Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca2+ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca2+ signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca2+ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.

Calpain 1 (microcalpain) and calpain 2 (millicalpain), the best characterized calpains, are known as intracellular calcium-dependent endoproteases and are expressed in different tissues of vertebrates. These ubiquitous cysteine proteases [1] play important roles in a large set of intracellular events [2][3][4][5], particularly in the selective proteolysis of factors involved in the cell cycle [6], during apoptosis in association with caspases [7], or in the cleavage of membrane-cytoskeleton complexes during cell motility phases [8]. Their activities are blocked by calpastatins (a specific inhibitor family largely expressed in the cell) and are regulated at the membrane level by phospholipids, which decrease the calcium requirements of calpains [1]. Calpain 1 (active in vitro at 50 lm Ca 2+ ions) and calpain 2 (active in vitro at 500 lm Ca 2+ ions) are composed of an 80 kDa and a 30 kDa subunit. The spatial structure of calpain 2 has recently been determined [9] and the organization of the six domains (dI-dIV in the 80 kDa subunit, and dV-dVI in the 30 kDa subunit) has been defined as well as the calcium-binding regions. In particular, it was found that dIV and dVI (calmodulin-like [2097][2098][2099][2100][2101][2102][2103][2104][2105][2106][2107]. Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK ⁄ N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl 2 and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.Abbreviations CP1 Ig, anti-(calpain 1) Ig; FITC, fluorescein isothiocyanate.

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca 2þ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca 2þ signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca 2þ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.

Purpose To provide whole-body physiologically based pharmacokinetic (PBPK) models of the potent clinical organic anion transporter (OAT) inhibitor probenecid and the clinical OAT victim drug furosemide for their application in transporter-based drug-drug interaction (DDI) modeling. Methods PBPK models of probenecid and furosemide were developed in PK-Sim®. Drug-dependent parameters and plasma concentration-time profiles following intravenous and oral probenecid and furosemide administration were gathered from literature and used for model development. For model evaluation, plasma concentration-time profiles, areas under the plasma concentration–time curve (AUC) and peak plasma concentrations (Cmax) were predicted and compared to observed data. In addition, the models were applied to predict the outcome of clinical DDI studies. Results The developed models accurately describe the reported plasma concentrations of 27 clinical probenecid studies and of 42 studies using furosemide. Furthermore, application of these models to predict the probenecid-furosemide and probenecid-rifampicin DDIs demonstrates their good performance, with 6/7 of the predicted DDI AUC ratios and 4/5 of the predicted DDI Cmax ratios within 1.25-fold of the observed values, and all predicted DDI AUC and Cmax ratios within 2.0-fold. Conclusions Whole-body PBPK models of probenecid and furosemide were built and evaluated, providing useful tools to support the investigation of transporter mediated DDIs.