Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700

L-type calcium (Ca²⁺) currents conducted by voltage-gated Ca²⁺ channel Ca_V1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca_V1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca²⁺ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the Ca_V1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca_V1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca²⁺ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.Upon membrane depolarization, Ca_V1.2 channels conduct L-type calcium (Ca²⁺) current into cardiomyocytes and initiate excitation–contraction coupling (1, 2). Ca²⁺ influx through Cav1.2 channels activates Ca²⁺ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation–contraction coupling, Ca²⁺ influx via Ca_V1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca_V1.2 channel (1, 3). Phosphorylation of the Ca_V1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca_V1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4–6). Under those pathological conditions, responsiveness of Ca_V1.2 channel activity to β-adrenergic receptors and PKA activation is severely blunted, resulting in diminished contractile reserve and impaired fight-or-flight response (6, 7). Enormous effort has been devoted to understanding how β-adrenergic regulation of the Ca_V1.2 channel is achieved, but the exact molecular mechanisms remain unresolved.Ca_V1.2 channels contain multiple subunits, including a pore-forming α₁1.2 subunit (also designated α_1C), β and α₂δ subunits that modulate expression of Ca_V1.2 at the cell surface, and possibly γ subunits (8). The closely related Ca_V1.1 and Ca_V1.2 channels in skeletal and cardiac muscle, respectively, are both proteolytically processed near the center of their large C-terminal domains (9, 10), and the distal C terminus (dCT) remains associated noncovalently with the proximal C terminus (pCT) and serves as a potent autoinhibitor (11, 12). Regulation of Ca_V1.2 channels by PKA was reconstituted in nonmuscle cells with a dynamic range of threefold to fourfold similar to native cardiomyocytes by building the autoinhibitory Ca_V1.2 complex through cotransfection of each of its components (13). Successful reconstitution required an A Kinase Anchoring Protein (AKAP), which recruits PKA to the dCT (13–15). Deletion of the dCT in vivo results in loss of regulation of the L-type Ca²⁺ current by the β-adrenergic pathway and embryonic death from heart failure (16, 17). These results suggest that the autoinhibited Ca_V1.2 signaling complex serves as the substrate for β-adrenergic regulation, and disruption of this complex leads to heart failure.PKA is responsible for phosphorylation of the Ca_V1.2 channel in response to β-adrenergic stimulation in cardiac myocytes (18–22). Although multiple PKA sites have been identified in α₁ subunits by in vitro phosphorylation (10, 23), none of these sites is required for regulation of Ca_V1.2 channels in vivo. For example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10, 24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca_V1.2 channel activity in transfected cells or cardiomyocytes (13, 25, 26). Two sites in the C terminus of the skeletal muscle Ca_V1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27). These sites are conserved in cardiac Ca_V1.2 channels as S1700 and T1704, and phosphoproteomics analysis revealed β-adrenergic–stimulated phosphorylation of S1700 by PKA (28). S1700 and T1704 reside at the interface between the pCT and dCT. In studies of the Ca_V1.2 signaling complex reconstituted in nonmuscle cells, phosphorylation of both sites was required for normal basal channel activity, whereas only S1700 was essential for PKA stimulation (13). Mutation of S1700 and T1704 to Ala in STAA mice reduced basal activity and Ca_V1.2 channel regulation by the β-adrenergic pathway in cardiomyocytes (29). To further dissect the contribution of S1700, we studied a mutant mouse line expressing Ca_V1.2 channel with the S1700A mutation (SA mice). Our results demonstrate that this single phosphorylation site is required for normal regulation of Ca_V1.2 channels, contraction of cardiac myocytes, exercise capacity, and cardiac homeostasis.     

Functional protein expression of multiple sodium channel α- and β-subunit isoforms in neonatal cardiomyocytes

Voltage-gated sodium channels are composed of pore-forming α- and auxiliary β-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel α-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na_v1.5 sodium channel α-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel α- and β-subunits. α-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel α-subunit isoforms Na_v1.1, Na_v1.2, Na_v1.3, Na_v1.4 and Na_v1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac α-subunit isoform, Na_v1.5. Each of the β-subunit isoforms (β1-β4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the α-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel α-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na_v1.5 α-subunit and they contribute to the total sodium current.     

