Global Thrombosis Test: Occlusion by Coagulation or SIPA?

The global thrombosis test (GTT) is a point of care device that tests thrombotic and thrombolytic status. The device exposes whole blood flow to a combination of both high and low shear stress past and between ball bearings potentially causing thrombin and fibrin formation. The question arises as to whether thrombosis in the GTT is dominated by coagulation-triggered red clot or high shear-induced white clot. We investigated the nature of the thrombus formed in the GTT, the device efficacy, human factors use, and limitations. The GTT formed clots that were histologically fibrin-rich with trapped red blood cells. The occlusion time (OT) was more consistent with coagulation than high shear white clot and was strongly lengthened by heparin and citrate, two common anticoagulants. The clot was lysed by tissue plasminogen activator (tPA), also consistent with a fibrin-rich red clot. Changing the bead to a collagen-coated surface and eliminating the low shear zone between the beads induced a rapid OT consistent with a platelet-rich thrombus that was relatively resistant to heparin or tPA. The evidence points to the GTT as occluding primarily due to fibrin-rich red clot from coagulation rather than high shear platelet aggregation and occlusion associated with arterial thrombosis.

Negatively charged nanoparticles of multiple materials inhibit shear-induced platelet accumulation.

Platelet accumulation by VWF under high shear rates at the site of atherosclerotic plaque rupture leads to myocardial infarction and stroke. Current anti-platelet therapies remain ineffective for a large percentage of the population, while presenting significant risks for bleeding. We explore a novel way to inhibit arterial thrombus formation. Theoretically, a negative charge may influence the tertiary structure of VWF to favor the globular configuration by biophysical means without the use of platelet inactivating drugs. We tested this hypothesis experimentally for charged nanoparticles (CNPs) to inhibit thrombus formation in a microfluidic thrombosis assay (MTA). Several different CNPs demonstrated the ability to retard thrombotic occlusion in the MTA. A preliminary study in mice shows that thrombus stability is weaker with CNP administration and bleeding times are not markedly prolonged. The CNPs tested here show promise as a new class of antithrombotic therapies that act by biophysical means rather than biochemical pathways.

Shear-induced platelet aggregation: 3D-grayscale microfluidics for repeatable and localized occlusive thrombosis.

Atherothrombosis leads to complications of myocardial infarction and stroke as a result of shear-induced platelet aggregation (SIPA). Clinicians and researchers may benefit from diagnostic and benchtop microfluidic assays that assess the thrombotic activity of an individual. Currently, there are several different proposed point-of-care diagnostics and microfluidic thrombosis assays with different design parameters and end points. The microfluidic geometry, surface coatings, and anticoagulation may strongly influence the precision of these assays. Variability in selected end points also persists, leading to ambiguous results. This study aims to assess the effects of three physiologically relevant extrinsic design factors on the variability of a single end point to provide a quantified rationale for design parameter and end-point standardization. Using a design of experiments approach, we show that the methods of channel fabrication and collagen surface coating significantly impact the variability of occlusion time from porcine whole blood, while anticoagulant selection between heparin and citrate did not significantly impact the variability. No factor was determined to significantly impact the mean occlusion time within the assay. Occlusive thrombus was found to consistently form in the first third (333 µm) of the high shear zone and not in the shear gradient regions. The selection of these factors in the design of point-of-care diagnostics and experimental SIPA assays may lead to increased precision and specificity in high shear thrombosis studies.

Inhibition of high shear arterial thrombosis by charged nanoparticles.

Platelet accumulation under high shear rates at the site of atherosclerotic plaque rupture leads to myocardial infarction and stroke. Current antiplatelet therapies remain ineffective within a large percentage of the population, while presenting significant risks for bleeding. We explore a novel way to inhibit arterial thrombus formation by biophysical means without the use of platelet inactivating drugs. Our computational multi-scale dynamics model has predicted that charged particles of a specific size may entangle von Willebrand Factor (vWF) polymers and reduce the amount of elongation at high shear rates. We tested this hypothesis experimentally for negatively charged nanoparticles (CNP) to inhibit arterial thrombus formation. CNP of a particular size and charge inhibited thrombus formation, with a 10-fold peak inhibition over control conditions of thrombotic occlusion. Particles of differing material composition, size, and charge had little effect as predicted by computational studies. Surprisingly, the dose response curve was not sigmoidal, but exhibited a peak at 1.5 CNP:vWF proteins, which was not predicted by the model. This study describes a new antithrombotic agent that may have a different mechanism of action than current pharmaceutical therapies.

