Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Synthesis and evaluation of (S)-[18F]-fluoroethylcarazolol for in vivo beta-adrenoceptor imaging in the brain

The beta-adrenergic receptor ligand (S)-4-(3-(2'-[18F]-fluoroethylamino)-2-hydroxypropoxy)-carbazol ((S)-[18F]-fluoroethylcarazolol) was prepared by reaction of [18F]-fluoroethylamine with the corresponding (S)-epoxide and was evaluated in rats by studying its pharmacokinetics and its binding profile both in vitro and in vivo. In vitro, (S)-fluoroethylcarazolol binds preferentially to beta-adrenoceptors (pK(i)=9.3 for beta(1) and 9.4 for beta(2)) and has less affinity to 5HT(1A) and 5HT(1D) receptors (pK(i)=6.7 and 5.2). In vivo, standard uptake values (SUVs) up to 0.63+/-0.07 in cortical regions were found after 60 min. Metabolites (90%) appeared within 10 min in plasma, whereas, in brain 70-75% parent compound was found after 60 min. Clearance from plasma occurred within 5 min. Cerebral uptake could be blocked by 'cold' fluoroethylcarazolol in every region, except medulla. Uptake was also blocked by propranolol and pindolol, but not by WAY 100635. ICI 89406 hardly lowered [18F] levels in brain. ICI 118551 reduced uptake of [18F] in cerebellum (mainly beta(2)) by 30%. Specific binding (tissue minus medulla values) in various brain regions corresponded with those observed for [18F]-fluorocarazolol (r(2)=0.95) and with in vitro beta-adrenoceptor densities (r(2)=0.76). Autoradiography using phosphor images of (S)-[18F]-fluoroethylcarazolol in rat brain showed the characteristic binding pattern of beta-antagonists, while propranolol treatment resulted in low and homogenous uptake. Regional tissue minus medulla values corresponded with in vitro beta-adrenoceptor densities (r(2)=0.77). We conclude that (S)-[18F]-fluoroethylcarazolol is a high affinity ligand that binds specifically to cerebral beta-adrenoceptors in vivo and may be of use for beta-adrenoceptor imaging in the brain with PET.     

Synthesis and evaluation of radiolabeled antagonists for imaging of beta-adrenoceptors in the brain with PET.

Five potent, lipophilic beta-adrenoceptor antagonists (carvedilol, pindolol, toliprolol and fluorinated analogs of bupranolol and penbutolol) were labeled with either carbon-11 or fluorine-18 and evaluated for cerebral beta-adrenoceptor imaging in experimental animals. The standard radioligand for autoradiography of beta-adrenoceptors, [125I]-iodocyanopindolol, was also included in this survey. All compounds showed either very low uptake in rat brain or a regional distribution that was not related to beta-adrenoceptors, whereas some ligands did display specific binding in heart and lungs. Apparently, the criteria of a high affinity and a moderately high lipophilicity were insufficient to predict the suitability of beta-adrenergic antagonists for visualization of beta-adrenoceptors in the central nervous system.     

Growth requirement for N as a criterion to assess the effects of physical manipulation on nitrate uptake fluxes in spinach

The effects of physical manipulation of hydroponically grown plants of spinach (Spinacia oleracea L., cvs Subito and Glares) on nitrate uptake fluxes were studied in a long-term experiment (3 days), and in short-term label experiments (2 h) with ¹³N-nitrate and ¹⁵N-nitrate. In the long-term experiment, net nitrate uptake rate (NNUR) was measured by following the nitrate depletion in the uptake solution, which was replaced at regular intervals. In the short-term experiments, NNUR and nitrate influx were measured by simultaneous application of ¹³N-nitrate and ¹⁵N-nitrate. Plants were gently transferred into the labelled uptake solution, as is usually done in nutrient uptake studies. In addition, a more severe physical manipulation was carried out, including blotting of the roots, to mimic pretreatments which involve more handling of the plants prior to uptake measurements. Nitrate influx was measured immediately after physical manipulation and after 2 h of recovery. To assess the impact of the physical manipulation the experimentally determined nitrate uptake fluxes were compared with the N demand for growth, defined as relative growth rate (RGR) times plant nitrogen concentration (PNC) of parallel plants, which were left undisturbed. Nitrate influx and efflux were both subject to changes after physical manipulation of the plants. Physical handling, however, did not always result in an alteration of NNUR, which complicates the determination of the length of the recovery period. The impact of the handling and the time course of the recovery depended on the severity of the disturbance and were independent of the light conditions during the experiments. Even after a gentle transfer of the plants, recovery, in most cases, was not complete within 2 h. The data emphasise the need for minimal disturbance of plants during the last hours prior to nutrient uptake measurements.     

Measuring drug-related receptor occupancy with positron emission tomography

Several techniques can be used to measure indirectly the effect of drugs (e.g., EEG, fMRI) in healthy volunteers and in patients. Although each technique has its merits, a direct link between drug efficacy and site of action in vivo usually cannot be established. In addition, when the specific mode of action of a drug has been determined from preclinical studies, it is often not known whether the administered dose is optimal for humans. Both industry and academia are becoming more and more interested in determining the dose-related occupancy of specific targets caused by administration of drugs under test. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are noninvasive imaging techniques that can give insight into the relationship between target occupancy and drug efficacy, provided a suitable radioligand is available. Although SPECT has certain advantages (e.g., a long half-life of the radionuclides), the spatial and temporal resolution as well as the labeling possibilities of this technique are limited. This review focuses on PET methodology for conducting drug occupancy studies in humans.     

