Ca-induced Ca Release Mechanism Research Articles

Alterations by age of calcium handling in rat resistance arteries.

This study investigated the alterations by age of Ca handling in rat small mesenteric arteries. The contractile responses to phenylephrine and caffeine in small mesenteric arteries from young and old rats were studied in Ca -containing and Ca -free medium. In Ca -containing medium the contraction to phenylephrine (100 micro M) but not to caffeine (10 mM) was greater in old than in young rats. Concentration-response curve to phenylephrine was affected to the same extent by nifedipine (1 micro M) in both age groups, whereas ryanodine (20 micro M) decreased the maximal response to phenylephrine only in young rats. These results suggest the participation of intracellular Ca handling on the observed differences by aging. In Ca -free medium, phenylephrine (10 micro M) but not caffeine (10 mM) induced a greater contraction in old than in young animals, corroborating the results obtained in Ca -containing solution. The greater response to phenylephrine observed in old rats cannot be explained by an increase in the inositol 1,4,5-trisphosphate (IP ) formation because the accumulation of inositol phosphates by phenylephrine was not affected by aging. Results obtained in Ca -free medium using caffeine after phenylephrine or vice versa suggest a common intracellular Ca pool. Pretreatment with ryanodine in Ca -free medium almost abolished contractile response to phenylephrine and caffeine in young rats but only partially decreased them in old animals, suggesting an impairment in the Ca -induced Ca release (CICR) mechanism leading to an increase in the stored Ca content. The greater amount of stored Ca could explain the higher contractile response to phenylephrine observed in aged rats. As a consequence of all these changes due to aging, an imbalance between the two Ca release mechanisms from sarcoplasmic reticulum was observed with a major role of Ca induced release by IP at the expense of an impairment of CICR mechanism. This observation will also help explain the results obtained in the presence of extracellular Ca, where phenylephrine induced a greater maximum response in old animals in spite of a decrease in the midrange sensitivity to this agonist.

Plasma albumin induces calcium waves in rat cortical astrocytes

Changes in intracellular calcium were monitored in cultured cortical astrocytes stimulated with albumin. Albumin elicited intracellular calcium mobilisation from intracellular stores, inducing repetitive intracellular calcium oscillations. The oscillations were not blocked by ryanodine, a blocker of the Ca-induced Ca release mechanism, and the release occurred from the same store as is accessed by glutamate and bradykinin, both of which release calcium by an IP3-dependent mechanism. Calcium signals induced by albumin appear therefore to occur via a pure IP3-dependent mechanism. When albumin was applied to confluent monolayers of astrocytes, the oscillations in individual cells were initially unsynchronised, but after several minutes of application, the Ca2 oscillations were observed to synchronise and spread through the astrocyte network as a wave. These intercellular calcium waves were inhibited by the gap junction blocker halothane. Using the fluorescence recovery after photobleaching (FRAP) technique, we demonstrate that the development of propagated waves with prolonged exposure to albumin does not result from an increase in cell coupling. The development of calcium waves on exposure to albumin may be important in the formation of glial scars in the CNS after breakdown of the blood-brain barrier.

Ca ions and potassium contracture in the retractor unguis muscle of the cockroach.

Potassium contracture consists of a phasic and a sustained component. Extracellular Ca ions are essential for both components. In the phasic component the Ca-induced Ca release mechanism is activated by the entry of the Ca ions.

Ca regulation in cardiac muscle

Contraction in cardiac muscle is activated by a combination of Ca influx via sarcolemmal Ca channels and release of Ca from the sarcoplasmic reticulum (SR). The release of Ca from the SR appears to be triggered by Ca influx via a Ca-induced Ca-release mechanism. The amount of Ca influx and Ca released from the SR vary with conditions and with different cardiac muscle preparations. In contrast to the Ca-induced Ca-release process in cardiac muscle, excitation-contraction coupling in skeletal muscle appears to be via a more direct coupling between sarcolemmal voltage sensors and the Ca release (sometimes referred to as depolarization-induced Ca-release). During cardiac relaxation Ca is removed from the cytoplasm by the SR Ca-pump and the sarcolemmal Na/Ca exchange (with the former being somewhat more dominant). Ca extrusion from the cell during both relaxation and diastole is predominantly via Na/Ca exchange, with the sarcolemmal Ca ATPase pump contributing very little. Thus, sarcolemmal Na/Ca exchange appears to be critically involved in the overall regulation of cardiac cellular Ca content.

