Components of cholinergic excitation coupling with smooth muscle contraction in the guinea pig taenia coli]

In concentrations of 10(-9)-10(-7) g/ml acetylcholine increased the tone of the smooth muscles of the longitudinal band of the large intestine of a guinea pig, increasing the permeability of the cellular membranes for the entering flux of 45Ca2+. In concentrations of 10(-6) g/ml and over acetylcholine caused a release of the membranous calcium and in the concentrations of 10(-5)-10(-3) g/ml markedly increased the permeability of the membranes of the smooth muscle cells for the 22Na+ ions causing depolarization and an increase in the frequency of the action potentials. It is supposed that the coupling of the cholinergic stimulus with the end effect (muscle contraction) included 3 components: intensification of the entrance of Ca2+ into the smooth muscle cells, release of the membrane calcium and adhesion mechanism.     

Inward current in single smooth muscle cells of the guinea pig taenia coli

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.     

Outward current in single smooth muscle cells of the guinea pig taenia coli

In single myocytes of the guinea pig taenia coli, dispersed by enzymatic digestion, the late outward current is carried by K+. It has both a Ca2+-activated component and a voltage-dependent component which is resistant to external Co2+. The reversal potential is -84 mV, and the channel(s) for it are highly selective to K+. At 33 degrees C, the activation follows n2 kinetics, with a voltage-dependent time constant of 10.6 ms at 0 mV, which shortens to 1.7 ms at +70 mV. Deactivation follows a single-exponential time course, with a voltage-dependent time constant of 11 ms at -50 mV, which lengthens to 33 ms at -20 mV. During a 4.5-s maintained depolarization, IK inactivates, most of it into two exponential components, but there is a small noninactivating residue. It is surmised that during an action potential under physiological conditions, there is sufficient IK to cause repolarization.     

Inhibitory synaptic potentials of the smooth muscle cells of guinea pig taenia coli

A single submaximal intramural application of rectangular stimuli (duration 0.2–0.5 msec) to an atropine-treated taenia coli muscle band evoked inhibitory postsynaptic potentials (IPSP) and a marked relaxation of the muscle band in the vast majority of muscle cells. The latency period of the IPSP was 122±16 msec; the times for a rise and fall of amplitude were 96±8 and 370±60 msec, respectively. The mean latency period of muscle relaxation was 800 msec. The latency period, and especially the amplitude of the IPSP depended on the intensity of the intramural stimulation. This indicates that one muscle cell is inhibited by several nerve fibers. IPSP evoked by threshold stimuli displayed a tendency toward summation, while the amplitude of the second and of subsequent IPSP evoked by low-frequency maximal stimuli was always less than that of the first IPSP. After periodic stimulation (frequency 10–60 impulses/min) was discontinued, a posttetanic decrease in IPSP amplitude was observed. Anodic polarization of the muscle band with a direct current raised the effectiveness of synaptic transmission, as was evidenced by the considerable increase in IPSP amplitude. When the muscle membrane was hyperpolarized with noradrenaline, IPSP inhibition was reversible. This is evidence that the unknown mediator and noradrenaline have a common ionic inhibitory mechanism.A. A. Bogomol'ts Institute of Physiology of the Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 544–551, September–October, 1970.     

Effect of ethacrynic acid on contractility and Ca flux of guinea pig taenia coli smooth muscle (author's transl)

The effects of ethacrynic acid (ETCA) which has been known as an -SH groups inhibitor on the contractility and the Ca flux of guinea pig taenia coli were investigated. The results obtained were as follow: 1) Contractures induced by 10(-4) M ACh, or the tonic component of 150 mM K-contractures were markedly suppressed by previous treatment with a low concentration (2 X 10(-4) M) of ETCA for 40 min. Conversely with the same treatment, the phasic component of this K-contracture was only slightly suppressed. The inhibitory effects of ETCA in both cases were reversed by the repetitive washing out of ETCA from taenia coli with normal tris-buffered solution. 2) ETCA, at concentrations higher than 10(-3) M, more markedly inhibited the ACh-, and the K-contractures. In this case these inhibitions were irreversible. 3) Cysteine in an equimolar concentration of ETCA prevented the inhibitory effects of ETCA on both contractures. 4) ETCA (10(-4) M) inhibited the ACh-contracture in Ca2+-free isotonic KCl solution to approximately the same degree as that in normal solution. 5) Inhibition of ACh-contracture by ETCA in Na+-free isotonic LiCl solution was less than that in normal solution. 6) ETCA (2 X 10(-4), or 10(-3) M) markedly stimulated 45Ca efflux from taenia coli in 20 mM Ca-EGTA tris-buffered solution. 7) 45Ca efflux acceleration by ETCA in Na+-free (replaced by Li+) 20 mM Ca-EGTA tris-buffered solution was less than that in 20 mM Ca-EGTA tris-buffered solution. These results may be explained by assuming that the inhibitory effect of ETCA on ACh-contracture can be attributed to the depletion of stored intracellular Ca and the acceleration of Ca efflux as a result of ETCA treatment.     

Relationship between vanadate induced relaxation and vanadium content in guinea pig taenia coli

Abstract: Vanadate has been known to induce a transient increase in high K+ induced contraction, and also gradually relax the high K+ contraction itself in guinea pig taenia coli. The relationship between the rate of relaxation and ion content of Na+, K+, and V ion at the cellular level was investigated when vanadate was applied to contracted muscle. Tissue Na+ and V ion content increased linearly, depending on the time after vanadate treatment, reaching maximum levels of approximately 50 mM x kg(-1) and 0.25 mM x kg(-1) wet weight, respectively. There was a positive correlation between the V ion and Na+ contents, while there was a negative correlation between both ions and the relaxed rate of the high K+ induced contraction. The uptake of V ion was affected by the external K+ concentration, and the maximum rate of V ion uptake decreased to 40% in the presence of 90 mM external K+. These results suggest that a small amount of V ion was enough to inhibit the Na+ pump activity and muscle contraction in the high K+ solution.     

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.     

Effects of magnesium pyrophosphate on mechanical properties of skinned smooth muscle from the guinea pig taenia coli.

Effects of the non-hydrolyzable nucleotide analogue magnesium pyrophosphate (MgPPi) on cross-bridge properties were investigated in skinned smooth muscle of the guinea pig Taenia coli. A "high" rigor state was obtained by removing MgATP at the plateau of an active contraction. Rigor force decayed slowly towards an apparent plateau of approximately 25-35% of maximal active force. MgPPi markedly increased the rate of force decay. The initial rate of the force decay depended on [MgPPi] and could be described by the Michaelis-Menten equation with a dissociation constant of 1.6 mM. The decay was irreversible amounting to approximately 50% of the rigor force. Stiffness decreased by 20%, suggesting that the major part of the cross-bridges were still attached. The results can be interpreted as "slippage" of PPi-cross-bridges to positions of lower strain. The initial rate of MgPPi-induced force decay decreased with decreasing ionic strength in the range 45-150 mM and was approximately 25% lower in thiophosphorylated fibers. MgADP inhibited the MgPPi-induced force decay with an apparent Ki of 2 microM. The apparent Km of MgATP for the maximal shortening velocity in thiophosphorylated fibers was 32 microM. This low Km of MgATP suggests that steps other than MgATP-induced detachment are responsible for the low shortening velocity in smooth muscle. No effects were observed of 4 mM MgPPi on the force-velocity relation, suggesting that cross-bridges with bound MgPPi do not constitute an internal load or that binding of MgPPi is weaker in negatively strained cross-bridges during shortening.     

Desensitization of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine

The role of tissue organization of smooth muscle in short-term desensitization to acetylcholine (ACh) was examined by studying the desensitization of isolated single cells from guinea pig taenia caecum. Cells were isolated by collagenase digestion. The conditions during cell isolation were adjusted to obtain cells that showed repeated contractions. The cells contracted on treatment with 10(-7)-10(-6) M ACh, showing an all-or-none response. Desensitized cells also showed an all-or-none response but required a higher concentration of ACh for induction of contraction; i.e., the magnitude of their maximal response was not changed appreciably but the threshold concentration of ACh for their contraction was raised. Incubation of the whole tissue with 10(-4) M ACh for 10 min also caused desensitization. This desensitization was accompanied by reduction of the contractile response at intermediate concentrations. The mode of desensitization of isolated cells determined from the average response of the isolated cells was almost the same as that of whole muscle. It is concluded that the desensitization occurred in each cell irrespective of its tissue organization and that the desensitization was due to an increase of the threshold for contraction to ACh of each cell.     

S-nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea pig

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.     

Vitamin PP, nicotinamide nucleotides and neuro-muscular transmission in guinea pig cecum longitudinal muscle (taenia coli)

The membrane potential, inhibition postsynaptic potentials, resistance of the guinea pig taenia coli membrane were studied as affected by exogenic vitamin PP and its derivatives of nucleotide nature. It is shown that nicotinic acid, nicotinamide, NAD+, NADH, NADP+, NADPH evoke the membrane hyperpolarization and a decrease in the amplitude of the inhibition postsynaptic potentials. Nicotinamid dinucleotides cause a decrease in the membrane resistance, whereas nicotinic acid and nicotinamide do not affect this parameter. The character of the observed effects does not depend on the degree of nicotinamide dinucleotides oxidation.     

Membrane mechanisms of activating depolarized smooth muscle contraction (guinea pig small intestine) during exposure to physiologically active compounds

The experiments were performed on the depolarized smooth muscle of taenia coli with the use of double sucrose-gap arrangement. Muscle contractions were induced by 1) application of hystamin or bradikynin; 2) rectangular long-lasting (10--20 s) pulses of hyperpolarizing current--the strong contraction appeared in response to the switching off the current (off-response). Both the on- and off-responses to the hyperpolarizing current recorded before, during and after hystamine (or bradikynin)-induced contration were, as a rule, very similar. Treatment of smooth muscle with local anaesthetics (procaine, trimecaine, QX-572) removed hystamine- and bradikynin-induced contrations and only decreased off-responses. The analysis of the data obtained suggested the existence of the independent electrically and chemically excitable systems (channels?) OF Ca2+ ion transport in the membrane of smooth muscle cells.     

The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli

Currents through single potassium channels were studied in cell-attached or inside-out patches from collagenase-dispersed smooth muscle cells of the guinea pig taenia coli. Under conditions mimicking the physiological state with [K+]i = 135 mM: [K+]o = 5.4 mM, three distinct types of K+ channel were identified with conductances around 0 mV of 147, 94, and 63 pS. The activities of the 94- and 63-pS channel were observed infrequently. The 147-pS channel was most abundant. It has a reversal potential of approximately -75 mV. It is sensitive to [Ca2+]i and to membrane potential. At -30 mV, the probability of a channel being open is at a minimum. At more positive voltages, the probability follows Boltzman distribution. A 10-fold change in [Ca2+]i causes a 25-mV negative shift of the voltage where half of the channels are open; an 11.3-mV change in membrane potential produces an e-fold increase in the probability of the channel being open when P is low. At voltages between -30 and -50 mV, the open probability increases in an anomalous manner because of a large decrease of the channel closed time without much change in the channel open time. This anomalous activity may play a regulatory role in maintaining the resting potential. The histograms of channel open and closed time fit well, respectively, with single and double exponential distributions. Upon step depolarizations by 100-ms pulses, the 147-pS channel opens with a brief delay. The delay shortens and both the number of open channels and the open time increase with increasing positivity of the potential. The averaged currents during the step depolarizations closely resemble the delayed rectifying outward K+ currents in whole-cell recordings.     

The action potential in the smooth muscle of the guinea pig taenia coli and ureter studied by the double sucrose-gap method

The configuration of the electrotonic potential and the action potential observed by the double sucrose-gap method was similar to that observed with a microelectrode inserted into a cell in the center pool between the gaps. In the taenia and the ureter, the evoked spike was larger in low Na or in Na-free (sucrose substitute) solution than in normal solution. However, the plateau component in the ureter was suppressed in the absence of Na. In Ca-free solution containing Mg (3–5 mM) and Na (137 mM), the membrane potential and membrane resistance were normal, but no spike could be elicited in both the taenia and ureter. Replacement of Ca with Sr did not affect the spike in the taenia, nor the spike component of the ureter but prolonged the plateau component. The prolonged plateau disappeared on removal of Na, while repetitive spikes could still be evoked. It was concluded that the spike activity in the taenia and in the ureter of the guinea pig is due to Ca entry, that the plateau component in the ureter is due to an increase in the Na conductance of the membrane, and that both mechanisms, for the spike and for the plateau, are separately controlled by Ca bound in the membrane.     

Selective utilization of L-isomer of lactate in the smooth muscle of the guinea pig taenia caeci

Utilization of D- and L-lactate in the isolated intestinal smooth muscle of the guinea pig taenia caeci was examined by measuring contractile tension, oxygen consumption, and adenosine triphosphate (ATP) and creatine phosphate (PCr) concentrations. In the absence of glucose in the medium, muscle contraction induced by a high concentration of K+ was inhibited and the rate of oxygen consumption and the concentrations of ATP and PCr were decreased. Addition of glucose, L-lactate, and D,L-lactate, but not D-lactate, led to recovery of muscle contraction, rate of oxygen consumption, and ATP and PCr concentrations when the tissue had been incubated in the high K+, glucose-free solution. These results suggest that the isolated guinea pig taenia caeci selectively utilizes the L-isomer of lactate as a substrate for energy metabolism.     

Caffeine and excitation-contraction coupling in the guinea pig taenia coli

The effects of caffeine (0.2–10 mM) on the electrical and mechanical activities of guinea pig taenia coli were investigated with the double sucrose-gap method. Caffeine evoked a small tension with a latency of 20–30 sec, then phasic contraction developed and finally relaxation. The initial tension development also appeared in the Na-free solution without any marked changes in the membrane potential and membrane resistance. The phasic contraction disappeared in the Na-free solution. The relaxation in the presence of caffeine was accompanied by depolarization block of the spike generation. The minimum concentration of Ca ion needed to evoke the tension development by the caffeine was 10^-7 M. Caffeine also potentiated the twitch tension below a concentration of 5 mM either in the Na-free solution or at low temperature (5°C). NO₃ ^- and Br^- showed a similar response to caffeine on the potentiation of the twitch tension at low temperature.