Dopamine D3 (Auto)receptors Inhibit Dopamine Release in the Frontal Cortex of Freely Moving Rats In Vivo

Abstract: In freely moving rats, the novel, selective dopamine (DA) D₃ receptor agonist PD 128,907 dose-dependently [effective dose (ED₂₅) = 0.07 mg/kg, s.c.] reduced dialysate levels of DA in the frontal cortex, a structure innervated by the ventral tegmental area (VTA). This action of PD 128,907 (0.16 mg/kg, s.c.) was abolished by a selective DA D₃ receptor antagonist S 14297 (1.25 mg/kg, s.c.), which alone did not modify levels of DA. In contrast to S 14297, its inactive distomer, S 17777, did not modify the actions of PD 128,907. In addition, PD 128,907 dose-dependently and potently inhibited the firing rate of VTA-localized neurons in anesthetized rats (ED₅₀ = 0.001 mg/kg, i.v.). S 14297, but not S 17777, completely reversed the actions of PD 128,907 (0.005 mg/kg, i.v.) with a 50% inhibitory dose of 0.03 mg/kg, i.v. and did not itself significantly modify the firing rate. In conclusion, these data provide the first direct evidence that DA D₃ (auto)receptors modulate (inhibit) the release of DA in the frontal cortex.     

Potent activation of dopamine D3/D2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole

Recombinant, human dopamine D3 and D2 receptors form functional heterodimers upon co-expression in COS-7 cells. Herein, actions of the antiparkinsonian agents, S32504, ropinirole and pramipexole, at D3/D2L heterodimers were compared to their effects at the respective monomers and at split, chimeric D3trunk/D2tail and D2trunk/D3tail receptors: the trunk incorporated transmembrane domains (TDs) I-V and the tail TDs VI and VII. In binding assays with the antagonist [3H]nemonapride, all agonists were potent ligands of D3 receptors showing, respectively, 100-, 18- and 56-fold lower affinity at D2L receptors, mimicking the selective D3 receptor antagonist, S33084 (100-fold). At D3trunk/D2tail receptors, except for ropinirole, all drugs showed lower affinities than at D3 sites, whereas for D2trunk/D3tail receptors, affinities of all drugs were higher than at D2L sites. The proportion of high affinity binding sites recognized by S32504, pramipexole and ropinirole in membranes derived from cells co-expressing D3 and D2L sites was higher than in an equivalent mixture of membranes from cells expressing D3 or D2L sites, consistent with the promotion of heterodimer formation. In contrast, the percentage of high and low affinity sites (biphasic isotherms) recognized by S33084 was identical. Functional actions were determined by co-transfection of a chimeric adenylyl cyclase (AC)-V/VI insensitive to D3 receptors. Accordingly, D3 receptor-transfected cells were irresponsive whereas, in D2L receptor-transfected cells, agonists suppressed forskolin-stimulated cAMP production with modest potencies. In cells co-transfected with D3 and D2L receptors, S32504, ropinirole and pramipexole potently suppressed AC-V/VI with EC50s 33-, 19- and 11-fold lower than at D2L receptors, respectively. S32504 also suppressed AC-V/VI activity at split D3trunk/D2tail and D2trunk/D3tail chimeras transfected into COS-7 cells. In conclusion, antiparkinson agents behave as potent agonists at D3/D2'heterodimers', though any role in their actions in vivo remains to be demonstrated.     

Specific cytosol binding proteins for 25(OH)D3 and 1.25(OH)2D3 in human breast cancers

Binding proteins for 1.25 (OH) 2D3 were investigated in thirty breast cancers. Human breast cancer was shown to contain specific, high affinity cytosol binding proteins for 1.25 (OH) 2D3 and 25 (OH) D3. The binding protein for 1.25 (OH) 2D3 sedimented at 3.7 S and the binding protein for 25 (OH) D3 at about 6.0 S on sucrose density gradient analysis containing 0.3 M KCl and 1 mM dithiothreitol in buffer. Kd for 1.25 (OH) 2D3 were from 0.1 x 10(-11) M to 7.1 x 10(-11) M measured by Scatchard plots. Competition binding studies indicated that the relative specificity of the binding protein for 1.25 (OH) 2D3 much greater than 25 (OH) D3 greater than 1 alpha (OH) D3, 24,25 (OH)2D3 greater than D3 much greater than Estradiol-17 beta. 1.25 (OH) 2D3 receptor-positive was detected in twenty-eight out of thirty breast cancers.     

