BACKGROUND
the brain is considered a fully aerobic organ as it requires about 20% of total oxygen consumption in humans  <cit> .
interruption of steady oxygen supply results in focal necrosis and causes severe dysfunction in the ischemic penumbra  <cit> .
numerous studies underlined the seminal role of hypoxia inducible factor-1α  in governing the hypoxic response in both neurons and glial cells  <cit> .
the neuroprotective role of hif-1α has been demonstrated in the ischemic penumbra through erythropoietin induction  <cit>  as well as in mediating a neuroprotective response to amyloid-β peptide  <cit> .
however, the regulation of central neurotransmission systems has not been thoroughly investigated under hypoxic conditions, although their inadequate adaptation might contribute to the development of cerebral palsy and abnormal behavioural patterns in patients affected by pre- or postnatal cerebral hypoxia, respectively  <cit> .
apart from its well-known functions in the nigro-striatal pathway, dopamine plays a very important role in the regulation of mood, affections, impulsivity and cognitive functions in the limbic system  <cit> .
dopaminergic neurotransmissison has been shown to be exquisitely vulnerable to ischemic-anoxic insults, and hypoxic derangements of the dopamine system have been implicated in the pathogenesis of cerebral palsy, schizophrenia and minimal brain dysfunction such as attention deficit hyperactivity disorder   <cit> .
on the other hand, hypoxia has been implicated in promoting differentiation of neuronal precursor cells to dopaminergic neurons through activation of hif-1α  <cit> .
our current understanding of dopaminergic signalling in hypoxia is further confounded by results of recent in vivo studies showing that hypoxic regulation of key dopaminergic genes is highly tissue-specific, and strongly influenced by the duration of hypoxic periods.
among these factors, most of attention has been attributed to the dopamine d <dig> receptor  due to its pathological role in schizophrenia.
drd <dig> mrna levels show an early and transient reduction in the striatum after hypoxia-ischemia in newborn rats  <cit> , and attenuation of drd <dig> mediated inhibition of calcium influx in pheochromocytoma cells has been reported in hypoxia  <cit> .
on the other hand, huey and powell revealed that hypoxia modulates drd <dig> expression in a tissue-dependent manner  <cit> .
for instance, drd <dig> mrna levels initially increased in the caudal nucleus tractus solitarius in rats in response to hypoxia, but then significantly decreased after  <dig> h  hypoxic treatment.
a similar tendency was unveiled in the rat carotid body, too.
in contrast, hypoxia profoundly increased drd <dig> mrna in the rostral nucleus tractus solitarius at all time points investigated  <cit> .
a study conducted on rabbit brains also revealed that hypoxic expression patterns of drd <dig> and drd <dig> in different brain areas are far from being uniform  <cit> .
moreover, widely accepted concepts like induction of the tyrosine hydroxylase gene by hypoxia  <cit>  have been challenged by recent studies finding practically unaltered or slightly decreased transcript and protein levels upon hypoxia  <cit> .
to our best knowledge, however, the hypoxic modulation of drd <dig> and drd <dig> receptors, two highly analyzed polymorphic determinants of psychiatric disorders  <cit> , has not been addressed yet experimentally.
previously we studied the functional effects of drd <dig> promoter polymorphisms on gene expression  <cit> , and reinforced the molecular function of a promoter variant characterized earlier  <cit> .
in the present study, we aimed to investigate the transcriptional regulation of a set of dopamine-specific genes by measuring their mrna and protein levels upon short-term hypoxic treatment of a neural  and a glial  cell line.
these cell lines were chosen since they correspond to the main cell types of the brain, neurons and astrocytes; moreover, both of them express drd <dig>  drd <dig> and drd <dig> receptors.
we found that the transcription of the type d <dig> and d <dig> postsynaptic dopamine receptors  was induced several fold upon 2-day hypoxia in a cell-specific manner.
