Objective— Inward rectifying K + (K IR ) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial K IR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results— A Ba 2+ -sensitive K IR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed K IR 2.1/K IR 2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of K IR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial K IR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle K IR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of K IR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized K IR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions— The data show for the first time that specific membrane lipid-K IR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.
Objective: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. Conclusions: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.
37Inward rectifying (KIR) K + channels are present in cerebral arterial smooth muscle and endothelial 38 cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. We 39 explored whether vascular KIR channels were uniquely modulated by membrane lipids and 40 hemodynamic stimuli. A KIR current was isolated in smooth muscle and endothelial cells of rat 41 cerebral arteries and molecular analyses confirmed KIR2.1/KIR2.2 mRNA and protein expression. 42Electrophysiology next revealed that endothelial KIR was sensitive to phosphatidylinositol 4,5-43 bisphosphate (PIP2), with depletion impairing flow-induced activation of the channel. In contrast, 44 smooth muscle KIR was sensitive to membrane cholesterol, with sequestration blocking pressure's 45 ability to inhibit this channel. Membrane lipids helped confer KIR mechanosensitivity to intact 46 arteries; virtual models then reconceptualised KIR as a dynamic regulator of basal tone 47 development. We conclude that specific membrane lipid-KIR interactions enable unique channel 48 populations to sense hemodynamic stimuli and set brain perfusion.
Doxorubicin remains one of the most widely used chemotherapeutic agents however its effect on healthy tissue, such as skeletal muscle, remains poorly understood. The purpose of the current study was to examine the accumulation of doxorubicin (DOX) and its metabolite doxorubicinol (DOXol) in skeletal muscle of the rat up to 8 days after the administration of a 1.5 or 4.5 mg kg-1 i.p. dose. Subsequent to either dose, DOX and DOXol were observed in skeletal muscle throughout the length of the experiment. Interestingly an efflux of DOX was examined after 96 hours, followed by an apparent re-uptake of the drug which coincided with a spike and rapid decrease of plasma DOX concentrations. The interstitial space within the muscle did not appear to play a significant rate limiting compartment for the uptake or release of DOX or DOXol from the tissue to the circulation. Furthermore, there was no evidence that DOX preferentially accumulated in a specific muscle group with either dose. It appears that the sequestering of drug in skeletal muscle plays an acute and important role in the systemic availability and metabolism of DOX which may have a greater impact on the clinical outcome than previously considered.
Skeletal muscle (SM) health and integrity is dependent on the dynamic balance between protein synthesis and degradation, and central to this process is the availability of amino acids (AA) in the amino pool. While Doxorubicin (DOX) remains one of the most widely used chemotherapeutic agents for the treatment of solid and hematological malignancies, little is known of the effect of the drug on SM, particularly its effect on the availability of amino acids in the tissue. The purpose of this study was to examine the effect of DOX administration on vascular, interstitial and intracellular concentrations of AA in SM of the rat up to 8 days after the administration of a 1.5 or 4.5 mg/kg i.p. dose of DOX. In the plasma, total amino acids (TAA) were significantly increased compared to control where greater (P<0.05) concentrations were observed following the 1.5 mg/kg dose compared to the 4.5 mg/kg dose. Compared to control, the 1.5 mg/kg dose resulted in an increase (P<0.05) in interstitial TAA whereas the 4.5 mg/kg resulted in a sustained decrease (P<0.05). Intracellular TAA, essential amino acids (EAA) and branched-chain amino acids (BCAA) where significantly increased in each muscle group analyzed, following the 1.5 and 4.5 mg/kg doses compared to control. This study provides important insight into the amino acid response following DOX chemotherapy and presents a substantial foundation for future studies focused on reducing SM damage and recovery by targeting amino acid metabolism.
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