Jesse Baumgold

A novel m2-selective muscarinic antagonist: binding characteristics and autoradiographic distribution in rat brain.

Although several m2-selective muscarinic antagonists have been described, they are not particularly potent. Thus, the development of potent m2-selective compounds remains an important goal. We now report that a bio-isoster of AQ-RA 741 is both one order of magnitude more potent and slightly more selective than previously described compounds. DIBA, a di-benzo derivative of AQ-RA 741, in which the pyridine of the tricycle is replaced with a benzene ring, had Ki values of 4, 0.3, 11 and 2 nM at m1 through m4 receptors, respectively. These values were determined in competition studies with [3H]N-methylscopolamine ([3H]NMS) in membranes from transfected A9 L cells (m1 and m3), rat heart (m2) and NG108-15 cells (m4). AQ-RA 741 had Ki values of 34, 4, 86 and 15 nM at each of these receptors. The autoradiographic distribution of DIBA binding sites was determined by competition studies of [3H]NMS in rat brain. At low concentration, DIBA reduced [3H]NMS binding most significantly from superior colliculi, thalamus, hypothalamus, pontine nucleus, and interpeduncular nucleus, and not appreciably from caudate nucleus, cerebral cortical regions, or hippocampus, consistent with its binding to m2 receptors. These data indicate that DIBA is the most potent, m2-selective muscarinic antagonist yet described. DIBA should therefore become a useful probe in future studies of muscarinic function.

Calcium Independence of Phosphoinositide Hydrolysis-Induced Increase in Cyclic AMP Accumulation in SK-N-SH Human Neuroblastoma Cells

Abstract: Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin‐sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)‐mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK‐N‐SH human neuroblastoma cells while blocking the IP3‐mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) abolished the carbachol‐mediated intracellular calcium release without abolishing the carbachol‐mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist‐induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor‐mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.

Modulation of Adenylylcyclase by Protein Kinase C in Human Neurotumor SK‐N‐MC Cells: Evidence that the α Isozyme Mediates Both Potentiation and Desensitization

Abstract: Exposure of human SK‐N‐MC neurotumor cells to 4β‐phorbol 12‐myristate 13‐acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down‐regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein Gi was unlikely. Cholate extracts of membranes from control and PMA‐treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc− membranes, which lack the stimulatory G protein subunit Gsα; thus, Gs did not appear to be the target of PMA action. Membranes from PMA‐treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn2+ and Mn2+ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK‐N‐MC cells. PMA treatment also resulted in a shift to less sensitivity in the Kact for isoproterenol but not for dopamine or CGP‐12177 (a β3‐adrenergic agonist) stimulation. Thus, the β1 but not the D1 or β3 receptors were being desensitized by PKC activation. Analysis of SK‐N‐MC cells by western blotting with antibodies against different PKC isozymes revealed that both the α and ζ isozymes were present in these cells. Whereas PKC‐α was activated and translocated from cytosol to membrane by phorbol esters, the ζ isozyme was not. Thus, PKC‐α, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.

Effects of verapamil on the binding characteristics of muscarinic receptor subtypes.

The calcium channel antagonist verapamil inhibited [3H]N-methyl scopolamine ([3H]NMS ) binding to muscarinic receptors from both rat brain cortex, an area rich in M1 receptors, and from pons-medulla, an area rich in M2 receptors. Verapamil reduced the dissociation kinetics of [3H]NMS binding in cortical receptors but had no detectable effects on the dissociation kinetics of receptors from the pons-medulla. These data suggest that receptors from the cortex have an allosteric site which regulates the binding kinetics, whereas receptors from the pons-medulla lack this site.

Binding of radioiodinated SPECT ligands to transfected cell membranes expressing single muscarinic receptor subtypes.

The equilibrium dissociation constant and the kinetic rate constants were determined for the binding of (R)-[3H]3-quinuclidinyl benzilate ([3H]QNB) and [125I]3-quinuclidinyl-4-iodobenzilate ((R,R)- and (R,S)-[125I]IQNB) to transfected cell membranes expressing one single muscarinic acetylcholine receptor (mAChR) subtype. The association and dissociation kinetics for the m2 subtype were more rapid than for the m1 and m3 subtypes. The differential kinetic properties may be useful for the single photon emission computed tomographic (SPECT) evaluation of regional mAChR subtype alterations in disease states.

