La Familia y su importancia en la Educación Inicial

2.3.8.1 Phospholemman (FXDY1) and NKA

Phospholemman phosphorylation is an important regulator of NKA activity in muscle (Crambert et al., 2002; Crambert et al., 2003; Rasmussen et al., 2008; Cirri et al., 2011). In Xenopus oocytes, Phospholemman associates with both the α1 and α2 isoform of NKA,

although a stronger association was seen with the α1 isoform (Crambert et al., 2002).

Phosphorylation of phospholemman caused a small but significant decrease in affinity of NKA to K+ _{and an approximate two-fold decrease in NKA affinity to Na}+ _{in Xenopus oocytes}

(Crambert et al., 2002), although Cirri et al., (2011) report only a 30% increase in affinity to Na+ _{in the α}

1β1 NKA heterodimer when phospholemman was associated with NKA in vitro,

with no changes in K+ _{affinity. Knockout or inactivation of phospholemman in mice led to a}

~50% decrease in NKA activity in two studies using homogenised heart and skeletal muscle (Jia et al., 2005; Reis et al., 2005), however, other studies have found contradictory results where NKA activity was increased compared to controls in myocytes harvested from phospholemman knockout mice (Despa et al., 2005; Han et al., 2006; Pavlovic et al., 2007).

Phospholemman is activated during muscle contraction is via upstream activation of Protein Kinase A (PKA) and Protein Kinase C (PKC) (Palmer et al., 1991; Walaas et al., 1999; Pavlovic et al., 2007; Bibert et al., 2008; Rasmussen et al., 2008). While it is clear that phospholemman has an important role in the short-term regulation of NKA, the contradictory results which report both an increase (Despa et al., 2005; Han et al., 2006; Pavlovic et al., 2007) and decrease (Jia et al., 2005; Reis et al., 2005) in NKA activation when phospholemman is knocked out/removed demonstrates that the upstream activators and phosphorylation status/site of phosphorylation of phospholemman may alter the effects on NKA activity. Bibert et al. (2008) demonstrated that activation of phospholemman from PKA or PKC and the resultant difference in phosphorylation state of phospholemman, had divergent effects on either NKA activity or affinity. In cardiac myocytes, phospholemman in its inactive state decreased NKA ionic affinity, while phosphorylated phospholemman increased the ionic affinity of NKA to Na+ _{and K}+ _{(Pavlovic et al., 2007). Hence the}

phosphorylation state and upstream activation of phospholemman may be of greater importance than the total phospholemman content. In humans, one-legged cycling caused a significant increase in phosphorylated phospholemman in only the exercising leg, demonstrating that the phosphorylation of phospholemman during exercise is caused by contractile activity and not systemic factors such as circulating epinephrine or insulin during exercise (Benziane et al., 2011). Further, various Na+ _{concentrations caused substantially}

increased NKA activation in muscle after exercise in humans, potentially due to the phosphorylation of phospholemman (Juel et al., 2013).

2.3.8.2 Translocation of NKA

Increased 22_Na+ _{efflux from rat soleus was reported when intracellular Na}+ _{was artificially}

increased in the muscle via cuts in the sarcolemma (Buchanan et al., 2002). Conversely, other studies have reported increased 42_K+ _or 86_Rb+ _{influx and} 22_Na+ _{efflux, synonymous via}

NKA activation, without any change in intracellular Na+ _{content using a variety of}

stimulation frequencies (Everts et al., 1992; Everts et al., 1994; Buchanan et al., 2002). In light of such findings, a potential mechanism for excitation-mediated activation of NKA is an increased sensitivity of NKA to Na+ _{ions (Crambert et al., 2000) A potential mechanism}

behind this phenomenon is the proposed translocation of NKA to the sarcolemma from intracellular stores. The first evidence of this phenomenon was reported when insulin incubation increased ouabain binding site content in frog muscle, which the investigators suspected as being due to an “unmasking” of inactive NKA (Grinstein et al., 1974; Erlij et al., 1976). This increased NKA content was then confirmed to occur during exercise and electrically stimulated muscle contraction (Sandiford et al., 2005b). In rat skeletal muscle, α1 and α2 isoforms increased in abundance by 20-58% in the fractionised membrane portion

by increase after exercise/contraction, accompanied by a concomitant decrease in α1 and

α2 abundance in the endosomal fraction of the homogenate (Sandiford et al., 2005b). This

suggests that the increased sarcolemmal NKA isoform abundance was due to translocation from an intracellular source, but may also reflect imprecision in membrane isolation. Other laboratories have also been able to confirm a translocation paradigm of NKA α2 subunit

using isolated giant sarcolemma vesicles in both rat and human muscle (Juel et al., 2000a; Juel et al., 2001). While centrifuge based fractionation and giant sarcolemma vesicle

methodologies have proven effective in demonstrating a translocation paradigm in NKA during muscle contraction, several studies using vanadate facilitated ouabain-binding site assessment have not been able to find an increased [3_{H]ouabain binding site content, after}

insulin incubation or muscle contraction (Clausen et al., 1977b; McKenna et al., 2003). Considering that [3_{H]ouabain binding site assessment reports only functional units}

compared to individual isoforms, such a finding does create some doubt over the legitimacy of the NKA translocation paradigm. Clausen and Kohn (1977b) argue that subcellular fractionation leads to a low yield of membrane which may not be representative of the entire sample, thus findings relying on subcellular fractionation are not a valid method to assess NKA translocation; they further asserted that the earlier frog muscle studies which used ouabain binding and supported the translocation paradigm did not adequately incubate/saturate the muscle sample, which caused an artificial increase in [3_H]ouabain

binding site content (Clausen et al., 1977b). Conversely, Benziane et al. (2008) contest that the ouabain binding methodology, while accurate for overall muscle ouabain binding sites, is not appropriate for detecting NKA translocation due to the long incubation time of muscle ex vivo which negates the timeframe when translocation could occur (Juel et al., 2001). Hence while there is substantial evidence for translocation of NKA in response to exercise and insulin (Tsakiridis et al., 1996; Juel et al., 2000a; Chibalin et al., 2001; Juel et al., 2001; Sandiford et al., 2005a; Benziane et al., 2008), the lack of data demonstrating changes in functional membrane NKA after exercise or insulin should be considered (Clausen et al., 1977b; McKenna et al., 2003). More research needs to be conducted to conclusively demonstrate the existence and potential mechanisms underlying NKA

translocation. In conclusion, the acute activation of NKA in skeletal muscle during muscle contraction may be caused by a numerous factors, including increased intracellular Na+

which directly stimulates NKA activity, increased Na+ _{sensitivity of NKA due to translocation}

of NKA to the sarcolemma from intracellular stores and the phosphorylation of the NKA associated protein phospholemman.