China Initiative for Multi-Domain Intervention (CHINA-IN-MUDI) to Prevent Cognitive Decline: Study Design and Progress.

BACKGROUND: Alzheimer's disease (AD), the most common type of irreversible dementia, is predicted to affect 152 million people by 2050. Evidence from large-scale preventive randomized controlled trials (RCTs) on modifiable risk variables in Europe has shown that multi-domain lifestyle treatments for older persons at high risk of dementia may be practical and effective. Given the substantial differences between the Chinese and European populations in terms of demographics and living conditions, direct adoption of the European program in China remains unfeasible. Although a RCT has been conducted in China previously, its participants were mainly from rural areas in northern China and, thus, are not representative of the entire nation.There is an urgent need to establish cohorts that represent different economic, cultural, and geographical situations in order to explore implementation strategies and evaluate the effects of early multi-domain interventions more comprehensively and accurately. MEDTODS: We developed an integrated intervention procedure implemented in urban neighborhood settings, namely China Initiative for Multi-Domain Intervention (CHINA-IN-MUDI). CHINA-IN-MUDI is a 2-year multicenter open-label cluster-randomised controlled trial centered around a Chinese-style multi-domain intervention to prevent cognitive decline. Participants aged 60-80 years were recruited from a nationally representative study, i.e. China Healthy Aging and Dementia Study cohort. An external harmonization process was carried out to preserve the original FINGER design. Subsequently, we standardized a series of Chinese-style intervention programs to align with cultural and socioeconomic status. Additionally, we expanded the secondary outcome list to include genomic and proteomic analyses. To enhance adherence and facilitate implementation, we leveraged an e-health application. RESULTS: Screening commenced in July 2022. Currently, 1,965 participants have been randomized into lifestyle intervention (n = 772) and control groups (n = 1,193). Both the intervention and control groups exhibited similar baseline characteristics. Several lifestyle and vascular risk factors were present, indicating a potential window of opportunity for intervention. The intervention will be completed by 2025. CONCLUSIONS: This project will contribute to the evaluation of the effectiveness and safety of intervention strategies in controlling AD risk and reducing clinical events, providing a basis for public health decision-making in China.

Depletion of substance P from rat primary sensory neurons by ATP, an implication of P2X receptor-mediated release of substance P.

Effects of ATP on substance P immunoreactivity were examined in cultured dorsal root ganglion neurons. We found that treatment of dorsal root ganglion neurons with ATP significantly depleted substance P immunoreactivity on the neurites and somata of the neurons. The effects of ATP were significantly inhibited by the purinergic P2 receptor antagonists suramin (30 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (10 microM). We also showed that ATP-induced depletion of substance P immunoreactivity from dorsal root ganglion neurons depended on the entry of Ca(2+). In a spinal cord slice preparation, we also found the internalization of neurokinin-1/substance P receptors in many dorsal horn neurons following the application of ATP or alpha,beta-methylene-ATP. Together these results indicate that activation of P2X receptors may result in release of substance P from primary afferent neurons.

ATP P2X receptor-mediated enhancement of glutamate release and evoked EPSCs in dorsal horn neurons of the rat spinal cord.

Presynaptic ATP P2X receptors have been proposed to play a role in modulating glutamate release from the first sensory synapse in the spinal cord. Using spinal cord slice preparations and patch-clamp recordings from dorsal horn neurons in lamina V of the rat spinal cord, we showed that the activation of P2X receptors by alpha,beta-methylene-ATP (alphabetam-ATP) resulted in a large increase in the frequency of spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs). The increases in mEPSC frequency by alphabetam-ATP were not blocked by the Ca(2+) channel blocker, 30 microm La(3+), but were abolished in a bath solution when Ca(2+) was omitted. The increases in mEPSC frequency by alphabetam-ATP were blocked completely by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) at 10 microm. Furthermore, the EPSCs evoked by dorsal root stimulation were potentiated by alphabetam-ATP as well as by the ecto-ATPase inhibitor ARL67156 and were depressed in the presence of P2 receptor antagonists PPADS (10 microm) and suramin (5 microm). The effects of these compounds on the evoked EPSCs were associated with the changes in glutamate release probability of primary afferent central terminals. Our results indicate that alphabetam-ATP-sensitive P2X receptors play a significant role in modulating excitatory sensory synaptic transmission in the spinal cord, and the potential role of endogenous ATP is suggested.

