Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) mediates many cellular responses to elevated cytosolic Ca²⁺ levels (Soderling et al. 2001). In vascular smooth muscle CaMKII is implicated in the regulation of cell migration, contraction and force maintenance, the Ca²⁺ sensitivity of MLCK, Ca²⁺ channel activity, Ca²⁺/ATPase activity, and MAP kinase activation. The membrane potential of proximal colon myocytes is modulated by CaMKII regulation of Ca²⁺-activated K⁺ channels.

CaMKII holoenzymes are multimers comprised of 6-12 kinase subunits arranged as a stacked pair of hexameric rings. Four genes encode the kinase subunit isoforms (α, β, γ, δ) and alternative splicing within a variable domain generates additional diversity. Ca²⁺/CaM activation of CaMKII results in rapid Thr286 phosphorylation on adjacent subunits. Thr286 autophosphorylation allows the kinase to maintain activity towards substrates in response to transient increases in cytosolic Ca²⁺ levels after Ca²⁺ levels decrease (independent or autonomous activity) in vivo (Braun & Schulman, 1995). Following release of Ca²⁺/CaM, Thr305 autophosphorylation prevents subsequent CaM binding, lowers total CaMKII activity, and prevents the generation of additional autonomous activity (Hashimoto et al. 1987). Assaying CaMKII autonomous activity in a tissue lysate is an indication of prior enzyme activation because Thr286 autophosphorylation only occurs after Ca²⁺/CaM binding to the holoenzyme. The holoenzyme structure of CaMKII and the effects of Thr286 autophosphorylation on enzyme activity, along with the different affinities of the subunit isoforms for CaM allows CaMKII to be activated to different levels in response to different Ca²⁺ oscillation frequencies (De Koninck & Schulman, 1998). We are investigating the characteristics of CaMKII expressed in murine proximal colon and fundus: representative phasic and tonic gastrointestinal smooth muscles, respectively, which are characterized by different Ca²⁺ signalling patterns (Somlyo & Himpens, 1989). Adult BalbC mice were killed by CO₂ inhalation followed immediately by cervical dislocation. This method of euthanasia is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. RT-PCR analysis identified the same four λ and δ isoforms in each tissue. However, total CaMKII activity in proximal colon smooth muscle lysates is 3-fold higher than in fundus (3.8 ± 0.51 vs. 1.2 ± 0.13 pmol min^-1 µg^-1; n = 3). The levels of autonomous activity in fundus and proximal colon are 29.2 ± 5.1 and 3.3 ± 0.4 %, (n = 3), respectively, of total CaMKII activity levels.

Autonomous CaMKII activity in proximal colon lysates can be increased to 52.3 ± 4.2 % of total CaMKII activity, while autonomous CaMKII activity in fundus lysates shows little or no increase (n = 3). However, alkaline phosphatase treatment of fundus lysates increased total CaMKII activity by 64.7 ± 10 %, decreased autonomous activity to 3.3 ± 0.4 % of total activity, and allowed subsequent generation of autonomous activity up to 25.8 ± 3.7 % of total activity (n = 3). Acetylcholine treatment of fundus and proximal colon smooth increased autonomous CaMKII activity to 44.7 ± 8.2 and 26.8 ± 0.5 %, respectively (n = 3). The ability of alkaline phosphatase to increase total and autonomous CaMKII activity in fundus smooth muscle lysates represents the first indication that Thr305 phosphorylation plays a role in regulating CaMKII activity in vivo.

Braun, A.P. & Schulman, H. (1995). Annu. Rev. Physiol. 57, 417-445.

De Koninck, P. & Schulman, H. (1998). Science 279, 227-230.

Hashimoto, Y., Schworer, C.M., Colbran, R.J. & Soderling, T.R. (1987). J. Biol. Chem. 262, 8051-8055.

Soderling, T.R., Chang, B. & Brickey, D.A. (2001). J. Biol. Chem. 276, 3719-3722.

Somlyo, A.P. & Himpens, B. (1989). FASEB J. 3, 2266-2276.