Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.     

Riochemical and physiological effects in farmed Baltic salmon fed lipids containing xenobiotics extracted from Baltic herring

During a 2-year experimental period female baltic salmon (Salmo salar) were fed pellets impregnated with oil extracted from Baltic herring (Clupea harengus). This extract contained lipid-soluble xenobiotics present in Baltic herring, which constitute a major part of the natural diet of Baltic salmon. The fish were examined at the time of ovulation in November each year. After 2 years of feeding, the load of polychlorinated dibenzo-paradioxins and furans in the exposed group was about twice that in the control group, but still low compared with concentrations in feral Baltic salmon. In spite of the relatively low exposure level, several vital biochemical functions were disturbed in the treated fish. Organic skeletal variables were affected indicating that the bone metabolism had been altered. Furthermore, the activities of enzymes involved in steroid biosynthesis were affected, which could lead to disturbances in reproductive functions. Splenocytes from exposed fish sampled in November 1990 showed a reduced mitogenic response, indicating that their immune system was suppressed. Feeding the salmon with pollutant-impregnated pellets also resulted in an induction of the hepatic ethoxyresorufin-O-deethylase (EROD) activity after only 6 weeks of exposure. Likewise, morphological abnormalities, i.e. hypertrophic hepatocytes and various stages of hepatic degeneration, were already apparent after 6 weeks of exposure. However, no EROD induction or morphological responses were recorded at the second and third sampling event, i.e. after one and 2 years of exposure, respectively. this could indicate that some physiological functions may adapt to a restricted xenobiotic load.     

Impacts of elevated terrestrial nutrient loads and temperature on pelagic food‐web efficiency and fish production

Both temperature and terrestrial organic matter have strong impacts on aquatic food‐web dynamics and production. Temperature affects vital rates of all organisms, and terrestrial organic matter can act both as an energy source for lower trophic levels, while simultaneously reducing light availability for autotrophic production. As climate change predictions for the Baltic Sea and elsewhere suggest increases in both terrestrial matter runoff and increases in temperature, we studied the effects on pelagic food‐web dynamics and food‐web efficiency in a plausible future scenario with respect to these abiotic variables in a large‐scale mesocosm experiment. Total basal (phytoplankton plus bacterial) production was slightly reduced when only increasing temperatures, but was otherwise similar across all other treatments. Separate increases in nutrient loads and temperature decreased the ratio of autotrophic:heterotrophic production, but the combined treatment of elevated temperature and terrestrial nutrient loads increased both fish production and food‐web efficiency. CDOM: Chl a ratios strongly indicated that terrestrial and not autotrophic carbon was the main energy source in these food webs and our results also showed that zooplankton biomass was positively correlated with increased bacterial production. Concomitantly, biomass of the dominant calanoid copepod Acartia sp. increased as an effect of increased temperature. As the combined effects of increased temperature and terrestrial organic nutrient loads were required to increase zooplankton abundance and fish production, conclusions about effects of climate change on food‐web dynamics and fish production must be based on realistic combinations of several abiotic factors. Moreover, our results question established notions on the net inefficiency of heterotrophic carbon transfer to the top of the food web.     

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Mice carrying a conditional Serca2 allele for the generation of Ca handling-deficient mouse models

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca²⁺ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2^null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2^flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2^flox mice with MLC-2v^wt/Cre mice and with αMHC-Cre transgenic mice. In heterozygous Serca2^wt/floxMLC-2v^wt/Cre mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2^flox/flox Tg(αMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2^flox mouse is the first murine conditional gene disruption model for the SERCA family of Ca²⁺ ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.