The effects of ATP on the contractions of rat and mouse fast skeletal muscle

Introduction: The aim of this study was to compare the effects of adenosine-5'-triphosphate (ATP) and adenosine on the contractility of rodent extensor digitorum longus (EDL) muscle at normal and low temperatures. Methods: Contractions of rat and mouse isolated EDL were induced by either electrical stimulation (ES) or exogenous carbachol and recorded in the presence of ATP or adenosine (both at 100 μM). Results: ATP at all temperatures caused a decrease of the contractions induced by carbachol in rat and mouse EDL and ES-induced contractions in rat EDL, while it potentiated the ES-induced contractions of mouse EDL. Adenosine reduced the contractility of rat and mouse EDL evoked by ES and did not affect the carbachol-induced contractions of rat and mouse EDL at any temperature. Discussion: Under various temperature conditions, ATP inhibits pre- but potentiates postsynaptic processes in the mouse EDL; in the rat EDL ATP causes only inhibition of neuromuscular conduction. Muscle Nerve 59:509–516, 2019     

Hypothermia at 35 °C Reduces the Time-Dependent Microglial Production of Pro-inflammatory and Anti-inflammatory Factors that Mediate Neuronal Cell Death

Background

Therapeutic hypothermia protects neurons after severe brain damage. This effect has been mainly achieved at the core temperatures of 32–34 °C; however, the optimum temperature of therapeutic hypothermia is not fully defined. Here we studied whether hypothermic culture at 35 °C had the same effects on the decrease of time-dependent expression of tumor necrosis factor (TNF)-α, interleukin (IL)-10, and nitric oxide (NO) by stimuli-activated microglia as that at 33 °C, as determined in our previous reports, and whether these factors directly induced neuronal cell death.

Methods

We determined the levels of cytokines and NO produced by microglia cultured with adenosine triphosphate (ATP), a toll-like receptor (TLR)2 agonist (N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3-lysine, Pam₃CSK₄), or a TLR4 agonist (lipopolysaccharide) under mild hypothermic (33 °C), minimal hypothermic (35 °C), and normothermic (37 °C) conditions. We also determined the viability of rat neuronal pheochromocytoma PC12 cells treated with recombinant TNF-α or IL-10 or (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3, an NO donor).

Results

Production of TNF-α, as well as that of IL-10 and NO were decreased by minimal hypothermia at 1.5–6, and 24–48 h, respectively, compared with normothermia, although some effects were diminished as compared with those by mild hypothermia. Exposure to TNF-α, IL-10, and NOR3 caused the death of PC12 cells in a concentration-dependent manner after 24 h.

Conclusion

Hypothermic culture at 35 °C decreased the production of early-phase TNF-α and late-phase IL-10 and NO from ATP- and TLR-activated microglia as observed at 33 °C, albeit with diminished effects. Moreover, these factors caused the death of neuronal cells in a concentration-dependent manner. These results suggest that the attenuation of microglial production of TNF-α, IL-10, and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death. Minimal hypothermia at 35 °C may be sufficient to elicit neuroprotective effect.     

Temperature dependency of human muscle velocity recovery cycles

Introduction: Velocity recovery cycles (VRCs) of human muscle action potentials have been proposed as a new technique for studying muscle membrane function. This study was undertaken to determine the temperature dependency of VRC parameters. Methods: VRCs with 1 conditioning stimulus were recorded repeatedly from brachioradialis muscle during cooling, and intramuscular temperature was recorded. VRCs in 6 normal volunteers were measured at 1°C intervals from 34°C to 28°C. The effects of temperature on relative refractory period and early and late supernormality were quantified. Results: Muscle VRC curves shift progressively upward and to the right with cooler temperatures. The most sensitive parameter to temperature changes was relative refractory period, whereas early supernormality was rather insensitive. Late supernormality was unaffected by temperature changes. Conclusions: Knowledge of temperature‐induced changes of muscle VRC parameters is essential for future studies. Thus, the results of this study provide a firm basis for clinical studies with this technique. Muscle Nerve 46: 264–266, 2012     