Defining the regulation of IL-1β- and CHOP-mediated β-cell apoptosis

Diabetes is a multifaceted metabolic disorder that can be caused by pancreatic β-cell destruction (type I diabetes) and/or heightened by β-cell failure (type II diabetes). The gross clinical and physiological characteristics of the disease are well characterized, and viable treatment options can drastically alter the course and effects of the disease. However, the molecular events occurring within the β-cell that cause or contribute to diabetes are not adequately understood, especially in terms of the interplay between the physiological signals that facilitate disease development. A recent report, focused on a mechanism by which IL-1β induces β-cell apoptosis, underscores the complexity of the molecular events that may cause or affect the progression of diabetes. This commentary summarizes aspects of this report, discusses an example of the complexity of β-cell regulation and proposes more frequent use of complex in vitro systems that more closely mimic in vivo conditions so that greater advances can be made toward understanding the molecular mechanisms contributing to diabetes. Understanding the molecular etiology of β-cell dysfunction will likely enhance the possibility of developing novel diabetes therapeutic interventions for diabetes.     

From the Cover: Cell-type–specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation

Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na⁺/K⁺ ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a β subunit required for its transport to the plasma membrane and for regulating its activity. We show that α and β subunits are expressed in Johnston''s organ (JO), the Drosophila auditory organ. We knocked down expression of α subunits (ATPα and α-like) and β subunits (nrv1, nrv2, and nrv3) individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among β subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell–specific support of the catalytic ATPα. Our studies provide an amenable model to investigate generation of endolymph-like extracellular compartments.     

Functional expression of β1 and β2 integrins on tumor infiltrating lymphocytes (TILs) in colorectal cancer

Integrins play an important role in various lymphocyte functions. In this study, tumor-infiltrating lymphocytes (TIL) were isolated from colorectal cancer tissues and the expression of β1 and β2 integrins on the TIL was quantitatively examined with two-color flow cytometry. In comparison with peripheral blood lymphocytes (PBL), TIL expressed a lower level of common β1 chain (CD29) in both CD4 and CD8 sub-populations. Among the associated α chains, the expressions of α1 (CD49a) and α2 (CD49b) were slightly higher in TIL than in PBL, whereas α4 (CD49d) and α6 (CD49f) were markedly downregulated in TIL. Both αL (CD11a) and β2 (CD18) were reduced in CD8(+) TIL but not in CD4(+) TIL. TIL with the CD8(+) cytotoxic phenotype showed significantly decreased binding to purified intracellular adhesion molecules (ICAM)-1, and vascular adhesion cell molecule (VCAM)-1, and HT29 colon cancer cells, compared with the in counterparts in PBL. The peculiar expression pattern and functional down regulation of these integrins may explain why TIL in colorectal cancer cannot eradicate the malignant cells. Received: August 24, 1998/Accepted: November 27, 1998     

Defining the regulation of IL-1β- and CHOP-mediated β-cell apoptosis

Diabetes is a multifaceted metabolic disorder that can be caused by pancreatic β-cell destruction (type I diabetes) and/or heightened by β-cell failure (type II diabetes). The gross clinical and physiological characteristics of the disease are well characterized, and viable treatment options can drastically alter the course and effects of the disease. However, the molecular events occurring within the β-cell that cause or contribute to diabetes are not adequately understood, especially in terms of the interplay between the physiological signals that facilitate disease development. A recent report, focused on a mechanism by which IL-1β induces β-cell apoptosis, underscores the complexity of the molecular events that may cause or affect the progression of diabetes. This commentary summarizes aspects of this report, discusses an example of the complexity of β-cell regulation and proposes more frequent use of complex in vitro systems that more closely mimic in vivo conditions so that greater advances can be made toward understanding the molecular mechanisms contributing to diabetes. Understanding the molecular etiology of β-cell dysfunction will likely enhance the possibility of developing novel diabetes therapeutic interventions.     

Exploring the relationship between β-adrenergic receptor kinase-1 and physical symptoms in heart failure

Background

The relationship between physical heart failure (HF) symptoms and pathophysiological mechanisms is unclear.