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells.

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.

Blockade by the local anaesthetic, tetracaine, of desensitization of Ca‐induced Ca release after muscarinic stimulation in smooth muscle

1. Desensitization of contractile responses dependent on release of intracellularly stored Ca elicited by carbachol, histamine or caffeine was measured after desensitizing treatment with carbachol or histamine in the presence or absence of local anaesthetics in Ca-free solution containing 2 mM EGTA in the smooth muscle of guinea-pig taenia caecum. 2. Histamine-induced homologous desensitization was inhibited by tetracaine and procainamide. Dibucaine did not exert an inhibitory effect on the desensitization. This is consistent with our previous findings concerning the effects of local anaesthetics on the desensitization of histamine H1-receptors measured under normal physiological conditions. 3. Carbachol induced a functional change of intracellular Ca stores which resulted in heterologous desensitization. Tetracaine completely blocked carbachol-induced desensitization of the caffeine-elicited contraction, but in the case of carbachol-induced desensitization of carbachol- and histamine-elicited contractions, this blocking effect of tetracaine was very weak and absent, respectively. The other local anaesthetics used did not affect the desensitization. These results suggest that the Ca-induced and inositol trisphosphate-induced Ca release mechanisms were both desensitized by carbachol and that the desensitization of the Ca-induced Ca release mechanism was selectively blocked by tetracaine.

P-362 - Potentiation of Ca-induced Ca release mechanism by methylxanthines. Caffeine vs. others.

P-362 - Potentiation of Ca-induced Ca release mechanism by methylxanthines. Caffeine vs. others.

Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci.

Ca2+ dependence of the inositol 1,4,5-trisphosphate (IP3)-induced Ca release was studied in saponin-skinned smooth muscle fiber bundles of the guinea pig taenia caeci at 20-22 degrees C. Ca release from the skinned fiber bundles was monitored by microfluorometry of fura-2. Fiber bundles were first treated with 30 microM ryanodine for 120 s in the presence of 45 mM caffeine to lock open the Ca-induced Ca release channels which are present in approximately 40% of the Ca store of the smooth muscle cells of the taenia. The Ca store with the Ca-induced Ca release mechanism was functionally removed by this treatment, but the rest of the store, which was devoid of the ryanodine-sensitive Ca release mechanism, remained intact. The Ca2+ dependence of the IP3-induced Ca release mechanism was, therefore, studied independently of the Ca-induced Ca release. The rate of IP3-induced Ca release was enhanced by Ca2+ between 0 and 300 nM, but further increase in the Ca2+ concentration also exerted an inhibitory effect. Thus, the rate of IP3-induced Ca release was about the same in the absence of Ca2+ and at 3 microM Ca2+, and was about six times faster at 300 nM Ca2+. Hydrolysis of IP3 within the skinned fiber bundles was not responsible for these effects, because essentially the same effects were observed with or without Mg2+, an absolute requirement of the IP3 phosphatase activity. Ca2+, therefore, is likely to affect the gating mechanism and/or affinity for the ligand of the IP3-induced Ca release mechanism. The biphasic effect of Ca2+ on the IP3-induced Ca release is expected to form a positive feedback loop in the IP3-induced Ca mobilization below 300 nM Ca2+, and a negative feedback loop above 300 nM Ca2+.

Calcium-induced calcium release mechanism in guinea pig taenia caeci.

