Repeated amphetamine couples norepinephrine transporter and calcium channel activities in PC12 cells.

Repeated intermittent amphetamine enhances efflux of dopamine through the dopamine transporter in rat basal ganglia and through the norepinephrine transporter in rat pheochromocytoma PC12 cells. Extracellular Ca2+ is required for the detection of this enhancement in the rat. In this study, we examined the role of Ca2+ and Ca2+ channels in the enhanced amphetamine-induced dopamine efflux that develops in PC12 cells following repeated intermittent amphetamine. Repeated pretreatment of PC12 cells with 1 microM amphetamine followed by a drug-free period increased amphetamine-induced efflux of dopamine compared with controls. The enhancement in amphetamine-induced dopamine efflux depended upon the presence of extracellular Ca2+ and was inhibited by the blockade of N-type and L-type Ca2+ channels. The enhanced dopamine efflux was not altered by tetanus toxin or reserpine, treatments that abrogate synaptic vesicle-mediated, exocytotic dopamine efflux. Measurement of intracellular Ca2+ concentrations using fura-2/acetoxymethyl ester revealed that amphetamine increased intracellular Ca2+ by a transporter-dependent mechanism. In amphetamine-pretreated cells, amphetamine elicited a greater increase in intracellular Ca2+; this increase depended upon the presence of extracellular Ca2+ and N- and L-type Ca2+ channel activity. The enhanced amphetamine-induced dopamine efflux requires Ca2+/calmodulin kinase activity. In vehicle-treated cells, 1 microM amphetamine inhibited the calmodulin kinase activity although it did not in amphetamine-pretreated cells. This study suggests that repeated intermittent amphetamine couples norepinephrine transporter activity and Ca2+ signaling.

Caspase-mediated calcineurin activation contributes to IL-2 release during T cell activation.

Calcineurin, a Ca(2+)/calmodulin-dependent Ser/Thr phosphatase (protein phosphatase 2B), plays a critical role in IL-2 production during T cell activation. It has been previously reported that IL-2 release in activated Jurkat T requires caspase-like activity (Posmantur et al. (1998) Exp. Cell. Res. 244, 302-309). We report here that the 60-kDa catalytic subunit of calcineurin A (Cn A) was partially cleaved to a 45-kDa form in phytohemagglutinin A (PHA) or phorbol ester + ionomycin (P + I)-activated Jurkat cells. In parallel, proteolytic activation of upstream caspases (caspase-8 and -9) as well as effector caspase-3 was also observed. Cn A cleavage was caspase mediated, since it was inhibitable by pan-caspase inhibitor Cbz-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB). Cn A cleavage was also observed when purified calcineurin was digested in vitro with caspase-3. Truncated Cn A was associated with enhanced phosphatase activity and reduced calmodulin sensitivity. Furthermore, in PHA or P + I-activated Jurkat cells, dephosphorylation of calcineurin substrate NFATc (a transcription factor known to be involved in transactivation of the IL-2 gene), was also suppressed by Z-D-DCB. Taken together, our results suggest that caspase-mediated cleavage of Cn A contributes to IL-2 production during T cell activation.

Protein kinase C and intracellular calcium are required for amphetamine-mediated dopamine release via the norepinephrine transporter in undifferentiated PC12 cells.

The role of protein kinase C and intracellular Ca(2+) on amphetamine-mediated dopamine release through the norepinephrine plasmalemmal transporter in undifferentiated PC12 cells was investigated. The selective protein kinase C inhibitor chelerythrine completely inhibited endogenous dopamine release elicited by 1 microM amphetamine. Direct activation of protein kinase C increased dopamine release in a Ca(2+)-insensitive, imipramine-sensitive manner and the release was not additive with amphetamine. Exocytosis was not involved since these events were not altered by either deletion of extracellular Ca(2+) or reserpine pretreatment. Down-regulation of protein kinase C activity by long-term phorbol ester treatment resulted in a dramatic decrease in amphetamine-mediated dopamine release with no apparent effect on [(3)H]dopamine uptake. To more completely examine a role for Ca(2+), intracellular Ca(2+) was chelated in the cells. Depletion of intracellular Ca(2+) considerably decreased dopamine release in response to 1 microM amphetamine compared with vehicle-treated cells, but had no effect on the [(3)H]dopamine uptake. Thus, our results suggest that amphetamine-mediated dopamine release through the plasmalemmal norepinephrine transporter is highly dependent on protein kinase C activity and intracellular but not extracellular Ca(2+). Furthermore, protein kinase C and intracellular Ca(2+) appear to regulate [(3)H]dopamine inward transport and amphetamine-mediated outward transport of dopamine independently in PC12 cells.