Intestinal muscle contractility during acute pancreatitis

Abstract Experiments were designed to study the effects of acute pancreatitis, induced by bile duct ligation, on the contractility of longitudinal muscle from the jejunum of rats. Forty animals were divided randomly into 5 groups, 8 in each. In three groups, the common bile duct was ligated at its entrance into the duodenum (low ligation), and their jejunal longitudinal muscle contractility was studied at 24, 48 or 72 hours after operation. In the fourth group, the common bile duct was ligated above the pancreas (high ligation). These animals were studied 72 hours after operation. In the fifth group, sham operation was performed as a control. At the time of study, two strips of longitudinal muscle were peeled from a segment of the jejunum from each animal, and were mounted in organ baths. Muscle contractions in response to carbachol (10^-7-10^-4m) and to KCL (30 mM) were measured, correlated to the cross-sectional area of the muscle strips, and expressed as stress. Animals with low ligation developed histological evidence of acute pancreatitis. Maximal stress to carbachol of intestinal muscle from these animals decreased progressively with time after operation. Forty-eight and 72 hours following low ligation, Maximal stresses in response to carbachol were 488 ± 33 g cm^-2 and 438 ± 28 g cm^-2, values significantly lower than those after sham operation (611 ± 24 g cm^-2). In contrast, median effective concentrations of carbachol (D-50s) were not significantly different among groups. The response to KCl, although lower than that to carbachol in each group, did not differ among groups. Animals with high ligation, although showing signs of bile stasis, did not develop histological evidence of pancreatitis, and maximal stresses to either carbachol or KCl developed by muscle from these animals did not differ significantly from control. We conclude that low ligation with the induction of pancreatitis leads to a decreased contractility of the jejunum. Also, the impairment responsible for the decreased contractility may reside in an excitation-contraction pathway step initiated by carbachol but not by KCl.     

Temperature-Related Effects of Adenosine Triphosphate-Activated Microglia on Pro-Inflammatory Factors

Background

Therapeutic hypothermia protects neurons after severe brain injury. Activated microglia produce several neurotoxic factors, such as pro-inflammatory cytokines and nitric oxide (NO), during neuron destruction. Hence, suppression of microglial release of these factors is thought to contribute partly to the neuroprotective effects of hypothermia. After brain insults, adenosine triphosphate (ATP) is released from injured cells and activates microglia. Here, we examined the acute effects of temperature on ATP-activated microglial production of inflammatory factors, and the possible involvement of p38 mitogen-activated protein kinase (p38) underlying such effects.

Methods

Microglia were cultured with ATP at 33, 37, and 39°C, or with ATP in the presence of a p38 inhibitor, SB203580, at 37°C. Cytokine and NO levels, and p38 activation were measured.

Results

Compared to 37°C, TNF-α was reduced at 33°C and augmented at 39°C for 1.5 h. IL-6 was reduced at 33°C for 6 h. NO was reduced at 33°C, but augmented at 39°C for 6 h. p38 was reduced at 33°C for 1 min. SB203580 inhibited ATP-induced TNF-α, IL-6, and NO production.

Conclusion

Lowering temperature rapidly reduced p38 activation and the subsequent p38-regulated production of pro-inflammatory cytokines and NO in ATP-activated microglia, suggesting that attenuation of early phase inflammatory responses via suppression of p38 in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Temperature elevation increased TNF-α and NO production in these cells. These temperature-dependent changes imply that monitoring of TNF-α and NO in the cerebrospinal fluid during the early phase might be useful as biomarkers for responses to therapeutic hypothermia and hyperthermia.

     

Lack of caspase‐3 attenuates immobilization‐induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

Introduction: Immobilization by casting induces disuse muscle atrophy (DMA). Methods: Using wild type (WT) and caspase‐3 knockout (KO) mice, we evaluated the effect of caspase‐3 on muscle mass, apoptosis, and inflammation during DMA. Results: Caspase‐3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P < 0.05)] and gastrocnemius twitch tension decrease (23 ± 4% in KO vs. 36 ± 3% in WT, P < 0.05) at day 14 in immobilized vs. contralateral hindlimb. Lack of caspase‐3 decreased immobilization‐induced increased apoptotic myonuclei (3.2‐fold) and macrophage infiltration (2.2‐fold) in soleus muscle and attenuated increased monocyte chemoattractant protein‐1 mRNA expression (2‐fold in KO vs. 18‐fold in WT) in gastrocnemius. Conclusions: Caspase‐3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. Muscle Nerve 47: 711–721, 2013     

