2.3.7.1 Excitation induced activation of NKA
While the NKA content is an important factor in the capacity of muscle to maintain Na+ and
K+ gradients required to maintain muscle excitability, the NKA activity can increase acutely
via activation of several factors including muscle excitation, increased intracellular [Na+],
hormones such as adrenaline and insulin, β2 agonists and via the phosphorylation of
phospholemman (PLM). The most rapid and prominent activation of NKA occurs with muscle excitation. In rat isolated muscle preparations, stimulation at 60 Hz caused a rise of [K+]
e from 4 to 10 mmol/L within 10 s in the soleus and within 2 s in the EDL muscle
(Clausen et al., 2004). While such a rapid increase in [K+]
e may not occur in humans during
exercise, a high magnitude of K+ efflux does occur in humans, with several studies reporting
interstitial [K+] concentration up to ~10-14 mmol/L after intense muscle contraction (Green
et al., 1999b; Green et al., 2000; Juel et al., 2000b; Mohr et al., 2004; Nielsen et al., 2004). Such rapid release of K+ from skeletal muscle and increase in [K+]
e requires prompt increase
of NKA activity in order to maintain muscle excitability (Clausen, 2003). In rat isolated soleus muscle, high frequency unloaded stimulation at 60 Hz for 10 s caused a 58% increase in [Na+]
i, which returned to baseline within 2 minutes (Everts et al., 1994). Importantly, the
recovery of baseline intracellular [Na+] after stimulation was prevented when ouabain, a
specific NKA inhibitor, was pre-incubated into the muscle, thus indicating that the NKA was the cause of the accelerated post-excitation Na+ efflux (Everts et al., 1994). The same study
also reported an approximate two-fold increase in labelled 86Rb+ influx and 22Na+ efflux at
contraction without increased Na+
i accumulation still activates NKA (Everts et al., 1994). The
NKA activity varies between fibre types in rats; when 2 Hz electrical stimulation was performed for 5 minutes, 86Rb+ influx was 2.5 fold higher in the soleus than the EDL muscle
(138% vs 58% respectively) and with only the EDL having a 70% increase in [Na+]
i content
(Everts et al., 1992). This finding implicates soleus NKA having a greater sensitivity to excitation than the EDL in vivo. The rate of activation of NKA is extremely rapid; 10 s of 120 Hz contractions elicited a ~22-fold increase in NKA activity (Nielsen et al., 1997). Because the frequency of electrical stimulation used by Nielsen et al. (1997) is non-physiological, it is unknown whether such activation of NKA can occur in humans. Human exercise studies do, however, strongly support excitation-induced NKA activation; rapid increases in plasma [K+]
to 5-8 mmol/L at the immediate completion of high intensity exercise is consistently followed by a rapid recovery of K+ to baseline levels, usually within ~2 minutes after
intensive exercise, using a variety of exercise intensities, durations and modalities (Medbø et al., 1990; Fraser et al., 2002; Medved et al., 2003; Petersen et al., 2005; Sostaric et al., 2006). Intriguingly, some studies have reported post-exercise hypokalemia where plasma venous [K+] can decrease by as much as 0.5-1.0 mmol/L below resting [K+] at 5-10 minutes
post-exercise (Hallen et al., 1994; Petersen et al., 2005), suggesting that the increased excitation induced activity of NKA is sustained well after exercise, potentially due to the combined effect of muscle contraction and exercise-induced sympathoadrenal activation and the subsequent release of epinephrine (Clausen et al., 1987; Everts et al., 1988). This is supported in vitro where intracellular Na+ was still reduced by ~30% below rest 10 minutes
2.3.7.2 Hormonal and β2 receptor activation of NKA
Another important regulator of acute NKA activity is β2 adrenoreceptors agonists, including
endogenously produced epinephrine and synthetic β2 agonists including isoproteranol and
salbutamol, which are used in the treatment of asthma. In denervated rat diaphragm, isoproteranol caused a 30% decrease in intracellular Na+ (Evans et al., 1973); additionally
isoproteranol caused hyperpolarisation in avian slow twitch muscle, which was preventable by pre-incubation with ouabain (Somlyo et al., 1969). In rat soleus muscle, isoproteranol caused a 12% increase in 42K+ influx, 40% increase in 22Na+ efflux and up to a 67% decrease
in intracellular Na+, which was suppressed by ouabain incubation (Clausen et al., 1977a).
Structurally similar to salbutamol and isoproteranol, epinephrine (adrenaline) is produced endogenously and has a marked effect on acute NKA activation (Clausen et al., 1977a). Epinephrine caused an ouabain-suppressible increase in 22Na+ efflux by 83% and 42K+ influx
by 34% in rat soleus muscle in vitro (Clausen et al., 1977a) and may induce a greater activation of NKA in soleus muscle compared to EDL muscle (Pfliegler et al., 1983; Everts et al., 1988). Importantly, epinephrine at concentrations commonly seen in plasma during intense exercise in humans (Galbo et al., 1975) caused hyperpolarisation and activation of NKA in isolated human intercostal muscles (Ballanyi et al., 1988). Brachial arterial infusion of the β2 agonist terbutaline into the forearm of humans caused a 7% decrease in arterial
plasma [K+], supporting that β
2 agonists activate NKA in humans in vivo. Adrenalectomy also
caused decreased tolerance to KCl infusion in cats (Lockwood et al., 1977) and rats (Bia et al., 1982), whilst the exercise induced hyperkalemia was enhanced when the β blocker propranolol was given before incremental cycling (Hallen et al., 1994), suggesting that
epinephrine via the activation of NKA has an important physiological role in muscle K+
uptake during and immediately after exercise. The common mechanism behind the activation of NKA by catecholamines and other β2 agonists is via activation of β2
adrenoreceptors, which cause downstream activation of cAMP and subsequent phosphorylation of protein kinase A (Clausen, 2003; Clausen, 2010). Unlike excitatory, ion- induced activation of NKA which occurs within seconds, epinephrine takes minutes to have effect (Clausen, 2010). Thus while epinephrine has an important role in regulating [K+]
during rest and exercise, excitatory activation of skeletal muscle NKA is likely to be the key regulator of K+ during sustained muscle contractions.
Insulin and other hormones such as insulin like growth factor 1 (IGF-1), calcitonin gene- related peptide (CGRP), calcitonin and amylin also regulates acute NKA activity. Insulin is a well-established acute activator of NKA, both in vivo and in vitro. In vitro, insulin caused a significant increase in 86Rb uptake in cultured human fibroblasts and a 70% increase in NKA
currant in rabbit cardiac myocytes (Hansen et al., 2000). Insulin induced increased NKA activity was suppressible using ouabain (Clausen et al., 1977b; Clausen et al., 1987). The effect of insulin on NKA is independent of translocation of potential NKA to the sarcolemma (Longo et al., 2001) and does not necessarily increase intracellular Na+ (Kitasato et al., 1980;
Lytton, 1985); thus insulin-mediated NKA activity is likely due to different mechanism(s) than muscle excitation or epinephrine. In vivo studies also support the role of insulin activating NKA; infusion of insulin into the forearm increased K+ uptake, which was reversed
when ouabain was infused (Ferrannini et al., 1988) and insulin has been extensively shown to decrease plasma [K+] (DeFronzo et al., 1980). Like epinephrine, the activation of NKA by
insulin is much slower than excitatory activation of NKA and while insulin activation of NKA has profound implications during basal conditions and in clinical populations requiring insulin, it is unlikely that the insulin-mediated NKA activation is important during or immediately post-exercise as plasma insulin decreases during heavy exercise (Pruett, 1970). Each of IGF-1, CGRP, calcitonin and amylin also cause acute NKA activation, but these peptide hormones do not heavily contribute to K+ regulation during or immediately after
(within 5-10 minutes) exercise and thus are beyond the scope of this review.