Ret heterozygous mice have enhanced intestinal adaptation after massive small bowel resection.

Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 ± 46.0 µm vs. WT 306.5 ± 7.7 µm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 µm vs. WT 82.4 ± 3.1 µm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P = 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon-like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P = 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome.

Asymmetric synthesis and evaluation of a hydroxyphenylamide voltage-gated sodium channel blocker in human prostate cancer xenografts.

Voltage-gated sodium channels are known to be expressed in neurons and other excitable cells. Recently, voltage-gated sodium channels have been found to be expressed in human prostate cancer cells. α-Hydroxy-α-phenylamides are a new class of small molecules that have demonstrated potent inhibition of voltage-gated sodium channels. The hydroxyamide motif, an isostere of a hydantoin ring, provides an active scaffold from which several potent racemic sodium channel blockers have been derived. With little known about chiral preferences, the development of chiral syntheses to obtain each pure enantiomer for evaluation as sodium channel blockers is important. Using Seebach and Frater's chiral template, cyclocondensation of (R)-3-chloromandelic acid with pivaldehyde furnished both the cis- and trans-2,5-disubsituted dioxolanones. Using this chiral template, we synthesized both enantiomers of 2-(3-chlorophenyl)-2-hydroxynonanamide, and evaluated their ability to functionally inhibit hNa(v) isoforms, human prostate cancer cells and xenograft. Enantiomers of lead demonstrated significant ability to reduce prostate cancer in vivo.

Temporal lobe epilepsy induces intrinsic alterations in Na channel gating in layer II medial entorhinal cortex neurons.

Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) channels play a critical role in AP generation and conduction we sought to determine if Na channel gating parameters and expression levels were altered in TLE. Na channel currents recorded from isolated mEC TLE neurons revealed increased Na channel conductances, depolarizing shifts in inactivation parameters and larger persistent (I(NaP)) and resurgent (I(NaR)) Na currents. Immunofluorescence experiments revealed increased staining of Na(v)1.6 within the axon initial segment and Na(v)1.2 within the cell bodies of mEC TLE neurons. These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.

The importance of serine 161 in the sodium channel beta3 subunit for modulation of Na(V)1.2 gating.

Voltage-gated sodium (Na) channels contribute to the regulation of cellular excitability due to their role in the generation and propagation of action potentials. They are composed of a pore-forming alpha subunit and are modulated by at least two of four distinct beta subunits (beta1-4). Recent studies have implicated a role for the intracellular domain of beta subunits in modulating Na channel gating and trafficking. In beta3, the intracellular domain contains a serine residue at position 161 that is replaced by an alanine in beta1. In this study, we have probed the functional importance of beta3S161 for modulating Na channel gating. Wild-type beta3 and point mutations beta3S161A or beta3S161E were individually co-expressed in HEK 293 cells stably expressing human Na(v)1.2. WTbeta3 expression increased Na current density, shifted steady-state inactivation in a depolarized direction, and accelerated the kinetics of recovery from inactivation of the Na current. Analogous effects were observed with beta3S161E co-expression. In contrast, beta3S161A abolished the shifts in steady-state inactivation and recovery from inactivation of the Na current, but did increase Na current density. Immunocytochemistry and Western blot experiments demonstrate membrane expression of WTbeta3, beta3S161E, and beta3S161A, suggesting that the differences in Na channel gating were not due to disruptions in beta subunit trafficking. These studies suggest that modification of beta3S161 may be important in modulating Na-channel gating.

2,4(5)-diarylimidazoles as inhibitors of hNaV1.2 sodium channels: pharmacological evaluation and structure-property relationships.

Sodium (Na) channels continue to represent an important target for the development of novel anticonvulsants. We have synthesized and evaluated a series of 2,4(5)-diarylimidazoles for inhibition of the human neuronal Na(V)1.2 Na channel isoform. Starting with the unsubstituted lead compound previously published 3, SAR studies were performed introducing substituents with different physico-chemical properties. Lipophilicity (log D(7.4)) and basicity (pK(a)) of the compounds were measured and submitted for QSPR investigations. Some of the active compounds described had IC(50) values that were considerably lower than our lead compound. In particular, the m-CF(3) disubstituted 22 was the most active compound, inhibiting hNa(V)1.2 currents within the nanomolar concentration range (IC(50)=200 nM). In comparison, lamotrigine and phenytoin, two clinically used anticonvulsant drugs known to inhibit Na channels, had IC(50)'s values that were greater than 100 microM.

Modulation of sodium channel inactivation gating by a novel lactam: implications for seizure suppression in chronic limbic epilepsy.

Epilepsy remains a devastating neurological disorder associated with recurrent, unprovoked, spontaneous epileptic seizures. Current treatments involve seizure suppression using antiepileptic drugs (AEDs); however, many patients remain refractory to current treatments or suffer serious side effects. In view of this continued need for more effective and safer AEDs, we have designed a novel compound, 3-hydroxy-3-(4-methoxyphenyl)-1-methyl-1,3-dihydro-indol-2-one (YWI92), based on a lactam structural class, and evaluated its modulation of human neuronal sodium channel isoform (hNa(v))1.2 currents and hippocampal neuron action potential firing. Furthermore, we have tested its AED activity using a chronic and acute rat seizure model. In a similar manner to lamotrigine, a clinically used AED, YWI92 exhibited tonic block of hNa(v)1.2 channels and caused a hyperpolarizing shift in the steady-state inactivation curve when using a 30-s inactivating prepulse. YWI92 also delayed the time constants of channel repriming after a 30-s inactivating prepulse and exhibited use-dependent block at 20-Hz stimulation frequency. In membrane excitability experiments, YWI92 inhibited burst firing in CA1 neurons of animals with temporal lobe epilepsy at concentrations that had little effect on CA1 neurons from control animals. These actions on neuronal activity translated into AED activity in the maximal electroshock acute seizure model (ED(50) = 22.96 mg/kg), and importantly, in a chronic temporal lobe epilepsy model, in which the mean number of seizures was reduced. Notably, YWI92 exhibited no sedative/ataxic side effects at concentrations up to 500 mg/kg. In summary, greater affinity for inactivated sodium channels, particularly after long depolarizing prepulses, may be important for both anticonvulsant activity and drug tolerability.

2,4(5)-Diarylimidazoles: synthesis and biological evaluation of a new class of sodium channel blockers against hNa(v)1.2.

A small family of novel 2,4(5)-diarylimidazoles were prepared through a simple and efficient synthesis and evaluated as potential inhibitors of hNa(v)1.2 sodium channel currents. One member of this series (4) exhibited profound inhibition of Na(v)1.2 currents, emerging as a promising lead compound for further structure-activity relationship studies for the development of novel sodium channel blockers.