Do opioid peptides modulate,at the level of the nerve endings,the release of neurohypophysial hormones?

Summary Rat neurointermediate lobes and neurohypophyses separated from the pars intermedia were stimulated in vitro in the presence of either D-Ala², D-Leu⁵-enkephalin (DADLE), a Leu-enkephalin stable analogue or FK 33-824 a Met-enkephalin stable analogue. Secretion of vasopressin (AVP) and oxytocin (OT) was produced by either a Ca²⁺-ionophore or with electrical stimulation or by K⁺-induced depolarization. These opioid peptides and their antagonist naloxone did not affect basal nor evoked hormone release. Furthermore, they did not affect the evoked calcium uptake induced with electrical stimulation. These findings were confirmed using a preparation of isolated neurosecretory nerve endings. Further, dopamine had no effect on the K⁺-induced AVP release although a crude extract of the pars intermedia abolished the electrically-evoked and reduced considerably the potassium-evoked AVP release. It is concluded that in the neurohypophysis neither Leu- and Met-enkephalin nor dopamine affect the secretion-coupling mechanism at the level of the neurosecretory nerve endings.     

Opiate binding differentially associated with oxytocin and vasopressin nerve endings from porcine neurohypophyses

Summary We examined opioid binding in fractions with disconnected nerve endings (secretosomes) which were prepared from porcine neurohypophyses by centrifugation in a discontinuous Percoll gradient. Specific (= displaceable) binding was observed with ³H-etorphine and with ³H-diprenorphine, two ligands with low selectivity for distinct opiate receptor sub-classes. No displaceable binding was found with the prototypic mu- and delta-ligands ³H-dihydromorphine and ³H-(D-Ala, D-Leu) enkephalin. Displacement of ³H-diprenorphine binding was almost absent with the selective mu- and delta-ligands morphiceptin and ICI-174864. Partial displacement occurred with the selective kappa-ligand U-50488 and with dynorphin (1–8). Binding of ³H-etorphine was stereo-specific. ³H-diprenorphine binding was saturable with a K_D between 2 and 4 nM. Maximum of opiate binding activity was detected in the fractions with accumulated secretosomes. By autoradiography specific ³H-diprenorphine binding is shown to be mainly associated with secretosomes. In imunocytochemical preparations an oxytocin antibody was immunoreactive in 85% of the secretosomes in the fraction with highest opiate binding. These fractions in radioimmunoassays exhibited the largest contents in oxytocin and low vasopressin levels. The data therefore suggest that in the porcine neurohypophysis opioid binding sites of the kappa-type occur in secretory endings presumably of the oxytocin type.     

Inflammation-induced increase in the density of neuropeptide-immunoreactive nerve endings in rat skeletal muscle

The density of substance P (SP)-, calcitonin gene-related peptide (CGRP)- and vasoactive intestinal polypeptide (VIP)-immunoreactive (ir) nerve endings was quantitatively evaluated in intact and inflamed gastrocnemius-soleus muscle of the rat. In persistently inflamed muscle (12 days after a single injection of Freund’s adjuvant into the muscle), the density of SP-ir fibres was significantly increased. CGRP- and VIP-ir fibres displayed an insignificant increase in density. The density of fibres ir for nerve growth factor (NGF) and for growth-associated protein 43 (GAP-43/B-50), a marker for axonal sprouting, regeneration and synaptic reorganisation, increased significantly in persistently inflamed muscle. The data are consistent with the established contribution of NGF on the expression of SP and GAP-43 in afferent neurones under the influence of a persistent inflammation. Received: 8 September 1997 / Accepted: 12 February 1998     

Pharmacologic characterization of opioid peptide release from chromaffin cell transplants using a brain slice superfusion method

A simple chamber and an inexpensive superfusion system for studying mammalian brain slices containing neural transplants is described. With this method, rat brain slices containing bovine chromaffin cell transplants can be maintained for several hours, allowing for the determination of neurochemical characteristics and pharmacologic responsiveness of the grafted cells. Using this technique, basal and nicotine-stimulated release of metenkephalin from rat periaqueductal gray slices containing bovine chromaffin cell transplants were measured. Results showed that met-enkephalin release can be increased by nicotinic stimulation in slices containing chromaffin cell, but not control implants, for at least 8 weeks postimplantation. Furthermore, this response was doserelated. These results are in good agreement with previous behavioral studies and provide corroborative evidence for the mechanism of pain reduction by the release of opioid peptides from chromaffin cell transplants in the periaqueductal gray. This study demonstrates that neurochemical and pharmacologic analyses of neural transplants using a superfused brain slice method can be a complementary approach in determining the underlying mechanisms of neural transplants in the central nervous system.     

