Differential regulation of nicotinic receptor-mediated neurotransmitter release following chronic (-)-nicotine administration

The objective of this study was to compare nAChR-mediated neurotransmitter release from slices of rat striatum, frontal cortex and hippocampus following chronic (-)-nicotine (Nic) administration (tartrate salt, 2 mg/kg twice daily for 10 days). Binding studies were also conducted to measure changes in receptor density. Relative to saline-treated animals, the number of nAChRs measured by [(3)H]-cytisine (CYT) binding was significantly increased in all brain regions examined by 15% to 25% following chronic Nic administration. Using a relatively high throughput method to measure neurotransmitter release, we found that Nic, CYT, and (+/-)-epibatidine (EB) evoked similar concentration-dependent striatal [(3)H]-dopamine (DA) and hippocampal [(3)H]-norepinephrine (NE) release from both saline (rank order of potency for [(3)H]-DA: EB>CYT>Nic; pEC(50) values, EB (9 +/- 0.1), CYT (8 +/- 0.13), Nic (7.3 +/- 0.19); rank order potency for [(3)H]-NE: EB>Nic=CYT; pEC(50) values, EB (8 +/- 0.18), Nic (5.5 +/- 0.09), CYT (5.12 +/- 0.1)) -and Nic-treated animals (pEC(50) values [(3)H]-DA, EB (9.5 +/- 0.15), Nic (8 +/- 0.16, CYT (6.6 +/- 0.52); [(3)H]-NE, EB (8.4 +/- 0.23), Nic (5.19 +/- 0.1), CYT (5.18 +/- 0.29)). Although no change in potency was detected between the two treatment groups, the agonist efficacies in both tissues were significantly reduced by approximately 17-54% following chronic Nic administration. In contrast to striatum, treatment with Nic did not affect the maximal [(3)H]-DA response (efficacy) in the frontal cortex. However, as observed in the striatum, no change in agonist potency was observed in the frontal cortex following chronic Nic administration (pEC(50) values, saline; EB (9.2 +/- 0.2), >CYT (6.95 +/- 0.75) = Nic (6.9 +/- 0.16); Nic-treated, EB (9 +/- 0.42)>CYT (6.88 +/- 0.27) = Nic (7.1 +/- 0.17)). Chronic Nic treatment did not significantly affect KCl-evoked [(3)H]-NE release from hippocampus or [(3)H]-DA release from frontal cortex or striatum. Since previous work has demonstrated that different nAChR subtypes display various sensitivities to chronic Nic exposure, we suggest that the subtypes of nAChRs involved in regulating [(3)H]-DA release may be different in the striatum and frontal cortex. These results support findings from earlier studies comparing the pharmacology of nAChR-evoked striatal versus cortical [(3)H]-DA release.     

Metabotropic glutamate 1 (mGlu1) receptor antagonists enhance GABAergic neurotransmission: a mechanism for the attenuation of post-ischemic injury and epileptiform activity?

Selective antagonists of mGlu1 metabotropic glutamate receptors attenuate neuronal death in models of cerebral ischemia. Because GABAergic mechanisms have recently been proposed to contribute to these neuroprotective effects, we examined the effects of selective mGlu1 antagonists characterized in our laboratory on GABAergic transmission in three different models of neuropathology. In rat organotypic hippocampal slices exposed to oxygen-glucose deprivation, the mGlu1 antagonists AIDA, CBPG and 3-MATIDA reduced CA1 pyramidal cell loss when added to the medium during the insult and the subsequent recovery period. This effect was mimicked by the GABA(A) and GABA(B) agonists muscimol and baclofen and partially prevented by the antagonists bicuculline and CGP 55845. In gerbils subjected to global ischemia, protection of CA1 pyramidal cells by transdialytic perfusion of AIDA and CBPG was associated with a significant increase in the basal and ischemic output of GABA and minor changes in the output of glutamate. In a mouse cortical wedge model, both muscimol and 3-MATIDA reduced the frequency of spontaneous bursts induced by 4-aminopyridine and this reduction was prevented by co-perfusion with bicuculline. Taken together, our results suggest that the release of GABA, and the subsequent activation of GABA receptors, may contribute to the attenuation of post-ischemic neuronal damage and epileptiform activity induced by mGlu1 receptor antagonists.     

