Inhibition and excitation in non-propulsive mammalian smooth muscle

Mechanisms underlying relaxation in response to inhibitory NANC nerve stimulation and putative neurotransmitters of these nerves have been examined in the guinea-pig internal anal sphincter (IAS) and compared with those in the bovine retractor penis muscle (ERP) and guinea-pig taenia caeci. Two types of techniques were employed. One which measured the effects of nerve stimulation and drugs on electrical membrane properties where intracellular microelectrode and simultaneous mechanical recording techniques were used. Drugs, for example ATP or cromakalim were applied by perfusion in the Krebs' solution, microinjection into the bath, or by hydrostatic pressure ejection. A second method assessed the underlying biochemical changes accompanying relaxation by measuring alterations in second messenger systems, for example cyclic AMP and cyclic GMP using radiomimmunoassay techniques. Electrical events were clearly an important accompaniment to mechanical inhibition in the IAS. Field stimulation (single pulse and 5 pulses at 5, 10 and 20 Hz; 0.5ms; supramaximal voltage) produced large inhibitory junction potentials of up to 15mV in amplitude which accompanied relaxation of 80% of muscle tone. Indeed, hyperpolarising electrotonic current passed into the IAS produced relaxation. The neurotransmitter which is released by field stimulation of the inhibitory nerves is probably ATP since exogenous application of purine by hydrostatic pressure ejection (5.8x10-4M; 10-55ms) produced a dose-dependent hyperpolarisation. The membrane potential change was similar in size, rate of decline and duration to the ijp. Neither hyperpolarisation nor relaxation could be achieved with the P2x-purinergic agonist, betaMeATP (10-5-10-3M) or the P2-purinergic agonist adenosine (10-3M) thus ATP was acting on the P2y-purinergic receptor. Inhibitory NANC neurotransmission was not peptidergic since VIP (10-7-10-5M), bradykinin (10-3M), neuropeptide Y (10-5M), bombesin (10-5M), leu-enkephalin (1.8x10-4M), met-enkephalin (1.8x10-5M), somatostatin (10-6-10-3M) and substance P (7.6x10-6 - 7.6x10-4M) each had no effect on the membrane potential of the IAS. There is also evidence that stimulation of -adrenoceptors by isoprenaline (10^-9 - 10^-5M) produced relaxation which was accompanied hyperpolarisation of the IAS. In all cases where hyperpolarisation and relaxation are associated in the IAS, the mechanism underlying the electrical change appeared to be an increase in K^+ conductance. Apamin (4.5 x 10^-6M) which blocks certain Ca^2+-mediated K^+ channels, antagonised the electrical and mechanical responses produced by field stimulation and ATP. Similarly, TEA (8x10^-2M), which blocks most K^+ channels, antagonised the hyperpolarisations and relaxations produced by field stimulation, ATP and isoprenaline. Indeed, the K^+ channel activator cromakalim (10^-9-10^-5M) produced hyperpolarisation and relaxation of the IAS suggesting that an increase in K^+ conductance is important in the mediation of mechanical inhibition of the IAS. Relaxation of the IAS was also produced without a significant change in membrane potential by altering the levels of cyclic nucleotides within the smooth muscle cells of the IAS. Forskolin (10^-9-10^-5M), which activates adenylate cyclase with a subsequent increase in cyclic AMP, relaxed the IAS. Similarly, sodium nitroprusside (10^-9-10^-4M) - a cyclic GMP phosphodiesterase inhibitor, and 8-bromo-cyclic GMP (10^-4M) each increased cyclic GMP and produced relaxation of the IAS. Direct measurement of cyclic nucleotide levels of the IAS showed that field stimulation (80 pulses at 8H_Z; 0.5ms; supramaximal voltage) and ATP (10^-4) elevated the cyclic AMP and cyclic GMP contents of the IAS. All other stimuli which produced slow, prolonged electrical and mechanical changes increased the level of only one cyclic nucleotide. Isoprenaline (10^-4M), cromakalim (10^-5M) and forskolin (10^-5) increased cyclic AMP content while sodium nitroprusside (10^-5M) increased the cyclic GMP content. Further investigation of other second messenger systems involved in relaxation of the IAS showed that increase in inositol phosphate turnover was not associated with stimulation of inhibitory P_2y-purinoceptors by ATP (10^-2M) in the IAS. However, an increase in inositol phosphate accumulation was produced by noradrenaline (10^-4M) and associated with contraction. A method was devised to measure the intraluminal pressure changes of the internal anal spincter in the anaesthetised guinea-pig using a Millar pressure transducer. Using this method the in vitro results were largely confirmed by this in vivo study. Basal intraluminal sphincter pressure was increased by noradrenaline acting on -adrenoceptors and decreased by isoprenaline acting on -adrenoceptors, ATP on P_2y-purinoceptors and 2-chloroadenosine on P_1-purinoceptors

