Further studies on opioids and hibernation: delta opioid receptor ligand selectively induced hibernation in summer-active ground squirrels

To examine the possible involvement of multiple opioid receptors in animal hibernation, we infused opioids selective for mu, kappa, and delta opioid receptors into summer-active ground squirrels (Citellus tridecemlineatus). The effects of those opioid treatments on the hibernation induced by HIT (Hibernation Induction Trigger) were also examined. Mu opioids morphine (1.50 mg/kg/day) and morphiceptin (0.82 mg/kg/day) and kappa opioid peptide dynorphin A (0.82 mg/kg/day) did not induce hibernation. On the contrary, morphine, morphiceptin and dynorphin A antagonized HIT-induced hibernation in summer-active ground squirrels. Infusion of delta opioid DADLE (D-Ala2-D-Leu5 enkephalin; 1.50 mg/kg/day), however, induced summer hibernation in a manner comparable to that induced by HIT. It is concluded therefore that delta opioid receptor and its ligand may be intimately involved in animal hibernation. In view of the fact that HIT was obtained from winter hibernating animals and might therefore be responsible for natural hibernation, our results also suggest that naturally occurring mu and kappa opioids may play an important role in the arousal state of hibernation.     

Opioids and hibernation. I. Effects of naloxone on bear HIT'S depression of guinea pig ileum contractility and on induction of summer hibernation in the ground squirrel

Summer hibernation in ground squirrels (Citellus tridecemlineatus) can be induced by intravenous injection of hibernation-induction trigger (HIT) from winter bear plasma or its albumin fraction. In this study, we show that bear HIT depresses electrically-induced contraction of the guinea pig ileum myenteric plexus-longitudinal muscle preparation, and that naloxone, at 100, 1,000, or even 4,000 nM, fails to reverse that effect. In a simultaneous study, four sets of ground squirrels were implanted with osmotic minipumps which delivered solutions at a controlled and continuous rate. Two of the groups had pumps delivering naloxone at 1 mg/kg body weight per hour. The other two groups had saline-filled pumps (controls). One set of squirrels from each of the saline- and naloxone-filled pump groups were then injected intravenously with winter bear plasma. The remaining two groups of squirrels were injected with winter bear albumin fraction. Hibernation frequency was determined by measurements of core temperature (from surgically-implanted radio capsules), respiratory rate, and bouts of activity. Squirrels with saline-filled pumps hibernated four times more frequently than the naloxone groups. To confirm these findings, three squirrels from each naloxone group were reinjected with bear HIT after removal of the pumps. These six squirrels then hibernated over four times their previous frequency. Results suggest that bear HIT is not itself an opioid (since naloxone did not reverse bear HIT's depression of electrically-induced contraction of guinea pig ileum). The fact that bear HIT's effect of inducing summer hibernation in ground squirrels is effectively blocked in vivo by naloxone leads to the speculation that HIT may be either a precursor of endogenous opioids or a potent releaser of them, which, in turn, induce hibernation.     

Delta opioid peptide [D-Ala2,D-Leu5]enkephalin promotes cell survival

By studying the hibernation in ground squirrels, a protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Further purification of HIT yielded an 88-kD peptide that is enriched in winter hibernator. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Delta opioid [D-Ala(2),D-Leu(5)]enkephalin (DADLE) also induced hibernation. HIT and DADLE were found to prolong survival of peripheral organs preserved en bloc or as a single preparation. These organs include the lung, the heart, liver and kidney. DADLE also promotes survival of neurons in the central nervous system. Methamphetamine (METH) is known to cause destruction of dopaminergic (DA) terminals in the brain. DADLE blocked and reversed the DA terminal damage induced by METH. DADLE acted against this effect of METH at least in part by attenuating the mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. DADLE also blocked the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In PC12 cells, DADLE blocked the cell death caused by serum deprivation in a naltrexone-sensitive manner. Thus, DADLE, and by extension the endogenous delta opioid peptides and delta opioid receptors, may play an important role in organ and neuronal survival. Here, critical developments concerning these fascinating cell protective properties of DADLE are reviewed.     

Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein

Previous studies show that infusion of hibernating woodchuck albumin (HWA) induces hibernation in summer-active ground squirrels and results in profound behavioral and physiological depression in primates. These effects are reversed by the administration of opiate antagonists, suggesting that the putative hibernation induction trigger (HIT) may act through opioid receptors. We have demonstrated that both HIT-containing plasma and the synthetic α opioid -Ala²- -Leu⁵-enkephalin (DADLE), which mimics the activity of HIT in hibernators, extend tissue survival time of a multi-organ autoperfusion system by 3-fold. In this study we present the first data showing biological activity with a much more highly purified plasma fraction from hibernating woodchucks, identified as the hibernation-related factor (HRF). Both the HRF and DADLE show opiate-like contractile inhibition in the mouse vas deferens (Mvd) bioassay. We also have preliminary evidence in an isolated rabbit heart preparation indicating that the HRF and DADLE act similarly to restore left ventricular function following global myocardial ischemia. Furthermore, we have partially sequenced an α 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.     

Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5,000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3.5 to 10.0 resulting in protein components having isoelectric points (pIs) of 4.5, 5.2, 5.5, 6.3, and 7.0. Fraction I (comprised of proteins having pIs of 4.5 and 5.2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5.5) and Fraction III (pI 6.3 and 7.0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation. Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3.5 to 6.0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.     

Absence of evidence for a hibernation "trigger" in blood dialyzate of Richardson's ground squirrel.

In order to examine evidence for a blood-borne “trigger” for mammalian hibernation, serum dialyzate from hibernating Richardson's ground squirrels (Spermophilus richardsonii) was injected into summer-active ground squirrels of the same species. Four independent trials involving 52 animals were performed. In all trials, no effect of the dialyzate was seen on nest building, weight gain or loss, or on occurrence of hibernation.     

Lipids of the liver microsomal fraction in the ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis> during hibernation

Winter sleep of the ground squirrel Spermophilus undulatus was accompanied by a 20% decrease in phospholipid content (µg phospholipid per 1 mg protein) in microsomal fractions of the liver as compared with summer-active squirrels. The phosphatidylcholine level (mol %) in hibernating squirrels was lower than in summer-active squirrels, and the content of sphingomyelin (mol %) during the torpor bout was higher than in winter- and summer-active squirrels. The cholesterol, fatty acid, monoglyceride, and diglyceride levels in the microsomal fraction of the liver were elevated during hibernation. Pronounced seasonal changes in the lipid/protein ratio implicate the lipids of the liver microsomal fraction in adaptation of the ground squirrel to hibernation.     

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.     

Effect of sodium ion on the affinity of naloxone for the kappa opioid receptor

Several investigators have observed that sodium ion enhances the binding of naloxone to opioid receptors. This effect has generally been attributed to allosteric modulation of the state of the mu receptor. However, a recent claim has been made that the enhancement does not involve a change in the mu receptor, but instead occurs because naloxone becomes a more kappa-specific drug when sodium ion is present in high concentration. Since the claim was not based on experimental evidence from binding studies involving known high-affinity kappa ligands, we have investigated the competition of naloxone for the kappa site using [3H]U-69593 as the marker for receptor binding. Assays were carried out in the presence and absence of 100 mM NaCl. The results of the study indicate that sodium ion does not increase the affinity of naloxone or U-69593 for the kappa receptor.     

Hemoglobin alterations of the 13-lined ground squirrel while in various activity states.

1. Characterization of fetal, winter-hibernating, winter-active, summer-active and summer-induced hibernating hemoglobins of 13-lined ground squirrels (Citellus tridecemlineatus) by isoelectric focusing (IEF) pH 7.0-9.0 indicated that this molecule is extremely responsive to the various activity states of this hibernator. 2. Major alterations of ground squirrel hemoglobin occur with the varying activity states as evidenced by the distinctive changes in the isoelectric points (pIs) of these protein components. 3. Hemoglobin from winter-hibernating or summer-induced hibernating ground squirrels does not revert to a fetal type of hemoglobin. 4. The presence of an additional hemoglobin peak pI 6.55 in the summer-induced hibernator may serve as a possible assay for hibernation inducing trigger(s) (HIT) molecules under study in our laboratory.     

Isolation of a Hibernation Inducing Trigger(s) From the Plasma of Hibernating Woodchucks

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5, 000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3. 5 to 10. 0 resulting in protein components having isoelectric points (pis) of 4. 5, 5. 2, 5. 5, 6. 3, and 7. O. Fraction I (comprised of proteins having pis of 4. 5 and 5. 2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5. 5) and Fraction III (pi 6. 3 and 7. 0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation.

Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3. 5 to 6. 0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when Injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.     

Characterization of kappa1-opioid receptor binding in human insular cortex.

Mesolimbic dopaminergic neurotransmission is modulated by dynorphin peptides binding to kappa-opioid receptors. The interaction between dynorphin and dopamine systems makes the kappa-opioid receptor a potential drug discovery target for the development of therapeutic agents for schizophrenia and drug abuse. This study reports the specificity and parameters of [3H]U69593 binding in the insular cortex, a representative corticolimbic area of the human brain. The results demonstrate that the radioligand [3H]U69593 labels a single population of receptors in human insular cortex with an affinity in the low nanomolar range. The pharmacological profile for inhibition of [3H]U69593 binding was determined in this brain region using drugs known to bind to mu, kappa and delta opioid receptors. The results show that kappa-opioid selective agonists and antagonists inhibit binding of this ligand in human brain with comparable affinities and rank order as previously described for rat and guinea pig brain and the cloned kappa1-opioid receptor subtype.     

Summer hibernation induced in ground squirrels (Citellus tridecemlineatus) by urine or plasma from hibernating bats (Myotis lucifugus or Eptesicus fuscus)

Summer hibernation induced in ground squirrels (Citellus tridecemlineatus) by urine or plasma from hibernating bats (Myotis lucifugus or Eptesicus fuscus). Summer hibernation in the thirteen-lined ground squirrel can be induced by intravenous injection of urine or blood plasma previously isolated from winter hibernating little brown bats (M. lucifugus) or big brown bats (E. fuscus). Urine- and plasma-injected ground squirrels kept at 8 °C hibernated earlier, longer, and deeper (as indicated by core temperature and respiratory rate measurements) than control ground squirrels injected with saline. This successful cross-order induction of hibernation demonstrates that the hibernation-inducing trigger (HIT) may be present in nonrodent mammals.     

Selective activation of opioid receptors differentially affects lordosis behavior in female rats

The effects of opioid peptides that are highly selective ligands for mu receptors (morphiceptin). delta receptors (delta-receptor peptide), kappa receptors (dynorphin 1-9), and the mu/delta complex (beta-endorphin), were tested on lordosis behavior in ovariectomized rats primed with estrogen and progesterone. Intracerebroventricular infusions of beta-endorphin or morphiceptin both inhibited and facilitated lordosis in a dose-dependent fashion whereas all doses of delta-receptor peptide facilitated lordosis. Dynorphin 1-9 had no significant effect at any dose, although a trend toward increased lordosis quotients was observed 30 min after infusion. The effects of beta-endorphin, morphiceptin, and delta-receptor peptide were reversed with naloxone, although naloxone alone had no effect on lordosis behavior. These results indicate that the specific activation of opioid receptor subtypes differentially affects lordosis behavior. It appears that binding to high-affinity mu 1 receptors exerts an inhibitory influence on lordosis, whereas binding to low-affinity mu 2 receptors or delta receptors exerts a facilitatory influence. Binding to kappa receptors does not appear to affect lordosis behavior.     

弓状核中阿片受体在细胞介素所致发热效应中的作用

本研究旨在了解弓状核内的阿片受体在体温调节中的作用。研究使用细胞介素ＩＬ１β做致热源。以自动推进器向ＳＤ雄性大鼠弓状核微量注射１μ１ＩＬ１β。在给药前３０ｍｉｎ分别向弓状核微量注射通常阿片受体拮抗剂纳洛酮（Ｎａｌ）、阿片受体μ、δ和κ各自特异性拮抗剂ＣＴＡＰ、ＮＴＩ和ｎｏｒＢＮＩ做预处理,用生理盐水（Ｓａｌ）做对照。结果表明：ＩＬ１β所致的升体温效应能被Ｎａｌ和ＣＴＡＰ阻断,提示弓状核中的阿片受体（主要是μ受体）参与或介导了ＩＬ１β的致热效应;δ和κ受体特异性拮抗剂阻断ＩＬ１β所致的体温升高效应不明显。提示δ和κ阿片受体参与体温调节的可能性较小。对照ＡＲＨ和ＰＯＡＨ中阿片受体在ＩＬ１β所致发热中的作用可发现：二者作用极为相似,这一结果有力地支持了弓状核是体温调节中枢重要组成部分的观点。     

Summer hibernation of infant (six week old) 13-lined ground squirrels, Citellus tridecemlineatus

