Contraction In Ca-free Solution Research Articles

Haloperidol differentiates smooth muscle contractions induced by release of intracellularly stored Ca and by influx of extracellular Ca

1. 1. The effects of haloperidol on smooth muscle contraction induced by carbachol, histamine, high K or caffeine in the presence or absence of extracellular Ca were investigated. 2. 2. In the presence of extracellular Ca, the maximal contraction induced by carbachol was reduced by haloperidol, while that by histamine or high K was much less affected. 3. 3. In Ca-free solution, contraction induced by histamine was extremely reduced by haloperidol, while that by carbachol was not affected. 4. 4. These results suggest that haloperidol selectively inhibited signal transduction processes from activation of muscarinic acetylcholine receptors to influx of extracellular Ca and from activation of histamine H 1-receptors to release of intracellularly stored Ca. 5. 5. Caffeine-induced contraction in Ca-free solution was markedly potentiated by haloperidol, although haloperidol did not elicit contraction in Ca-free solution by itself. 6. 6. These results suggest that haloperidol increased the sensitivity of Ca-induced Ca release channels to caffeine.

TPA- and agonist-induced force development in myometrium from pregnant and non-pregnant rats

In myometrium from pregnant rats, 100 nM-TPA elevated resting tension and initially slightly enhanced the contraction induced by 138 mM-KCl. After 20 min this force development significantly declined. In saponin-treated skinned myometrial cells from pregnant rats, 100 nM-TPA enhanced the contraction induced by 0.3 microM-Ca2+, but reduced that induced by 1 microM-Ca2+. These findings suggest that the excitatory and inhibitory actions of TPA on the myometrium are probably due to its action on the contractile proteins. In myometrium from non-pregnant rats, TPA affected neither the resting tension, nor the amplitude of the evoked contractions, nor the Ca2+-induced contractions in skinned myometrium. While TPA only affected tension development in pregnant rats, both 1 mM-carbachol and 90 nM-oxytocin induced a tonic contraction in Ca-free solution independently of the hormonal status of the rats. The latter finding makes it unlikely that activation of protein kinase C is involved in the agonist-induced tonic force development in Ca-free solution.

Calcium requirement of uterine contraction induced by PGE 1: Importance of intracellular calcium stores

The contraction of the rat uterus in response to PGE 1 in high K + medium and in Ca-free solution which contained EDTA has been investigated in order to examine whether excitation-contraction coupling involves the release of Ca from an intracellular store. In uterus maximally contracted by K +, cumulative concentrations of PGE 1 (1.25 – 20 ng/ml) caused maintained concentration-dependent contraction. PGE 1 induced sustained contraction of rat uterus in Ca-free medium after incubation with 3mM EDTA for 50 min. In these conditions the involvement of extracellular Ca is highly unlikely. The PGE 1-induced contraction could be repeated without exposure to external Ca ions and with only slight reduction in magnitude. The PGE 1 concentrations required to elicit uterine contraction in Ca-free solution were about 1000 times higher than the effective doses in KCl-depolarized uterus. In conclusion, the present investigation shows that Ca influx is not essential for PGE 1-induced contraction of rat uterus, although extracellular Ca enhances it presumably by increasing the free Ca levels in the cytosol.

Receptor Operated Intracellular Calcium Stores in the Smooth Muscle of the Guinea Pig Bladder

Study of the in vitro behavior of strips of guinea pig bladder and taenia coli demonstrated that:1.Both bladder and taenia temporarily retain their ability to contract in Ca-free solutions, but the magnitude of this response decays with time.2.Carbachol is capable of producing contraction in Ca-free solution for a longer period of time than K depolarisation.3.Once lost, the ability of carbachol (but not K) to contract the tissues in Ca-free solution can be temporarily restored by a brief application of high Ca.4.The size of the carbachol contraction in Ca-free solution is reduced in Na-free solution, suggesting that membrane-bound Ca may not play a major role in this response.5.In depolarised bladder exposed to nifedipine 2xl0−7M, carbachol can only elicit 1 large contraction, suggesting depletion of an intracellular source.6.It is concluded that whereas the response of the bladder to depolarisation depends primarily on extracellular Ca, the response to carbachol may also involve release of stored Ca and that the bladder, like other smooth muscles, appears to contain agonist-releasable intracellular Ca stores.

