromatography (HPLC). Gel filtration of I-Dy from these tissues revealed the major component in the position of Dy(1-17) and other minor components with large molecular weight forms. HPLC profile of this major component from gel filtration showed a large peak corresponding to the position of Dy(1-17) and small peaks corresponding to the positions of Dy (1-13), (1-12) and other unknown peptides. These results strongly suggest the presence of Dy(1-17) as the major component, and Dy (1-13), (1-12) or other unknown peptides as the minor components in these human tissues.     

Multiple forms of immunoreactive dynorphin in rat pituitary and brain

Multiple forms of immunoreactive dynorphin (I-dynorphin) in rat anterior pituitary (AP), intermediate-posterior pituitary (IP) and medial basal hypothalamus (MBH) were examined. Three I-dynorphin peaks were observed on gel filtration. The first peak (big form) was eluted near the position of beta-LPH, and was predominant in AP. The second peak (middle form) was eluted near the position of ACTH. The third peak (small form) was eluted at the position of dynorphin (1-13), ane was predominant in IP and MBH. The heterogeneity of this small form was examined by ion exchange chromatography and reverse phase high performance liquified chromatography (HPLC). I-dynorphin peaks were observed at the positions of dynorphin(1-17), (1-13), (1-11), (1-10) and other peptides. These results strongly suggest (i) the presence of dynorphin(1-17), (1-13), (1-11) and (1-10) in rat IP, (ii) dynorphin(1-11) and (1-10) as the major components in this small form, (iii) the difference of I-dynorphin processing in AP, IP and MBH.     

Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.     

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin.

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.     

Pupillary effects of leucine and methionine enkephalin in rats after intraperitoneal administration

Changes in pupil size after peripheral administration of met-enkephalin, leu-enkephalin, or morphine were studied in the rat. With a simple pupillographic technique, the pupil diameter of male, S.D. rats (250–300 g) was measured by a series of photographs taken every 60 sec for at least 45 min after the last drug injection. Morphine (8 mg/kg, SC) caused mydriasis characterized by rapid and marked fluctuations of pupil size. Mydriasis also occurred after leu-enkephalin (5 and 10 mg/kg, IP) and met-enkephalin (20 mg/kg, IP). Both peptides induced morphine-like fluctuations. When given 15 min after morphine, leu-enkephalin (5 and 10 mg/kg) increased the mydriatic effect of morphine from 172 percent of control to 224 and 272 percent, respectively. Met-enkephalin (20 mg/kg, but not 10 mg/kg) also enhanced the mydriatic response of morphine, to 244 percent of control. These interactions appear to represent simple addition rather than potentiation. The effects of both peptides were reversed by naloxone (1 mg/kg, SC), suggesting an opiate receptor interaction for the pupillary effects of the enkephalins. The rat pupil thus provides one of the few in vivo models permitting quantification of enkephalin action after parenteral administration.     

Light and electron-microscopic study of leucine enkephalin immunoreactivity in the cat claustrum

The claustrum is a complex telencephalic structure owing to its reciprocal connectivity with most—if not all—cortical areas. However, there is a paucity of data in the literature concerning its histochemical components, including opioid peptide neurotransmitters. The aim of the present study was to examine the morphology, distribution and ultrastructure of leucine-enkephalin-immunoreactive (Leu-enk-ir) neurons and fibers in the dorsal claustrum (DC) of the cat. Seven healthy, adult male and female cats were used in our study. All animals received humane care. They were irreversibly anesthetized and transcardially perfused with fixative. Brains were removed, postfixed, blocked and sectioned. Sections were incubated with polyclonal anti-Leu-enk antibodies using the Avidin–Biotin–Peroxidase Complex method. Leu-enk-ir neurons and fibers were distributed throughout the DC. Some of the neurons were lightly-stained, while others were darkly-stained. Light-microscopically, they varied in shape: oval, fusiform, multipolar and irregular. With regard to size, they were categorized as small (15?μm or less in diameter), medium (16–20?μm in diameter) and large (21?μm or more in diameter). No specific pattern of regional distribution was found. On the electron microscope level, immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of Leu-enk-ir neurons differ in their ultrastructural features, including two types of synaptic boutons. No gender-specific features were observed. In conclusion, it is our hope that our study will serve to contribute to a better understanding of the functional neuroanatomy of the DC in the cat, and that it can be extrapolated and applied to other mammals, including humans.     

Corticosteroidogenesis modulation by beta-endorphin and dynorphin1-17 in isolated rat adrenocortical cells

Two opioid peptides, beta-endorphin and dynorphin1-17 were bioassayed with isolated rat adrenocortical cells. beta-Endorphin increases basal production of corticosterone as well as the adrenal responsiveness to low doses of ACTH, these effects being partially reversed by naloxone. Dynorphin1-17, without affecting basal corticosterone synthesis, increases adrenocortical responsiveness to ACTH; naloxone does not influence this effect. It is suggested that peripheral opioid peptides may participate in the maintenance of the homeostatic balance by modulating adrenal corticosteroidogenesis.     

Co-existence of enkephalin and adrenalin in the frog adrenal gland

Summary By means of immunohistochemistry combined with formaldehyde-induced fluorescence microscopy, metenkephalin-like immunoreactivity was found to occur in the adrenalin-cells of frog adrenal glands. Immunoelectron microscopy demonstrated that the immunoreactive material is confined to the secretory granules. These findings suggest the concomitant release of enkephalin and adrenalin by exocytosis.     