Pharmacokinetics of lovastatin extended-release dosage form (Lovastatin XL) in healthy volunteers

The purpose of this study was to evaluate pharmacokinetics and dose proportionality of lovastatin extended-release dosage form (ER-lovastatin) in the dosage levels of 10, 20 and 40 mg in 9 healthy male subjects. Each subject was randomized to receive a single oral dose of ER-lovastatin either 10, 20 or 40 mg in a three-way crossover design with a washout period of 7 days between the treatments. Subjects were served dinner at approximately 5:30 PM followed by dosing at approximately 10:00 PM in each study period. Serial plasma samples were collected up to 48 h after dosing and assayed for lovastatin and its active metabolite lovastatin acid using an LC/MS/MS method. The plasma concentration-time profiles of lovastatin and its active metabolite lovastatin acid exhibited delayed- and extended-release characteristics at each dose. Mean (+/-) values for the C(max) of lovastatin were 1.04+/-0.43, 2.03+/-0.65 and 4.03+/-3.02 ng/ml for the 10, 20 and 40 mg dosage, respectively. The corresponding values for the AUC(0-48 h) of lovastatin were 14.6+/-7.8, 34.1 +/-13.7, and 53.9+/-35.6 ng h/ml. The same tendency was also found for C(max) and AUC(0-48 h) values of lovastatin acid. Results from this study demonstrated as the dose of ER-lovastatin increased from 10 to 40 mg, the C(max) and AUC(0-48 h) values of lovastatin as well as lovastatin acid appeared to increase linearly.     

Novel gastroretentive dosage forms: evaluation of gastroretentivity and its effect on riboflavin absorption in dogs

Purpose. The purpose of this study was to design novel gastroretentive dosage forms (GRDFs) based on unfolding multilayer polymeric films, to investigate the mechanism of their gastroretentivity in dogs, and to assess the effect of compounding a narrow absorption window drug in a GRDF on the drug's absorption properties. Methods. Dosage forms (DFs) with different dimensions and mechanical properties were administered to beagle dogs with acidic buffer (pH=1.5), whose gastric retention time (GRT) was then determined by X-ray pictures. Concurrent administration of radiopaque markers was used to assess the effect of the GRDF and/or acidic buffer on GRT. The absorption of riboflavin from a prototype GRDF was compared with a nongastroretentive controlled-release DF and to an oral solution of the drug. Results. Large DFs (2.5 × 2.5 cm) containing rigid frame had prolonged GRT (>4 h). Administration of 400 mL of acidic buffer (or water) prolonged GRT whereas the GRDF did not cause additional prolongation. The extended absorption phase (>48 h) of riboflavin administered in a GRDF led to 4-fold increased bioavailability. Conclusion. The combination of large dimensions with rigidity produce gastroretentivity that can be used to improve absorption properties of a model of narrow absorption window drugs in the gastrointestinal tract.     

Pharmaceutical Evaluation of Gas-Filled Microparticles as Gene Delivery System

Purpose. To produce and characterize a nonviral ultrasound-controlled release system of plasmid DNA (pDNA) encapsulated in gas-filled poly(D,L-lactide-co-glycolide) microparticles (PLGA-MPs). Methods. Different cationic polymers were used to form pDNA/polymer complexes to enhance the stability of pDNA during microparticle preparation. The physico-acoustical properties of the microparticles, particle size, pDNA integrity, encapsulation efficiency and pDNA release behavior were studied in vitro. Results. The microparticles had an average particle size of around 5 m. More than 50% of all microparticles contained a gas core, and when exposed to pulsed ultrasound as used for color Doppler imaging create a signal that yields typical color patterns (stimulated acoustic emission) as a result of the ultrasound-induced destruction of the microparticles. Thirty percent of the pDNA used was successfully encapsulated and approximately 10% of the encapsulated pDNA was released by ultrasound within 10 min. Conclusions. Plasmid DNA can be encapsulated in biodegradable gas-filled PLGA-MPs without hints for a structural disintegration. A pDNA release by ultrasound-induced microparticle-destruction could be shown in vitro.     