Role of the β1 integrin molecule in T-cell activation and migration

β1 integrins play crucial roles in a variety of cell processes such as adhesion, migration, proliferation, and differentiation of lymphocytes. To understand the molecular mechanisms of these various biological effects, it is particularly important to analyze cell signaling through the β1 integrins. Our previous study showed that PLC-γ, pp125FAK (focal adhesion kinase), pp105, paxillin, p59fyn, p56lck, and ERK1/2 are phosphorylated in their tyrosine residues upon engagement of β1 integrins. We identified pp105 as Cas (Crk-associated substrate)-related protein and successfully cloned its cDNA. pp105 is a Cas homologue predominantly expressed in the cells of lymphoid lineage, which led us to designate it Cas-L. Like p130Cas, Cas-L contains a single SH3 domain and multiple SH2-binding sites (YXXP motif), which are suggested to bind SH2 domains of Crk, Nck, and SHPTP2. Subsequent studies revealed that pp125FAK binds Cas-L on its SH3 domain and phosphorylates its tyrosine residues upon β1 integrin stimulation. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. We have shown that Cas-L is involved in the T-cell receptor (TCR)/CD3 signaling pathway as well as the β1 integrin signaling pathway. Cas-L is transiently phosphorylated following CD3 crosslinking and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant (Cas-LΔSH3), which lacks the binding site for FAK, is still tyrosine-phosphorylated upon CD3 crosslinking but not upon β1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Finally, we have identified a crucial role of Cas-L in β1 integrin-mediated T-cell co-stimulation. We have found that this co-stimulatory pathway is impaired in the Jurkat T-cell line, and that the expression level of Cas-L is reduced in the Jurkat cells compared to peripheral T-cells. The transfection of Cas-L cDNA into Jurkat cells restored the β1 integrin-mediated co-stimulation, while the transfection of Cas-LΔSH3 mutant failed to do so, which contrasts with the case of CD3-mediated signaling. These results indicate that Cas-L plays a key role, through the association and phosphorylation by FAK, in β1 integrin-mediated T-cell co-stimulation. Moreover, tyrosine phosphorylation of Cas-L is critical for T-cell receptor and β1 integrin-induced T-lymphocyte migration. Taken together, Cas-L might be the bi-modal docking protein which assembles the signals through β1 integrins and TCR/CD3, and which participates in a variety of T-cell functions. Received: August 24, 1999 / Accepted: August 31, 1999     

Interplay between the E2F pathway and β-adrenergic signaling in the pathological hypertrophic response of myocardium

The E2F/Pocket protein (Rb) pathway regulates cell growth, differentiation, and death by modulating gene expression. We previously examined this pathway in the myocardium via manipulation of the unique E2F repressor, E2F6, which is believed to repress gene activity independently of Rb. Mice with targeted expression of E2F6 in postnatal myocardium developed dilated cardiomyopathy (DCM) without hypertrophic growth. We assessed the mechanisms of the apparent failure of compensatory hypertrophic growth as well as their response to the β-adrenergic agonist isoproterenol. As early as 2 weeks, E2F6 transgenic (Tg) mice present with dilated thinner left ventricles and significantly reduced ejection fraction and fractional shortening which persists at 6 weeks of age, but with no apparent increase in left ventricle weight: body weight (LVW:BW). E2F6-Tg mice treated with isoproterenol (6.1 mg/kg/day) show double the increase in LVW:BW than their Wt counterparts (32% vs 16%, p-value: 0.007). Western blot analysis revealed the activation of the adrenergic pathway in Tg heart tissue under basal conditions with ~ 2-fold increase in the level of β₂-adrenergic receptors (p-value: 8.9E − 05), protein kinase A catalytic subunit (PKA-C) (p-value: 0.0176), activated c-Src tyrosine-protein kinase (p-value: 0.0002), extracellular receptor kinase 2 (ERK2) (p-value: 0.0005), and induction of the anti-apoptotic protein Bcl2 (p-value 0. 0.00001). In contrast, a ~ 60% decrease in the cardiac growth regulator: AKT1 (p-value 0.0001) and a ~ four fold increase in cyclic AMP dependent phosphodiesterase 4D (PDE4D), the negative regulator of PKA activity, were evident in the myocardium of E2F6-Tg mice. The expression of E2F3 was down-regulated by E2F6, but was restored by isoproterenol. Further, Rb expression was down-regulated in Tg mice in response to isoproterenol implying a net activation of the E2F pathway. Thus the unique regulation of E2F activity by E2F6 renders the myocardium hypersensitive to adrenergic stimulus resulting in robust hypertrophic growth.These data reveal a novel interplay between the E2F pathway, β₂–adrenergic/PKA/PDE4D, and ERK/c-Src axis in fine tuning the pathological hypertrophic growth response. E2F6 deregulates E2F3 such that pro-hypertrophic growth and survival are enhanced via β₂-adrenergic signaling however this response is outweighed by the induction of anti-hypertrophic signals so that left ventricle dilation proceeds without any increase in muscle mass.     

Convergence of IRBIT,phosphatidylinositol (4,5) bisphosphate,and WNK/SPAK kinases in regulation of the Na+-HCO3? cotransporters family

Fluid and HCO₃⁻ secretion is a vital function of secretory epithelia, involving basolateral HCO₃⁻ entry through the Na⁺-HCO₃⁻ cotransporter (NBC) NBCe1-B, and luminal HCO₃⁻ exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl⁻/HCO₃⁻ exchangers. HCO₃⁻ secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP₂). Activation of the transporters by IRBIT and PIP₂ is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO₃⁻ transporters and thus in the regulation of epithelial fluid and HCO₃⁻ secretion.Fluid and HCO₃⁻ secretion is a vital function of secretory epithelia that involves basolateral HCO₃⁻ entry through the Na⁺-HCO₃⁻ cotransporter (NBC) NBCe1-B and luminal HCO₃⁻ exit mediated by the concerted activity of cystic fibrosis transmembrane conductance regulator (CFTR) and members of the solute carrier family 26 (SLC26) transporter family (1). HCO₃⁻ secretion is osmotically active owing to an influx of Na⁺-2HCO₃⁻ (2, 3) and the exchange of Cl⁻/2HCO₃⁻ by Slc26a6 (4, 5), resulting in net osmolyte secretion in the form of HCO₃⁻. HCO₃⁻ secretion is a highly regulated activity, with several signaling pathways converging to regulate key transporters activity to tune the secretion (1); however, very little is known about the molecular mechanisms that regulate fluid and HCO₃⁻ secretion, particularly the regulation of NBCe1-B.NBCe1-B was originally designated pancreatic NBC 1 (6) and was later renamed NBCe1-B as a member of the electrogenic NBCe1 subfamily of the Na⁺-coupled bicarbonate transporter (NCBT) superfamily (2, 3). NBCe1-B is is expressed in the basolateral membrane of most secretory epithelia, including the pancreas, salivary glands, airway, and intestines (1). The NCBTs encompass 13 transmembrane domains with varying cytoplasmic N and C termini among the isoforms (3). Most members of the NCBT superfamily have a unique N terminus (the first 85 residues in NBCe1-B) (7). This domain has been shown to function as an autoinhibitory domain (AID) in NBCe1-B (8–10). Very little is known about the regulation of NBCe1-B or other members of the superfamily beyond that NBCe1-B may be modestly activated by cAMP (11, 12), although inhibition of NBCe1-B by cAMP was subsequently reported by the same group (13). NBCe1-B appears to be constitutively phosphorylated by protein kinase A in Thr49 (11); however, its role in activation of the transporter is not clear, given that both the T49A and T49D mutations were found to prevent activation by cAMP (11). Activation of NBCe1-B and NBCe1-C by intracellular Ca²⁺ through an unknown mechanism was reported recently (5). NBCe1-A is activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) (14), but direct activation of NBCe1-B and NBCe1-C by PIP₂ has not been examined. The site of interaction of PIP₂ in regulating the activity of any NCBT family member remains to be determined.NBCe1-B (9, 10, 15, 16) and NBCe1-C (17) are potently activated by the inositol 1,4,5-triphosphate (IP₃) receptors binding protein released with IP₃ (IRBIT) and are inhibited by the with no lysine kinase (WNK) and Ste20-related proline alanine rich kinase (SPAK) (15). IRBIT also regulates CFTR (15, 16) and sodium-hydrogen exchanger 3 (NHE3) (18). IRBIT activates NBCe1-B by recruiting protein phosphatase 1 (PP1), reversing inhibition by the WNK/SPAK pathway through dephosphorylation of NBCe1-B (15) and relief of inhibition by the AID (9, 10, 15, 16). The WNKs function as scaffolds to recruit SPAK to NBCe1-B, which in turn phosphorylates the transporter at unknown sites. Similarly, the site of IRBIT–AID interaction is unknown. Deletion of the first 16 residues of NBCe1-B prevents activation by IRBIT (9), although the effect of this truncation on IRBIT binding is unclear. On the other hand, an in vitro binding assay revealed binding of IRBIT to NBCe1-B(1-62), but not to NBCe1-B(1-37) (10). This finding suggests that the IRBIT binding site may be located within NBCe1-B(37-62); however, the possible binding of IRBIT to NBCe1-B(37-62) or a site within has not yet been examined.The extent to which these pathways regulate other members of the NBC family is unknown, although IRBIT may activate an unspecified member of the NBCn1 subfamily (2). The NBCn1 subfamily was established with the discovery of NBC3, later renamed NBCn1-A. NBCn1-A is a widely expressed electroneutral NBC (3, 7) that mediates HCO₃⁻ salvage in secretory epithelia (19, 20). Given that sequence analysis has shown significant conservation of the N termini of NBCe1-B and NBCn1-A, we deemed it useful to compare the regulation of these NBCs by IRBIT/PP1, PIP₂, and SPAK to evaluate the generality of this regulation. In the present studies, we also investigated whether these multiple regulatory pathways converge on the same domain to regulate the transporters.We have identified a positively charged domain within NBCe1-B(37-65) that is conserved in NBCn1-A and most members of the NCBT superfamily and is required for interaction and activation of the transporters by IRBIT. The same domain mediates regulation of NBCe1-B and NBCn1-A by PIP₂. Importantly, activation of the transporters by IRBIT and PIP₂ is nonadditive but complementary. Phosphorylation of Ser65 within this domain mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 within NBCe1-B(37-65) is required for regulation by the activator IRBIT and the inhibitor SPAK. Moreover, a sequence search using the conserved module identified a similar module in Slc26a6 and CFTR that is required for regulation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in the AID of the NBCs and thus in the regulation of epithelial fluid and HCO₃⁻ secretion.     

