Spontaneous Contractile Waves Research Articles

Alteration in propagating colonic contractions by dairy proteins in isolated rat large intestine

Gastrointestinal conditions in which the transit of contents is altered may benefit from nutritional approaches to influencing health outcomes. Milk proteins modulate the transit of contents along different regions, suggesting that they have varying effects on neuromuscular function to alter gastrointestinal motility. We tested the hypothesis that bovine whey and casein milk protein hydrolysates could have direct modulatory effects on colonic motility patterns in isolated rat large intestine. Casein protein hydrolysate (CPH), whey protein concentrate (WPC), whey protein hydrolysate (WPH), and a milk protein hydrolysate (MPH; a hydrolyzed blend of 60% whey to 40% casein) were compared for their effects on spontaneous contractile waves. These contractions propagate along the length of the isolated intact large intestine (22 cm) between the proximal colon and rectum and were detected by measuring activity at 4 locations. Milk proteins were perfused through the tissue bath, and differences in contraction amplitude and frequency were quantified relative to pretreatment controls. Propagation frequency was decreased by CPH, increased by MPH, and unaffected by intact whey proteins. The reduced motility with CPH and increased motility with MPH indicate a direct action of these milk proteins on colon tissue and provide evidence for differential modulation by hydrolysate type. These findings mirror actions on lower gastrointestinal transit reported in vivo, with the exception of WPH, suggesting that other factors are required.

Curcumin Alleviates the Functional Gastrointestinal Disorders of Mice In Vivo.

Curcumin is a natural polyphenol extracted from the turmeric rhizome, which has a wide range of biological activities, but until now the effects of curcumin on the gastrointestinal peristalsis have not been fully understood. In vivo study, we observed the effects of curcumin on gastric emptying and intestinal propulsion rates of mice in normal state and in delayed state by atropine (ATR) or nitric oxide precursor L-arginine (L-Arg). An in vitro study explored the direct effects of curcumin on the intestinal contractility, but were studied through measuring spontaneous contraction of isolated jejunum of mice. Our results showed that intragastric administration of curcumin (200 mg/kg/day) for 10-20 days significantly improved gastric emptying and intestinal propulsion rates of mice delayed by ATR. Moreover, intragastric administration of curcumin (200 mg/kg/day) for 15 days also significantly improved mice gastric emptying and intestinal propulsion rates delayed by L-Arg. There was no significant effect on normal gastrointestinal propulsion of mice after intragastric administration of curcumin (200 mg/kg/day) for 1-20 days. When normal isolated jejunum of mice were incubated with curcumin in vitro, the amplitude of the spontaneous contractile waves of jejunum was reduced in a concentration-dependent manner. Moreover, curcumin reduced the amplitude of the contractile waves of jejunum in both contracted and relaxed state induced by acetylcholine or ATR individually. Taken together, our results suggest that curcumin has quite different effects on gastrointestinal peristalsis in vivo and in vitro. Moderate dose of curcumin by intragastric administration for more than 10 days can alleviate the functional gastrointestinal disorders of mice, but cannot affect normal gastrointestinal propulsion.

Inhibitory Effects of Botulinum Toxin Type A on Pyloric Cholinergic Muscle Contractility of Rat.

Botulinum toxin type A (BTX-A) selectively cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) and results in inhibition of the fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane to undergo exocytosis and release. The aim of this study was to investigate whether BTX-A inhibited the pyloric smooth muscle contractility induced by acetylcholine (ACh) after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin (TSN). Three groups of rat pyloric muscle strips were studied in vitro. All strips were allowed to equilibrate for 52 min under a basal loading tension of 1 g in Krebs solution and spontaneous contractile waves were recorded as their own controls before adding each drug. According to experimental protocols, 100 μM ACh, 1 μM atropine, 29.6 μM TSN and 10 U/ml BTX-A was added, respectively. BTX-A directly inhibited pyloric spontaneous contraction and ACh-induced contractile response. Addition of 10 U/ml BTX-A still inhibited pyloric smooth muscle contractility following incubation of TSN, while subsequent administration of 100 μM ACh had no effect. BTX-A inhibits pyloric smooth muscle contractility in our study suggesting BTX-A inhibits not only ACh release from cholinergic nerves but also muscarinic cholinergic muscular transmission.