RESULTS
expression and transcriptional activity of hif-1α in sk-nf-i and ccf-sttg <dig> cells
hypoxia-dependent transcriptional activation of genes is mostly governed by hif-1α.
this fact prompted us to analyze the expression patterns of hif-1α in the sk-nf-i human neuroblastoma and ccf-sttg <dig> human astrocytoma cell line.
to this end, cells were challenged either with 1% hypoxia or with  <dig> μm desferrioxamine , a hypoxia-mimicking agent that is known to stabilize hif-1α through blocking the activity of proline hydroxylases  <cit> .
as it can be seen in fig.
<dig>  the hif-1α protein was undetectable in normoxic cultures, while both short-term hypoxic or dfo treatment dramatically upregulated the protein levels of this hypoxia dependent transcription factor.
importantly, two separate bands were recognized by the specific hif-1α antibody that might correspond to differentially spliced variants  <cit> .
in neuroblastoma cells, both bands were induced equally, while in ccf-sttg <dig> cells the staining of the upper band was more pronounced .
in order to directly monitor the transcriptional activity of hif-1α in neuroblastoma cells, we generated a luciferase reporter vector containing multiple hypoxia responsive elements inserted in the sv <dig> strong promoter .
this construct was transiently transfected into sk-nf-i cells which were subsequently subjected to 1% hypoxia or treated with dfo as described in methods.
notably, both hypoxia and dfo treatment enhanced the activity of this reporter construct several fold over basal levels .
these results proved that the hypoxic signalling pathway is intact and readily inducible in sk-nf-i cells.
induction of dopamine receptors  <dig> and  <dig> upon hypoxia
we aimed to study the expression of a set of dopaminergic neurotransmission specific genes under hypoxia in the sk-nf-i and astrocytoma model system.
to this end, parallel cultures were maintained both in hypoxia and normoxia, and the temporal pattern of gene expression was followed by quantitative reverse transcription pcr using gene-specific taqman probes.
finding a stable endogenous control gene is the cornerstone of the validation of qrt-pcr data.
in order to select an optimal hypoxia-insensitive reference gene, we sought to screen for amplification efficiency and overall stability the mrna levels of the following five candidate genes widely used in qrt-pcr studies: β-actin, hydroxymethylbilane synthase , hypoxanthine guanine phosphorybosyltransferase , p <dig> large ribosomal protein  and rna polymerase ii .
data indicate the ratios of 2-Δct values of corresponding gene pairs.
the ratios were calculated by dividing the greater value with the smaller one so as to get quotients exceeding  <dig>  ct values were determined by calculating the means of three parallel pcr amplifications from three independent cdna samples  prepared from sk-nf-i cultures kept for  <dig> hrs in 1% hypoxia.
no significant changes in the mrna levels of dopamine d <dig>  d <dig> and d <dig> receptors , the dopamine transporter , monoamino oxidase a , catechol-o-methyltransferase  and vascular endothelial growth factor  were revealed in samples kept for 0– <dig> hrs in normoxia .
on the contrary, both drd <dig> and drd <dig> receptor mrna levels were upregulated upon long-term  incubation of sk-nf-i and ccf-sttg <dig> cells in 1% hypoxia .
in neuroblastoma cells, the induction of drd <dig> was much more pronounced  than that of drd <dig> .
in contrast, the drd <dig> was much more inducible than drd <dig> in astrocytoma cells, and its mrna levels were highly elevated already after  <dig> hr hypoxia .
however, the induction pattern of both receptors differed profoundly from that of vegf, a positive control gene known to be directly activated by hif-1α: transcription of the vegf gene was induced already after  <dig> hr hypoxia and its mrna levels dynamically increased over the entire incubation period in both cell lines.
importantly, the drd <dig> gene did not prove to be hypoxia sensitive at all in either cell lines .
furthermore, no remarkable alterations were revealed in the mrna levels of comt, maoa and dat upon hypoxia in either cell lines investigated .
parallel cell cultures were incubated in 1% hypoxia for the indicated time periods.
cdna samples were prepared from three independent cultures and amplified in three parallel pcr reactions .
relative expression levels were calculated as means and standard deviations of  values normalized to expression levels of the rplp <dig> internal control gene.
