Effects of ATP and adenosine on contraction amplitude of rat soleus muscle at different temperatures

Introduction: The aim of this study was to evaluate the effects of adenosine 5′‐triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. Methods: Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30–14°C) and recorded in the presence of ATP, adenosine, suramin, and 8‐(p‐sulfophenyl)‐theophylline (8‐SPT). Results: At 37°C, incubation of the muscles with ATP inhibited ES‐induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES‐induced contractions at all temperature levels; 8‐SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol‐induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. Conclusions: ATP is involved in both pre‐ and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55 : 417–423, 2017     

Effects of sympathetic histamine on vasomotor responses of blood vessels in rabbit ear to electrical stimulation

Objective

To investigate the effects of histamine receptor antagonists on vasoconstriction induced by electrical stimulation (ES) on posterior auricular nerve, and to explore the pre- and post-synaptic effects of sympathetic histamine on the vasomotor responses of vascular smooth muscle in rabbit ear.

Methods

ES was applied to posterior auricular nerves of the whole rabbit ear at 10 Hz, 20 Hz and 40 Hz, respectively. Besides, the whole ear was perfused with different histamine receptor antagonists under constant perfusion pressure, and the changes in the flow rate of perfusate were observed.

Results

The flow rate of venous outflow was decreased by ES at all the 3 frequencies. The ES-induced vasoconstriction at 20 Hz and 40 Hz could be partly inhibited by H₁ receptor antagonist chlorpheniramine (P < 0.05). After exhaustion of histamine in mast cells by pretreatment with specific mast cell degranulator compound 48/80, chlorpheniramine could still inhibit the ES-induced flow rate reduction. In contrast, H₂ receptor antagonist cimetidine could enhance the 40-Hz ES-induced flow rate reduction (P < 0.05). Moreover, ES-induced vasoconstriction at the 3 frequencies could all be enhanced by H₃ receptor antagonist thioperamide (P < 0.05).

Conclusion

Stimulation on the auricular nerve may evoke histamine release from sympathetic nerves rather than from mast cells. Moreover, the functions of sympathetic histamine vary from pre-synaptic modulation to post-synaptic vasoconstriction or vasodilatation, via activation of different histamine receptors.     

Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 μM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to α,β-methylene ATP (α,β-mATP) (10 μM) from 97.0% to 43.4% (p < 0.05). Treatment with the cholinesterase inhibitor eserine (10 μM) also significantly decreased the NANC response to 21.1% (p < 0.0001). The purinergic receptor antagonist suramin (100 μM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with α,β-mATP) decreased the NANC contractions and the direct contractile response to α,β-mATP. When the electrically evoked contractions were facilitated by the L-type Ca²⁺ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.     

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.     

Functional study on TRPV1-mediated signalling in the mouse small intestine: involvement of tachykinin receptors

Abstract Afferent nerves in the gut not only signal to the central nervous system but also provide a local efferent‐like effect. This effect can modulate intestinal motility and secretion and is postulated to involve the transient receptor potential of the vanilloid type 1 (TRPV1). By using selective TRPV1 agonist and antagonists, we studied the efferent‐like effect of afferent nerves in the isolated mouse jejunum. Mouse jejunal muscle strips were mounted in organ baths for isometric tension recordings. Jejunal strips contracted to the TRPV1 agonist capsaicin. Contractions to capsaicin showed rapid tachyphylaxis and were insensitive to tetrodotoxin, hexamethonium, atropine or l ‐nitroarginine. Capsaicin did not affect contractions to electrical stimulation of enteric motor nerves and carbachol. Tachykinin NK₁, NK₂ and NK₃ receptor blockade by RP67580, nepadutant plus SR‐142801 reduced contractions to capsaicin to a similar degree as contractions to substance P. The effect of the TRPV1 antagonists capsazepine, SB‐366791, iodo‐resiniferatoxin (iodo‐RTX) and N‐(4‐tertiarybutylphenyl)‐4‐(3‐cholorphyridin‐2‐yl)tetrahydropyrazine‐1(2H)‐carbox‐amide (BCTC) was studied. Capsazepine inhibited contractions not only to capsaicin but also those to carbachol. SB‐366791 reduced contractions both to capsaicin and carbachol. Iodo‐RTX partially inhibited the contractions to capsaicin without affecting contractions to carbachol. BCTC concentration‐dependently inhibited and at the highest concentration used, abolished the contractions to capsaicin without affecting those to carbachol. From these results, we conclude that activation of TRPV1 in the mouse intestine induces a contraction that is mediated by tachykinins most likely released from afferent nerves. The TRPV1‐mediated contraction does not involve activation of intrinsic enteric motor nerves. Of the TRPV1 antagonists tested, BCTC combined strong TRPV1 antagonism with TRPV1 selectivity.     