A New Mathematical Model Quantifying Drug Release from Bioerodible Microparticles Using Monte Carlo Simulations

Purpose. The major objectives of this study were to 1) develop a new mathematical model describing all phases of drug release from bioerodible microparticles; 2) evaluate the validity of the theory with experimental data; and 3) use the model to elucidate the release mechanisms in poly(lactide-co-glycolide acid)-based microspheres. Methods. 5-Fluorouracil-loaded microparticles were prepared with an oil-in-water solvent extraction technique and characterized in vitro. Monte Carlo simulations and sets of partial differential equations were used to describe the occurring chemical reactions and physical mass transport phenomena during drug release. Results. The new mathematical model considers drug dissolution, diffusion with nonconstant diffusivities and moving boundary conditions, polymer degradation/erosion, time-dependent system porosities, and the three-dimensional geometry of the devices. In contrast with previous theories, this model is able to describe the observed drug release kinetics accurately over the entire period of time, including 1) initial burst effects; 2) subsequent, approximately zero-order drug release phases; and 3) second rapid drug release phases. Important information, such as the evolution of the drug concentration profiles within the microparticles, can be calculated. Conclusions. A new, mechanistic mathematical model was developed that allows further insight into the release mechanisms in bioerodible microparticles.     

Subtype-specific roles of cAMP phosphodiesterases in regulation of atrial natriuretic peptide release

cAMP is known to control the release of atrial natriuretic peptide. To define the roles of cyclic nucleotide phosphodiesterase subtypes in the regulation of atrial natriuretic peptide (ANP) release, experiments were done with perfused beating rabbit atria. Phosphodiesterase 3 subtype-specific inhibitors, milrinone and cilostamide, inhibited myocytic ANP release with a concomitant increase in cAMP efflux. Similarly, trequinsin, another phosphodiesterase 3 inhibitor, decreased ANP release. A phosphodiesterase 4 subtype-specific inhibitor, rolipram, did not significantly change ANP release but increased AMP efflux. Also, 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (Ro 20-1724), another phosphodiesterase 4 inhibitor, did not significantly change ANP release. The cAMP efflux was higher in the atrium treated with rolipram than in the atrium treated with milrinone or cilostamide. The data show that the cAMP pool, which is metabolized by phosphodiesterase 3, but not phosphodiesterase 4, is closely related to the basal regulation of atrial ANP release. The results suggest that intracellular cAMP is compartmentalized in the regulation of atrial ANP release, and that the release is controlled by a phosphodiesterase subtype-specific mechanism.     

Protein kinase C regulation of glycine and gamma-aminobutyric acid release in brain stem auditory nuclei

Protein kinase C (PKC) can regulate transmitter release in several brain areas. We determined if PKC could regulate the electrically evoked release of radiolabeled glycine (Gly) and gamma-aminobutyric acid (GABA) in dissected samples of several brain stem auditory nuclei, such as the major subdivisions of the cochlear nucleus (CN) and the main nuclei of the superior olivary complex (SOC). The PKC activators, phorbol 12,13-diacetate (PDA) or phorbol 12,13-dibutyrate (PDBu) (3 microM), elevated the release by 1.4- to 2.0-fold. The PKC inhibitor, Ro31-8220 (50 nM), did not alter the release in most of the tissues but blocked the stimulatory effects of PDA and PDBu. This suggested that PKC positively regulates glycinergic and GABAergic release in the sampled nuclei. In the dorsal CN (DCN), Ro31-8220 elevated the release of [(14)C]Gly by 23%, suggesting that PKC negatively regulates glycinergic release in a proportion of DCN synapses. We also determined if PKC could regulate release after unilateral cochlear ablation (UCA). In the anteroventral (AVCN) and posteroventral (PVCN) CN and in the lateral (LSO) and medial (MSO) superior olive, the stimulatory effects of PDBu declined after this lesion and Ro31-8220 failed to alter release. Since UCA failed to alter release in these tissues, the stability of the release correlated with the lack of regulatory capacity of PKC. In the DCN and the medial nucleus of the trapezoid body (MNTB), the stimulatory effects of PDBu persisted after UCA. We previously demonstrated a postablation decline of Gly release in the DCN and elevated GABA release in the MNTB. Treatment of these tissues with Ro31-8220 reversed these changes in release. These findings suggested that PKC regulation persisted in the DCN and MNTB after UCA. Moreover, endogenous regulatory mechanisms activated after UCA probably act through PKC to alter release in these tissues. Thus, limiting PKC activation or activity might ameliorate pathological symptoms that accompany hearing loss and that stem from these plasticities in the DCN and MNTB.     

