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Introduction Medial epicondyle fracture displacement is notoriously difficult to determine on conventional radiography, and follow-up computed tomography (CT) is often obtained to measure precise displacement. Another option for fracture characterization is digital tomosynthesis (DT), a technology providing high in-plane resolution of bony anatomy by acquiring multiple low-dose images in a linear arc. Advantages of DT include lower radiation exposure and lower cost than CT, rapid image acquisition, and a similar patient experience to conventional radiography. The digital application of tomosynthesis is relatively new and is integrated as an add-on feature with modern radiography equipment. This study compares DT, CT and conventional radiography for measurement accuracy in medial epicondyle fractures with the goal of determining relative accuracy in measuring medial epicondyle fracture displacement. Methods Medial epicondyle fractures were created in 5 cadaveric elbow specimens. Each specimen was imaged with conventional radiography, DT, and CT. True displacement measured by digital calipers was compared with "measured" displacement for each image acquisition. CT images included axial, sagittal, and coronal reformats. DT images of the elbow included anteroposterior (AP) longitudinal and transverse, lateral longitudinal and transverse, and axial longitudinal and transverse. Conventional radiographs included AP, lateral, and axial distal humerus images. Four physicians reviewed all images 3 months later. Each reviewer independently measured maximum apparent fracture displacement to the nearest 0.1 mm. Measurement accuracy was calculated as percent difference [(measured displacement-actual displacement)/actual displacement] for each acquisition. Mean, median, and SD for measurement accuracy were calculated. Two-tailed paired t tests were performed on each acquisition to compare the measurement accuracy. Results Compared with conventional radiographs, accuracy of DT was superior in AP longitudinal (P=0.03), AP transverse (P=0.01), axial longitudinal (P=0.0001), and axial transverse projections (P=0.001). Accuracy of CT was superior to conventional radiography in the AP projection (P=0.03), but was equivalent in the axial projection (P=0.9). Accuracy of CT was similar to DT in AP longitudinal (P=0.6), AP transverse (P=0.5), and axial longitudinal projections (P=0.07). Accuracy of DT in the axial transverse projection was superior to CT (P=0.03). Conclusion DT is more accurate than conventional radiography (both AP and axial views) and as accurate as CT in assessing millimeters of displacement of medial epicondyle fracture fragments. Level of evidence Level IV-diagnostic study.

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.

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.

Environmental planners seek techniques that will enable them to analyze impervious cover to develop sound management plans for coastal regions. The spatial scale in which impervious cover has traditionally been widely analyzed is mismatched to the fine-scale resolution needed for local environmental management. This study examines impervious cover in New Hanover County, North Carolina using LiDAR derived subwatersheds and United States Geological Survey (USGS) 14-digit hydrologic unit watersheds to evaluate potential scale-dependency of impervious cover estimates. Spatial analysis of impervious cover across multi-scale watersheds indicates that fine scale subwatersheds exhibit patterns not revealed with coarser watersheds. Spatial and cartographic analyses suggest that localized impervious development and its expansion in first-order drainages originating in coastal lagoon watersheds is more appropriately analyzed using fine-scale, LiDAR-derived watersheds. Results stress the importance of using scale in watershed management and hydrogeomorphic context to aid planners when making decisions involving impervious cover.

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.

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.

Climate change and sea-level rise will have severe impacts on coastal water resources around the world. However, whereas the influence of marine inundation is well documented in the literature, the impact of groundwater inundation on coastal communities is not well known. Here, core analysis, groundwater monitoring, and ground penetrating radar are utilized to assess the groundwater regime of the surficial aquifer on Bogue Banks Barrier Island (USA). Then, geospatial techniques are used to assess the relative roles and extents of groundwater and marine inundation on the dune-dominated barrier island under sea-level rise scenarios of 0.2, 0.5, and 1.0 m above current conditions by 2100. Additionally, the effects of rising water tables on onsite wastewater treatment systems (OWTS) are modelled using the projected sea-level rise scenarios. The results indicate that the surficial aquifer comprising fine to medium sands responds quickly to precipitation. Water-level measurements reveal varying thicknesses of the vadose zone (>3 to 0 m) and several groundwater mounds with radial flow patterns. Results from projected sea-level rise scenarios suggest that owing to aquifer properties and morphology of the island, groundwater inundation may occur at the same rate as marine inundation. Furthermore, the area inundated by groundwater may be as significant as that affected by marine inundation. The results also show that the proportion of land in the study area where OWTS may be perpetually compromised by rising water tables under worst case scenarios may range from ~43 to ~54% over an 86-year-period. Copyright © 2014 John Wiley & Sons, Ltd.