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.     

Extracellular ATP induces retinal photoreceptor apoptosis through activation of purinoceptors in rodents

We have previously demonstrated that photoreceptors express P2X₇ purinoceptors. These excitatory receptors are activated by extracellular adenosine 5′‐triphosphate (ATP) and have been implicated in neurodegeneration in other parts of the central nervous system (CNS). In this study we examined whether extracellular ATP could contribute to photoreceptor degeneration in rodents through excessive activation of P2 purinoceptors. Intravitreal injection of high concentrations of extracellular ATP into normal rat eyes induced extensive and selective apoptosis of photoreceptors within 18 hours of injection. Five days after injection the outer nuclear layer was severely degenerated and electroretinographic responses were impaired. Preinjection of the purinergic antagonist pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) protected against ATP‐mediated apoptosis. The initial phase of ATP‐induced photoreceptor death did not temporally coincide with retinal pigment epithelium degeneration or microglial activation, suggesting that cell death was due to direct activation of purinergic receptors on photoreceptors. Finally, we demonstrate that intravitreal injection of PPADS results in a 30% increase in photoreceptor survival in the rd1 mouse, a model of human recessive retinitis pigmentosa (RP). These findings highlight the importance of extracellular ATP in retinal neurodegeneration and provide a potential new avenue for therapeutic intervention in RP. J. Comp. Neurol. 513:430–440, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanosensitive release of adenosine 5′‐triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: A mechanism for purinergic involvement in chronic strain

As adenosine 5′‐triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1–3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1^?/? mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling‐induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg‐MYOC^Y437H mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma. GLIA 2014;62:1486–1501     

Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 microM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to alpha,beta-methylene ATP (alpha,beta-mATP) (10 microM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 microM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100 microM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with alpha,beta-mATP) decreased the NANC contractions and the direct contractile response to alpha,beta-mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.     

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.     

An octaguanidine-morpholino oligo conjugate improves muscle function of mdx mice

Introduction: Skeletal muscles of mdx mice lack functional levels of dystrophin due to a mutation in Dmd exon 23. Morpholino antisense oligomers can induce expression of a truncated dystrophin by redirecting splicing to skip processing of exon 23. Methods: We tested whether systemic administration of Vivo‐Morpholino, an octaguanidine delivery moiety–Morpholino conjugate that targets exon 23 (VMO23), restored function to muscles of mdx mice. Results: Extensor digitorum longus (EDL) muscles of mdx mice were weaker, less powerful, and showed greater functional deficits after eccentric contractions than normal. VMO23 treatment normalized EDL force and power of mdx mice and eliminated their exaggerated sensitivity to eccentric contractions. Diaphragm muscle strips from mdx mice also produced lower‐than‐normal force and power, and these variables were restored to normal, or near‐normal, levels by VMO23 treatment. Conclusion: These results provide a functional basis for continuing development of VMO23 as a treatment for Duchenne muscular dystrophy. Muscle Nerve, 2011     

Adenosinergic modulation of caffeine-induced c-fos mRNA expression in mouse brain

The adenosine receptor antagonist, caffeine, transiently induced proto-oncogene c-fos mRNA in mouse brain in a dose-dependent fashion. In situ hybridization revealed that caffeine-induced c-fos expression was high in caudate-putamen and olfactory tubercle at both subconvulsive and convulsive doses. The pattern of c-fos mRNA distribution following caffeine administration differs from that reported after seizures induced by electroconvulsive shock (ECS) or other chemical convulsants, and closely parallels the distribution of adenosine A₂ receptors. Furthermore, the potent adenosine A₂ receptor agonist,5′-N-ethylcarboxamide adenosine (NECA) blocked caffeine-induced c-fos expression whereas the adenosine A₁ receptor ligand, N⁶-cyclohexyladenosine (CHA), had no effect. This study suggests that the caffeine-induced expression of c-fos mRNA may be mediated by the adenosine A₂ receptor in mouse brain.     