Positron emission tomography: the conceptual idea using a multidisciplinary approach

Positron emission tomography (PET) is a method for quantitatively measuring biochemical and physiological processes in vivo by using radiopharmaceuticals labeled with positron-emitting radionuclides such as 11C, 13N, 15O, and 18F and by measuring the annihilation radiation using a coincidence technique. This technique is also used for measurement of the pharmacokinetics of labeled drugs and measurement of the effects of drugs on metabolism. Deviations from normal metabolism can be measured and insight into biological processes responsible for diseases can be obtained.     

Dynamics of multidrug resistance: P-glycoprotein analyses with positron emission tomography

Multidrug resistance (MDR) is characterized by the occurrence of cross-resistance to a broad range of structurally and functionally unrelated drugs. Several mechanisms are involved in MDR. One of the most well-known mechanisms is the overexpression of P-glycoprotein (P-gp), encoded by the MDR1 gene in humans and by the mdr1a and mdr1b genes in rodents. P-gp is extensively expressed in the human body, e.g., in the blood-brain barrier and also in solid tumor tissue. Overexpression of P-gp on tumor membranes might result in MDR of human tumors. To circumvent this resistant phenotype, several P-gp modulators such as cyclosporin A (CsA) are available. Competition between P-gp drugs and modulators results in decreased transport of the drug out of tumor tissue and an increased cellular level of these drugs. For effective clinical treatment it is important to have knowledge about P-gp functionality in tumors. Therefore, we have developed a method to measure the P-gp functionality in vivo with PET and [(11)C]verapamil as a positron-emitting P-gp substrate. The results obtained in rodents and in cancer patients are described in this article.     

18F]FHPG positron emission tomography for detection of herpes simplex virus (HSV) in experimental HSV encephalitis

Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial. Subtle brain infections with HSV may be causally related to neuropsychiatric disorders such as Alzheimer's dementia. We investigated the feasibility of a noninvasive positron emission tomography (PET) imaging technique using [(18)F]FHPG as a tracer for the detection of HSE. For this purpose, rats received HSV-1 (infected group) or phosphate-buffered saline (control group) by intranasal application, and dynamic PET scans were acquired. In addition, the distribution of tracer accumulation in specific brain areas was studied with phosphor storage imaging. The PET images revealed that the overall brain uptake of [(18)F]FHPG was significantly higher for the infected group than for control animals. Phosphor storage images showed an enhanced accumulation of [(18)F]FHPG in regions known to be affected after intranasal infection with HSV. High-performance liquid chromatography metabolite analysis showed phosphorylated metabolites of [(18)F]FHPG in infected brains, proving that the increased [(18)F]FHPG uptake in infected brains was due to HSV thymidine kinase-mediated trapping. Freeze lesion experiments showed that damage to the blood-brain barrier could in principle induce elevated [(18)F]FHPG uptake, but this nonspecific tracer uptake could easily be discriminated from HSE-derived uptake by differences in the tracer kinetics. Our results show that [(18)F]FHPG PET is a promising tool for the detection of HSV encephalitis.     

Quantification of an C-labelled β-adrenoceptor ligand, S-(−)CGP 12177, in plasma of humans and rats

β-Adrenoceptors in human lungs and heart can be imaged with the radioligand 4-[3-[(1,1-dimethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-¹¹C-one (CGP 12177, [¹¹C]I). For quantification of receptor density with compartment models by adjustment of rate constants, an ‘input function’ is required which consists of the integral of the concentration of unmodified ligand in arterial plasma over time. A discrepancy in the literature regarding metabolic stability of [¹¹C]I prompted us to study metabolism in rats by reversed-phase HPLC (RP-HPLC) of trichloroacetic acid extracts of arterial plasma after i.v. injection of [¹¹C]I (> 11.1 TBq/mmol, 11 MBq/kg). Some plasma samples were also directly applied to an internal-surface reversed-phase (ISRP) column. In parallel experiments, tritiated [¹¹C]I was employed and methanol extracts of arterial plasma were analyzed by straight-phase TLC. The three methods were in excellent agreement. Unmodified [¹¹C]I decreased from > 98.5% (³H) or > 99.9% (¹¹C) initially to 57 ± 7% at 80 min post injection to formation of two polar metabolites. Using the RP-HPLC method, no metabolism was detectable in humans up to 30 min after injection of [¹¹C]I (1851 MBq). Deproteinization of plasma with acetonitrile resulted in the formation of a radioactive species (artifact) which eluted immediately after the void volume in RP-HPLC and which could be mistakenly interpreted as a metabolite. Plasma protein binding was low (ca. 30%) in both humans and rats. Association of the radioligand to blood cells suggested that equilibrium between receptor-bound and free radioligand was reached within 15 min after high-specific-activity injections, but only after more than 30 min after low-specific-activity injections.     

Efficiency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with 13N- and 15N-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plants were grown at different nitrate concentrations (0.8 and 4 mM), with mineral composition of growth and uptake solutions identical. Nitrate influx, efflux and net nitrate uptake rate (NNUR) were independent of the external nitrate concentration, despite differences in internal nitrate concentration. At both N regimes, NNUR was adequate to meet the N demand for growth. RGR-related signals predominantly determined the nitrate fluxes. At high RGR (0.25 g g-1 day-1), nitrate influx was 20 to 40% lower and nitrate efflux was 50 to 70% lower than at lower RGR (0.17 g g-1 day-1); efflux:influx ratio (E:I) declined from 0.5 at low RGR to 0.2 at higher RGR. Thus, the efficiency of NNUR substantially increased with increasing RGR. Differences in nitrate translocation between morning and afternoon coincided with differences in nitrate efflux, which is in accordance with the suggested regulation of nitrate efflux by the root cytoplasmic nitrate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.