Pig Striatum Research Articles

The DaNeX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson's disease: preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.

A multicenter study is under way to investigate the efficacy of allografting of embryonic mesencephalic neurons in a pig model of Parkinson's disease. We have first established that a stable parkinsonian syndrome can be established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of adult male Göttingen minipigs. We are now using positron emission tomography (PET) methods for testing the physiological responses to MPTP intoxication and the time course of the response to several treatment strategies. We now report preliminary results obtained in 11 pigs employed in the initial phase of the study; the completed study shall ultimately include 30 pigs. Animals were randomly assigned to one of five groups: 1) Control, 2) MPTP intoxication, 3) MPTP intoxication followed by allograft, 4) MPTP intoxication followed by allograft with immunosuppression, and 5) MPTP intoxication followed by allograft with immunosuppression and co-grafting of immortalized HiB5 cells, which had been manipulated to secrete glia cell line-derived neurotrophic factor (GDNF) (approximately 2 ng GDNF/h/10(5) cells). MPTP was administered (1 mg/kg/day, SC) for 7-10 days until the pigs had developed mild parkinsonian symptoms of muscle rigidity, hypokinesia, and impaired coordination, especially of the hind limbs. Approximately 2 weeks after the last MPTP dose, animals received a T1-weighted magnetic resonance imaging (MRI) scan, and a series of dynamic PET recordings. After the first series of PET scans, four grafts of porcine embryonic mesencephalic tissue (E28 days) were placed in each striatum of some MPTP-intoxicated pigs, using MRI-based stereotactic techniques. Immunosuppression of some animals with cyclosporin and prednisolone began just prior to surgery. Two more series of PET scans were performed at 4-month intervals after surgery. After the last scans, pigs were killed and the brains were perfused for unbiased stereological examination of cytological and histochemical markers in striatum and substantial nigra. The behavioral impairment of the animals (the "Parkinson's score") had been evaluated throughout the 8-month period. Kinetic analysis of the first set of PET scans has indicated that the rate constant for the decarboxylation of FDOPA in catecholamine fibers was reduced by 33% in striatum of the mildly parkinsonian pigs. The rate of association of [11C]NS-2214 to catecholamine uptake sites was reduced by 62% in the same groups of pigs. No significant difference was found in the binding potential of [11C]raclopride to the dopamine D2-like receptors in striatum of the MPTP-intoxicated versus control pigs. These preliminary results are suggestive that the activity of DOPA decarboxylase may be upregulated in the partially denervated pig striatum.

Regional differences in asphyxia- and reperfusion-induced cytotoxic and vasogenic brain edema formation in newborn pigs

Neonatal asphyxia may induce brain injuries. Development of cytotoxic and vasogenic edema was measured in frontal, parietal, occipital cortex, hippocampus, striatum, thalamus, internal capsule, cerebellum, pons, and medulla of newborn pigs in the following groups: control, asphyxia, and 3-h reperfusion. Water content was significantly (P < 0.05) increased in parietal cortex and hippocampus during asphyxia, and in cortical regions, hippocampus, and striatum during recovery. Asphyxia-reperfusion resulted in increased Na+ and Ca2+-, but decreased K+-concentrations in several brain regions. Blood-brain barrier permeability for sodium fluorescein (mw: 376) was significantly increased in all regions but internal capsule and medulla in asphyxia, and in each region during reperfusion. Evan's blue-albumin (mw: 67,000) efflux was unchanged in asphyxia, but significantly increased after reperfusion in all regions. Increased permeability was also demonstrated by confocal laser scanning microscopy. Edema formation in the early postasphyxial period in porcine brain corresponded to the specific patterns of cerebral damage in human neonates.