Early osmoregulatory signals in the control of water intake and neurohypophyseal hormone secretion

Dehydrated dogs inhibit secretion of vasopressin (VP) within minutes after drinking water, before plasma osmolality (pOsm) diminishes. In recent studies, we found that water ingestion by rats similarly inhibits VP and oxytocin (OT) secretion rapidly, before pOsm is diluted. Adult male rats were infused with 1 M NaCl (2 ml/h iv) for 240 min to stimulate VP and OT secretion. After 220 min of infusion, rats were given water or isotonic saline (IS) to drink for 5 min, and blood samples were taken 5 and 15 min later. Plasma levels of VP (pVP) and OT (pOT) were much lower when rats ingested water instead of IS, even though rats drank comparable amounts of both fluids ( approximately 5.5 ml) and pOsm was not significantly affected in either case. In another study, rats were infused with 1 M NaCl (2 ml/h iv) for 120 min before receiving 4-ml gastric loads of either 0.5 M NaCl (HS) or IS; blood samples taken 25 min later showed that pVP and pOT were much higher when rats were given gastric loads of HS instead of IS, even though pOsm was not significantly altered. Comparable results were obtained when gastric loads of HS or IS were given to rats that had been deprived of drinking water overnight. Other dehydrated rats treated similarly but given access to drinking water consumed much more when they had been given gastric loads of HS instead of IS. Collectively, these and other findings suggest the importance of early signals, perhaps from hepatoportal osmoreceptors or Na(+) receptors, in the control of VP and OT secretion and water intake in rats.     

Lack of response in the release of oxytocin and vasopressin from isolated neurohypophyses to dopamine,met-enkephalin and leu-enkephalin

Summary We investigated the effects of dopamine, met-enkephalin and leu-enkephalin on basal and ouabain-stimulated release of oxytocin and vasopressin from isolated neurointermediate lobes. The present study revealed that neurohypophyseal hormone release was not affected by dopamine, neither from lobes of untreated rats nor from those of rats with dopamine-deficiency (pretreated with -methyl-p-tyrosine-methylester). Likewise, metoclopramide, a dopamine antagonist, was unable to alter the neurohypophyseal hormone release. Our results also indicate that the opioid peptides met-enkephalin and leu-enkephalin do not influence spontaneous or ouabain-stimulated oxytocin and vasopressin release, which is in accordance with our findings that naloxone under our experimental conditions is also ineffective.     

Nerve endings in the pulmonary trunk, ductus arteriosus and aorta of intact and decapitated pig fetuses

Summary Nerve fibres reactive to acetyl-thiocholine, and tissues showing catecholamine fluorescence were examined in the pulmonary trunk, ductus arteriosus and aorta of 28 pig fetuses between 31 and 113 days of gestation (term=114±1 days). Eight additional fetuses, which had been decapitated in utero at 40–43 days, were also studied at ages between 51 and 114 days of gestation. Spherical micro-networks of nervous tissue reactive to acetyl-thiocholine are present in the adventitia on the cranial aspect of the pulmonary trunk and ductus arteriosus, between the aorta and pulmonary trunk, and on the caudal aspects of the pulmonary trunk and the pulmonary arteries. These fibres invest spherical clusters of catecholamine containing cells which are well supplied with blood vessels. Nerve fibres which fluoresce are also found in association with these cells. Decapitation in utero does not appear to affect the distribution or morphology of these structures. The observations show that structures are present in the major arteries of the fetal pig which may act as sensory receptors, and that these structures are unaffected by chronic vagotomy of the fetus produced by decapitation early in gestation.     

