Humoral endorphin: A new endogenous factor with opiate-like activity

Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$ attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.     

Regulation of cyclopropane fatty acid biosynthesis by variations in enzyme activities

It is proposed that cyclopropane fatty acid biosynthesis in $\text{Lactobacillus plantarum}$ is regulated by $\text{in vivo}$ variations in the activities of two enzymes acting sequentially. S-adenosylhomocysteine hydrolase relieves the end-product inhibition of cyclopropane synthetase by degrading a product (S-adenosyl-homocysteine) of the latter enzyme activity. Both enzymes show an abrupt increase and subsequent decrease in activity at a time during the bacterial growth cycle which corresponds to the period of most rapid synthesis of cyclopropane fatty acid $\text{in vivo}$.     

Inhibitory effects of dithiothreitol and sodium bisulfite on isolated rat ileum and atrium

The utility of sodium bisulfite and dithiothreitol as reducing agents in $\text{in}$$\text{vitro}$ preparations such as isolated rat atrium and ileum has been investigated. The right atria and 3 cm long ileal segments from male Sprague Dawley rats (250–350 g, sacrificed by cervical dislocation) were suspended in a 10 ml Magnus bath containing Tyrode's solution maintained at 37°C. Sodium bisulfite (0.955 - 9.55 mM) and dithiothreitol (5–50 μM) significantly depressed (P < .05) the duration of spontaneous atrial beating and the magnitude of methacholine-induced ileal contractions. The ileal inhibitory effects of these antioxidants became more pronounced with successive concentration-responses in the presence of methacholine but not with duration of exposure in the absence of methacholine, suggesting depletion of cellular energy stores by the antioxidants with successive ileal contractions. Depression of heart and intestinal tissues by bisulfite and dithiothreitol severelylimits their use as antioxidants to protect readily air oxidizable drugs during pharmacological testing with these standard tissue preparations.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and 5′-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome $\text{c}$ reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADHPH cytochrome $\text{c}$ reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome $\text{c}$ reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Enkephalins increase dopamine levels in the CNS of a marine mollusc.

Intracardiac administration of methionine enkephalin and leucine enkephalin increased dopamine but not serotonin levels in the CNS of the marine mollusc $\text{Mytilus}$$\text{edulis}$. Naloxone blocked the effects of the enkephalins. These responses displayed a time dependent desensitization to methionine enkephalin. The study suggests the presence of an opiate receptor mechanism in this invertebrate species.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

Biosynthesis of beta-phenethyl alcohol in Candida guilliermondii.

$\text{Candida guilliermondii}$ produced β-phenethyl alcohol and β-phenyllactic acid when grown in a synthetic medium containing L-phenylalanine as sole source of nitrogen. The cell-free preparations from these cells showed the following enzymes: phenylalanine aminotransferase, phenylpyruvate decarboxylase, phenylpyruvate reductase and phenylacetaldehyde reductase. The cell-free preparations of $\text{C. guilliermondii}$ grown in medium with ammonium sulfate, lacked these enzyme activities, indicating the inducible nature of these enzymes. The results indicate the role of β-phenylpyruvate as a key intermediate in the pathway of biosynthesis of β-phenethyl alcohol and β-phenyllactic acid from L-phenylalanine.     

Inhibition of acyl coenzyme A:lysolecithin acyltransferases by local anesthetics, detergents and inhibitors of cyclic nucleotide phosphodiesterases.

Acyl coenzyme A:lysolecithin acyltransferase plays a major role in regulating the amount of lysolecithin in cell membranes. The acyltransferase activity in microsomal preparations from rat liver, rat heart and rabbit gastric mucosa is inhibited by a series of tertiary amine local anesthetics, detergents, and some inhibitors of cyclic nucleotide phosphodiesterases. Aspirin and indomethacin cause elevated lysolecithin/lecithin ratios in the stomachs of mice after oral administration. Inhibition of acyltransferase activity in microsomal preparations by local anesthetics correlates with reported anesthetic potencies at approximately $\text{1}\text{100}$ reported therapeutic dosages. In BHK-13 cells acyltransferase activity is inhibited at $\text{1}\text{3}$ to $\text{1}\text{10}$ the concentrations that have been reported to cause alterations in the mobility and topography of cell surface receptors.     