Contribution of L-type Ca2＋ channel to the regulation of coronary arterial smooth muscle contraction is different in rats and mice

Background L-type calcium channel participates in the regulation of a variety of physical and pathological process. In vasculature, it mainly mediated agonist-induced vascular smooth muscle contraction. However, it is not clear whether there are differences in L-type calcium channel mediated vessel responses to certain vasoconstrictors among different species. Methods The coronary arteries were dissected from the heart of rats and mice respectively. The coronary arterial ring contraction was measured by Multi Myograph System. Results Endothelin-1, U46619 and 5-HT could produce concentration-dependent vasoconstriction of coronary arterial rings from rats and mice. Compared with rats, the vessel rings of mice were more sensitive to ET-1 and U46619, and less sensitive to 5-HT. The L-type Ca2~ channel blocker nifedipine could significantly inhibit the coronary artery contractions induced by ET-1, U46619 and 5-HT. The inhibitory effect of i ixM nifedipine on ET-1 and 5-HT-induced coronary artery contractions were stronger in mice than in rats, but its effect on U46619 induced-vessel contractions was much weaker in mice than in rats. Conclusions L-type Ca2＋ channel plays an important role in the coronary arterial contraction, but the responses to vasoconstrictor and L-type Ca2＋ channel blocker are different between rats and mice, thus suggesting that the coronary arteries of rats and mice have different biological characteristics.     

Interferon β-1a and Atorvastatin in the Treatment of Multiple Sclerosis

Background: Statins, widely used cholesterol-lowering agents, have also been demonstrated to have anti-inflammatory and immunomdulatory effects. Objective: To evaluate the effects of atorvastatin in combination with Interferon-β in the treatment of multiple sclerosis (MS) in a randomized controlled clinical trial. Methods: Multiple sclerosis patients were randomized independently, in a double blind design, into one of two treatment groups. Control group (n=45) received 30 μg/week interferon β-1a via intra-muscular injection. Atorvastatin-treated group (n=50) received interferon β-1a similar to control group in addition to atorvastatin (40 mg/day) for 18-months. All clinical and immunological variables were measured at the baseline and at the end of the study. Results: There was no significant difference between the two groups in the expanded disability status scale scores and the number of gadolinium-enhancing lesions during the 18-month treatment period. After 18 months, the levels of interleukin (IL)-4, IL-10, transforming growth factor-β and serum ferric reducing antioxidant power in the atorvastatin treatment group were significantly higher than the control group. Levels of IL-17, TNF-α and lymphocyte proliferation in the atorvastatin treatment group were significantly lower than the control group. Conclusion: Although combined atorvastatin and interferon-β do not change the clinical course of MS, atorvastatin might have beneficial effects in MS treatment possibly through inducing anti-inflammatory responses.     