Calcium/calmodulin-dependent protein kinase inhibition potentiates thapsigargin-mediated cell death in SH-SY5Y human neuroblastoma cells.

We previously demonstrated a loss in Ca(2+)/Calmodulin-dependent protein kinase (CaM kinase) activity in SH-SY5Y undergoing thapsigargin-mediated apoptosis. To extend that finding we report that CaM kinase inhibition potentiates thapsigargin-mediated cell death. CaM kinase inhibitor KN93 on its own exhibits little toxicity up to 10 mM, as measured by release of lactate dehydrogenase (LDH) into the culture medium. In SH-SY5Y cells pretreated with KN93 and the non-selective protein kinase inhibitor k252a and then treated with 2 mM thapsigargin, loss of viability is significantly greater than in cells treated with thapsigargin alone. Pretreatment with the pan-caspase inhibitor Z-D-DCB prevented the thapsigargin-mediated increase in LDH release. Furthermore, thapsigargin-induced caspase-3-like activation, demonstrated by poly(ADP)ribose polymerase cleavage and pro-caspase-3 processing, was elevated in the presence of KN93.

Caspase-mediated proteolytic activation of calcineurin in thapsigargin-mediated apoptosis in SH-SY5Y neuroblastoma cells.

We previously demonstrated a loss in calmodulin (CaM)-dependent protein kinase activity in SH-SY5Y cells undergoing thapsigargin-mediated apoptosis, (K. M. McGinnis et al., 1998, J. Biol. Chem. 273, 19993-20000). Here we demonstrate that the large subunit of the CaM-dependent protein phosphatase 2B (calcineurin) is fragmented during SH-SY5Y cell apoptosis to a major fragment of 45 kDa in a caspase inhibitor-sensitive manner. A 45-kDa fragment was also produced when purified calcineurin was digested with recombinant caspase-3. The major cleavage site was identified to be DFGD* G(386)ATAA, which removes the C-terminal CaM-binding and autoinhibitory regions from the catalytic domain. Phosphatase activity increased progressively with caspase-3 digestion, coupled with the eventual loss of CaM-dependency. Calcineurin-mediated dephosphorylation of NFATc was also detected in thapsigargin-treated cells. Last, calcineurin inhibitors FK506 and cypermethrin provided partial protection against thapsigargin-mediated apoptosis, suggesting that calcineurin overactivation contributes to thapsigargin-induced apoptosis.

Differential regulation of calmodulin content and calmodulin messenger RNA levels by acute and repeated, intermittent amphetamine in dopaminergic terminal and midbrain areas.

Repeated doses of psychoactive drugs often produce adaptive responses that differ from the initial drug application and additional adaptive processes occur following cessation of the drug. The relationship between alterations in calmodulin protein and messenger RNA produced by an initial versus a repeated dose of amphetamine was examined, as well as changes following drug cessation. Calmodulin protein and messenger RNA of the three individual calmodulin genes were measured in rat dopaminergic cell body and terminal areas following acute or repeated amphetamine. Rats were either injected once with 2.5mg/kg amphetamine or saline and decapitated after 3h, or given 10 injections of amphetamine three to four days apart and decapitated 3h after the final injection. Calmodulin messenger RNA and protein were also measured three and seven days after ceasing drug treatment. Acute amphetamine increased calmodulin 1.7-fold in the striatum and threefold in the ventral mesencephalon, with corresponding elevations in calmodulin messenger RNAs. In response to the 10th dose of amphetamine, however, the degree of increase in calmodulin was diminished in the striatum and ablated in the ventral mesencephalon. Correspondingly, select species of calmodulin messenger RNA were decreased from control levels. In the frontal cortex or nucleus accumbens, calmodulin levels were basically unaltered by the first or 10th doses of amphetamine, but both calmodulin and its messenger RNA were altered with time upon cessation of the drug. Three days later, both calmodulin protein and messenger RNA were decreased in select brain areas. By seven days after the 10th injection, calmodulin content was altered compared to saline controls in all areas, but the change in messenger RNA no longer paralleled the change in protein.Our findings demonstrate that both calmodulin protein and select species of calmodulin messenger RNA are altered by acute amphetamine, but this effect is attenuated after repeated, intermittent amphetamine. There are further time-dependent changes after cessation of repeated amphetamine, which may reflect compensatory neuronal responses. The alterations in calmodulin content and synthesis could contribute to changes in patterns or duration of behaviors that occur upon cessation of repeated amphetamine.

