Hiromitsu Morita

Transient Receptor Potential Channels in Cardiovascular Function and Disease

Sustained elevation in the intracellular Ca2+ concentration via Ca2+ influx, which is activated by a variety of mechanisms, plays a central regulatory role for cardiovascular functions. Recent molecular biological research has disclosed an unexpectedly diverse array of Ca2+-entry channel molecules involved in this Ca2+ influx. These include more than ten transient receptor potential (TRP) superfamily members such as TRPC1, TRPC3–6, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and polycystin (TRPP2). Most of them appear to be multimodally activated or modulated and show relevant features to both acute hemodynamic control and long-term remodeling of the cardiovascular system, and many of them have been found to respond not only to receptor stimulation but also to various forms of stimuli. There is good evidence to implicate TRPC1 in neointimal hyperplasia after vascular injury via store-depletion–operated Ca2+ entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca2+ mobilizations, being involved in vasoconstrictor and myogenic responses and pulmonary arterial proliferation and its associated disease (idiopathic pulmonary arterial hypertension). Considerable evidence has also been accumulated for unique involvement of TRPV1 in blood flow/pressure regulation via sensory vasoactive neuropeptide release. New lines of evidence suggest that TRPV2 may act as a Ca2+-overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg2+ entry channel, and TRPP2 as a Ca2+-entry channel requisite for vascular integrity. This review attempts to provide an overview of the current knowledge on TRP proteins and discuss their possible roles in cardiovascular functions and diseases.

Cyclic GMP-dependent but G-kinase-independent inhibition of Ca2+-dependent Cl- currents by NO donors in cat tracheal smooth muscle.

1 The effects of NO donors on Ca2+‐dependent Cl− currents (ICl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole‐cell patch clamp technique. 2 With nystatin‐perforated whole‐cell recording, carbachol (CCh, ≥ 1 μm) induced a transient inward current (ICCh) with a reversal potential of about ‐20 mV. Activation of ICCh probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) greatly inhibited this current, while 11‐(2‐(diethylamino)methyl)‐1‐piperidinylacetyl)‐5,11‐dihydro‐6H‐pyrido (2,3β) (1,4)benzodiazepine‐6‐one (AF‐DX 116) or pirenzepine at concentrations of up to 1 μm were almost ineffective. 3 Chloride channel/transporter blockers such as DIDS (100 μm), anthracene‐9‐carboxylic acid (9‐AC, 100 μm) and niflumic acid (100 μm) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 μm), Zn2+ (500 μm) or Gd3+ (500 μm), were without effect. 4 Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 μm) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml−1) in the pipette abolished a substantial part of ICCh. These results suggest that ICCh is ICl(Ca), which is activated by inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ release. 5 The nitric oxide donor S‐nitroso‐N‐acetyl penicillamine (SNAP) reduced the amplitude of ICCh dose dependently (IC50, ≈10 μm). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (±)‐E‐methyl‐2‐(E‐hydroxyimitol)‐5‐nitro‐6‐methoxy‐3‐hexeneamide (NO‐R). 6 SNAP‐induced ICCh inhibition was effectively antagonized by Methylene Blue (1‐100 nM), and mimicked by dibutyryl cGMP (db‐cGMP) (0.5‐1 mM), whereas two structurally distinct types of cGMP‐dependent (G)‐kinase inhibitor, N‐(2‐aminoethyl)‐5‐isoquinilinesulphonamide (H‐8, 2.5 μm) and KT5823 (1 μm), failed to counteract the inhibitory effects of SNAP or db‐cGMP. Another G‐kinase‐specific inhibitor Rp‐8‐(para‐chlorophenylthio)guanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cGMPS; 1 μm) itself caused a marked reduction in ICCh. 7 SNAP (100 μm) or db‐cGMP (100 μm) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current. 8 These results suggest that inhibition of ICCh by NO donors involves a cGMP‐dependent but G‐kinase‐independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.

