Nicotine Impairs Smooth Muscle cAMP Signaling and Vascular Reactivity.

OBJECTIVE: This study aimed to determine nicotine's impact on receptor-mediated cyclic adenosine monophosphate (cAMP) synthesis in vascular smooth muscle (VSM). We hypothesize that nicotine impairs ß adrenergic-mediated cAMP signaling in VSM, leading to altered vascular reactivity. METHODS: The effects of nicotine on cAMP signaling and vascular function were systematically tested in aortic VSM cells and acutely isolated aortas from mice expressing the cAMP sensor TEpacVV (Camper), specifically in VSM (e.g., CamperSM). RESULTS: Isoproterenol (ISO)-induced ß-adrenergic production of cAMP in VSM was significantly reduced in cells from second-hand smoke (SHS)-exposed mice and cultured wild-type VSM treated with nicotine. The decrease in cAMP synthesis caused by nicotine was verified in freshly isolated arteries from a mouse that had cAMP sensor expression in VSM (e.g., CamperSM mouse). Functionally, the changes in cAMP signaling in response to nicotine hindered ISO-induced vasodilation, but this was reversed by immediate PDE3 inhibition. CONCLUSIONS: These results imply that nicotine alters VSM ß adrenergic-mediated cAMP signaling and vasodilation, which may contribute to the dysregulation of vascular reactivity and the development of vascular complications for nicotine-containing product users.

Spatiotemporal Control of Vascular CaV1.2 by α1C S1928 Phosphorylation.

BACKGROUND: L-type CaV1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the CaV1.2 pore-forming subunit α1C at S1928 mediates vascular CaV1.2 cooperativity during diabetic hyperglycemia. METHODS: A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine CaV1.2 cooperativity, α1C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels. RESULTS: CaV1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1C clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y11 inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. CaV1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca2+ influx and contractility and in vivo reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice. CONCLUSIONS: These results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1C into "superclusters" upon hyperglycemia and diabetes. This triggers CaV1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct CaV1.2 and arterial function during diabetic hyperglycemia.

Cellular and molecular effects of hyperglycemia on ion channels in vascular smooth muscle.

Diabetes affects millions of people worldwide. This devastating disease dramatically increases the risk of developing cardiovascular disorders. A hallmark metabolic abnormality in diabetes is hyperglycemia, which contributes to the pathogenesis of cardiovascular complications. These cardiovascular complications are, at least in part, related to hyperglycemia-induced molecular and cellular changes in the cells making up blood vessels. Whereas the mechanisms mediating endothelial dysfunction during hyperglycemia have been extensively examined, much less is known about how hyperglycemia impacts vascular smooth muscle function. Vascular smooth muscle function is exquisitely regulated by many ion channels, including several members of the potassium (K+) channel superfamily and voltage-gated L-type Ca2+ channels. Modulation of vascular smooth muscle ion channels function by hyperglycemia is emerging as a key contributor to vascular dysfunction in diabetes. In this review, we summarize the current understanding of how diabetic hyperglycemia modulates the activity of these ion channels in vascular smooth muscle. We examine underlying mechanisms, general properties, and physiological relevance in the context of myogenic tone and vascular reactivity.

α1C S1928 Phosphorylation of CaV1.2 Channel Controls Vascular Reactivity and Blood Pressure.

BACKGROUND: Increased vascular CaV1.2 channel function causes enhanced arterial tone during hypertension. This is mediated by elevations in angiotensin II/protein kinase C signaling. Yet, the mechanisms underlying these changes are unclear. We hypothesize that α1C phosphorylation at serine 1928 (S1928) is a key event mediating increased CaV1.2 channel function and vascular reactivity during angiotensin II signaling and hypertension. METHODS AND RESULTS: The hypothesis was examined in freshly isolated mesenteric arteries and arterial myocytes from control and angiotensin II-infused mice. Specific techniques include superresolution imaging, proximity ligation assay, patch-clamp electrophysiology, Ca2+ imaging, pressure myography, laser speckle imaging, and blood pressure telemetry. Hierarchical "nested" and appropriate parametric or nonparametric t test and ANOVAs were used to assess statistical differences. We found that angiotensin II redistributed the CaV1.2 pore-forming α1C subunit into larger clusters. This was correlated with elevated CaV1.2 channel activity and cooperativity, global intracellular Ca2+ and contraction of arterial myocytes, enhanced myogenic tone, and altered blood flow in wild-type mice. These angiotensin II-induced changes were prevented/ameliorated in cells/arteries from S1928 mutated to alanine knockin mice, which contain a negative modulation of the α1C S1928 phosphorylation site. In angiotensin II-induced hypertension, increased α1C clustering, CaV1.2 activity and cooperativity, myogenic tone, and blood pressure in wild-type cells/tissue/mice were averted/reduced in S1928 mutated to alanine samples. CONCLUSIONS: Results suggest an essential role for α1C S1928 phosphorylation in regulating channel distribution, activity and gating modality, and vascular function during angiotensin II signaling and hypertension. Phosphorylation of this single vascular α1C amino acid could be a risk factor for hypertension that may be targeted for therapeutic intervention.

Secondhand Smoke Exposure Impairs Ion Channel Function and Contractility of Mesenteric Arteries.

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 h/d, 5 d/wk) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 wk but not for 4 or 8 wk. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 wk of SHS exposure. Moreover, 12 wk SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke.

AKAP5 complex facilitates purinergic modulation of vascular L-type Ca2+ channel CaV1.2.

The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia.