David S. Bruce

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.

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.

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.

Summer hibernation in ground squirrels (Citellus tridecemlineatus) induced by injection of whole or fractionated plasma from hibernating black bears (Ursus americanus)

Abstract 1. 1. Summer hibernation in the thirteen-lined ground squirrel can be induced by the intravenous injection of plasma or its albunmin fraction previously isolated from winter torpid black bears. Squirrels injected with winter bear plasma or fraction and at 8°C hibernated (as measured by core temperature, respiratory rate, and bouts of activity), whereas those injected with summer bear plasma or fraction did not. 2. 2. A comparison of protein banding patterns of summer and winter bear plasma is also made. 3. 3. This successful induction of summer hibernation indicates that the hibernation-induction trigger (HIT) found in hibernating rodents (woodchuck, ground squirrel) and bats may also be present in a carnivore, the black bear, despite its less profound winter torpid state.

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

Abstract 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.

Hibernation-induction trigger. I. Opioid-like effects of prairie dog plasma albumin on induced contractility of guinea pig ileum

Studies have shown that plasma albumin fractions (PAFs) from hibernating mammals can inhibit induced contractility of the guinea pig ileum similarly to morphine. This study examined PAFs from two species of prairie dogs, one that undergoes natural seasonal hibernation (white-tailed, WT) and one that does not but can be induced to hibernate (black-tailed, BT). Dose-response curves of lyophilized PAF yielded IC50 values (mg) of 20.23 for summer WT, 15.53 for hibernating WT, 15.45 for summer BT, and 13.16 for winter-active BT. Winter samples from both species have IC50s lower than samples from summer animals, indicating greater potency of winter PAFs in suppressing guinea pig ileum contractility and therefore the presence of more opioid ligands in winter prairie dog plasma. Studies to elucidate receptor selectivity of PAF continue.

Opioid-Like Hibernation Factors Provide Protection to the Ischemic Myocardium

The purpose of the present study was to test the hypothesis that a single 24 hr infusion of HIT, DADLE, or a highly specific delta2 opioid receptor agonist, ZGI-04, can provide extended pharmacological ischemic protection to the rat myocardium by a process similar to that which occurs in ischernia preconditioning (IPC). This effect involves delta2 opioid receptor activation that is mediated by opening of KATP channels in the isolated perfused rat heart model. Rats (n=6 per group) were subjected to 7 different infusion protocols. Controls received 0.5 ml of saline while treated groups received either 0.5 ml of summer active woodchuck plasma (SAWP) or hibernating woodchuck plasma (HIT) or 2 mg/Kg of DADLE or ZGI-04. Two additional groups were infused with the KATP channel blocker, Glibenclamide, at either 30 min prior to ZGI-04 infusion or 30 min prior to cardiectomy. After 24 hr rats were anesthetized and cardiectomy was performed. Hearts were perfused with a Krebs-Henseleit buffer in Langendorff mode. All hearts under went 20 min ischemia followed by 120 min reperfusion. Infarct size was determined by triphenyltetrazolium (TTC) staining, and left ventricular functional parameters were determined. HIT, DADLE and ZGI-04 treatment significantly decreased infarct size compared to controls. SAWP did not significantly decrease infarct size. Glibenclamide at both treatment times effectively blocked ZGI-04-induced ischemic cardioprotection. HIT and ZGI-04 preinfusions also enhanced left ventricular function. These results indicate that delta2 opioid receptor stimulation is responsible for mediating the long-term ischemic protection. In the case of ZGI-04 the effect is mediated via an opioid receptor-KATP channel linked mechanism since Glibenclamide abolished its protective effects.

Induction of summer hibernation in the 13-lined ground squirrel, Citellus tridecemlineatus.

The induction of summer hibernation in the 13-lined ground squirrel (Citellus tridecemlineatus) by intravenous injection of plasma obtained from winter hibernating ground squirrels was confirmed. Hibernation was also induced by injection of urine from arousing winter ground squirrels. Results support the “trigger” theory of hibernation proposed by Dawe and Spurrier (3) and also suggest that tissues are set free from “trigger” influence during winter arousal by the excretion of “trigger.”

Effects of the beta-adrenergic blocker, propranolol, on the hamster (Mesocricetus auratus) rewarming from induced hypothermia☆

Abstract Hamsters were made hypothermic (core temperature of 7 °C) by a modification of the Giaja technique involving hypoxic, hypercapnic cold exposure. One group of hypothermic hamsters was injected with the beta-adrenergic blocker, propranolol. A control hypothermic group of hypothermic hamsters was injected with saline, and a third group, consisting of expired animals, was also studied. At the final temperature measurement (160 min of rewarming) the control hamsters had the highest mean temperature, the propranolol-treated hamsters were intermediate, and the expired group lowest, the latter leveling off at room temperature. The propranol-treated hamsters rewarmed at a rate significantly slower than the control subjects, thereby demonstrating the importance of the sympathetic beta-adrenergic system in rewarming from induced hypothermia.