Inhibition of cortisol production in isolated guinea-pig adrenal cells

Cortisol, added to 1 ml incubation medium containing 3-4 X 10(5) isolated guinea-pig adrenal cells, provoked a decrease in basal and ACTH (250 pg)-stimulated cortisol production, in correlation with the amounts used (50 ng-2,000 ng). A decrease in aldosterone production could be seen when cortisol concentrations reached or exceeded 1,000 ng/ml. There were no variations in either androgens (delta 4-androstenedione, dehydropiandrosterone) or 17-hydroxyprogesterone. Only 11-deoxycortisol was slightly increased. Using increasing concentrations of ACTH (50-250 pg), both in the absence and in the presence of 1,000 ng cortisol, it was noted that the inhibition induced by cortisol was of a competitive type and could be overcome by ACTH. This decrease in cortisol was concomitant with an increase in 11-deoxycortisol. Neither corticosterone nor dexamethasone reduced cortisol production. In addition, it was shown that the conversion of tritiated 11-deoxycortisol to radioactive cortisol increased significantly under the influence of 250 pg ACTH (mean relative variation of 21.7% +/- 7.7 (SEM), n = 6, P less than 0.05); but decreased significantly under the combined effect of 1,000 ng exogenous cortisol and the same dose of ACTH: (mean relative variation of 4.3% +/- 1 (SEM), n = 8, P less than 0.005). There is therefore reason to believe that the concentrations of cortisol at the adrenal level modulate the stimulation induced by ACTH and that this self-adjustment forms part of the control mechanisms involved in corticosteroidogenesis.     

ACTH regulation of cholesterol movement in isolated adrenal cells

Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The in vitro release of immunoreactive dynorphin and alpha-neoendorphin from the perfused rat duodenum

The release of immunoreactive (ir) dynorphin (DYN) and alpha-neoendorphin (ir-ANEO) from the isolated perfused rat duodenum was demonstrated using specific radioimmunoassays (RIAs). Depolarization of the tissue by increasing the potassium (K+) concentration up to 108 mM enhanced the release of ir-DYN and ir-ANEO in Ca2+-dependent manner. Administration of the serotonin-releasing agent fenfluramine (10(-6) M) and the serotonin receptor agonist m-chlorophenylpiperazine (m-CPP, 10(-6) M) stimulated the release of ir-DYN and ir-ANEO from the duodenum. A subsequent study revealed that serotonin (5-HT, 10(-6)-10(-4) M) induced a dose-dependent increase in the release of ir-DYN and ir-ANEO from the duodenum. The effect of 5-HT on the release of ir-DYN and ir-ANEO from the duodenum was antagonized by 5-HT antagonist cyproheptadine (10(-6) M). The presence of dynorphin and the related peptides in the gastrointestinal tract (GIT) and their release from the duodenum in vitro indicate that these peptides may act as transmitters involved in some GIT functions. Furthermore, our results suggest that at least part of 5-HT effects on the GIT may be mediated by the release of dynorphin and the related peptides.     

Differential regulation of the brain and gut immunoreactive dynorphin by the serotonin system

Administration of drugs such as fenfluramine, 20-40 mg/kg, and m-chlorophenylpiperazine (m-CPP), 2.5-5 mg/kg, which release serotonin or activate postsynaptic serotonin receptors, respectively, induced a dramatic decrease in the duodenal content of immunoreactive dynorphin (ir-DYN). The effect was antagonized by cyproheptadine, 1 mg/kg. Similarly, acute administration of the specific serotonin reuptake blockers fluvoxamine, 15 mg/kg, or femoxetine, 10 mg/kg, and 5-hydroxytryptophan (5-HTP), 40-160 mg/kg, evoked a marked decrease in the duodenal content of ir-DYN. A combined administration of fluvoxamine or femoxetine and 5-HTP failed to potentiate the effect of individual treatment. Only a higher dose of fenfluramine, 40 mg/kg, increased the ir-DYN content in the hypothalamus. These results suggest that the brain and gut ir-DYN is independently regulated by the serotonin system and that a serotonin mechanism might stimulate release of the gut dynorphin content.     

Pressor effects of systemic administration of methionine and leucine enkephalin in the conscious rat

Experiments were designed using conscious Sprague-Dawley rats to determine the blood pressure (BP) and heart rate (HR) responses to intravenous doses of (1) the adrenal catecholamines noradrenaline (NA) and adrenaline (A), (2) adrenal pentapeptides methionine enkephalin (ME) and leucine enkephalin (LE), (3) combination (i.v.) injections of both ME or LE with NA or A that modulate the hemodynamic responses when the adrenal catecholamines were given alone, and (4) the possible receptor mechanisms mediating the resultant BP and HR response to i.v. pentapeptide administration. NA (0.48 and 2.4 nmol) and A (0.3 and 1.5 nmol) given i.v. evoked potent, dose-related pressor responses associated with reflex bradycardia. ME and LE (1.6 - 48 nmol) elicited transient (10-20 s) increases in mean arterial pressure (MAP), which was associated either with no change in mean heart rate (MHR), such as ME, or with slight bradycardia (i.e., LE). Combining ME or LE (16 nmol) with NA (2.4 nmol) or A (0.3 or 1.5 nmol) did not change MAP and MHR from when these respective doses of NA or A were given alone. However, 16 nmol of ME or LE with a low dose of NA (0.48 nmol) increased the pressor response compared with NA (0.48 nmol) given alone. Other experiments whereby specific receptor blockers (naloxone, diprenorphine, atropine, propranolol, phentolamine or guanethidine) were given i.v. 5 min before subsequent i.v. administration of LE or ME (16 nmol) indicated that only phentolamine or guanethidine could completely suppress the pressor responses of LE and ME. Naloxone and diprenorphine pretreatment attenuated the pressor response of LE but did not affect the BP response to ME.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.