Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Genetic ablation of K IR 2.1 does not eliminate inward K + currents in cerebral arterial smooth muscle cells (SMCs). Whole-cell patch clamp electrophysiology was used to measure K IR current with voltage ramps from −100 to +20 mV in the absence and presence of Ba 2+ in 60 mM K + . (a,b) Representative recordings of whole-cell and Ba 2+ -subtracted K IR currents in myocytes isolated from SMC K IR 2.1 −/− mice and non-induced Cre SMC controls. (c) Summary data compare peak inward current at −100 mV between groups ( n = 9 SMCs from 6 mice in control group and n = 9 SMCs from 8 mice in knockout group; nested t -test).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Patch Clamp, Isolation, Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K I R 2.1 is negligibly expressed in cerebral vascular smooth muscle cells (SMCs) while K IR 2.2 is highly expressed at the cell membrane. Tamoxifen-induced K IR 2.1 knockout significantly reduced subunit expression but levels remained detectable by immunofluorescence. (a) Fluorescent anti-K IR 2.1 (green) exhibited a faint labeling pattern in cerebral arterial myocytes from SMC K IR 2.1 −/− and control mice with nuclei stained with DAPI (blue). (b) Summary of data compares fluorescence intensity (background subtracted) of K IR 2.1 signal between groups ( n = 10 cells from 5 animals in control group and n = 9 cells from 5 animals in knockout group; unpaired t -test). (c) Immunofluorescence labeling of SMCs for K IR 2.2. (d) Summary of data compares background-subtracted fluorescence intensity of K IR 2.2 signal between groups ( n = 8 cells pooled from 4 animals/group; unpaired t -test). Two cells were analyzed per animal with background signal subtracted using 2° antibody control.

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Knock-Out, Expressing, Immunofluorescence, Labeling, Staining, Fluorescence

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K IR 2.2 protein expression is unaltered in smooth muscle cell (SMC) K IR 2.1 −/− cerebral arteries. (a) Western blot of intact cerebral arteries confirmed that protein levels of K IR 2.2 are not impacted by deletion of SMC K IR 2.1. (b) Summary of data compares K IR 2.2 protein levels between control and knockout mice (normalized to actin; n = 6 mice; unpaired t -test).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Expressing, Western Blot, Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K IR 2.1 and K IR 2.2 subunits are inversely expressed in cerebral endothelial and vascular smooth muscle cells. (a) K IR 2.1 and (b) K IR 2.2 subunit expression is shown as average cellular transcript counts per cell, as determined by single-cell RNA sequencing of the mouse brain vasculature. Data highlight differences in the dominant subunit between cell types. Abbreviations: PC – pericytes; SMC – smooth muscle cells; EC – endothelial cells; v – venous; c – capillary; a – arterial; aa – arteriolar. Figures provided by http://betsholtzlab.org/VascularSingleCells/database.html . 33,34

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Expressing, RNA Sequencing Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Myogenic responses and K + -induced dilation are intact in cerebral arteries of smooth muscle cell (SMC) K IR 2.1 −/− mice. Cerebral arteries from control and SMC K IR 2.1 −/− mice were cannulated and intravascular pressure was elevated stepwise while vasomotor responses were measured. (a) Representative diameter traces from endothelium-denuded vessels of control and SMC K IR 2.1 −/− mice show the effect of increasing pressure on myogenic tone. (b) Summary of data highlights limited impact of smooth muscle K IR 2.1 knockout on myogenic tone development. Paired t -test was performed for 0 vs. 100 μM Ba 2+ treatment ( n = 10 vessels in the control group and n = 11 vessels in the SMC K IR 2.1 −/− group with 1 vessel/mouse; *P < 0.05). (c) K + -induced dilation, elicited by increasing extracellular K + from 5 mM to 10 mM before and after treatment with 100-μM Ba 2+ , was intact in the knockout group ( n = 6 vessels in the control group and SMC K IR 2.1 −/− group; one-way ANOVA with Sidak’s multiple comparisons test). (d) K + -induced dilation (5 mM K + to 10 mM K + ) was abrogated with exposure to low concentrations of Ba 2+ , implicating K IR 2.2 as the mediator of this response based on its Ba 2+ sensitivity profile ( n = 7 vessels from control mice; repeated measures one-way ANOVA with Sidak’s multiple comparisons test). Myogenic tone (%) was calculated as: [(passive diameter – active diameter)/(passive diameter – minimal diameter)] × 100 at each pressure step. K + -induced dilation (%) was calculated as difference between diameter at 10 mM [K + ] and 5 mM [K + ] divided by the dilatory range (passive diameter – minimal diameter).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Region-specific brain perfusion is not altered in smooth muscle cell (SMC) K IR 2.1 −/− mice at rest and with increased systemic blood pressure. (a) Representative arterial spin-labeled MR brain perfusion maps. Scans were done in a posterior-to-anterior direction and the volume of brain scanned was divided into 5 coronal slices. Resting cerebral blood flow was measured in control and SMC K IR 2.1 −/− mice. Scans were repeated after blood pressure challenge with an intraperitoneal phenylephrine injection. Figure shows slices from 2 regions of the brain (red boxes) spanning cerebral nuclei, hippocampus, thalamus, and hypothalamus. (b) Baseline perfusion in several major brain structures was not significantly different between control and tamoxifen-induced mice. The blood pressure challenge caused a modest but significant rise in cerebral blood flow to a similar extent in control and SMC K IR 2.1 −/− animals. Unpaired t -test was performed for control ( n = 7 mice) vs. SMC K IR 2.1 −/− ( n = 11 mice) comparison; paired t -test was performed for baseline vs. phenylephrine-treatment. *P < 0.05 compared to baseline control.

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Labeling, Injection