Multiple effects of caffeine on contraction and cytosolic free Ca2+ levels in vascular smooth muscle of rat aorta

Summary 1. Effects of caffeine on cytosolic Ca²⁺ level ([Ca²⁺]_cyt), measured simultaneously with muscle tension using fura-2-Ca²⁺ fluorescence, were examined in isolated smooth muscle of rat aorta. 2. Caffeine (20 mmol/l) induced a large transient increase in [Ca²⁺]_cyt followed by a plateau which was higher than resting level. However, muscle tension showed a transient increase followed by a decrease to or below the resting level. In Ca²⁺-free solution, caffeine induced only a transient increase in both [Ca²⁺]_cyt, and muscle tension. 3. At low temperature (22°C), high K⁺ (72.7 mmol/l) induced sustained increase in both [Ca²⁺]_cyt and muscle tension which were smaller than those observed at 37°C. At 22°C, however, caffeine-induced transient changes were greater than those observed at 37°C. 4. Ryanodine (10 mol/l) inhibited the transient changes due to caffeine but showed little effects on the sustained changes due to high K⁺. 5. During the sustained increase in [Ca²⁺]_cyt induced by noradrenaline (10 gmmol/l) or high K⁺ (140 mmol/l), addition of caffeine transiently increased [Ca²⁺]_cyt followed by a decrease to a level slightly lower than that before the addition of caffeine. In contrast to this, muscle tension transiently increased and then decreased to or below the resting level. 6. These results suggest that caffeine-induced contraction is due to the release of Ca²⁺ from cellular store. Caffeine also has an inhibitory effect which is partly attributable to decrease in [Ca²⁺ _cyt, and partly to the decrease in the sensitivity to Ca²⁺ of the contractile elements.Send offprint requests to H. Ozaki at the above address     

K+ channel openers,cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery

Summary The effects of K⁺ channel openers, cromakalim and an acetoxyl derivative of KRN 2391 (Ki 4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca²⁺]_i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP₃) production induced by the thromboxane A₂ analogue, U46619, in canine coronary arteries. Upon single dose applications of U46619 at 300 nmol/l, phasic and tonic increases in [Ca²⁺]_i and force were seen, which were almost abolished by cromakalim (10 mol/l) and Ki4032 (100 mol/l).In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction. Cromakalim (0.01–10 mol/l) and Ki4032 (0.1–100 mol/l) concentration-dependently inhibited the increases in [Ca²⁺]_i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K⁺ channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/l KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K⁺ channel openers inhibits the production of IP₃ and Ca ²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.Correspondence to T. Yanagisawa at the above address     

Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle

Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157–2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 M) decreased MLC phosphorylation and the amplitude of contractions induced by high K⁺ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic Ca²⁺ levels ([Ca²⁺]_i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 M) and prostaglandin F₂ (PGF₂, 10 M) without a change in the [Ca²⁺]_i. On the other hand, wortmannin (1 or 10 M) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF₂ to a level seen at rest. In the absence of external Ca²⁺, caffeine (20 mM) induced a transient increase in [Ca²⁺]_i and force with an increase in MLC phosphorylation. Wortmanmn completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca²⁺]_i. In the absence of external Ca²⁺, phenylephrine induced a small transient increase in [Ca²⁺]_i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca²⁺]_i and MLC phosphorylation. Wortmannin (1 M) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca²⁺]_i nor the sustained contraction. Wortmannin inhibited the Ca²⁺-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca²⁺-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca²⁺ or through the release of Ca²⁺ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF₂. induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.     

Inhibitory effects of caffeine on Ca2+ influx and histamine secretion independent of cAMP in rat peritoneal mast cells

• 1.1. Caffeine did not evoke Ca²⁺ mobilization and histamine secretion.
• 2.2. Caffeine, as well as other methylxanthines but not forskolin or 8 bromo-cAMP, inhibited Ca²⁺ responses from compound 48/80.
• 3.3. Evoked histamine secretion was severely reduced by caffeine but not by cAMP analogs.
• 4.4. In β-escin-permeabilized cells, caffeine did not affect resting and IP3-stimulated ⁴⁵Ca²⁺ release, but it inhibited Ca²⁺-induced histamine secretion.
• 5.5. These results indicate that caffeine inhibits Ca²⁺ influx and Ca²⁺ efficacy in the secretory apparatus independent of cAMP, resulting in the inhibition of secretagogs-evoked histamine secretion from rat mast cells.