Fura-2 was used to measure the amount of Ca released from the intracellular Ca store of a saponin-skinned smooth muscle fiber bundle of the guinea pig taenia caeci (width, 150-250 microns) placed in a capillary cuvette at 20-22 degrees C. The amount of Ca actively loaded into the store was assayed when released by the application of 50 mM caffeine and/or 10 microM inositol 1,4,5-trisphosphate (IP3) in the absence of ATP, and was found to have a biphasic dependence on the loading [Ca2+] with a peak near pCa 6. After Ca loading at pCa 6, IP3 released almost all the releasable Ca, whereas caffeine discharged Ca from only approximately 40% of the store. The maximum amount of Ca in the store was some 220 mumol/liter cell water. Ca in the caffeine-releasable store was released approximately exponentially to zero with time when Ca2+ was applied in the absence of ATP, and the rate constant of the Ca-induced Ca release (CICR) increased steeply with the concentration of Ca2+ applied. Increase in [Mg2+] (0.5-5.0 mM) or decrease in pH (7.3-6.7) shifted the relation between pCa and the rate of CICR roughly in parallel toward the lower pCa. An adenine nucleotide increased the rate of the CICR, but it did not change the range of effective [Ca2+]. 5 mM caffeine greatly enhanced the CICR mechanism, making it approximately 30 times more sensitive to [Ca2+]. However the drug had no Ca-releasing action in the absence of Ca2+. Procaine in millimolar concentrations inhibited the rate of the CICR. These properties are similar to those of the skeletal muscle CICR and ryanodine receptor channels. Rates of the CICR under a physiological ionic milieu were estimated from the results, and a [Ca2+] greater than 1 microM was expected to be necessary for the activation of the Ca release. This Ca sensitivity seems too low for the CICR mechanism to play a primary physiological role in Ca mobilization, unless assisted by other mechanisms.

Ca-induced Ca release from the sarcoplasmic reticulum of isolated myofibrillar bundles of barnacle muscle fibres

Isometric tension and aequorin light were recorded from isolated myofibrillar bundles (diameter 0.2 mm) of barnacle muscle fibres to examine Ca release from the sarcoplasmic reticulum (SR). Transfer of a bundle from a pCa 6.7 solution to a pCa 5.8 solution, both buffered with 0.1 mM EGTA, resulted in a phasic increase in myofibrillar free Ca2+ which was superimposed on a slow rise to a steady level and a fast rise in tension. The peak of the free Ca2+ response was higher than the free Ca2+ in the bulk solution. Treatment of the bundle with the detergent Brij to destroy the SR membranes abolished the phasic rise in Ca2+ and considerably reduced the amplitude of contraction. A second challenge of a bundle to the pCa 5.8 solution without prior reloading of the SR Ca store gave a much reduced phasic component. When a pCa 5.8 solution with 1.0 mM EGTA buffering was used, the phasic rise in myofibrillar free Ca2+ could not be detected and the rise in tension was four times slower than with 0.1 mM EGTA. The results are consistent with the operation of a Ca-induced Ca release mechanism in the SR membrane of this crustacean muscle.

Ca-induced Ca release in malignant hyperthermia-susceptible pig skeletal muscle.

To provide information regarding the cause of the muscle rigidity in malignant hyperthermia-susceptible (MHS) pigs, the Ca-induced Ca-release mechanism of the sarcoplasmic reticulum (SR), the Ca uptake by the SR, and the Ca-activated tension production of the contractile system were examined in skinned skeletal muscle fibers from MHS and normal pigs. In muscles of MHS pigs, the rate of Ca-induced Ca release was significantly higher than in normal muscle. The potentiation effect on Ca-induced Ca release by halothane and caffeine did not differ appreciably between MHS and normal fibers. The rate of Ca uptake by the SR and the Ca sensitivity of the contractile system of MHS fibers were not different from those of normal fibers, and halothane in an anesthetic concentration exerted no effect on them. Dantrolene inhibited the Ca-induced Ca release at 38 degrees C. These results suggest that the principal cause of malignant hyperthermia (MH) in MHS pigs is due to the enhancement of the Ca-induced Ca-release mechanism of the SR of the skeletal muscle.