Influence of 5‐HT4 receptor activation on acetylcholine release in human large intestine with endometriosis

Background The 5‐HT₄ receptor agonist prucalopride enhances large intestinal contractility by facilitating acetylcholine release through activation of 5‐HT₄ receptors on cholinergic nerves and is effective in patients with constipation. Patients with intestinal endometriosis can present with constipation. We investigated in vitro whether large intestinal endometriotic infiltration influences contractility and facilitation of acetylcholine release by prucalopride. Methods Sigmoid colon or rectum circular muscle strips were obtained at the level of an endometriotic nodule with infiltration of the Auerbach plexus, and at a macroscopically healthy site at least 5 cm cranially from the nodule, in patients undergoing laparoscopic colorectal resection because of symptomatic bowel endometriosis. Responses to muscarinic receptor stimulation and to electrical field stimulation (EFS), and the facilitating effect of prucalopride on acetylcholine release were evaluated. Key results The EC50 and E_max of the contractile responses to the muscarinic receptor agonist carbachol did not differ between healthy and lesioned strips. EFS‐induced on‐contractions were not different between the healthy and lesioned strips, while the non‐nitrergic relaxant responses induced by EFS were decreased in the lesioned strips. The facilitating effect of prucalopride on acetylcholine release in healthy strips was similar to that reported before in macroscopically healthy colon tissue of patients with colon cancer; in lesioned strips, the effect of prucalopride was fully maintained in 6/8 patients and absent in two. Conclusions & inferences Large intestinal endometriosis does not lead to a systematic interference with the cholinergic facilitating effect of prucalopride.     

Effect of caffeine ingestion on torque and muscle activity during resistance exercise in men

Introduction: We examined the effect of caffeine ingestion on muscle torque production and muscle activity at different contraction speeds in trained men. Methods: 10 men (mean age ± SD = 22 ± 1.1 years) volunteered to participate. A double‐blind, randomized cross‐over design was used. Sixty minutes postingestion of caffeine (6 mg kg^?1) or placebo, participants completed 6 repetitions of isokientic knee extension at 3 angular velocities (30°s^?1, 150°s^?1, 300°s^?1) from which peak torque was determined. Electromyographic activity of the vastus medialis was also collected. Results: Repeated measures analysis of variance indicated that muscle torque production was significantly higher (P = 0.02) with caffeine compared with placebo. A significant (P = 0.02) substance by velocity interaction for muscle activity indicated significantly higher vastus medialis muscle activity in the presence of caffeine versus placebo, and this difference was amplified as angular velocity increased. Conclusions: Acute caffeine ingestion improves muscle performance and increases muscle activity during short‐duration maximal dynamic contractions. Muscle Nerve 50: 523–527, 2014     

In Vivo Study of Flow-Rate Accuracy of the MedStream Programmable Infusion System

Objective: Flow‐rate accuracy and precision are important parameters to optimizing the efficacy of programmable intrathecal (IT) infusion pump delivery systems. Current programmable IT pumps are accurate within ±14.5% of their programed infusion rate when assessed under ideal environmental conditions and specific flow‐rate settings in vitro. We assessed the flow‐rate accuracy of a novel programmable pump system across its entire flow‐rate range under typical conditions in sheep (in vivo) and nominal conditions in vitro. Materials and Methods: The flow‐rate accuracy of the MedStream Programmable Pump was assessed in both the in vivo and in vitro settings. In vivo flow‐rate accuracy was assessed in 16 sheep at various flow‐rates (producing 90 flow intervals) more than 90 ± 3 days. Pumps were then explanted, re‐sterilized and in vitro flow‐rate accuracy was assessed at 37°C and 1013 mBar (80 flow intervals). Results: In vivo (sheep body temperatures 38.1°C–39.8°C), mean ± SD flow‐rate error was 9.32% ± 9.27% and mean ± SD leak‐rate was 0.028 ± 0.08 mL/day. Following explantation, mean in vitro flow‐rate error and leak‐rate were −1.05% ± 2.55% and 0.003 ± 0.004 mL/day (37°C, 1013 mBar), respectively. Conclusions: The MedStream Programmable Pump demonstrated high flow‐rate accuracy when tested in vivo and in vitro at normal body temperature and environmental pressure as well as when tested in vivo at variable sheep body temperature. The flow‐rate accuracy of the MedStream Programmable Pump across its flow‐rate range, compares favorably to the accuracy of current clinically utilized programmable IT infusion pumps reported at specific flow‐rate settings and conditions.     

