Effect of chronic morphine exposure on the synaptic plasma‐membrane subproteome of rats: a quantitative protein profiling study based on isotope‐coded affinity tags and liquid chromatography/mass spectrometry

Abstract

The effect of chronic morphine exposure on the synaptic plasma-membrane subproteome in rats was studied by the isotope-coded affinity tag (ICAT) method coupled with capillary reversed-phase liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. ICAT-labeled tryptic peptides of synaptic membrane proteins were successfully identified using tandem mass spectrometry in conjunction with protein database searching. Several important synaptic plasma-membrane proteins displayed significant regulation changes as a result of chronic morphine exposure in vivo. In particular, an integral membrane protein Na(+)/K+ ATPase (alpha-subunit) involved in regulation of the cell membrane potential by controlling sodium and potassium ion permeability was downregulated by 39 +/- 2%. This result was in excellent agreement with the reduction in electrogenic Na+, K+ pumping due to about 40% downregulation of Na(+)/K+ ATPase alpha3-isoform in myenteric S-neurons of morphine-exposed guinea-pigs measured by others via immunohistochemistry. The decrease in the abundance of non-erythroid alpha II-spectrin in the synaptic plasma-membrane fraction was also observed, which was hypothetically associated with the breakdown of the protein due to the upregulation of the proteolytic enzyme caspase-3 upon chronic morphine exposure.

An Integrated Quantitative Proteomics and Systems Biology Approach to Explore Synaptic Protein Profile Changes During Morphine Exposure

“…In addition, a number of studies also observed consistent and significant morphine-regulated alterations in the expression of chaperone proteins, specifically heat shock proteins including Hsp60 and Hsp70 (Prokai et al, 2005;Shui et al, 2007;Yang et al, 2007;Suder et al, 2009;Bu et al, 2012). Heat shock proteins such as HSP70 and HSP90 often serve as molecular chaperones, and both iTRAQ þ MS/MS analysis and western Blotting analyses have confirmed the presence of significantly elevated levels of HSP70 in the striatal PSD fraction following the induction of morphine dependence.…”

“…Conversely, Li et al (2009) documented an increase in HSP72 in dorsal root ganglion cells subsequent to a 4-day escalating dependence paradigm, whereas the abundance of several heat shock proteins (HSC71, HSP1B, and HSP86) was upregulated in mouse hippocampal PSD fractions after short-term morphine treatment (Moron et al, 2007). Furthermore, a 7-day chronic morphine administration resulted in significantly elevated levels of HSP60 relative to saline controls (Prokai et al, 2005). Yet, HSP70 was found to be significantly downregulated in the primate NAc during the development of morphine dependence as well as during withdrawal while treated with either methadone or clonidine (Bu et al, 2012), and in the spinal cord of rats after repeated intrathecal morphine injections (Shui et al, 2007).…”

“…A series of additional studies have utilized differential isotopic labeling to investigate alterations in synaptic protein profiles in response to varying paradigms of morphine administration (Prokai et al, 2005;Moron et al, 2007). In the first of these studies, a 7-day paradigm of morphine exposure (continuously delivered via osmotic pump) was used to induce morphine dependence.…”

“…In the first of these studies, a 7-day paradigm of morphine exposure (continuously delivered via osmotic pump) was used to induce morphine dependence. Animals from the saline-and morphine-treatment groups were killed on day 8 and cortical synaptic fractions were then prepared, subjected to labeling with the isotopic reagent (isotopecoded affinity tag (ICAT)) coupled with LC-ESI-MS/MS (Prokai et al, 2005). The authors reported that 74 proteins were identified in total, of which 27 (10 increased and 17 decreased) exhibited significant morphine-dependent regulation.…”

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis

“…For example, the use of subcellular fractionation, which allows for the separation and generation of protein fractions selectively enriched in either pre-or postsynaptic proteins (25,27), in combination with proteomic analysis has revealed that an acute escalating dose of morphine administration produces significant alterations in the postsynaptic density (PSD) 1 associated protein network in the hippocampus. This includes significant redistribution of endocytic proteins such as clathrin (20,22) and glutamatergic ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (12,13). Proteomic analysis coupled with tandem mass spectrometry (MS/MS) followed by the computational analysis that placed the proteomics results within known protein-protein interaction (PPI) networks also revealed a novel function for heat shock proteins and their co-chaperones in the presynaptic active zone of the morphine tolerant striatum.…”