Dopamine transporter antagonists block phorbol ester-induced dopamine release and dopamine transporter phosphorylation in striatal synaptosomes.

We have reported that inhibition of protein kinase C blocks the Ca(2+)-independent reverse transport of dopamine mediated by amphetamine. In this study we investigated whether activation of protein kinase C by 12-O-tetradecanoyl phorbol-13-acetate (TPA) would mediate dopamine release through the plasmalemmal dopamine transporter. TPA, at 250 nM, increased the release of dopamine from rat striatal slices and synaptosomes while the inactive phorbol ester, 4alpha-phorbol, was ineffective. The TPA-mediated dopamine release was independent of extracellular calcium and was blocked by a selective protein kinase C inhibitor, Ro31-8220. The dopamine transporter antagonists, cocaine and GBR 12935 blocked the TPA-mediated dopamine release. In addition, cocaine blocked TPA-mediated phosphorylation of the plasmalemmal dopamine transporter. These results suggest that activation of protein kinase C results in reverse transport of dopamine through the plasmalemmal dopamine transporter and the phosphorylated substrate could be the dopamine transporter.

Ca2+/calmodulin signaling in NMDA-induced synaptic plasticity.

Repeated experiences induce a synaptic plasticity in neurons that can be very long lasting. The neurotransmitter, glutamate, acting through N-methyl-D-aspartate (NMDA) receptors is integrally involved in eliciting persistent changes in synaptic function resulting in learning and memory. The permeability of NMDA receptors to Ca2+ implies the close involvement of Ca2+ and the Ca2+-binding protein, calmodulin, in NMDA-induced synaptic plasticity. A notable example of NMDA-induced synaptic plasticity is long-term potentiation in the hippocampal CA1 region. The involvement of Ca2+ and calmodulin in the induction and expression of LTP has been intensively investigated and documented. Less well studied are neurochemical adaptations in another example of NMDA-induced synaptic plasticity, stimulant-induced behavioral sensitization. Although amphetamine and cocaine increase synaptic monoamines, glutamate is involved in the induction and expression of the sensitization. Activating NMDA receptors in dopamine midbrain cell bodies is required for inducing stimulant sensitization, implying a role for Ca2+ in this plasticity. The purpose of this review is to examine the role of Ca2+ and calmodulin in two examples of NMDA-based plasticity, LTP, and stimulant-induced behavioral sensitization. There are similarities in the neuroadaptations, although the role of Ca2+ and calmodulin has not been thoroughly investigated in the stimulant-induced plasticity.

Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates.

We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment.

Enhanced amphetamine- and K+-mediated dopamine release in rat striatum after repeated amphetamine: differential requirements for Ca2+- and calmodulin-dependent phosphorylation and synaptic vesicles.