Nitroglycerine- and isoprenaline-induced vasodilatation: assessment from the actions of cyclic nucleotides.

To investigate the vasodilator actions of nitroglycerine and isoprenaline, the effects of these agents, dibutyryl cyclic AMP (db cyclic AMP) and 8-bromo cyclic GMP (8-Br cyclic GMP) on intact muscle tissue, and cyclic AMP and cyclic GMP on skinned muscle of the rabbit mesenteric artery were investigated. In porcine coronary artery, nitroglycerine (greater than 0.1 microM) increased the production of cyclic GMP with no change in the amount of cyclic AMP, while isoprenaline (greater than 0.1 microM) significantly increased the production of cyclic AMP with no change in the amount of cyclic GMP. In the rabbit mesenteric artery, nitroglycerine or isoprenaline inhibited the tonic component of the 39 mM [K]o-induced contraction to a greater extent than the phasic component. Nitroglycerine and 8-Br cyclic GMP showed a stronger inhibitory action on the K-induced contraction than did isoprenaline and db cyclic AMP. The sources of Ca utilized for the generation of contraction by noradrenaline and caffeine were estimated to be the same as those determined from the amplitudes of contractions evoked in Ca-free solution by various concentrations of noradrenaline or caffeine. In intact muscle tissues, the effects of nitroglycerine or 8-Br cyclic GMP on the amount of Ca stored in cells were estimated from the caffeine-induced contraction in Ca-free solution. Both agents inhibited the contractions due to a reduction in the amount of Ca in the cells. When the effects of isoprenaline or db cyclic AMP were observed, both agents inhibited the caffeine-induced contraction but the accumulation of Ca into cells was greater than the control. In saponin skinned muscles, the pCa-tension relationship in the presence of cyclic AMP and cyclic AMP-dependent protein kinase (cyclic AMP-PK) shifted to the right and to a lower level in comparison with the control. Applications of cyclic GMP with cyclic GMP-dependent protein kinase (cyclic GMP PK) also inhibited the contraction induced by low concentrations of Ca. In skinned muscles, cyclic AMP exhibited dual actions on Ca store sites, i.e. in the presence of high concentrations of Ca or prolonged superfusion of Ca, cyclic AMP reduced the amount of Ca due to activation of the Ca-induced Ca release mechanism by excess accumulation of Ca. On the other hand, cyclic GMP consistently inhibited the amplitude of the caffeine-induced contraction due to a reduction in the amount of Ca in the store sites. 8 These results indicate that nitroglycerine and isoprenaline increase the amount of cyclic GMP and cyclic AMP, respectively. The main effect of cyclic GMP is activation of Ca extrusion, thus reducing the amount of Ca stored in the cell, while the main effect of cyclic AMP is to increase the amount of Ca stored in the cell. Both cyclic AMP with cyclic AMP-PK and cyclic GMP with cyclic GMP-PK inhibit the phosphorylation of myosin. Consequently both cyclic nucleotides reduce the free Ca in the myoplasm and promote relaxation, but by different mechanisms.

Contraction of smooth muscle in Ca-free solution.

