Haruko Iida

Nonselective Cation Currents Regulate Membrane Potential of Rabbit Coronary Arterial Cell Modulation by Lysophosphatidylcholine

Background—The effects of lysophosphatidylcholine (LPC) on electrophysiological activities and intracellular Ca2+ concentration ([Ca2+]i) were investigated in coronary arterial smooth muscle cells (CASMCs). Methods and Results—The patch clamp techniques and Ca2+ measurements were applied to cultured rabbit CASMCs. The membrane potential was −46.0±5.0 mV, and LPC depolarized it. Replacement of extracellular Na+ with NMDG+ hyperpolarized the membrane and antagonized the depolarizing effects of LPC. In Na+-, K+-, or Cs+-containing solution, the voltage-independent background current with reversal potential (Er) of approximately +0 mV was observed. Removal of Cl− failed to affect it. When extracellular cations were replaced by NMDG+, Er was shifted to negative potentials. La3+ and Gd3+ abolished the background current, but nicardipine and verapamil did not inhibit it. In Na+-containing solution, LPC induced a voltage-independent current with Er of approximately +0 mV concentration-dependently. Similar current was recorded in K+- and Cs+-containing solution. La3+ and Gd3+ inhibited LPC-induced current, but nicardipine and verapamil did not inhibit it. In cell-attached configurations, single-channel activities with single-channel conductance of ≈32pS were observed when patch pipettes were filled with LPC. LPC increased [Ca2+]i as the result of Ca2+ influx, and La3+ completely antagonized it. Conclusions—These results suggest that (1) nonselective cation current (INSC) contributes to form membrane potentials of CASMCs and (2) LPC activates INSC, resulting in an increase of [Ca2+]i. Thus, LPC may affect CASMC tone under various pathophysiological conditions such as ischemia.

Voltage-gated sodium channel expressed in cultured human smooth muscle cells: involvement of SCN9A

Voltage‐gated Na+ channel (INa) is expressed under culture conditions in human smooth muscle cells (hSMCs) such as coronary myocytes. The aim of this study is to clarify the physiological, pharmacological and molecular characteristics of INa expressed in cultured hSMCs obtained from bronchus, main pulmonary and coronary artery. INa, was recorded in these hSMCs and inhibited by tetrodotoxin (TTX) with an IC50 value of approximately 10 nM. Reverse transcriptase/polymerase chain reaction (RT‐PCR) analysis of mRNA showed the prominent expression of transcripts for SCN9A, which was consistent with the results of real‐time quantitative RT‐PCR. These results provide novel evidence that TTX‐sensitive Na+ channel expressed in cultured hSMCs is mainly composed of Nav1.7.

Effects of low-intensity resistance exercise with blood flow restriction on coagulation system in healthy subjects

Recent studies have demonstrated that even a low‐intensity resistance exercise can effectively induce muscle hypertrophy and strength increase when combined with moderate blood flow restriction (BFR) into the exercising muscle. Although serious side effects of low‐intensity resistance exercise with BFR have not been reported, a concern of thrombosis has been suggested, because this type of exercise is performed with restricted venous blood flow and pooling of blood in extremities. Thus, the purpose of this study was to investigate the effects of low‐intensity resistance exercise with BFR on coagulation system in healthy subjects. Ten healthy men (25·1 ± 2·8 year) performed four sets of leg press exercises with and without BFR (150–160 mmHg) at an intensity of 30% of one‐repetition maximum (1RM). In each exercise session, one set with 30 repetitions was followed by three sets with 15 repetitions. Blood samples were taken before, and 10 min, 1, 4 and 24 h after the exercise. Prothrombin fragment 1 + 2 (PTF) and thrombin–antithrombin III complex (TAT) were measured as markers of thrombin generation, whereas D‐dimer and fibrin degradation product (FDP) were measured as markers of intravascular clot formation. Changes in plasma volume (PV) were calculated from haemoglobin and hematocrit values. PV reduction was significantly greater after the exercise with BFR than without (P<0·05). However, neither markers of thrombin generation nor intravascular clot formation increased after the exercises. These results suggest that low‐intensity resistance exercise with BFR does not activate coagulation system in healthy subjects.