Cerebral 6-[18F]fluoro-L-DOPA (FDOPA) metabolism in pig studied by positron emission tomography

We measured 6-[(18)F]fluoro-L-DOPA (FDOPA) uptake and metabolism in the brain of 4-month-old female pigs (n = 8) using a high-resolution positron emission tomograph (PET) in 3D mode. The mean net blood-brain clearance of FDOPA (K(i)(D)) to striatum was 0.011 ml g(-1) min(-1). Correcting for the elimination of decarboxylated metabolites from striatum (k(loss) = 0.004 min(-1)) increased the apparent magnitude of the estimate of K(i)(D) by 50%, at the expense of doubling the variance of the mean estimate. The mean decarboxylation rate of FDOPA in striatum relative to the cerebellum input (k(3)(s)) was 0.008 min(-1). For multicompartmental analyses, the FDOPA partition volume (V(e)(D)) was constrained to the individual value observed in cerebellum (mean = 0.53 ml g(-1)), with correction for the presence in brain of the plasma metabolite 3-O-methyl-FDOPA (OMFD). Using the first 60 min of the dynamic PET scans, the rate constant of FDOPA decarboxylation (k(3)(D)) was estimated to be 0.037 min(-1 )in striatum, but was not significantly different than zero in frontal cortex. Fitting of a compartmental model correcting for elimination of decarboxylated metabolites to the complete PET frame-sequence (120 min) increased the variance of the estimate of k(3)(D) in striatum. The magnitude of k(3)(D) in striatum of young pig was less than values estimated previously in neonatal piglet, adult monkey, and human. MRI-based simulations predicted that recovery of radioactivity from pig striatum was highly sensitive to the volume of interest. We conclude that the spatial resolution of our tomograph reduces the apparent magnitude of k(3)(D) in striatum. However, anaesthetised pigs are an appropriate experimental model for PET studies of DOPA decarboxylation in striatum.

Serotonin 5-HT 1B/1D agonist-stimulated [ 35S]GTPγS binding in rat and guinea pig striatal membranes

Serotonin (5-hydroxytryptamine; 5-HT) receptor ligands were used to assess agonist-stimulated [ 35S]GTPγS binding in rat and guinea pig striatal membranes. The assay contained 45–60 μg protein, 300 μM GDP and 0.1 nM [ 35S]GTPγS, incubated at 37°C for 20 min. The non-selective agonists 5-HT, 5-CT (5-carboxyamidotryptamine), and L-694,247, and the selective 5-HT 1B receptor agonist CP 93,129 produced concentration-dependent increases in [ 35S]GTPγS binding in rat striatum, whereas the selective 5-HT 1A receptor agonist R(+)-8-OH-DPAT [R(+)-8-hydroxy-2-(di- n-propylamino)tetralin] was inactive. Cyanopindolol, a 5-HT 1A/1B receptor antagonist, completely blocked the effect of 5-HT. Methiothepin, yohimbine and cyanopindolol also blocked 5-CT-stimulated [ 35S]GTPγS binding with the following rank order of potency: cyanopindolol ≥ methiothepin >>> yohimbine, consistent with rat 5-HT 1B receptor pharmacology. Neither cyanopindolol nor methiothepin altered basal [ 35S]GTPγS binding by themselves while yohimbine had weak partial agonist activity. Furthermore, cyanopindolol shifted the 5-CT concentration-response curve rightward, increasing the EC 50 and decreasing the maximal response, but did not affect L-694,247-stimulated [ 35S]GTPγS binding. The ability of cyanopindolol or spiperone (a 5-HT 1A/1D receptor antagonist) to alter CP 93,129-stimulated [ 35S]GTPγS binding was determined. Cyanopindolol produced a rightward shift in the CP 93,129 concentration-response curve, while spiperone had no affect. Finally, in guinea pig striatum and hippocampus, L-694,247 produced a concentration dependent increase in [ 35S]GTPγS binding. In conclusion, these studies indicate that 5-HT 1B receptor function can be assessed using agonist-stimulated [ 35S]GTPγS binding in rat striatal membranes using CP 93,129, 5-HT or 5-CT, but not L-694,247.