Induction of the allantoin degradative enzymes by allophanic acid, the last intermediate of the pathway

$\text{Saccharomyces cerevisiae}$ can utilize allantoin as a sole nitrogen source by degrading it in five steps to ammonia, “CO₂”, and glyoxylate. We have previously shown that allophanic acid is the inducer of the urea carboxylase: allophanate hydrolase multienzyme complex. Since these enzymes catalyse the last two steps of allantoin degradation, experiments were performed to determine if allophanate was also the inducer of any other enzymes in the pathway. Our data demonstrate that allophanate induces synthesis of at least five of the seven purine degradative enzymes.     

Characterization of the passive and active transport mechanisms for bile acid uptake into rat isolated intestinal epithelial cells

The unstirred water layer has been shown to lead to an underestimation of apparent passive permeability coefficients ($\text{P(}\text{app}\text{)}$) and cause a significant overestimation of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{(K}{}_{\mathrm{<mtext>m</mtext>}}\text{(}\text{app}\text{))}$ values for active transport processes in intestinal whole tissue preparations. Isolated cells offer several potential advantages in the study of transport processes including a decreased diffusion layer of water adjacent to their absorptive membranes. Initial studies in cells isolated from rat intestine involving measurements of CO₂ and lactate production and O₂ consumption showed that overall metabolic pathways were functioning. Next, unidirectional uptake rates of bile acids across the isolated cell membrane were determined following correction for extracellular fluid contamination with a non-absorbable marker. Using epithelial cells isolated from jejunum $\text{P(}\text{app}\text{)}$ for eight bile acid monomers varied from 24.9 (taurocholate) to 1563 (deoxycholate) nmol/min/100 mg protein/mM. From these data the incremental free energy changes for the addition of a hydroxyl, glycine and taurine group to the bile acid molecule were calculated to be 982, 1040 and 1464 cal/mol, respectively, values similar to those obtained after correction for unstirred water layer resistance in whole tissue preparations. Following subtraction of the passive component in isolated ileal cells complete kinetic curves for taurocholate and taurodeoxycholate yielded $\text{V(}\text{app}\text{)}$ values of 109 and 70 nmol/min per 100 mg, respectively. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{(}\text{app}\text{)}$ values of 0.24 mM (taurocholate) and 0.10 mM (taurodeoxycholate) are lower than usually recorded in whole tissue. Bile acid uptake into cells from ileum, but not jejunum, was affected by temperature, metabolic and competitive inhibition. These studies indicate that isolated epithelial cells are a metabolically viable, relatively purified intestinal preparation which discriminates between active and passive transport processes for bile acids under conditions where unstirred water layer artifacts are are minimized.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Phosphatidylethanolamine methylation in membranes from rat cerebral cortex: Effect of exogenous phospholipids and S-adenosylhomocysteine

In acellular membrane preparations from rat brain, two phosphatidylethanolamine-N-methylase activities were found: a first with a Km ranging near 10^?6M, a second with a Km higher than 10^?3M. Methylase I accepted exogenous phospholipids as substrate. Both enzymes were strongly inhibited by S-adenosylhomocysteine. Our results are compared to those obtained by Crews with synaptosomal preparations and paralleled with $\text{SAH}\text{SAM}$ binding to rat brain membranes.     

Induction,modification and inhibition of rat liver microsomal benzo(a)pyrene hydroxylase; Correlation with the S-9-mediated mutagenicity of benzo(a)pyrene

The effects of various pretreatments $\text{in vivo}$ (3MC, PB, 2 and 4FAA) and of various inhibitors $\text{in vitro}$ (7,8 BF, SKF525A and MN ^R) on the activity of rat liver microsomal BP hydroxylase were analyzed and correlated with the S-9 mediated mutagenicity of BP. 3MC is the only treatment which both induces and modifies the hydroxylase activity; it also specifically increases the enzyme mediated mutagenicity. Miconazole ^R which inhibits all the tested microsomal preparations, also reduces the mutagenicity mediated by all the S-9 preparations whereas the inhibitory effects of 7,8 BF and SKF525A are limited respectively to enzyme preparations from 3MC induced and control or PB treated rats.     