H‐reflexes reduce fatigue of evoked contractions after spinal cord injury

Introduction: Neuromuscular electrical stimulation (NMES) over a muscle belly (mNMES) generates contractions predominantly through M‐waves, while NMES over a nerve trunk (nNMES) can generate contractions through H‐reflexes in people who are neurologically intact. We tested whether the differences between mNMES and nNMES are present in people with chronic motor‐complete spinal cord injury and, if so, whether they influence contraction fatigue. Methods: Plantar‐flexion torque and soleus electromyography were recorded from 8 participants. Fatigue protocols were delivered using mNMES and nNMES on separate days. Results: nNMES generated contractions that fatigued less than mNMES. Torque decreased the least when nNMES generated contractions, at least partly through H‐reflexes (n = 4 participants; 39% decrease), and torque decreased the most when contractions were generated through M‐waves, regardless of NMES site (nNMES 71% decrease, n = 4; mNMES, 73% decrease, n = 8). Conclusions: nNMES generates contractions that fatigue less than mNMES, but only when H‐reflexes contribute to the evoked contractions. Muscle Nerve 50:224–234, 2014     

Inhibition of p38 MAPK improves intestinal disturbances and oxidative stress induced in a rabbit endotoxemia model

Background Lipopolysaccharide (LPS) decreases intestinal contractility and induces the release of reactive oxygen species, which play an important role in the pathogenesis of sepsis. p38 mitogen‐activated protein kinase (MAPK) can be activated by a variety of stimuli such as LPS. The aims of this study were: (i) to investigate the role of p38 MAPK in the effect of LPS on (a) the acetylcholine, prostaglandin E₂ and KCl‐induced contractions of rabbit duodenum and (b) the oxidative stress status; (ii) to localize the active form of p38 in the intestine. Methods Rabbits were injected with (i) saline, (ii) LPS, (iii) SB203580, a specific p38 MAPK inhibitor or (iv) SB203580 + LPS. Duodenal contractility was studied in an organ bath. SB203580 was also tested in vitro. The protein expression of p‐p38 and total p38 was measured by Western blot and p‐p38 was localized by inmunohistochemistry. The formation of products of oxidative damage to proteins (carbonyls) and lipids (MDA+4‐HDA) was quantified in intestine and plasma. Key Results ACh, PGE₂ and KCl‐induced contractions decreased with LPS. LPS increased phospho‐p38 expression and the levels of carbonyls and MDA+4‐HDA. SB203580 blocked the effect of LPS on the ACh, PGE₂ and KCl‐induced contractions in vivo and in vitro and the levels of carbonyls and MDA+4‐HDA. P‐p38 was detected in neurons of the myenteric plexus and smooth muscle cells of duodenum. Conclusions & Inferences Lipopolysaccharide decreases the duodenal contractility in rabbits and increases the production of free radicals. p38 MAPK is a mediator of these effects.     

Heat‐Stress effects on the myosin heavy chain phenotype of rat soleus fibers during the early stages of regeneration

Introduction: We investigated heat‐stress effects on the adult myosin heavy chain (MyHC) profile of soleus muscle fibers at an early stage of regeneration. Methods: Regenerating fibers in adult rats were analyzed 2, 4, or 6 days after bupivacaine injection. Rats were heat stressed by immersion in water (42 ± 1°C) for 30 minutes 24 hours after bupivacaine injection and every other day thereafter. Results: No adult MyHC isoforms were observed after 2 days, whereas some fibers expressed only fast MyHC after 4 days. Heat stress increased fast and slow MyHC in regenerating fibers after 6 days. Regenerating fibers expressing only slow MyHC were observed only in heat‐stressed muscles. Bupivacaine injection increased the number of Pax7⁺ and MyoD⁺ satellite cells in regenerating fibers, more so in heat‐stressed rats. Conclusion: The results indicate that heat stress accelerates fast‐to‐slow MyHC phenotype conversion in regenerating fibers via activation of satellite cells. Muscle Nerve 52 : 1047–1056, 2015     