Spontaneous diastolic contractions and phosphorylation of the cardiac ryanodine receptor at serine-2808 in congestive heart failure in rat

The role of phosphorylation of the ryanodine receptor at serine-2808 (RyRS2808) in congestive heart failure (CHF) is controversial, and effects of RyRS2808 phosphorylation on contraction are unclear. It has been reported that diastolic sarcomere length (SL) fluctuations accompany propagating contractile waves due to propagating SR Ca2+ release in trabeculae from rats with CHF. Here, we studied the influence of RyR destabilization by FK506 and isoproterenol on twitch force (Ftw) and SL fluctuations in right ventricular (RV) trabeculae. We measured phosphorylation of RyRS2808 in rats with myocardial infarction (MI) with or without beta-blockade and in rats during isoproterenol stimulation in order to assess the role of RyRS2808 phosphorylation in SL fluctuations in failing hearts. Five groups of male Lewis Brown-Norway rats were studied 3 months after MI: i) Sham; ii) MI with CHF (cMI); iii) MI without CHF; iv) metoprolol-treated MI, with and without CHF. The root mean square (RMSSL) of SL fluctuations in RV trabeculae was calculated. RMSSL increased strongly both following a short train of stimuli at 2.5 Hz and following catecholamine activation in trabeculae from MI with CHF, resulting in a decrease in Ftw in proportion to RMSSL. RyRS2808 phosphorylation was increased significantly in the left ventricle (LV; approximately 58%, P<0.05) but not in the RV (n.s.) in MI rats with CHF. FK506 tripled high frequency stimulation-induced RMSSL in nonfailing trabecula but did not further enhance RMSSL in failing trabecula. Isoproterenol increased RMSSL in nonfailing trabeculae only modestly despite a substantial increase in RyRS2808 phosphorylation in the RV (approximately 60%, P<0.05). Isoproterenol induced SL fluctuation without an increase in RV-RyRS2808 phosphorylation in failing trabeculae. Chronic beta-blockade decreased high frequency and catecholamine stimulation-induced RMSSL while RyRS2808 phosphorylation in the RV was indistinguishable from that in cMI. Acute RyRS2808 phosphorylation by itself does not cause spontaneous contractile waves owing to RyR2 destabilization. Spontaneous contractile waves in CHF are not caused by RyRS2808 phosphorylation alone, suggesting that factors other than RyRS2808 phosphorylation affect RyR function.

Reduced threshold for myocardial cell calcium intolerance in the rat heart with aging.

To determine whether advancing age is accompanied by a reduced Ca2+ tolerance, we measured Ca(2+)-dependent diastolic pressure, prolonged relaxation and systolic functional deterioration, spontaneous sarcoplasmic reticulum (SR)-generated Ca2+ oscillations [detected as scattered laser light intensity fluctuations (SLIF)], aftercontractions, and ventricular fibrillation in isolated, isovolumic, atrioventricular-blocked intact hearts from 24- to 26-mo (old) and 6- to 8-mo (young) male Wistar rats. In enzymatically isolated single cardiac myocytes, the likelihood of the occurrence of spontaneous contractile waves driven by spontaneous SR Ca2+ release was also determined. In response to stepwise increase in perfusate Ca2+ concentration (Cao), a reduction in the maximum developed pressure accompanied by an elevation in end-diastolic pressure and a prolonged contraction duration was observed at lower Cao in old vs. young hearts (P < 0.01 for each parameter). Furthermore, Ca(2+)-dependent ventricular fibrillation occurred during pacing in six old but in no young hearts (P < 0.01), aftercontractions were observed in seven old vs. one young heart (P < 0.01), and SLIF increased to a greater extent in old vs. young hearts. In single cardiac myocytes, spontaneous contractile waves occurred more frequently with increasing age (P < 0.01). These results indicate that aging is associated with an increased likelihood for the occurrence of SR-generated Ca2+ oscillations and functional abnormalities that result from these oscillations.

Effect of Halothane on Arrhythmogenic Responses Induced by Sympathomimetic Agents in Single Rat Heart Cells

The combination of catecholamines and halothane has long been recognized as arrhythmogenic. The purpose of this study was to evaluate whether the mechanism of this interaction originates at the single cell level. The incidence of spontaneous contractile waves occurring between stimulated beats (interbeat waves), early aftercontractions, and late aftercontractions was measured in rat myocytes exposed to sympathomimetics with and without halothane. Each of these endpoints in single cells has the potential to produce arrhythmias in multicellular preparations. Interbeat waves and late aftercontractions were observed with isoproterenol (1 X 10(-7) M) and norepinephrine (1-3 X 10(-7) M). The incidence of these phenomena was significantly reduced in the presence of 0.30 mM halothane. Early aftercontractions occurred in the presence of isoproterenol (1 X 10(-7) M), norepinephrine (1-3 X 10(-7) M), and phenylephrine (5-10 X 10(-6) M). There was a statistically significant decrease in the incidence of early aftercontractions in the presence of 0.30 mM halothane. These results indicate that the mechanism behind the clinically observed increased arrhythmogenicity of catecholamines with halothane does not arise at the level of single ventricular cells because halothane inhibited sympathomimetic-induced arrhythmogenic activity in this model. The probable mechanisms rather include altered impulse propagation, which might lead to phenomena such as reentry.