κ Opioid Receptor Stimulation of [ 35S] GTPγS Binding in Guinea Pig Brain

Although only one gene for κ opioid receptors has been cloned to date, κ 1 and κ 2 receptors have been defined pharmacologically, with drugs such as bremazocine binding to both putative κ receptor subtypes. To examine whether κ receptor subtypes can be distinguished at the level of the G-protein, the ability of the κ 1 agonist ( trans-(dl)-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) methane sulfonate (U-50488H) to stimulate [ 35S]guanosine-5′-O-(γ-thio)-triphosphate ([ 35S]GTPγS) binding in guinea pig brain was compared with that of bremazocine and dynorphin. In membranes prepared from guinea pig striatum, both bremazocine and U-50488H stimulated [ 35S]GTPγS binding with the same relative efficacy, while dynorphin produced at least two-fold greater efficacy than the other two agonists. In vitro autoradiography of agonist-stimulated [ 35S]GTPγS binding revealed similar regional distributions of bremazocine- and U-50488H-activated G-proteins. In striatal membranes, the κ antagonist nor-binaltorphimine (nor-BNI) blocked both bremazocine- and U-50488H-stimulated [ 35S]GTPγS binding with similar K e values. In agonist additivity experiments, the stimulation of [ 35S]GTPγS binding by the δ agonist [ d-pen 2,5, p-Cl-Phe 4]enkephalin (p-Cl-DPDPE) was approximately additive with the two κ agonists. Stimulation of [ 35S]GTPγS binding by the μ agonist [ d-Ala 2, N-Me 4, Gly 5-ol]-enkephalin (DAMGO) was additive with U-50488H, but not with bremazocine, reflecting the μ antagonist properties of this compound. The combination of bremazocine and U-50488H together produced no greater stimulation of binding than either agonist alone, indicating that they were binding to the same site. These results demonstrate that bremazocine and U-50488H activate G-proteins in guinea pig brain through the same receptor, and suggest that κ 2 receptors are not coupled through the same signal transduction mechanisms as κ 1 receptors.

Structural requirements for roxatidine in the stimulant effect of rat gastric mucin synthesis and the participation of nitric oxide in this mechanism.

1. The structural requirements of the histamine H2-receptor antagonist, roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride), for the stimulant effect on mucin biosynthesis and their relation to histamine H2-receptor antagonism were identified by considering the structural analogues of this drug using an organ culture system of the rat stomach and competition studies with [125I]iodoaminopotentidine ([125I]-APT) binding to membranes of the guinea pig striatum. 2. [3H]Glucosamine incorporation into mucin during 5 h incubation period was stimulated by roxatidine and its structural analogues A (2-hydroxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide) and B (N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide). This effect was seen in mucosal cultures of the corpus, but not antrum, region. 3. Structural analogues, in which the length of the flexible chain between the benzene ring and the amide structure differs from that of roxatidine, failed to activate mucin synthesis. No significant change in mucus synthesis occurred with the addition of analogues in which the piperidine ring attached to the benzene ring via a methylene bridge was changed. 4. Specific [125I]-APT binding to the histamine H2 receptor of guinea pig brain membranes was inhibited by roxatidine and all structural analogues used in this study, except F (N-(3-[m-(N, N-dimethyl-aminomethyl)phenoxy]-propyl)acetamide). 5. Ranitidine at 10(-4) M did not suppress the roxatidine-induced increase in [3H]glucosamine incorporation into mucin. 6. Roxatidine-induced stimulation of [3H]glucosamine incorporation into mucin was completely blocked by the addition of either NG-nitro-L-arginine (10(-5) M) or 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide sodium salt (10(-5) M). The inhibitory action of NG-nitro-L-arginine was totally reversed by L-arginine (5 x 10(-3) M). 7. These results suggest that the cardinal chemical features of roxatidine for the activation of mucin biosynthesis in the corpus region of the rat stomach are the appropriate length of the flexible chain between the amide structure and the aromatic ring system bearing the methylpiperidinyl group at the meta position. The activity of roxatidine and its analogues to stimulate mucin synthesis is not related to their histamine H2 receptor antagonistic activity. Roxatidine-induced activation of mucin biosynthesis in the corpus tissue is mediated by nitric oxide.