High‐frequency gamma oscillations coexist with low‐frequency gamma oscillations in the rat visual cortex in vitro

Synchronization of neuronal activity in the visual cortex at low (30–70 Hz) and high gamma band frequencies (> 70 Hz) has been associated with distinct visual processes, but mechanisms underlying high‐frequency gamma oscillations remain unknown. In rat visual cortex slices, kainate and carbachol induce high‐frequency gamma oscillations (fast‐γ; peak frequency ～ 80 Hz at 37°C) that can coexist with low‐frequency gamma oscillations (slow‐γ; peak frequency ～ 50 Hz at 37°C) in the same column. Current‐source density analysis showed that fast‐γ was associated with rhythmic current sink‐source sequences in layer III and slow‐γ with rhythmic current sink‐source sequences in layer V. Fast‐γ and slow‐γ were not phase‐locked. Slow‐γ power fluctuations were unrelated to fast‐γ power fluctuations, but were modulated by the phase of theta (3–8 Hz) oscillations generated in the deep layers. Fast‐γ was spatially less coherent than slow‐γ. Fast‐γ and slow‐γ were dependent on γ‐aminobutyric acid (GABA)_A receptors, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors and gap‐junctions, their frequencies were reduced by thiopental and were weakly dependent on cycle amplitude. Fast‐γ and slow‐γ power were differentially modulated by thiopental and adenosine A₁ receptor blockade, and their frequencies were differentially modulated by N‐methyl‐d ‐aspartate (NMDA) receptors, GluK1 subunit‐containing receptors and persistent sodium currents. Our data indicate that fast‐γ and slow‐γ both depend on and are paced by recurrent inhibition, but have distinct pharmacological modulation profiles. The independent co‐existence of fast‐γ and slow‐γ allows parallel processing of distinct aspects of vision and visual perception. The visual cortex slice provides a novel in vitro model to study cortical high‐frequency gamma oscillations.     

Apigenin inhibits sciatic nerve denervation–induced muscle atrophy

Introduction: Apigenin (AP) has been reported to elicit anti‐inflammatory effects. In this study, we investigated the effect of AP on sciatic nerve denervation–induced muscle atrophy. Methods: Sciatic nerve–denervated mice were fed a 0.1% AP‐containing diet for 2 weeks. Muscle weight and cross‐sectional area (CSA), and the expression of atrophic genes and inflammatory cytokines in the gastrocnemius were analyzed. Results: Denervation significantly induced muscle atrophy. However, values for muscle weight and CSA were greater in the denervated muscle of the AP mice than the controls. AP suppressed the expression of MuRF1, but upregulated both myosin heavy chain (MHC) and MHC type IIb. AP also significantly suppressed expression of tumor necrosis‐alpha in the gastrocnemius and soleus muscles, and interleukin‐6 expression in the soleus muscle. Discussion: AP appears to inhibit denervation‐induced muscle atrophy, which may be due in part to its inhibitory effect on inflammatory processes within muscle. Muscle Nerve 58 : 314–318, 2018     

Prior heat stress effects fatigue recovery of the elbow flexor muscles

Introduction: Long‐lasting alterations in hormones, neurotransmitters, and stress proteins after hyperthermia may be responsible for the impairment in motor performance during muscle fatigue. Methods: Subjects (n = 25) performed a maximal intermittent fatigue task of elbow flexion after sitting in either 73° or 26°C to examine the effects of prior heat stress on fatigue mechanisms. Results: The heat stress increased the tympanic and rectal temperatures by 2.3° and 0.82°C, respectively, but there was full recovery prior to the fatigue task. Although prior heat stress had no effects on fatigue‐related changes in volitional torque, electromyographic (EMG) activity, torque relaxation rate, motor evoked potential (MEP) size, and silent period (SP) duration, prior heat stress acutely increased the pre‐fatigue relaxation rate and chronically prevented long‐duration fatigue (P < 0.05). Conclusions: These findings indicate that prior passive heat stress alone does not alter voluntary activation during fatigue, but prior heat stress and exercise produce longer‐term protection against long‐duration fatigue. Muscle Nerve 44: 115–125, 2011     

