K⁺ channels permit more than one ion within their conducting pathway at any given moment and show a saturating single-file behavior. The conduction of Rb⁺ shows an unusual behavior, a so-called “Rb⁺ anomaly,” and it has been used to probe the mechanism of the ion conduction through K⁺-selective channels. Under the bi-ionic condition of K⁺ and Rb⁺, we carried out patch-clamp single-channel current measurements in MaxiK⁺ channels from mouse submandibular acinar cells. Keeping only K⁺ on one side of the membrane while varying fractional Rb⁺ concentration on the opposite, we had a series of current-voltage relationships. It showed a characteristic inflection at which the ion conductance was divided into two components, one ascribed to pure K⁺ conduction and the other to K⁺ and Rb⁺ bi-ionic conduction. By analyzing the latter, we depicted that (1) the bi-ionic conductance showed a characteristic reduction curve as the Rb⁺ fractional concentration increased; (2) Rb⁺ can bind the channel more tightly when it accesses from the outside than from the inside. Thus we conclude that such asymmetry of the Rb⁺ binding determines the pattern of bi-ionic conductance reduction in K-selective channels.

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Anoikis is a specific type of apoptosis caused by the detachment of anchorage-dependent cells from their supportive matrix. Aggregation of suspended cells is believed to suppress anoikis. Here we describe the effects of cellular aggregation on anoikis in Madin-Darby canine kidney (MDCK) cells. Suspension cultures of MDCK cells grown under conditions known to induce extensive cellular aggregation were less able to reattach to culture dishes, exhibited higher caspase-8 activity, and contained more sub-G₁ cells than suspension cultures did with less cellular aggregation. When suspension cultures of MDCK cells were separated into aggregated cells and single cells, the aggregated cells had low caspase-8 activity regardless of suspension conditions, whereas the single cells had higher caspase-8 activity that increased with an increasing degree of aggregation. These results suggest that cell-cell interactions in cellular aggregates of suspended MDCK cells facilitate anoikis, causing more apoptosis in individual cells than when these interactions are absent.

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Although muscarinic K⁺ (K_ACh) channels contribute to a rapid heart rate (HR) response to vagal stimulation, whether background sympathetic tone affects the HR control via the K_ACh channels remains to be elucidated. In seven anesthetized rabbits with sinoaortic denervation and vagotomy, we estimated the dynamic transfer function of the HR response by using random binary vagal stimulation (0–10 Hz). Tertiapin, a selective K_ACh channel blocker, decreased the dynamic gain (to 2.3 ± 0.9 beats·min^–1·Hz^–1, from 4.6 ± 1.1, P < 0.01, mean ± SD) and the corner frequency (to 0.05 ± 0.01 Hz, from 0.26 ± 0.04, P < 0.01). Under 5 Hz tonic cardiac sympathetic stimulation (CSS), tertiapin decreased the dynamic gain (to 3.6 ± 1.0 beats·min^–1·Hz^–1, from 7.3 ± 1.1, P < 0.01) and the corner frequency (to 0.06 ± 0.02 Hz, from 0.23 ± 0.06, P < 0.01). Two-way analysis of variance indicated significant interaction between the tertiapin and CSS effects on the dynamic gain. In contrast, no significant interactions were observed between the tertiapin and CSS effects on the corner frequency and the lag time. In conclusion, although a cyclic AMP-dependent mechanism has been well established, an accentuated antagonism also occurred in the direct effect of ACh via the K_ACh channels. The rapidity of the HR response obtained by the K_ACh channel pathway was robust during the accentuated antagonism.

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Our previous study showed that electro-acupuncture (EA) pretreatment protects the heart from injury of ischemia. The present study explored further whether adenylate cyclase (AC), protein kinase A (PKA), and L-type Ca²⁺ channel, theβ₁-AR signaling components modulating intracellular Ca²⁺ ([Ca²⁺]_i), are involved in the mediation of the antiarrhythmic effect of EA pretreatment in the rats from which the hearts were subsequently isolated and subjected to simulative global ischemia and reperfusion (SGIR). SGIR was performed by perfusing the isolated heart at a low flow followed by normal perfusion. Adult rats were randomized into four groups, namely, normal control (NC), SGIR, EA, and NC plus EA (NCEA) groups. The rats in the EA and NCEA groups were given EA pretreatment at bilateral Neiguan points (PC6) for 30 min once a day in 3 consecutive days before the hearts were isolated and perfused. The arrhythmia score and the response of [Ca²⁺]_i to the activators of AC, PKA, and L-type Ca²⁺ channel in single ventricular myocyte isolated from the hearts subjected to SGIR were compared among the groups. The results showed that the arrhythmia score was significantly higher in the SGIR group as compared with the NC and NCEA groups. The SGIR-enhanced arrhythmia score was significantly attenuated in the EA group. More interesting, EA pretreatment also attenuated the SGIR-enhanced response of [Ca²⁺]_i to the activators of AC, PKA, and the L-type Ca²⁺ channel in the myocytes isolated from the hearts subjected to SGIR. In conclusion, EA pretreatment can produce an antiarrhythmic effect in the rat of SGIR. AC, PKA and the L-type Ca²⁺ channel are involved in the mediation of the antiarrhythmic effect of EA pretreatment.

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The effect of functional overloading on the regenerating process of injured skeletal muscle was investigated in 10-week-old male mice (C57BL/6J). Functional overloading on soleus of both hindlimbs was performed by cutting the distal tendons of plantaris and gastrocnemius muscles for 2 weeks before cardiotoxin (CTX) injection as the preconditioning and also during 10 weeks of recovery. To activate the necrosis- regeneration cycle, 0.1 ml of 10-μM CTX was injected into soleus muscle. The mean values of absolute muscle weight and the percentage of Pax7-positive nuclei in soleus were increased by the preconditioning. These values, as well as total muscle protein content, in the group with CTX injection plus overloading were larger than in the group with CTX injection alone. Fibers with central nucleus were noted in the group with CTX injection with or without overloading. The rate of disappearance of fibers having central nucleus during recovery was stimulated by overloading. Histological analyses revealed that the regeneration of injured soleus muscle with overloading proceeded more rapidly than the muscle without overloading. These results, in combination with previous lines of evidence, strongly suggest that functional overloading may facilitate the regeneration of injured skeletal muscles.

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Endothelial dysfunction often precedes Type 2 diabetes–associated cardiovascular complications. One important cause of endothelial dysfunction is oxidative stress, which can lead to reduced nitric oxide (NO) bioavailability. In this study, we examined the effects of ramipril (an angiotensin-converting enzyme inhibitor, ACEI) on reactive oxygen species (ROS) production and endothelium-dependent vasodilation using a Type 2 diabetic (db/db) murine model. Plasma concentration of 8-isoprostane ([8-isoP]) was measured and used as an indication of the amount of ROS production. Six weeks of ramipril (10 mg/kg/day) treatment significantly reduced [8-isoP] and improved acetylcholine(ACh)-induced vasodilation in db/db mice without altering responses in wild-type (WT) mice. Responsiveness of smooth muscle cells to NO, assessed by sodium nitroprusside–induced vasodilation, was not different between db/db and WT mice regardless of ramipril or vehicle treatment. Our results suggest that ramipril specifically improved endothelium-dependent vasodilation in Type 2 diabetic mice, possibly by reducing ROS levels.

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Gene expression levels of heat shock proteins (HSPs) in the slow-twitch soleus and fast-twitch plantaris muscles of rats were determined after hindlimb suspension or spaceflight. Male rats were hindlimb-suspended for 14 d or exposed to microgravity for 9 d. The mRNA expression levels of HSP27, HSP70, and HSP84 in the hindlimb-suspended and microgravity-exposed groups were compared with those in the controls. The mRNA expression levels of the 3 HSPs in the soleus muscle under normal conditions were higher compared with those in the plantaris muscle. The mRNA expression levels of the 3 HSPs in the soleus muscle were inhibited by hindlimb suspension and spaceflight. The mRNA expression levels of the 3 HSPs in the plantaris muscle did not change after hindlimb suspension. It is suggested that the mRNA expression levels of the 3 HSPs are regulated by the mechanical and neural activity levels, and therefore the decreased mRNA expression levels of HSPs in the slow-twitch muscle following hindlimb suspension and spaceflight are related to a reduction in the mechanical and neural activity levels.

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We examined the fail-safe responses against low-dose botulinum intoxication (botulinum neurotoxin serotype A; 0.05 ng/35 g body weight) in electrically activated in vitro phrenic nerve-diaphragm preparations, since sustained ventilation is critical for the prognosis of clinical botulinum intoxication. At 0, 1, 2 and 4 wks after the peritoneal injection of the toxin, both contractility and neurotransmitter release were measured. There was an increase in directly induced twitch force without affecting directly induced tetanus throughout the observation period. Indirectly induced twitch force decreased by 60% at 1 wk, which gradually recovered only during the 4-wk observation period. Spontaneous neurotransmitter release, evaluated as the frequency of miniature end plate potentials, was largely abolished 1 wk after the injection and recovered only slightly during the 4-wk period. The effects on spontaneous release were independent of medium Ca²⁺ concentration. Evoked release, evaluated as quantal content, was also mostly inhibited at 1 wk, but it recovered to approximately 50% of controls at 4 wks. The recovery of quantal content was more prominent at low medium Ca²⁺ concentration. These results indicated two functional fail-safe responses that compensate for the acute inhibitory effect of low dose of botulinum toxin on neuromuscular transmission; increased contractility of muscle, and improved efficiency of evoked quantum release. The increased contractility probably reflects remodeling of muscle fiber composition of the diaphragm. The improved efficiency of evoked quantum release probably involves remodeling of voltage-gated Ca²⁺ channels, intracellular Ca²⁺ store sites, or transmitter-releasing apparatuses.

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