Effects of the selective kappa-opioid agonist U50,488 upon the electrical activity of supraoptic neurones in morphine-tolerant and morphine-naive rats

Spontaneous electrical activity of oxytocin-secreting neurones in the rat supraoptic nucleus is depressed by the mu-opiate receptor agonist morphine, leading to a reduction in plasma oxytocin concentration. In the present experiments, the electrical activity of single neurones was recorded from the supraoptic nucleus of urethane-anaesthetized rats. For 5 days prior to the experiments the rats had received a continuous infusion of either morphine or vehicle into a lateral cerebral ventricle; this regimen of morphine treatment results in tolerance to, and dependence upon, morphine in the central mechanisms controlling oxytocin secretion. Intravenous injection of the specific kappa- opioid agonist U50,488 at low doses resulted in small but significant increases in the electrical discharge activity of some putative oxytocin neurones in both morphine-treated and morphine- naive rats. At higher doses, the kappa-agonist consistently inhibited almost all cells tested. Morphine-treated rats, despite showing tolerance to morphine itself, showed no cross-tolerance to the inhibitory actions of U50,488 upon the oxytocin system. In separate experiments both morphine and U50,488 were effective in inhibiting supraoptic neuronal activation evoked by stimulation of the region anterior and ventral to the third ventricle, and activation following systemic injection of cholecystokinin, suggesting that both opioids act upon the final common pathway — the oxytocin neurone itself.     

Membrane recapture after hormone release from nerve endings in the neural lobe of the rat pituitary gland

The magnocellular neurosecretory system of mammalia, which produces oxytocin and vasopressin and releases these hormones at a neurohaemal contact area in the neural lobe of the pituitary gland, provides a useful model for the study of release of granule-packaged material from both neurones and endocrine cells. Isolated neural lobes from rats were stimulated to release hormone by incubation for 15min in a depolarizing sodium-free medium containing 56 mM potassium ions. Controls were incubated in a sodium-free medium containing 5.6 mM potassium ions. The amounts of vasopressin and oxytocin released by depolarization together comprised ~83 mU. At the end of the stimulation the glands were fixed for electron microscopy and stereological analysis. Loss of neurosecretory granules from stimulated glands was restricted to the nerve endings. From knowledge of the amount of hormone stored in a single neurosecretory granule, the number of granules lost from the gland could be calculated to contain approximately the amount of hormone released. After stimulation, the nerve endings were unaltered in size or membrane area. There was also no change in the total microvesicle population of the endings, though the microvesicles were redistributed towards the basement membrane contact zone. The vacuole population of the endings was, however, increased three-fold after stimulation.We conclude that, after acute stimulation of hormone release from the neural lobe, neurosecretory granules are lost specifically from the nerve endings and that the excess membrane that results from their exocytosis is to be found primarily in vacuoles.     

Topography and ultrastructure of sensory nerve endings in the joint capsules of the Kowari (Dasyuroides byrnei), an Australian marsupial

Summary The present investigation is concerned with the topography and ultrastructure of sensory nerve endings in the joint capsules of the Kowari (Dasyuroides byrnei), an Australian marsupial. Material for light and electron microscopy was obtained from shoulder, elbow and knee joint capsules.On the basis of differences in the organization of the connective tissue belonging to the fibrous layer, 3 variants of capsule structure have been distinguished: a rigid, a flaccid and an intermediate type. Whilst the rigid type is characterized by dense connective tissue in the clearly demarcated fibrous layer, the flaccid type shows loose, irregularly arranged connective tissue in the fibrous layer which merges into the synovial layer of the joint capsule. the morphology of the intermediate type corresponds to an intermediate stage between the former two types.In the fibrous layer of the joint capsules three different types of sensory nerve endings were observed: free nerve endings, Ruffini corpuscles and lamellated corpuscles. The free nerve endings are supplied by myelinated afferent axons (1–2 m in diameter); the terminal thickenings of which are incompletely surrounded by a terminal Schwann cell. Ruffini corpuscles are present in three different varieties: 1. small corpuscles without a perineural capsule predominantly within the flaccid part of the capsule; 2. slightly larger corpuscles with an incomplete perineural capsule and 3. large corpuscles resembling Golgi tendon organs which predominantly occur in the rigid parts of the capsule. The afferent myelinated axons measure 2–4 m in diameter. The lamellated corpuscles show two variants: 1. small corpuscles with a 2 to 4-layered perineural capsule in the rigid parts of the joint capsules and 2. large corpuscles with two longitudinal clefts of the inner core in the flaccid parts. Both types are supplied by myelinated axons of 3–5 m in diameter. Thus, in the fibrous layer of the rigid type of joint capsules large Ruffini and small lamellated corpuscles predominate, whereas the fibrous layer of the flaccid type coincides with small Ruffini and large lamellated corpuscles. The present data, therefore, corroborate the concept that the morphology of mechanoreceptors depends upon the texture of the surrounding connective tissue.Supported by the Verein zur Förderung der Erforschung und Bekämpfung rheumatischer Krankheiten e.V. in Bad Bramstedt, and by the Deutsche Forschungsgemeinschaft (Ha 1194/2-1)     