The tonic contractions which are extremely resistant to removal of the external Ca were investigated in the rat vas deferens and myometrium. Both the noradrenaline response in the vas deferens and the oxytocin response in the myometrium could be repeatedly produced without appreciable diminution in Ca-free solution for more than 24 hrs. On the other hand, the tissue Ca content decreased exponentially after Ca-removal with a half time of 130-180 min. When Ca was readmitted, no indication of the early transient contraction was observed in the subsequent response in Ca-free solution, but the response was reduced compared with the response before Ca readmission. Verapamil suppressed the response in the presence of Ca, while it had very weak inhibitory effect even at 10 microM. Calmodulin antagonists of phenothiazine derivatives had a strong inhibitory effect on Ca-induced contractions, whereas they had very weak effects on the receptor-mediated contraction in Ca-free solution. Another calmodulin antagonist, W-7 suppressed both Ca-induced contraction and the contractions independent of external Ca. HA-1004, a vasodilator which has a structure similar to W-7, reduced the receptor-mediated contraction in Ca-free solution without much effect on Ca-induced contractions. These results may suggest that the receptor-mediated contractions resistant to Ca-removal are caused by some process without a contribution of the Ca-calmodulin system.

Nisoldipine-induced relaxation in intact and skinned smooth muscles of rabbit coronary arteries.

In smooth muscles of the rabbit coronary artery, nisoldipine inhibited the phasic and tonic responses of the contraction induced by 128 mM K (the IC50 values were 4 X 10(-8) M and 1 X 10(-13) M, respectively). This agent also inhibited the tonic response of the acetylcholine (ACh) (10(-5) M)-induced contraction (the IC50 value was 3 X 10(-10) M), but only slightly inhibited the phasic response (in 10(-7) M, 0.86 times the control). Nisoldipine (less than 10(-7) M) had no effect on the K-induced depolarization of the membrane at any given concentration. This drug (5 X 10(-8) M) did inhibit the oscillatory potential changes and spike potential evoked on the ACh-induced slow depolarization. After depletion of stored Ca from the polarized muscles (5.9 mM K), muscle cells accumulated Ca by application of 2.6 mM Ca without generation of contraction, i.e. a subsequently applied 20 mM caffeine produced the contraction in Ca-free solution. Nisoldipine (less than 10(-7) M) had little effect on this accumulation of Ca. The rate of rise and time to reach the maximum amplitude of the 128 mM K- or ACh-induced contraction (in 2.6 mM Ca) depended on the amount of stored Ca in cells. Nisoldipine (10(-8) M) consistently inhibited the Ca-induced contraction evoked in depolarized muscles (128 mM K), regardless of the amount of stored Ca. However, this agent (10(-8) M) did not inhibit the Ca release from storage sites evoked by activation of the muscarinic receptor. After prolonged superfusion (over 120 min) with Na- and Ca-free solution (guanethidine and atropine were present), application of 2.6 mM Ca produced contraction which was inhibited by 10(-8) M nisoldipine, while the depolarization induced by application of these solutions was not inhibited by nisoldipine. In saponin-skinned muscles, nisoldipine had no effect on the contractile proteins, as estimated from the pCa-tension relationship, or on the Ca accumulation into the Ca release from the Ca storage sites, as estimated from the caffeine-induced contraction. It is concluded that nisoldipine possesses a selective inhibitory action on voltage-dependent Ca influx, when the Ca channel is activated by depolarization.

Effects of noradrenaline and acetylcholine on electro-mechanical properties of the guinea-pig portal vein.

Effects of acetylcholine (ACh) and noradrenaline (NA) on mechanical properties of smooth muscle cells of the guinea-pig portal vein were investigated using intact and skinned muscle preparations. In some preparations, the electrical activity was also recorded. In addition to ACh and NA, the effects of caffeine, procaine, excess concentrations of [K]o and MnCl2 were investigated. NA enhanced the mechanical response due to increase in the spike generation, receptor activated depolarization and release of Ca stored in the cell. ACh also enhanced the mechanical response, but this agent had little effect on the Ca release from the storage sites. Caffeine and procaine (10 mM) depolarized the membrane and enhanced the electrical activity. Caffeine enhanced the mechanical activity due to an increase in the membrane activity and the release of Ca stored in the cell, while procaine inhibited the contraction. Procaine inhibited and caffeine accelerated the Ca-induced Ca-release mechanism in the cell. NA released the Ca from the storage sites to a greater extent than did caffeine or ACh. MnCl2 inhibited the spontaneous membrane activity and contraction; however, low concentrations of MnCl2 increased the caffeine- or NA-induced contraction in Ca-free solution. When the Ca-tension relationship was observed in saponin-treated skinned muscles, the minimum concentration of Ca required to produce the contraction was 10(-7)M, as observed in other vascular tissues. Caffeine released the stored Ca, Na and ACh had no effect on the Ca release and procaine inhibited the caffeine-induced Ca release in skinned muscles. It was concluded that the membrane properties of smooth muscle cells in the portal vein are much the same as observed in other spontaneously active visceral muscles. Differences observed in the actions of NA and ACh on mechanical properties seems to be mainly due to different receptor-operated Ca release mechanisms.