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112±3 and 110±3 nMvs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation

Recent advances in vasoactive intestinal peptide physiology and pathophysiology: focus on the gastrointestinal system.

Vasoactive intestinal peptide (VIP), a gut peptide hormone originally reported as a vasodilator in 1970, has multiple physiological and pathological effects on development, growth, and the control of neuronal, epithelial, and endocrine cell functions that in turn regulate ion secretion, nutrient absorption, gut motility, glycemic control, carcinogenesis, immune responses, and circadian rhythms. Genetic ablation of this peptide and its receptors in mice also provides new insights into the contribution of VIP towards physiological signaling and the pathogenesis of related diseases. Here, we discuss the impact of VIP on gastrointestinal function and diseases based on recent findings, also providing insight into its possible therapeutic application to diabetes, autoimmune diseases and cancer

Role of endothelin-1 in the gastrointestinal tract of horses in health and disease

Gastrointestinal tract disease is the leading natural cause of death in horses and horses with ischemic intestinal disease have the greatest mortality. We hypothesized there is basal synthesis of endothelin-1 (ET-1) in the intestinal tract of healthy horses that is likely involved in regulating vasomotor tone, secretion and motility and that ET-1 synthesis increases with gastrointestinal tract disease, which may be involved in the pathophysiology of these disorders. Plasma ET-like immunoreactivity was increased in horses with naturally-acquired gastrointestinal disease, compared with normal horses; values were greatest in horses with large intestinal strangulation obstruction, enterocolitis and peritonitis. There was an association between ET-1 levels and survival, PCV and duration of signs of pain. Immunohistochemical staining for ET-1 was present in surface epithelium, villi, muscularis and serosa of numerous intestinal segments in healthy horses. Staining was also present in submucosal vessels with veins staining more intense than arteries. Staining appeared more diffuse and intense in samples from horses with intestinal strangulation obstruction. Polymerase chain reaction analysis revealed the presence of ET-1 gene expression in numerous intestinal segments of normal horses. These findings suggest ET-1 is involved in physiologic functions such as regulation of secretion, vasomotor tone and motility, and that increased ET-1 with strangulation obstruction may be involved in the pathophysiology of these disorders. ET-1 caused sustained, concentration-dependent increases in cecal longitudinal smooth muscle tone in vitro, but the magnitude of contraction was less than that induced by carbachol. Pre-incubation of tissues with ETA (BQ-123) and ETB (IRL-1038) receptor antagonists alone did not inhibit ET-1 induced contraction. However, contractile responses were inhibited when tissues were incubated with both antagonists (10-5 M) together, suggesting both ETA and ETB receptors mediate the contraction. Electric field stimulation did not change the contractile response. These studies indicate a physiologic role of ET-1 in the equine gastrointestinal tract and that increased synthesis and release occurs with gastrointestinal tract disease, especially ischemic conditions, and may contribute to the pathophysiology of these disorders. Further studies involving ET-1 and ET antagonists appear warranted

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+ level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112+/-3 and 110+/-3 nM vs. 150+/-7 and 135+/-5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454+/-41 muM/s vs. 144+/-24 muM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610+/-60 vs. 377+/-64 muM/s) and the amount of calcium released (843+/-75 muM vs. 576+/-80 muM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation