Professor Matthew Stewart: asbestosis research 1929-1934

The chronic continuous infusion of cocaine produces partial behavioral tolerance to cocaine and tolerance to the inhibition of dopamine uptake by cocaine, without changing dopamine transporter binding. In order to examine more closely the dopaminergic contribution to this effect, the selective dopamine uptake inhibitor GBR 12,909 (30 mg/kg/day), cocaine (50 mg/kg/day), or vehicle, were continuously infused via osmotic minipump, and their effects on the dopamine transporter examined. Drug and vehicle pumps were implanted into male Sprague-Dawley rats and removed after seven days. [3H]WIN 35,428 binding and [3H]dopamine uptake were measured in caudate putamen and nucleus accumbens at varying intervals after pump removal. The Bmax for [3H]WIN 35,428 binding was decreased by approximately 75% in the caudate putamen and by 40% in the nucleus accumbens of GBR 12,909-treated rats both 1 and 4 days after pump removal, and was still significantly decreased after 10 days, but had returned to normal by 20 days post-treatment. In contrast, cocaine did not significantly alter [3H]WIN 35,428 binding. GBR 12,909 produced both tolerance to the inhibition of [3H]dopamine uptake by cocaine, and a decrease in total uptake of dopamine, in the caudate putamen, with no change in the nucleus accumbens. The persistent reduction of [3H]WIN 35,428 binding following continuous GBR 12,909 does not appear to result from residual drug binding. These findings suggest that GBR 12,909 and cocaine may bind to and regulate the dopamine transporter in different ways.     

Comparison of [3H]WIN 35,428 binding, a marker for dopamine transporter, in embryonic mesencephalic neuronal cultures with striatal membranes of adult rats

In contrast to striatal membranes of adult rats, where high- (KD1 = 34 nM) and low- (KD2 = 48,400 nM) affinity binding sites for [3H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found (KD = 336,000 nM). The binding of [3H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [3H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [3H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [3H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [3H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation (r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [3H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC50 values of drugs that inhibited [3H]WIN 35,428 binding and [3H]DA uptake in CVMNs. The cytosolic [3H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

Measurement of serum isopropanol and the acetone metabolite by proton nuclear magnetic resonance: application to pharmacokinetic evaluation in a simulated overdose model

The regional distribution of [11C]d-threo-methylphenidate in mouse brain was very similar to that of [3H]WIN 35,428 ((-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane), and the two radioligands were displaced from striatum similarly after administration of the potent cocaine analog RTI-55 ((-)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane). However, while striatal [3H]WIN 35,428 increased between 5 and 30 min, striatal [11C]d-threo-methylphenidate halved. Thus [11C]d-threo-methylphenidate binds similarly to but more reversibly than [3H]WIN 35,428. The methyl ester of L-DOPA (L-3,4-dihydroxyphenylalanine; 200 mg/kg) plus benserazide plus clorgyline, which markedly elevates rat striatal extracellular dopamine (Wachtel and Abercrombie, 1994, J. Neurochem. 63, 108), decreased the mouse striatum-to-cerebellum ratio for [11C]d-threo-methylphenidate at 30 min by 13% (P < 0.05). In positron emission tomographic (PET) baboon studies [11C]d-threo-methylphenidate binding was insensitive to drugs expected to lower endogenous dopamine. These experiments suggest that normal synaptic dopamine does not compete for binding with [11C]d-threo-methylphenidate, and will not affect PET measures of dopamine transporter availability.     

WIN 35,428 and mazindol are mutually exclusive in binding to the cloned human dopamine transporter

It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.     

Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Nature of methamphetamine-induced rapid and reversible changes in dopamine transporters

The nature of methamphetamine-induced rapid and transient decreases in dopamine transporter activity was investigated. Regional specificity was demonstrated, since [3H]dopamine uptake was decreased in synaptosomes prepared from the striatum, but not nucleus accumbens, of methamphetamine-treated rats. Differences among effects on dopamine transporter activity and ligand binding were also observed, since a single methamphetamine administration decreased [3H]dopamine uptake without altering [3H]WIN35428 ([3H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate) binding in synaptosomes prepared 1 h after injection. Moreover, multiple methamphetamine injections caused a greater decrease in [3H]dopamine uptake than [3H]WIN35428 binding in synaptosomes prepared I h after dosing. Finally, decreases in [3H]dopamine uptake, but not [3H]WIN35428 binding, were partially reversed 24 h after multiple methamphetamine injections. Western blotting indicated that saline- and methamphetamine-affected dopamine transporters co-migrated on sodium dodecyl sulfate (SDS) gels at approximately 80 kDa, and that acute, methamphetamine-induced decreases in [3H]dopamine uptake were not due to loss of dopamine transporter protein. These findings demonstrate heretofore-uncharacterized features of the acute effect of methamphetamine on dopamine transporters.     