The Action of Halo thane on Spontaneous Contractile Waves and Stimulated Contractions in Isolated Rat and Dog Heart Cells

The cell length of single, isolated rat and dog heart cells was monitored during exposure to halothane-containing solution to define the cellular mechanism of halothane's negative inotropic effect. Spontaneous contractile waves, which reflect spontaneous Ca release from the sarcoplasmic reticulum (SR) in resting rat heart cells, exhibited a significant increase in frequency and a decrease in amplitude in the presence of halothane 0.27 mM (0.9 vol%) and 0.55 mM (1.7 vol%). Electrically stimulated dog and rat heart cells abruptly exposed to halothane (0.47-0.55 mM or 1.5-1.7 vol%) revealed a transient increase in twitch amplitude (significantly different from control). Twitch amplitude then declined to values significantly below control as halothane exposure continued. This decrease in twitch reached 42 +/- 13% (mean +/- SD) of control in rat cells and 50 +/- 14% in dog cells beating at 60 beats per min. In dog cells the magnitude of the transient increase in twitch amplitude was greater at faster beating rates compared with lower rates in the same cells (P less than 0.01) and the transient increase was insensitive to verapamil. Halothane 0.55 mM (1.7 vol%) also significantly accelerated the rate of decline in the twitch amplitude of successive beats in rat cells stimulated after a rest interval (negative staircase). The findings regarding spontaneous contractile waves indicate a direct effect of halothane at the SR in resting cells, occurring independently of any changes in the slow inward current. The halothane-induced changes in beating cells can be explained by an enhancement of Ca release from the SR with an eventual reduction of SR Ca stores.

Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+-dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium.

We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneous contractile wave. In rat myocytes exposed to increasing bathing [Ca2+] (Cao) from 0.25 to 10 mM, the Cao at which the peak twitch amplitude occurred in a given cell was not unique but varied with the rate of stimulation or the presence of drugs: in cells stimulated at 0.2 Hz in the absence of drugs, the maximum twitch amplitude occurred in 2 mM Cao; a brief exposure to 50 nM ryanodine before stimulation at 0.2 Hz shifted the Cao of the maximum twitch amplitude to 7 mM. In cells stimulated at 1 Hz in the absence of drugs, the maximum twitch amplitude occurred in 4 mM Cao; 1 microM isoproterenol shifted the Cao of the maximum twitch amplitude to 3 mM. Regardless of the drug or the stimulation frequency, the Cao at which the twitch amplitude saturated varied linearly with the Cao at which spontaneous Ca2+ release first occurred, and this relationship conformed to a line of identity (r = 0.90, p = less than 0.001, n = 25). The average peak twitch amplitude did not differ among these groups of cells. In other experiments, (a) the extent of rest potentiation of the twitch amplitude in rat myocytes was also limited by the occurrence of spontaneous Ca2+ release, and (b) in both rat and rabbit myocytes continuously stimulated in a given Cao, the twitch amplitude after the addition of ouabain saturated when spontaneous contractile waves first appeared between stimulated twitches. A mathematical model that incorporates this interaction between action potential-mediated SR Ca2+ release and the occurrence of spontaneous Ca2+ release in individual cells predicted the shape of the Cao-twitch relationship observed in other studies in intact muscle. Thus, the occurrence of spontaneous SR Ca2+ release is a plausible mechanism for the saturation of the inotropic response to Ca2+ in the intact myocardium.

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium.

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.

Single adult rabbit and rat cardiac myocytes retain the Ca2+- and species-dependent systolic and diastolic contractile properties of intact muscle.

