Elevated levels of beta-Amyloid (Abeta) are present in the brains of individuals with either the sporadic or familial form of Alzheimer's disease (AD), and the deposition of Abeta within the senile plaques that are a hallmark of AD is thought to be a primary cause of the cognitive dysfunction that occurs in AD. Recent evidence suggests that Abeta induces neuronal apoptosis in the brain and in primary neuronal cultures, and that this Abeta-induced neuronal death may be responsible in part for the cognitive decline found in AD patients. In this study we have characterized one mechanism by which Abeta induces neuronal death. We found that in cortical neurons exposed to Abeta, activated c-Jun N-terminal kinase (JNK) is required for the phosphorylation and activation of the c-Jun transcription factor, which in turn stimulates the transcription of several key target genes, including the death inducer Fas ligand. The binding of Fas ligand to its receptor Fas then induces a cascade of events that lead to caspase activation and ultimately cell death. By analyzing the effects of mutations in each of the components of the JNK-c-Jun-Fas ligand-Fas pathway, we demonstrate that this pathway plays a critical role in mediating Abeta-induced death of cultured neurons. These findings raise the possibility that the JNK pathway may also contribute to Abeta-dependent death in AD patients.

The inositol 1,4,5-trisphosphate (InsP3) receptor acts as an InsP3-gated Ca2+ release channel in a variety of cell types. Type 1 InsP3 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Here we report that most IP3R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP3R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP3R1-deficient mice were not severely impaired.

Both ACh and levcromakalim evoke smooth muscle cell hyperpolarization and associated relaxation in rat mesenteric resistance arteries. We investigated if they could evoke conducted vasodilatation along isolated arteries, whether this reflected spreading hyperpolarization and the possible mechanism involved. Focal micropipette application of either ACh, to stimulate endothelial cell muscarinic receptors, or levcromakalim, to activate smooth muscle K ATP channels, each evoked a local dilatation (88 ± 14%, n = 6 and 92 ± 6% reversal of phenylephrine-induced tone, n = 11, respectively) that rapidly spread upstream (at 1.5 mm 46 ± 19%, n = 6 and 57 ± 13%, n = 9) to dilate the entire isolated artery. The local dilatation to ACh was associated with a rise in endothelial cell [Ca 2+ ] i (F/F t=0 = 1.22 ± 0.33, n = 14) which did not spread beyond 0.5 mm (F/F t=0 = 1.01 ± 0.01, n = 14), while the local dilatation to levcromakalim was not associated with any change in endothelial cell [Ca 2+ ] i . In contrast, ACh and levcromakalim both stimulated local (12.7 ± 1.2 mV, n = 10 and 13.5 ± 4.7 mV, n = 10) and spreading (at 2 mm: 3.0 ± 1.1 mV, n = 5 and 4.1 ± 0.7 mV, n = 5) smooth muscle hyperpolarization. The spread of hyperpolarization could be prevented by cutting the artery, so was not due to a diffusible agent. Both the spreading dilatation and hyperpolarization were endothelium dependent. The injection of propidium iodide into either endothelial or smooth muscle cells revealed extensive dye coupling between the endothelial cells, but limited coupling between the smooth muscle cells. Some evidence for heterocellular spread of dye was also evident. Together, these data show that vasodilatation can spread over significant distances in mesenteric resistance arteries, and suggest this reflects an effective coupling between the endothelial cells to facilitate [Ca 2+ ] i -independent spread of hyperpolarization.

Membrane potential recordings, made from the circular smooth muscle layer of the gastric antrum taken from mutant mice which lacked the inositol trisphosphate (InsP3) type 1 receptor, were compared with those obtained from the stomach of control (wild‐type) mice. Immunostaining of gastric muscles indicated that the distribution and form of c‐kit positive cells were similar in wild‐type and mutant mice. Smooth muscles from wild‐type mice generated slow waves that in turn initiated spike potentials, while those from mutant mice were either quiescent or generated irregular bursts of spike potentials. In the presence of nifedipine, slow waves with reduced amplitude were generated in wild‐type mice, while all electrical activity was abolished in mutant mice. Acetylcholine depolarized and sodium nitroprusside hyperpolarized the membrane in muscles from both types of mice, being more effective in wild‐type mice. Noradrenaline produced similar hyperpolarizations in both types of mice. Transmural nerve stimulation evoked inhibitory junction potentials (IJPs) in both wild‐type and mutant mice. In wild‐type mice, the IJPs were reduced in amplitude by nitroarginine and converted to a cholinergic excitatory junction potential (EJP) by apamin. In mutant mice, the IJPs were unaffected by nitroarginine or atropine but were abolished by apamin. It is concluded that in antral smooth muscle, the expression of InsP3 type 1 receptors may be causally related to the generation of slow waves but not to the generation of action potentials. A lack of InsP3 receptors attenuates cholinergic excitatory and nitrergic inhibitory responses but does not alter the response to noradrenaline.

The origin and propagation of waves of spontaneous excitation in bundles of smooth muscle of the guinea‐pig bladder were examined using intracellular recording techniques and visualization of the changes in the intracellular calcium concentration ([Ca2+]i). Bladder smooth muscle cells exhibited spontaneous transient increases in [Ca2+]i which originated along a boundary of each smooth muscle bundle and then spread to the other boundary with a conduction velocity of 2.0 mm s−1. Spontaneous increases in [Ca2+]i were always preceded by action potentials. Nifedipine (10 μM) abolished increases in both [Ca2+]i and action potentials. Caffeine (10 mM), ryanodine (50 μM) and cyclopiazonic acid (10 μM) reduced the amplitude of the associated increases in [Ca2+]i without preventing the generation of action potentials. Spontaneous action potentials had conduction velocities of 40 mm s−1 in the axial direction and 1.3 mm s−1 in the transverse direction. The electrical length constants of the bundles of muscle were 425 μm in the axial direction and 12.5 μm in the transverse direction. Neurobiotin, injected into an impaled smooth muscle cell, spread more readily to neighbouring cells located in the axial direction than those located in the transverse direction. The spread of neurobiotin was inhibited by 18β‐glycyrrhetinic acid (18β‐GA, 40 μM), a gap junction blocker. Immunohistochemistry for Connexin 43 showed abundant punctate staining on the smooth muscle cell membranes. These results suggested that spontaneous action potentials and associated calcium waves occur almost simultaneously along the boundary of bladder smooth muscle bundles and then propagate to the other boundary probably through gap junctions.