Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

Structural-functional relations of short analogs of enkephalin

The similarity of action of narcotic analgesics and opioid peptides is due to activation of a common opiate receptor as the primary step in initiating biochemical chains responsible for diverse morphine-like effects. The most widely used assays for opioid and analgesic activities are presented and evaluated. Approximately 180 short enkephalin analogues (di-, tri- and tetrapeptides), described in the literature, are systematized and their opioid and systemic analgesic activities compared with methionine-enkephalin and morphine as the reference compounds, respectively. The analysis of structure-opioid activity relationships among these enkephalin analogues substantiates the hypothesis that only a limited N-terminal region of the peptide molecule is essential for the binding of opioid peptides to the subclass of opiate receptors interacting with narcotic alkaloids (mu-receptors). An attempt has been made to identify minimal structural elements responsible for the mu-receptor activation. Shortening of the molecule and modification of its elements are examined with regard to the mu- and delta-receptor selectivity. It is emphasized that the aromatic structure of the C-terminal region of the peptide is not obligatory for the mu-receptor binding. Modifications of short enkephalin analogues which might confer them antagonistic properties are reviewed. The correlation between the ability of short enkephalin analogues to interact with mu-receptors and their antinociceptive properties is discussed along with some structural features pertinent to the analgesic effect after systemic administration of peptides. On the basis of this analysis, peptides containing no more than four amino acids are considered as the most probable morphine-like analgesics.     

Narcotic antagonists attenuate drinking induced by water deprivation in a primate

Pure narcotic antagonists such as naloxone and naltrexone have consistently been shown to attenuate drinking in the rat after periods of water deprivation. One objective of this study was to extend observations to a primate species, the squirrel monkey. Whereas naloxone and naltrexone have a greater relative affinity for opiate receptors preferentially binding morphine and other opiate alkaloids than for those with high affinity for the endogenous opioid peptides, diprenorphine, another pure opiate antagonist, binds with equally high affinity to both receptor subtypes. Therefore, a second objective was to determine the actions of diprenorphine on drinking in water-deprived rats and squirrel monkeys and to compare the effects of this drug to those of naloxone and naltrexone. All three narcotic antagonists suppressed water consumption of monkeys and rats deprived of water for 18 and 24 hr, respectively. Diprenorphine was the most potent compound tested in both species, producing significant reductions in water consumption of monkeys and rats at systemic doses as low as 0.01 and 0.1 mg/kg respectively. Moreover, diprenorphine was the longest acting of the three drugs in the monkey. These results demonstrate that the narcotic antagonists attenuate drinking in primates as well as in rodents and support the hypothesis that these drugs reduce water intake by interrupting the activity of endogenous opioid pathways mediating drinking behavior.     

Effect of myelopeptides on physiological and pathological pain

The action of bone marrow low-molecular peptides (myelopeptides) was studied in the models of physiologic and pathologic pain. Myelopeptides were demonstrated to have a pronounced analgetic effect: they increased the latent period of the rats' response in the hot plate test (physiologic pain) and suppressed severe spinal pain syndrome induced by the generator of pathologically enhanced excitation in the dorsal horn of the spinal cord (pathologic pain). In the experiments with naloxone (an opiate receptor blocker) the data on the opiate properties of myelopeptides were further substantiated. The analgetic effect of myelopeptides can be compared to that of morphine and promedol. Myelopeptides even in considerable doses did not have the side effects characteristic of the majority of opiate analgesics. Therefore, they may be recommended for clinical trials.     

Effect of bone marrow mediator myelopeptides on the summation-threshold index and behavioral reactions of rats

Transmitter peptides having immunostimulant and endorphine-like properties were isolated from supernatant of medullary cell culture. Bioregulatory peptides were called myelopeptides. Myelopeptides provoked changes of the summation-threshold index in rats, which augmented in time. These changes pointed to the realization of the analgetic effect of myelopeptides via the spinal-stem structures of the central nervous system. Having a remarkable analgesic effect myelopeptides administered in the doses tested did not produce any action on the behavioral responses. The latter circumstance makes them differ from narcotic analgesics and known endorphines.     

Peptide dose, affinity, and time of differentiation can contribute to the Th1/Th2 cytokine balance.