S1-2 - The pharmacological properties of the Ca-induced Ca release mechanism and its role in Ca mobilization

S1-2 - The pharmacological properties of the Ca-induced Ca release mechanism and its role in Ca mobilization

Change in intracellular calcium ion concentration induced by caffeine and rapid cooling in frog skeletal muscle fibres.

In a single skeletal muscle fibre treated with concentrations of caffeine below threshold for caffeine contracture, rapid lowering of the temperature of the bathing solution from 18 degrees C to below 5 degrees C induced a contracture (rapid cooling contracture). Intracellular Ca2+ concentration ([Ca2+]i) was recorded during rapid cooling contracture using aequorin. Low concentrations of caffeine often caused a slight elevation of the light signal in resting muscle without detectable tension. During rapid cooling contracture, the change in light signal occurred in three phases. The first phase was a transient change of [Ca2+]i accompanying slight tension. During the second phase, the light signal slowly increased as cooling produced maximum tension development. The third phase was an additional light signal induced after the second phase, even though the tension was saturated. The second and third phases were more sensitive to low concentrations of procaine (0.2-0.5 mM) than the first phase. Synchronous oscillations of light and tension were often observed during the second phase. The light signal during rapid cooling contracture was only slightly affected by long incubation in Ca-free or Ca-rich solutions. These results are interpreted as follows. A low concentration of caffeine elevates cytoplasmic resting Ca2+ level without tension development. The oscillations of light and tension often observed in the second phase might represent a cyclic release of Ca2+ from the sarcoplasmic reticulum (s.r.). The third phase is considered to be due to a massive Ca2+ release by a Ca-induced Ca-release mechanism which might be similar to that in skinned fibres. The second phase is probably essential for generation of rapid cooling contracture tension and the third phase represents an excess Ca2+ for tension development.

Calcium-induced calcium release mechanism in vascular smooth muscles--assessments based on contractions evoked in intact and saponin-treated skinned muscles.

This article was concerned with the role of Ca in triggering the contraction in vascular smooth muscles. Whenever Ca influx is activated, this Ca does not directly activate the contractile proteins, but rather triggers the release of Ca from the SR to activate calmodulin. This release of Ca by Ca is dependent on the amount of Ca stored within the cells. Voltage dependent Ca influx activated by excess concentrations of K, electrical depolarization and Ca spikes is required to produce the contraction through activation of the Ca-induced Ca release mechanism. The elucidation of the contribution of the P-I response for Ca mobilization through activation of receptors under physiological conditions hopefully will lend support to our hypothesis.

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.

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.

Effects of ATP and related compounds on the Ca-induced Ca release mechanism of the Xenopus SR.

The effects of ATP and related compounds on the Ca2+ release mechanism of the sarcoplasmic reticulum (SR) was studied by using skinned skeletal muscle fibers of Xenopus laevis. ATP evoked marked Ca2+ release at very low level of Mg2+. beta, gamma-Methylene analogue of ATP was almost as effective as ATP, which suggests Ca2+ release evoked by ATP is elicited without ATP hydrolysis. ADP and AMP also evoked Ca2+ release from the SR, but the effect of them became gradually weaker than that of ATP as the number of phosphates decreased. CTP, UTP and ITP were less potent than ATP. Adenosine also evoked more effective Ca2+ release than inosine. The compounds with adenine base, therefore, seem to elicit more potent Ca2+ release than those which have the same number of phosphates but do not consist of adenine base. AMP and Ca2+ ion evoked Ca2+ release synergistically, and the Ca2+ release responses evoked by ATP and related compounds showed the same pharmacological characteristics as Ca-induced Ca release. So, these Ca2+ release responses are construed as the manifestation of the same mechanism as Ca-induced Ca release. Effective concentration range of ATP and the effect of pyrophosphate on Ca2+ release evoked by ATP suggest that neither the high affinity ATP catalytic site of (Ca2+ + Mg2+) ATPase of the SR nor the low affinity ATP binding site, reported by Dupont (1977), is implicated in the enhancement of these Ca2+ release responses from the SR.

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.