Investigating the Effects of Peripheral Electrical Stimulation on Corticomuscular Functional Connectivity Stroke Survivors

Background: Electrical stimulation (ES) in the periphery can induce brain plasticity and has been used clinically to promote motor recovery in patients with central nervous system lesion. Electroencephalogram (EEG) and electromyogram (EMG) are readily applicable in clinical settings and can detect real-time functional connectivity between motor cortex and muscles with EEG–EMG (corticomuscular) coherence.

Objective: The purpose of this study was to determine whether EEG–EMG coherence can detect changes in corticomuscular control induced by peripheral ES.

Methods: Fifteen healthy young adults and 15 stroke survivors received 40-min electrical stimulation session on median nerve. The stimulation (1-ms rectangular pulse, 100 Hz) was delivered with a 20-s on–20-s off cycle, and the intensity was set at the subjects’ highest tolerable level without muscle contraction or pain. Both before and after the stimulation session, subjects performed a 20-s steady-hold thumb flexion at 50% maximal voluntary contraction (MVC) while EEG and EMG were collected.

Results: Our results demonstrated that after ES, EEG–EMG coherence in gamma band increased significantly for 22.1 and 48.6% in healthy adults and stroke survivors, respectively. In addition, after ES, force steadiness was also improved in both groups, as indicated by the decrease in force fluctuation during steady-hold contraction (?1.7% MVC and ?3.9%MVC for healthy and stroke individuals, respectively).

Conclusions: Our results demonstrated that EEG–EMG coherence can detect ES-induced changes in the neuromuscular system. Also, because gamma coherence is linked to afferent inputs encoding, improvement in motor performance is likely related to ES-elicited strong sensory input and enhanced sensorimotor integration.     

Purine modulation of cholinomimetic responses in the rat hippocampal slice

Iontophoretic application of carbachol caused excitation of CA1 neurones and decreased the amplitude of antidromic CA1 population spikes recorded extracellularly. Adenosine, adenosine triphosphate (ATP) and the purine analogues N-ethylcarboxamide adenosine (NECA) and R- and S-phenylisopropyladenosine (PIA) reduced the effects of carbachol on single cell firing and on the population spike. Responses to excitatory amino acids were unaffected by adenosine except for a small depression of kainate and N-methyl-D-aspartate responses at high concentrations. The rank order of potency for the purine reduction of carbachol responses was R-PIA = S-PIA = NECA much greater than adenosine greater than ATP. The actions of purines and purine analogues were antagonized by 8-phenyltheophylline (8-PT) and other xanthine antagonists. Application of 8-PT and other xanthines without prior exposure to purines frequently resulted in marked potentiation of carbachol responses. Thus in the hippocampus, responses to the cholinomimetic carbachol are markedly and selectively reduced by purines acting at the P1 purine receptor type and it appears that endogenous levels of adenosine limit the effects of cholinergic agents.     

Long‐lasting in vivo inotropic effects of the K+ channel blocker 3,4‐diaminopyridine during fatigue‐inducing stimulation

Blocking K⁺ channels with aminopyridines enhances muscle contractile performance in vitro, but the improvements are relatively short‐lasting during fatigue‐inducing stimulation. We hypothesized that in vivo inotropic actions persist over long periods of fatigue‐inducing stimulation. The effects of 3,4‐diaminopyridine (DAP) were evaluated for rat extensor digitorum longus (EDL) muscle. DAP increased twitch force by 105%. There was a significant leftward shift in the force–frequency relationship, with force values being increased at frequencies up to and including 20 HZ . During repetitive fatigue‐inducing 20‐HZ stimulation, DAP‐induced force increases were large and persisted significantly for at least 30 minutes. Thus, DAP substantially improves contractile performance of EDL muscle in vivo for much longer periods during fatigue‐inducing contractions than in vitro. These data provide support for a potential role for aminopyridines as inotropic agents in applications such as functional electrical stimulation, in which low to medium stimulation frequencies are typically utilized. Muscle Nerve 38: 1616–1622, 2008     

