Leukotriene B₄(LTB₄), a potent leukocyte chemoattractant derived from the 5-lipoxygenase metabolism of arachidonic acid, exerts its action by means of specific cell surface receptors, denoted BLT₁and BLT₂. In this study, BLT₁receptor proteins were detected in human carotid artery atherosclerotic plaques, colocalizing with markers for macrophages, endothelial cells, and vascular smooth muscle cells (SMC). Challenge of human coronary artery SMC with either LTB₄or U75302, a partial agonist that is selective for the BLT₁receptor, induced an ≈4-fold increase of whole-cell currents by using the patch-clamp technique, indicating that these cells express functional BLT₁receptors. LTB₄induced migration and proliferation of SMCin vitro, and treatment with the BLT receptor antagonist BIIL 284 (10 mg/kg, once daily) for 14 days after carotid artery balloon injuryin vivoinhibited intimal hyperplasia in rats. In the latter model, SMC derived from the intima exhibited increased levels of BLT₁receptor mRNA compared with medial SMC. BLT receptor up-regulation in the intimain vivo, as well as that induced by IL-1βin vitro, were prevented by transfection with a dominant-negative form of Iκ kinase β carried by adenovirus, indicating that BLT₁receptor expression depends on NF-κΒ. These results show that LTB₄activates functional BLT₁receptors on vascular SMC, inducing chemotaxis and proliferation, and that BLT₁receptors were up-regulated through an Iκ kinase β/NF-κB-dependent pathway. Inhibition of LTB₄/BLT₁signaling during the response to vascular injury reduced intimal hyperplasia, suggesting this pathway as a possible target for therapy.

Long term exposure to elevated levels of long chain free fatty acids decreases glucose-induced insulin secretion from pancreatic islets and clonal pancreatic β-cells. The mechanism for this loss of glucose sensitivity is at present not known. In this study, we evaluated the possibility that increases in long chain acyl-CoA esters (LC-CoA), the metabolically active form of free fatty acids, might mediate the loss of glucose sensitivity. We observed that cellular levels of LC-CoA increased more than 100% in response to overnight incubation with 0.5 mM palmitic acid complexed to albumin. In the same studies, the total CoA pool increased by about 40%. Patch-clamp studies demonstrated that saturated and unsaturated LC-CoA, but not malonyl-CoA or free CoASH, induced a rapid and slowly reversible opening of ATP-sensitive K channels. The effect was concentration-dependent between 10 nM and 1 μM. These findings indicate that the ATP-regulated K channel is a sensitive target for LC-CoA and suggest that high levels of LC-CoA, which accumulate in response to hyperglycemia or prolonged exposure to free fatty acids, may prevent channel closure and contribute to the development of β-cell glucose insensitivity.

Interaction of syntaxin 1 with the α _1D subunit of the voltage-gated L type Ca ²⁺ channel was investigated in the pancreatic β cell. Coexpression of the enhanced green fluorescent protein-linked α _1D subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the α _1D subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the α _1D subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca ²⁺ channel activity because there was a faster Ca ²⁺ current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K ⁺ or exposure to high Ca ²⁺ . Exchanging Ca ²⁺ for Ba ²⁺ abolished the effect of antisyntaxin 1 on both Ca ²⁺ channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca ²⁺ -independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure–function relationship between the α _1D subunit of the L type Ca ²⁺ channel and the exocytotic machinery in the pancreatic β cell.

Parafibromin is a protein product derived from the hyperparathyroidism 2(HRPT2) tumor suppressor geneand its inactivation has been coupled to familial and sporadic forms of parathyroid malignancy. In this study, we have conducted immunohistochemistry on 33 parathyroid carcinomas (22 unequivocal and 11 equivocal) using four parafibromin antibodies directed to different parts of the protein. Furthermore, for a fraction of cases, the immunohistochemical results were compared with known HRPT2 mutational status. Our findings show that 68% (15 out of 22) of the unequivocal carcinomas exhibited reduced expression of parafibromin while the 25 sporadic adenomas used as controls were entirely positive for parafibromin expression. Additionally, three out of the six carcinomas with known HRPT2 mutations showed reduced expression of parafibromin. Using all four antibodies, comparable results were obtained on the cellular level in individual tumors suggesting that there exists no epitope of choice in parafibromin immunohistochemistry. The results agree with the demonstration of a ~60 kDa product preferentially in the nuclear fraction by western blot analysis. We conclude that parafibromin immunohistochemistry could be used as an additional marker for parathyroid tumor classification, where positive samples have low risk of malignancy, whereas samples with reduced expression could be either carcinomas or rare cases of adenomas likely carrying an HRPT2 mutation.

Background Sporadic multiglandular disease (MGD) has been reported in literature in 8–33 % of patients with primary hyperparathyroidism (pHPT). This paper aimed to review controversies in the pathogenesis and management of sporadic MGD. Methods A literature search and review was made to evaluate the level of evidence concerning diagnosis and management of sporadic MGD according to criteria proposed by Sackett, with recommendation grading by Heinrich et al. and Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Results were discussed at the 6th Workshop of the European Society of Endocrine Surgeons entitled ‘Hyperparathyroidism due to multiple gland disease: An evidence-based perspective’. Results Literature reports no prospective randomised studies; thus, a relatively low level of evidence was achieved. Appropriate surgical therapy of sporadic MGD should consist of a bilateral approach in most patients. Unilateral neck exploration guided by preoperative imaging should be reserved for selected patients, performed by an experienced endocrine surgeon and monitored by intraoperative parathormone assay (levels of evidence III–V, grade C recommendation). There is conflicting or equally weighted levels IV–V evidence supporting that cure rates can be similar or worse for sporadic MGD than for single adenomas (no recommendation). Best outcomes can be expected if surgery is performed by an experienced parathyroid surgeon working in a high-volume centre (grade C recommendation). Levels IV–V evidence supports that recurrent/persistence pHPT occurs more frequently in patients with double adenomas hence in situations where a double adenoma has been identified, the surgeon should have a high index of suspicion during surgery and postoperatively for the possibility of a four-gland disease (grade C recommendation). Conclusions Identifying preoperatively patients at risk for MGD remains challenging, intraoperative decisions are important for achieving acceptable cure rates and long-term follow-up is mandatory in such patients.

The ATP-dependent potassium (K_ATP) channel in the pancreatic β-cell is a complex of two proteins, the pore-forming Kir6.2 and the sulfonylurea receptor type 1 (SUR1). Both subunits are required for functional K_ATP channels because expression of Kir6.2 alone does not result in measurable currents. However, truncation of the last 26 or 36 amino acids of the C terminus of Kir6.2 enables functional expression of the pore-forming protein in the absence of SUR1. Thus, by using the truncated form of Kir6.2, expressed in the absence and presence of SUR1, it has been shown that the site at which ATP mediates channel inhibition is likely to be situated on Kir6.2. We have now examined the effects of long chain acyl-CoA (LC-CoA) esters on the C-terminally truncated mouse Kir6.2Δ365–390 (Kir6.2ΔC26) in inside-out patches isolated from Xenopus laevis oocytes. LC-CoA esters, saturated (C14:0, C16:0) and unsaturated (C18:1), increased Kir6.2ΔC26 currents, whereas short and medium chain CoA esters (C3:0, C8:0, C12:0) were unable to affect channel activity. The LC-CoA esters were also able to counteract the blocking effect of ATP on Kir6.2ΔC26. The stimulatory effect of the esters could be explained by the induction of a prolonged open state of Kir6.2ΔC26. In the presence of the esters, channel open time was increased approximately 3-fold, which is identical to what was obtained in the native mouse K_ATP channel. Coexpression of SUR1 together with Kir6.2ΔC26 did not further increase the ability of LC-CoA esters to stimulate channel activity. We conclude that Kir6.2 is the primary target for LC-CoA esters to activate the K_ATP channel and that the esters are likely to induce a conformational change by a direct interference with the pore-forming subunit, leading to openings of long duration.