Regarding the mechanism of action of AAP10, it was assumed that AAP10 increases cellular coupling because an enhancement of dispersion normally is ascribed to a reduction in cellular coupling and vice versa. The present study provides more evidence for this mechanism of action because it was possible to demonstrate a reduction in coupling time in papillary muscles without reduction in maximum upstroke velocity. Thus, the reduction in coupling time cannot be caused by an inhibition of the fast sodium current, which is also related to propagation velocity , and therefore likely depends on enhancement of cellular coupling. This is supported by previous experiments of our group using the double-cell voltage clamp described by Weingart . These experiments showed in freshly isolated adult guinea pig cardiomyocyte pairs an enhancement of gap junction conductivity in the order of 25% (six experiments) by 0.01 |JM AAP10, which was added to the superfusion solution. In these experiments one cell was stepped from a holding potential of -40 to -90 to +10 mV in 10 mV steps for 200 ms, thereby stabilizing a transjunctional potential of -50 to +50 mV (sampling rate 1 kHz; filter 400 Hz). In addition, we demonstrated deposition of 14C-labelled AAP10 in the vicinity of the intercalated disks. In step 21 of the purification protocol there are membrane-bound proteins and only some intracellular proteins in the pellet. The bulk of intracellular proteins, especially all contractile proteins, are separated in an earlier step, as stated by Manjunath et al . Because activity was lost in the next purification step, we suggest that AAP10 is probably acting via an acceptor protein and not binding directly to connexin 43.
With regard to structure, we conclude from the NMR spectra that AAP10 in its preferred structure is a semicyclic molecule. The OH moiety of 4-Hyp seems to be essential for its effect, as shown by the lack of effect of AAP8TT, possibly by stabilizing the semicyclic structure. Future studies will focus on the structure-activity relationship by replacing the various amino acids in order to allow definition of further structural characteristics that are essential for the effect. Visit the best pharmacy that thousands of international customers already chose to be their favorite one and see how easy it can be for you to get that Xopenex Inhaler buy here in the amount required for your treatment spending a lot less money than otherwise.