The systolic and diastolic properties of single myocytes and intact papillary muscles isolated from hearts of adult rats and rabbits were examined at 37 degrees C over a range of stimulation frequencies and bathing [Ca2+]o (Cao). In both rabbit myocytes and intact muscles bathed in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted in a positive staircase of twitch performance. During stimulation at 2 min-1, twitch performance also increased with increases in Cao up to 20 mM. In the absence of stimulation, both rabbit myocytes and muscles were completely quiescent in less than 15 mM Cao. Further increases in Cao caused the appearance of spontaneous asynchronous contractile waves in myocytes and in intact muscles caused scattered light intensity fluctuations (SLIF), which were previously demonstrated to be caused by Ca2+-dependent spontaneous contractile waves. In contrast to rabbit preparations, intact rat papillary muscles exhibited SLIF in 1.0 mM Cao. Two populations of rat myocytes were observed in 1 mM Cao: approximately 85% of unstimulated cells exhibited low-frequency (3-4 min-1) spontaneous contractile waves, whereas 15%, during a 1-min observation period, were quiescent. In a given Cao, the contractile wave frequency in myocytes and SLIF in intact muscles were constant for long periods of time. In both intact rat muscles and myocytes with spontaneous waves, in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted, on the average, in a 65% reduction in steady state twitch amplitude. Of the rat myocytes that did not manifest waves, some had a positive, some had a flat, and some had a negative staircase; the average steady state twitch amplitude of these cells during stimulation at 120 min-1 was 30% greater than that at 6 min-1. In contrast to rabbit preparations, twitch performance during stimulation at 2 min-1 saturated at 1.5 mM Cao in both intact rat muscles and in the myocytes with spontaneous waves. We conclude that the widely divergent, Ca2+-dependent systolic and diastolic properties of intact rat and rabbit cardiac muscle are retained with a high degree of fidelity in the majority of viable single myocytes isolated from the myocardium of these species, and that these myocytes are thus a valid model for studies of Ca2+-dependent excitation-contraction mechanisms in the heart.

The interaction of electrically stimulated twitches and spontaneous contractile waves in single cardiac myocytes.

Spontaneous myofilament motion that propagates within cells as a contractile wave is a manifestation of localized Ca2+ release from sarcoplasmic reticulum (SR). At 37 degrees C, when bathing [Ca2+] (Cao) is 1.0 mM, rat myocytes exhibit contractile waves at rest and the interwave interval averages 9.1 +/- 1.5 s (n = 6). We determined whether there was an interaction between this type of SR Ca2+ release and that induced by electrical stimulation to cause a twitch, and whether such an interaction had functional significance. Progressive decreases in SR Ca2+ loading effected by graded concentrations of caffeine produced proportional decreases in the mechanical amplitude of the twitch and of the spontaneous contractile wave. Regular electrical stimulation in physiologic Cao abolished the waves and, after stimulation, waves did not reappear for a period of time (delay interval). Over a range of stimulation frequencies (6-72 min-1), the delay interval ranged from 11.4 +/- 3.6 to 12.4 +/- 1.7 s and was usually greater than the interwave interval at rest. The delay interval for a wave to occur after a twitch was reduced in the presence of increased Cao, glycosides, or catecholamines. When the interstimulus interval exceeded the delay interval, waves could appear between twitches and had a marked effect of shortening the duration of the action potential and decreasing the amplitude of the subsequent twitch. The magnitude of this effect varied inversely with time (up to 2 s) between the onset of the spontaneous diastolic wave and the subsequent stimulated twitch. A reduction of the interstimulus interval to less than the delay interval prevented the occurrence of diastolic waves. These results demonstrate the presence of an interaction between spontaneous and action potential-mediated Ca2+ release, which can be interpreted on the basis of a common Ca2+ pool and perhaps common release mechanisms. This interaction can explain many of the known effects of electrical stimulation on phenomena that are thought to result from spontaneous Ca2+ oscillations in intact tissue.

Frequency, amplitude, and propagation velocity of spontaneous Ca++-dependent contractile waves in intact adult rat cardiac muscle and isolated myocytes.

Spontaneous contractile waves due to spontaneous calcium cycling by the sarcoplasmic reticulum occur in unstimulated bulk rat papillary muscle and single rat cardiac myocytes with intact sarcolemmal function. We used video analytic techniques to quantify the wave characteristics in both bulk muscle and myocytes; laser-light scattering techniques were also employed in muscle. In muscle bathed in physiological concentrations of calcium, the true periodicity of these waves was a fraction of 1 Hz and increased up to several hertz with increases in cell calcium. This was paralleled by an increase in the frequency of scattered laser light intensity fluctuations. In myocytes, a range of spontaneous contractile wave frequencies similar to that which occurred in the muscle was observed; it could be demonstrated that an increase in superfusate calcium concentrations (2-15 mM at 23 degrees C) increases the oscillation frequency but not amplitude. In both myocytes and muscle, low concentrations of caffeine (0.5 mM) and higher temperature increased the oscillation frequency but diminished their amplitude. However, the scattered light fluctuations did not change with temperature and decreased with caffeine. These results demonstrate that (1) the true frequency of spontaneous sarcoplasmic reticulum oscillations in the unstimulated rat muscle and single myocytes with intact sarcolemmal function is low, i.e., a fraction of a hertz; (2) with cell calcium loading, the oscillation frequency accelerates to those frequencies measured previously in the "calcium overload" state; (3) while scattered light fluctuations which sample myofilament motion are a sensitive, noninvasive method of detecting the oscillations in bulk muscle, they can be insensitive to the divergent changes in oscillation amplitude and frequency.