Distribution of P2X1, P2X2, and P2X3 receptor subunits in rat primary afferents: relation to population markers and specific cell types.

We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.

Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents.

We used a "current signature" method to subclassify acutely dissociated dorsal root ganglion (DRG) cells into nine subgroups. Cells subclassified by current signature had uniform properties. The type 1 cell had moderate capsaicin sensitivity (25.9 pA/pF), powerful, slowly desensitizing (tau = 2,300 ms), ATP-activated current (13.3 pA/pF), and small nondesensitizing responses to acidic solutions (5.6 pA/pF). Type 1 cells expressed calcitonin gene-related peptide immunoreactivity (CGRP-IR), manifested a wide action potential (7.3 ms), long duration afterhyperpolarization (57.0 ms), and were IB4 positive. The type 2 cell exhibited large capsaicin activated currents (134.9 pA/pF) but weak nondesensitizing responses to protons (15.3 pA/pF). Currents activated by ATP and alphabeta-m-ATP (51.7 and 44.6 pA/pF, respectively) had fast desensitization kinetics (tau = 214 ms) that were distinct from all other cell types. Type 2 cells were IB4 positive but did not contain either substance P (SP) or CGRP-IR. Similar to capsaicin-sensitive nociceptors in vivo, the afterhyperpolarization of the type 2 cell was prolonged (54.7 ms). The type 3 cell expressed, amiloride-sensitive, rapidly desensitizing (tau = 683 ms) proton-activated currents (127.0 pA/pF), and was insensitive to ATP or capsaicin. The type 3 cell was IB4 negative and contained neither CGRP nor SP-IR. The afterhyperpolarization (17.5 ms) suggested nonnociceptive function. The type 4 cell had powerful ATP-activated currents (17.4 pA/pF) with slow desensitization kinetics (tau = 2, 813 ms). The afterhyperpolarization was prolonged (46.5 ms), suggesting that this cell type might belong to a capsaicin-insensitive nociceptor population. The type 4 cell did not contain peptides. The type 7 cell manifested amiloride-sensitive, proton-activated currents (45.8 pA/pF) with very fast desensitization kinetics (tau = 255 ms) and was further distinct from the type 3 cell by virtue of a nondesensitizing amiloride-insensitive component (6.0 pA/pF). Capsaicin and ATP sensitivity were relatively weak (4.3 and 2.9 pA/pF, respectively). Type 7 cells were IB4 positive and contained both SP and CGRP-IR. They exhibited an exceptionally long afterhyperpolarization (110 ms) that was suggestive of a silent (mechanically insensitive) nociceptor. We concluded that presorting of DRG cells by current signatures separated them into internally homogenous subpopulations that were distinct from other subclassified cell types.

P2X1 receptor subunit immunoreactivity and ATP-evoked fast currents in adult rat dorsal root ganglion neurons.

Triple fluorescent staining for P2X1 and P2X3 subunits and isolectin I-B4 (IB4) were performed on acutely dissociated rat DRG neurons. Immunoreactivity for P2X1 and P2X3 subunits was present separately or together in DRG neurons. P2X1 immunoreactivity was present in both IB4-positive and IB4-negative cells. When combining patch-clamp recordings with immunostaining for the P2X1 and P2X3 subunits on single recorded cells, ATP-evoked fast currents were shown to be present on DRG neurons that have immunoreactivity for the P2X3 subunit only, the P2X1 subunit only, or both P2X1 and P2X3 subunits. These results raised a possibility that, in addition to the P2X3 receptor subunit, the P2X1 subunit may also contribute to functional P2X receptors with fast kinetics in DRG neurons.

Distribution patterns of different P2x receptor phenotypes in acutely dissociated dorsal root ganglion neurons of adult rats.

P2x receptors may be used to detect ATP release from tissues during physiological and pathological conditions. We used whole-cell patch clamp recordings to study the expression of P2x receptor phenotypes, their distribution patterns, and their sensitivity to alphabetamATP and suramin in dorsal root ganglion (DRG) neurons acutely dissociated from adult rats. Based on the onset and decay rates of 10 microM ATP-evoked currents, we showed three types of P2x currents: fast, slow, and mixed. Each of these P2x receptor phenotypes had a distinct distribution pattern among DRG neurons. The fast P2x currents were predominantly expressed in small-diameter, isolectin-B4 (IB4)-positive, and capsaicin-sensitive DRG neurons. The slow P2x currents were expressed in both small and medium DRG neurons, and about half of them were IB4 positive. The mixed P2x currents were also expressed in both small and medium-sized DRG neurons, and most of these neurons were IB4-positive neurons. The slow and mixed P2x current groups had both capsaicin-sensitive and -insensitive DRG neurons. All phenotypes revealed with 10 microM ATP could be inhibited by 30 microM suramin. All DRG neurons with fast or mixed P2x currents were also sensitive to 10 microM alphabetamATP, and alphabetamATP evoked currents similar to those induced by ATP. The group expressing slow P2x currents could be further divided into alphabetamATP-sensitive and -insensitive groups. Thus, the relationships among P2x receptor phenotypes, cell sizes, IB4 positivity, and capsaicin sensitivity are more complicated than previously thought, and different P2x receptors may be involved in both nociceptive and non-nociceptive functions.

Effects of the P2-purinoceptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid on glutamatergic synaptic transmission in rat dorsal horn neurons of the spinal cord.

The effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 microM significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 microM had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P2x receptors. When whole-cell (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2x receptor function at excitatory synapses mediated by AMPA receptors.

Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses.

Painful stimuli to the skin initiate action potentials in the peripheral terminals of dorsal root ganglion (DRG) neurons. These action potentials propagate to DRG central terminals in the dorsal horn of the spinal cord, evoking release of excitatory transmitters such as glutamate onto postsynaptic dorsal horn neurons. P2X receptors, a family of ligand-gated ion channels activated by the endogenous ligand ATP, are highly expressed by DRG neurons. Immunoreactivity to P2X receptors has been identified in the dorsal horn superficial laminae associated with nociceptive DRG central terminals, suggesting the presence of presynaptic P2X receptors. Here we have used a DRG-dorsal horn co-culture system to show that P2X receptors are localized at presynaptic sites on DRG neurons; that activation of these receptors results in increased frequency of spontaneous glutamate release; and that activation of P2X receptors at or near presynaptic DRG nerve terminals elicits action potentials that cause evoked glutamate release. Thus activation of P2X receptors at DRG central terminals can modify sensory signal throughput, and might even initiate sensory signals at central synapses without direct peripheral input. This putative central modulation and generation of sensory signals may be associated with physiological and pathological pain sensation, making presynaptic P2X receptors a possible target for pain therapy.

ATP P2X receptors mediate fast synaptic transmission in the dorsal horn of the rat spinal cord.

ATP has been proposed to mediate synaptic transmission in the spinal cord dorsal horn, particularly in the pathway carrying nociceptive information. Using transverse spinal cord slices from postnatal rats, we show that EPSCs mediated by P2X receptors, and presumably activated by synaptically released ATP, are evoked in a subpopulation of spinal cord lamina II neurons, a region known to receive strong input from nociceptive primary afferents. The P2X receptors on acutely dissociated dorsal horn neurons are nondesensitizing, insensitive to alphabeta methylene ATP, and show strong but variable sensitivity to the antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These characteristics are consistent with a heterogeneous population of P2X receptors, the composition of which includes P2X2, P2X4, and P2X6 receptor subtypes. Our results suggest that ATP-activated P2X receptors in lamina II of the rat spinal cord may play a role in transmitting or modulating nociceptive information.