Half-of-the-sites reactivity of the membrane-bound Electrophorus electricus acetylcholine receptor

Equilibrium binding studies of the interaction of activators (decamethonium, carbamylcholine) and inhibitors (d-tubocurarine, α-bungarotoxin) of membrane electrical potential changes in electroplax membrane preparations from $\text{Electrophorus electricus}$ have been carried out at 4°C, in cel Ringer solution, pH 7.0. The properties of the interaction of these chemical mediators with the membrane-bound receptor appear to be similar to those observed with regulatory enzymes which exhibit an allosteric mechanism involving ligand-induced conformational changes. The data presented here show that activators and inhibitors compete for only one-half the available membrane sites. The experiments also provide additional support for the interpretation of kinetic studies which indicated that electroplax membranes contain two different binding sites, one for activators and one for inhibitors of electrical membrane potential changes.     

Differential inhibition of mammalian aminopropyltransferase activities

Rat ventral prostate spermine synthetase was inhibited by 5′-methylthioadenosine and by S-adenosylhomocysteine at concentrations which did not inhibit spermidine synthetase from the same tissue. S-Adenosylethionine inhibited both enzymes to an equal extent. These aminopropyltransferases were also inhibited by diamines not normally present in mammalian cells. All the α,ω-diamines with 3 to 12 C atoms had inhibitory activity, but 1,3-diaminopropane and 1,5-diaminopentane were most active. Spermine synthetase was more sensitive than spermidine synthetase to the effects of these diamines. These results suggest that the relative rates of spermidine and spermine formation $\text{in}$,$\text{vivo}$ might be affected by the intracellular concentration of nucleosides such as S-adenosylhomocysteine. They also raise the possibility that these rates of synthesis could be selectively affected by administration of one or the other of these inhibitors.     

Changes in liver and adipose tissue enzymes and lipogenic activities during the onset of hypothalamic obesity in mice

Introduction of hyperphagia by injection of aurothioglucose resulted in rapid deposition of tissue lipid. The changes in tissue enzyme levels and $\text{in vivo}$ rates of lipogenesis from U-¹⁴C glucose were measured at 2, 4, and 8-week intervals post-aurothioglucose injection. Rapid increases of both enzyme activity and $\text{in vivo}$ lipogenesis were observed during the onset of obesity. The elevated levels of adipose tissue enzyme activities were restored to normal levels 8 weeks post-injection. However, some lipogenic enzymes in liver tissue remained elevated throughout the experimental period. Liver tissue enzymes normally associated with glucogenesis were slightly elevated during the onset of obesity.     

Evidence against the release of prostaglandin-like material from isolated intestinal tissue by pure cholera toxin

Prostaglandin-like material was released from finely cut guinea-pig ileum or human intestinal mucosa during incubation with Krebs solution. The tissue inactivated some of the released material and added PGE₂. There was no significant change in release of prostaglandin-like material when pure cholera toxin was incubated with guinea-pig ileum or human intestinal mucosa. The work is discussed in relation to the action of cholera toxin $\text{in vivo}$.     

D-Ala2, deltaZPhe4-methionine enkephalin amide, a dehydropeptide hormone.

The enkephalin analog, D-Ala², Δ^zPhe⁴ amide, has been prepared and shown to be five times as active as D-Ala²-methionine enkephalin amide $\text{in}\text{vitro}$ and the dehydrophenylalanine moiety conferred complete stability to chymotrypsin on the peptide.     

Electrically induced narcotic-like dependence in the isolated guinea-pig ileum

Segments of guinea-pig ileum stimulated at 10 Hz (0.5 msec, supramaximal voltage) for periods of 15 min respond with an intense, dose-dependent contraction to the $\text{in}$$\text{vitro}$ administration of naloxone. The antagonist produced only a very modest contraction in control segments (0.1 Hz stimulation). Comparison of dose-response curves for naloxone indicated that the sensitizing effect of electrical stimulation at 10 Hz was on the order of 100-fold. The addition of naloxone to the bath immediately before the stimulatory period at 10Hz completely prevented the development of this effect. Moreover, atropine produced a dose-dependent inhibition of the contraction. It was also found that the magnitude of the naloxone-induced contraction is a function of the duration of the stimulatory period and is maximal after about 15 min. The data presented indicate that the contraction induced by naloxone in ilea stimulated at 10 Hz has many similarities to the response produced by the same antagonist in narcotic-dependent preparations. Thus, it is possible that electrical stimulation at high frequencies induces a state of narcotic-like dependence in this tissue. Acetylcholine may be the mediator of the naloxone-induced contraction.