Characterization of L-type Calcium Channel Binding-Site of a new class of Calcium modulators by a Multidisciplinary approach

Many potential diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transient and on recombinant L-VDCC. Twenty particular compounds were selected by the ligand-based virtual screening procedure (LBVS). From these compounds, five of them (5b, M2, M7, M8 and P1) showed a potent and selective inotropic activity on guinea-pig left atria driven 1 Hz. Further assays displayed an interesting negative inotropic effect of M2, M8, P1 and M7 on guinea pig isolated left papillary muscle driven at 1 Hz, a relevant vasorelaxant activity of 5b, M2, M7, M8 and P1 on K+-depolarized guinea-pig ileum longitudinal smooth muscle and a significant inhibition of contraction of 5b, M2, M8 and P1 on carbachol stimulated ileum longitudinal smooth muscle. Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus Leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding site for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this findings, the relative order of increased rates of contraction and relaxation at lower concentrations s(≤10-6M) in unpaced hearts was M7>M2>M8>P1. Similar increases in Ca2+ transient were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5b had no significant effect. Diltiazem blocks Ca2+current in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8, M2 showed ~ 10% tonic block; in addition, M8, M2 and P1 shifted the steady state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6 ± 11.5 ms, 226 ± 14.5 vs. 269 ± 12.9 vs. 199.28 ± 8.19 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. Only P1 showed a significantly decrease of the time for the recovery from inactivation. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8, M2 and P1 might directly decrease the binding affinity or allow rapid dissociation from the benzothiazepine binding site.

Role of nitric oxide and endocannabinoids in synaptic plasticity in the perirhinal cortex and in visual recognition memory

Introduction and aims of the research Nitric oxide (NO) and endocannabinoids (eCBs) are major retrograde messengers, involved in synaptic plasticity (long-term potentiation, LTP, and long-term depression, LTD) in many brain areas (including hippocampus and neocortex), as well as in learning and memory processes. NO is synthesized by NO synthase (NOS) in response to increased cytosolic Ca2+ and mainly exerts its functions through soluble guanylate cyclase (sGC) and cGMP production. The main target of cGMP is the cGMP-dependent protein kinase (PKG). Activity-dependent release of eCBs in the CNS leads to the activation of the Gαi/o-coupled cannabinoid receptor 1 (CB1) at both glutamatergic and inhibitory synapses. The perirhinal cortex (Prh) is a multimodal associative cortex of the temporal lobe, critically involved in visual recognition memory. LTD is proposed to be the cellular correlate underlying this form of memory. Cholinergic neurotransmission has been shown to play a critical role in both visual recognition memory and LTD in Prh. Moreover, visual recognition memory is one of the main cognitive functions impaired in the early stages of Alzheimer’s disease. The main aim of my research was to investigate the role of NO and ECBs in synaptic plasticity in rat Prh and in visual recognition memory. Part of this research was dedicated to the study of synaptic transmission and plasticity in a murine model (Tg2576) of Alzheimer’s disease. Methods Field potential recordings. Extracellular field potential recordings were carried out in horizontal Prh slices from Sprague-Dawley or Dark Agouti juvenile (p21-35) rats. LTD was induced with a single train of 3000 pulses delivered at 5 Hz (10 min), or via bath application of carbachol (Cch; 50 μM) for 10 min. LTP was induced by theta-burst stimulation (TBS). In addition, input/output curves and 5Hz-LTD were carried out in Prh slices from 3 month-old Tg2576 mice and littermate controls. Behavioural experiments. The spontaneous novel object exploration task was performed in intra-Prh bilaterally cannulated adult Dark Agouti rats. Drugs or vehicle (saline) were directly infused into the Prh 15 min before training to verify the role of nNOS and CB1 in visual recognition memory acquisition. Object recognition memory was tested at 20 min and 24h after the end of the training phase. Results Electrophysiological experiments in Prh slices from juvenile rats showed that 5Hz-LTD is due to the activation of the NOS/sGC/PKG pathway, whereas Cch-LTD relies on NOS/sGC but not PKG activation. By contrast, NO does not appear to be involved in LTP in this preparation. Furthermore, I found that eCBs are involved in LTP induction, but not in basal synaptic transmission, 5Hz-LTD and Cch-LTD. Behavioural experiments demonstrated that the blockade of nNOS impairs rat visual recognition memory tested at 24 hours, but not at 20 min; however, the blockade of CB1 did not affect visual recognition memory acquisition tested at both time points specified. In three month-old Tg2576 mice, deficits in basal synaptic transmission and 5Hz-LTD were observed compared to littermate controls. Conclusions The results obtained in Prh slices from juvenile rats indicate that NO and CB1 play a role in the induction of LTD and LTP, respectively. These results are confirmed by the observation that nNOS, but not CB1, is involved in visual recognition memory acquisition. The preliminary results obtained in the murine model of Alzheimer’s disease indicate that deficits in synaptic transmission and plasticity occur very early in Prh; further investigations are required to characterize the molecular mechanisms underlying these deficits.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.