The effects of calcium channel agonists and antagonists on the release of endogenous glutamate from cerebellar slices

The effects of compounds acting at the calcium channel on neurotransmitter release are equivocal. We report here the effects of the antagonists, verapamil, diltiazem and nifedipine; the agonists, bay K8644 and the calcium ionophore, A23187 on the release of endogenous glutamate from rat cerebellar slices. Of these compounds, only verapamil and diltiazem modified glutamate release and these were effective at relatively high concentrations (greater than 1 x 10(-5) M). It is suggested that the high-affinity binding sites found in neuronal tissue for the dihydropyridine-like compounds are not involved in neurotransmitter release.     

The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction

Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from large muscle fibers by means of patch clamp electrodes. Compared to usual extracellular recordings, better signal/noise ratio and temporal stability were achieved. In the range of extracellular calcium concentrations [Ca]₀ between 2.7 and 13.5 mmol/l (normal), the average amplitude of the EPSC increased more than proportional to [Ca]₀. The unit quantum current,C ₁, and the average release rate,m, were determined from EPSCs and also from spontaneous sEPSCs, using both Poisson and binomial statistics. The main effect of [Ca]₀ was onm: at different synaptic sitesm depended on the second to fourth power of [Ca]₀. In terms of binomial parameters, the release probabilityp is the [Ca]₀-dependent one. In addition, reduction of [Ca]₀ from 13.5 to 2.7 mmol/l decreased the unit quantumC ₁ consistently to 60%; simultaneously the rise and decay of EPSCs and sEPSCs were shortened by 10–20%. [Ca]₀ thus has strong presynaptic effects on the release probability, but in addition smaller ones on teh postsynaptic channel characteristics.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Effects of kainic acid lesions on calcium uptake and dopamine release in nerve endings isolated from rat striata

Rats were injected intrastriatally with kainic acid and the viability of dopaminergic terminals two days following the injection was determined by comparing voltage dependent calcium uptake and dopamine release in isolated nerve endings. Evoked dopamine release remained normal following the lesion, but the initial rate of potassium stimulated calcium entry decreased by approximately 1/3. These results suggest that the excitotoxic lesion caused by acute intrastriatal injection of kainic acid results in retention of functional dopaminergic terminals.     

Mu and kappa opioid receptor expression in the mediobasal hypothalamus and effectiveness of selective antagonists on prolactin release during lactation

Endogenous opioid peptides are involved in prolactin release during lactation, in part by decreasing tuberoinfundibular dopaminergic (TIDA) neuronal activity. Both mu (μ) and kappa (κ) opioid receptors have a role in the suckling-induced prolactin rise after 4–5 h up deprivation. The aim of this study was to investigate effects of μ opioid receptor antagonist, β-funaltrexamine (β-FNA), and κ opioid receptor antagonist, nor-binaltorphimine (nor-BNI), on prolactin secretion and TIDA neuronal activity in lactating rats after 18 h pup deprivation. After 4 h separation from pups, the suckling-induced prolactin rise was abolished by 16 μg nor-BNI and 5 μg β-FNA, coincident with increased dihydroxyphenylacetic acid (DOPAC):dopamine ratio in the stalk-median eminence (SME). However, after 18 h pups separation, these same doses of nor-BNI and β-FNA did not alter the prolactin surge or DOPAC:dopamine ratios in the SME. Higher doses of nor-BNI (32 μg) and β-FNA (10 μg) were required to inhibit suckling-induced prolactin secretion. β-FNA (10 μg) increased the DOPAC:dopamine ratio in the SME, whereas nor-BNI (32 μg) treatment had no effect. The μ and κ opioid receptor mRNA levels in the mediobasal hypothalamus were similar to suckled control rats after 4 h pup deprivation, but increased 1.4-fold after 18 h pup deprivation. These data support involvement of endogenous opioidergic systems in the suckling-induced prolactin rise after a prolonged (18 h) period of pup deprivation, as well as the shorter (4 h) pup deprivation period previously reported. Suppression of TIDA neuronal activity likely played a part in μ opioid receptor input to the suckling-induced prolactin rise after both 4 h and 18 h separation, whereas non-dopaminergic input was implicated with κ opioid receptors after 18 h pup deprivation. Increased μ and κ opioid receptors gene expression in the mediobasal hypothalamus may contribute to reduced effectiveness of opioid receptor antagonists to block suckling-induced prolactin release after 18 h pup deprivation.     