Characterization of inhibitor-sensitive and -resistant adenosine transporters in cultured human fetal astrocytes.

The kinetic characteristics of [3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of KT and Vmax, and the sensitivities with which nucleoside transport inhibitors blocked [3H] adenosine accumulations were determined in cultured human fetal astrocytes. KT and Vmax values for accumulations of [3H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and the adenosine kinase inhibitor 5'-iodotubercidin (ITU) were 6.2 microM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components, respectively. In the presence of EHNA and ITU, where < 4% of accumulated [3H] adenosine was metabolized, transport per se was measured, and kinetic values for KT and Vmax were 179 microM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [3H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of "concentrative" uptake in that the intracellular levels of [3H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [3H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H]-adenosine transport; for the inhibitor-sensitive components the IC50 values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC50 values were 2.5 microM for NBI, 5.1 microM for dilazep, and 39.0 microM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.

Synaptic strengthening through activation of Ca2+-permeable AMPA receptors.

Postsynaptic Ca2+ elevation during synaptic transmission is an important trigger for short- and long-term changes in synaptic strength in the vertebrate central nervous system. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors, a subfamily of glutamate receptors, mediate much of the excitatory synaptic transmission in the brain and spinal cord. It has been shown that a subtype of the AMPA receptor is Ca2+-permeable and is present in the subpopulations of neurons. When synaptically localized, these receptors should mediate postsynaptic Ca2+ influx, providing a trigger for changes in synaptic strength. Here we show that Ca2+-permeable AMPA receptors are synaptically localized on a subpopulation of dorsal horn neurons, and that they provide a synaptically gated route of Ca2+ entry, and that activation of these receptors strengthens synaptic transmission mediated by AMPA receptors. This pathway for postsynaptic Ca2+ influx may provide a new form of activity-dependent modulation of synaptic strength.

Involvement of bidirectional adenosine transporters in the release of L-[3H]adenosine from rat brain synaptosomal preparations.

Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with L-[3H]adenosine, our objectives here were to determine (1) if L-[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological D-adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. L-[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. L-[3H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 microM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with L-[3H]adenosine alone or I-[3H]adenosine plus unlabeled L-adenosine, D-adenosine, or uridine, L-[3H]adenosine release was inhibited 42% by L-adenosine, 69% by uridine, and 81% by D-adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorotyrosine exerts renal effects and antagonizes renal and gastric responses to atrial natriuretic peptide.

3-Chloro-L-tyrosine (3CT) is an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis. In vivo inhibition of tyrosine hydroxylase results in lower catecholamine levels. 3CT (0.5 mg/kg), administered as a bolus i.v. to anesthetized uninephrectomized rats, elicited increases of 72% and 44% in urinary sodium concentration and volume, respectively, whereas a dose of 1 mg/kg caused increases of 27% and 29%. 3CT, 1 mg/kg, resulted in a 2-fold increase in plasma aldosterone (ALD); 0.5 mg/kg was without significant effect. At a dose of 1 mg/kg 3CT significantly antagonized the renal effects of atrial natriuretic peptide (ANP) (1.5 micrograms kg-1 min-1 by intrarenal infusion), expressed as an enhanced excretion of urine volume (102 +/- 14 vs. 70 +/- 11 microliters/min) and sodium (16.1 +/- 1.8 vs. 11.5 +/- 1.7 microEq/min) and increased osmolar clearance (171 +/- 12 vs. 144 +/- 13 microliters/min). A dose of 0.5 mg/kg of 3CT did not produce these same responses to ANP. The increased urine flow caused by 3CT may reflect reduced norepinephrine synthesis. The inverse dose-effect relationship of 3CT on urine flow rate may result from concomitant depletion of dopamine (DA) and elevated circulating ALD. The antagonism of 3CT on responses to ANP is not at the receptor level, because 3CT did not compete for [125I] ANP binding or inhibit ANP-stimulated guanylate cyclase in kidney cell membranes. It was proposed that the reduced basal sympathetic and renal DA tone, together with the elevated ALD level, account for this antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of diadenosine polyphosphates on sodium nitroprusside-induced soluble guanylate cyclase activity in rat cerebellum.