Effects of low‐intensity, elastic band resistance exercise combined with blood flow restriction on muscle activation

We examined the effects of blood flow‐restricted, low‐intensity resistance exercise (termed kaatsu) using an elastic band for resistance on muscle activation. Nine men performed triceps extension and biceps flexion exercises (four sets respectively) using an elastic band for resistance with blood flow restriction (BFR) or CON (unrestricted blood flow). During a BFR session, subjects wore pressure cuffs inflated to 170–260 mmHg on the proximal region of both arms. Surface electromyography (EMG) was recorded from the triceps brachii and biceps brachii muscles, and mean integrated EMG (iEMG) was analyzed. Blood lactate concentration was obtained before (Pre) and immediately after two exercises (Post). During triceps extension and biceps flexion exercises, muscle activation increased progressively (P < 0.05) under BFR (46% and 69%, respectively) but not under CON (12% and 23%, respectively). Blood lactate concentration at Post was higher (P < 0.05) under BFR than under CON (3.6 and 2.1 mmol/L, respectively). Blood lactate concentration at Post was significantly correlated with increased iEMG in both triceps extension (r = 0.65, P < 0.01) and biceps flexion exercises (r = 0.52, P < 0.05). We conclude that kaatsu training using elastic bands for resistance enhances muscle activation and may be an effective method to promote muscle hypertrophy in older adults or patients with a low level of activity.

Effects of Low-Load, Elastic Band Resistance Training Combined With Blood Flow Restriction on Muscle Size and Arterial Stiffness in Older Adults

We examined the effect of low-load, elastic band resistance training with blood flow restriction (BFR) on muscle size and arterial stiffness in older adults. Healthy older adults (aged 61-85 years) were divided into BFR training (BFR-T, n = 9) or non-BFR training (CON-T, n = 8) groups. Both groups performed low-load arm curl and triceps down exercises (four sets, total 75 repetitions for each) using an elastic band, 2 d/wk for 12 weeks. The BFR-T group wore inflated pneumatic elastic cuffs (120-270 mm Hg) on both arms during training. Magnetic resonance imaging-measured muscle cross-sectional area of the upper arm, maximum voluntary isometric contraction of the elbow flexors and extensors, cardio-ankle vascular index testing, and ankle-brachial pressure index were measured before and 3-5 days after the final training session. Muscle cross-sectional area of the elbow flexors (17.6%) and extensors (17.4%) increased, as did elbow flexion and elbow extension maximum voluntary isometric contraction (7.8% and 16.1%, respectively) improved (p < .05) in the BFR-T group, but not in the CON-T group. In cardio-ankle vascular index and ankle-brachial pressure index testing, there were no changes between pre- and post-results in either group. In conclusion, elastic band BFR-T improves muscle cross-sectional area as well as maximal muscle strength but does not negatively affect arterial stiffness in older adults.

Muscle size and arterial stiffness after blood flow-restricted low-intensity resistance training in older adults

Previous studies have shown that blood flow‐restricted low‐intensity resistance training (BFR‐RT) causes muscle hypertrophy while maintaining arterial function in young adults. We examined the effects of BFR‐RT on muscle size and arterial stiffness in older adults. Healthy subjects (ages 61–84 years) were divided into BFR‐RT (n = 9) or non‐training control (CON; n = 10) groups. The BFR‐RT group performed 20% and 30%, respectively, of one‐repetition maximal (1‐RM) knee extension and leg press exercises, 2 days/wk for 12 weeks. The BFR‐RT group wore elastic cuffs (120–270 mmHg) on both legs during training. Magnetic resonance imaging‐measured muscle cross‐sectional area (CSA), 1‐RM strength, chair stand (CS) test, and cardio‐ankle vascular index testing (CAVI), an index of arterial stiffness, were measured before and 3–5 days after the final training session. Muscle CSA of the quadriceps (8.0%), adductors (6.5%), and gluteus maximus (4.4%), leg extension and leg press 1‐RM strength (26.1% and 33.4%), and CS performance (18.3%) improved (P < 0.05) in the BFR‐RT group, but not in the CON group. In CAVI testing, there were no changes in both two groups. In conclusion, BFR‐RT improves muscle CSA as well as maximal muscle strength, but does not negatively affect arterial stiffness or humeral coagulation factors in older adults.

Function and role of voltage-gated sodium channel NaV1.7 expressed in aortic smooth muscle cells

Voltage-gated Na(+) channel currents (I(Na)) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of I(Na), its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, I(Na) was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular Na(+) with N-methyl-d-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding Na(V)1.7, as determined by quantitative RT-PCR. I(Na) was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type Ca(2+) channel current was recorded, and I(Na) was not observed in freshly isolated rASMCs, whereas TTX-sensitive I(Na) was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for Na(V)1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that I(Na) is expressed in cultured and diseased conditions but not in normal aorta. The Na(V)1.7 plays an important role in cell migration, endocytosis, and secretion. Na(V)1.7 is also expressed in aorta after balloon injury, suggesting a potential role for Na(V)1.7 in the progression of intimal hyperplasia.