Role of superior laryngeal nerve and Fos staining following dehydration and rehydration in the rat

Immunohistochemistry for Fos was used to determine the role of the superior laryngeal nerve in conscious rats following water deprivation and rehydration. Adult male rats were subjected to either unilateral superior laryngeal nerve section (SLNX) or sham surgery. Two weeks later rats from each surgical group were water deprived for 48 h or water deprived for 46 h and given access to water for 2 h prior to perfusion. Controls were allowed ad libitum access to water. Brains were processed for Fos using a commercially available antibody. Changes in plasma osmolality and hematocrit were not significantly different between SLNX and sham following any of the treatments. Water intake in rats was not significantly affected by SLNX. In the supraoptic nucleus (SON) of sham rats, water deprivation significantly increased Fos staining while water intake following dehydration prevented this increase. Water deprivation significantly increased Fos staining in the SON of SLNX rats. Following water intake after 46 h water deprivation in SLNX rats, Fos staining in the ipsilateral SON was significantly greater than the contralateral SON and significantly lower than 48 h water deprivation. In the nucleus of the solitary tract (NTS) of sham rats, both water deprivation and water intake produced significant increases in Fos staining bilaterally compared to euhydrated controls. In SLNX rats, water deprivation significantly increased Fos in both ipsilateral and contralateral NTS that was not different from sham rats. SLNX significantly decreased Fos staining in the ipsilateral NTS of rats given access to water after dehydration compared to the corresponding sham treated rats. Fos staining was not affected in the contralateral NTS of SLNX rats given access to water after dehydration. This suggests that the superior laryngeal nerve contributes to changes in Fos staining in the NTS and SON following water intake in dehydrated rats.     

Afferent nerve endings in the tracheal muscle of guinea-pigs and rats

Summary The trachea of guinea-pigs was stained as a whole-mount preparation with the zinc iodide-osmium technique. A distinct class of nerve endings was observed associated with the tracheal muscle. The endings, issued from myelinated fibres of the vagus nerve via the recurrent laryngeal nerve, are distributed on either side of the midline and ventral to the tips of cartilages. They are interpreted as afferent nerve endings that may correspond to slow adapting stretch receptors identified by physiological studies. Each nerve contributes predominantly, but not exclusively, to the receptors of the ipsilateral side. There are 120–180 receptors along the full length of the guinea-pig trachea, their density being higher at the cranial end. The receptors are variable in size and structural complexity, and, to some extent, also in spatial orientation, but distinct subtypes are not recognizable. Receptors of similar morphology and distribution are found also in the rat trachea. The receptors can also be visualized with a cytochrome oxidase method for nerve endings, but they do not stain with immunohistochemistry for the neuropeptides substance P, calcitonin gene-related peptide, vasointestinal polypeptide and neurotensin.     