Recovery of strength is dependent on mTORC1 signaling after eccentric muscle injury

Introduction: Eccentric contractions may cause immediate and long‐term reductions in muscle strength that can be recovered through increased protein synthesis rates. The purpose of this study was to determine whether the mechanistic target‐of‐rapamycin complex 1 (mTORC1), a vital controller of protein synthesis rates, is required for return of muscle strength after injury. Methods: Isometric muscle strength was assessed before, immediately after, and then 3, 7, and 14 days after a single bout of 150 eccentric contractions in mice that received daily injections of saline or rapamycin. Results: The bout of eccentric contractions increased the phosphorylation of mTORC1 (1.8‐fold) and p70s6k1 (13.8‐fold), mTORC1's downstream effector, 3 days post‐injury. Rapamycin blocked mTORC1 and p70s6k1 phosphorylation and attenuated recovery of muscle strength (～20%) at 7 and 14 days. Conclusion: mTORC1 signaling is instrumental in the return of muscle strength after a single bout of eccentric contractions in mice. Muscle Nerve 54 : 914–924, 2016     

The Effect of Fatigue on the Timing of Electrical Stimulation‐Evoked Muscle Contractions in People with Spinal Cord Injury

This study investigated the activation dynamics of electrical stimulation‐evoked muscle contractions performed by individuals with spinal cord injury (SCI). The purpose was to determine whether electrical stimulation (ES) firing patterns during cycling exercise should be altered in response to fatigue‐induced changes in the time taken for force to rise and fall with ES. Seven individuals with SCI performed isometric contractions and pedaled a motorized cycle ergometer with stimulation applied to the quadriceps muscles. Both exercise conditions were performed for five minutes while the patterns of torque production were recorded. ES‐evoked knee extension torque fell by 75% under isometric conditions, and the rate of force rise and decline decreased in proportion to torque (r = 0.91, r = 0.94, respectively). There was no change in the time for torque to rise to 50% of maximum levels. The time for torque to decline did increase slightly, but only during the first minute of exercise. Cycling power output fell approximately 50% during the five minutes of exercise, however, there was no change in the time taken for torque to rise or fall. The magnitude of ES‐evoked muscle torques decline substantially with fatigue, however, the overall pattern of torque production remained relatively unchanged. These results suggest there is no need to alter stimulation firing patterns to accommodate fatigue during ES‐evoked exercise.     

Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca(2+) sensitization in intestinal smooth muscle

Background Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI‐17 and MYPT1, primarily based on studies with tonic‐type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic‐type intestinal smooth muscle. Methods Phosphorylation levels, tension, and Ca²⁺ sensitization was detected in rat ileal smooth muscle. Key Results In rat ileal smooth muscle, phosphorylation level of CPI‐17 at Thr³⁸ and MYPT1 at Thr⁸⁵³, but not MYPT1 at Thr⁶⁹⁶, were increased with carbachol (1 μmol L^?1) accompanied with muscle contraction. The PKC inhibitor Go6976 (1 μmol L^?1) inhibited the carbachol‐induced phosphorylation of CPI‐17, whereas the Rho‐associated kinase (ROCK) inhibitor, Y‐27632 (10 μmol L^?1) inhibited the carbachol‐induced phosphorylation of both CPI‐17 and MYPT1. Application of Go6976 or Y‐27632 alone inhibited the carbachol‐induced contraction; however, the combined application of these inhibitors did not inhibit the contraction in an additive manner. In β‐escin‐permeabilized ileal strip, treatment with antiphosphorylated antibodies for CPI‐17 at Thr³⁸ and MYPT1 at Thr⁸⁵³ and Thr⁶⁹⁶ alone almost completely abolished the Ca²⁺ sensitization due to carbachol with GTP. Conclusions & Inferences In conclusion, receptor stimulation increases the Ca²⁺ sensitivity of contractile elements through CPI‐17 phosphorylation via the PKC/ROCK pathways and MYPT1 phosphorylation via the ROCK pathway, when these mechanisms operate cooperatively and/or synchronously in intestinal smooth muscle.