Myogenic tone is impaired at low arterial pressure in mice deficient in the low‐voltage‐activated CaV3.1 T‐type Ca2+ channel

Using mice deficient in the CaV3.1 T‐type Ca2+ channel, the aim of the present study was to elucidate the molecular identity of non‐L‐type channels involved in vascular tone regulation in mesenteric arteries and arterioles.

T-channel-like pharmacological properties of highvoltage-activated, nifedipine-insensitive Ca2+ currents inthe rat terminal mesenteric artery

Pharmacological properties of nifedipine‐insensitive, high voltage‐activated Ca2+ channels in rat mesenteric terminal arteries (NICCs) were investigated and compared with those of α1E and α1G heterologously expressed in BHK and HEK293 cells respectively, using the patch clamp technique. With 10 mM Ba2+ as the charge carrier, rat NICCs (unitary conductance: 11.5 pS with 110 mM Ba2+) are almost identical to those previously identified in a similar region of guinea‐pig, such as in current‐voltage relationship, voltage dependence of activation and inactivation, and divalent cation permeability. However, these properties are considerably different when compared with α1E and α1G. SNX‐482(200 nM and sFTX3.3 (1 μM), in addition to ω‐conotoxin GVIA (1 μM) and ω‐agatoxin IVA (100 nM), were totally ineffective for rat NICC currents, but significantly suppressed α1E (by 82% at 200 nM; IC50=11.1 nM) and α1G (by 20% at 1 μM) channel currents, respectively. A non‐specific T‐type Ca2+ channel blocker nimodipine (10 μM) differentially suppressed these three currents (by 40, 3 and 85% for rat NICC, α1E and α1G currents, respectively). Mibefradil, the widely used T‐type channel blocker, almost equally inhibited rat NICC and α1G currents in a voltage‐dependent fashion with similar IC50 values (3.5 and 0.3 μM and 2.4 and 0.14 μM at −100 and −60 mV, respectively). Furthermore, other organic T‐type channel blockers such as phenytoin, ethosuximide, an arylpiperidine derivative SUN N5030 (IC50=0.32 μM at −60 mV for α1G) also exhibited comparable inhibitory efficacies for NICC currents (inhibited by 22% at 100 μM; IC50=27.8 mM; IC50=0.53 μM, respectively). These results suggest that despite distinctive biophysical properties, the rat NICCs have indistinguishable pharmacological sensitivities to many organic blockers compared with T‐type Ca2+ channels.

Multiple regulation by external ATP of nifedipine-insensitive, high voltage-activated Ca2+ current in guinea-pig mesenteric terminal arteriole

We investigated the receptor‐mediated regulation of nifedipine‐insensitive, high voltage‐activated Ca2+ currents in guinea‐pig terminal mesenteric arterioles (ImVDCC) using the whole‐cell clamp technique. Screening of various vasoactive substances revealed that ATP, histamine and substance P exert modulatory effects on ImVDCC. The effects of ATP on ImVDCC after complete P2X receptor desensitization exhibited a complex concentration dependence. With 5 mm Ba2+, ATP potentiated ImVDCC at low concentrations (∼1–100 μm), but inhibited it at higher concentrations (>100 μm). The potentiating effects of ATP were abolished by suramin (100 μm) and PPADS (10 μm) and by intracellular application of GDPβS (500 μm), whereas a substantial part of ImVDCC inhibition by milimolar concentrations of ATP remained unaffected; due probably to its divalent cation chelating actions. In divalent cation‐free solution, ImVDCC was enlarged and underwent biphasic effects by ATPγS and ADP, while 2‐methylthio ATP (2MeSATP) exerted only inhibition, and pyrimidines such as UTP and UDP were ineffective. ATP‐induced ImVDCC potentiation was selectively inhibited by anti‐Gαs antibodies or protein kinase A (PKA) inhibitory peptides and mimicked by dibutyryl cAMP. In contrast, ATP‐induced inhibition was selectively inhibited by Gαq/11 antibodies or protein kinase C (PKC) inhibitory peptides and mimicked by PDBu. Pretreatment with pertussis toxin was ineffective. The apparent efficacy for ImVDCC potentiation with PKC inhibitors was: ATPγS > ATP≥ADP and for inhibition with PKA inhibitors was: 2MeSATP > ATPγS > ATP > ADP. Neither ImVDCC potentiation nor inhibition showed voltage dependence. These results suggest that ImVDCC is multi‐phasically regulated by external ATP via P2Y11‐resembling receptor/Gs/PKA pathway, P2Y1‐like receptor/Gq/11/PKC pathway, and metal chelation.