Modulatory effect of oxytocin and arginine-vasopressin on functional properties of three types of acetylcholine receptors in molluscan neurons.

It was found that 10(-7)-10(-8) mol/l oxytocin (OT) or arginine-vasopressin (AVP) applications produced effects on functional properties of three types of acetylcholine (ACh) receptors on various neurons identified in the ganglia of Helix pomatia under voltage clamp conditions. OT and AVP depressed ACh-induced sodium-potassium-calcium current in neuron RBc3 without shift of reversal potential. Our data show that there are two types (subtypes) of ACh receptors which are connected with chloride current in neurons of Helix pomatia. OT decreased ACh-induced chloride current in neuron D4 and enhanced ACh-induced chloride current in neuron D5. These effects of OT were mimicked by the intracellular injection of cyclic AMP or application of isobutylmethylxanthine and an active cyclic AMP analog. AVP as a rule mimicked the effects of OT on functional properties of ACh receptors, but in neuron F8 effects of OT and AVP were independent. The present results suggest that cyclic AMP may be the second messenger mediating the OT- and AVP-induced modulations of functional properties of three types of ACh-receptors.     

Experimental study of the effects of amiridin and tacrine on learning and memory

The authors studied the influence of amiridin and tacrine on learning and memory in mice and rat by passive avoidance conditioning test at norm and under scopolamine induced amnesia as well as of their effect on acetylcholine esterase (AChE) activity in brain cortex homogenates. Amiridin in doses 0.1 and 0.2 mg/kg showed a beneficial action on conditioning in untreated animals, its effect being comparable with that of piracetam. Tacrine was ineffective. In scopolamine treated animals amiridin and tacrine showed anti-amnestic action at dose of 0.1 mg/kg which was found ineffective with respect to AChE activity. The data suggests that the ameliorating effect of amiridin and tacrine on cognitive abilities in patients with senile dementia is not related their anticholinesterase properties.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Doses of prostaglandin F(2)alpha effective for induction of estrus in goats

A total of 11 cycling does weighing between 24 and 50 kg were injected with varying dosages of prostaglandin F(2)alpha (PGF(2)alpha) between 7 and 10 days into each estrous cycle. Five injections each of 1.25, 2.5, 5.0, or 7.5 mg PGF(2)alpha were alternated with five injections of 1.0 ml saline. Saline treated does served as controls. All does were teased twice daily with a buck and observed for signs of estrus for 5 days post-injection. Daily systemic concentrations of progesterone (P(4)) were determined by radioimmunoassay. The mean (+/- S.E.) hours from injection to estrus was 47 +/- 3.3, 42 +/- 4.3, 44 +/- 8.5, and 43 +/- 5.5 for does receiving 1.25, 2.5, 5.0, and 7.5 mg PGF(2)alpha, respectively. None of the does receiving saline exhibited estrus in the 5-day post-injection observation period. Mean (+/- S.E.) concentrations of systemic P(4) in all does on the day of injection was 4.22 +/- 0.45 ng/ml. Concentrations 24 hours post-injection were 0.21 +/- 0.02, 0.15 +/- 0.05, 0.17 +/- 0.04, 0.16 +/- 0.04, and 4.5 +/- 1.36 ng/ml for does receiving 1.25, 2.5, 5.0, and 7.5 mg PGF(2)alpha, and 1.0 ml saline, respectively. The results suggested that 1.25 mg PGF(2)alpha was effective for induction of estrus in the cycling goat.     

p38 MAPK associated with stereoselective priming by grepafloxacin on O2- production in neutrophils

Grepafloxacin is an asymmetric fluoroquinolone derivative which possesses high tissue penetrability as well as strong, broad-spectrum antimicrobial activities. We recently found that grepafloxacin induced a priming effect on neutrophil respiratory burst induced by N-formylmethionylleucylphenylalanine. In this report, we elucidate the precise mechanism of the priming by grepafloxacin. The R(+) enantiomer of grepafloxacin induced a more potent priming effect than did S(-)-grepafloxacin. R(+)-Grepafloxacin also produced a more potent translocation of both p47- and p67-phox proteins to membrane fractions of neutrophils. Grepafloxacin-induced primed superoxide generation was significantly inhibited by pretreatment with PD169316 and SB203580, p38 mitogen-activated protein kinase (MAPK) inhibitors, but not with PD98059, a specific inhibitor of the upstream kinase that activates p44/42 MAPK, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Grepafloxacin strongly phosphorylated p38 MAP kinase but not p44/42 MAPK or JNK. R(+)-Grepafloxacin showed more potent phosphorylation of p38 MAPK than did S(-)-grepafloxacin, in a time- and concentration-dependent manner. PD169316 significantly inhibited R(+)-grepafloxacin-induced translocation of p47-phox protein to the membrane fraction. Interestingly, grepafloxacin stereospecifically bound to the membrane fractions of neutrophils. These results strongly suggest that grepafloxacin stereospecifically primes neutrophil respiratory burst, and p38 MAPK activation is closely related to the grepafloxacin priming.     

