Long-term changes in glutamatergic synaptic transmission in phenylketonuria.

The cellular mechanisms that underlie impaired brain function during phenylketonuria (PKU), the most common biochemical cause of mental retardation in humans, remain unclear. Acute application of L-Phe at concentrations observed in the PKU brain depresses glutamatergic synaptic transmission but does not affect GABA receptor activity in cultured neurons. If these depressant effects of L-Phe take place in the PKU brain, then chronic impairment of the glutamate system, which may contribute to impaired brain function, could be detected as changes in postsynaptic glutamate receptors. This hypothesis was tested by using a combination of liquid chromatography-mass spectrometry, patch-clamp, radioligand binding and western blot approaches in forebrain tissue from heterozygous and homozygous (PKU) Pah(enu2) mice. Brain concentrations of L-Phe were nearly six-fold greater in PKU mice (863.12 +/- 17.96 micromol/kg) than in their heterozygous counterparts (149.32 +/- 10.23 micromol/kg). This concentration is significantly higher than the K(B) of 573 microM for L-Phe to compete for N-methyl-D-aspartate (NMDA) receptors. Receptor binding experiments with [3H]MK-801 showed significant up-regulation of NMDA receptor density in PKU mice. Consistent with the depressant effects of L-Phe, expression of NMDA receptor NR2A and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Glu1 and Glu2/3 subunits was significantly increased, whereas expression of the NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Given the role of the glutamatergic system in brain development and function, these changes may, at least in part, explain the brain disorders associated with PKU.

Structure-affinity profile of 8-hydroxycarbostyril-based agonists that dissociate slowly from the beta2-adrenoceptor.

Several carbostyril-based beta-agonists have been shown to bind tightly to and slowly dissociate from the beta2-adrenoceptor (beta2AR). In the present study, the structural features of 8-hydroxy-5-[2-[(1-phenyl-2-methylprop-2-yl)amino]-1-hydroxyethyl] -carbostyril (11a) which contribute to its binding properties at the beta2AR were investigated using a series of synthesized analogs. The k(off), estimated by the rate of cAMP decline in DDT1 MF-2 (DDT) cells with a reduced receptor density, Ki and ligand-induced receptor reductions were determined. All of the derivatives stimulated cAMP accumulation in DDT cells in the sub to mid nanomolar range and elicited the same maximal stimulation as (-)isoproterenol. Derivatives of 11a with side chain N-substitutions comprising 2-methylbutyl, phenylethyl and isopropyl had higher k(off)-values and lower affinities as compared to 11a. Increasing the number of methylenes between the side chain tertiary alpha carbon and phenyl from 1 in 11a to 3 or reducing the number to 0 also resulted in derivatives with higher k(off)- and Ki-values. In addition, replacement of the 8-hydroxycarbostyril nucleus of 11a with catechol reduced the affinity of the compound for the beta2AR by 48-fold and increased its k(off). Only those derivatives with the lowest k(off)-values induced a decrease in the receptor density of DDT cell membranes following a preincubation and extensive washing. The data show that the 8-hydroxycarbostyril nucleus in conjunction with substitutions on the tertiary alpha carbon of the side chain and positioning of the phenyl group are important characteristics determining the high affinity and slow dissociation of 11a from the beta2AR.

Irreversible binding of a carbostyril-based agonist and antagonist to the beta-adrenoceptor in DDT1 MF-2 cells and rat aorta.

1. The chemoreactive ligands 5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)-amino) -2-methylpropyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl+ ++)amino)-2-methylprop-2-yl)amino-1-hydroxyethyl)-carbostyril++ + (HCITC) were synthesized and shown to be potent irreversible antagonist and agonist ligands, respectively, for the beta-adrenoceptor in DDT1 MF-2 (DDT) cells and the rat isolated aorta. 2. In DDT cell membranes DCITC and HCITC inhibited (-)[125I]-iodocyanopindolol (CYP) binding to the beta-adrenoceptor with IC50 values of 1.1 and 18 nM, respectively. (-)-Isoprenaline inhibited [125I]-CYP binding with an IC50 of 355 nM. Pretreatment of membranes with either chemoreactive ligand produced a time- and concentration-dependent decrease in the beta-adrenoceptor content, indicating irreversible receptor binding. DCITC at concentrations up to 10 microM did not stimulate cyclic AMP accumulation in DDT cells nor did it amplify forskolin-stimulated cyclic AMP accumulation. 3. In the rat isolated aorta, DCITC (0.1 microM) did not affect either the phenylephrine-mediated tissue contraction or the acetylcholine-mediated relaxation. DCITC attenuated the maximal (-)-isoprenaline-mediated relaxation of a phenylephrine contracted aorta in a concentration-dependent manner and shifted the dose-response curves for (-)-isoprenaline to the right. The DCITC-induced decrease in maximal response was not reversed by extensive tissue washing. By use of the operational model of agonism, the calculated dissociation constant for (-)-isoprenaline ws 286 nM and the estimated receptor reserve for this agonist was 23% at the maximal response. 4. HCITC and (-)-isoprenaline stimulated cyclic AMP accumulation in DDT cells with pD2 values (negative logarithm to base 10 of EC50) of 7.95 and 7.97, respectively, and both mediated the same maximal stimulation. In the rat isolated aorta, HCITC produced a concentration-dependent relaxation of the tissue with a pD2 value of 6.62, whereas the pD2 for (-)-isoprenaline was 7.03. However, HCITC produced a greater maximal relaxation of the tissue than (-)-isoprenaline. The HCITC-mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the beta-antagonist propranolol was added concurrently. In contrast, once the HCITC-mediated responses were established, the addition of propranolol did not result in any attenuation indicating that HCITC is an irreversible beta-agonist.

