Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A.

This study explored the mechanism by which Ca2+-activated Cl- channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl- currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

Molecular and functional significance of Ca(2+)-activated Cl(-) channels in pulmonary arterial smooth muscle.

Increased peripheral resistance of small distal pulmonary arteries is a hallmark signature of pulmonary hypertension (PH) and is believed to be the consequence of enhanced vasoconstriction to agonists, thickening of the arterial wall due to remodeling, and increased thrombosis. The elevation in arterial tone in PH is attributable, at least in part, to smooth muscle cells of PH patients being more depolarized and displaying higher intracellular Ca(2+) levels than cells from normal subjects. It is now clear that downregulation of voltage-dependent K(+) channels (e.g., Kv1.5) and increased expression and activity of voltage-dependent (Cav1.2) and voltage-independent (e.g., canonical and vanilloid transient receptor potential [TRPC and TRPV]) Ca(2+) channels play an important role in the functional remodeling of pulmonary arteries in PH. This review focuses on an anion-permeable channel that is now considered a novel excitatory mechanism in the systemic and pulmonary circulations. It is permeable to Cl(-) and is activated by a rise in intracellular Ca(2+) concentration (Ca(2+)-activated Cl(-) channel, or CaCC). The first section outlines the biophysical and pharmacological properties of the channel and ends with a description of the molecular candidate genes postulated to encode for CaCCs, with particular emphasis on the bestrophin and the newly discovered TMEM16 and anoctamin families of genes. The second section provides a review of the various sources of Ca(2+) activating CaCCs, which include stimulation by mobilization from intracellular Ca(2+) stores and Ca(2+) entry through voltage-dependent and voltage-independent Ca(2+) channels. The third and final section summarizes recent findings that suggest a potentially important role for CaCCs and the gene TMEM16A in PH.

Regulation of gastric electrical and mechanical activity by cholinesterases in mice.

BACKGROUND/AIMS: Gastric peristalsis begins in the orad corpus and propagates to the pylorus. Directionality of peristalsis depends upon orderly generation and propagation of electrical slow waves and a frequency gradient between proximal and distal pacemakers. We sought to understand how chronotropic agonists affect coupling between corpus and antrum. METHODS: Electrophysiological and imaging techniques were used to investigate regulation of gastric slow wave frequency by muscarinic agonists in mice. We also investigated the expression and role of cholinesterases in regulating slow wave frequency and motor patterns in the stomach. RESULTS: Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to var-icose processes of motor neurons. Inhibition of AChE in the absence of stimulation increased slow wave frequency in corpus and throughout muscle strips containing corpus and antrum. CCh caused depolarization and increased slow wave frequency. Stimulation of cholinergic neurons increased slow wave frequency but did not cause depolarization. Neostigmine (1 µM) in-creased slow wave frequency, but uncoupling between corpus and antrum was not detected. Motility mapping of contractile activity in gastric muscles showed similar effects of enteric nerve stimulation on the frequency and propagation of slow waves, but neostigmine (> 1 µM) caused aberrant contractile frequency and propagation and ectopic pacemaking. CONCLUSIONS: Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and sug-gest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns.

Combined administration of RG7652, a recombinant human monoclonal antibody against PCSK9, and atorvastatin does not result in reduction of immune function.

RG7652 is a human IgG1 monoclonal antibody designed to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) binding to hepatic low density lipoprotein receptor (LDL-r), thereby blocking PCSK9-mediated degradation of LDL-r. This therapeutic candidate is under development for the prevention of cardiovascular mortality and morbidity in dyslipidemic patients. The primary objective of this study was to evaluate the potential immunotoxicological effects of RG7652 when given to cynomolgus monkeys either alone or in combination with a daily oral dose of atorvastatin. Administration of RG7652 via subcutaneous injection every other week for 12 weeks (a total of seven doses), daily oral doses of atorvastatin (total of 85 doses), and combinations of each up to 15 and 20 mg/kg/dose, respectively, were well tolerated and there was no evidence of alteration in immune function. Administration of pharmacologically relevant doses of RG7652 in combination with atorvastatin to healthy monkeys does not result in clinically meaningful immunosuppression as measured by T-cell dependent antibody responses, natural killer cell activity, immunophenotype, or delayed type hypersensitivity. The only pharmacologically mediated changes observed during the dosing period were the anticipated changes in circulating cholesterol.

Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 µM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 µM, with or without 10 µM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 µM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease.

Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange.

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 µmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 µmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 µmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 µmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 µmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.

Expression profile and protein translation of TMEM16A in murine smooth muscle.

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-µm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that I(ClCa) generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to I(ClCa) recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca(2+)-activated Cl(-) channel in this cell type.

Intricate interaction between store-operated calcium entry and calcium-activated chloride channels in pulmonary artery smooth muscle cells.

Ca(2+)-activated Cl-() channels (Cl(Ca)) represent an important excitatory mechanism in vascular smooth muscle cells. Active accumulation of Cl-() by several classes of anion transporters results in an equilibrium potential for this ion about 30 mV more positive than the resting potential. Stimulation of Cl(Ca) channels leads to membrane depolarization, which enhances Ca(2+) entry through voltage-gated Ca(2+) channels and leads to vasoconstriction. Cl(Ca) channels can be activated by distinct sources of Ca(2+) that include (1) mobilization from intracellular Ca(2+) stores (ryanodine or inositol 1,4,5-trisphosphate [InsP(3)]) and (2) Ca(2+) entry through voltage-gated Ca(2+) channels or reverse-mode Na(+)/Ca(2+) exchange. The present study was undertaken to determine whether Ca(2+) influx triggered by store depletion (store-operated calcium entry, SOCE) activates Cl(Ca) channels in rabbit pulmonary artery (PA) smooth muscle. Classical store depletion protocols involving block of sarcoplasmic reticular Ca(2+) reuptake with thapsigargin (TG; 1 microM) or cyclopiazonic acid (CPA; 30 microM) led to a consistent nifedipine-insensitive contraction of intact PA rings and rise in intracellular Ca(2+) concentration in single PA myocytes that required the presence of extracellular Ca(2+). In patch clamp experiments, TG or CPA activated a time-independent nonselective cation current (I (SOC)) that (1) reversed between -10 and 0 mV; (2) displayed the typical "N"-shaped current-voltage relationship; and (3) was sensitive to the (I (SOC)) blocker by SKF-96365 (50 microM). In double-pulse protocol experiments, the amplitude of I (SOC) was varied by altering membrane potential during an initial step that was followed by a second constant step to +90 mV to register Ca(2+)-activated Cl(-) current, I (Cl(Ca)). The niflumic acid-sensitive time-dependent I (Cl(Ca)) at +90 mV increased in proportion to the magnitude of the preceding hyperpolarizing step, an effect attributed to graded membrane potential-dependent Ca(2+) entry through I (SOC) and confirmed in dual patch clamp and Fluo-5 experiments to record membrane current and free intracellular Ca(2+) concentration simultaneously. Reverse-transcription polymerase chain reaction (RT-PCR) experiments confirmed the expression of several molecular determinants of SOCE, including transient receptor potential canonical (TRPC) 1, TRPC4, and TRPC6; stromal interacting molecule (STIM) 1 and 2; and Orai1 and 2, as well as the novel and probable molecular candidates thought to encode for Cl(Ca) channels transmembrane protein 16A (TMEM16A) Anoctamin 1 (ANO1) and B (ANO2). Ourpreliminary investigation provides new evidence for a Ca(2+) entry pathway consistent with store-operated Ca(2+) entry signaling that can activate Ca(2+)-activated Cl-() channels in rabbit PA myocytes. We hypothesize that this mechanism may be important in the regulation of membrane potential, Ca(2+) influx, and tone in these cells under physiological and pathophysiological conditions.

Complex phosphatase regulation of Ca2+-activated Cl- currents in pulmonary arterial smooth muscle cells.