With the possibility of future fresh water shortages increasing, a methodology that incorporates climatic and anthropogenic factors is needed. This research estimates future water availability in the Lower Cape Fear basin using changes in climate, land use, and population growth. The USGS Thornthwaite monthly water balance model is used with estimates of climate change and land use change parameters to assess future water resources based on predicted monthly fluxes of the water balance. The southern United States is a rapidly growing region. Trends present in the population data are used to produce future estimates of population for the basin. Precipitation and temperature estimates based on Intergovernmental Panel on Climate Change (IPCC) predictions and current climatology are inputs to the model. Projected increases in impervious surface cover due to population growth and urbanization are incorporated through the model runoff factor. Water stress indicators are used to categorize the sub-watersheds as water rich, water stressed, or water scarce. Scenarios incorporating regional predictions of climate change indicate a decrease in summer soil moisture minima and increases in summer water deficits. Ensemble runs indicate a shift toward water stress in the Lower Cape Fear River basin, due to a warming climate as well as increased demand. While climate change has a significant impact on water resources, population growth was found to have the most substantial impact. The methods and findings have application to water managers at local and regional levels.

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 non-suction, partial, gradual or severe entrainment, and ventricular collapse. The parabolic-tip cannula was found to be significantly less sensitive to placement position (p-values < 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 in order to maximize unloading of the left ventricle in ideal and non-optimal conditions.

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.

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 ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M(2) muscarinic receptor knockout mice. Forskolin (5.0 micro M) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M(2) muscarinic receptor knockout mice. This reduction in contractile activity was characterized by decreases in potency or maximal response, but not always both. Similar results were obtained with isoproterenol (1.0 micro M). The relaxant effects of forskolin in ileum, trachea, and urinary bladder from M(2) receptor knockout mice were approximately 3- to 9-fold greater than those observed in the same tissues from wild-type mice. Similar results were obtained with isoproterenol in ileum and urinary bladder, although the differences between wild-type and M(2) receptor knockout tissues were less than those observed with forskolin. In contrast, there was no significant difference between the relaxant effect of isoproterenol in trachea from wild-type and M(2) receptor knockout mice. In contrast to the results observed with oxotremorine-M as the contractile agent, forskolin and isoproterenol did not exhibit greater relaxant activity against KCl-induced contractions in M(2) receptor knockout mice compared with wild-type mice. These results suggest that a component of the contractile response to muscarinic agonists in smooth muscle involves an M(2) muscarinic receptor-mediated inhibition of the relaxant effects of agents that increase cAMP levels.

Irreversible ligands are useful tools for investigating the function of receptor subtypes in various physiological processes. The mechanism for alkylation involves the formation of a reversible receptor complex followed by a covalent reaction. The extent of receptor alkylation is determined by the dissociation constant of the reversible complex and the rate constant for conversion to the covalent complex. Selectivity can be achieved if the irreversible ligand exhibits a difference in its dissociation constants for receptor subtypes. Selective alkylation can also be achieved using a selective competitive inhibitor to protect the desired receptor subtype. By using the non-M2-selective irreversible antagonist, 4-DAMP mustard, in combination with the competitive M2-selective antagonist, AF-DX 116, it has been possible to achieve a highly selective inactivation of all non-M2 subtypes of the muscarinic receptors in smooth muscle and has enabled the discovery of the functional role of M2 receptors in smooth muscle.