     

Intracellular mechanism of quinidine action on muscle contraction

Summary The mechanism of quinidine action on rabbit cardiac and skeletal muscle was examined with functionally skinned muscle-fiber preparations. By using these preparations we could correlate measurements of muscle tension with the effect of quinidine on the Ca²⁺ activation of the contractile proteins and on the Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR). Effect of quinidine on the contractile proteins. Quinidine concentrations above 0.5 mmol/l increased the maximal Ca²⁺-activated tension development 12% for papillary muscle and 5% for soleus (slow-twitch). Adductor magnus (fast-twitch) showed no significant change. Quinidine (0.1–1.0 mmol/l) also increased the submaximal Ca²⁺-activated tension development for the three muscle types (papillary muscle=soleus>adductor magnus) and shifted the [Ca²⁺]-tension curves to the left in a dose-dependent fashion. Effects of quinidine on the Ca ²⁺ uptake and release from the SR. Sarcoplasmic reticulum of skinned fibers was loaded with Ca²⁺ (uptake phase), then Ca²⁺ was released by 25 mmol/l caffeine (release phase) giving a tension transient. The area under the tension transient was used to estimate the amount of Ca²⁺ released. Quinidine (>0.5 mmol/l) decreased the Ca²⁺ uptake (soleus>adductor magnus>papillary muscle) and increased the Ca²⁺ release [papillary muscle=soleus adductor magnus (only at 1.5 mmol/l, the highest concentration tested)] from the SR of all three muscles in a dose-dependent manner. Quinidine at low concentration (0.1 and 0.5 mmol/l) increased the caffeine-induced tension transient of papillary muscle and higher quinidine concentrations (1.0 and 1.5 mmol/l) decreased the caffeine-induced tension transient of soleus and adductor magnus during both the uptake and release phases. The decreased Ca²⁺ uptake of papillary muscle in 1.5 mmol/l quinidine was antagonized by increasing the free Mg²⁺ from 0.032 to 0.32 mmol/l.In summary, quinidine has similar mechanisms of action in all three muscles: increased Ca²⁺ activation of the contractile proteins, decreased Ca²⁺ uptake and increased Ca²⁺ release from the SR in functionally skinned muscle fibers. We conclude that quinidine-induced decreases in Ca²⁺ uptake by the SR could be responsible for quinidine-induced myocardial depression and that quinidine-induced increases in Ca²⁺ activation of the contractile proteins and Ca²⁺ release from the SR could be responsible for the increases in skeletal muscle contraction caused by quinidine.     

Doxorubicin-induced vasomotion and [Ca^2＋]i elevation n vascular smooth muscle cells from C57BL/6 mice

Aim:

To explore the action of doxorubicin on vascular smooth muscle cells.

Methods:

Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca²⁺]_i in isolated aortic smooth muscle cells was measured by using Fluo-3.

Results:

Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca²⁺]_i in single muscle cells. Treatment with 10 μmol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca²⁺ or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 μmol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 μmol/L increased the frequency of the RC only. In the presence of 100 μmol/L caffeine, however, 100 μmol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 μmol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca²⁺-free solution, doxorubicin induced a transient [Ca²⁺]_i increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca²⁺]_i was suppressed by nifedipine and potentiated by ryanodine and charybdotoxin.

Conclusion:

Doxorubicin not only releases Ca²⁺ from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca²⁺ into vascular smooth muscle cells.     