Hemodynamic studies of platelet thrombosis using microfluidics.

Platelets contribute to thrombus formation in a variety of ways. Platelet adhesion, activation, and thrombus growth depend greatly on the type of hemodynamic environment surrounding an inciting event. Microfluidic systems may be used to explore these relationships. In this review, we describe some important considerations required in the design of a microfluidic system and identify some limitations that may require use of a macroscale system.

Ex Vivo Assessment of a Parabolic-Tip Inflow Cannula for Pediatric Continuous-Flow VADs.

To address the challenge of unloading the left ventricle during pediatric mechanical circulatory support using next-generation rotary blood pumps, a novel inflow cannula was developed. This unique inflow cannula for pediatric, continuous-flow, left ventricular assist devices (VADs) with a parabolic-shaped inlet entrance was evaluated alongside a bevel-tip and fenestrated-tip cannula via an ex vivo, isolated-heart experimental setup. Performance was characterized using two clinical scenarios of over-pumping and hypovolemia, created by varying pump speed and filling preload pressure, respectively, at ideal and off-axis cannula placement to assess ventricular unloading and positional sensitivity. Quantitative and qualitative assessments were performed on the resultant hemodynamics and intra-ventricular boroscopic images to classify conditions of nonsuction, partial, gradual or severe entrainment, and ventricular collapse. The parabolic-tip cannula was found to be significantly less sensitive to placement position (p < 0.001) than the bevel-tip cannula under all conditions, while not statistically different from the fenestrated cannula. Visual analysis of the parabolic-tip cannula showed complete resistance to entrainment, whereas the fenestrated-tip had partial entrainment in 90% and 87% of the over-pumping and hypovolemic studies, respectively. We conclude that future pediatric VAD designs may benefit from incorporating the parabolic-tip inflow cannula design to maximize unloading of the left ventricle in ideal and nonoptimal conditions.

Evolution of multi-well pad development and influence of well pads on environmental violations and wastewater volumes in the Marcellus shale (USA).

A majority of well pads for unconventional gas wells that are drilled into the Marcellus shale (northeastern USA) consist of multiple wells (in some cases as many as 12 wells per pad), yet the influence of the evolution of well pad development on the extent of environmental violations and wastewater production is unknown. Although the development of multi-well pads (MWP) at the expense of single well pads (SWP) has been mostly driven by economic factors, the concentrated nature of drilling activities from hydraulic fracturing and horizontal drilling operations on MWP suggests that MWP may create less surface disturbance, produce more volumes of wastewater, and generate more environmental violations than SWP. To explore these hypotheses, we use geospatial techniques and statistical analyses (i.e., regression and Mann-Whitney tests) to assess development of unconventional shale gas wells, and quantify environmental violations and wastewater volumes on SWP and MWP in Pennsylvania. The analyses include assessments of the influence of different types of well pads on potential, minor and major environmental events. Results reveal that (a) in recent years, a majority of pads on which new wells for unconventional gas were drilled are MWP, (b) on average, MWP have about five wells located on each pad and thus, had the transition to MWP not occurred, between two and four times as much land surface disturbance would have occurred per year if drilling was relegated to SWP, (c) there were more environmental violations on MWP than SWP, but when the number of wells were taken into account, fewer environmental violations per well were observed on MWP than on SWP, (d) there were more wastewater and recycled wastewater volumes per pad and per well produced on MWP than on SWP, and (e) the proportion of wastewater that was recycled was higher on MWP than SWP. This study sheds light on how the evolution from SWP to MWP has influenced environmental violations and wastewater production in a field that has undergone rapid development in recent years.

Estimation of ligand affinity constants for receptor states in functional studies involving the allosteric modulation of G protein-coupled receptors: implications for ligand bias.

INTRODUCTION: The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. METHODS: Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands - including inverse agonists - and the efficacy of the unoccupied receptor (i.e., constitutive activity). RESULTS AND DISCUSSION: We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration-response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor.

Muscarinic agonists and antagonists: effects on gastrointestinal function.