data presented in the table were generated by dividing these normalized expression levels by that of the 0-hr control .
drrf transcription is repressed by hypoxia
drrf  is a zinc finger transcription factor that is considered a key regulator of post-synaptic dopamine receptors.
drrf has reportedly modulated drd <dig>  drd <dig> and drd <dig> promoter activities in a cell specific manner  <cit> .
we were prompted to check whether activation of drd <dig> and drd <dig> promoters might be due to altered drrf expression in hypoxia.
it turned out that 1% hypoxia repressed drrf levels in a time-dependent manner in sk-nf-i cells.
its concentration was the lowest after  <dig> hr hypoxia and then slightly elevated up to  <dig> hrs .
in conclusion, there seems to be an inverse correlation between drd3/drd <dig> and drrf levels in the context of sk-nf-i neuroblastoma cells.
no elevation of drd <dig> and drd <dig> protein levels by hypoxia
having demonstrated the hypoxia sensitivity of the drd <dig> and drd <dig> genes, we aimed to examine whether elevated mrna levels of both genes correlate well with protein expression.
to this end, neuroblastoma and astrocytoma cells were cultured parallel for  <dig> hrs in normoxia or in 1% hypoxia, respectively, then fixed and immunostained with specific anti-drd <dig> and anti-drd <dig> antibodies.
cells were homogenously stained with marked cortical enrichment , a pattern characteristic of membrane surface receptors  <cit> .
we quantified staining intensities of corresponding normoxic and hypoxic samples by subjecting 500- <dig> cells to densitometry; however, no significant changes were revealed .
one can conclude that the transcriptional activation of drd <dig> and drd <dig> genes was not followed by elevation of their protein levels in these two cell lines.
discussion
since we chose human neural tumour cell lines as experimental model systems, it was mandatory to verify that the hypoxic signalling pathway is intact and functional in these cells.
checking the mere expression of hif-1α by western blot is, however, not sufficient to claim that sk-nf-i and ccf-sttg <dig> cells express a functional hif-1α variant .
for instance, others found that the transcriptional activity of hif- <dig> can be strongly impaired without simultaneous reduction in hif-1α protein levels under certain conditions  <cit> , and accumulation of non-functional hif-1α has also been reported in normoxic cells  <cit> .
results presented in fig.
<dig> clearly demonstrated that hif-1α was highly functional in our experimental system as it transactivated an artificial hypoxia-responsive promoter construct several fold upon hypoxia.
importantly, the desferrioxamine treatment used as a positive control did not elicit a transactivation commeasurable with that of 1% hypoxia , although the expression patterns of hif-1α in sk-nf-i cells were highly similar in both cases as evaluated by western blot .
this marked difference might be attributed to the fact that hif-1α is modified post-transcriptionally both by proline and asparagine hydroxylases in its oxygen-dependent degradation domain and in its c-terminal transactivation domain, respectively  <cit> .
proline hydroxylation destabilizes hif-1α by facilitating its interaction with the von hippel-lindau protein, while asparagine hydroxylation by fih- <dig> abrogates its interaction with the coactivator protein cbp/p <dig>  no hydroxylation takes place in hypoxia, therefore hif-1α is fully functional, while dfo treatment might not block fully asparagine hydroxylation, leading to the accumulation of hif-1α with compromised transactivation potential.
in the rt-pcr assays we ab ovo excluded gapdh, one of the most frequently employed internal control genes, due to its explicit hypoxia sensitivity  <cit> , although early studies with hypoxia relied upon this conventional control gene  <cit> .
in accordance with data of zhong and simons  <cit> , β-actin did not prove to be a reliable internal control gene in the context of our cells too .
although the positive control gene vegf was induced in both cell lines in hypoxia as expected, its expression patterns were slightly different .
this observation might be related to the fact that ccf-sttg <dig> cells predominantly express the higher molecular weight variant of hif-1α  that might bind the vegf promoter with slightly different affinity.
regarding the hypoxic induction of the dopamine receptor genes, two important conclusions could be drawn.
