Suppression of guinea pig ileum induced contractility by plasma albumin of hibernators.

Previous studies suggest that hibernation may be regulated by internal opioids and that the putative "hibernation induction trigger" (HIT) may itself be an opioid. This study examined the effect of plasma albumin (known to bind HIT) on induced contractility of the guinea pig ileum muscle strip. Morphine (400 nM) depressed contractility and 100 nM naloxone restored it. Ten milligrams of lyophilized plasma albumin fractions from hibernating ground squirrels, woodchucks, black bears, and polar bears produced similar inhibition, with partial reversal by naloxone. Five hundredths mg of D-Ala2-D-Leu5-enkephalin (DADLE) also inhibited contractility and naloxone reversed it. Conclusions are that hibernating individuals of these species contain an HIT substance that is opioid in nature and summer animals do not; an endogenous opioid similar to leu-enkephalin may be the HIT compound or give rise to it.

Two-day preservation of major organs with autoperfusion multiorgan preparation and hibernation induction trigger. A preliminary report.

A new autoperfusion multiorgan preparation was studied in which the heart and lungs were removed with the liver, pancreas, duodenum, and both kidneys en bloc while being perfused by the heart and oxygenated by the lungs. A respirator with 50% oxygen was used for ventilation. Fresh blood, glucose, electrolytes, mannitol, and antibiotics were given through the portal vein. Fifteen mongrel dogs were used. In the study group (seven dogs), 10 ml of plasma containing hibernation induction trigger, obtained from deeply hibernating woodchucks, was given intravenously 2 hours before the operation, and 4 ml was given every 4 hours during the preservation period. In the control group (eight dogs), no hibernation induction trigger was used. Survival time in the study group ranged from 33 to 56 hours (mean 43.4 +/- 4.1 hours), longer than that of the control group, which was 9 to 31 hours (mean 16.2 +/- 2.6 hours, p less than 0.001). In the study group aortic systolic pressure ranged from 64 +/- 5 to 92 +/- 7 mm Hg, arterial oxygen tension from 180 +/- 35 to 285 +/- 66 mm Hg. Urine output ranged from 15 to 70 ml/hour. Blood urea nitrogen declined from 15.6 +/- 2.5 to 6.6 +/- 1.3 mg/dl (p less than 0.01); creatinine declined from 0.8 +/- 0.03 to 0.3 +/- 0.01 mg/dl (p less than 0.01). Severe liver congestion and premature renal failure occurred in the control group but did not occur in the study group. In the study group one lung was transplanted after 33 hours of preservation with simultaneous contralateral pulmonary artery ligation. Good lung function was maintained after transplantation. Although the exact mechanism by which hibernation induction trigger extends tissue survival time is still not clear, its effect on organ preservation is profound. This study also produced one of the longest average survival times for organ preservation.

Is the polar bear (Ursus maritimus) a hibernator? Continued studies on opioids and hibernation.

Polar bear behavior and biochemistry suggest they may have the ability to hibernate year-round, even though this species is not considered to be a true hibernator. This observation, plus the discovery of a hibernation-induction trigger (HIT) in the blood of black bears, prompted the examination of polar bear blood collected throughout the year for evidence of HIT, and to determine if it displayed opioid activity, as black bear blood does. A bioassay was conducted by injecting summer 13-lined ground squirrels with serum collected from polar bears at different seasons. One group of squirrels was previously implanted with osmotic pumps containing naloxone. The rest had pumps containing saline. Squirrels with saline pumps all hibernated significantly more than those with naloxone, except the group receiving blood from a November polar bear, observed to be highly active and hyperphagic. An in vitro study, using guinea pig ileum, showed that 400 nM morphine inhibited induced contractions and 100 nM naloxone reversed the inhibition. Ten mg of winter polar bear serum albumin fraction (to which HIT binds in ground squirrels and woodchucks) had a similar inhibiting effect, but naloxone, even at 4,000 nM, didn't reverse it. It is concluded that polar bear blood contains HIT, that it has an inhibiting effect, but naloxone, even at 4,000 nM, didn't reverse it. It is concluded that polar bear blood contains HIT, that it has an opioid effect, but may not itself be an opioid.

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.

Evidence for repetitive structure of the large acyl-carrier protein subunit of Escherichia coli citrate lyase.

The amino acid composition of the unusually large acyl-carrier protein subunit of citrate lyase from Escherichia coli is characteristic of a protein with a highly repetitive structure. Peptide mapping studies provide further evidence of repetitive sequences within the subunit. Only a single Pauly-positive spot is detected in the tryptic peptide map although the subunit contains 8 histidine residues. The 4 prosthetic groups covalently bound to the subunit are recovered in a single tryptic fragment in almost quantitative yield. These structural features of the large acyl-carrier protein subunit probably reflect internal gene duplications.

Purification and properties of citrate lyase from Escherichia coli.

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)