Muscarinic agonists and antagonists are used to treat a handful of gastrointestinal (GI) conditions associated with impaired salivary secretion or altered motility of GI smooth muscle. With regard to exocrine secretion, the major muscarinic receptor expressed in salivary, gastric, and pancreatic glands is the M3 with a small contribution of the M1 receptor. In GI smooth muscle, the major muscarinic receptors expressed are the M2 and M3 with the M2 outnumbering the M3 by a ratio of at least four to one. The antagonism of both smooth muscle contraction and exocrine secretion is usually consistent with an M3 receptor mechanism despite the major presence of the M2 receptor in smooth muscle. These results are consistent with the conditional role of the M2 receptor in smooth muscle. That is, the contractile role of the M2 receptor depends on that of the M3 so that antagonism of the M3 receptor eliminates the response of the M2. The physiological roles of muscarinic receptors in the GI tract are consistent with their known signaling mechanisms. Some so-called tissue-selective M3 antagonists may owe their selectivity to a highly potent interaction with a nonmuscarinic receptor target.

Analysis of agonism and inverse agonism in functional assays with constitutive activity: estimation of orthosteric ligand affinity constants for active and inactive receptor states.

We describe a modification of receptor theory for the estimation of observed affinities (K(obs)) and relative efficacies of orthosteric ligands in functional assays that exhibit constitutive activity. Our theory includes parameters for the fractions of the occupied receptor population in the active (intrinsic efficacy, ε) and inactive (ε(i)) states and analogous parameters for the fractions of the free receptor population in the active (ε(sys)) and inactive (ε(i-sys)) states. The total stimulus represents the summation of the active states of the free and occupied receptor populations. A modified operational model is developed that expresses the response as a logistic function of the total stimulus. This function includes the standard parameters related to affinity and efficacy (K(obs) and τ) as well as a parameter proportional to the activity of the free receptor complex, τ(sys). Two related parameters are proportional to the fraction of the free (τ(i-sys)) and occupied (τ(i)) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (K(b)) and inactive (K(a)) states of the receptor are equivalent to τK(obs)/τ(sys) and τ(i)K(obs)/τ(i-sys), respectively. We verify our method with computer simulation techniques and apply it to the analysis of M(2) and M(3) muscarinic receptors. Our method is applicable in the analysis of ligand bias in drug discovery programs.

Analysis of functional responses at G protein-coupled receptors: estimation of relative affinity constants for the inactive receptor state.

We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (K(obs)) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, ε(i)). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes K(obs) and the parameter τ, which is proportional to ε. We introduce the parameter τ(i), which is proportional to ε(i). We have previously shown that the product, K(obs)τ, of one agonist, expressed relative to that of another (intrinsic relative activity, RA(i)), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, K(obs)τ(i), of one agonist, expressed relative to that of another (intrinsic relative inactivity, RI(i)), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M(3) muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RA(i)/RI(i)).

Quantifying agonist activity at G protein-coupled receptors.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (τ). The estimate of τK(obs) of one agonist, divided by that of another, is a relative measure of K(b) (RA(i)). For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τ(sys)). In this case, the K(b) value of an agonist is equivalent to τK(obs)/τ(sys). Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

Selectivity of agonists for the active state of M1 to M4 muscarinic receptor subtypes.

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M(4) receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M(1) to M(4) muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride] for the M(1) and M(4) subtypes and selectivity of pilocarpine for the M(1) and M(3) subtypes. The other agonists tested lacked marked selectivity among M(1) to M(4) receptors. Finally, we estimated RA(i) values from published literature on M(1), M(2), and M(3) muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.

Two-state models and the analysis of the allosteric effect of gallamine at the M2 muscarinic receptor.

We measured the influence of gallamine on the functional responses and binding properties of selected agonists at the M(2) muscarinic receptor and analyzed the data within the context of the allosteric ternary complex model. Our analysis showed that gallamine modified agonist affinity without influencing efficacy. To explain this behavior, we investigated the allosteric ternary complex model at a deeper level of analysis to assess allosterism in terms of the differential affinity of gallamine for ground and active states of the receptor. Our simulations showed that two-state models based on a single orthosteric site for the agonist linked to an allosteric site for gallamine could not account for affinity-only modulation, even if multiple conformations of ground and active states were considered. We also expanded the tandem two-site model (J Biol Chem 275:18836-18844, 2000) within the context of the allosteric ternary complex model and analyzed the resulting hybrid model at the level of receptor states. This model posits that the agonist first binds to a relay site and then shuttles to the activation site to turn on the receptor. If it is assumed that allosterism occurs at the relay site and not the activation site, then this model can account for affinity-only modulation in a manner consistent with the allosteric ternary complex model.