first, the profound difference between the induction patterns of drd <dig> and drd <dig> and the direct hif-1α target gene vegf implies that the drd <dig> and drd <dig> promoters might not be activated directly by hif-1α but other slow-reacting hypoxia-sensitive transcription factors might be involved in their transcriptional regulation .
apart from the hif family, hypoxia activates a cohort of other well-known transcription factors, such as nf-κb  <cit> , ap- <dig>   <cit> , the tumour suppressor p <dig>  <cit>  and c-myc  <cit> , among others.
these factors activate target promoters alone or in concert with hif family members; moreover, they might modulate hif-1α expression, eliciting a protracted transcriptional activation of hif-1α target genes in hypoxia  <cit> .
on the other hand, p <dig>  c-myc and nf-κb have been shown to induce micrornas that may reflect another level of control of the hypoxic response  <cit> .
unfortunately, the promoters of drd <dig> and drd <dig> receptors have not yet been characterized thoroughly, and further investigations such as chromatin immunoprecipitation assays are needed to explore whether the above mentioned transactivators can really be recruited to these promoters in vivo.
interestingly, although drd <dig> is a known target gene of the hypoxia-inducible nf-κb  <cit> , we did not observe any hypoxia-related alterations in its expression in the context of our cell lines .
a similar contradiction has been revealed upon studying the expression pattern of drrf in hypoxia.
although there are consensus binding sites for ap- <dig> in the drrf promoter  <cit> , repressed drrf mrna levels were detected upon hypoxia .
on the other hand, it is tempting to assume that drrf might repress directly or indirectly the drd <dig> and drd <dig> promoters in cells as the expression patterns of these genes were inverse: drrf levels were the lowest after 16– <dig> hr hypoxia when drd <dig> and drd <dig> mrna concentrations started to elevate .
second, the observation that sk-nf-i cells preferentially expressed drd <dig> while drd <dig> was mostly activated in ccf-sttg <dig> cells might be due to different expression of critical, gene-specific transcription factors, or to epigenetic differences in chromatin structure or in hypoxia-responsive remodelling of chromatin .
this issue is particularly interesting in the light of recent reports claiming that hif-1α is capable of interacting both with histone acetyltransferases and deacetylases  <cit> .
we currently try to address the epigenetic regulation of dopaminergic neurotransmission related genes by using specific modulators of chromatin modifying enzymes.
in spite of the profound transcriptional activation of drd <dig> and drd <dig> promoters in hypoxia, we could not detect elevated protein levels by immunostaining of either sk-nf-i or ccf-sttg <dig> cultures .
one can speculate that the duration of hypoxic treatment  might not have been enough for protein synthesis, although drd <dig> mrna levels were elevated already upon  <dig> hrs incubation under hypoxic conditions in astrocytoma cells .
on the other hand, it can be assumed that translation of these transcripts was strongly blocked by hypoxia.
a large body of experimental evidence suggests that hypoxia can reduce cellular energy levels, leading to activation of the amp-activated protein kinase  that downregulates mammalian target of rapamycine  activity, a critical stimulator of translation  <cit> .
moreover, hypoxia has been reported to activate perk  that inactivates the eif2α translation initiation factor by phosphorylation  <cit> .
however, particular mention must be made of the results of  <cit>  who reported that 2-hr oxygen and glucose deprivation increases drd <dig> and drd <dig> protein expression in rat oligodendrocytes.
unfortunately, they did not study the effect of hypoxia alone on the expression of these receptors, therefore their results are not directly comparable with ours.
CONCLUSIONS
in the present study we reported for the first time the hypoxia-induced transcriptional activation of the dopamine d <dig> and d <dig> receptor genes.
however, the molecular mechanism of transactivation remains to be elucidated as our data indicate that these promoters might not be targeted directly by hif-1α.
nevertheless, modulation of postsynaptic dopamine receptor genes by hypoxia might play a role both in the formation of dopaminergic circuitries in the developing brain and in the adaptation of neurons to post-ischemic conditions.

