Synthesis of carbon-11-labeled isonicotinamides as new potential PET agents for imaging of GSK-3 enzyme in Alzheimer’s disease

The authentic standards 2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide (4a) and 2-(cyclopropanecarboxamido)-N-(4-(4-methoxyphenyl)pyridin-3-yl)isonicotinamide (7a), and their corresponding precursors 2-(cyclopropanecarboxamido)-N-(4-hydroxypyridin-3-yl)isonicotinamide (4b) and 2-(cyclopropanecarboxamido)-N-(4-(4-hydroxyphenyl)pyridin-3-yl)isonicotinamide (7b) were synthesized from methyl 2-aminoisonicotinate and cyclopropanecarbonyl chloride with overall chemical yield 47% in three steps, 22% in four steps, 40% in three steps, and 17% in four steps, respectively. The target tracers 2-(cyclopropanecarboxamido)-N-(4-[¹¹C]methoxypyridin-3-yl)isonicotinamide ([¹¹C]4a) and 2-(cyclopropanecarboxamido)-N-(4-(4-[¹¹C]methoxyphenyl)pyridin-3-yl)isonicotinamide ([¹¹C]7a) were prepared from the precursors (4b and 7b) with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ～40-min from EOB.     

Repizine concurrently enhances and inhibits [3H] dextromethorphan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

Ropizine (10 μM) produces a simultaneous enhancement and inhibition of [³H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [³H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [³H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [³H]DM binding sites. However, this study does not rule out thet the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart,cerebral cortex,and ileum of rats

The binding properties of the calcium channel antagonist, [³H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [³H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1 ? 0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [³H]nitrendipine with K_i's that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [³H]nitredipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [³H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La⁺³ and several divalent cations caused an inhibition of [³H]nitrendipine with the rank order of potency being Cd⁺² > La⁺³ > Ni⁺² > Co⁺² ? Mn⁺² > Mg⁺² ? Ba⁺² > Ca⁺².     

Deoxycytidine Transport and Metabolism in the Central Nervous System

Abstract: The mechanisms by which deoxycytidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [³H]deoxycytidine intraarterially, intravenously, and intraventricularly. After intracarotid injection of deoxycytidine (1.0 μM) into rats, deoxycytidine did not pass through the blood-brain barrier at a faster rate than sucrose. [³H]Deoxycytidine, either alone or together with unlabeled deoxycytidine, was infused at a constant rate into conscious adult rabbits. At 130 min, [³H]deoxycytidine readily entered CSF, choroid plexus, and brain. In brain, approx. 60% of the nonvolatile radioactivity was attributable to [³H]deoxycytidine phosphates. The addition of 0.22 mmol/kg unlabeled deoxycytidine to the infusion syringe decreased the phosphorylation of [³H]deoxycytidine in brain by approx. 50%; the addition of 2.2 mmol/kg of unlabeled deoxycytidine to the infusion syringe decreased the relative entry of [³H]deoxycytidine into CSF and brain by approx. 50 and 75%, respectively. Two hours after the intraventricular injection of [³H]deoxycytidine, [³H]deoxycytidine was rapidly cleared from CSF, in part, to brain, where approx. 65% of the [³H]deoxycytidine was converted to [³H]deoxycytidine phosphates. The intraventricular injection of unlabeled deoxycytidine with the [³H]deoxycytidine decreased the phosphorylation of [³H]deoxycytidine in the brain significantly and also decreased the clearance of [³H]deoxycytidine from the CSF. These results were interpreted as showing that the entry of deoxycytidine from blood into CSF occurs by a saturable transport system within the choroid plexus. Once within the CSF, the deoxycytidine can enter brain, undergo phosphorylation to deoxycytidine phosphates, and subsequently be incorporated into DNA.     

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H?2CO]⁺ ions derived from isoflavones and [M+H?B-ring?CO]⁺ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1′,2′,3′,4′,5′,6′,2,3,4-¹³C₉] and [2′,3′,5′,6′,2-D₅] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied ¹³C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H?2CO]⁺ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H?B-ring?CO]⁺ ion is demonstrated to contain a C₂H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSⁿ.     

3H] prostaglandins binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors.

Suspensions of dispersed bovine luteal cells prepared by collagenase digestion of luteal tissue specifically bound [³H]Prostaglandin (PG) E₁ and [³H]PGF_2α. While the number of sites per cell (～ 1.8 × 10⁵) were about the same for both [³H]PGs, the apparent Kds were different: [³H]PGE₁ ? 2.4 nM; [³H]PGF_2α ? 11 nM. The [³H]PGs binding was inhibited in a dose-dependent manner in the presence of increasing concentrations of unlabeled PGs. Potency order for inhibition of [³H]PGE₁ binding was: PGE₂ > PGE₁ > PGF_2α > PGF_1α. The corresponding data for [³H]PGF_2α was: PGF_2α > PGF_1α > PGE₂ > PGE₁. While [³H]PGE₁ and [³H]PGF_2α bind to their own receptors with high affinity, their affinities for each other's binding were extremely low. Thus, these results demonstrate that luteal cells, like plasma membranes isolated from luteal tissue, contain receptors for PGEs and PGF_2α which are discrete with respect to specificity and affinity.     