Newly emerging Ca2+ entry channel molecules that regulate the vascular tone

Local blood flow is critically determined by the arterial tone in which sustained Ca2+ influx, activated by a variety of mechanisms, plays a central regulatory role. Recent progress in molecular biological research has disclosed unexpectedly diverse and complex facets of Ca2+ entry channel molecules involved in this Ca2+ influx. Candidates include several transient receptor potential (TRP) superfamily members such as TRPC1, TRPC4, TRPC6, TRPV2, TRPV4 and TRPM4, none of which exhibit simple properties attributable to a single particular role. Rather, they appear to be multimodally activated or modulated by receptor stimulation, temperature, mechanical stress or lipid second messengers generated from various sources, and may be involved in both acute vasomotor control and long-term vascular remodelling. This paper provides an overview of existing knowledge of TRP proteins, and their possible relationships with principal factors regulating the arterial tone (i.e., autonomic nerves, various autocrine and paracrine factors, and intravascular pressure).

Endogenous Cardiac Troponin T Modulates Ca2+-Mediated Smooth Muscle Contraction

Mechanisms linked to actin filaments have long been thought to cooperate in smooth muscle contraction, although key molecules were unclear. We show evidence that cardiac troponin T (cTnT) substantially contributes to Ca2+-mediated contraction in a physiological range of cytosolic Ca2+ concentration ([Ca2+]i). cTnT was detected in various smooth muscles of the aorta, trachea, gut and urinary bladder, including in humans. Also, cTnT was distributed along with tropomyosin in smooth muscle cells, suggesting that these proteins are ready to cause smooth muscle contraction. In chemically permeabilised smooth muscle of cTnT+/− mice in which cTnT reduced to ~50%, the Ca2+-force relationship was shifted toward greater [Ca2+]i, indicating a sizeable contribution of cTnT to smooth muscle contraction at [Ca2+]i < 1 μM. Furthermore, addition of supplemental TnI and TnC reconstructed a troponin system to enhance contraction. The results indicated that a Tn/Tn-like system on actin-filaments cooperates together with the thick-filament pathway.

Involvement of IP3-receptor activation in endothelin-1-induced Ca2+ influx in rat pulmonary small artery

We examined the endothelin-1 (ET-1)-induced increase in the intracellular free Ca(2+) concentration ([Ca(2+)]i) in fura-2-loaded rat pulmonary small arteries. ET-1 (30 nM) elicited a long-lasting increase in [Ca(2+)]i in physiological salt solution (PSS). In subsequent experiments, arteries were pretreated with BQ-788, an ETB-specific blocker, to allow us to focus on responses mediated via the ETA receptor, the existence of which was confirmed by immunohistochemistry. In Ca(2+)-free PSS, ET-1 evoked a small transient increase in [Ca(2+)]i, indicating Ca(2+) release from the SR (sarcoplasmic reticulum). After a switch to PSS (containing 2mM CaCl2), ET-1 elicited a long-lasting increase in [Ca(2+)]i that was not inhibited by 1 μM nicardipine, an L-type Ca(2+)-channel inhibitor, suggesting involvement of a Ca(2+)-influx pathway independent of that channel. In arteries preincubated with 30 μM cyclopiazonic acid (CPA) or 2 μM thapsigargin (TG), the ET-1-induced Ca(2+)-release was greatly reduced, and the induced Ca(2+)-influx was attenuated. U-73122, a phospholipase C (PLC) inhibitor, had inhibitory effects similar to those of CPA and TG on the ET-1-induced Ca(2+)-release and Ca(2+)-influx, whereas U-73343, an inactive analogue of U-73122, had no such effects. Two putative membrane-permeable IP3-receptor blockers, 2-aminoethoxydiphenyl borate (2APB, 50 μM) and Xestospongin C (20 μM), (a) almost completely inhibited the ET-1-induced Ca(2+)-release and Ca(2+)-influx, and (b) reduced the ET-1-induced contraction. These results indicate that in rat pulmonary small arteries, ET-1 induces receptor-operated Ca(2+) influx via the ETA receptor, and that this influx interacts with InsP3-receptor activation.