Effect of pinaverium bromide on electrical and mechanical activity of smooth muscle cells.

Pinaverium bromide exerts antagonistic effects on the contractions induced by BaCl2 in intestinal smooth muscle. We have therefore investigated its effects on the electrical and mechanical activity and on the 45Ca exchange of guinea-pig taenia coli and ileum. In the concentration range 10−7 to 10−5 M this drug does not exert an effect on the resting potential of either preparation. At 10−6 M no effects could be observed on the spontaneous electrical activity in taenia coli. However, in the ileum the frequency of the action potentials, as well as their amplitude and maximum rate of rise and of repolarization were significantly reduced, causing inhibitory effects on the concomitant mechanical responses. This effect is more pronounced at 10−5 M, and in some tissues the spontaneous activity is completely abolished. Similar effects have been observed in taenia coli at this concentration of pinaverium bromide. The contractions induced by K-depolarization and by 10−5 M carbachol are inhibited in both preparations in a dose-dependent way. Also the contraction induced by carbachol in Ca free solution, as well as the restoration of that contraction by exposing the tissues to Ca containing solutions are reduced in the presence of 10−5 M pinaverium bromide. Pinaverium also inhibits the stimulation of the 45Ca efflux induced by K-depolarization and by stimulation with 10−5 M carbachol. In rabbit ear artery it was found that the contraction induced by noradrenaline is much less sensitive to pinaverium bromide than that induced by depolarization. It was also observed that this substance does not significantly affect the noradrenaline induced contraction in Ca free solution, neither does it prevent the restoration of that contraction by exposure of the tissue to Ca containing solutions. These effects are similar to those observed with other Caentry blocking agents, and therefore suggest that pinaverium bromide mainly acts by blocking voltage-dependent Ca-channels.

Effects of calcium and manganese ions on mechanical properties of intact and skinned muscles from the guinea-pig stomach.

To investigate the mechanism of generation of contractions in tissues from the guinea-pig stomach, the effects of caffeine procaine, acetylcholine(ACh) diltiazem or MnCl2 on the contraction evoked from small bundles of intact or skinned muscles were observed. The antrum possesses multiple Ca channels for induction of the Ca influx. In physiological solution, Mn and diltiazem inhibited the spontaneously generated contraction presumably due to suppression of the voltage-dependent Ca-influx. In the Ca-free polarized and depolarized muscles after depletion of stored Ca, the influx of Ca by addition of Ca may relate to at least three different channels. Furthermore, some of the Ca influx directly increases the amount of free Ca, while a part of Ca is sequestered into the store site and may activate the Ca-induced Ca-release mechanism, thus increasing the amount of free Ca. These results indicate that the mode of Ca in the muscle cell of the antrum is not the same as that in the mesenteric artery. Reduction in [Na]o transiently enhanced the amplitude and prolonged the duration of the Ca- and caffeine-induced contraction in Ca-free solution. Presumably these effects may relate to accelerated leakages of Ca from the store sites, as estimated from skinned muscles and inhibition of the Ca extrusion from the cell. MnCl2 acts on the surface membrane and suppresses most of the Ca influxes, except for some fractions directly sequestered into the store site. If MnCl2 penetrates the cell membrane, such as occurs in Ca-free solution, the interaction of Ca and its receptors of contractile proteins is accelerated, and the contractile protein is activated.

