Muscarinic M1 and M4 receptor subtypes in normal and pathological conditions in the central nervous system : studies on human and animal tissues using subtype selective ligands

Abstract

Snake venoms from different mamba species contain toxins that bind to muscarinic acetylcholine receptors (mAChR). The toxins MT-1 (selective for M1 after iodination), M1 toxin-1 (also known as MT7, selective for MI) and M4 toxin-1 (also known as MT3, selective for M4 receptors) were isolated and used to study M1 and M4 receptor subtypes in normal and pathological conditions. The pharmacological profile of M1 toxin-1 was determined in functional assays using cloned human M1- M4 receptors expressed in CHO cells. It was clearly demonstrated that M1 toxin-1 acts as a selective noncompetitive antagonist of the muscarinic M1 receptors by binding stably to an allosteric site.

Age-related loss of mAChRs in rat hippocampus and neocortex is still controversial. The effect of ageing on the level of M1 and M4 mAChR subtypes was investigated in the hippocampus and entorhinal cortex of young (21 days), adult (3 months) and old (25 months) rats. A significant increase in muscarinic M1 receptor binding in all areas of the hippocampus and a significant loss in M4 binding only in the CA1 region and entorhinal cortex have been observed in 25 months compared to 21-day-old rats. The increase of M1 receptors in (old aged) rats could be due to compensatory processes as a result of the changes in M4 receptors.

The decrease in M4 receptors in the entorhinal cortex and in the CA1 area of the hippocampus of (old aged) rats could be one of the factors leading to impaired cognitive function. Administration of mAChR agonists or acetylcholinesterase inhibitors produces effective pain relief However, the mAChR subtype(s) involved in the spinal cord are not fully defined. The levels of M1 and M4 receptor subtypes in spinal cord of acute and chronic arthritic rats were investigated. No M1- toxin 1 binding was observed, indicating an absence of M1 receptors. However, the binding Of M4- toxin 1 was reduced between 87-90 % in the Rexed laminae I to X of the spinal cord both in acute and chronic pain as compared to controls. These findings suggest that the mAChR M4 subtype may have a role in cholinergic mechanisms of analgesia. Alzheimers disease (AD) related loss of mAChR subtype has been controversial. Muscarinic M1, M2 and M4 receptor subtype changes in the hippocampus of AD and control brains were evaluated. A significant decrease in M4 receptors was observed in the dentate gyrus and CA4 regions of brain sections from AD patients compared to controls. These findings suggest that, relative to other mAChR subtype, the M4 receptor could be the subtype that is selectively compromised in AD.

Long-term adrenalectomy (ADX) has been reported to cause significant loss of cells in the dentate gyrus and CA1-CA4 fields of the hippocampus resulting in impairment of cognitive functions. The effect of ADX on muscarinic M1 and M4 receptors levels in the hippocampus and entorhinal cortex of 3, 14, 30, 90 and 150 days adrenalectomized male Wistar rats was studied. A significant loss Of M4 but not M1 receptors was observed in the different areas of the hippocampus and entorhinal cortex which varied with time after ADX. The vulnerability is in the order entorhinal cortex > dentate gyrus, CA4 > CA3 > CA2, CAI. Our results show that M1 and M4 receptors are differentially affected after ADX and indicate that the M4 receptor subtype is influenced by adrenal hormones and suggest that the M4 receptor might be the subtype linked to memory functions in the hippocampus.

In conclusion, the studies from the five papers (Papers I - V) constituting this thesis demonstrate that selective ligands like the muscarinic toxins are valuable tools to establish the roles of the different mAChR subtypes involved in normal and pathological conditions in the CNS. These findings suggest that, relative to the M1, the M4 receptor could be the subtype that is selectively compromised in Alzheimer's disease and in pain response. These findings might lead to new therapeutic strategies in the treatment of pain and Alzheimer's disease.