In an endeavor to more clearly define the physiological action of hibernation trigger (previously identified by the authors) an experiment was devised using infant ground squirrels. In one case, pregnant ground squirrel mothers were transfused intravenously with cold dialysate of serum of hibernating woodchuck, and in another case, infant (1 week to 3 week) squirrels were transfused intraperitoneally with the same material. When isolated and placed in a cold room (15 °C) at 6 weeks post partum, both the infant born of the transfused mothers, as well as the infant which has been transfused directly, hibernated in the summer. This was in contrast to controls of several kind, i.e., animals not injected, or receiving active or arousing woodchuck serum or heat-treated hibernation serum. Some details for a theory, which had been advanced in a previous article (4), were essentially substantiated by this experiment. It is our belief that this represents the first time mammalian hibernation has been observed in infant hibernators.     

The "hibernation induction trigger": specificity and validity of bioassay using the 13-lined ground squirrel

Even though the existence of the blood-borne "hibernation induction trigger" has been reported in the 13-lined ground squirrel, transfusion of plasma from hibernating rodents with other hibernating species as the recipients failed to induce the occurrence of summer hibernation. In order to verify whether the response to the "trigger" substance is species specific, the present study was carried out to compare the effect of plasma from hibernating Richardson's ground squirrels on the incidence of summer hibernation in both juvenile Richardson's and adult 13-lined ground squirrels. In two series of experiments, 13-lined ground squirrels entered hibernation quite readily independent of the treatment. The rate of occurrence of hibernation ranged from 78% after sham injection to 86% after warm saline, fresh summer active plasma, and fresh hibernating plasma, respectively. There were no differences in the number of hibernation bouts and the number of days in hibernation after each treatment. In contrast, none of the juvenile Richardson's ground squirrels entered hibernation after any of the treatments up to the end of the 8-week observation period. These results not only argue against the existence of blood-borne "trigger" substance, at least in the Richardson's ground squirrel, but also caution against the use of the 13-lined ground squirrel as a standard test animal for the bioassay of the "trigger" substance.     

State-dependent variation in the inhibitory effect of [D-Ala2, D-Leu5]-enkephalin on hippocampal serotonin release in ground squirrels.

Accumulated evidence has suggested that increased endogenous opioid activities may facilitate the onset of hibernation either directly or possibly through modulation of other neurotransmitter systems. The seasonal change of [D-Ala2, D-Leu5]-enkephalin (DADLE), a delta receptor agonist, in modulating K+ (35 mM)-induced [3H]-5-hydroxytryptamine (5-HT) release from the hippocampal and hypothalamic slices of euthermic and hibernating Richardsons' ground squirrels was therefore investigated. DADLE (0.1-10 microM) had no effect on 5-HT release in the hypothalamic slices but elicited a dose-related inhibition on [3H]-5-HT release from the hippocampal slices of the euthermic ground squirrel. The inhibitory effect of DADLE was completely reversed by naloxone (10 microM), but not by tetrodotoxin (1 microM). In contrast, DADLE failed to alter the K(+)-induced 5-HT release from the hippocampal slices of the hibernating ground squirrel. This state-dependent reduction in responsiveness to an opioid is consistent with the hypothesis that enhanced endogenous opioid activity in the hibernating phase could lead to down regulation of the opioid receptors and minimize its inhibition on hippocampal serotonergic activity. A high 5-HT activity would inhibit midbrain reticular activating system indirectly through non-serotonergic fibers, which in turn facilitate the onset or maintenance of hibernation.     

Different effects of methionine-enkephalin on paw edema in two inbred rat strains

The effect of intraplantarly (i.pl.)-injected methionine-enkephalin (ME) on Concanavalin A (Con A)-induced paw edema in Dark Agouti (DA) and Albino Oxford (AO) rats was investigated. ME suppressed edema in DA rats, which was antagonized with naloxone (non-selective opioid receptor antagonist) and naltrindole (delta opioid receptors antagonist). Potentiating effect of ME in AO rats was blocked by naloxone, nor-binaltorphimine (kappa opioid receptors antagonist) and beta-funaltrexamine (mu opioid receptors antagonist). Dexamethasone suppressed edema in both rat strains. These findings suggest that strain-dependent differences in the effects of ME on inflammation in DA and AO rats could be related to diversity in opioid receptors expression in these strains.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.