Nestin-Cre mice have a significant metabolic phenotype that is hard to discern from current literature. Indeed, the Cre-lox system has numerous problems that can affect physiological parameters, and these are missed when the correct control strains are not used. Despite the increasing use of the Cre-lox system, these issues were not visible to the scientific community previously and may have affected published work. This makes it important to highlight the issues and raise awareness of the pitfalls of the Cre-lox system. Therefore, this perspective will discuss the impact of CNS and peripheral "off-target" Cre recombination on metabolic systems and describe the development of new approaches to obviate the difficulties.

Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon.

Background and purpose: The small and intermediate conductance, Ca 2 þ -sensitive K þ channels (SK Ca and IK Ca , respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IK Ca and SK Ca channels was investigated. Experimental approach: The effect of the caveolae-disrupting agent methyl-b-cyclodextrin (MbCD) on IK Ca and SK Ca localization and function was determined. Key results: EDHF-mediated, SK Ca -dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries (following blockade of IK Ca with TRAM-34) were inhibited by MbCD. Hyperpolarizations evoked by direct SK Ca channel activation (using NS309 in the presence of TRAM-34) were also inhibited by MbCD, an effect reversed by cholesterol. In contrast, IK Ca -dependent hyperpolarizations (in the presence of apamin) were unaffected by MbCD. Similarly, in porcine coronary arteries, EDHF-mediated, SK Ca -dependent (but not IK Ca -dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MbCD. In mesenteric artery homogenates subjected to sucrose-density centrifugation, caveolin-1 and SK3 (SK Ca ) proteins but not IK1 (IK Ca ) protein migrated to the buoyant, caveolin-rich fraction. MbCD pretreatment redistributed caveolin-1 and SK3 proteins into more dense fractions. In immunofluorescence images of porcine coronary artery endothelium, SK3 (but not IK1) and caveolin-1 were co-localized. Furthermore, caveolin-1 immunoprecipitates prepared from native porcine coronary artery endothelium contained SK3 but not IK1 protein.Conclusions and Implications: These data provide strong evidence that endothelial cell SK Ca channels are located in caveolae while the IK Ca channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade.

Background and purpose:Previous studies have shown that endothelium-dependent hyperpolarization of myocytes is reduced in resistance arteries from spontaneously hypertensive rats (SHRs). The aim of the present study was to determine whether this reflects down-regulation of endothelial K + channels or their associated pathways. Experimental approach: Changes in vascular K+ channel responses and expression were determined by a combination of membrane potential recordings and Western blotting. Key results: Endothelium-dependent myocyte hyperpolarizations induced by acetylcholine, 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) (opens small-and intermediate-conductance calcium-sensitive K + channels, SKCa and IKCa, respectively) or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (SKCa opener) were reduced in mesenteric arteries from SHRs. After blocking SKCa channels with apamin, hyperpolarizations to acetylcholine and NS309 in SHR arteries were similar to those of controls. Hyperpolarization to 5 mM KCl was reduced in SHR arteries due to loss of the Ba 2+ -sensitive, inward-rectifier channel (KIR) component; the contribution of ouabain-sensitive, Na + /K + -ATPases was unaffected. Protein expression of both SKCa and KIR channels was reduced in SHR arteries; the caveolin-1 monomer/dimer ratio was increased. Conclusions and implications:In SHRs, the distinct pathway that generates endothelium-dependent hyperpolarization in vascular myocyte by activation of IKCa channels and Na + /K + -ATPases remains intact. The second pathway, initiated by endothelial SKCa channel activation and amplified by KIR opening on both endothelial cells and myocytes is compromised in SHRs due to down-regulation of both SKCa and KIR and to changes in caveolin-1 oligomers. These impairments in the SKCa-KIR pathway shed new light on vascular control mechanisms and on the underlying vascular changes in hypertension.

Background and purpose: The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K þ channels (IK Ca ) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. Experimental approach: Pressure myography, western blotting, sharp microelectrode and K þ -selective electrode recordings were used to investigate the functional expression of the CaR and IK Ca in rat mesenteric arteries. Key results: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K þ ] around the myocytes, and inhibition of this 'K þ cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK Ca expression was also diminished, but IK Cagenerated hyperpolarizations mediated by 1-EBIO were unaffected. Conclusions and implications:The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK Ca channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.