Biotin transport and metabolism in the central nervous system

The mechanisms by which biotin enters and leaves brain, choroid plexus and cerebrospinal fluid (CSF) were investigated by injecting [³H]biotin either intravenously or intraventricularly into adult rabbits. [³H]biotin, either alone or together with unlabeled biotin was infused at a constant rate into conscious rabbits. At 180 minutes, [³H]biotin had entered CSF, choroid plexus, and brain. In brain, CSF, and plasma, greater than 90% of the nonvolatile³H was associated with [³H]biotin. The addition of 400 mol/kg unlabeled biotin to the infusion syringe decreased the penetration of [³H]biotin into brain and CSF by approximately 70 percent. Two hours after an intraventricular injection, [³H]biotin was cleared from the CSF more rapidly than mannitol and minimal metabolism of the [³H]biotin had occurred in brain. However, 18 hours after an intraventricular injection, approximately 35% of the [³H]biotin remaining in brain had been covalently incorporated into proteins, presumably into carboxylase apoenzymes. These results show that biotin enters CSF and brain by saturable transport systems that do not depend on metabolism of the biotin. However, [³H]biotin is very slowly incorporated covalently into proteins in brain in vivo.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Nicotinic cholinergic receptor in brain detected by binding of α-[3H]bungarotoxin

α-[³H]Bungarotoxin was prepared by catalytic reduction of iodinated α-bungarotoxin with tritium gas. Crude mitochondrial fraction from rat cerebral cortex bound 40 · 10^?15 ?60 · 10^?15 moles of α-[³H]bungarotoxin per mg of protein. This binding was reduced by 50% in the presence of approx. 10^?6 M d-tubocurarine or nicotine, 10^?5 M acetylcholine, 10^?4 M carbamylcholine or decamethonium or 10^?3 M atropine. Hexamethonium and eserine were the least effective of the drugs tested. Crude mitochondrial fraction was separated into myelin, nerve endings, and mitochondria. The highest binding of toxin per mg of protein was found in nerve endings, as well as the greatest nhibition of toxin binding of d-tubocurarine. Binding of α-[³H]bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction indicates that the receptor for the toxin is membrane bound. ¹²⁵I-Labeled α-bungarotoxin, prepared with Na ¹²⁵I and chloramine T, was highly specific for the acetylcholine receptor in diaphragm, however, it was less specific and less reliable than α-[³H]bungarotoxin in brain. We conclude that a nicotinic cholinergic receptor exists in brain, and that α-[³H]bungarotoxin is a suitable probe for this receptor.     

Nucleoside transport in choroid plexus: Mechanism and specificity

In vitro the transport into and release of [³H]thymidine, [³H]deoxyuridine, and [³H]nitrobenzylthioinosine (NBTI) from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. Using the ability of NBTI to inhibit nucleoside efflux from the choroid plexus, the transport of [³H]thymidine and [³H]deoxyuridine into the choroid plexus at 37 °C was measured. Like thymidine, deoxyuridine was transported into the choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production but not intracellular binding or metabolism. The Michaelis-Menten constants (K_T) for the active transport of thymidine and deoxyuridine into the choroid plexus were 13.6 and 7.2 μM, respectively. Deoxyuridine and adenosine were competitive inhibitors of thymidine transport into the choroid plexus with inhibitor constants (K_I) of 6.8 and 14.5 μM, respectively. [³H]NBTI was also transported into the choroid plexus at 37 °C; unlike [³H]thymidine and [³H]deoxyuridine, the release of [³H]NBTI was not inhibited by NBTI itself. These studies provide evidence that the choroid plexus contains an active nucleoside transport system of low specificity for nucleosides, and a separate, saturable efflux system for nucleosides that is very sensitive to inhibition by NBTI. In vivo these systems transport nucleosides from blood into cerebrospinal fluid.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Evidence for Different States of the Dopamine Dl Receptor: Clozapine and Fluperlapine May Preferentially Label an Adenylate Cyclase-Coupled State of the Dl Receptor

