Synthesis of 1-[11C]methylpiperidin-4-yl propionate ([11C]PMP) for in vivo measurements of acetylcholinesterase activity.

Synthesis of 1-[11C]methylpiperidin-4-yl propionate ([11C]PMP), an in vivo substrate for acetylcholinesterase, is reported. An improved preparation of 4-piperidinyl propionate (PHP), the immediate precursor for radiolabeling, was accomplished in three steps from 4-hydroxypiperidine by (a) protection of the amine as the benzyl carbamate, (b) acylation with propionyl chloride, and (c) deprotection of the carbamate by catalytic hydrogenation. The final product was obtained in an overall 82% yield. Reaction of the free base form of PHP with [11C]methyl trifluoromethanesulfonate at room temperature in N,N-dimethylformamide, followed by high performance liquid chromatography (HPLC) purification, provided [11C]PMP in 57% radiochemical yield, > 99% radiochemical purity, and > 1500 Ci/mmol at the end of synthesis. The total synthesis time from end-of-bombardment was 35 min. [11C]PMP can thus be reliably prepared for routine clinical studies of acetylcholinesterase in human brain using positron emission tomography.

Synthesis of 11C-labeled desipramine and its metabolite 2-hydroxydesipramine: potential radiotracers for PET studies of the norepinephrine transporter.

The antidepressant desipramine (DMI) and its principal metabolite 2-hydroxydesipramine (HDMI) have been radiolabeled with 11C for PET studies. The normethyl precursors of DMI and HDMI were synthesized from iminodibenzyl in 35% and 11% overall yield, respectively. Direct methylation of the normethyl precursor with [11C]CH3I, followed by HPLC purification, provided [11C]DMI and [11C]HDMI in 18-30% and 15-23% decay-corrected radiochemical yields, respectively, in a 45 min synthesis time from end of bombardment. The specific activities of the two radiotracers were >1459 Ci/mmol at the end of synthesis. [11C]DMI and [11C]HDMI have potential utility as PET radiotracers for the norepinephrine transporter.

Synthesis and biological evaluation in mice of (2-[11C]methoxy)-6',7'-dihydrorotenol, a second generation rotenoid for marking mitochondrial complex I activity.

Evidence has accumulated suggesting that impairment of the function of the complexes of the mitochondrial respiratory chain might be involved in the pathology of neurological diseases including Parkinson's and Huntington's diseases. Recently we reported the synthesis of (2-[11C]methoxy)rotenone ([11C]ROT) as a tool for in vivo studies of complex I. In an effort to develop a complex I imaging radiotracer which might be easier to synthesize and less likely to be metabolized, we prepared (2-[11C]methoxy)-6',7'-dihydrorotenol ([11C]DHROT). The radiotracer was synthesized by [11C]methylation of 2-O-desmethyl-6',7'-dihydrorotenol under basic [11C]alkylation conditions. (2-[11C]Methoxy)-6',7'-dihydrorotenol was produced in 30-35% radiochemical yields (decay corrected), with synthesis times shorter than 35 min. Radiochemical purities were over 95% and specific activities averaged 1000 Ci/mmol. The brain distributions of [11C]ROT and [11C]DHROT were investigated in mice after intravenous injections. For both radiotracers, distribution of radioactivity was similar in all brain regions examined. However, significantly higher uptake was observed with [11C]DHROT than with [11C]ROT, indicating that the alterations introduced in the structure of rotenone during the design of [11C]DHROT resulted in a tracer with greater brain barrier permeability.

The oocyte nucleus isolated in oil retains in vivo structure and functions.

We describe a technique for isolating the nucleus of the giant amphibian oocyte under paraffin oil. The method precludes the losses of small solutes and proteins that accompany isolation of nuclei into aqueous media. An individual oocyte is blotted, placed under oil, punctured near the animal pole and then squeezed to gently extrude the nucleus into the oil, thereby avoiding exposure to any aqueous environment. Light and electron microscopy of the oil-isolated nucleus demonstrate that its in vivo morphology is preserved. We also describe techniques that facilitate the study of nuclear functions under oil. Oil-isolated oocyte nuclei retain many in vivo functions for several hours, including size-selective envelope permeability, RNA synthesis and the ability to break down in response to cdc2/cyclin meiotic maturation promoting factor.

Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange.

We previously showed that gonadotropin increases the K+ activity in Xenopus oocytes and that this is a signal for increased translation. However, K+ need not act to control synthesis directly but may act through an unidentified downstream effector. Using microinjection to vary the salt content of oocytes and concomitantly measuring [3H]leucine incorporation, we found that small changes in Mg2+ greatly affect translation rates. (Ca2+ had little influence.) By measuring intracellular ion activities, we found that oocyte cations existed in a buffer-like (ion-exchange) equilibrium in which K+ and Mg2+ are the preponderant monovalent and divalent cations. Hence, increasing cellular K+ activity might increase translation by causing Mg2+ activity to rise. If so, the increased translation rates produced by hormone treatment or K+ injection would be prevented by EDTA, a Mg2+ chelating agent. This prediction was tested and confirmed. We conclude that, when gonadotropin increases K+ activity, the cell's internal ion-exchange equilibrium is altered thereby increasing Mg2+ activity and this up-regulates translation.

Water, potassium, and sodium during amphibian oocyte development.

Water, K+, and Na+ were measured in Rana pipiens oocytes during growth and prematurational development using low-temperature microdissection. Whole oocytes were analyzed during previtellogenic and vitellogenic growth. Ooplasm and germinal vesicle (nucleus) were analyzed at the onset and conclusion of vitellogenic growth. In previtellogenic oocytes (less than 40 micrograms), water, K+, and Na+ concentrations resembled those in somatic cells and were independent of cell size. With the onset of yolk deposition, water and K+ concentrations progressively decreased and Na+ progressively increased. These changes were restricted to ooplasm, the site of yolk deposition. In full-grown oocytes, vegetal ooplasm, with greater yolk density than animal ooplasm, contained less water and K+ and more Na+ than animal ooplasm. Collectively, the data indicate that yolk is poorer in water and K+ and richer in Na+ than yolk-free ooplasm (cytoplasm) or nucleoplasm. Yolk concentrations were estimated to be approximately 32%, water, approximately 69 meq K+/liter H2O, and approximately 94 meq Na+/liter H2O. Several nonyolk parameters, such as cation activities and nucleoplasmic binding, also appear to change during oogenesis.

Reference phase analysis of free and bound intracellular solutes. I. Sodium and potassium in amphibian oocytes.

A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.