The present study was undertaken to determine whether the two ubiquitously expressed Ca(2+)-independent phosphatases PP1 and PP2A regulate Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) elicited by 500 nM [Ca(2+)](i) in rabbit pulmonary artery (PA) myocytes dialyzed with or without 3 mM ATP. Reverse transcription-PCR experiments revealed the expression of PP1alpha, PP1beta/delta, PP1gamma, PP2Aalpha, PP2Abeta, PP2Balpha (calcineurin (CaN) Aalpha), and PP2Bbeta (CaN Abeta) but not PP2Bgamma (CaN Agamma) in rabbit PA. Western blot and immunofluorescence experiments confirmed the presence of all three PP1 isoforms and PP2A. Intracellular dialysis with a peptide inhibitor of calcineurin (CaN-AIP); the non-selective PP1/PP2A inhibitors okadaic acid (0.5, 10, or 30 nM), calyculin A (10 nM), or cantharidin (100 nM); and the selective PP1 inhibitor NIPP-1 (100 pM) potently antagonized the recovery of I(Cl(Ca)) in cells dialyzed with no ATP, whereas the PP2A-selective antagonist fostriecin (30 or 150 nM) was ineffective. The combined application of okadaic acid (10 nM) and CaN-autoinhibitory peptide (50 microM) did not potentiate the response of I(Cl(Ca)) in 0 ATP produced by maximally inhibiting CaN or PP1/PP2A alone. Consistent with the non-additive effects of either classes of phosphatases, the PP1 inhibitor NIPP-1 (100 pM) antagonized the recovery of I(Cl(Ca)) induced by exogenous CaN Aalpha (0.5 microM). These results demonstrate that I(Cl(Ca)) in PA myocytes is regulated by CaN and PP1 and/or PP2A. Our data also suggest the existence of a functional link between these two classes of phosphatases.

Prostaglandin regulation of gastric slow waves and peristalsis.

Gastric emptying depends on functional coupling of slow waves between the corpus and antrum, to allow slow waves initiated in the gastric corpus to propagate to the pyloric sphincter and generate gastric peristalsis. Functional coupling depends on a frequency gradient where slow waves are generated at higher frequency in the corpus and drive the activity of distal pacemakers. Simultaneous intracellular recording from corpus and antrum was used to characterize the effects of PGE(2) on slow waves in the murine stomach. PGE(2) increased slow-wave frequency, and this effect was mimicked by EP(3), but not by EP(2), receptor agonists. Chronotropic effects were due to EP(3) receptors expressed by intramuscular interstitial cells of Cajal because these effects were not observed in W/W(V) mice. Although the integrated chronotropic effects of EP(3) receptor agonists were deduced from electrophysiological experiments, no clear evidence of functional uncoupling was observed with two-point electrical recording. Gastric peristalsis was also monitored by video imaging and spatiotemporal maps to study the impact of chronotropic agonists on propagating contractions. EP(3) receptor agonists increased the frequency of peristaltic contractions and caused ectopic sites of origin and collisions of peristaltic waves. The impact of selective regional application of chronotropic agonists was investigated by use of a partitioned bath. Antral slow waves followed enhanced frequencies induced by stimulation of the corpus, and corpus slow waves followed when slow-wave frequency was elevated in the antrum. This demonstrated reversal of slow-wave propagation with selective antral chronotropic stimulation. These studies demonstrate the impact of chronotropic agonists on regional intrinsic pacemaker frequency and integrated gastric peristalsis.

Neural regulation of slow-wave frequency in the murine gastric antrum.

Gastric peristaltic contractions are driven by electrical slow waves modulated by neural and humoral inputs. Excitatory neural input comes primarily from cholinergic motor neurons, but ACh causes depolarization and chronotropic effects that might disrupt the normal proximal-to-distal spread of gastric slow waves. We used intracellular electrical recording techniques to study cholinergic responses in stomach tissues from wild-type and W/W(V) mice. Electrical field stimulation (5 Hz) enhanced slow-wave frequency. These effects were abolished by atropine and the muscarinic M(3)-receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. ACh released from nerves did not depolarize antral muscles. At higher rates of stimulation (10 Hz), chronotropic effects were mediated by ACh and a noncholinergic transmitter and blocked by muscarinic antagonists and neurokinin (NK(1) and NK(2))-receptor antagonists. Neostigmine enhanced slow-wave frequency, suggesting that the frequency of antral pacemakers is kept low by efficient metabolism of ACh. Neostigmine had no effect on slow-wave frequency in muscles of W/W(v) mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). These muscles also showed no significant chronotropic response to 5-Hz electrical field stimulation or the cholinergic agonist carbachol. The data suggest that the chronotropic effects of cholinergic nerve stimulation occur via ICC-IM in the murine stomach. The capacity of gastric muscles to metabolize ACh released from enteric motor neurons contributes to the maintenance of the proximal-to-distal slow-wave frequency gradient in the murine stomach. ICC-IM play a critical role in neural regulation of gastric motility, and ICC-IM become the dominant pacemaker cells during sustained cholinergic drive.