Contribution of nifedipine-insensitive voltage-dependent Ca2+ channel to diameter regulation in rabbit mesenteric artery.

We investigated a possible role of nifedipine-insensitive high voltage-activated (NI-HVA) Ca2+ channels in arterial diameter regulation in the semi-terminal branches of rabbit mesenteric artery (RMA). From these branches, NI-HVA Ca2+ currents showing almost identical properties to those previously identified in a similar region of guinea-pig [Circulation Research 1999;85:596-605] were recorded with whole-cell patch clamp recording. With video-microscopic measurement, the diameter of RMA segments perfused intraluminally at a constant rate (2-6 mL/h; 269 +/- 9 micro m, n = 27) decreased by 50-60% by raising the external K+ concentration ([K+]o) to 75 mM, a substantial part of which remained after addition of 1-10 micro M nifedipine (44 +/- 5% of initial diameter, n = 27). This nifedipine-insensitive diameter decrease (NI-DD) appeared to consist of initial transient and subsequent tonic phases (this separation was, however, not always clear), was resistant to tetrodotoxin, and was completely abolished in Ca2+-free or 100 micro M Cd2+-containing bath solutions. The magnitude of NI-DD increased depending on [K+]o with a threshold concentration of 20-40 mM. Raising the external Ca2+ concentration dose-dependently increased the magnitude of NI-DD, the extent being more prominent in the late tonic phase. Combined application of caffeine (10 mM) with ryanodine (3 micro M) produced a large transient NI-DD, which strongly attenuated the NI-DD evoked by a subsequent elevation in [K+]o. Using the fura-2 spectrofluorimetric Ca2+ imaging technique, a nifedipine-insensitive [Ca2+]i increase showing similar [K+]o-dependence and Cd2+ sensitivity to NI-DD was observed. These properties of NI-DD accord with those of NI-HVA Ca2+ channels, thus suggesting their contribution to small arterial diameter regulation in RMA.

Inhibitory Effect of Enterococcus faecium WB2000 on Volatile Sulfur Compound Production by Porphyromonas gingivalis

Volatile sulfur compounds (VSCs) produced by oral anaerobes are the major compounds responsible for oral malodor. Enterococcus faecium WB2000 is recognized as an antiplaque probiotic bacterium. In this study, the effect of E. faecium WB2000 on VSC production by Porphyromonas gingivalis was evaluated, and the mechanism of inhibition of oral malodor was investigated. P. gingivalis ATCC 33277 was cultured in the presence of four lactic acid bacteria, including E. faecium WB2000. Subsequently, P. gingivalis ATCC 33277, W50, W83, and two clinical isolates were cultured in the presence or absence of E. faecium WB2000, and the emission of VSCs from spent culture medium was measured by gas chromatography. The number of P. gingivalis ATCC 33277 in mixed culture with E. faecium WB2000 decreased at 6 h, and the rate of decrease was higher than that in mixed cultures with the other lactic acid bacteria. The numbers of five P. gingivalis strains decreased at similar rates in mixed culture with E. faecium WB2000. The concentration of methyl mercaptan was lower in spent culture medium from P. gingivalis and E. faecium WB2000 cultures compared with that from P. gingivalis alone. Therefore, E. faecium WB2000 may reduce oral malodor by inhibiting the growth of P. gingivalis and neutralizing methyl mercaptan.