Effect of isoprenaline on intracellular Ca uptake and on Ca influx in arterial smooth muscle.

We have investigated the effect of isoprenaline on the amplitude of the transient contraction of isolated arteries induced by releasing the intracellular Ca with an agonist during incubation in a Ca-free solution. Under some conditions this force development is an indication of the amount of Ca in the intracellular store. Loading the store in high K+ solution in the presence of isoprenaline increases the amplitude of the subsequent contraction in Ca-free solution by 6 to 16% as compared to the control. The presence of isoprenaline during the loading procedure in the control solution with 5.9 mM K+ does not affect the amplitude of the subsequent contraction of the rabbit ear artery. In the rabbit coronary artery such pretreatment with isoprenaline inhibits the contraction to 40% of the control. These results indicate that beta-agonists may stimulate the Ca uptake in the intracellular store and, depending on the tissue, inhibit the influx of Ca.

The action of acetylcholine and catecholamines on an intracellular calcium store in the smooth muscle cells of the guinea-pig taenia coli.

1. The role of an intracellular Ca store in excitation-contraction coupling was studied by recording isometric tension development of thin strips (100-150 micron in diameter) of taenia coli incubated in Ca-free solution containing 2 mM-EGTA. 2. The smooth muscle cells of taenia coli do not contract during exposure to a K+-rich and Ca-free solution. However a contractile response can be elicited by acetylcholine or carbachol at concentrations exceeding 10(-6) M. These contractions are probably induced by a release of intracellular Ca. Ca is also released from the same store, although less effectively, by histamine and caffeine. 3. The amount Ca in the intracellular store, as revealed by the magnitude of the carbachol contraction in Ca-free solution, increases after contractions have been induced by high (K+)0 or by solutions containing low concentrations of carbachol. This contraction amplitude decreases after stimulation with a high concentration of carbachol. The amount of Ca in the filled store is sufficient for a near-maximal contraction. 4. The activation of beta-receptors during a K+-depolarization reduces the height of the contracture and induces a carbachol response in Ca-free solution which is higher than that obtained after a preceding K+ depolarization without isoprenaline. This observation indicates that an increased uptake of Ca into the carbachol-sensitive store contributes to the relaxing effect of beta-agonists. 5. In the tissues which have been loaded with 45Ca in a K+-depolarizing solution, a release of Ca into Ca-free solution is observed when the muscle is stimulated with carbachol, but not when it is stimulated with Ca-free high K+. The release is larger when isoprenaline was present during the loading with 45Ca. 6. The removal of Na+ from the solution exerts a complex and unexplained action on the Ca store. Substitution of Na+ by Tris+ and by K+ have similar effects. 7. It is concluded that the smooth muscle cells of the guinea-pig taenia coli have an intracellular store of Ca which participates in excitation-contraction coupling. The store is sensitive to muscarinic agonists, beta-agonists and monovalent ions, but is not affected by depolarization of the outer membrane.

Mobilization of stored calcium for phasic contraction induced by norepinephrine in rabbit aorta

In Ca-free solution norepinephrine (NE) produced only a phasic contraction in the media-intima layer of rabbit aorta. The second application of NE was almost ineffective. Incubation of the muscle with Ca for a short period (Ca loading) restored the ability to produce a phasic contraction in Ca-free solution. Various ions and compounds were applied to the muscle prior to the Ca loading or prior to NE application and it was found that these treatments variously affected the NE-induced phasic contraction. The data suggest that the NE-induced phasic contraction in Ca-free solution results from Ca release from cellular sites and that a hyperbolic relationship exists between the amount of Ca at these sites and the magnitude of the contraction. These sites take up extracellular Ca by a process sensitive to La 3+ but not to D600; the Ca influx stimulated by high K solution, which is sensitive to D600 also contributes to refilling of these sites; NE releases this stored Ca by a process sensitive to procaine, caffeine and theophylline and, in this manner, elicits a phasic contraction.