After cessation of repeated, intermittent amphetamine, we detected an emergent Ca2+-dependent component of amphetamine-induced dopamine release and an increase in calmodulin and Ca2+- and calmodulin-dependent protein kinase activity in rat striatum. This study examined the involvement of calmodulin-dependent protein kinase II (CaM kinase II) and synaptic vesicles in the enhanced Ca2+-dependent dopamine release in response to amphetamine or K+ in rat striatum. Rats were pretreated for 5 d with 2.5 mg/kg amphetamine or saline and withdrawn from drug for 10 d. The selective CaM kinase II inhibitor KN-93 (1 microM), but not the inactive analog KN-92, attenuated the Ca2+-dependent amphetamine-mediated dopamine release from amphetamine-pretreated rats but had no effect in saline-pretreated controls. [3H]Dopamine uptake was unaltered by repeated amphetamine or KN-93 and was Ca2+ independent. Striatal dopamine release stimulated by 50 mM KCl was enhanced twofold after repeated amphetamine compared with that in saline controls but was unaffected by KN-93. To examine the requirement for dopaminergic vesicles in the Ca2+-dependent dopamine release, we administered reserpine to saline- and amphetamine-pretreated rats 1 d before killing. Reserpine pretreatment did not affect amphetamine-mediated dopamine release from either pretreatment group but completely ablated K+-mediated dopamine release. Reserpine did not disrupt the ability of 1 microM KN-93 to block the Ca2+-dependent amphetamine-mediated dopamine release from amphetamine-pretreated rats. The results indicate that the enhanced dopamine release elicited by amphetamine from chronically treated rats is dependent on Ca2+- and calmodulin-dependent phosphorylation and is independent of vesicular dopamine storage. On the contrary, the enhanced depolarization-mediated vesicular dopamine release is independent of Ca2+- and calmodulin-dependent phosphorylation.

Continuous phosphorylation of GAP-43 and MARCKS by long-term TPA treatment in SK-N-SH human neuroblastoma cells.

Long-term treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulates select protein kinase C (PKC) isozymes and may differentially affect PKC substrates. We investigated the role of PKC down-regulation on phosphorylation of two PKC substrates, the 43 kDa growth-associated protein (GAP-43) and the myristoylated alanine-rich C-kinase substrate (MARCKS) in SK-N-SH human neuroblastoma cells. Cells were treated with 70 nM TPA for 15 min, 17 or 72 h. Phosphorylation of MARCKS and GAP-43 was elevated throughout 72 h of TPA. The magnitude and peptidic sites of phosphorylation in GAP-43 and MARCKS were similar after all TPA treatments. GAP-43, but not MARCKS, content was increased after 17 and 72 h of TPA. The ratio of GAP-43 phosphorylation to content was elevated throughout 17 h but returned to control by 72 h as content increased. PKC epsilon and alpha isozyme content was greatly reduced after 72 h of TPA but membranes retained 23% of PKC activity. Only PKC epsilon translocated to membranes after 15 min TPA. GAP-43 content after 72 h of TPA was increased in subcellular fractions in which significant PKC epsilon isozyme concentration remained. These results demonstrate that continuous TPA differentially affected phosphorylation of PKC substrate proteins and regulation of PKC isozyme content in SK-N-SH cells.

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells.

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis.

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.

Injection of the protein kinase C inhibitor Ro31-8220 into the nucleus accumbens attenuates the acute response to amphetamine: tissue and behavioral studies.

The ability of amphetamine to produce heightened locomotor activity is thought to be due to its ability to enhance dopamine release from mesolimbic dopamine neurons. The mechanism by which amphetamine increases dopamine release is not well understood, but is thought to involve exchange diffusion with synaptosomal dopamine through the dopamine transporter. We recently reported that amphetamine-mediated dopamine release in the striatum is also dependent on protein kinase C activity. In the current study, we investigated the role of protein kinase C activity in the acute neurochemical and behavioral response to amphetamine in the nucleus accumbens. Consistent with previous results in the striatum, amphetamine-stimulated dopamine release from nucleus accumbens tissue was inhibited by the specific protein kinase C inhibitor Ro31-8220, but not by the relatively inactive analog bisindoylmaleimide V. In addition, the effects of protein kinase C activity on the acute behavioral response to amphetamine was examined by injecting Ro31-8220 into the nucleus accumbens 15 min prior to intra-accumbens amphetamine. Pretreatment with Ro31-8220 attenuated the motor-stimulant effects of intra-accumbens amphetamine relative to control subjects pretreated with vehicle. Bisindoylmaleimide V did not significantly inhibit the motor-stimulant effects of intra-accumbens amphetamine. These results suggest that the action of amphetamine in the nucleus accumbens in increasing dopamine release and locomotor activity is dependent on protein kinase C activity.

