Myofiber injury induces capillary disruption and regeneration of disorganized microvascular networks

Myofibers regenerate following injury, however the microvasculature must also recover to restore skeletal muscle function. We aimed to define the nature of microvascular damage and repair during skeletal muscle injury and regeneration induced by BaCl2. To test the hypothesis that microvascular disruption occurred secondary to myofiber injury in mice, isolated microvessels were exposed to BaCl2 or the myotoxin was injected into the gluteus maximus (GM) muscle. In isolated microvessels, BaCl2 depolarized smooth muscle cells and endothelial cells while increasing [Ca2+]i, but did not elicit cell death. At 1 day post injury (dpi) of the GM, capillary fragmentation coincided with myofiber degeneration while arteriolar and venular networks remained intact; neutrophil depletion before injury did not prevent capillary damage. Perfused capillary networks reformed by 5 dpi in association with more terminal arterioles and were dilated through 10 dpi; with no change in microvascular area or branch point number in regenerating networks, fewer capillaries aligned with myofibers and capillary networks were no longer organized into microvascular units. By 21 dpi, capillary orientation and organization had nearly recovered to that in uninjured GM. We conclude that following their disruption secondary to myofiber damage, capillaries regenerate as disorganized networks that remodel while regenerated myofibers mature.

Purinergic and nitrergic neuromuscular transmission mediates spontaneous neuronal activity in the rat colon

Nitric oxide (NO) and ATP mediate smooth muscle relaxation in the gastrointestinal tract. However, the involvement of these neurotransmitters in spontaneous neuronal activity is unknown. The aim of the present work was to study spontaneous neuromuscular transmission in the rat midcolon. Microelectrode experiments were performed under constant stretch both in circular and longitudinal directions. Spontaneous inhibitory junction potentials (sIJP) were recorded. Tetrodotoxin (1 μM) and apamin (1 μM) depolarized smooth muscle cells and inhibited sIJP. Nω-nitro-l-arginine (l-NNA, 1 mM) depolarized smooth muscle cells but did not modify sIJP. In contrast, the P2Y1 antagonist MRS-2500 (1 μM) did not modify the resting membrane potential (RMP) but reduced sIJP (IC50 = 3.1 nM). Hexamethonium (200 μM), NF-023 (10 μM), and ondansetron (1 μM) did not modify RMP and sIJP. These results correlate with in vitro (muscle bath) and in vivo (strain gauges) data where l-NNA but not MRS-2500 induced a sustained increase of spontaneous motility. We concluded that, in the rat colon, inhibitory neurons regulate smooth muscle RMP and cause sIJP. In vitro, the release of inhibitory neurotransmitters is independent of nicotinic, P2X, and 5-hydroxytryptamine type 3 receptors. Neuronal NO causes a sustained smooth muscle hyperpolarization that is responsible for a constant inhibition of spontaneous motility. In contrast, ATP acting on P2Y1 receptors is responsible for sIJP but does not mediate inhibitory neural tone. ATP and NO have complementary physiological functions in the regulation of gastrointestinal motility.

Ca2+- and Myosin Phosphorylation–independent Relaxation by Halothane in K+-depolarized Rat Mesenteric Arteries

Background Volatile anesthetics inhibit vascular smooth muscle contraction, but the mechanisms responsible are uncertain. In this study, the effects of halothane on Ca2+ signaling and Ca2+ activation of contractile proteins were examined in high K+-depolarized smooth muscle from rat mesenteric resistance arteries. Methods Vessels were cannulated and held at a constant transmural pressure (40 mmHg). Image analysis and microfluorimetry were used to simultaneously measure vessel diameter and smooth muscle intracellular [Ca2+] concentration ([Ca2+]i). Myosin light chain (MLC) phosphorylation was measured using the Western blotting technique. Results Step increases in extracellular [Ca2+] concentration (0-10 mM) in high K+ (40 mM)-depolarized smooth muscle produced incremental increases in [Ca2+]i, MLC phosphorylation, and contraction. Halothane (0.5-4.5%) inhibited contraction in a concentration-dependent manner, but the decrease in [Ca2+]i was small, and there was a marked shift in the [Ca2+]i-contraction relationship to the right, indicating an important Ca2+ desensitizing effect. Halothane (0.5-4.5%) did not affect MLC phosphorylation or the [Ca2+]-MLC phosphorylation relationship, but the MLC phosphorylation-contraction relationship was also shifted rightward, indicating an "MLC phosphorylation" desensitizing effect. In contrast, control relaxations produced by the Ca2+ channel blocker nifedipine were accompanied by decreases in both [Ca2+]i and MLC phosphorylation, and nifedipine had no affect on the [Ca2+]i-contraction, [Ca2+]i-MLC phosphorylation, and MLC phosphorylation-contraction relationships. Conclusions In high K+-depolarized vascular smooth muscle, halothane relaxation is largely mediated by a Ca2+ and MLC phosphorylation desensitizing effect. These results suggest that the relaxing action of halothane is independent of the classic Ca2+-induced myosin phosphorylation contraction mechanism.

Chronic hypoxia-induced spontaneous and rhythmic contractions in the rat main pulmonary artery

The effect of chronic hypoxia (CH; 1–4 wk) on the electromechanical properties of the rat main pulmonary artery (MPA) was investigated. MPA rings obtained from rats exposed for 14 days to hypobaric (50.5 kPa) CH exhibited spontaneous and rhythmic contractions (SRCs) that were never observed in control (normoxic) rats. SRCs were unaffected by tetrodotoxin, phentolamine, BQ-123 and BQ-788, N-nitro-l-arginine methyl ester, or endothelium removal. CH depolarized smooth muscle cells from −58.8 ± 9 to −38.6 ± 5.4 mV and increased the resting cytosolic Ca2+ concentration from 67.3 ± 11.9 to 112.5 ± 16.4 nM. CH also induced spontaneous spikelike depolarizations. All of these effects were inhibited by external Ca2+ removal or nifedipine (1 μM). Moreover, depletion of intracellular Ca2+ stores with ryanodine (1–5 μM) or cyclopiazonic acid (3 μM) progressively attenuated SRCs. This study demonstrates that CH switches the MPA from a quiescent to a spontaneously active mechanical state. Finally, the fact that SRCs precede the development of right ventricle hypertrophy and disappear when this hypertrophy reaches a maximal value (after 3–4 wk of CH) suggests that SRCs may play a role in the adaptive process of the pulmonary circulation to CH.