Abstract:

A-type voltage-gated K⁺ channels auto-regulate their function by undergoing fast inactivation. Independent of molecular mechanisms, this inactivation can proceed after channel opening (open-state inactivation, OSI) or from a closed state prior to opening (closed-state inactivation, CSI). We hypothesize that the specific neurophysiological roles of A-type Kv channels depend on whether they undergo OSI, CSI or both (CSI+OSI). To explore these possibilities, we introduced Markov kinetic schemes of the A-type Kv4 conductance into a computational model of the hippocamcal CA1 neuron assuming either CSI or CSI+OSI and compared the properties of the somatic and dendritic action potentials (APs). Relative to the impact of CSI, the main differential effects of CSI+OSI are:

1. 15% less attenuation of back-propagating APs

2. Shorter latency to the first somatic spike

3. Exaggerated activity-dependent spike broadening and peak attenuation in somatic AP trains

4. The inter-spike intervals of AP trains initially increases before it is shortened (CSI generates monotonic shortening).

The outcome of these simulations thus motivated the development of a simple, modeling-independent method to conclusively elucidate the preferred pathways of inactivation in two distinct A-type Kv channels -Kv3.4 and Kv4.2- expressed in heterologous cells and specific neurons. We applied two voltage-clamp pulse protocols- single- and double-pulse (modest conditioning step followed by a strong test step) - to obtain three pieces of critical information: development macroscopic single-pulse inactivation, the rate of double-pulse inactivation and the voltage-dependence of the time constant of macroscopic inactivation. Consistent with OSI, the rate of Kv3.4 inactivation precisely superimposes on the profile of the Kv3.4 current evoked by a single-pulse and the time constant vs. voltage relation decreases monotonically and levels off. By contrast, in ternary Kv4.2 channels, the rate of Kv4.2 inactivation is asynchronous, peaking earlier relative to the profile of the Kv4.2 single-pulse current and the time constant vs. voltage relation displays a 'J-shape' profile. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, removing KChIP1 from the Kv4 ternary complex or adding DPP10a to Kv4.2 channels produces a CSI+OSI phenotype. This procedure unambiguously establishes contrasting pathways of inactivation in neuronal A-type Kv channels, and provides a simple tool to correlate regulation of ionic conductance and neurophysiological activity.

A recent X-ray crystallographic study revealed an allosteric interaction between PP1C and the N-terminal ankyrin repeat domain of MYPT1 that confers the substrate specificity of the enzyme.2 MP activity is suppressed when Thr696 or Thr853 of MYPT1 is phosphorylated by various kinases, such as ROCK, ZIPK, ILK, and PAK.1,3 However, it is still unclear how the phosphorylation of MYPT1 inhibits MP activity. The amino acid sequence around Thr696 of MYPT1 is highly conserved among MYPT1 family members including MYPT2 and MBS85. Therefore, structural insights into the inhibitory domain of MYPT1 are expected to provide new clues to fully elucidate the mechanism that controls phosphatase activity via the phosphorylation of MYPT1 or other family members involved in kinase-phosphatase crosstalk in cytoskeletal regulation.

Here, we prepared a bacterial recombinant fragment of MYPT1 corresponding to residues 658 to 714, including the phosphorylation site Thr696, and determined its three-dimensional structure through the use of computer-assisted distance geometry and a simulated annealing protocol combined with stable-isotope-aided multi-dimensional NMR techniques.

METHODS: Matched NAF and plasma were collected from 46 high risk women who were administered celecoxib twice daily for two weeks, 20 subjects receiving 200 mg and 26 subjects 400 mg of the agent. NAF and plasma samples were collected before and 2 weeks after taking celecoxib.

RESULTS: In women taking 400 mg bid celecoxib, plasma concentrations of the agent correlated inversely with the change in NAF PGE2 levels from pre- to posttreatment. Nonsignificant trends toward higher celecoxib levels were observed in post- compared to premenopausal women. There was a significant decrease in NAF but not plasma PGE2 concentrations in postmenopausal women who took 400 mg celecoxib (p = 0.03).

CONCLUSION: In high risk women taking 400 mg celecoxib twice daily, plasma concentrations of celecoxib correlated with downregulation of PGE2 production by breast tissue. Strategies synergistic with celecoxib to downregulate PGE2 are of interest, in order to minimize the celecoxib dose required to have an effect.

Celecoxib inhibits PGE2 production in cancerous tissue. We previously reported that PGE2 levels in nipple aspirate fluid (NAF) and plasma were not decreased in women at increased breast cancer risk who received celecoxib 200 mg twice daily (bid). The endpoints of the current study were to determine if a short course of celecoxib 400 mg bid would decrease PGE2 levels in women 1) at increased breast cancer risk, and 2) with established breast cancer.

Methods

NAF and plasma samples were collected before, 2 weeks after taking celecoxib 400 mg bid, and two weeks after washout from 26 women who were at increased breast cancer risk. From 13 women with newly diagnosed breast cancer, NAF from the incident breast and plasma were collected before and on average 2 weeks after taking celecoxib. Additionally, in nine of the 13 women with breast cancer, NAF was collected from the contralateral breast.

Results

No consistent change in NAF or plasma PGE2 levels was noted in high risk premenopausal women. NAF PGE2 levels decreased after celecoxib administration in postmenopausal high risk women (p = 0.02), and in both the NAF (p = 0.02) and plasma (p = 0.03) of women with breast cancer.

Conclusion

Celecoxib 400 mg bid taken on average for 2 weeks significantly decreased NAF, but not plasma, PGE2 levels in postmenopausal high risk women, and decreased both NAF and plasma PGE2 levels in women with newly diagnosed breast cancer. PGE2 levels may predict celecoxib breast cancer prevention and treatment efficacy. Our observations are preliminary, and larger studies to confirm and extend these findings are warranted.