Opposing viewpoints exist regarding how Ag dose and affinity modulate Th1/Th2 differentiation, with data suggesting that both high and low level stimulation favors Th2 responses. With transgenic T cells bearing a single TCR, we present novel data, using peptides differing in affinity for the TCR, that show that the time period of differentiation can determine whether Th1 or Th2 responses predominate as the level of initial stimulation is altered. Over the short term, IFN-gamma-producing cells were induced by lower levels of stimulation than IL-4-producing cells, although optimal induction of both was seen with the same high level of stimulation. Over the long term, however, high doses of high affinity peptides led selectively to IFN-gamma-secreting cells, whereas IL-4- and IL-5-secreting cells predominated with lower levels of initial signaling, brought about by moderate doses of high affinity peptides. In contrast, too low a level of stimulation at the naive T cell stage, with low affinity peptides at any concentration, promoted only IL-2-secreting effectors or was not sufficient for long term T cell survival. These results demonstrate that the level of signaling achieved through the TCR is intimately associated with the induction of distinct cytokine-secreting T cells. We show that dose, affinity, time over which differentiation occurs, and initial production of IL-4 and IFN-gamma all can contribute to which T cell subset will predominate. Furthermore, these data reconcile the two opposing views on the effects of dose and affinity and provide a unifying model of Th1/Th2 differentiation based on strength of signaling and length of response.     

Analysis of the effect of increasing electrodermal stimulation on the somatovegetative reactions evoked by stimulation of the ventromedial hypothalamus

The influence of increasing electrodermal stimulation (EDS) on the dynamics of the somatovegetative reactions evoked by electrical stimulation of negative emotiogenic regions of the hypothalamus was studied. EDS produced a blocking effect on the somatovegetative reactions evoked by stimulation of the ventromedial hypothalamus (VMH). Pretreatment with naloxone prevented the effects of EDS. Injection of beta-endorphine in a dose of 10 micrograms to the lateral ventricules of the animal brain also blocked the somatovegetative reactions during VMH stimulation. Injection of beta-endorphine in doses of 50-100 micrograms enhanced and prolonged the somatovegetative reactions evoked by VMH stimulation. Elevated arterial blood pressure, pronounced bradycardia, extrasystoles, muscle tremor, and pathologic respiration were recorded. These disorders were completely reversed by EDS. It is assumed that both opiate peptides and their receptors are involved in the mechanism of the somatovegetative reactions evoked by VMH stimulation, experiencing the influence of EDS.     

Inhibition of ganglionic transmission by narcotic analgesics; an effect antagonized by naloxone, nalorphine and calcium

Methadone, azidomorphine, oxycodone and fentanyl inhibit synaptic transmission in isolated sympathetic ganglia of the frog and rat, just as did morphine and pethidine in our previous investigations. This inhibitory effect can be antagonized not only by naloxone and nalorphine but also by increasing calcium concentration of the perfusion fluid of the ganglia. The inhibitory effect on transmission of narcotic analgesics takes place on specific opiate receptors of the peripheral ganglia.     

Stereospecific binding of narcotics to brain cerebrosides

Cerebrosides were shown to bind etorphine and naloxone stereo-specifically with high affinity. The relative potency of several narcotic analgesics in preventing the binding of etorphine and naloxone to cerebrosides correlated well with their reported intraventricular analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Explanations for the apparent proteo-like behavior of the opiate receptor are provided.     

Evidence that opiate receptors mediate suppression of hypertonic saline-induced drinking in the mouse by narcotic antagonists

The effects of naloxone, its $\text{dextro}$-stereisomer, and five other narcotic antagonists were determined on water intake induced by intracellular dehydration in the mouse. The intraperitoneal administration of a 2M sodium chloride solution served as the model for intracellular dehydration. $\text{1}$-Naloxone (0.01-10 mg/kg) reduced drinking in a dose-dependent fashion with an ED₅₀ of 0.55 mg/kg. In contrast, $\text{d}$-naloxone failed to suppress water consumption at doses up to 10 mg/kg. The other narcotic antagonists tested --- naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine --- also produced dose-dependent decreases in water consumption. The order of potency of these narcotic antagonists in suppressing water intake was highly correlated with their orders of potency in other procedures involving the opiate receptor. The stereoselectivity and order of potency suggest that the suppressant effects of the narcotic antagonists on drinking induced by hypertonic saline administration in the mouse are mediated through an opiate receptor-dependent mechanism.     

Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction $\text{in vitro}$.     

A pool of central memory-like CD4 T cells contains effector memory precursors

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.     

MIF-1 and Tyr-MIF-1 augment GABA-stimulated benzodiazepine receptor binding

Behavioral evidence in laboratory animals and human beings indicates possible links between the endogenous opiate and gamma-aminobutyric acid (GABA)-benzodiazepine receptor systems, especially with regard to antagonistic properties. To assess possible interactions between endogenous opiate antagonists and benzodiazepine receptor binding, we evaluated the effects of the peptides MIF-1 and Tyr-MIF-1 on benzodiazepine receptor binding in mouse brain membranes. Neither peptide affected receptor binding in cortex over a broad dose range, but both peptides significantly augmented GABA-stimulated benzodiazepine receptor binding at GABA concentrations of 10(-8) and 10(-7) M. Rosenthal-Scatchard analysis indicated that the increase in binding was largely due to increased apparent affinity. Both peptides augmented GABA-enhanced binding at low doses (MIF-1 10(-11) M, Tyr-MIF-1 10(-13) M) with decreased effects at higher doses. In cerebellum and brainstem, MIF-1 tended to enhance GABA-stimulated binding but Tyr-MIF-1 was inactive. These results indicate benzodiazepine-opiate and benzodiazepine-peptide interactions.     

Narcotic antagonist-induced hypotension in the spontaneously hypertensive rat

Intravenous naloxone or naltrexone produced transient, dose-related reductions in the mean arterial pressure (MAP) and heart rate (HR) of urethane-anesthetized spontaneously hypertensive rats (SHRs). Yet these same doses of narcotic antagonists reduced HR but not MAP of normotensive Wistar-Kyoto rats (WKYs). Such effects were not observed upon administration to SHRs of increasing doses of methylnaltrexone, which possesses no central activity. (+)-Naloxone, which does not block opiate receptors, reduced HR but not MAP of both SHRs and WKYs. These findings indicate that SHRs and WKYs differ in their MAP and HR responses to narcotic antagonists. The high doses required for effect plus the brevity of the responses suggest that these drug effects are perhaps not mu-opiate receptor-mediated; however, the methylnaltrexone and (+)-naloxone findings clearly implicate a central specificity of action. We conclude that narcotic antagonist-induced changes in MAP and HR in SHRs are possibly specific and central in origin yet not mediated by mu-opiate receptors.     

Morphine-like ligand for opiate receptors in human CSF.

Human CSF was fractionated chromatographically and tested for affinity to opiate receptors in synaptic plasma membrane preparations from rat brain. A unique fraction of an apparent molecular weight of 1000 to 1200 dalton, probably identical with that previously found by us in brain extracts, showed affinity. Since the active factor behaved similar to morphinomimetic analgesics in its interaction with the receptors and not like the narcotic antagonist naltrexone, it is termed MLF (morphine-like factor). The level of MLF varied between different individuals and was apparently lower in patients with trigeminal neuralgia than in other patients.     

Targeting peroxynitrite driven nitroxidative stress with synzymes: A novel therapeutic approach in chronic pain management