 the brain is considered a fully aerobic organ as it requires about 20% of total oxygen consumption in humans. interruption of steady oxygen supply results in focal necrosis and causes severe dysfunction in the ischemic penumbra. numerous studies underlined the seminal role of hypoxia inducible factor-1α in governing the hypoxic response in both neurons and glial cells. the neuroprotective role of hif-1α has been demonstrated in the ischemic penumbra through erythropoietin induction as well as in mediating a neuroprotective response to amyloid-β peptide. however, the regulation of central neurotransmission systems has not been thoroughly investigated under hypoxic conditions, although their inadequate adaptation might contribute to the development of cerebral palsy and abnormal behavioural patterns in patients affected by pre- or postnatal cerebral hypoxia, respectively. apart from its well-known functions in the nigro-striatal pathway, dopamine plays a very important role in the regulation of mood, affections, impulsivity and cognitive functions in the limbic system. dopaminergic neurotransmissison has been shown to be exquisitely vulnerable to ischemic-anoxic insults, and hypoxic derangements of the dopamine system have been implicated in the pathogenesis of cerebral palsy, schizophrenia and minimal brain dysfunction such as attention deficit hyperactivity disorder. on the other hand, hypoxia has been implicated in promoting differentiation of neuronal precursor cells to dopaminergic neurons through activation of hif-1α. our current understanding of dopaminergic signalling in hypoxia is further confounded by results of recent in vivo studies showing that hypoxic regulation of key dopaminergic genes is highly tissue-specific, and strongly influenced by the duration of hypoxic periods. among these factors, most of attention has been attributed to the dopamine d receptor due to its pathological role in schizophrenia. drd mrna levels show an early and transient reduction in the striatum after hypoxia-ischemia in newborn rats, and attenuation of drd mediated inhibition of calcium influx in pheochromocytoma cells has been reported in hypoxia. on the other hand, huey and powell revealed that hypoxia modulates drd expression in a tissue-dependent manner. for instance, drd mrna levels initially increased in the caudal nucleus tractus solitarius in rats in response to hypoxia, but then significantly decreased after h hypoxic treatment. a similar tendency was unveiled in the rat carotid body, too. in contrast, hypoxia profoundly increased drd mrna in the rostral nucleus tractus solitarius at all time points investigated. a study conducted on rabbit brains also revealed that hypoxic expression patterns of drd and drd in different brain areas are far from being uniform. moreover, widely accepted concepts like induction of the tyrosine hydroxylase gene by hypoxia have been challenged by recent studies finding practically unaltered or slightly decreased transcript and protein levels upon hypoxia. to our best knowledge, however, the hypoxic modulation of drd and drd receptors, two highly analyzed polymorphic determinants of psychiatric disorders, has not been addressed yet experimentally. previously we studied the functional effects of drd promoter polymorphisms on gene expression, and reinforced the molecular function of a promoter variant characterized earlier. in the present study, we aimed to investigate the transcriptional regulation of a set of dopamine-specific genes by measuring their mrna and protein levels upon short-term hypoxic treatment of a neural and a glial cell line. these cell lines were chosen since they correspond to the main cell types of the brain, neurons and astrocytes; moreover, both of them express drd drd and drd receptors. we found that the transcription of the type d and d postsynaptic dopamine receptors was induced several fold upon 2-day hypoxia in a cell-specific manner.  expression and transcriptional activity of hif-1α in sk-nf-i and ccf-sttg cells hypoxia-dependent transcriptional activation of genes is mostly governed by hif-1α. this fact prompted us to analyze the expression patterns of hif-1α in the sk-nf-i human neuroblastoma and ccf-sttg human astrocytoma cell line. to this end, cells were challenged either with 1% hypoxia or with μm desferrioxamine, a hypoxia-mimicking agent that is known to stabilize hif-1α through blocking the activity of proline hydroxylases. as it can be seen in fig. the hif-1α protein was undetectable in normoxic cultures, while both short-term hypoxic or dfo treatment dramatically upregulated the protein levels of this hypoxia dependent transcription factor. importantly, two separate bands were recognized by the specific hif-1α antibody that might