Diadenosine polyphosphates (ApnAs,n = 1-6 phosphates) have been shown to interact with heme containing proteins and in the present study we determined for guanylate cyclase (GC), a heme containing enzyme whose activity is modulated by adenine nucleotides and nitric oxide, the degree to which ApnAs affect sodium nitroprusside (SNP, a nitric oxide donor)-induced GC activity using cytosolic fractions of rat cerebellum and radioimmunoassays for cGMP. SNP-stimulated GC activity was inhibited by up to 60% by ApnAs; rank order of potency was Ap6A > Ap5A > Ap4A > Ap3A = Ap2A. At concentrations ranging from 0.01 to 100 microM, only Ap6A potentiated in a concentration-dependent manner (apparent EC50 value 1 microM), by up to two-fold, SNP-induced GC activity. The effects of Ap6A on GC activity were neither to changes in SNP degradation nor due to metabolites of Ap6A. These results suggest that ApnAs through its effects on GC activity may play a role in regulating a variety of CNS functions.

[3H]adenosine transport in synaptoneurosomes of postmortem human brain.

[3H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor-stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [3H]adenosine. For 5-s incubations, two kinetically distinguishable processes were identified, i.e., a high-affinity adenosine transport system with Kt and Vmax values of 89 microM and 0.98 nmol/min/mg of protein, respectively, and a low-affinity adenosine transport system that did not appear to be saturable. For incubations with 1 microM [3H]adenosine as substrate, intrasynaptoneurosomal concentrations of [3H]adenosine were 0.26 microM at 5 s and 1 microM at 600 s. Metabolism of accumulated [3H]adenosine to adenine nucleotides was 15% for 5-s, 23% for 15-s, 34% for 30-s, 43% for 60-s, and 80% for 600-s incubations. The concentrations (microM) of total accumulated 3H-purines ([3H]adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of 3H-purines was higher than the initial concentration of 1 microM [3H]adenosine in the reaction medium. For 5-, 15-, 30-, 60-, and 600-s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5'-iodotubercidin, metabolism of the transported [3H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total 3H-purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport and metabolism of D-[3H]adenosine and L-[3H]adenosine in rat cerebral cortical synaptoneurosomes.

The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D-[3H]adenosine and L-[3H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent KT and Vmax values for adenosine transport. For D-[3H]adenosine, high- and low-affinity accumulation processes were present. For the high-affinity system an inverse relationship was found between transport reaction times and KT and Vmax values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D-[3H]adenosine to nucleotides, apparent KT values were 9.4, 8.4, and 4.5 microM, respectively, and Vmax values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an adenosine deaminase inhibitor, and 5'-iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D-[3H]adenosine to 6% with 5-s and 9% with 15-s incubations. This resulted in significantly higher KT values: 36 microM at 5 s and 44 microM at 15 s. At 10-min incubations in the presence of these inhibitors, metabolism of accumulated D-[3H]adenosine was 32%, and apparent KT and Vmax values at this time were not significantly different from those obtained without inhibitors. For L-[3H]adenosine, apparent KT and Vmax values for 20-s incubations were 38.7 microM and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L-[3H]adenosine was observed only at incubations of greater than 30 s.(ABSTRACT TRUNCATED AT 250 WORDS)

L-[3H]adenosine, a new metabolically stable enantiomeric probe for adenosine transport systems in rat brain synaptoneurosomes.

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.