Dopamine transporter development in postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428

The dopamine transporter mediates the reinforcing effects of cocaine, thus playing a central role in human cocaine addiction, and perhaps providing the mechanism for inducing the effects of prenatal cocaine exposure. This possibility has stimulated growing interest in the normal and abnormal development of this transporter. [3H]WIN 35,428 is a cocaine analog that is useful for studying the distribution and density of the dopamine transporter in striatum and other brain regions. The postnatal development of the dopamine transporter in the rat striatum was measured by quantitative autoradiography with [3H]WIN 35,428. Dopamine transporter levels were low at birth, increased through day 15, followed by much more rapid growth in late postnatal development. The majority of the transporter sites appeared after day 15. Lateral to medial and anterior to posterior gradients in transporter density were established early during development, and there was also an early concentration of transporter in striosomes that became difficult to identify by day 15. Differences between the developmental patterns described here and studies using other ligands for the dopamine transporter suggest there are significant differences in the transporter binding sites for these drugs. These differences in transporter ligand binding characteristics may reflect developmental changes in post-translational modification of the transporter and/or changes in the functional activity rather than simply the presence of the transporter.     

The vesicular monoamine transporter, in contrast to the dopamine transporter, is not altered by chronic cocaine self-administration in the rat

Although much evidence suggests that the brain dopamine transporter (DAT) is susceptible to dopaminergic regulation, only limited information is available for the vesicular monoamine transporter (VMAT2). In the present investigation, we used a chronic, unlimited-access, cocaine self-administration paradigm to determine whether brain levels of VMAT2, as estimated using [3H]dihydrotetrabenazine (DTBZ) binding, are altered by chronic exposure to a dopamine uptake blocker. Previously, we showed that striatal and nucleus accumbens DAT levels, as estimated by [3H]WIN 35,428 and [3H]GBR 12,935 binding, are altered markedly using this animal model (Wilson et al., 1994). However, in sequential sections from the same animals, [3H]DTBZ binding was normal throughout the entire rostrocaudal extent of the basal ganglia (including striatum and nucleus accumbens), cerebral cortex, and diencephalon, as well as in midbrain and brainstem monoamine cell body regions, both on the last day of cocaine access and after 3 weeks of drug withdrawal. These data provide additional evidence that VMAT2, unlike DAT, is resistant to dopaminergic regulation.     

Which form of dopamine is the substrate for the human dopamine transporter: the cationic or the uncharged species?

The question of which is the active form of dopamine for the neuronal dopamine transporter is addressed in HEK-293 cells expressing the human dopamine transporter. The Km value for [3H]dopamine uptake fell sharply when the pH was increased from 6.0 to 7.4 and then changed less between pH 7.4 and 8.2. The KI for dopamine in inhibiting the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane binding displayed an identical pH dependence, suggesting that changes in uptake result from changes in dopamine recognition. Dopamine can exist in the anionic, neutral, cationic, or zwitterionic form, and the contribution of each form was calculated. The contribution of the anion is extremely low (相似文献   

Novel N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues: selective ligands for the dopamine transporter

A series of N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues has been prepared that function as dopamine uptake inhibitors. The N-methylated analogue of this series had a significantly higher affinity for the dopamine transporter than the parent compound, N-methyl-3 alpha- (diphenylmethoxy)tropane (benztropine, Cogentin). Yet like the parent compound, it retained high affinity for muscarinic receptors. A series of N-substituted compounds were prepared from nor-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane via acylation followed by hydride reduction of the amide or by direct alkylation. All compounds containing a basic tropane nitrogen displaced [3H]WIN 35,428 at the dopamine transporter (Ki range = 8.5-634 nM) and blocked dopamine uptake (IC50 range = 10-371 nM) in rat caudate putamen, whereas ligands with a nonbasic nitrogen were virtually inactive. None of the compounds demonstrated high binding affinity at norepinephrine or serotonin transporters. Importantly, a separation of binding affinities for the dopamine transporter versus muscarinic m1 receptors was achieved by substitution of the N-methyl group with other N-alkyl or arylalkyl substituents (eg. n-butyl, allyl, benzyl, 3-phenylpropyl, etc.). Additionally, the most potent and selective analogue in this series at the dopamine transporter, N-(4"-phenyl-n-butyl)-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogue failed to substitute for cocaine in rats trained to discriminate cocaine from saline. Potentially, new leads toward the development of a pharmacotherapeutic for cocaine abuse and other disorders affecting the dopamine transporter may be discovered.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Phorbol esters increase dopamine transporter phosphorylation and decrease transport Vmax

Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK1, cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.     

