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Supplemental Digital Content is available in the text., Background: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. Methods: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14–16 weeks) and old (57–60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. Results: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant’s rare allele is significantly associated with increased BP in middle-aged men. Conclusions: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.

The distribution of functionally active, inwardly rectifying K (KIR) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca2+-free solution, endothelial cells displayed a KIR current with a maximum amplitude of 190 ± 16 pA at –150 mV and sensitivity to block with 30 µM Ba2+ (n = 7). In smooth muscle cells, outward K current was activated at around –47 ± 3 mV, but there was no evidence of KIR current (n = 6). Furthermore, raising extracellular [K+] to either 60 or 140 mM, or applying the α1-adrenoceptor agonist phenylephrine (PE; 30 µM), failed to reveal an inwardly rectifying current in the smooth muscle cells, although PE did stimulate an iberiotoxin-sensitive outward K current (n = 4). Exogenous K+ (10.8–16.8 mM) both relaxed and repolarized endothelium-denuded segments of the mesenteric artery contracted with PE. These effects were depressed by 100 µM ouabain but unaffected by either 30 µM BaCl2 or 3 µM glibenclamide. These data suggest that functional, inwardly rectifying Ba2+-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.

1. In rat small mesenteric arteries contracted with phenylephrine, 1-ethyl-2-benzimidazolinone (1-EBIO; 3-300 microM) evoked concentration-dependent relaxation that, above 100 microM, was associated with smooth muscle hyperpolarization. 2. 1-EBIO-evoked hyperpolarization (maximum 22.1+/-3.6 mV with 300 microM, n=4) was endothelium-dependent and inhibited by charybdotoxin (ChTX 100 nM; n=4) but not iberiotoxin (IbTX 100 nM; n=4). 3. In endothelium-intact arteries, smooth muscle relaxation to 1-EBIO was not altered by either of the potassium channel blockers ChTX (100 nM; n=7), or IbTX (100 nM; n=4), or raised extracellular K(+) (25 mM). Removal of the endothelium shifted the relaxation curve to the right but did not reduce the maximum relaxation. 4. In freshly isolated mesenteric endothelial cells, 1-EBIO (600 microM) evoked a ChTX-sensitive outward K-current. In contrast, 1-EBIO had no effect on smooth muscle cell conductance whereas NS 1619 (33 microM) stimulated an outward current while having no effect on the endothelial cells. 5. These data show that with concentrations greater than 100 microM, 1-EBIO selectively activates outward current in endothelial cells, which presumably underlies the smooth muscle hyperpolarization and a component of the relaxation. Sensitivity to block with charybdotoxin but not iberiotoxin indicates this current is due to activation of IK(Ca). However, 1-EBIO can also relax the smooth muscle by an undefined mechanism, independent of any change in membrane potential.

Intercellular communication between cells within the blood vessel wall plays an important role in the control of artery diameter. The endothelial cells lining the lumen of arteries can evoke smooth muscle hyperpolarization both by the release of a factor (EDHF) and by direct cell-cell coupling through gap junctions. Hyperpolarizing current can spread rapidly to cause widespread vasodilatation, and thus increase blood flow to that segment. In addition to the spread of current, small molecules, such as Ca2+, can also pass between cells, but at a much reduced rate. Instead of co-ordinating changes in diameter, intercellular Ca2+ signalling acts to amplify and, in special cases, modulate vascular responses. Together, direct cell-cell communication enables the blood vessel wall to act as a functional syncytium, which is influenced by surrounding tissues and nerves, and blood constituents.

Crude extracts of both fresh and dry ginger induced the perfused rat hindlimb to consume oxygen in association with increases in perfusion pressure and lactate production. The principles responsible for these observations, the gingerols and shogaols, were isolated and tested for relative thermogenic activity. The gingerol homologues possessed greater molar potency than their shogaol counterparts. (6)-Gingerol was the most potent principle isolated, causing a mean maximal increase in oxygen consumption of 1.4 +/- 0.1 mumol/g/h (21%), an increase in lactate efflux of 4.7 +/- 0.6 mumol/g/h (87%) with a perfusion pressure increase of 7.7 +/- 0.7 mmHg (30%). Increases in alkyl chain length within each homologous series led to decreased molar potency. Specific nitro-vasodilation using glyceryl trinitrate demonstrated that thermogenesis was at least partly associated with vasoconstriction. Concurrent infusion of alpha or beta antagonists showed that neither adrenergic receptors nor secondary catecholamine release were responsible for the observed effects. Increasing doses of the ginger principles ultimately led to inhibition of steady state oxygen consumption, although perfusion pressure continued to increase. Removal of high ginger principle doses was followed by apparent increases in oxygen uptake unaccompanied by elevated perfusion pressure. As a consequence, the effective concentration ranges of the ginger principles were relatively narrow. The cause of high dose effects is as yet undetermined but may have been due in part to disruption of mitochondrial function.