Tadao Tomita

Effects of phosphodiesterase inhibitors on spontaneous electrical activity (slow waves) in the guinea‐pig gastric muscle.

1. The effects of the phosphodiesterase inhibitors caffeine, theophylline, isobutylmethylxanthine (IBMX) and rolipram on spontaneous electrical activity (slow waves) were studied in the circular muscle of the guinea‐pig gastric antrum. 2. All the inhibitors reduced slow wave frequency without changing the membrane potential and the slow wave configuration, but at higher concentrations they blocked the slow waves and caused membrane hyperpolarization. In the presence of the inhibitors a low level of irregular electrical activity could be observed in many preparations. 3. Isoprenaline, forskolin, dibutyryl cAMP and 8‐bromo‐cAMP all produced effects essentially similar to those of phosphodiesterase inhibitors. K+ (12 mM) and removal of K+ both depolarized the membrane and these were not affected by IBMX (1‐3 microM). A decrease in frequency caused by IBMX was also not significantly affected by 12 mM K+ or K+ removal and only partially antagonized by TEA or 4‐aminopyridine. 4. These results suggest that an increase in intracellular cAMP inhibits pacemaker activity of slow waves. An increase in K+ conductance does not seem to be a major factor in this inhibition. Slow waves appear to be a compound electrical activity in a group of muscle cells and are likely to be disintegrated by xanthine derivatives.

Effects of verapamil on the response of the guinea‐pig tracheal muscle to carbachol

1 The effects of verapamil on the contraction of the guinea‐pig tracheal smooth muscle induced by calcium (Ca2+) or barium (Ba2+) were investigated in three different conditions: (a) in excess K solution, (b) in the presence of carbachol, and (c) in excess K solution containing carbachol. In order to clarify the contractions, the effects of removal and readdition of the divalent cations were also investigated. 2 In Ca2+‐loaded tissues, application of carbachol in Ca‐free medium produced a transient contraction, the magnitude of which decreased the longer the duration of exposure to Ca2+‐free solution. 3 In Ca2+‐depleted, Ba2+‐loaded tissues, application of carbachol in a Ba2+‐ and Ca2+‐free medium produced a transient contraction the magnitude of which decreased the longer the duration of exposure to the Ba2+‐ and Ca2+‐free solution. 4 After exposure to Ca2+‐free solution for 40 min, the sensitivity of the tissue to Ca2+ was greater in the presence of 30 μM carbachol (ED50 = 0.06 mM) than in the presence of 40 mM K+ (ED50 = 0.3 mM). The Ca2+‐sensitivity in the presence of 30 μM carbachol plus K+ (40 mM) was not different from that in the presence of 30 μM carbachol alone. 5 In Ca2+‐free solution, the sensitivity of the tissue to Ba2+ in the presence of 40 mM K+ (ED50 = 1.4 mM) was not different from that observed in the presence of 30 μM carbachol (ED50 = 1.3 mM). 6 After exposure to Ca2+‐free solution, verapamil produced a parallel rightward shift in the concentration‐response curves to added Ca2+ and Ba2+ in the presence of either 40 mM K+, 30 μM carbachol or 40 mM K+ plus 30 μM carbachol. 7 The pA2 values of verapamil against Ca2+ responses in the presence of 40 mM K+, 30 μM carbachol and 40 mM K+ plus 30 μM carbachol were 7.0, 6.5 and 6.5, respectively. The pA2 values of verapamil against Ba2+ responses under these conditions were 7.1, 7.0 and 7.1, respectively. 8 It is concluded that the sustained contraction produced by carbachol requires the influx of Ca2+ and that Ba2+ can substitute for Ca2+ in this process. Furthermore, the ionic channels which admit Ca2+ may be modified by carbachol to different degrees depending on the presence of Ca2+ or Ba2+. Such changes alter the affinity of the channel to verapamil.

Pacemaker phase shift in the absence of neural activity in guinea‐pig stomach: a microelectrode array study

Gastrointestinal (GI) motility is well organized. GI muscles act as a functional syncytium to achieve physiological functions under the control of neurones and pacemaker cells, which generate basal spontaneous pacemaker electrical activity. To date, it is unclear how spontaneous electrical activities are coupled, especially within a micrometre range. Here, using a microelectrode array, we show a spatio‐temporal analysis of GI spontaneous electrical activity. The muscle preparations were isolated from guinea‐pig stomach, and fixed in a chamber with an array of 8 × 8 planar multielectrodes (with 300 μm in interpolar distance). The electrical activities (field potentials) were simultaneously recorded through a multichannel amplifier system after high‐pass filtering at 0.1 Hz. Dihydropyridine Ca2+ channel antagonists are known to differentiate the electrical pacemaker activity of interstitial cells of Cajal (ICCs) by suppressing smooth muscle activity. In the presence of nifedipine, we observed spontaneous electrical activities that were well synchronized over the array area, but had a clear phase shift depending on the distance. The additional application of tetrodotoxin (TTX) had little effect on the properties of the electrical activity. Furthermore, by constructing field potential images, we visualized the synchronization of pacemaker electrical activities resolving phase shifts that were measurable over several hundred micrometres. The results imply a phase modulation mechanism other than neural activity, and we postulate that this mechanism enables smooth GI motility. In addition, some preparations clearly showed plasticity of the pacemaker phase shift.

Some properties of calcium-induced contraction in the isolated human and guinea-pig tracheal smooth muscle

We studied the effects of the cyclooxygenase inhibitors, indomethacin and aspirin, the leukotriene antagonist, FPL 55712 and the Ca antagonist, verapamil on basal tone and Ca-induced contractions in the human and guinea-pig tracheal muscle. Indomethacin and aspirin usually increased spontaneous tone or Ca-induced contractions in human strips, while consistently decreased tension development in the guinea-pig muscle. FPL 55712 strongly reduced contractions in the human, whereas it had a very weak effect on the guinea-pig muscle. Verapamil had a small inhibitory effect in the human trachea, either at 5.9 or 40 mM K, but markedly suppressed Ca-induced contractions of guinea-pig trachea in 40 mM K and had little effect on contractions in 5.9 mM K in this tissue. It is concluded that active tone in the two different tracheal muscles were controlled by different processes; that different arachidonate by-products may exert an effect on basal tone in the two different muscles; and that different plasma membrane Ca channels may be operative under basal conditions in the different muscles.

Consequences of metabolic inhibition in smooth muscle isolated from guinea-pig stomach

1 In smooth muscle isolated from the guinea‐pig stomach, cyanide (CN) and iodoacetic acid (IAA) were applied to block oxidative phosphorylation and glycolysis, respectively. Effects of IAA on generation of spontaneous mechanical and electrical activities were systematically investigated by comparing those of CN. Spontaneous activity ceased in 10–20 min during applications of 1 mm IAA. On the other hand, application of 1 mm CN also reduced the spontaneous activity, but never terminated it. In the presence of CN the negativity of the resting membrane potential was slightly reduced. 2 When spontaneous activity ceased with IAA, the resting membrane potential was not significantly affected. Also, before ceasing, the amplitude and duration of the spontaneous electrical activity were significantly reduced. The amplitude of the electrotonic potential was, however, not changed by IAA. Further, glibenclamide did not prevent the effects of IAA. These results suggest that, unlike cardiac muscle, activation of metabolism‐dependent K+ channels in stomach smooth muscle does not seem to play a major role in reducing and terminating spontaneous activity during metabolic inhibition. 3 Carbachol‐induced contraction transiently increased, and subsequently decreased gradually during application of IAA. 4 After 50 min application of IAA, when there was no spontaneous activity, the concentrations of phosphocreatine (PCr) and ATP measured with 31P nuclear magnetic resonance decreased to 60 and 80% of the control, respectively, while inorganic phosphate (Pi) concentration paradoxically fell to below detectable levels. During subsequent prolonged application of IAA, high‐energy phosphates steadily decreased. On the other hand, after 50 min CN application, [PCr] and [ATP] decreased to approximately 30 and 80% of the control, respectively, while [Pi] increased by 2.6‐fold. 5 In the presence of either CN or IAA, spontaneous mechanical and electrical activities were reduced or eliminated, although amounts of high‐energy phosphates sufficient to contract smooth muscle remained. It can be postulated that some mechanism(s) related to energy metabolism, but not including ATP‐sensitive K+ channels, plays an important role in generating spontaneous activity in guinea‐pig stomach smooth muscle. During metabolic inhibition the energy metabolism‐dependent mechanism(s) would preserve high‐energy phosphates, and consequently cell viability, by stopping spontaneous activity.