Voltage -dependent suppression of calcium current by caffeine in single smooth muscle cells of the guinea-pig urinary bladder

The suppressive action of caffeine on l,-type Ca current (Ica) in smooth muscle cells of the guinea-pig urinary bladder was investigated using the whole-cell patch clamp technique. Caffeine (5–30 mM) suppressed Ica, the effect having two phases: a rapid and transient suppression of Ica, which was followed by a sustained suppression. When intracellular Ca⁺ was strongly buffered by the Ca⁺ chelator EGTA (20 mM) or BAPTA (5 mM) in the patch pipette, the transient suppression of Ica was abolished, whereas the sustained effect remained. Similarly, inclusion of both 10 mM procaine and 1 mg/ml heparin in the patch pipette blocked the transient suppression of Ica, but did not block the sustained effect. The degree of the sustained effect of caffeine on Ica was dose-dependent with a _d of 20 mM. Application of the cyclic AMP analogue, 8-bromo-cyclic AMP (100 M) or forskolin (10 M) to the bath failed to mimick the sustained suppression of Ica, suggesting that inhibition of phosphodiesterase activity was not involved in the caffeine action. The steady-state activation curve remained unchanged by 10 mM caffeine but the steady-state inactivation curve was significantly shifted in the negative direction by 15.6 mV in 1.8 mM Ca²⁺ solution or by 10 mV in 1.8 mM Ba²⁺ solution. From these results it appears that caffeine inhibits L-type Ica via two mechanisms: (1) it releases Ca²⁺ from an internal store causing a transient Ca²⁺-mediated inactivation of the Ca channel; (2) it inhibits Ca channel via a mechanism that does not require such a Ca²⁺ release. It is possible that caffeine suppresses Ica through a preferential binding to the inactivated state of l-type Ca channel.     

Effects of 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on skinned myocardial fibres of the rat: reversible inhibition of calcium release from the sarcoplasmic reticulum

The effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is reported to inhibit the release of intracellularly stored Ca²⁺ in skeletal and smooth muscles, were examined in ventricular myocardia of the adult rat. In skinned papillary muscle fibres with functional sarcoplasmic reticulum (SR) preserved, application of 100 or 300 M TMB-8 during the Ca²⁺ loading period had no significant effect on the peak tension of subsequent caffeine-induced contraction, but when applied during exposure to caffeine, concentration-dependent reduction of the peak tension was observed. At 1000 M, TMB-8 reduced the peak tension of caffeine-induced contraction when applied either during Ca²⁺ loading or during exposure to caffeine. TMB-8 had no substantial influence on the Ca⁺-tension of skinned fibres without functional SR. In isolated papillary muscle preparations, TMB-8 prolonged the action potential duration and decreased the maximum rate of rise of potential, leading to abolition of contraction at 100 M. In conclusion, TMB-8 may be a useful pharmacological tool for inhibiting Ca²⁺ release from SR, but only in skinned myocardial preparations.     

Evidence for a non-precursor dopamine pool in noradrenergic neurones of the dog mesenteric artery

Summary Isolated tracheal strip-chain preparation of the guinea-pig was used to study the effect of temperature on carbachol-induced contraction. The preparation was suspended in the organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of bath temperature from 37°C to 20°C (cooling) caused a transient increase in tension and thereafter inhibited the contractile response of the trachea caused by carbachol (30 nmol/l–3 mol/l). Isosmotic potassium chloride (KCl, 64.7 mmol/l)-induced contraction or calcium chloride (CaCl₂, 0.1–3 mmol/l)-induced contraction in K⁺-depolarized muscle was markedly inhibited by cooling. Verapamil in concentrations of 1 mol/l or greater, which markedly depressed the CaCl₂-induced contraction, caused partial depression of the contractile response to carbachol.On the other hand, carbachol-induced contraction of the trachea which was incubated with K⁺-rich, verapamil (3 mol/l) containing Krebs solution and with Ca²⁺-free, EGTA (0.4 mmol/l) containing Krebs solution were both augmented at 20°C. From these observations, it is concluded that decreased responsiveness of the guinea-pig airway smooth muscle to carbachol with lowered temperature may be due to an inhibition of Ca²⁺ influx through voltage-dependent Ca²⁺ channels which involves part of the contraction.     