correspond to differentially spliced variants. in neuroblastoma cells, both bands were induced equally, while in ccf-sttg cells the staining of the upper band was more pronounced. in order to directly monitor the transcriptional activity of hif-1α in neuroblastoma cells, we generated a luciferase reporter vector containing multiple hypoxia responsive elements inserted in the sv strong promoter. this construct was transiently transfected into sk-nf-i cells which were subsequently subjected to 1% hypoxia or treated with dfo as described in methods. notably, both hypoxia and dfo treatment enhanced the activity of this reporter construct several fold over basal levels. these results proved that the hypoxic signalling pathway is intact and readily inducible in sk-nf-i cells. induction of dopamine receptors and upon hypoxia we aimed to study the expression of a set of dopaminergic neurotransmission specific genes under hypoxia in the sk-nf-i and astrocytoma model system. to this end, parallel cultures were maintained both in hypoxia and normoxia, and the temporal pattern of gene expression was followed by quantitative reverse transcription pcr using gene-specific taqman probes. finding a stable endogenous control gene is the cornerstone of the validation of qrt-pcr data. in order to select an optimal hypoxia-insensitive reference gene, we sought to screen for amplification efficiency and overall stability the mrna levels of the following five candidate genes widely used in qrt-pcr studies: β-actin, hydroxymethylbilane synthase, hypoxanthine guanine phosphorybosyltransferase, p large ribosomal protein and rna polymerase ii. data indicate the ratios of 2-Δct values of corresponding gene pairs. the ratios were calculated by dividing the greater value with the smaller one so as to get quotients exceeding ct values were determined by calculating the means of three parallel pcr amplifications from three independent cdna samples prepared from sk-nf-i cultures kept for hrs in 1% hypoxia. no significant changes in the mrna levels of dopamine d d and d receptors, the dopamine transporter, monoamino oxidase a, catechol-o-methyltransferase and vascular endothelial growth factor were revealed in samples kept for 0– hrs in normoxia. on the contrary, both drd and drd receptor mrna levels were upregulated upon long-term incubation of sk-nf-i and ccf-sttg cells in 1% hypoxia. in neuroblastoma cells, the induction of drd was much more pronounced than that of drd. in contrast, the drd was much more inducible than drd in astrocytoma cells, and its mrna levels were highly elevated already after hr hypoxia. however, the induction pattern of both receptors differed profoundly from that of vegf, a positive control gene known to be directly activated by hif-1α: transcription of the vegf gene was induced already after hr hypoxia and its mrna levels dynamically increased over the entire incubation period in both cell lines. importantly, the drd gene did not prove to be hypoxia sensitive at all in either cell lines. furthermore, no remarkable alterations were revealed in the mrna levels of comt, maoa and dat upon hypoxia in either cell lines investigated. parallel cell cultures were incubated in 1% hypoxia for the indicated time periods. cdna samples were prepared from three independent cultures and amplified in three parallel pcr reactions. relative expression levels were calculated as means and standard deviations of values normalized to expression levels of the rplp internal control gene. data presented in the table were generated by dividing these normalized expression levels by that of the 0-hr control. drrf transcription is repressed by hypoxia drrf is a zinc finger transcription factor that is considered a key regulator of post-synaptic dopamine receptors. drrf has reportedly modulated drd drd and drd promoter activities in a cell specific manner. we were prompted to check whether activation of drd and drd promoters might be due to altered drrf expression in hypoxia. it turned out that 1% hypoxia repressed drrf levels in a time-dependent manner in sk-nf-i cells. its concentration was the lowest after hr hypoxia and then slightly elevated up to hrs. in conclusion, there seems to be an inverse correlation between drd3/drd and drrf levels in the context of sk-nf-i neuroblastoma cells. no elevation of drd and drd protein levels by hypoxia having demonstrated the hypoxia sensitivity of the drd and drd genes, we aimed to examine whether elevated mrna levels of both genes correlate well with protein expression. to this end, neuroblastoma and astrocytoma cells were cultured parallel for hrs in normoxia or in 1% hypoxia, respectively, then fixed and immunostained with specific anti-drd and anti-drd antibodies. cells were homogenously stained