These data suggest that the initial acute DVT or PE event is associated with high total health care costs and that these costs are further increased by subsequent events such as recurrent DVT or PE and PTS. Early detection and appropriate treatment of this high-risk population have the potential for both clinical and economic benefits.

β1-Adrenergic receptors (β1ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β1ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β1AR activation enhanced the L-type Ca2+ current, intracellular Ca2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β1ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β1AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β1AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β1AR and EPR responses. They also suggest that β1AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca2+ current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser16. Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

(7), unlike AC1 and AC8.AC1 is closely related in sequence to the Ca 2ϩ /CaM-stimulable rutabaga AC from Drosophila, which is important in Drosophila learning tasks (8 -10). AC1 and the other Ca 2ϩ /CaMstimulable mammalian AC, AC8, have also been implicated in learning and memory (11). As a means of establishing their proposed roles, single and/or double AC1 and AC8 knockout mice have been generated. Mouse models have demonstrated that Ca 2ϩ /CaM-stimulable ACs are involved in long-term potentiation and long-term memory (12). However, despite the general view that AC1 and AC8 can behave similarly, discrete physiological actions of each isoform are becoming apparent. Recent studies by Zhuo's group demonstrated that AC1 specifically participates in N-methyl-D-aspartic acid receptor-induced neuronal excitotoxicity (13) and an increase in GluR1 synthesis induced by blocking AMPA receptors (14). Furthermore, Nicol and colleagues (15, 16) showed a contribution of AC1, but not AC8, in axon terminal refinement in the retina. On the other hand, AC8 specifically was seen to be responsible for retrieval from adaptive presynaptic silencing (17) and the acquiring of new spatial information (18). These differences in physiological roles must reflect not only differences in their distributions but also presumably in their regulatory properties. Both enzymes are expressed in brain; AC1 is neuro-specific, whereas the expression of AC8 is more widespread (1, 12). Within the central nervous system, AC1 is abundant in the hippocampus, the cerebral cortex, and the granule cells of the cerebellum, whereas AC8 has a high expression level in the thalamus and the cerebral cortex (19). Studies of mouse brain revealed that AC1 is distributed pre-synaptically and AC8 post-synaptically (18,20).Although physiological differences in the roles of these two enzymes are suggested from the studies outlined above, the regulatory mechanisms that might underlie these differences are not. AC1 is more sensitive to Ca 2ϩ than is AC8 in vitro (21, 22), yet details on how these two enzymes are regulated by Ca 2ϩ /CaM are sparse. In non-excitable cells, a Ca 2ϩ elevation * The work was supported in part by the Wellcome Trust (Grant RG31760).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 3 All ACs have the following structure: a variable N terminus is followed by two transmembrane cassettes each of which is followed by highly conserved C1 and C2 domains (2). C1 and C2 domains are subdivided into C1a and C1b, and C2a and C2b. C1a and C2a domains are required for the catalytic activity (3).

No abstract

Because of clerical errors in preparing the figures, in Fig. 1A, the last portions of the mouse and human TILRR sequences are not aligned with the consensus sequence, and the human form is mislabeled as 716 amino acids (aa). In Fig. 2C (and on page 7227, right column, line 4), the most potent form of the human protein is mislabeled as 710 aa. Supplemental Fig. S1 correctly shows the alignment and the length of the human TILRR protein as 715 aa, with the most potent form, lacking the N-terminal 6 aa, as 709 aa. The amino acid sequence is correct as shown in all figures, and the clerical errors have no impact on any of the results, including the function of the protein, the probes used, or the numbering of the mutants.

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca(2+) channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn(2+) binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca(2+) channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.

The ubiquitous Ca2+-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca2+ acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the α-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca2+-bound, but not Ca2+-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.