Dopamine release rather than content in the caudate putamen is associated with behavioral changes in the iron rat model of Parkinson's disease

The effects of intranigral iron injection on dopamine (DA) release and content in the caudate putamen (CPu) and their relationship to DA-related behavioral response were investigated in rats. Different concentrations of FeCl(3) (10, 20, and 40 microg) and saline were injected separately into the left substantia nigra. In some experiments, rats were pretreated with desferrioxamine or saline before iron injection. After 3 weeks, changes in behavioral response, DA release, and DA content in the CPu were determined. In all iron injection groups (10, 20, and 40 microg), DA content in the lesioned side of the brain was significantly decreased, showing a significant linear correlation (R(2) = 0.981, P = 0.01), and DA turnover ratio significantly increased (both P = 0.01, 0.01 and 0.001 vs unlesioned sides, respectively). However, injection dosages of 10 or 20 microg of iron did not lead to significant changes in DA release in the CPu or in behavioral response. At the 40-microg dosage, it was found that DA release in the lesioned side and rearing activity both were significantly reduced (all P = 0.01 vs unlesioned side or control) and apomorphine-induced rotation was observed. Pretreatment with desferrioxamine significantly inhibited the effect of iron on DA release and content. These results demonstrate that iron injection can damage dopaminergic neurons and suggest that DA release, rather than DA content, in the CPu is associated with DA-related behavioral changes in this PD model.     

Subcellular relationships between cholinergic terminals and estrogen receptor-alpha in the dorsal hippocampus

Cholinergic septohippocampal neurons are affected by circulating estrogens. Previously, we found that extranuclear estrogen receptor-alpha (ERalpha) immunoreactivity in presynaptic profiles had an overlapping distribution with cholinergic afferents in the rat hippocampal formation. To determine the subcellular relationships between cholinergic presynaptic profiles and ERalpha, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and ERalpha and examined by electron microscopy. Within the hippocampal formation, immunoreactivities for VAChT and ERalpha both were presynaptic, although their subcellular targeting was distinct. VAChT immunoreactivity was found exclusively within presynaptic profiles and was associated with small synaptic vesicles, which usually filled axon terminals. VAChT-labeled presynaptic profiles were most concentrated in stratum oriens of the hippocampal CA1 region and dentate inner molecular layer and hilus. In contrast, ERalpha immunoreactivity was found in clusters affiliated either with select vesicles or with the plasmalemma within preterminal axons and axon terminals. ERalpha-immunoreactive (IR) presynaptic profiles were more evenly distributed between hippocampal lamina than VAChT-IR profiles. Quantitative ultrastructural analysis revealed that VAChT-IR presynaptic profiles contained ERalpha immunoreactivity (ranging from 3% to 17%, depending on the lamina). Additionally, VAChT-IR presynaptic profiles apposed ERalpha-IR dendritic spines, presynaptic profiles, and glial profiles; many of the latter two types of profiles abutted unlabeled dendritic spines that received asymmetric (excitatory-type) synapses from unlabeled terminals. The presence of ERalpha immunoreactivity in cholinergic terminals suggests that estrogen could rapidly and directly affect the local release and/or uptake of acetylcholine. The affiliation of cholinergic terminals with excitatory terminals near ERalpha-labeled dendritic spines or glial profiles suggests that alterations in acetylcholine release could indirectly affect estrogen-modulated structural plasticity.