Effects of walking with blood flow restriction on limb venous compliance in elderly subjects

Venous compliance declines with age and improves with chronic endurance exercise. KAATSU, an exercise combined with blood flow restriction (BFR), is a unique training method for promoting muscle hypertrophy and strength gains by using low‐intensity resistance exercises or walking. This method also induces pooling of venous blood in the legs. Therefore, we hypothesized that slow walking with BFR may affect limb venous compliance and examined the influence of 6 weeks of walking with BFR on venous compliance in older women. Sixteen women aged 59–78 years were partially randomized into either a slow walking with BFR group (n = 9, BFR walk group) or a non‐exercising control group (n = 7, control group). The BFR walk group performed 20‐min treadmill slow walking (67 m min−1), 5 days per week for 6 weeks. Before (pre) and after (post) those 6 weeks, venous properties were assessed using strain gauge venous occlusion plethysmography. After 6 weeks, leg venous compliance increased significantly in the BFR walk group (pre: 0·0518 ± 0·0084, post: 0·0619 ± 0·0150 ml 100 ml−1 mmHg−1, P<0·05), and maximal venous outflow (MVO) at 80 mmHg also increased significantly after the BFR walk group trained for 6 weeks (pre: 55·3 ± 15·6, post: 67·1 ± 18·9 ml 100 ml−1 min−1, P<0·01), but no significant differences were observed in venous compliance and MVO in the control group. In addition, there was no significant change in arm compliance in the BFR walk group. In conclusion, this study provides the first evidence that 6 weeks of walking exercise with BFR may improve limb venous compliance in untrained elder female subjects.

Involvement of CaV3.1 T-type calcium channels in cell proliferation in mouse preadipocytes

Voltage-gated Ca(2+) channels (Ca(V)) are ubiquitously expressed in various cell types and play vital roles in regulation of cellular functions including proliferation. However, the molecular identities and function of Ca(V) remained unexplored in preadipocytes. Therefore, whole cell voltage-clamp technique, conventional/quantitative real-time RT-PCR, Western blot, small interfering RNA (siRNA) experiments, and immunohistochemical analysis were applied in mouse primary cultured preadipocytes as well as mouse 3T3-L1 preadipocytes. The effects of Ca(V) blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold Ca(V), which could be inhibited by mibefradil, Ni(2+) (IC(50) of 200 muM), and NNC55-0396. Dominant expression of alpha(1G) mRNA was detected among Ca(V) transcripts (alpha(1A)-alpha(1I)), supported by expression of Ca(V)3.1 protein encoded by alpha(1G) gene, with immunohistochemical studies and Western blot analysis. siRNA targeted for alpha(1G) markedly inhibited Ca(V). Dominant expression of alpha(1G) mRNA and expression of Ca(V)3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of alpha(1G) mRNA and Ca(V)3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type Ca(V) blocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for alpha(1G) also prevented cell cycle entry/progression. The present study demonstrates that the Ca(V)3.1 T-type Ca(2+) channel encoded by alpha(1G) subtype is the dominant Ca(V) in mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development.

Comparative effects of azelnidipine and other Ca2+-channel blockers on the induction of inducible nitric oxide synthase in vascular smooth muscle cells.

Overproduction of nitric oxide by inducible nitric oxide synthase contributes to the progression of cardiovascular disease. We investigated the effects of azelnidipine and other Ca2+-channel blockers on nitric oxide production by cultured aortic smooth muscle cells isolated from Wistar rats and human umbilical vein endothelial cells (HUVECs), using the Griess reaction and oxyhemoglobin method. Release of lactic dehydrogenase (LDH) was measured to evaluate cell damage, and immunohistochemistry was performed to examine the expression of inducible nitric oxide synthase and nitrotyrosine protein. Azelnidipine and other Ca2+-channel blockers inhibited the release of nitric oxide induced by lipopolysaccharide plus interferon-γ. Azelnidipine inhibited it most potently among the Ca2+-channel blockers tested (azelnidipine, amlodipine, nifedipine, diltiazem, verapamil, and nicardipine) at a concentration of 10 μM. Longer stimulation with these agents induced the expression of inducible nitric oxide synthase and nitrotyrosine, with an increase of lactic dehydrogenase release, whereas azelnidipine suppressed these changes. In human umbilical vein endothelial cells, azelnidipine enhanced basal nitric oxide production by endothelial nitric oxide synthase. In conclusion, azelnidipine potently inhibited the induction of inducible nitric oxide synthase and then nitric oxide production in vascular smooth muscle cells, while enhancing constitutive nitric oxide production by endothelial cells. Azelnidipine may inhibit nitrotyrosine expression and cell damage caused by overproduction of nitric oxide, suggesting a mechanism for its cardiovascular protective effect.