Release of [3H]dopamine from guinea pig striatal slices is modulated by sigma1 receptor agonists.

Sigma receptors are found in motor and limbic areas in the brains of humans, non-human primates, and rodents. The most extensive pharmacological studies of ligand binding to sigma receptors have utilized brain tissue from guinea pigs, where two subtypes of sigma receptor, designated sigma1 and sigma2, have been identified. Few functional roles for sigma receptors have been described. Their location in guinea pig striatum, a terminal field of dopaminergic projections arising from the substantia nigra, suggested that this tissue would be a logical choice in which to examine physiological properties of sigma receptor activation. We found that sigma1 receptor agonists inhibited N-methyl-D-aspartate-stimulated [3H]dopamine release from guinea pig striatal slices in a concentration-dependent manner. The inhibition by sigma1 receptor agonists was reversed by a selective sigma1 receptor antagonist, as well as by a non-subtype-selective sigma receptor antagonist. The ability of agonists working through sigma1 receptors, but not through sigma2 receptors, to inhibit the stimulated release of catecholamines appears to be a unique characteristic of guinea pig striatum. We have previously reported that in rat striatum and hippocampus, as well as in guinea pig nucleus accumbens, prefrontal cortex, and hippocampus, activation of either sigma receptor subtype inhibits such release. Stimulated release of [3H]dopamine from guinea pig striatum was also inhibited by the phencyclidine receptor agonist dizocilpine, but this inhibition was not reversed by the sigma receptor antagonists. Therefore, the inhibition produced by sigma receptor agonists was not mediated via the phencyclidine binding site within the N-methyl-D-aspartate-operated cation channel. Our findings support the hypothesis that sigma receptor activation provides a mechanism of modulating dopamine release from striatum, and that striatal tissue from guinea pigs appears to be an appropriate model for characterizing sigma1 receptor-mediated effects.

Effect of Serotonin (5-HT)3-Receptor Antagonists YM060, YM114 (KAE-393), Ondansetron and Granisetron on 5-HT4 Receptors and Gastric Emptying in Rodents

We investigated the effects of YM060 {(R)-5-[(1-methyl-3-indolyl)carbonyl]-4,5,6,7-tetrahydro-1//-benzimidazole hydrochloride} and YM114 (KAE-393) {(R)-5-[(2,3-dihydro-1-indolyl)-carbonyl]-4,5,6,7-tetrahydro-lH-benzimidazole hydrochloride} on 5-HT4 receptors and gastric emptying in normal and cisplatin-treated rats and compared results with those for ondansetron and granisetron. YM060, YM114, ondansetron and granisetron dose-dependently inhibited the specific binding of [3H]-GR113808 {[[l-[(2-methylsulphonyl)amino]ethyl]-4-piperidin-yl]methyl 1-methyl-lH-indole-3-carboxylate} in guinea pig striatum, with pKi values of 5.53, 5.13, 5.21 and 5.63, respectively. According to the pKi values reported in 5-HT3-receptor binding of [3H]GR65630 to rat cortical membranes, the affinity of YM060, YM114, ondansetron and granisetron for 5-HT4 receptors was approximately 5, 5, 3.5 and 3.5 log units lower than that for 5-HT3 receptors, respectively. In the guinea pig longitudinal muscle with myenteric plexus and rat esophageal tunica muscularis mucosae, YM060 and YM114 showed neither 5-HT4-agonistic nor antagonistic properties. Although ondansetron produced concentration-dependent increases in the magnitude of the twitch response in longitudinal muscle, it did not possess 5-HT3- and 5-HT4-agonistic activity. Granisetron antagonized 5-HT-induced relaxation of the rat esophagus with an apparent pA2 value of 5.39. Intravenous YM060, YM114, ondansetron and granisetron significantly enhanced gastric emptying of glass beads and improved cisplatin-induced slowing of gastric emptying in rats. These results indicate that the selectivity of YM060 and YM114 for 5-HT3 receptors is higher than that of ondansetron and granisetron and that these 5-HT3 antagonists have gastroprokinetic activity in normal and cisplatin-treated rats without affecting 5-HT4 receptors.

