John Lehmann

Characterization of dopamine autoreceptor and [3H]spiperone binding sites in vitro with classical and novel dopamine receptor agonists.

The specific D2 receptor agonist, LY 141865, but not the specific D1-receptor agonist, SK&F 38393, potently inhibited electrically evoked [3H]dopamine release from slices of the cat caudate. Similarly, LY 141865, but not SK&F 38393, inhibited [3H]spiperone binding to membranes of the cat caudate. The inhibition by dopamine receptor agonists of electrically evoked [3H]dopamine release was antagonized by the specific D2-receptor antagonist S-sulpiride. The inhibition of the electrically evoked release of [3H]dopamine by apomorphine was not, however, antagonized by the specific D1-receptor antagonist, bulbocapnine. Similarly, S-sulpiride but not bulbocapnine potently inhibited [3H]spiperone binding to membranes of the cat caudate. These results suggest that the dopamine autoreceptor modulating the depolarization-evoked release of [3H]dopamine, and the binding site of [3H]spiperone, are valid in vitro models for D2-dopamine receptors. Contrary to some previous reports, DPI was inactive in both in vitro dopamine receptor models. The IC50 values of a series of dopamine receptor agonists correlated very well in the two in vitro dopamine receptor models. One exception to this correlation was bromocriptine, which was more potent at [3H]spiperone binding sites than at the dopamine autoreceptor. With the exception of bromocriptine, all dopamine receptor agonists had one-hundred fold higher potency at the dopamine autoreceptor than at [3H]spiperone binding sites. [3H]Spiperone binding sites are localized primarily postsynaptic to dopamine terminals. Possible differences between the pharmacological properties of pre- and postsynaptic dopamine receptors should become apparent in the comparison of the two in vitro dopamine receptor models. However, the order of potency of dopamine receptor agonists with both in vitro models, dopamine autoreceptor and [3H]spiperone binding, was the same: N-n-propylnorapomorphine greater than TL-99 = 7-HAT greater than M-7 greater than Apomorphine greater than LY 141865.

Stereoisomers of apomorphine differ in affinity and intrinsic activity at presynaptic dopamine receptors modulating [3H]dopamine and [3H]acetylcholine release in slices of cat caudate.

We investigated the effects of R(-)-apomorphine and S(+)-apomorphine on dopamine receptors modulating electrically evoked [3H]dopamine and [3H]acetylcholine release from slices of cat caudate nucleus. R(-)-Apomorphine inhibited the release of both [3H]dopamine and [3H]acetylcholine with an IC50 of 20 nM, while S(+)-apomorphine was without inhibitory action on the electrically evoked release of either neurotransmitter at concentrations up to 1 microM. At a concentration of 1 microM, however, S(+)-apomorphine antagonized the inhibition by R(-)-apomorphine, producing a parallel five-fold shift to the right in the concentration-response curve to R(-)-apomorphine. These results indicate that S(+)-apomorphine is devoid of intrinsic activity to stimulate presynaptic dopamine receptors modulating the electrically evoked release of dopamine and acetylcholine. In addition, S(+)-apomorphine has an approximately ten-fold lower affinity for presynaptic dopamine receptors compared to R(-)-apomorphine.

NMDA- and endothelin-1-induced increases in blood-brain barrier permeability quantitated with Lucifer yellow

At 48 h following intrastriatal injection of N-methyl-D-aspartate (NMDA; 100 nmol/microliter) or endothelin-1 (ET-1; 143 pmol/microliter), significant increases in brain penetration of the highly polar, fluorescent tracer Lucifer yellow were observed. The competitive NMDA receptor antagonist selfotel (CGS-19755; 30 nmol/microliter, i.c.) significantly reduced the NMDA-induced increases in blood-brain barrier permeability, but not those induced by ET-1. These results suggest that NMDA receptors can mediate increases in blood-brain barrier permeability but do not primarily mediate increases in blood-brain barrier permeability caused by ET-1. This is the first study to our knowledge investigating the relationship between excitotoxicity and disruption of the blood-brain barrier, a major pathophysiological event in stroke and traumatic brain injury.