Morphine sulfate and other opiate/narcotic analgesics are the most effective treatments for acute and chronic severe pain. However, their clinical utility is often hampered by the development of analgesic tolerance. This complex pathophysiological cycle contributes significantly to decreased quality of life in the growing population of subjects with chronic pain due to oversedation, reduced physical activity, respiratory depression, constipation, potential for addiction, and other side-effects. Accordingly, there is growing interest in new approaches that would maintain opiate efficacy during repetitive dosing without engendering tolerance or unacceptable side-effects. Considerable evidence implicates nitroxidative stress in the development of pain of several etiologies and importantly in opiate antinociceptive tolerance, caused by the presence of superoxide, O₂^?, (SO) nitric oxide, NO (NO) and more recently peroxynitrite, ONOO^? or its conjugate acid ONOOH, (PN) that is the product of their interaction. To this end, several antioxidant synthetic enzymes (synzymes) have been developed to effectively prevent the formation of PN (superoxide dismutase mimetics, SODms) or to decompose PN once it is formed (PN decomposition catalysts). The objectives of this mini-review written on PN and morphine antinociceptive tolerance are to 1) summarize recent advances made in the development of novel synzymes as therapeutics, 2) discuss the importance of nitroxidative stress in opiate anatinociceptive tolerance and 3) argue that PN is a rational target for therapeutic intervention in pain management. These concepts provide a pharmacological basis for developing inhibitors of PN biosynthesis as novel non-narcotic analgesics, thus addressing a large and currently unmet medical need with major socioeconomic consequences.     

Pharmacology of amphibian opiate peptides

In 1980 the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective mu-opioid agonists, and named dermorphins. Since 1987 deltorphins, a family of highly selective delta-opioid peptides were identified either by cloning of the cDNA from frog skins or isolation of the peptides. The distinctive feature of opioid peptides is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe. The discovery of the amphibian opiate peptides, provided new insights into the functional role of the mu- and delta-opiate systems. It also provided models for novel analgesics with enhanced therapeutic benefits and reduced toxicity.     

Narcotic dependence, narcotic action and dopamine receptors.

Acute systematic administration of narcotic analgesics increases the firing rate of nerve cells in the zona compacta of the substantia nigra, causes an increase in the rate of dopamine turnover in striatal and mesolimbic areas of the brain, stimulates prolactin release, inhibits brain self-stimulation and discriminated shock-avoidance, blocks cardiovascular effects of systemically injected dopamine, blocks aggression as well as compulsive jumping in mice treated with DOPA and amphetamine, antagonizes stereotypy induced by apomorphine or amphetamine, and blocks apomorphine-induced vomiting in dogs. Chronic administration of narcotic analgesics results in withdrawal signs upon the cessation of the drug administration. These signs include, tolerance to the increase in striatal dopamine turnover caused by narcotic analgesics or haloperidol, aggressive behaviors which are further stimulated by directly or indirectly acting dopamine-receptor agonists and are blocked by dopamine-receptor blockers, facilitation of recovery from the “lateral hypothalamic syndrome”, an increase in basal levels of striatal adenylate cyclase which shows greater sensitivity to dopamine, and, an enhanced sensitivity to apomorphine-induced reduction of dopamine turnover. It is therefore, concluded that acute administration of narcotic drugs results in an inhibition of dopamine-receptor activity while chronic administration of these drugs results in an increased response of these dopamine receptors to dopamine agonists. Recent experiments on the interaction of other drugs with narcotic analgesics suggest that, unlike the direct action of neuroleptics on the dopamine receptors, the narcotic action on dopamine receptors is indirect.     

Withdrawal symptoms in morphine-dependent rats intracerebroventricularly injected with ACTH1-24 and with beta-MSH

The injection of ACTH_1–24 and of β_mMSH into a brain lateral ventricle of morphine dependent rats, at doses of 10 and 50 μ/animal, precipitates a typical opiate withdrawal syndrome. The data strenghten the idea that melanocortin peptides (corticotropin and melanotropins) play an important role in nociception and in opiate tolerance and withdrawal.     

GABA in morphine analgesia and tolerance

Drugs affecting various steps of GABA transmission exhibit analgesia in a variety of experimental models in animals; this analgesic response generally requires high doses of the drugs and does not appear to be opiate-like since the GABAergic analgesia is naloxone-insensitive and lacks dependence liability. The outcome of the analgesia response is variable when opiate and GABAergic drugs are administered together; however, directly acting GABA receptor stimulants and GABA-transaminase inhibitors generally enhance the analgesic effect of opiates. The development of newer GABAergic drugs with greater potency and specificity may offer an alternative to opiate analgesics. The results obtained over the years, on the possible involvement of the GABA system in morphine tolerance and dependence are equivocal. Studies on region-specific changes in opiate-GABA interaction as well as opiate-GABA-benzodiazepine interaction are needed to further elucidate the role of GABA on opiate system.