The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca 2+ release is altered in mdx fibres. Action potential-evoked Ca 2+ -dependent fluorescence transients were recorded, using both low and high affinity Ca 2+ indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N -benzyl-p-toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca 2+ transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca 2+ transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca 2+ release flux is responsible for the decrease in the peak of the Ca 2+ transient in mdx fibres. Since the myoplasmic Ca 2+ concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features trans-differentiation of tissue-resident pancreatic stellate cells (PSCs) into activated cancer-associated fibroblasts (CAFs), a process induced by PDAC cells but of unclear significance for PDAC progression. Here we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound healing mediators to stimulate their own growth and migration.

Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation-contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca 2+ release by measuring Ca 2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca 2+ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca 2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca 2+ may be altered in the mdx mouse.

Mutant p53s (mutp53) increase cancer invasiveness by upregulating Rab-coupling protein (RCP) and diacylglycerol kinase-α (DGKα)-dependent endosomal recycling. Here we report that mutp53-expressing tumour cells produce exosomes that mediate intercellular transfer of mutp53’s invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53’s ability to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment in distant organs in a way that can support invasive growth.