The selective protein kinase C inhibitor, NPC 15437, induces specific deficits in memory retention in mice

We studied the effects of a selective inhibitor of protein kinase C (PKC), 2,6-diamino-N-[(1-(1-oxotridecyl)-2-piperidinyl]methyl)hexamide (NPC 15437), on acquisition and memory retention of a Y-maze avoidance task in mice. Post-training administration of NPC 15437 (0.1-10 mg/kg i.p.) induced a dose-dependent deficit in retention of the temporal but not the spatial component of the task. This selective amnesia does not reflect state dependence and NPC 15437 (1 mg/kg) had no effect on acquisition and memory retrieval. Our results suggest that this new PKC inhibitor interferes with mechanisms underlying memory consolidation. This is in agreement with recent findings suggesting that PKC is involved in memory processes.

Cerebral uric acid increases following experimental traumatic brain injury in rat

Following traumatic brain injury (TBI), cortical and thalamic areas were analyzed histologically and by high-performance liquid chromatography with electrochemical detection for uric acid at various survival times. Following TBI, cortical uric acid was elevated by ten-fold at 24 and 48 h, but not at 1 h post-TBI. Histological evidence of neurodegeneration was found not only in cortex but also in the anteroventral thalamus. These data suggest that as in stroke, uric acid measurements may be a convenient and sensitive method for measuring peroxidative status in TBI.

Cortical norepinephrine release elicited in situ by N-methyl-d-aspartate (NMDA) receptor stimulation: a microdialysis study

Norepinephrine (NE) release measured by microdialysis from probes positioned in the prefrontal cortex of anesthetized rats was increased when N-methyl-D-aspartate (NMDA) was contained in the microdialysis medium. NE release increased by a factor of 3.6 compared to prior baseline levels when 1 mM NMDA was applied for 30 min. This increase was largely reversible, and when a second stimulation was applied using aconitine (0.3 mM, 30 min), which acts on voltage-sensitive sodium channels, a second evoked release of NE was observed, of a similar magnitude as that evoked by NMDA. Dizocilpine (300 nM), which blocks cation channels associated with NMDA receptors, prevented the NMDA-elicited NE increase but not the aconitine-elicited increase.

7-Chlorokynurenate prevents NMDA-induced and kainate-induced striatal lesions.

NMDA-induced lesions of striatal cholinergic interneurons were attenuated by 7-chlorokynurenate (7-ClKyn), an antagonist of the glycine site of the NMDA receptor complex. However, it was not possible to demonstrate clearly that the mechanism of action of 7-ClKyn was in fact antagonism of the glycine site. Thus, the agonists at the glycine site, D-serine and 1-aminocyclopropane-1-carboxylic acid, failed to reverse the protection afforded by 7-ClKyn. Finally, 7-ClKyn also protected against lesions produced by kainate. The selectivity of 7-ClKyn under intracerebral administration is apparently insufficient for determining the role of the glycine site in NMDA-receptor mediated excitotoxicity.

Glutamate and glycine co-activate while polyamines merely modulate the NMDA receptor complex

1. Agonists may act at any one of three sites on the N-methyl-D-aspartate (NMDA) receptor-effector complex to promote opening of the associated ion channel. The three sites are activated by i) NMDA, L-glutamate, aspartate, and other dicarboxylic amino acids; ii) glycine, D-serine, D-cycloserine, and others; iii) the polyamines spermine or spermidine, but not cadaverine or putrescine. 2. This opening by exogenous agonists is reflected by an enhanced binding of the phencyclidine-like dissociative anesthetic [3H]MK-801 to rat cortical membranes (well washed to remove endogenous agonists, e.g., L-glutamate, glycine). 3. The effects of adding combinations of agonists yielded stimulation approximately equal to the sum of each agonists effect, suggesting that in the first approximation the three classes act at independent sites. 4. When the glutamate (E) site was antagonized with D-2-amino-5-phosphonopentanoate (D-AP5), no stimulation in binding could be elicited by agonists at the two other sites. Activation of the E site is therefore necessary but not sufficient for channel opening. 5. When the glycine (G) site was antagonized with 7-chlorokynurenate, no stimulation in binding could be elicited by agonists at the other two sites. Activation of the G site is therefore necessary but not sufficient for channel opening. 6. Of the two putative antagonists for the polyamine (PA) site, ifenprodil fails to completely inhibit the binding of [3H]MK-801, whereas arcaine inhibited [3H]MK-801 binding completely. We present data which question the selectivity of arcaine for the polyamine site, and propose that the polyamine site is merely modulatory, but neither necessary nor sufficient, for channel opening.