with marked cortical enrichment, a pattern characteristic of membrane surface receptors. we quantified staining intensities of corresponding normoxic and hypoxic samples by subjecting 500- cells to densitometry; however, no significant changes were revealed. one can conclude that the transcriptional activation of drd and drd genes was not followed by elevation of their protein levels in these two cell lines. discussion since we chose human neural tumour cell lines as experimental model systems, it was mandatory to verify that the hypoxic signalling pathway is intact and functional in these cells. checking the mere expression of hif-1α by western blot is, however, not sufficient to claim that sk-nf-i and ccf-sttg cells express a functional hif-1α variant. for instance, others found that the transcriptional activity of hif- can be strongly impaired without simultaneous reduction in hif-1α protein levels under certain conditions, and accumulation of non-functional hif-1α has also been reported in normoxic cells. results presented in fig. clearly demonstrated that hif-1α was highly functional in our experimental system as it transactivated an artificial hypoxia-responsive promoter construct several fold upon hypoxia. importantly, the desferrioxamine treatment used as a positive control did not elicit a transactivation commeasurable with that of 1% hypoxia, although the expression patterns of hif-1α in sk-nf-i cells were highly similar in both cases as evaluated by western blot. this marked difference might be attributed to the fact that hif-1α is modified post-transcriptionally both by proline and asparagine hydroxylases in its oxygen-dependent degradation domain and in its c-terminal transactivation domain, respectively. proline hydroxylation destabilizes hif-1α by facilitating its interaction with the von hippel-lindau protein, while asparagine hydroxylation by fih- abrogates its interaction with the coactivator protein cbp/p no hydroxylation takes place in hypoxia, therefore hif-1α is fully functional, while dfo treatment might not block fully asparagine hydroxylation, leading to the accumulation of hif-1α with compromised transactivation potential. in the rt-pcr assays we ab ovo excluded gapdh, one of the most frequently employed internal control genes, due to its explicit hypoxia sensitivity, although early studies with hypoxia relied upon this conventional control gene. in accordance with data of zhong and simons, β-actin did not prove to be a reliable internal control gene in the context of our cells too. although the positive control gene vegf was induced in both cell lines in hypoxia as expected, its expression patterns were slightly different. this observation might be related to the fact that ccf-sttg cells predominantly express the higher molecular weight variant of hif-1α that might bind the vegf promoter with slightly different affinity. regarding the hypoxic induction of the dopamine receptor genes, two important conclusions could be drawn. first, the profound difference between the induction patterns of drd and drd and the direct hif-1α target gene vegf implies that the drd and drd promoters might not be activated directly by hif-1α but other slow-reacting hypoxia-sensitive transcription factors might be involved in their transcriptional regulation. apart from the hif family, hypoxia activates a cohort of other well-known transcription factors, such as nf-κb, ap-, the tumour suppressor p and c-myc, among others. these factors activate target promoters alone or in concert with hif family members; moreover, they might modulate hif-1α expression, eliciting a protracted transcriptional activation of hif-1α target genes in hypoxia. on the other hand, p c-myc and nf-κb have been shown to induce micrornas that may reflect another level of control of the hypoxic response. unfortunately, the promoters of drd and drd receptors have not yet been characterized thoroughly, and further investigations such as chromatin immunoprecipitation assays are needed to explore whether the above mentioned transactivators can really be recruited to these promoters in vivo. interestingly, although drd is a known target gene of the hypoxia-inducible nf-κb, we did not observe any hypoxia-related alterations in its expression in the context of our cell lines. a similar contradiction has been revealed upon studying the expression pattern of drrf in hypoxia. although there are consensus binding sites for ap- in the drrf promoter, repressed drrf mrna levels were detected upon hypoxia. on the other hand, it is tempting to assume that drrf might repress directly or indirectly the drd and drd promoters in cells as the expression patterns of these genes were inverse: drrf levels were the lowest after 16– hr hypoxia when drd