Characterization of ouabain-induced noradrenaline and acetylcholine release from in situ cardiac autonomic nerve endings

Aim: Although ouabain modulates autonomic nerve ending function, it is uncertain whether ouabain‐induced releasing mechanism differs between in vivo sympathetic and parasympathetic nerve endings. Using cardiac dialysis, we examined how ouabain induces neurotransmitter release from autonomic nerve ending. Methods: Dialysis probe was implanted in left ventricle, and dialysate noradrenaline (NA) or acetylcholine (ACh) levels in the anaesthetized cats were measured as indices of neurotransmitter release from post‐ganglionic autonomic nerve endings. Results: Locally applied ouabain (100 μm ) increased in dialysate NA or ACh levels. The ouabain‐induced increases in NA levels remained unaffected by cardiac sympathetic denervation and tetrodotoxin (Na⁺ channel blocker, TTX), but the ouabain‐induced increases in ACh levels were attenuated by TTX. The ouabain‐induced increases in NA levels were suppressed by pretreatment with desipramine (NA transport blocker) and augmented by reserpine (vesicle NA transport blocker). In contrast, the ouabain‐induced increases in ACh levels remained unaffected by pretreatment with hemicholinium‐3 (choline transport blocker) but suppressed by vesamicol (vesicle ACh transport blocker). The ouabain‐induced increases in NA levels were suppressed by pretreatment with ω‐conotoxin GVIA (N‐type Ca²⁺ channel blocker), verapamil (L‐type Ca²⁺ channel blocker) and TMB‐8 (intracellular Ca²⁺ antagonist). The ouabain‐induced increases in ACh levels were suppressed by pretreatment with ω‐conotoxin MVIIC (P/Q‐type Ca²⁺ channel blocker), and TMB‐8. Conclusions: Ouabain‐induced NA release is attributable to the mechanisms of regional exocytosis and/or carrier‐mediated outward transport of NA, from stored NA vesicle and/or axoplasma, respectively, while the ouabain‐induced ACh release is attributable to the mechanism of exocytosis, which is triggered by regional depolarization. At both sympathetic and parasympathetic nerve endings, the regional exocytosis is because of opening of calcium channels and intracellular calcium mobilization.     

A comparison of the effect of calcium channel ligands and GABAB agonists and antagonists on transmitter release and somatic calcium channel currents in cultured neurons

Glutamate release has been examined from cultured cerebellar granule neurons in the rat using the technique of prelabelling the releasable pool of glutamate with [3H]glutamine. Glutamate release was stimulated in control neurons by 2-min incubation with 50 mM K+, or in neurons continuously depolarized in Ca2(+)-free 50 mM K+ medium, by 2-min incubation with medium containing 5 mM Ca2+. The ability of the Ca2(+)-channel agonist (+)-202-791 to increase the stimulated release of [3H]glutamate was approximately doubled in the depolarized condition. The antagonist enantiomer (-)-202-791 produced a small inhibition of K(+)-stimulated release, whereas (-)-202-791 completely inhibited Ca2(+)-stimulated release from depolarized neurons at concentrations greater than 10 nM. (-)-Baclofen (100 microM) inhibited transmitter release similarly (25-30%) under the two conditions. Calcium-channel currents were recorded from cultured dorsal root ganglion neurons under control conditions at a holding potential of -80 mV, or in neurons depolarized to -30 mV. (-)-202-791 produced a greater effect at -30 than at -80 mV although even at -30 mV the inhibition was slow in onset and incomplete. (-)-Baclofen (100 microM) inhibited the amplitude of the calcium-channel current at both holding potentials by 30-50%, although it did not clearly slow activation of the current at the depolarized holding potential. The GABAB receptors associated with inhibition of glutamate release and of calcium-channel currents were both markedly blocked by phaclofen but not by 2-OH-saclofen. These findings suggest that the GABAB receptor associated with inhibitory modulation of transmitter release, and that associated with inhibition of calcium-channel currents show pharmacological similarities, and are able to exert their action even at levels of steady depolarization at which most N-type channels should be inactivated.     

Differential antiepileptic effects of the organic calcium antagonists verapamil and flunarizine in neurons of organotypic neocortical explants from newborn rats

Summary Effects of the organic calcium antagonists verapamil and flunarizine on pentylenetetrazol induced paroxysmal depolarizations were tested in organotypic neocortical explants taken from neonatal rats. In these in vitro experiments the papaverin derivative verapamil depressed, and finally abolished, epileptic discharges in all cases. The piperazine derivative flunarizine, however, which is known to suppress epileptic discharges in hippocampal CA3 neurons (Bingmann and Speckmann 1986), showed no significant antiepileptic effects in the explanted neocortical neurons. Thus, the present findings may indicate that the suppressive action of flunarizine on the generation of paroxysmal depolarizations is restricted to distinct populations of neurons.