Determination of Acetylcholine Dynamics

During the last 15 years, the methods proposed to study the regulation of cholinergic transmission m viva have required the labeling of the acetylcholine (ACh) and choline stored in neurons by injecting labeled precursors of ACh, and by measuring simultaneously the changes with time in the specific activities of choline and ACh (Cheney et al., 1974, 1975a; Costa et al. 1975; Dross and Kewitz, 1966; Hanin et al., 1973; Jenden et al., 1974; Racagm et al., 1974; Saelens et al., 1973; Schuberth et al., 1969; Zsilla et al., 1977). In addition to these rsotopic methods, various nonisotopic approaches have been suggested to estimate the fractional rate constant of ACh efflux (Bartholmi et al., 1976; Celesia and Jasper, 1966; Hemsworth and Neal, 1968; Mitchell, 1960; Pepeu et al., 1975; Smith, 1972; Szerb, 1975; Yaksh and Yamamura, 1975). Some of the nonisotopic methods have used drugs to instantaneously inhibit synthesis, metabolrsm, or transport of ACh, its precursor, or metabolites (Costa and Neff, 1966). A flaw common to all the methods involving the use of inhibitors derives from the widely spread assumption that drugs are so specific that, even in high doses, they selectively inhibit a given metabolic step. Obviously, such specificity is exceptional; more often, drugs that inhibit ACh metabolism exert collateral actions on other processes that directly or indirectly regulate a number of other metabolic steps. The isotopic methods that use tracer doses of a precursor of ACh are not based on this assumption, but may be plagued by other conceptual flaws. Therefore, they are not devoid of problems; in fact, they include a number of tacrt assumptions concerning the dynamics and compartmentation of ACh and its precursors that are open to critique. The assumption implicit in all of the techniques, whether isotopic or nonisotopic, is that at steady-state the newly taken up pool of choline is m rapid equilibrium with the pool of choline that

Phenoxybenzamine blocks dopamine autoreceptors irreversibly: Implications for multiple dopamine receptor hypotheses

Abstract Phenoxybenzamine in μM concentrations increased the electrically evoked overflow of recently taken up [3H]dopamine from superfused slices of cat caudate nucleus, an effect which is also observed for dopamine receptor antagonists. The magnitude of the increase in electrically evoked [3H]dopamine release caused by 1 μM phenoxybenzamine was equal to that elicited by maximally effective concentrations of the specific dopamine receptor antagonist, S-sulpiride. Phenoxybenzamine (1 μM) completely antagonized the inhibition of [3H]dopamine release caused by the dopamine receptor agonist pergolide (10 nM). The α-adrenoceptor antagonist phentolamine (1 μM) had no effect on the electrically evoked overflow of [3H]dopamine, ruling out the possibility that the effect of phenoxybenzamine could be attributed to α-adrenoceptor blockade. A 20 min exposure to 1 μM phenoxybenzamine increased the electrically evoked [3H]dopamine overflow even after the tissue had been washed for two and a half hours. When the caudate slices were exposed for 30 min to the reversible dopamine receptor antagonist S-sulpiride (1 μM) and washed for two and a half hours, no similar increase in stimulation-evoked [3H]dopamine overflow was observed. When sulpiride (1 μM) was present during the exposure to phenoxybenzamine (1 μM), no increase in electrically evoked [3H]dopamine overflow was observed after the washout period. Thus phenoxybenzamine at 1 μM appears to block irreversibly the dopamine autoreceptor in the caudate nucleus. Phenoxybenzamine has been previously reported to block irreversibly dopamine-stimulated adenylate cyclase (D1) and neuroleptic receptor binding (D2). The present demonstration that phenoxybenzamine also blocks the dopamine autoreceptor irreversibly thus supports the view that all currently well-established dopamine receptors are sensitive to phenoxybenzamine.