Galanin modulates vagally induced contractions in the mouse oesophagus

Abstract Nitrergic myenteric neurons co‐innervating motor endplates were previously shown to inhibit vagally induced contractions of striated muscle in the rodent oesophagus. Immunohistochemical demonstration of putative co‐transmitters, e.g. galanin, in enteric neurons prompted us to study a possible role of galanin in modulating vagally mediated contractions in an in vitro vagus nerve‐oesophagus preparation of the mouse. Galanin (1–16) (1–100 nmol L^?1), in the presence of the peptidase inhibitor, phenanthroline monohydrate, inhibited vagally induced contractions in a concentration‐dependent manner (control: 100%; galanin 1 nmol L^?1: 95.6 ± 1.6%; galanin 10 nmol L^?1: 57.3 ± 6.5%; galanin 100 nmol L^?1: 31.2 ± 8.1%, n = 5). The non‐selective galanin receptor antagonist, galantide (100 nmol L^?1), blocked the inhibitory effect of galanin (10 nmol L^?1) while the selective non‐galanin receptor 1 and galanin receptor 3 antagonists, M871 (1 μmol L^?1) and SNAP37889 (100 nmol L^?1), respectively, and the nitric oxide synthase inhibitor, NG‐nitro‐l ‐arginine methyl ester (l ‐NAME) (200 μmol L^?1), failed to affect this galanin‐induced response. Simultaneous application of galantide (100 nmol L^?1) and l ‐NAME (200 μmol L^?1) significantly reduced the inhibitory effect of capsaicin (30 μmol L^?1) on vagally induced contractions when compared with its effect in the presence of l ‐NAME alone or in combination with the selective galanin receptor 2 or 3 antagonists. An inhibitory effect of piperine on vagally induced contractions was reduced neither by galantide nor by l ‐NAME. Immunohistochemistry revealed galanin immunoreactive myenteric neurons and nerve fibres intermingling with cholinergic vagal terminals at motor endplates. These data suggest that galanin from co‐innervating enteric neurons co‐operates with nitric oxide in modulating vagally induced contractions in the mouse oesophagus.     

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.     

Potassium and caffeine contractures of mouse muscles before and after fatiguing stimulation

To assess the impairment of muscle membrane excitation, excitation–contraction (E–C) coupling, and contractility during muscle fatigue, we monitored the contracture responses of resting and fatigued muscles on exposure to high potassium and caffeine. On exposure to 140 mmol/L potassium, mouse extensor digitorum longus (EDL) developed a contracture which was 15.7% of tetanic tension before fatigue and 31.7% after fatigue, while soleus developed 59.4% contracture before and 68.8% after fatigue. Potassium causes contractures by depolarizing the muscle fiber membrane. Hence, membrane excitation is reduced in fatigued EDL and soleus. On exposure to 32 mmol/L caffeine, the contracture was 7.1% in resting EDL, 8.5% in fatigued EDL, 50.1% in resting soleus, and 43.7% in fatigued soleus. On exposure to 1 mmol/L caffeine followed by rapid cooling, the contracture was 3.0% in resting EDL, 3.2% in fatigued EDL, 21.5% in resting soleus, and 10.3% in fatigued soleus. Caffeine causes contracture by releasing Ca^{+ +} from the sarcoplasmic reticulum. Our results indicate reduced E–C coupling attributable to reduced membrane excitation in fatigued EDL, and reduced contractility in fatigued soleus. © 1994 John Wiley & Sons, Inc.     

Muscle contractility dysfunction precedes loss of motor unit connectivity in SOD1(G93A) mice

Introduction: Electrophysiological measurements are used in longitudinal clinical studies to provide insight into the progression of amyotrophic lateral sclerosis (ALS) and the relationship between muscle weakness and motor unit (MU) degeneration. Here, we used a similar longitudinal approach in the Cu/Zn superoxide dismutase (SOD1[G93A]) mouse model of ALS. Methods: In vivo muscle contractility and MU connectivity assays were assessed longitudinally in SOD1(G93A) and wild type mice from postnatal days 35 to 119. Results: In SOD1(G93A) males, muscle contractility was reduced by day 35 and preceded MU loss. Muscle contractility and motor unit reduction were delayed in SOD1(G93A) females compared with males, but, just as with males, muscle contractility reduction preceded MU loss. Discussion: The longitudinal contractility and connectivity paradigm employed here provides additional insight into the SOD1(G93A) mouse model and suggests that loss of muscle contractility is an early finding that may precede loss of MUs and motor neuron death. Muscle Nerve 59 :254–262, 2019