Further, it was reported that 30 days of OB treatment caused in the rat colon a significant decrease in the neuronal variant of NOS in the myenteric plexus associated with an up-regulation of excitatory neurotransmission in the CM. It was concluded that chronic OB treatment modifies the balance between inhibitory and excitatory neurotransmission. All these findings indicate that OB owns a broader spectrum of BIX-01294 actions than expected by the in vitro results. In summary, chronic administration of OB indicates that: i) the colonic CM seems to be the main target of OB actions; ii) most OB actions are likely involved in the storage and handling of calcium; and iii) there is an increase in excitatory neurotransmission. Kinetic experiments have shown that OB is poorly systemically absorbed and accumulates in the muscle wall of the large intestine where it exerts its spasmolytic activity. In the JNJ-42041935 present study we investigated the colonic CM of rats chronically treated with OB, with particular regard to the inner portion of this layer. At first, the expression of markers related to the organelles involved in calcium availability, such as caveolae, SER and mitochondria, was investigated and compared to that of the outer portion. Second, by transmission electron microscopy, we ascertained changes in the features of these organelles; by immunohistochemistry and western blot, changes in the expression of markers related to the above organelles and of muscarinic receptors; by functional experiments, the possible involvement of the cholinergic system in the increased excitatory neurotransmission. The present findings demonstrate that in control animals the inner portion of the CM shows a higher expression of several markers compared to the outer portion, and is richer in organelles involved in calcium handling and storage. After chronic OB treatment, both CM portions undergo similar ultrastructural and immunohistochemical changes, but with a different time course. An increase in calcium-related organelles and in Cav-1 and eNOS content is observed after 10 days of treatment.