Roy L. Sutliff

Fibroblast growth factor 2 control of vascular tone

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FCF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 ± 5.6% in wild type, 35.4 ± 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.

Phospholamban regulation of bladder contractility: evidence from gene-altered mouse models

1 Phospholamban (PLB) is an inhibitor of the sarcoplasmic reticulum (SR) Ca2+‐ATPase (SERCA). Its presence and/or functional significance in contractility of bladder, a smooth muscle tissue particularly dependent on SR function, is unknown. We investigated this by measuring the effects of carbachol (CCh) on force and [Ca2+]i in bladder from mice in which the PLB gene was ablated (PLB‐KO mice). In the PLB‐KO bladder, the maximum increases in [Ca2+]i and force were significantly decreased (41.5 and 47.4 % of WT), and the EC50 values increased. 2 Inhibition of SERCA with cyclopiazonic acid (CPA) abolished these differences between WT and PLB‐KO bladder, localizing the effects to the SR. 3 To determine whether these effects were specific to PLB, we generated mice with smooth‐muscle‐specific expression of PLB (PLB‐SMOE mice), using the SMP8 α‐actin promoter. Western blot analysis of PLB‐SMOE mice showed approximately an eightfold overexpression of PLB while SERCA was downregulated 12‐fold. 4 In PLB‐SMOE bladders, in contrast, the response of [Ca2+]i and force to CCh was significantly increased and the EC50 values were decreased. CPA had little affect on the CCh‐induced increases in [Ca2+]i and force in PLB‐SMOE bladder. 5 These results show that alteration of the PLB:SERCA ratio can significantly modulate smooth muscle [Ca2+]i. Importantly, our data show that PLB can play a major role in modulation of bladder contractility.

Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice

1 The Ca2+ sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor‐1 (I‐1) is the classic type‐1 phosphatase inhibitor, but its presence and role in cAMP‐dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I‐1 in vivo, we investigated smooth muscle function in a mouse model lacking the I‐1 protein (I‐1(‐/‐) mice). 2 Significant amounts of I‐1 protein were detected in the wild‐type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by 32P‐labelled aortic extracts from WT mice. 3 Despite the significant presence of I‐1 in WT aorta, phenylephrine and KCl concentration‐ isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I‐1(‐/‐) aorta. cGMP‐dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC20), measured in aortic extracts, were nearly identical between WT and I‐1(‐/‐) mice. 4 In the portal vein, I‐1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC50 of isoproterenol relaxation (EC50= 10.4 ± 1.4 nm) compared to the WT value (EC50= 3.5 ± 0.2 nm). Contraction in response to acetylcholine as well as Ca2+ sensitivity were similar between WT and I‐1(‐/‐) aorta. 5 Despite the prevalence of I‐1 and its activation by PKA in the aorta, I‐1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I‐1 may play a role as a fine‐tuning mechanism involved in regulating portal vein responsiveness to β‐adrenergic agonists.