Relaxant actions of isoprenaline on guinea‐pig isolated tracheal smooth muscle

1 The effects of isoprenaline on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in guinea‐pig isolated tracheal muscle were studied by use of intracellular micro‐electrodes and fura‐2 signals respectively. Measurements of membrane potential were carried out in the presence of spontaneously‐generated muscle tone, whereas fura‐2 signals were measured during contraction produced by exogenous prostaglandin E2 (100 nm). The potency of isoprenaline in causing relaxation was the same in these two different situations. 2 Isoprenaline (0.01 μm) produced relaxation accompanied by 5 mV hyperpolarization. A combination of tetraethylammonium (TEA, 10 μm) and verapamil (3 μm) did not alter the effects of isoprenaline. Removal of external K+ did not increase the degree of hyperpolarization produced by isoprenaline. 3 In the presence of TEA (10 μm) and verapamil (3 μm), isoprenaline (0.03‐1 μm) reduced [Ca2+]i concentration‐dependently. A similar degree of inhibition was observed when isoprenaline was applied during the maintained contraction induced by prostaglandin E2 and against the contraction evoked by the addition of Ca2+ to tissues bathed in a Ca2+‐free medium and pretreated with both isoprenaline and prostaglandin E2. 4 It is concluded that activation of TEA‐sensitive Ca2+‐dependent K+channels does not play a significant role in isoprenaline‐induced relaxation. We propose that, in the guinea‐pig tracheal muscle, isoprenaline may produce relaxation mainly by inhibiting a receptor‐operated pathway for Ca2+ influx across the plasma membrane which is normally activated by prostaglandins.

Mechanical and electrical responses to α-adrenoceptor activation in the circular muscle of guinea-pig stomach

1 In the circular muscle of the corpus region of the guinea‐pig stomach, the effects of catecholamines on mechanical activity were studied with simultaneous recording of membrane potential by use of intracellular microelectrodes. In order to investigate responses mediated through α‐adrenoceptors, the β‐adrenoceptors were blocked by propranolol (10−6 m) in most experiments. 2 Adrenaline (< 10−6 m) produced a monophasic contraction with little change in membrane potential. At higher concentrations (> 10−5 m), adrenaline usually produced an early transient contraction followed by a slow tonic contraction. During the early phase, the membrane was hyperpolarized and slow waves were reduced in amplitude. 3 The phasic contractions superimposed on the late slow phase of the contractile response were higher in frequency and larger in amplitude than in controls before adrenaline application. These changes were associated with an increase in the amplitude and frequency of slow waves, and with a spike‐like component appearing on the top of each slow wave. 4 Adrenaline‐induced changes in mechanical and electrical activity were inhibited by prazosin, but largely unaffected by yohimbine. In some preparations, there was a transient weak inhibition of phasic contraction before muscle tone was increased by adrenaline, and this became more pronounced in the presence of yohimbine. 5 Phenylephrine, selective for α1‐receptors, produced responses very similar to those of adrenaline, whereas agonists selective for α2‐receptors, clonidine and B‐HT 920, produced only a small slow contraction without any clear change in membrane potential. 6 It is concluded that in the circular muscle of guinea‐pig stomach, the contractile response and changes in the electrical slow wave activity produced by adrenaline are both mediated mainly through the activation of α1‐receptors.

Depletion of intracellular free Mg2+ in Mg2(+)- and Ca2(+)-free solution in the taenia isolated from guinea-pig caecum.