Role of Asp193 in chromophore-protein interaction of pharaonis phoborhodopsin (sensory rhodopsin II)

Pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. By spectroscopic titration of D193N and D193E mutants, the pK(a) of the Schiff base was evaluated. Asp193 corresponds to Glu204 of bacteriorhodopsin (bR). The pK(a) of the Schiff base (SBH(+)) of D193N was approximately 10.1-10.0 (at XH(+)) and approximately 11.4-11.6 (at X) depending on the protonation state of a certain residue (designated by X) and independent of Cl(-), whereas those of the wild type and D193E were >12. The pK(a) values of XH(+) were approximately 11.8-11.2 at the state of SB, 10.5 at SBH(+) state in the presence of Cl(-), and 9.6 at SBH(+) without Cl(-). These imply the presence of a long-range interaction in the extracellular channel. Asp193 was suggested to be deprotonated in the present dodecyl-maltoside (DDM) solubilized wild-type ppR, which is contrary to Glu204 of bR. In the absence of salts, the irreversible denaturation of D193N (but not the wild type and D193E) occurred via a metastable state, into which the addition of Cl(-) reversed the intact pigment. This suggests that the negative charge at residue 193, which can be substituted by Cl(-), is necessary to maintain the proper conformation in the DDM-solubilized ppR.     

Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers.

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel benzopyrano[3,4-c]pyrrole derivatives as potent and selective dopamine D3 receptor antagonist.

A new series of benzopyrano[3,4-c]pyrrole derivatives were synthesized and evaluated for their interaction with dopamine D3 versus D2 receptors. Amongst these compounds, 4x (S 33084) was found to be a potent and selective dopamine D3 receptor antagonist.     

Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D)

The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein. TRIAL REGISTRATION: ClinicalTrials.gov NCT00307021.     

Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the post-natal subventricular zone

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) where dopamine D3 receptors are expressed. Here, we demonstrate that addition of 1 microm apomorphine increases cell numbers in post-natal SVZ cell cultures. This effect was prevented by a co-treatment with haloperidol, sulpiride or U-99194A, a D3-preferring antagonist, and mimicked by the dopamine D3 receptor selective agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT). EC50 values were 4.04 +/- 1.54 nm for apomorphine and 0.63 +/- 0.13 nm for 7-OH-DPAT, which fits the pharmacological profile of the D3 receptor. D3 receptors were detected in SVZ cells by RT-PCR and immunocytochemistry. D3 receptors were expressed in numerous beta-III tubulin immunopositive cells. The fraction of apoptotic nuclei remained unchanged following apomorphine treatment, thus ruling out any possible effect on cell survival. In contrast, proliferation was increased as both the proportion of nuclei incorporating bromo-deoxyuridine and the expression of the cell division marker cyclin D1 were enhanced. These findings provide support for a regulatory role of dopamine over cellular dynamics in post-natal SVZ.     

Effects of a memory enhancing peptide on cognitive abilities of brain-lesioned mice: additivity with huperzine A and relative potency to tacrine.

Alzheimer's disease (AD) and related dementing disorders having cognitive manifestations represent an increasing threat to public health. In the present study, the effects of a memory enhancing NLPR tetra-peptide (MEP), huperzine A (Hup A), or a combination of the two on the cognitive abilities of brain-lesioned mice were evaluated and compared with tacrine in the passive avoidance and Y-water maze tests for the acquisition and retention aspects of cognitive functions. MEP at microg kg(-1) doses, and Hup A or tacrine at mg kg(-1) doses significantly reversed the cognition deficits induced by scopolamine. For acquisition ability, it was observed that mice administered with MEP (4.0 microg kg(-1)) spent less time escaping onto the platform in the water maze than those treated with tacrine (1.5 mg kg(-1)); whereas for memory retention, tacrine-administration resulted in a higher step-through latency in mice at the tested dose regime. In addition, co-administration of MEP (2.0 microg kg(-1)) and Hup A (0.1 mg kg(-1)) exhibited an additive effect resulting in considerable improvements in both acquisition and retention abilities of brain-lesioned mice. The results demonstrated that MEP was highly efficient in the rescue of cognitive abilities of brain-lesioned mice and in particular, the effective doses of MEP were about two orders of magnitude lower than that of tacrine, a therapeutic currently used in the treatment of AD. Moreover, MEP and Hup A were effective at reduced doses when the two were co-administered, providing a rationale for their combined usage in the treatment of cognitive deficits.     

Are the receptors of 1,25-dihydroxyvitamin D3 vitamin K dependent?

Alimentary deficiency of vitamin K in rats causes a decrease in the level of in vivo occupied nuclear 1,25 (OH)2D3 receptors in small intestinal mucosa and an 2-2.5-fold increase in the ability of cytosolic 1,25 (OH)2D3-receptor complexes to bind to heterologous DNA. The 1,25 (OH)2D3 binding by the receptors is thereby unaffected. Preincubation of kidney and intestinal cytosol of rats with the secondary K-avitaminosis induced by vitamin K antagonist with the microsomal vitamin K-dependent gamma-carboxylation system sharply decreases the binding of the 1.25 (OH)2D3-receptor complexes to DNA. In rats treated with the vitamin K antagonist in combination with a low calcium diet, the subsequent maintenance on a high calcium diet does not cause, in contrast with vitamin K-repleted animals, a sharp decrease of the level of the in vivo occupied 1,25 (OH)2D3 receptors. In vitro Ca2+ cations decrease the binding of the 1,25 (OH)2D3-receptor complexes to DNA only in vitamin K-repleted rats (ED50 = 2.5 x 10(-6) M). The existence of a vitamin K-dependent Ca-sensitive mechanism regulating the binding of the 1,25 (OH)2D3 receptor to DNA has been postulated for the first time.     

Regulation of 1,25(OH)2D3 (calcitriol) receptor by vitamin B6

In vitamin B6-deficient rats the concentration of in vivo occupied nuclear and total cellular receptors of 1.25(OH)2D3 increases 1.3-1.7 times, whereas the binding of in vitro occupied receptors to DNA-cellulose increases by 40%. Pyridoxal-5'-phosphate (PLP) added in vitro to solubilized receptors of 1.25(OH)2D3 lowers the ligand binding by 15-25% but causes no dissociation of hormone-receptor complexes formed in vivo. The association of in vitro occupied receptors of 1.25(OH)2D3 with DNA-cellulose is suppressed by PLP (3.5-4.5-fold). It has been shown for the first time that vitamin B6 is a physiological regulator of 1.25(OH)2D3 receptor binding by chromatin and DNA which diminish the concentration of occupied receptors and thus suppress the hormonal response.     

Unique ionotropic receptors for D-aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica

The non-L-glutamate (L-Glu) receptor component of D-aspartate (D-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of D-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to D-Asp. These currents, however, were pharmacologically distinct from D-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (D-TC), while D-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while D-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked D-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, D-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with L-Glu, however, suggested some overlap between L-Glu and D-Asp receptors. Ten minute exposure to 5-HT induced facilitation of D-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.     

Protein kinase A and Ca(v)1 (L-Type) channels are common targets to facilitatory adenosine A2A and muscarinic M1 receptors on rat motoneurons

At the rat motor endplate, pre-synaptic facilitatory adenosine A2A and muscarinic M1 receptors are mutually exclusive. We investigated whether these receptors share a common intracellular signalling pathway. Suppression of McN-A-343-induced M1 facilitation of [3H]ACh release was partially recovered when CGS21680C (an A2A agonist) was combined with the cyclic AMP antagonist Rp-cAMPS. Forskolin, rolipram and 8-bromo-cyclic AMP mimicked CGS21680C blockade of M1 facilitation. Both Rp-cAMPs and nifedipine reduced augmentation of [3H]ACh release by McN-A-343 and CGS21680C. Activation of M1 and A2A receptors enhanced Ca2+ recruitment through nifedipine-sensitive channels. Nifedipine inhibition revealed by McN-A-343 was prevented by chelerythrine (a PKC inhibitor) and Rp-cAMPS, suggesting that Ca(v)1 (L-type) channels phosphorylation by PKA and PKC is required. Rp-cAMPS inhibited [3H]ACh release in the presence of phorbol 12-myristate 13-acetate, but PKC inhibition by chelerythrine had no effect on release in the presence of 8-bromo-cyclic AMP. This suggests that the involvement of PKA may be secondary to M1-induced PKC activation. In conclusion, competition of M1 and A2A receptors to facilitate ACh release from motoneurons may occur by signal convergence to a common pathway involving PKA activation and Ca2+ influx through Ca(v)1 (L-type) channels.     