Estimation of agonist activity at G protein-coupled receptors: analysis of M2 muscarinic receptor signaling through Gi/o,Gs, and G15.

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA(i)), is most applicable for the analysis of responses at G protein-coupled receptors. RA(i) is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M(2) receptor, including stimulation of phosphoinositide hydrolysis through G(alpha15) in HEK 293T cells, inhibition of cAMP accumulation through G(i) in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through G(s) in CHO cells treated with pertussis toxin. The RA(i) values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy]oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was approximately 10-fold greater at M(2) receptors coupled to G(alpha15) in HEK 293T cells compared with M(2) receptors coupled to G(i) in the same cells or in CHO cells. Our results show that the RA(i) estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling.

Comparison of the antimuscarinic action of p-fluorohexahydrosiladifenidol in ileal and tracheal smooth muscle.

We investigated the ability of the muscarinic antagonist p-fluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea. We also investigated the ability of p-fluorohexahydrosiladifenidol to affect the inhibition of contraction caused by the known competitive muscarinic antagonist atropine. Using resultant analysis to analyze this interaction, we found that the true dissociation constant of p-fluorohexahydrosiladifenidol for competitively antagonizing oxotremorine-M-induced contractions in the ileum exhibited significantly lower potency than when calculated assuming a simple competitive model. In contrast, resultant analysis showed little difference between the true and observed potencies of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced contractions in the trachea. Using a simple competitive model, we found little difference in the observed dissociation constant of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced phosphoinositide hydrolysis in guinea pig ileum and bovine trachea. We also noted that p-fluorohexahydrosiladifenidol (0.3-1.0 microM) moderately inhibited histamine-induced contractions of ileum but not of trachea. Our results suggest that p-fluorohexahydrosiladifenidol does not discriminate markedly between M(3) muscarinic receptors in the ileum and trachea and that it may posses a more potent, nonmuscarinic inhibitory effect on contraction in the ileum.

The M2 muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder.

We investigated the contractile role of M2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M3 response in urinary bladder from wild-type and M2 knockout (KO) mice. Evidence for a minor M2 receptor-mediated contraction was revealed by a comparison of responses in M3 knockout and M2/M3 double knockout mice. Treatment of wild-type and M2 knockout urinary bladder with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard) caused a large inhibition of the muscarinic contractile response. The residual contractions were much smaller in M2 knockout bladder compared with wild type, suggesting that M2 receptors rescue the muscarinic contractile response in wild-type bladder following inactivation of M3 receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F2alpha and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M3 KO mice. This response exhibited an M2 profile in competitive antagonism studies and was completely absent in M2/M3 KO mice. Following 4-DAMP mustard treatment, oxotremorine-M elicited a contractile response in wild-type urinary bladder in the presence of KCl and isoproterenol or forskolin, and this response was diminished in M2 KO mice. Our results show that the M2 receptor mediates contractions indirectly in the urinary bladder by enhancing M3 receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M2 receptor function in competitive antagonism studies under conditions where a simultaneous activation of M2 and M3 receptors occurs.

Muscarinic agonist-mediated heterologous desensitization in isolated ileum requires activation of both muscarinic M2 and M3 receptors.

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 microM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2alpha (PGF2alpha) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF2alpha was prevented in both M2 and M3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M2- and M3-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M2 and M3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization.

Comparison of the pharmacological antagonism of M2 and M3 muscarinic receptors expressed in isolation and in combination.

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M(2) and M(3) muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [3H]N-methylscopolamine binding to CHO cells expressing both M(2) and M(3) muscarinic receptors (CHO M(2)+M(3) cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M(2) cells, whereas the other site exhibited properties like those of CHO M(3) cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M(3) and CHO M(2)+M(3) cells, but not in CHO M(2) cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M(3) and CHO M(2)+M(3) cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M(2) and CHO M(2)+M(3) cells and a less robust inhibition in CHO M(3) cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M(3) and CHO M(2)+M(3) cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M(2), CHO M(3), and CHO M(2)+M(3) cells. The pharmacological antagonism of the cAMP response in CHO M(2)+M(3) cells resembled that expected for a response mediated independently by both M(2) and M(3) receptors.