BACKGROUND AND PURPOSET16Ainh-A01, CaCCinh-A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. EXPERIMENTAL APPROACHIsolated resistance arteries were maintained in a myograph and tension recorded, in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as a charge carrier. KEY RESULTSRodent arteries preconstricted with noradrenaline or U46619 were concentration-dependently relaxed by T16Ainh-A01 (0.1-10 μM): IC50 and maximum relaxation were equivalent in ±chloride (30 min aspartate substitution) and the T16Ainh-A01-induced vasorelaxation ±chloride were accompanied by membrane hyperpolarization and lowering of intracellular calcium. However, agonist concentration-response curves ±chloride, with 10 μM T16Ainh-A01 present, achieved similar maximum constrictions although agonist-sensitivity decreased. Contractions induced by elevated extracellular potassium were concentration-dependently relaxed by T16Ainh-A01 ±chloride. Moreover, T16Ainh-A01 inhibited VDCCs in A7r5 cells in a concentration-dependent manner. CaCCinh-A01 and MONNA (0.1-10 μM) induced vasorelaxation ±chloride and both compounds lowered maximum contractility. MONNA, 10 μM, induced substantial membrane hyperpolarization under resting conditions. CONCLUSIONS AND IMPLICATIONST16Ainh-A01, CaCCinh-A01 and MONNA concentration-dependently relax rodent resistance arteries, but an equivalent vasorelaxation occurs when the transmembrane chloride gradient is abolished with an impermeant anion. These compounds therefore display poor selectivity for TMEM16A and inhibition of CaCC in vascular tissue in the concentration range that inhibits the isolated conductance.

TMEM16A is essential for Ca -activated Cl conductance in vascular smooth muscle. The importance of TMEM16A for agonist-induced vascular constriction and blood pressure control is, however, under debate. Previous studies suggested that TMEM16A might have a complex cellular function beyond being essential for the Ca -activated Cl conductance, for example modulation of Ca channel expression. Mice with constitutive, smooth muscle-specific expression of siRNA directed against Tmem16a (transgenic mice, TG) were generated. Isometric constrictions of isolated aorta, mesenteric, femoral and tail arteries from TG mice were compared with wild-types. Protein expression was analysed by Western blots. Blood pressure and heart rate were studied telemetrically. Significant TMEM16A down-regulation was seen in aorta and tail arteries, while no changes were detected in mesenteric and femoral arteries. Contractile responses of mesenteric and femoral arteries from TG and wild-type mice were not different. Aorta from TG mice showed reduced agonist-induced constriction, while their responses to elevated K were unchanged. Tail arteries from TG mice also constricted less to adrenergic stimulation than wild-types. Surprisingly, tail arteries from TG mice constricted less to elevated K too and were more sensitive to nifedipine-induced relaxation. Consistently, TMEM16A down-regulation in tail arteries was associated with reduction in CACNA1C protein (i.e. vascular L-type Ca channel) expression. No differences in blood pressure and heart rate between the groups were seen. This study suggests a complex contribution of TMEM16A in vascular function. We suggest that TMEM16A modulates arterial contractility, at least in part, indirectly via regulation of CACNA1C expression.

Waldenstrom's macroglobulinamia (WM) is a rare malignant lymphoproliferative disorder, characterized by monoclonal IgM paraproteinemia and neoplastic proliferation of malignant lymphoplasmacytoid cells in the bone marrow. Traditionally, WM has been treated with modalities similar to those used in the management of other indolent lymphomas. Just recently, based on impressive clinical trial results in heavily pretreated WM patients, a new Bruton Tyrosine Kinase-inhibitor, Ibrutinib, has been approved for the treatment of this disorder. As the use of Ibrutinib in WM outside clinical trials is still limited, only few clinical reports illustrating treatment side effects are currently available. Here we review the current literature specific on Ibrutinib-associated rash in hematologic patients, and report on an elderly patient with WM, who developed a red maculopapular non-pruritic rash 12 weeks after starting Ibrutinib therapy. Without modifications of the ongoing Ibrutinib schedule, the rash regressed within two weeks of treatment with topical steroid-containing dermatological compounds.

Diabetes is associated with altered contractile responses of small arteries. In the streptozotocin (STZ) induced rat model of diabetes enhanced contractility several groups have reported an increased contractility. In this study we tested the hypothesis that chloride is important for the enhanced contractility in the STZ rats.Diabetes was induced in 6 weeks old rats by a single intraperitoneal injection of 60 mg/kg STZ, control rats received vehicle injection. Eight weeks later blood glucose was significantly increased in the STZ rats compared to control rats (24.6±1.2 mmol/L and 6.7±1.1 mmol/L, STZ (n=9) and control (n=9), respectively). The rats were killed and mesenteric small arteries were isolated. The contractile response to increasing concentrations of norepinephrine of mesenteric small arteries was assessed in isometric myographs in physiological salt solution and in physiological salt solution where Cl− was substituted with aspartate. Expression of the Ca2+ activated Cl− channel ANO1 was assessed with qPCR and Western blotting in mesenteric small arteries.The maximal contraction to norepinephrine in physiological salt solution was higher in arteries from STZ rats compared to control rats (5.12±0.33 N/m and 4.07±0.24 N/m, STZ (n=14) and control (n=13), respectively). In Cl− free solutions the maximal response to norepinephrine was reduced and no difference between STZ and control rats was found (3.76±0.26 N/m and 3.23±0.22 N/m, STZ (n=14) and control (n=13), respectively). Furthermore the effect of Cl− free solution on the area under the NE concentration response curve was bigger in arteries from STZ rats (2.47±0.42 and 1.39±0.26 STZ (n=14) and control (n=13), respectively). Expression of ANO1 mRNA was higher in arteries from STZ rats (181±10% and 100±18%, STZ (n=4) and control (n=4), respectively). Expression of ANO1 protein was higher in arteries from STZ rats (195±24% and 100±18%, STZ (n=9) and control (n=8), respectively).This study provides evidence that the enhanced contractility of mesenteric arteries from STZ rats is dependent on the chloride gradient over the cell membrane, and suggests that this may be consequent to enhanced expression of the Ca2+ activated Cl− channel ANO1.Support or Funding InformationDanish Council for Independent Research (7016‐00013B)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.