cAMP- and diacylglycerol-mediated pathways elevate cytosolic free calcium concentration via dihydropyridine-sensitive, ω-conotoxin-insensitive calcium channels in normal rat anterior pituitary cells

Summary The effect of 8-bromocyclic AMP (8-Br-cAMP) and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, on cytosolic free calcium concentration ([Ca²⁺]_i) in normal rat anterior pituitary cells was examined. [Ca²⁺]_i was monitored directly by the fluorescent indicator fura-2. 8-Br-cAMP as well as PMA elevated [Ca²⁺]_i in a concentration-dependent manner. Forskolin (10 mol/l), which activates adenylate cyclase, and 1-oleoyl-2-acetyl-glycerol (10 mol/l), another activator of protein kinase C, also increased [Ca²⁺]_i. Both the 8-Br-cAMP (2 mmol/l)- and the PMA (100 nmol/l)-induced increase in [Ca²⁺]_i was dependent on the presence of extracellular calcium and could be inhibited by the calcium channel blockers Mg²⁺ and nifedipine, but not by onotoxin (100 nmol/l). The half-maximally inhibitory concentrations of Mg²⁺ and nifedipine were about 12 mmol/l and 160 nmol/l, respectively, for the [Ca²⁺]_i response to 8-Br-cAMP (2 mmol/l), and were about 6 mmol/l and 400 nmol/1, respectively, for the PMA (100 nmol/1)-induced increase in [Ca²⁺]_i. The sodium channel blocker tetrodotoxin (5 mol/l) had no influence on the effect of 8-Br-cAMP (2 mmol/l) or PMA (100 nmol/l) on [Ca²⁺]_i. After pretreatment for 3 min with PMA (100 nmol/l), the subsequent K⁺ (100 mmol/l)- or arachidonic acid (3 mol/l)-induced increase in [Ca²⁺]_i was decreased by about 50%. By contrast, pretreatment (3 min) with 8-Br-cAMP (2–10 mmol/1) markedly enhanced the subsequent [Ca²⁺]_i response to K⁺ (100 mmol/l), and left the effect of arachidonic acid (3 mol/l) on [Ca²⁺]_i unimpaired. These data indicate that both cAMP- and diacylglycerol-mediated pathways increase [Ca²⁺ _i in normal rat anterior pituitary cells via an influx of extracellular Ca²⁺ through dihydropyridine-sensitive, -conotoxin-insensitive voltage-dependent calcium channels. These second messengers may thus be involved in Ca²⁺ channel activation by hypothalamic releasing hormones. Effects of cAMP- or diacylglycerol-induced pathways on anterior pituitary function may not be independent of but be mediated also by changes in [Ca²⁺]_i. However, substantial differences appear to exist in how cAMP and diacylglycerol influence voltage-dependent calcium channels.     

Inhibition of protein kinase C-mediated contraction by Rho kinase inhibitor fasudil in rabbit aorta

Protein kinase C (PKC) activation by a phorbol ester increases myosin light chain (MLC₂₀) phosphorylation through inhibition of MLC phosphatase (MLCP) and enhances contraction of vascular smooth muscle. We investigated whether Rho kinase, which is known to inhibit MLCP, is involved in the MLC₂₀ phosphorylation caused by a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), in rabbit aortas. DPB (1 M) increased MLC₂₀ phosphorylation and tension. The Rho kinase inhibitor fasudil (10 M) inhibited the DPB-induced contraction and decreased the MLC₂₀ phosphorylation at Ser19, a site phosphorylated by MLC kinase, although it did not affect the phosphorylation of total MLC₂₀. Rinsing a 65.4 mM KCl-contracted aorta with Ca²⁺-free, EGTA solution caused rapid dephosphorylation of MLC₂₀ and relaxation. When DPB was present in the rinsing solution, the MLC₂₀ dephosphorylation and the relaxation were inhibited. In this protocol, Ro31-8220 (10 M), a PKC inhibitor, suppressed the phosphorylation of total MLC₂₀ and Ser19 induced by DPB. Fasudil also inhibited the Ser19 phosphorylation to a degree similar to Ro31-8220 and accelerated relaxation, which was less than the relaxation caused by Ro31-8220. The phospholipase A₂ inhibitor ONO-RS-082 (5 M) inhibited the DPB-induced Ser19 phosphorylation but only transiently decreased the tension, suggesting the involvement of arachidonic acid in the phosphorylation and the existence of a MLC₂₀ phosphorylation-independent mechanism. When fasudil was combined with ONO-RS-082, fasudil exerted additional inhibition of the tension without further inhibition of the Ser19 phosphorylation. DPB phosphorylated the 130 kDa myosin binding subunit (MBS) of MLCP and fasudil inhibited the phosphorylation. These data suggest that the inhibition by fasudil of DPB-induced contraction and phosphorylation of MLC₂₀ at the MLC kinase-targeted site is a result of inhibition of Rho kinase. Thus, the PKC-dependent Ca²⁺-sensitization of vascular smooth muscle involves Rho kinase. A MLC₂₀ phosphorylation-independent mechanism is also involved in the Ca²⁺-sensitization.     