Species and age-dependent differences of functional coupling between opioid δ-receptor and G-proteins and possible involvement of protein kinase C in striatal membranes

[ d-Ser 2,Leu 5]-enkephalin-Thr 6 (DSLET) and [ d-Pen 2,5]-enkephalin (DPDPE), both δ-agonists, stimulated the high affinity GTPase in the rat striatal membranes in a naltrindole-reversible manner. Similar stimulation was also observed in the striatal membrane preparations of 16-week-old guinea pigs, while not in those preparations of 4-week-old ones. When calphostin C, a protein kinase C inhibitor, was added to the reaction mixture, DSLET showed a marked stimulation in this activity in 4-week guinea pig striatal membranes. There was no effect of KT5720, a cyclic AMP-dependent protein kinase inhibitor, on such δ-opioid-mediated responses. These findings suggest that protein kinase C is locally involved in the functional uncoupling of δ-receptors to G-proteins in the striatum of young guinea pigs.

Localization of aminopeptidase N and dipeptidyl peptidase IV in pig striatum and in neuronal and glial cell cultures.

The subcellular distribution of the plasma membrane ectoenzymes, aminopeptidase N (aminopeptidase M) and dipeptidyl peptidase IV, has been examined by fractionating homogenates of porcine striata by a discontinuous Percoll gradient centrifugation procedure which distinguishes fractions containing pre- and post-synaptic elements. The two enzymes showed different distributions--dipeptidyl peptidase IV did not show a significant pre-synaptic location, whereas aminopeptidase N was present on both pre- and post-synaptic fractions. Immunofluorescent staining on mixed and neuron-enriched primary cultures of pig striatal tissue using affinity purified antibodies to the aminopeptidase and to the dipeptidyl peptidase revealed the ectoenzymes on distinct populations of cells. The astrocytic identity of the aminopeptidase N-staining cells was established by correlation with immunostaining for glial fibrillary acidic protein and for vimentin by confocal microscopy. Ultracryosections of striatum immunostained with gold-labelled immunoglobulins of differing diameters demonstrated aminopeptidase N on pericytes and confirmed its location on endothelial and astrocytic glial cells. Thus, several independent approaches indicated that aminopeptidase N, in addition to being present on endothelial and synaptic membranes, is found on astrocytes and pericytes in the perivascular neuropil, whereas dipeptidyl peptidase IV is less widely distributed on microvessels and appears not to have a synaptosomal location.

Dopamine metabolism in the guinea pig striatum: role of monoamine oxidase A and B

These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig affects the actions of (−)-deprenyl on dopamine metabolism in whole tissue or in extracellular fluid. The administration of (−)-deprenyl in doses that do not affect monoamine oxidase A activity (1–4 mg kg −1, 2 h) increases striatal 2-phenylethylamine and dopamine concentrations and reduces 3,4-dihydroxyphenylacetic acid. No effects were observed on striatal homovanillic acid, 5-HT and 5-hydroxyindole acetic acid. Inhibition of monoamine oxidase A with clorgyline with doses up to 8 mg kg −1 (2 h) does not affect striatal 2-phenylethylamine but increases dopamine and 5-HT concentrations and reduces 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid. (−)-Deprenyl (2–4 mg kg −1) did not change the extracellular concentrations of dopamine but the higher dose produced a limited reduction in extracellular 3,4-dihydroxyphenylacetic acid. Inhibition of monoamine oxidase A and monoamine oxidase B with pargyline (75 mg kg −1, 2 h) significantly increased the levels of extracellular dopamine and reduced those of their acid metabolites. These results show that in the guinea pig striatum inhibition of monoamine oxidase B by (−)-deprenyl impairs the metabolism of dopamine in the whole tissue but does not produce a marked increase in extracellular dopamine.