Alterations in calmodulin mRNA expression and calmodulin content in rat brain after repeated, intermittent amphetamine.

To assess whether calmodulin (CaM) gene expression could have a role in behavioral sensitization induced by repeated, intermittent amphetamine, CaM protein and mRNA of the three separate CaM genes were measured in several different brain areas from rats repeatedly administered saline or amphetamine. Rats were injected twice weekly for five weeks, followed by one week of withdrawal. CaM protein and mRNA were measured in dorsal striatum, limbic forebrain, prefrontal cortex, ventral mesencephalon and piriform cortex. There were increases of CaM protein content and decreases of CaM I mRNA in the dorsal striatum and prefrontal cortex. CaM II mRNA was also decreased in the dorsal striatum. A decrease of CaM protein and an increase of CaM I mRNA were found in the ventral mesencephalon. There was no change of CaM protein in the limbic forebrain, although a decrease of CaM I mRNA was detected. CaM protein and mRNA were not altered in the piriform cortex. Our findings demonstrate that both CaM content and mRNA are altered after an amphetamine regimen leading to sensitization. The fact that the changes in CaM content and mRNA are in dopaminergic brain areas associated with sensitization suggests that CaM could contribute to neurochemical events underlying behavioral sensitization to amphetamine.

Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis.

We have previously demonstrated cleavage of alpha-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

The effect of environment on the changes in calmodulin in rat brain produced by repeated amphetamine treatment.

Rats were given repeated infusions of i.v. amphetamine in association with placement in a novel test environment, a protocol that produces behavioral sensitization, or in the home cage, a protocol that fails to induce sensitization. In several brain areas amphetamine altered calmodulin content, but only in the group treated in a novel environment, suggesting that amphetamine-induced alterations in calmodulin may occur only when drug treatments induce behavioral sensitization.

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.

Cytosolic calmodulin is increased in SK-N-SH human neuroblastoma cells due to release of calcium from intracellular stores.

Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 +/- 18% of control values for carbachol treatment alone vs. 163 +/- 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 +/- 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca2+ by pretreatment with the cell-permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 +/- 37% of control without BAPTA/AM vs. 136 +/- 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 +/- 30% of control). The mechanism for the CaM release may involve activation of the alpha isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.

Amphetamine increases the phosphorylation of neuromodulin and synapsin I in rat striatal synaptosomes.

Amphetamine is taken up through the dopamine transporter in nerve terminals and enhances the release of dopamine. We previously found that incubation of rat striatal synaptosomes increases phosphorylation of the presynaptic neural-specific protein, neuromodulin (Gnegy et al., Mol. Brain Res. 20:289-293, 1993). Using a state-specific antibody, we now demonstrate that incubation of rat striatal synaptosomes with amphetamine increases levels of neuromodulin phosphorylated at ser41, the protein kinase C substrate site. Phosphorylation was maximal at 5 min at 37 degrees C at concentrations from 100 nM to 10 microM amphetamine. The effect of amphetamine on the phosphorylation of synapsin I at a site specifically phosphorylated by Ca2+/calmodulin-dependent protein kinase II (site 3), was examined using a state-specific antibody for site 3-phosphosynapsin I. Incubation with concentrations of amphetamine from 1 to 100 nM increased the level of site 3-phospho-synapsin I at times from 30 sec to 2 min. The effect of amphetamine on synapsin I phosphorylation was blocked by nomifensine. The presence of calcium in the incubating buffer was required for amphetamine to increase the level of site 3-phospho-synapsin I. The amphetamine-mediated increase in the content of phosphoser41-neuromodulin was less sensitive to extrasynaptosomal calcium. The amphetamine-mediated increase in the content of site 3-phospho-synapsin I persisted in the presence of 10 microM okadaic acid and was not significantly altered by D1 or D2 dopamine receptor antagonists. Preincubation of striatal synaptosomes with 10 microM of the protein kinase C inhibitor, Ro-31-8220, blocked the amphetamine-mediated increases in the levels of both phosphoser41-neuromodulin and site 3-phospho-synapsin I. Our results demonstrate that amphetamine can alter phosphorylation-related second messenger activities in the synaptosome.