Interplay between FAK, PKCδ, and p190RhoGAP in the regulation of endothelial barrier function

Disruption of either intercellular or extracellular junctions involved in maintaining endothelial barrier function can result in increased endothelial permeability. Increased endothelial permeability, in turn, allows for the unregulated movement of fluid and solutes out of the vasculature and into the surrounding connective tissue, contributing to a number of disease states, including stroke and pulmonary edema (Ermert et al., 1995; Lee and Slutsky, 2010; van Hinsbergh, 1997; Waller et al., 1996; Warboys et al., 2010). Thus, a better understanding of the molecular mechanisms by which endothelial cell junction integrity is controlled is necessary for development of therapies aimed at treating such conditions. In this review, we will discuss the functions of three signaling molecules known to be involved in regulation of endothelial permeability: focal adhesion kinase (FAK), protein kinase C delta (PKCδ), and p190RhoGAP (p190). We will discuss the independent functions of each protein, as well as the interplay that exists between them and the effects of such interactions on endothelial function.     

Control of transient,resurgent, and persistent current by open-channel block by Na channel β4 in cultured cerebellar granule neurons

Voltage-gated Na channels in several classes of neurons, including cells of the cerebellum, are subject to an open-channel block and unblock by an endogenous protein. The Na_Vβ4 (Scn4b) subunit is a candidate blocking protein because a free peptide from its cytoplasmic tail, the β4 peptide, can block open Na channels and induce resurgent current as channels unblock upon repolarization. In heterologous expression systems, however, Na_Vβ4 fails to produce resurgent current. We therefore tested the necessity of this subunit in generating resurgent current, as well as its influence on Na channel gating and action potential firing, by studying cultured cerebellar granule neurons treated with siRNA targeted against Scn4b. Knockdown of Scn4b, confirmed with quantitative RT-PCR, led to five electrophysiological phenotypes: a loss of resurgent current, a reduction of persistent current, a hyperpolarized half-inactivation voltage of transient current, a higher rheobase, and a decrease in repetitive firing. All disruptions of Na currents and firing were rescued by the β4 peptide. The simplest interpretation is that Na_Vβ4 itself blocks Na channels of granule cells, making this subunit the first blocking protein that is responsible for resurgent current. The results also demonstrate that a known open-channel blocking peptide not only permits a rapid recovery from nonconducting states upon repolarization from positive voltages but also increases Na channel availability at negative potentials by antagonizing fast inactivation. Thus, Na_Vβ4 expression determines multiple aspects of Na channel gating, thereby regulating excitability in cultured cerebellar granule cells.     

PAI-1 and IFN-γ in the regulation of innate immune homeostasis during sublethal yersiniosis

Plasminogen activator inhibitor type 1 (PAI-l), a key part of the fibrinolytic system, plays a critical host protective role during the acute phase of infection by regulating interferon(IFN)-γ release. IFN-γ regulates PAI-1 expression, which suggests an intricate interplay between PAI-1 and IFN-γ. Here, using the notion of a feedback loop, we report the complicated regulatory relationship between PAI-1 and IFN-γ. Mice were inoculated intravenously with 1 × 10³ colony forming units of Yersinia enterocolitica; PAI-1 deficiency enhanced lethality (p < 0.0001) and increased bacterial growth and dissemination (p = 0.08 on day 3, p = 0.004 on day 5, respectively). PAI-1 significantly increased the levels IFN-γ mRNA (p < 0.005), which may increase survival and decrease bacterial burden. Simultaneously, we showed that IFN-γ increased PAI-1 mRNA levels in vivo (p < 0.05). Next, we investigated the transduction signal pathway. After mice were inoculated intraperitoneally with 50 μg lipopolysaccharide (LPS), both levels of IFN-γ mRNA (p = 0.05) and levels of PAI-1 mRNA (p < 0.0001) decreased in MyD88-deficient mice. The same trend was also found in mice treated with 1000 μg LPS. As a result of correlations of IFN-γ and PAI-1 in wild-type mice, we delineated the transduction signal pathway, namely MyD88-IFN-γ-PAI-1. The in vivo LPS-injected animal model further confirmed that PAI-1 feedback controlled IFN-γ in a direct or indirect manner. New perspectives of the relationship between PAI-1 and IFN-γ should help in understanding the complex and often conflicting results that have been reported in different infection models. Thus, the feedback loop between PAI-1 and IFN-γ is part of the dynamic equilibrium of coagulation and inflammation that helps maintain innate immune homeostasis.