Striatal dopaminergic abnormalities in human cocaine users

OBJECTIVE: Previous human postmortem experiments have shown an abnormally high number of dopamine uptake sites in the striatum of chronic cocaine users, which might contribute to cocaine withdrawal symptoms such as depression and suicidality. Previous inconsistencies in results were perhaps related to selective radioligand affinity changes or a coexisting loss of dopamine neurons. METHOD: In the present study, binding of the cocaine analog [3H]WIN 35428 to the dopamine transporter was assayed in postmortem striatal samples from 15 cocaine-using subjects and 15 matched comparison subjects to determine whether there were differences in number of binding sites or in affinity. Binding to the vesicular monoamine transporter, a measure of total dopaminergic terminals, was also assessed by using the radioligand (+)-[3H]dihydrotetrabenazine (DTBZ). RESULTS: Striatal [3H]WIN 35428 binding sites were significantly more numerous in the cocaine users: the mean Bmax value was 9.0 fmol bound/microg protein (SD = 2.8) for the cocaine users but only 6.0 (SD = 1.7) for the comparison subjects. Severity of chronic cocaine use was significantly related to [3H]WIN 35428 binding level. [3H]DTBZ binding was significantly lower in the cocaine users (mean = 330 nCi/mg, SD = 42) than in the comparison subjects (mean = 374, SD = 68). CONCLUSIONS: The present results confirm that cocaine users have a high number of dopamine transporter binding sites on dopaminergic neurons, despite an apparent low number of total dopamine terminals. These abnormalities may contribute to the abnormalities in subjective experience and behavior characteristic of chronic cocaine abusers.     

Protein kinase C inhibitors block amphetamine-mediated dopamine release in rat striatal slices

The stimulant drug amphetamine is postulated to enhance dopamine release through the plasmalemmal dopamine transporter by an exchange diffusion with synaptosomal dopamine. Because protein kinase C has been shown to have an effect on dopamine transporter activity, we examined the effect of protein kinase C inhibitors on endogenous dopamine release stimulated by amphetamine in perfused rat striatal slices. At concentrations of 1 microM, the selective protein kinase C inhibitors chelerythrine, Ro31-8220 and calphostin C nearly completely inhibited endogenous dopamine release elicited by 1 microM amphetamine. The inactive analog bisindoylmaleimide V had no effect. Extracellular Ca++ was not required for the effect of the inhibitors. The importance of vesicular dopamine release was examined by determining inhibitor activity in reserpine-treated rats. Dopamine release elicited by 1 microM amphetamine was not significantly altered in reserpine-treated rats compared with control animals. Ro31-8220 at 1 microM completely blocked amphetamine-induced dopamine release in reserpine-treated rats. Activation of protein kinase C with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased dopamine release, and the release was not additive with 1 microM amphetamine. Both chelerythrine and Ro31-8220 at 1 microM increased [3H]dopamine uptake by 17% and 30%, respectively, whereas a brief exposure to 12-O-tetradecanoylphorbol 13-acetate slightly inhibited [3H]dopamine uptake. Our results suggest that amphetamine-mediated dopamine release through the plasmalemmal transporter is highly dependent on protein kinase C activity.     

Long-acting blockade of biogenic amine transporters in rat brain by administration of the potent novel tropane 2beta-propanoyl-3beta-(2-Naphthyl)-tropane

2beta-Propanoyl-3beta-(2-naphthyl)-tropane (WF-23) is a potent cocaine analog with activity at dopamine and serotonin transporters. The purpose of these experiments was to characterize the time course of effects of acute administration of WF-23 on spontaneous locomotion and biogenic amine transporters. Rats received injections i.p. with WF-23 (1 mg/kg), cocaine (30 mg/kg) or vehicle and locomotor activity was measured at various times postinjection. Animals were killed immediately after behavioral activity. Locomotor activity was significantly increased by WF-23 administration, reaching maximum at 4 hr and persisting for 24 hr. Cocaine-elicited elevations in locomotor activity occurred only at the earliest times. WF-23 decreased DA transporter binding in striatal membranes ([125I]RTI-55 binding), with >50% loss in binding for up to 49 hr postinjection. WF-23 increased the Kd of the high affinity site, with no effect on Bmax. Cocaine depressed binding (20%) only at the earliest times. WF-23 decreased levels of [3H]WIN 35,428 binding sites up to 95% of control in both dorsal and ventral striatum with a similar time-course when assessed autoradiographically. WF-23 also reduced [3H]citalopram binding to serotonin transporter sites throughout the brain. The slow onset and very long duration of action of WF-23, taken together with its actions at dopamine and serotonin transporters, suggest a potential role for treatment of disorders characterized by their involvement of these neural systems.     

Binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binding

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.     

Synthesis, structure, dopamine transporter affinity, and dopamine uptake inhibition of 6-alkyl-3-benzyl-2-[(methoxycarbonyl)methyl]tropane derivatives

A series of 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl)methyl]tropane analogues were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter (DAT). The in vitro affinity (Ki) for the DAT of the 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl) methyl]tropane analogues was determined by inhibition of [3H]WIN 35,428 in rat caudate putamen tissue. The inhibition of dopamine uptake (IC50) was also measured for selected compounds which demonstrated moderate affinity for the dopamine transporter. The unsubstituted enantiopure analogues (-)-19a (Ki = 33 nM) and surprisingly (+)-20a (Ki = 60 nM) were found to be almost equipotent with the high-affinity binding components of cocaine and WIN 35,065-2 and exhibited slightly more potent dopamine uptake inhibition than both cocaine and WIN 35,065-2. In general, substitution at the 6-position of racemic 19a and 20a with alkyl groups was found to result in decreased activity relative to increased chain length of the substituent. The 3 beta-benzyl-2 beta-[(methoxycarbonyl)methyl]-6 beta-methyltropane (21b; Ki = 57 nM) was the only 6-alkyl derivative to exhibit moderately potent activity. The 6 beta-isomer 21b was 4-fold more potent than the 6 alpha-isomer 19b (Ki = 211 nM) and was nearly equipotent with (-)-19a and (+)-20a as well as with cocaine and WIN 35,065-2. The results of this study further demonstrate the steric constraints associated with the C(6)-C(7) methylene bridge of the tropane ring system for molecular recognition of cocaine analogues at the cocaine binding site(s) on the DAT.     

Angoline and chelerythrine, benzophenanthridine alkaloids that do not inhibit protein kinase C

Starting with an extract derived from the stem of Macleaya cordata (Papaveraceae) that was active in the process of inhibiting phorbol 12,13-dibutyrate binding to partially purified protein kinase C (PKC), the benzophenanthridine alkaloid angoline was isolated and identified. This discovery appeared in context, as a related benzophenanthridine alkaloid, chelerythrine, has been reported to mediate a variety of biological activities, including potent and selective inhibition of protein kinase C (PKC). However, in our studies, angoline was not observed to function as a potent inhibitor of PKC. Moreover, we were unable to confirm the reported inhibitory activity of chelerythrine. In a comprehensive series of studies performed with various PKC isozymes derived from a variety of mammalian species, neither chelerythrine nor angoline inhibited activity with high potency. To the contrary, chelerythrine stimulated PKC activity in the cytosolic fractions of rat and mouse brain in concentrations up to 100 microM. In addition, chelerythrine and angoline did not inhibit [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC at concentrations up to 40 microg/ml, and no significant alteration of PKC-alpha, -beta, or -gamma translocation was observed with human leukemia (HL-60) cells in culture. Further, chelerythrine did not inhibit 12-O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity with cultured mouse 308 cells, but angoline was active in this capacity with an IC50 value of 1.0 microg/ml. A relatively large number of biological responses have been reported in studies conducted with chelerythrine, and alteration of PKC activity has been considered as a potential mechanism of action. In light of the current report, mechanisms independent of PKC inhibition should be considered as responsible for these effects.     

Protein kinase C regulates nutrient uptake and growth in hepatoma cells

BACKGROUND: Human hepatoma cells extract glutamine at rates severalfold greater than normal hepatocytes through a high-affinity transporter encoded by the ATB0 gene, which contains two putative phosphorylation sites for protein kinase C (PKC). The studies presented here were undertaken to determine whether System B0-mediated glutamine uptake regulates hepatoma growth and whether PKC regulates the activity of this transporter. METHODS: SK-Hep cells were treated with the PKC activator phorbol 12-myristate 13-acetate (PMA) and the initial-rate transport of glutamine and other nutrients measured at specific times thereafter. Growth rates were monitored during culture +/- PMA or an excess of system B0 substrates relative to glutamine. RESULTS: PMA treatment exerted a rapid (half-life approximately 15 minutes) concentration-dependent inhibition of glutamine uptake rates to 50% of control values via a posttranslational mechanism that decreased transporter maximum velocity. This effect persisted after 24 hours and was abrogated by the PKC inhibitor staurosporine. PMA also significantly decreased amino acid transport System y+ and System L activities but no System A. Chronic treatment with PMA (PKC depletion) inhibited SK-Hep growth, as did attenuation of System B0-mediated glutamine uptake with other B0 substrates. CONCLUSIONS: System B0-mediated glutamine uptake regulates hepatoma cell growth, whereas PKC influences both processes.