Intracellular Ca release in skinned smooth muscle.

The release of internal Ca from saponin-treated skinned smooth muscle of guinea pig taenia caecum was studied. The amount of Ca released was estimated by the area under the contraction curve during treatment with 25 mM caffeine in the presence of 0.1 mM EGTA. The magnitude of the caffeine response in skinned muscle, after loading with 10(-6) M Ca for 3 min, was similar to that in the depolarized muscle in the presence of EGTA before treatment with saponin. This suggests that Ca in the skinned muscle was in a physiological range after loading. The release of Ca from the storage site could be facilitated by Ca itself when the skinned muscle was exposed to Ca above 3 x 10(-6) M. An increase in environmental MG concentration suppressed the Ca-induced Ca release mechanism. Sudden replacement of propionate with Cl in the bathing solution made it possible to release Ca from the storage site. This "depolarization"-induced Ca release occurred only immediately after the application of Cl; thereafter, the Ca release mechanism seemed to be inactivated by the prolonged presence of Cl. These results suggest that two mechanisms of Ca release operate in smooth muscle: (a) release induced by Ca itself, and (b) release by "depolarization".

Mechanisms of relaxation induced by activation of β‐adrenoreceptors in smooth muscle cells of the guinea‐pig mesenteric artery

Relaxation of smooth muscle cells induced by activation of beta-adrenoceptors was investigated in intact and skinned muscles of the guinea-pig mesenteric artery.1. In concentrations over 10(-7) M, isoprenaline reduced the resting tone of intact preparations and also the amplitude of K contractions. When Ca was applied after previous superfusion with Ca-free solution, the amount of Ca accumulated into storage sites was increased by isoprenaline in polarized and depolarized ([K](o) 128 mM) muscles. The amount of Ca stored increased even further when procaine and isoprenaline were applied simultaneously during store loading.2. Isoprenaline increased the concentration of cyclic AMP as determined by radioimmunoassay. Application of isoprenaline at a concentration of 10(-7) M increased cyclic AMP from 2.2+/-0.3 to 2.8+/-0.6 p-mole/mg wet weight and at 10(-6) M increased it to 4.5+/-0.8 p-mole/mg wet weight after 5 min incubation (n = 4).3. Application of cyclic AMP (3 x 10(-6) M) with cyclic AMP-dependent protein kinase (50 mug/ml.) had no effect on the pCa-tension relationship in the skinned muscles. However, an increased concentration of cyclic AMP (> 10(-5) M) suppressed the Ca-induced concentration only in the presence of protein kinase. This protein kinase (50 mug/ml.) alone had no effect on the Ca-induced contraction.4. In skinned fibres, the Ca store could be loaded by applying low concentrations of Ca. If cyclic AMP (3 x 10(-6) M) with protein kinase (50 mug/ml.) was applied during the loading procedure, the amount of Ca accumulated by the store increased if the loading solution contained 10(-6) M-Ca applied for 2 min or less, but if the loading solution was applied for 3 min, or if higher Ca concentrations were used, the presence of cyclic AMP with protein kinase decreased the store size, suggesting that a Ca-induced Ca-release mechanism was also being activated.5. In skinned muscles, accumulation of Ca into the store site in the presence of cyclic AMP (3 x 10(-6) M) with protein kinase (50 mug/ml.) was further accelerated by simultaneous applications of procaine (5 mM), as here the Ca-induced Ca-release mechanism was suppressed.6. These results indicate that activation of beta-adrenoceptors by isoprenaline increases the amount of cyclic AMP in the intact muscles, and leads to an increase in Ca accumulation into the store site. In the skinned muscles, the Ca-induced Ca-release mechanism is activated by cyclic AMP and the Ca receptor for contraction (leiotonin C or calmodulin) is somewhat suppressed. These effects of exogenously applied cyclic AMP require the presence of protein kinase. The relaxation following beta-adrenoceptor activation is more likely to involve Ca extrusion from the cell and accumulation of Ca in internal storage sites than suppression of the binding of calmodulin with the myosin light chain kinase.