Agents that stimulate phosphoinositide turnover also elevate cAMP in SK-N-SH human neuroblastoma cells

Stimulation of m1 and of m3 muscarinic receptors has previously been shown to increase intracellular cAMP levels in a variety of cells. Although the mechanism underlying this response is not fully understood, it has been hypothesized to be secondary to the IP3-mediated rise in intracellular calcium. In order to determine whether other means of elevating intracellular calcium also raise cAMP levels, we stimulated SK-N-SH human neuroblastoma cells with bradykinin or with maitotoxin. Both of these agents stimulated phospholipase C, stimulated inositol phosphate release and elevated cAMP levels, thus demonstrating that this cAMP response is not unique to muscarinic receptor stimulation.

Comparison of second-messenger responses to muscarinic receptor stimulation in M1-transfected A9 L cells

A9 L cells stable transfected with m1 muscarinic receptors were stimulated with the full agonist carbachol and with the partial agonist pilocarpine. The EC50 values and maximal activation from PI hydrolysis, calcium mobilization, membrane hyperpolarization, cell proliferation and arachidonic acid release were compared. Pilocarpine was approximately half as effective in eliciting PI hydrolysis, calcium mobilization and arachidonic acid release, but was almost as effective as carbachol in the other assays. These findings suggest that the intracellular signals leading to receptor-mediated cell proliferation and to membrane hyperpolarization are amplified, and that therefore these assays are not suitable for determining whether a compound is a partial agonist.

Muscarinic receptor-mediated increase in ζ-PKC expression in SK-N-SH human neuroblastoma cells

Anti-peptide antibodies specific for each protein kinase C (PKC) isozyme were used to screen SKN-SH human neuroblastoma cells. These cells were found to express only α- and ζ-PKC. Stimulation of these cells with phorbol esters caused α-but not ζ-PKC to translocate from cytosolic to membrane fractions. Stimulation of these cells with carbachol, which releases inositol trisphosphate and diacylglycerol, caused a transient translocation of α-PKC but not of ζ-PKC. Carbachol did, however, cause a gradual increase in immunoreactive ζ-PKC which reached maximal values 10–20 min after stimulation. These results implicate ζ-PKC in a receptor-mediated signalling event.

3-α-Chlorimperialine: an M2-selective muscarinic receptor antagonist that penetrates into brain

Abstract Muscarinic M 2 receptors have been found to be severely depleted in post-mortem brains of Alzheimers patients. This loss of receptor may represent a useful diagnostic marker, if it could be quantitatively imaged with single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. In order to develop a radioligand with selectivity for muscarinic M 2 receptors, we now report that 3-α-chlorimperialine is a potent M 2 receptor antagonist with a K i of 0.32 nM at M 2 receptors, a 12-fold selectivity for M 2 over M 1 receptors, and a 5-fold selectivity for M 2 over M 4 receptors. Furthermore, 2% of the injected dose of 3-α-chlorimperialine per gram tissue penetrates into brain within 30 min, then washes out gradually. Taken together, these studies demonstrate that 3-α-chlorimperialine is a potent M 2 -selective muscarinic antagonist that penetrates into brain and may be a useful substrate for radioiodination and subsequent imaging of brain muscarinic M 2 receptors.

Muscarinic receptors in brain from four strains of inbred mice.

The density of brain muscarinic receptors from four strains of inbred mice was determined. C57BL/6J mice had a significantly higher density of muscarinic receptors in the forebrain than Balb/cJ or C57BL/10J mice. In the midbrain, C57BL/6J mice also had the highest density of receptors and in the hindbrain, C57BL/6J and AKR/J mice had a two fold higher receptor density compared to the other two strains. These findings demonstrate that inbred strains of mice which exhibit a range of genetically-determined behaviors, have varying densities of muscarinic receptors.