Novel vitamin D3 derivatives, 26-homo-delta 22-dehydro-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-dehydro-1 alpha,25(R)-dihydroxyvitamin D3: preferential activity in c-myc mRNA production and in induction of phenotypic differentiation of HL-60 cells

Novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-1 alpha,25(R)-dihydroxyvitamin D3 were compared with the native hormone, 1,25-dihydroxyvitamin D3, and with other vitamin D3 derivatives, in inhibition of cell growth, induction of phenotypic differentiation, and c-myc mRNA reduction of HL-60 cells. The degree of inhibition in cell growth caused by 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 was the greatest followed by 26-homo-delta 22-1 alpha,25(R)-(OH)2D3. The ability to reduce NBT was parallel to that to inhibit cellular proliferation. 26-homo-delta 22-1 alpha,25(S)-(OH)2D3, 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, 24-homo-24-F2-1 alpha,25-(OH)2D3, and 1 alpha,24(R)-(OH)2-26-Cl-D3 were more active than 1 alpha,25-(OH)2D3 in the induction of OK-M1+ and OK-Mo-2+ HL-60 cells. Using two color flow cytometric analysis, the percentages of OK-M5(+)- and OK-DR(+)-HL-60 cells were 33% in the treatment with 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 plus interferon-gamma (IFN-gamma) but 14% in the treatment with 1 alpha,25-(OH)2D3 plus IFN-gamma. 26-Homo-delta 22-1 alpha,25(S)-(OH)2D3 has an inhibitory effect on c-myc reduction in treated HL-60 cells. These results suggest that the novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 and 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, have preferential activity in inducing phenotypic differentiation and in inducing cell proliferation related c-myc mRNA activity.     

Binding characteristics and sensitivity to endogenous dopamine of [11C]-(+)-PHNO, a new agonist radiotracer for imaging the high-affinity state of D2 receptors in vivo using positron emission tomography

[11C]-(+)-PHNO (4-propyl-9-hydroxynaphthoxazine) is a new agonist radioligand that provides a unique opportunity to measure the high-affinity states of the D2 receptors (D2-high) using positron emission tomography (PET). Here we report on the distribution, displaceablity, specificity and modeling of [11C]-(+)-PHNO and compare it with the well characterized antagonist D2 radioligand, [11C]raclopride, in cat. [11C]-(+)-PHNO displayed high uptake in striatum with a mean striatal binding potential (BP) of 3.95 +/- 0.85. Pre-treatment with specific D1 (SCH23390), D2 (raclopride, haloperidol) and D3 receptor (SB-277011) antagonists indicated that [11C]-(+)-PHNO binding in striatum is specific to D2 receptors. Within-subject comparisons showed that [11C]-(+)-PHNO BP in striatum was almost 2.5-fold higher than that measured with [11C]-(-)-NPA ([11C]-(-)-N-propyl-norapomorphine). Comparison of the dose-effect of amphetamine (0.1, 0.5 and 2 mg/kg; i.v.) showed that [11C]-(+)-PHNO was more sensitive to the dopamine releasing effect of amphetamine than [11C]raclopride. Amphetamine induced up to 83 +/- 4% inhibition of [11C]-(+)-PHNO BP and only up to 56 +/- 8% inhibition of [11C]raclopride BP. Scatchard analyses of [11C]-(+)-PHNO and [11C]raclopride bindings in two cats showed that the Bmax obtained with the agonist (29.6 and 32.9 pmol/mL) equalled that obtained with the antagonist (30.6 and 33.4 pmol/mL). The high penetration of [11C]-(+)-PHNO in brain, its high signal-to-noise ratio, its favorable in vivo kinetics and its high sensitivity to amphetamine shows that [11C]-(+)-PHNO has highly suitable characteristics for probing the D2-high with PET.