Peter R. Oeltgen

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.

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 alpha opioid D-Ala2-D-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 alpha 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.

Postprandial serum glucose, insulin, and lipoprotein responses to high- and low-fiber diets.

The effects of high-fiber (HF) and low-fiber (LF) meals on postprandial serum glucose, insulin, lipid, lipoprotein, and apolipoprotein concentrations of 10 hypercholesterolemic men were examined using a random-order, cross over design. HF and LF meals provided 15% of energy as protein, 40% as carbohydrate, and 45% as fat, 200 mg cholesterol/1,000 kcal, and 25 g fiber/1,000 kcal for HF or 3 g fiber/1,000 kcal for LF. Responses over a 15-hour period after multiple meals (MM) and over a 10-hour period after a single meal (SM) were compared. HF meals were associated with a significant reduction in postprandial serum glucose (P < .0005 after SM) and insulin (P < .0005 after SM). Serum free fatty acid (FFA) levels decreased significantly after MM and SM, but differences between HF and LF meals were insignificant. Although serum triglyceride responses did not differ significantly (ANOVA) between HF and LF meals, values were higher at 2 and 3 hours after a HF SM than after a LF SM and at 16 hours after HF MM than after LF MM. Although serum cholesterol values did not differ significantly (ANOVA) between HF and LF meals, values were higher after a HF SM than after a LF SM. Other subtle differences in responses of high-density lipoprotein (HDL) cholesterol, HDL2, and HDL3 concentrations were noted. These studies indicate that large increases in dietary fiber intake are accompanied by small changes in postprandial serum lipoprotein concentrations.

Hibernation-like state induced by an opioid peptide protects against experimental stroke

BackgroundDelta opioid peptide [D-ala2,D-leU5]enkephalin (DADLE) induces hibernation in summer ground squirrels, and enhances preservation and survival of isolated or transplanted lungs and hearts. In the present study, we investigated the protective effect of DADLE in the central nervous system.ResultsAdult Sprague-Dawley rats were pretreated with DADLE (4 mg/kg every 2 h × 4 injections, i.p.) or saline prior to unilateral occlusion of the middle cerebral artery (MCA). Daily behavioral tests revealed that ischemic animals treated with DADLE did not show any significant behavioral dysfunctions compared with saline-treated ischemic animals. Opioid antagonists only transiently inhibited the protective effect of DADLE, indicating the participation of non-opioid mechanisms in DADLE neuroprotection. Histological examination using triphenyltetrazolium chloride (TTC) revealed that brains from ischemic animals treated with DADLE, either alone or with adjuvant opioid blockers, exhibited almost completely intact striata. In contrast, brains from ischemic animals that received saline showed significant infarction in the lateral striatum. Analyses of apoptotic cell death revealed a significant increase in the p-53 mRNA expression in the striatum of ischemic animals that received saline, while those that received DADLE exhibited near normal striatal p-53 expression. This protective effect was accompanied by significant increments in protein levels of glial cell line-derived neurotrophic factor in the striatum of DADLE-treated ischemic animals.ConclusionThese results indicate that DADLE protected against necrotic and apoptotic cell death processes associated with ischemia-reperfusion injury. The present study demonstrates that delta opioids are crucially involved in stroke, suggesting that the opioid system is important in the study of brain injury and protection.

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 HITs depression of electrically-induced contraction of guinea pig ileum). The fact that bear HITs 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.

Activation of peripheral δ2 opioid receptors increases cardiac tolerance to ischemia/reperfusion injury Involvement of protein kinase C, NO-synthase, KATP channels and the autonomic nervous system

AIMS This study aims to investigate the role of peripheral delta(2) opioid receptors in cardiac tolerance to ischemia/reperfusion injury and to examine the contribution of PKC, TK, K(ATP) channels and the autonomic nervous system in delta(2) cardioprotection. MAIN METHODS Deltorphin II and various inhibitors were administered in vivo prior to coronary artery occlusion and reperfusion in a rat model. The animals were monitored for the development of arrhythmias, infarct development and the effects of selected inhibitors. KEY FINDINGS Pretreatment with peripheral and delta(2) specific opioid receptor (OR) antagonists completely abolished the cardioprotective effects of deltorphin II. In contrast, the selective delta(1) OR antagonist 7-benzylidenenaltrexone (BNTX) had no effect. The protein kinase C (PKC) inhibitor chelerythrine and the NO-synthase inhibitor L-NAME (N-nitro-L-arginine methyl ester) also reversed both deltorphin II effects. The nonselective ATP-sensitive K+ (K(ATP)) channel inhibitor glibenclamide and the selective mitochondrial K(ATP) channel inhibitor 5-hydroxydecanoic acid only abolished the infarct-sparing effect of deltorphin II. Inhibition of tyrosine kinase (TK) with genistein, the ganglion blocker hexamethonium and the depletion of endogenous catecholamine storage with guanethidine reversed the antiarrhythmic action of deltorphin II but did not change its infarct-sparing action. SIGNIFICANCE The cardioprotective mechanism of deltorphin II is mediated via stimulation of peripheral delta(2) opioid receptors. PKC and NOS are involved in both its infarct-sparing and antiarrhythmic effects. Infarct-sparing is dependent upon mitochondrial K(ATP) channel activation while the antiarrhythmic effect is dependent upon TK activation. Endogenous catecholamine depletion reduced antiarrhythmic effects but did not alter the infarct-sparing effect of deltorphin II.

Opioid preconditioning: myocardial function and energy metabolism.