In both shaker-flask and mesocosm-scale experiments, a commercial oleophilic bioremediation agent containing biostimulation (nutrients) and bioaugmentation (bacterial inocula) properties was more effective in enhancing oil biodegradation rates than that of no treatment and/or periodic inorganic nutrient addition. However, similar results were not obtained from a subsequent 129-day field trial conducted in a sand beach environment. In this case, periodic additions of inorganic nutrients, with and without the commercial bioremediation agent, enhanced the number of heterotrophic bacteria and microbial respiration rates within the oiled sediments. The commercial product appeared to elevate the number of oil-degrading bacteria within the oiled sediment between days 17 and 89. However, the addition of inorganic nutrients alone, on a periodic basis, was the most effective means of enhancing the extent of oil biodegradation within the residual oil and of reducing sediment toxicity. By retaining residual oil and altering the physical and chemical characteristics of the treated sediment, the oleophilic product suppressed both the rate and extent of oil loss by tidal activity and biodegradation. This is not to say that the use of the product was ineffective in protecting the environment or was detrimental to it; the product does enhance natural biodegradation rates, and it limits the transport of beached oil to more sensitive areas. This study clearly illustrates the complexity associated with the selection of bioremediation agents, the need for improved experimental protocols for evaluating the performance and toxicity of bioremediation agents, and the potential of nutrient enrichment as a bioremediation strategy.

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.

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.

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.

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.

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.

We explored whether the M2 muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M3 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 M3 selectivity. Then, we used 4-DAMP mustard to inactivate M3 responses in the guinea pig ileum to attempt to reveal direct, M2 receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M2 receptor knockout, and M3 receptor knockout mice characterized by negative log EC50 (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 M2 receptor knockout mice were approximately the same, but that in the M3 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 M3 knockout mouse in terms of its pEC 50 , E max, and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M3 responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M3 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 M3 response. The guinea pig ileum, therefore, lacks a direct, highly potent, M2-contractile component but may have a direct, lower potency M2 component.

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.

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.

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.

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.

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.

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.

The effects of oxotremorine-M (oxo-M), a muscarinic agonist, on cyclic AMP (cAMP) accumulation in slices of the rat peripheral lung were investigated. Oxo-M stimulated cAMP accumulation in a concentration-dependent manner with an EC50 value of 4.2 microM and a maximal effect of 2.4 +/- 0.39-fold over basal. In the presence of forskolin (25 microM), the maximal effect of oxo-M was increased to 14.1 +/- 4.0-fold over basal. Forskolin alone caused a 5.9 +/- 2.2-fold increase in cAMP relative to basal; therefore, the combination of both drugs was more than additive. The effects of oxo-M on cAMP accumulation were unaffected by tetrodotoxin, indicating that the action of oxo-M was not mediated by neuronal release of neurotransmitters. Oxo-M had a small inhibitory effect on cAMP in a homogenate preparation, indicating that the stimulatory response to oxo-M in slices of the lung is not due to direct stimulation of adenylyl cyclase. Characterization of the oxo-M potentiation of forskolin-stimulated cAMP accumulation using different muscarinic antagonists yielded calculated pKB values that agreed with binding affinities for the M3 subtype. Oxo-M elicited phosphoinositide hydrolysis in the lung, and the nature of the antagonism of this response was also consistent with that expected for an M3-mediated response. cAMP accumulation in the presence of oxo-M (100 microM), forskolin (12 microM), or both drugs combined was inhibited by indomethacin (1 microM). These results demonstrate that the M3 receptor stimulates cAMP accumulation and phosphoinositide hydrolysis in the rat peripheral lung, and the mechanism for cAMP stimulation may involve arachidonic acid metabolites.

Chronic etorphine treatment of neuroblastoma X glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (Ni) and stimulatory (NS) components. Inactivation of Ni by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of NS, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E1, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through Ni resulting in the unimpeded expression of NS activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.

A soluble cytosolic factor from neuroblastoma x glioma NG108-15 hybrid cells stimulates adenylate cyclase activity in isolated membrane preparations. This cytosolic component is heat stable, pronase insensitive, has a molecular weight less than 350 daltons and an absorbance peak at 260 nm. The stimulation is immediate, independent of Ca++ and exhibits a sigmoidal concentration dependency curve. The cytosolic factor stimulated adenylate cyclase activity in etorphine treated cells (100 nM etorphine, 16 hrs) to a greater extent than in control cells. In addition, cytosolic factor derived from etorphine treated cells, as compared to control cells, displayed an increased capacity to stimulate adenylate cyclase. It is suggested that the observed cytosolic factor may be adenosine and that cells chronically treated with an opiate exhibit an increase in both concentration and sensitivity to this agent.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.