Abstract: It has been shown previously that typical neuroleptics have higher affinities for 3,4-dihydroxyphenyl-ethylamine (dopamine) Dl receptors as labeled by(R)- (+)- 8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 -N-3-benzazepine-7-ol ([³H]SCH 23390) than for inhibiting dopamine-stimulated adenylate cyclase. We now report that the atypical neuroleptics, clozapine and fluperlapine, exhibit characteristics opposite to typical neuroleptics, i.e., they have higher affinity for inhibiting dopamine-stimulated adenylate cyclase than [³H]SCH 23390 binding. A variety of compounds, i.e., clozapine, fluperlapine, and dopamine, were tested for their capacity to affect the rate constants of [³H]SCH 23390 binding; these experiments revealed no effect of any tested compound on on-rate or off-rate of [³H]SCH 23390 binding. Treatment of striatal membranes with phospholipase A₂ (PLA₂) caused a rapid decrease in the B_max value of the [³H]SCH 23390 binding with no effect on the K_d value. The adenylate cyclase, both the unstimulated, the dopamine-, fluoride-, and forskolin-stimulated activity, was far less sensitive than [³H]SCH 23390 binding to PLA₂. Treatment of striatal membranes with filipine and (NH₄SO₄ produced, as did PLA₂ treatment, a rapid decline in [³H]SCH 23390 binding. However, opposite to PLA₂ treatment, these agents stimulated the adenylate cyclase. In conclusion, a comparison of the pharmacological characteristics of [³H]SCH 23390 binding and dopamine-stimulated adenylate cyclase suggests the existence of two different Dl binding sites. The rate experiments exclude the possibility of allosterically coupled sites. Instead our results favor that the Dl receptor exists in different states/conformations, i.e., both adenylate cyclase-coupled and uncoupled, and further, that the atypical neuroleptics clozapine and fluperlapine may have adenylate cyclase-coupled dopamine Dl receptors as target.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Evidence for the Involvement of Docosahexaenoic Acid in Cholinergic Stimulated Signal Transduction at the Synapse

[4,5-³H]Docosahexaenoic acid ([³H]DHA) or [9,10-³H]palmitic acid ([³H]PAM) was infused intravenously for 5 min to awake, adult male rats before and after treatment with arecoline (15 mg/kg, i.p.), a cholinergic agonist. Animals were killed 15 min post-infusion, the brains were rapidly removed and subcellular fractions were obtained after sucrose density centrifugation. In control animals, [³H]DHA and [³H]PAM were incorporated into the synaptosomal fractions, representing 50%–60% of total membrane label. Most remaining membrane label (30%–40%) was in the microsomal fraction. Both fractions contained the synaptic marker synaptophysin. The remaining 10% of radioactivity was in the myelin and mitochondrial fractions. Arecoline significantly increased [³H]DHA entry into the synaptosomal fractions by 100% and into the microsomal fraction by 50%. In these fractions 60%–65% of the [³H]DHA was in phospholipid, the rest corresponding to free fatty acid and diacylglycerol. In contrast, arecoline did not change [³H]PAM incorporation into any brain fraction. These results demonstrate that plasma [³H]DHA incorporation is selectively increased into synaptic membrane phospholipids of the rat brain in response to cholinergic activation. The increased incorporation of DHA but not of PAM into synaptic membranes in response to cholinergic stimulation indicates a primary role for DHA in phospholipid mediated signal transduction at the synapse involving activation of phospholipase A₂ and/or C.     

The biosynthesis of glucagon in perfused rat pancreas

The biosynthesis of glucagon was studied by using the recirculated, isolated perfused rat pancreas. [³H]Tryptophan was initially incorporated into acid–ethanol-extractable protein, which on gel filtration was eluted with a molecular weight of about 9000 and contained a small amount of glucagon immunoreactivity. With longer incubation [³H]tryptophan incorporation into a second peak was obtained in an identical position with that of the majority of rat glucagon immunoreactivity. This peak of labelled protein exhibited migration characteristics on polyacrylamide-gel electrophoresis identical with those of rat glucagon and was identified as newly synthesized glucagon by demonstration of specific binding and dissociation behaviour with glucagon antibodies. The incorporation of [³H]tryptophan into acid–ethanol-extractable protein was inhibited by cycloheximide. High concentrations of glucose increased [³H]tryptophan incorporation into high-molecular-weight protein but decreased incorporation into proteins smaller than cytochrome c. The pattern of [³H]leucine incorporation into protein was similar to that of [³H]tryptophan.     

The disposition of [24-3H]cerebrosterol in developing rat brain.

—The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-¹⁴C]cholesterol, [24-³H]cerebrosterol, and [24-³H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-¹⁴C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-³H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-³H]24-epicerebrosterol was essentially the same as that of [24-³H]cerebrosterol. Ratios of radioactivities of [24-³H]cerebrosterol and [4-¹⁴C]cholesterol accentuated the early accumulation of [24-³H]cerebrosterol in myelin, nerve endings, and microsomes, and declining ³H:¹⁴C ratios disclosed the rapid elimination of [24-³H]cerebrosterol and [24-³H]24-epicerebrosterol relative to [4-¹⁴C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-³H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.