Effects of cyanide in vitro on the activity of monoamine oxidase in striatal tissue from rat and pig

We have shown previously in the rat that lethal, acute cyanide intoxication dramatically decreased the levels of dopamine (DA) in the striatum, while the synthesis of DA was increased. The main brain metabolite of DA, homovanillic acid, was also diminished. However, the levels of the oxidatively deaminated metabolite of DA, 3,4-dihydroxyphenylacetic acid, were not significantly changed. In order to elucidate further these findings we examined the effects in vitro of sodium cyanide on rat and pig brain monoamine oxidase (MAO; EC 1.4.3.4). The MAO activity was measured radiochemically using [ 14C]5-hydroxytryptamine (5-HT; 100 μM), [ 14C]phenethylamine (PEA; 20μM) and [ 14C]DA (100 μM) as substrates. The amounts of cyanide added were comparable to those tissue concentrations of cyanide usually considered to be fatal in rats. The effect of cyanide on MAO was immediate. In rat, as well as pig, striatal tissue we found that cyanide produced a dose-dependent increase in the activity of MAO-A (as measured with 5-HT), but not MAO-B (as measured with PEA). The change in MAO activity was also seen with DA as substrate (MAO-A and -B). Kinetic constants, K m and V max were determined. In both rat and pig striatum the V max values for 5-HT were significantly increased, but the values for PEA were not affected. A significant decrease in the K m value for PEA was, however, found in the presence of high concentrations of cyanide.

Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation.

1. A series of adenosine receptor agonists of varying degrees of selectivity induced concentration-dependent contraction of the rat isolated spleen. With the exception of the response to the selective A2A receptor agonist, 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS 21680), responses to each ligand were blocked surmountably and to a broadly similar extent by 8-p-sulphophenyltheophylline (10(-5) M). 2. There was a significant correlation between the pEC50 values obtained on the spleen and the binding affinities (pKD; measured with [3H]-NECA) for the A1 receptor of pig striatum (r = 0.98, P < 0.001) but not the A2A receptor (r = 0.14, NS). 3. The antagonist potencies of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and 9-chloro-2-furyl [1,2,3]triazolo[1,5-C]quinazoline-5-amine (CGS 15943) were measured against the prototype selective A1 receptor agonist, R-N6-phenylisopropyladenosine (R-PIA). The resulting pKB values of 8.67 and 7.70, respectively are consistent with the A1 receptor subtype mediating splenic contraction. 4. The response to R-PIA was unaltered in the presence of a concentration (10(-7) M) of CGS 21680 which is 6 fold its KD concentration at the A2A binding site in pig striatum but below the threshold for causing contraction per se; thus, A2A receptors inhibitory to contraction appear to be absent. 5. The response to R-PIA was resistant to blockade by prazosin (10(-7) M) and by nifedipine (10(-6) M) but partially blocked by indomethacin (10(-6) M). 6. The results show that the rat isolated spleen responds to adenosine receptor agonists with contraction. Both the relative potencies of agonists and the effects of antagonists indicate mediation by the A1 receptor subtype. alpha1-Adrenoceptor activation is not involved in contraction but a role for products of cyclo-oxygenase and calcium from a source not dependent on entry through L-channels is implicated.

TRANSPLANTATION OF POLYMER ENCAPSULATED PC12 CELLS: USE OF CHITOSAN AS AN IMMOBILIZATION MATRIX.

Polymer capsules were fabricated to encapsulate PC12 cells within a semipermeable and immunoprotective barrier. The inclusion of precipitated chitosan as an immobilization matrix within the polymer capsules increased the survival and physiological functioning of the PC12 cells. In an initial study, HPLC analysis revealed that the inclusion of a chitosan matrix resulted in an increased output of catecholamines from the encapsulated PC12 cells under both basal conditions, and following high potassium depolarization at 2 and 4 wk following encapsulation in vitro. Furthermore, implantation of cohort PC12 cell-loaded capsules into guinea pig striata revealed that chitosan enhanced PC 12 cell survival after 6 wk. A second study determined that 12 wk after implantation into guinea pig striatum, abundant tyrosine hydroxylase-positive PC12 cells were evenly distributed within capsules containing chitosan. The long-term biocompatibility of these implants was good as determined by the absence of inflammatory or immune cells, and minimal GFAP reactivity surrounding the implant site. In contrast, implantation of unencapsulated PC12 cells resulted in a marked host tissue reaction, and destruction of the implanted cells within 4 wk. It is concluded that the inclusion of precipitated chitosan as an immobilization matrix enhanced the viability of encapsulated PC12 cells, and that altering the internal milieu of polymeric capsules may represent an effective transplant strategy for ameliorating human diseases characterized by secretory cell dysfunction.

Membrane-bound proteases involved in neuropeptide degradation in the brain

The action of neuropeptides at the synapse is terminated through enzymatic degradation by membrane-bound proteases. We defined and purified membrane-bound proteases functioning at the initial stage of degradation of four neuropeptides. 1. Substance P-degrading endopeptidases isolated from the rat brain and pig striatum showed similar properties to those of endopeptidase-24.16 (neurolysin) except for cleavage sites of substance P. 2. LHRH fragment (1-5)-generating endopeptidases isolated from the neuroblastoma cells and rat brain showed similar properties to those of endopeptidase-24.15 (thimet oligopeptidase). 3. One of two dynorphin-degrading cysteine proteases isolated from neuroblastoma cells showed strict specificity toward the Arg-Arg residues. 4. Endopeptidase-24.11 (neprilysin) isolated from the rat brain was identified as a somatostatin-degrading enzyme.

Dopamine D 2 synthesis-modulating receptors are present in the striatum of the guinea pig

Dopamine and selective agonists of D 1 [(1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride, SKF 38393] and D 2 [(3-[2-[ N-(3-hydroxyphenylethyl)- N-propylamino]ethyl] phenol, RU 24926] receptors were examined as inhibitors of the activity of tyrosine hydroxylase in the striatum of the guinea pig. In soluble enzyme preparations, the agonists were weak inhibitors of the activity of tyrosine hydroxylase. However, the catechol-containing agonists dopamine (EC 50 = 44.7 μM) and SKF 38393 (EC 50 = 35.5 μM) were more potent than the non-catechol agonist RU 24926 (EC 50 = 447 μM). All of the agonists were much more potent in synaptosome-rich preparations of guinea pig striatum, where stimulation of autoreceptors mediated inhibition of the enzyme (SKF 38393, D 1, EC 50 = 27 nM; RU 24926, D 2, EC 50 = 30nM; dopamine, non-selective, EC 50 = 1.5 μM). The D 1 antagonist, SCH 23390 [(R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-(-−)-3-benzazepine hydrochloride], did not significantly reduce the action of SKF 38393 or dopamine. Furthermore, the D 2 antagonist, (—)-sulpiride, significantly antagonized the inhibitory activity of both RU 24926 and dopamine. Studies in synaptosome-rich preparations from the striatum of the rat showed that both SKF 38393 (EC 50 = 398 nM) and RU 24926 (EC 50 = 58 nM) were also effective autoreceptor-mediated inhibitors of the activity of tyrosine hydroxylase in the rat. However, in the rat, SCH 23390 and (−)-sulpiride were equally effective in attenuating the inhibitory actions of dopamine. These results suggest that: (1) dopamine autoreceptors, similar to the D 2 type, modulate tyrosine hydroxylase in the striatum of the guinea pig. (2) In the striatum of the rat, dopamine synthesis-modulating autoreceptors are either a heterogeneous population of D 1, and D 2 receptors or a single population of receptors, with combined features of classic D 1 and D 2 receptors.

Galanin message associated peptide

These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig affects the actions of (−)-deprenyl on dopamine metabolism in whole tissue or in extracellular fluid. The administration of (−)-deprenyl in doses that do not affect monoamine oxidase A activity (1–4 mg kg−1, 2 h) increases striatal 2-phenylethylamine and dopamine concentrations and reduces 3,4-dihydroxyphenylacetic acid. No effects were observed on striatal homovanillic acid, 5-HT and 5-hydroxyindole acetic acid. Inhibition of monoamine oxidase A with clorgyline with doses up to 8 mg kg−1 (2 h) does not affect striatal 2-phenylethylamine but increases dopamine and 5-HT concentrations and reduces 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid. (−)-Deprenyl (2–4 mg kg−1) did not change the extracellular concentrations of dopamine but the higher dose produced a limited reduction in extracellular 3,4-dihydroxyphenylacetic acid. Inhibition of monoamine oxidase A and monoamine oxidase B with pargyline (75 mg kg−1, 2 h) significantly increased the levels of extracellular dopamine and reduced those of their acid metabolites. These results show that in the guinea pig striatum inhibition of monoamine oxidase B by (−)-deprenyl impairs the metabolism of dopamine in the whole tissue but does not produce a marked increase in extracellular dopamine.

Transient expression of opioid receptors in defined regions of developing brain: are embryonic receptors selective?

The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term cross-species brain transplantation of a polymer-encapsulated dopamine-secreting cell line

Cross-species transplantation of dopamine-releasing cell lines protected against immune rejection by a semipermeable synthetic membrane may provide a source of neurotransmitters for the treatment of Parkinson's disease. Experiments were carried out to assess whether polymer-encapsulated PC12 cells, a catecholaminergic cell line derived from a rat pheochromocytoma, could survive in vitro as well as in vivo after implantation in the striatum of adult guinea pigs. When maintained in vitro, the encapsulated PC12 cells exhibited good survival, proliferated, and spontaneously released dopamine for at least 6 months. They also retained the capacity for depolarization-elicited dopamine release. In vivo, well-preserved tyrosine hydroxylase-positive PC12 cells were observed in capsules implanted for 4, 8, and 12 weeks. Unencapsulated PC12 cells or cells in nonintact capsules did not survive transplantation at any of these time periods. The survival of encapsulated PC12 cells transplanted across species suggests that polymer encapsulation may provide an alternative for xenotransplantation of secretory cells in the absence of systemic immunosuppression.

In Vivo Autoxidation of Dopamine in Guinea Pig Striatum Increases with Age

Catechols are known to react readily with molecular oxygen to form the corresponding quinones together with reduced oxygen species. These products have been shown to be toxic in in vivo and in vitro systems. 5-S-Cysteinyl adducts of catechols are believed to be formed through the spontaneous reaction between quinones and thiol-containing compounds, like cysteine and glutathione (GSH). Thus, the brain levels of these adducts probably indicate the autoxidation rate of catechols in vivo. In the present study, the striatal concentrations of 5-S-cysteinyldopamine (5-S-cysteinyl-DA), 5-S-cysteinyl-3,4-dihydroxyphenylalanine (5-S-cysteinyl-DOPA), and 5-S-cysteinyl-3,4-dihydroxyphenylacetic acid (5-S-cysteinyl-DOPAC) were determined in 2-week-, 2-month- and 3-year-old guinea pigs. In addition, brain levels of DA, the DA metabolite DOPAC, and GSH were assessed. The concentration of 5-S-cysteinyl-DA increased markedly with age. The 3-year-old guinea pigs had the highest level, i.e., 248% of the concentration in the 2-week-old animals and 219% of the concentration in the 2-month-old animals. Furthermore, the striatal 5-S-cysteinyl-DOPA level in the 3-year-old group was 68% higher than in the 2-week-old group and 46% higher than in the 2-month-old group. No age difference in the striatal concentration of DA was found. In contrast, the concentration of DOPAC increased with age: The DOPAC level in the 3-year-old animals was 153% of the level in the 2-week-old animals and 116% of the level in the 2-month-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)