Endogenous opiates: 1979

Since the discovery in 1971 of opiate receptors and later of the opiate-like peptides, there has been widespread interest in determining their exact localization, number and kinds, nature, and physiological and pharmacological functions. Between 1971 and 1978, vast amounts of research investigated these problem areas, but in 1979 alone the literature on the opiate peptides nearly doubled. This review is the second of an annual series and summarizes the highlights of the work published during 1979.     

Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.     

Developmental Parameters of Mother-Offspring Interactions in Acomys cahirinus

Developmental parameters of mother-infant interactions in Acomys cahirinus were investigated in a series of observational and experimental studies. The uniquely precocial offspring associated closely with both parents beyond the time of weaning and birth of the next litter. The survival rate of foster pups was dependent upon both the age of the pups and the physiological stare of the foster mothers. While 1 and 8-day post partum ♂♂ nursed unfamiliar neonates as frequently as their own pups, they interacted with unfamiliar 8-day-old offspring less than with their own.     

The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized α-subunits and from preformed cross-reacting protein

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.     

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical,kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K_m values for NADPH and cytochrome c were: 20 and 29 μm or 14 and 28 μm for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.     

Early events following amputation of adult newt limbs given regeneration inhibitory doses of X irradiation

X irradiation, (2000 R) prevented regeneration but had no apparent effect on morphological dedifferentiation. DNA synthesis and mitotic activity increased significantly in irradiated limbs after amputation but not as much as in regenerating unirradiated controls. Even though fewer mitotic cells were seen in irradiated limbs, those that occurred were distributed normally throughout the stump. The results indicate that morphological dedifferentiation and entry into the cell cycle are dissociable events and that X rays act by interfering with the latter. They also show that some cells in irradiated amputated limbs not only synthesize DNA but also divide.     

Nippostrongylus brasiliensis: Peripheral leukocyte responses and correlation of basophils with blood histamine concentration during infection in rats

Rats infected on Day 0 with 3000 infective L₃ larvae of Nippostrongylus brasiliensis, and uninfected controls, were monitored daily through Day 23 postinfection for changes in peripheral leukocytes and blood histamine concentrations. A generalized leukocytosis was observed between Days 7 and 18, the period leading up to and immediately following the time of expulsion of adult worms from the small intestine. The total number of lymphocytes was elevated between Days 11 and 17 post-infection; however, there was no change in the percentage of lymphocytes relative to other white blood cell types. The total number and percentage of monocytes were no different from controls, with the exception of Day 5 postinfection. On that day, there was a significant elevation in the number (614/mm³ blood in infected rats, as compared to 160/mm³ blood in controls) and relative proportion (2.7% of total leukocytes in infected animals, compared to 0.8% in controls) of monocytes, coinciding with the termination of the pulmonary migration of larvae. A period of moderate neutrophilia occurred between Days 7 and 12, but this was not accompanied by any changes in the proportion of neutrophils. A biphasic eosinophil response was observed. An early elevation of eosinophils occurred between Days 3 and 5, corresponding to the period of larval migration through the lungs. A second period of eosinophilia began on Day 11, when worm expulsion was beginning, and continued through Day 19, i.e., beyond the period of worm expulsion. Basophilia was observed as early as Day 6 after infection, rising to a peak on Day 13 (6.8% of total leukocytes in the infected animals, as compared to 0.5% in controls), and declining thereafter, but remaining above control levels until termination of the experiment on Day 23. The histamine content of blood samples, as determined by an enzymic-isotopic assay, closely paralleled the development and decline of basophilia; histamine levels also peaked on Day 13 postinfection (422.5 pg histamine/mm³ blood in infected rats, compared to 66.0 pg histamine/mm³ blood in controls). As basophilia progressed during the course of infection, there was a decline in the amount of histamine per basophil. In uninfected rats and during the first week after infection, basophils contained about 1.5–2.0 pg histamine per cell. In the third week of infection, there was about 0.6 pg histamine per basophil. The time course of the basophilia suggests that these cells may be involved in the expression of immunity to N. brasiliensis.     

Intracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg

Fertilization of the sea urchin egg leads to a sequence of changes at the egg surface and the interior cytoplasm. Among these changes are the transient elevation of internal calcium levels, alkalization of the cytoplasm and development of new K⁺-conductance. In the series of experiments reported here, we separate the effects on potassium activation of the calcium release and the rise in the intracellular pH. The development of new K⁺-conductance was dependent on alkalization of the egg cytoplasm, and not on a rise of internal calcium levels. The effects of 2,4-dinitrophenol, N-ethylmalemide, antimycin A and oligomycin suggest that the maintenance of the alkaline internal pH of fertilized eggs appears to be dependent on membrane ATPase activity.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.