and drd mrna concentrations started to elevate. second, the observation that sk-nf-i cells preferentially expressed drd while drd was mostly activated in ccf-sttg cells might be due to different expression of critical, gene-specific transcription factors, or to epigenetic differences in chromatin structure or in hypoxia-responsive remodelling of chromatin. this issue is particularly interesting in the light of recent reports claiming that hif-1α is capable of interacting both with histone acetyltransferases and deacetylases. we currently try to address the epigenetic regulation of dopaminergic neurotransmission related genes by using specific modulators of chromatin modifying enzymes. in spite of the profound transcriptional activation of drd and drd promoters in hypoxia, we could not detect elevated protein levels by immunostaining of either sk-nf-i or ccf-sttg cultures. one can speculate that the duration of hypoxic treatment might not have been enough for protein synthesis, although drd mrna levels were elevated already upon hrs incubation under hypoxic conditions in astrocytoma cells. on the other hand, it can be assumed that translation of these transcripts was strongly blocked by hypoxia. a large body of experimental evidence suggests that hypoxia can reduce cellular energy levels, leading to activation of the amp-activated protein kinase that downregulates mammalian target of rapamycine activity, a critical stimulator of translation. moreover, hypoxia has been reported to activate perk that inactivates the eif2α translation initiation factor by phosphorylation. however, particular mention must be made of the results of who reported that 2-hr oxygen and glucose deprivation increases drd and drd protein expression in rat oligodendrocytes. unfortunately, they did not study the effect of hypoxia alone on the expression of these receptors, therefore their results are not directly comparable with ours.  in the present study we reported for the first time the hypoxia-induced transcriptional activation of the dopamine d and d receptor genes. however, the molecular mechanism of transactivation remains to be elucidated as our data indicate that these promoters might not be targeted directly by hif-1α. nevertheless, modulation of postsynaptic dopamine receptor genes by hypoxia might play a role both in the formation of dopaminergic circuitries in the developing brain and in the adaptation of neurons to post-ischemic conditions. 
in order to clarify the hypoxic sensitivity of key dopaminergic genes, we aimed to study their transcriptional regulation in the context of neuroblastoma and astrocytoma cell lines exposed to 1% hypoxia. in light of the relatively delayed transcriptional activation of the drd and drd genes, we propose that slow-reacting hypoxia sensitive transcription factors might be involved in the transactivation of drd and drd promoters in hypoxia. quantitative rt-pcr assays revealed that the transcription of both type d and d postsynaptic dopamine receptors was induced several fold upon 2-day hypoxia in a cell-specific manner, while the vascular endothelial growth factor gene was activated after 3-hr incubation in hypoxia. on the other hand, mrna levels of type dopamine receptor, dopamine transporter, monoamino oxidase and catechol-o-methyltransferase were unaltered, while those of the dopamine receptor regulating factor were decreased by hypoxia. notably, 2-day hypoxia did not result in elevation of protein levels of drd and drd in contrast, hypoxia promotes the differentiation of dopaminergic neurons.
 dopaminergic pathways that influence mood and behaviour are severely affected in cerebral hypoxia. in contrast, hypoxia promotes the differentiation of dopaminergic neurons. in order to clarify the hypoxic sensitivity of key dopaminergic genes, we aimed to study their transcriptional regulation in the context of neuroblastoma and astrocytoma cell lines exposed to 1% hypoxia.  quantitative rt-pcr assays revealed that the transcription of both type d and d postsynaptic dopamine receptors was induced several fold upon 2-day hypoxia in a cell-specific manner, while the vascular endothelial growth factor gene was activated after 3-hr incubation in hypoxia. on the other hand, mrna levels of type dopamine receptor, dopamine transporter, monoamino oxidase and catechol-o-methyltransferase were unaltered, while those of the dopamine receptor regulating factor were decreased by hypoxia. notably, 2-day hypoxia did not result in elevation of protein levels of drd and drd  in light of the relatively delayed transcriptional activation of the drd and drd genes, we propose that slow-reacting hypoxia sensitive transcription factors might be involved in the transactivation of drd and drd promoters in hypoxia. 