1. In isolated strips of the taenia of guinea‐pig caecum removal of Mg2+ alone from the external solution had no clear effects on contractions produced by carbachol. However, after treatment with Mg2(+)‐ and Ca2(+)‐free solution, readmission of 2.4 mM‐Ca2+ caused only limited recovery, and addition of Mg2+ was necessary for full recovery. 2. When Mg2+ was removed in the absence of Ca2+, oxygen consumption increased, but gradually decreased again in the prolonged absence of the divalent cations. The increase in O2 consumption was blocked by ouabain or by decreasing the external sodium concentration to 20 mM. 3. Under normal conditions, the intracellular free Mg2+ concentration [( Mg2+]i) was estimated to be 310 +/‐ 30 microM (n = 17) from the chemical shift of the ATP peaks obtained with 31P nuclear magnetic resonance (NMR), assuming the dissociation constant of MgATP to be 41 microM. 4. Removal of external Mg2+ did not alter [Mg2+]i within 100 min. However, when both Mg2+ and Ca2+ were omitted, [Mg2+]i decreased to 8.3 +/‐ 3.6 microM (n = 12) in 100 min. The [Mg2+]i recovered completely on readmission of 1.2 mM‐Mg2+. 5. When Mg2+ and Ca2+ were omitted, the phosphocreatine (PCr) content of the tissue slowly decreased to about 90% and the ATP concentration was reduced to about 60% of the control in 100 min. On Mg2+ readmission the ATP levels recovered partially, whereas PCr decreased further. 6. It is concluded that free [Mg2+]i slowly decreases when both Mg2+ and Ca2+ are removed from the external solution, due to an increased permeability of the plasma membrane, and that when [Mg2+]i is reduced to less than about 10% of the normal internal concentration, energy metabolism, membrane transport, and contraction are impaired.

Phosphorous compounds studied by 31P nuclear magnetic resonance spectroscopy in the taenia of guinea-pig caecum.

1. In the isolated taenia (0.4‐0.6 g) of guinea‐pig caecum, the intracellular phosphorous compounds and pH were investigated using 31P nuclear magnetic resonance (NMR) under various metabolic conditions. 2. The ratios of the intracellular concentration of phosphocreatine ([ PCr]) and inorganic phosphate [( Pi]) to nucleotide triphosphate ([ NTP]) were 1.71 +/‐ 0.14 and 0.58 +/‐ 0.11 (n = 25), respectively, in normal solution (32 degrees C). The intracellular pH estimated from the chemical shift of Pi was 7.05 +/‐ 0.06 (n = 25), agreeing well with those previously obtained. 3. In the absence of glucose, the [PCr] and [NTP] were decreased to almost a half after 150 min exposure to 40 mM‐K+ solution, while [Pi] was increased 3‐fold. These changes were much faster than the rate of decline in tension. When glucose was readmitted, the contractile response to K+ fully recovered in 50 min. However, this was accompanied with only a partial recovery of [PCr] and [Pi], but no recovery of [NTP]. The intracellular pH was lowered by about 0.2 of a unit, suggesting an increase in glycolysis. 4. In Ca2+‐free solution, respiratory inhibition with hypoxia or CN (1 mM) only decreased [PCr], leaving [NTP] nearly unchanged. On the other hand, respiratory inhibition in excess‐K+ solution containing Ca2+ (2.4 mM) severely depleted PCr and decreased [NTP] to 40%. Increasing glucose to 50 mM did not prevent these changes, although it increased tension development. 5. The simultaneous decrease of [NTP] and [PCr] during K+ contracture suggests that the activity of creatine phosphokinase is low. The recovery from respiratory inhibition was much better for [PCr] than for [NTP]. Slow, but perfect, recovery of all NTP peaks was produced by adding 1 mM‐adenosine to normal solution. 6. It was suggested that tension development is closely related to the turnover rate of ATP, and not to its concentration, and that deamination of adenosine is a limiting factor in the recovery of ATP after excessive consumption.

Sustained contraction produced by caffeine after ryanodine treatment in the circular muscle of the guinea-pig gastric antrum and rabbit portal vein.

1 Caffeine inhibited spontaneous mechanical activity at 0.3‐1 mM, but produced a tonic contraction at concentrations higher than 3 mM in the circular muscle of the guinea‐pig gastric antrum. In the circular muscle of the rabbit portal vein, caffeine at concentrations higher than 1 mM produced an early phasic contraction followed by a small tonic component. The caffeine‐induced contraction was abolished by removal of the external Ca2+ more rapidly in the gastric antrum than the portal vein.