Persistent activation by and receptor reserve for an irreversible A1-adenosine receptor agonist in DDT1 MF-2 cells and in guinea pig heart.

The p- and m-isothiocyanate adenosine derivatives N6-[4-[[[4-[[[[2-[[[(p-(m)-isothiocyanatophenyl)amino]thiocarbonyl ]am ino]ethyl]amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl] adenosine (p- and m-DITC-ADAC) were examined for irreversible agonist effects at the A1-adenosine receptor (A1-AdoR) in DDT1 MF-2 (DDT) cells and a functional A1-AdoR response in the guinea pig isolated heart. The p- and m-DITC-ADAC inhibited (-)-isoproterenol stimulated cAMP accumulation in DDT cells in the low nanomolar range, and the maximal responses elicited by both compounds were similar to that for N6-cyclopentyladenosine. Once established, the p-DITC-ADAC-mediated inhibition of cAMP accumulation in DDT cells was not affected by the addition of the AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX). Pretreatment of DDT cells with p-DITC-ADAC (1 microM), followed by washing, reduced [3H]CPX binding to the A1-AdoR by 44% without altering the Kd value for the radioligand to the remaining receptors. The relationship between irreversible A1-AdoR occupancy by p-DITC-ADAC and inhibition of cAMP accumulation revealed a relatively large receptor reserve (64%) for the maximal response. In guinea pig isolated hearts, m-DITC-ADAC (5 microM) prolonged the stimulus to His bundle (SH) interval by 2.1-fold; this response could be prevented by the antagonist 8-cyclopentyltheophylline (5 microM). However, after the SH interval prolongation was established, extensive washout or the addition of 8-cyclopentyltheophylline had little reversal effect on the m-DITC-ADAC response. Binding of [3H]CPX to the guinea pig ventricular membranes after m-DITC-ADAC treatment and washing was reduced by 35%. The A1-AdoR occupancy response relationship for m-DITC-ADAC to prolong the SH interval indicated a small (10-20%) receptor reserve. Both p -and m-DITC-ADAC seem to be irreversible full agonists at the A1-AdoR and may prove to be useful probes to further investigate A1-AdoR structure-function relationships.

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization, veratridine, or A23187 in rat cerebral cortical synaptosomes.

The effect of 4-aminopyridine on [3H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [3H]acetylcholine release in a concentration-dependent manner (5-10 mM); a high concentration (10 mM) also elevated [3H]choline efflux. However, the 35 mM K+ induced release of [3H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K+ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 microM). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K+ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.

Effects of acetylethylcholine mustard on [3H]quinuclidinyl benzilate binding and acetylcholine release in rat brain synaptosomes.

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.

Aging decreases the sensitivity of rat cortical synaptosomes to calcium ionophore-induced acetylcholine release.

The capacity of calcium ions to trigger acetylcholine release was studied in cerebral cortical synaptosomes from adult (6-month-old) and senescent (24-month-old) rats, using a calcium ionophore, A23187, that bypasses voltage-sensitive calcium channels. The potency but not the efficacy of the A23187 was reduced with respect to releasing acetylcholine (ACh) in the aged animals. There was no age-related difference in the synthesis of ACh or potency of the ionophore with respect to increasing 45calcium uptake. These results suggest that aging reduces the sensitivity of cerebral cortical nerve terminals to calcium-triggered ACh-release.

Effects of 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183) on rat cortical synaptosome choline uptake, acetylcholine storage and release.

The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.

Pharmacological and ionic characterizations of the muscarinic receptors modulating [3H]acetylcholine release from rat cortical synaptosomes.

The muscarinic receptors that modulate acetylcholine release from rat cortical synaptosomes were characterized with respect to sensitivity to drugs that act selectively at M1 or M2 receptor subtypes, as well as to changes in ionic strength and membrane potential. The modulatory receptors appear to be of the M2 type, since they are activated by carbachol, acetylcholine, methacholine, oxotremorine, and bethanechol, but not by pilocarpine, and are blocked by atropine, scopolamine, and gallamine (at high concentrations), but not by pirenzepine or dicyclomine. The ED50S for carbachol, acetylcholine, and oxotremorine are less than 10 microM, suggesting that the high affinity state of the receptor is functional. High ionic strength induced by raising the NaCl concentration has no effect on agonist (oxotremorine) potency, but increases the efficacy of this compound, which disagrees with receptor-binding studies. On the other hand, depolarization with either KCl or with veratridine (20 microM) reduces agonist potencies by approximately an order of magnitude, suggesting a potential mechanism for receptor regulation.