Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

No description available.

Background: Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) constitute rare chronic inflammatory biliary diseases which likely comprise genetic, environmental and autoimmune factors. Specific inhibitory (auto-) antibodies against the muscarinic acetylcholine receptor type 3 (mAChR3 auto-ab) may contribute to the pathogenesis of chronic biliary inflammation by modulating mAChR3− mediated signaling. Aims: The aim of this study was to analyze the prevalence and relevance of inhibitory mAChR3 auto-ab (mAChR3inh+ auto-ab) in a large cohort of PBC patients from two independent tertiary centers in Berlin and Leipzig in comparison to a large PSC cohort. Baseline parameters and response rates to standard treatment with ursodeoxycholic acid (UDCA) were characterized with respect to the individual mAChR3 auto-ab status. Methods: In total, the study population comprised 437 PBC patients, 187 PSC patients and 80 healthy controls. Clinical and laboratory baseline characteristics were retrieved from medical records. The response to ursodeoxycholic acid (UDCA) therapy after 12 months of treatment was available in 176 PBC and 45 PSC patients. Results: The prevalence of mAChR3inh+ auto-ab was significantly higher among PBC patients (11.2%, 49/437; p = 0.008 vs. healthy controls) and PSC patients (33.6%, 63/187; p < 0.0001 vs. healthy controls) compared to healthy controls (2.5%, 2/80), respectively. PBC patients with mAChR3inh+ auto-ab exhibited significantly higher levels of alkaline phosphatase (ALP) and bilirubin, which constitute established parameters for PBC risk stratification. Moreover, mAChR3inh+ PBC patients tended to show decreased response rates to UDCA therapy compared to PBC patients without mAChR3inh+ auto-ab (mAChR3− PBC). In contrast, PSC patients with mAChR3inh+ auto-ab showed no significant differences in laboratory findings compared to mAChR3 auto-ab negative (mAChR3−) PSC patients. Conclusion: MAChR3inh+ auto-ab might be involved in the pathogenesis and treatment response of chronic biliary inflammation in patients with PBC but not in patients with PSC.

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.