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Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.

Zolpidem is a nonbenzodiazepine GABA(A) receptor modulator that binds in vitro with high affinity to GABA(A) receptors expressing alpha(1) subunits but with relatively low affinity to receptors expressing alpha(2), alpha(3), and alpha(5) subunits. In the present study, it was investigated whether this subtype selectivity could be detected and quantified in vivo. Three doses (1.25, 5, and 25 mg) of zolpidem were administered to rats in an intravenous infusion over 5 min. The time course of the plasma concentrations was determined in conjunction with the change in the beta-frequency range of the EEG as pharmacodynamic endpoint. The concentration-effect relationship of the three doses showed a dose-dependent maximum effect and a dose-dependent potency. The data were analyzed for one- or two-site binding using two pharmacodynamic models based on 1) the descriptive model and 2) a novel mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for GABA(A) receptor modulators that aims to separates drug- and system-specific properties, thereby allowing the estimation of in vivo affinity and efficacy. The application of two-site models significantly improved the fits compared with one-site models. Furthermore, in contrast to the descriptive model, the mechanism-based PK/PD model yielded dose-independent estimates for affinity (97 +/- 40 and 33,100 +/- 14,800 ng x ml(-1)). In conclusion, the mechanism-based PK/PD model is able to describe and explain the observed dose-dependent EEG effects of zolpidem and suggests the subtype selectivity of zolpidem in vivo.

A mechanism-based pharmacokinetic-pharmacodynamic (PK/PD) model for neuroactive steroids, comprising a separate characterization of 1) the receptor activation process and 2) the stimulus-response relationship, was applied to various nonsteroidal GABAA receptor modulators. The EEG effects of nine prototypical GABAA receptor modulators (six benzodiazepines, one imidazopyridine, one cyclopyrrolone, and one beta-carboline) were determined in rats in conjunction with plasma concentrations. Population PK/PD modeling revealed monophasic concentration-EEG effect relationships with large differences in potency (EC50) and intrinsic activity between the compounds. The data were analyzed on the basis of the mechanism-based PK/PD model for (synthetic) neuroactive steroids on the assumption of a single and unique stimulus-response relationship. The model converged yielding estimates of both the apparent in vivo receptor affinity (KPD) and the in vivo intrinsic efficacy (ePD). The values of KPD ranged from 0.41 +/- 0 ng.ml(-1) for bretazenil to 436 +/- 72 ng.ml(-1) for clobazam and the values for e(PD) from -0.27 +/- 0 for methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate to 0.54 +/- 0.02 for diazepam. Significant linear correlations were observed between KPD for unbound concentrations and the affinity in an in vitro receptor bioassay (r = 0.93) and between e(PD) and the GABA-shift in vitro (r = 0.95). The findings of this investigation show that the in vivo effects of nonsteroidal GABAA receptor modulators and (synthetic) neuroactive steroids can be described on the basis of a single unique transducer function. In this paradigm, the nonsteroidal GABAA receptor modulators behave as partial agonists relative to neuroactive steroids.

The objective of the present investigation was to characterize the in vivo EEG effects of (synthetic) neuroactive steroids on the basis of a recently proposed mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model. After intravenous administration, the time course of the EEG effect of pregnanolone, 2beta-3alpha-5alpha-3-hydroxy-2-(2,2-dimethylmorpholin-4-yl)-pregnan-11,20-dione (ORG 21465), 2beta-3alpha-5alpha-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one (ORG 20599), and alphaxalone was determined in conjunction with plasma concentrations in rats. For each neuroactive steroid the PK/PD correlation was described on the basis of a two-compartment pharmacokinetic model with an effect compartment to account for hysteresis. The observed concentration EEG effect relationships were biphasic and characterized with a mechanism-based pharmacodynamic model, which is based on a separation between the receptor activation process and the stimulus-response relationship. A single unique biphasic stimulus-response relationship could be identified for all neuroactive steroids, which was successfully described by a parabolic function. The receptor activation process was described by a hyperbolic function. Estimates for the maximum activation (e(PD)) were similar for the different neuroactive steroids but values of the potency estimate (K(PD)) ranged from 157 +/- 16 ng. ml(-1) for pregnanolone, 221 +/- 83 ng. ml(-1) for ORG 20599, and 483 +/- 42 ng. ml(-1) for alphaxalone to 1619 +/- 208 ng. ml(-1) for ORG 21465. A statistically significant correlation was observed between the in vivo potency and the IC(50) in an in vitro [(35)S]t-butylbicyclophosphorothionate binding assay (r = 0.91). It is concluded that the new PK/PD model constitutes a new mechanism-based approach to the quantification of the effects of (synthetic) neuroactive steroids in vivo effects. The results show that the neuroactive steroids differ in potency but not in intrinsic efficacy at the GABA(A) receptor in vivo.

The neuroactive steroid alphaxalone reveals a complex biphasic concentration-effect relationship using the 11.5 to 30 Hz frequency band of the electroencephalogram (EEG) as biomarker. The purpose of the present investigation was to develop a mechanism-based pharmacokinetic-pharmacodynamic model to describe this observation. The proposed model is based on receptor theory and aims to separate the drug-receptor interaction from the transduction of the initial stimulus into the observed biphasic response. Individual concentration-time courses of alphaxalone were obtained in combination with continuous recording of the EEG parameter. Alphaxalone was administered intravenously in various dosages. The pharmacokinetics were described by a two-compartment model, and parameter estimates for clearance, intercompartmental clearance, volume of distribution 1 and 2 were 158 +/- 29 ml. min(-1). kg(-1), 143 +/- 31 ml. min(-1). kg(-1), 122 +/- 20 ml. kg(-1) and 606 +/- 48 ml. kg(-1), respectively. Concentration-effect relationships exhibited a biphasic pattern and delay in onset of effect. The hysteresis was described on the basis of an effect-compartment model with C(max) as covariate. The pharmacodynamic model consisted of a receptor model, featuring a monophasic saturable receptor activation model in combination with a biphasic stimulus-response model. The in vivo affinity (K(PD)) was estimated at 432 +/- 26 ng. ml(-1). Unique parameter estimates were obtained that were independent of the dose and the duration of the infusion. In conclusion, we have shown that this mechanism-based approach, which separates drug- and system-related properties in vivo, was successfully applied for the characterization of the biphasic effect versus time patterns of alphaxalone. The model should be of use in the characterization of other biphasic responses.