BACKGROUND Opioid receptor agonists are involved in ischemic preconditioning and natural hibernation. The aim of this study was to determine whether pretreatment with D-Ala2-Leu5-enkephalin or morphine confers cardioprotection in large mammalian hearts. We assessed myocardial functional recovery and global energy metabolism after ischemic cold storage. METHODS After pretreatment with D-Ala2-Leu5-enkephalin, morphine sulfate, or saline (n = 6 each), swine hearts were excised and stored for 75 minutes at 4 degrees C, then reperfused in a four-chamber isolated working heart apparatus. Serial myocardial biopsies were performed to assess cellular energy metabolism. RESULTS Improved systolic (cardiac output, contractility) and diastolic (tau) left ventricular functions were observed in hearts pretreated with D-Ala2-Leu5-enkephalin or morphine. These benefits were not correlated with changes in high-energy phosphate levels. Cardiac enzyme leakage (creatine kinase, troponin-I) was similar among treated and control groups. Lactate efflux increased significantly in controls, but not in opioid-pretreated hearts (p < 0.01) at 75 minutes of reperfusion. CONCLUSIONS D-Ala2-Leu5-enkephalin and morphine pretreatments improve postischemic function after cold storage of swine hearts. Postischemic lactate reduction, but not high-energy phosphate levels, may account for the observed cardioprotective effects.

Delta2‐Specific Opioid Receptor Agonist and Hibernating Woodchuck Plasma Fraction Provide Ischemic Neuroprotection

OBJECTIVES The authors present evidence that the delta opioid receptor agonist Deltorphin-D(variant) (Delt-D(var)) and hibernating woodchuck plasma (HWP), but not summer-active woodchuck plasma (SAWP), can provide significant neuroprotection from focal ischemia in mice by a mechanism that relies in part on reducing nitric oxide (NO) release in ischemic tissue. METHODS Cerebral ischemia was produced in wild-type and NO synthase-deficient (NOS(-/-)) mice by transient, 1-hour middle cerebral artery occlusion (MCAO). Behavioral deficits were determined at 22 hours and infarct volume was assessed at 24 hours after MCAO. Mice were treated with saline or Delt-D(var) at 2.0 and 4.0 mg/kg, or 200 microL of HWP or SAWP. NOS(-/-) mice were treated with either saline or Delt-D(var) at 4.0 mg/kg. NO release was determined using an N9 microglial cell line pretreated with delta- or mu-specific opioids and HWP or SAWP prior to activation with lipopolysaccharide and interferon-gamma. Nitrate in the medium was measured as an indicator of NO production. RESULTS Infusion of Delt-D(var) or HWP (but not SAWP) decreased infarct volume and improved behavioral deficits following 1 hour of MCAO and 24 hours of reperfusion. In NOS(-/-) mice, endothelial NOS(+/+) is required to provide Delt-D(var)-induced neuroprotection. Delt-D(var) and HWP dose-dependently decreased NO release in cell culture, while SAWP and other delta- and mu-specific opioids did not. CONCLUSIONS Delt-D(var) and HWP, but not SAWP, are effective neuroprotectant agents in a mouse model of transient MCAO. In cell culture, the mechanism of this ischemic neuroprotection may rely in part on their ability to block NO release.

Effect of Oxysterol-Enriched Low-Density Lipoprotein on Endothelial Barrier Function in Culture

High levels of plasma low-density lipoproteins (LDL) are known to be a risk factor for developing coronary artery disease although the specific mechanism involved is unknown. It may be related to effects of oxidized lipid components of LDL on vascular endothelial barrier function (EBF). This study addressed the hypothesis that LDL-associated products of cholesterol oxidation, oxysterols, decrease EBF resulting in increased penetration of blood components such as LDL into the arterial wall. LDL from human volunteers and rabbits was enriched with cholesterol or cholestan-3 beta,5 alpha,6 beta triol (Triol) by in vitro incubation. Exposure of cultured vascular endothelial cell monolayers to LDL enriched with Triol reduced EBF, measured as an increase in transendothelial albumin transfer, whereas cholesterol enrichment, like un-enriched LDL, had no effect on EBF. In a second experimental series, rabbits were gavaged with 100 mg of cholesterol or Triol/kg body weight, and LDL was isolated from serum 24 h after gavage. As was seen with the in vitro experiments, Triol-enriched LDL markedly decreased EBF. Similarly, LDL from cholesterol-gavaged rabbits reduced EBF, while LDL from vehicle treated rabbits had no effect. These results suggest that LDL-associated oxysterols are detrimental to normal barrier function of the vascular endothelium. Disruption of this barrier function may serve as an initiating factor in atherosclerotic lesion formation.

Circannual variations in bear plasma albumin and its opioid-like effects on guinea pig ileum

Previous studies suggest that hibernation is controlled by an opioid system. In this study we examined the effect of plasma albumin fractions drawn from black bears at timed intervals while in hibernation or during the awake state in fall and winter, on induced contractility of the guinea pig ileum. Four hundred nM morphine produced typical suppression of contractility and 400 or 1000 nM naloxone (an opiate antagonist) restored it. Twenty mg of lyophilized albumin fraction from the winter hibernating bear caused similar suppression, the effect being greater than that of either summer bear or winter-active bear plasma albumin. Naloxone reversed the suppression in all cases. Strong suppression of contractility was also demonstrated with 2.5 nM [D-Pen2.5]-enkephalin (DPDPE), a delta agonist, but only minor suppression with 2.5 nM dynorphin A, a kappa agonist. Results support the opioid nature of the albumin-bound hibernation-induction trigger substance, that it binds to the delta opiate receptor, and that delta agonist opioid production may increase during the hibernation season.