Mechanisms of vasorelaxation induced by N-allylsecoboldine in rat thoracic aorta

N-Allylsecoboldine was shown to be the most effective of several boldine derivatives that were tested for their vasorelaxing effect on the rat aorta. In KCl (60 mmol/l) medium, Ca²⁺ (0.03–3 mmol/l)-induced vasoconstriction was inhibited, concentration-dependently, by N-allylsecoboldine. The IC₅₀ for N-allylsecoboldine was calculated to be about 4 mol/l (for a Ca²⁺ concentration of 1 mmol/1). The vasorelaxant effect on KCl-induced responses was more pronounced at 60 mmol/l KCl than at 15 mmol/1 KCI. Contraction of rat aorta in response to phenylephrine (0.01-100 mol/l) was concentration-dependently inhibited by N-allylsecoboldine and by verapamil (3–30 mol/l), while contraction in response to B-HT 920, serotonin or PGF₂ was not affected. This relaxing effect of N-allylsecoboldine persisted in endothelium-denuded aorta. In cultured A 10 vascular smooth muscle cells, N-allylsecoboldine and verapamil displaced the binding of [³H]-prazosin (K _i values = 0.4±0.2 and 0.6±0.2 mol/l, respectively). The increase of inositol monophosphate caused by phenylephrine in rat aorta was completely suppressed by chloroethylclonidine, but only slightly inhibited by N-allylsecoboldine and by verapamil. Glibenclamide or charybdotoxin did not affect the relaxation induced by N-allylsecoboldine of aortic rings precontracted with phenylephrine. Neither the cGMP nor the cAMP content was changed by N-allylsecoboldine. We conclude that N-allylsecoboldine relaxes the rat aorta by blocking Ca²⁺ channels and that it also has an antagonistic effect at ₁-adrenoceptors. Correspondence to: C.M. Teng at the above address     

Cooling-induced contraction of trachea isolated from normal and sensitized guinea-pigs

Summary Fast (–7°C/min) cooling of guinea-pig isolated trachea produced a rapidly developing, transient contraction followed by relaxation. Cooling-induced contraction was dependent on temperature (30, 20 or 10°C) and responses in trachea obtained from actively sensitized guinea pigs were significantly greater (20 and 10°-C) than those observed in normal trachea. Cooling to 20°C was selected for subsequent experiments. Pre-treatment with sufficient concentrations of atropine, clemastine, cromoglycate, indomethacin, or nordihydroguaiaretic acid did not depress contraction to cooling in either normal or sensitized trachea. This indicates a direct effect of cooling. The contraction. produced by cooling was resistant to verapamil (1 mol/l) or dantrolene (0.3 mmol/l). Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium; all of them at 10–100 mol/l) inhibited contraction in sensitized and normal trachea. Activators of protein kinase C (phorbol 12,13-diacetate, 1 mol/l) enhanced while inhibitors (H-7, 20 mol/l; staurosporine, 10 mol/l) depressed cooling-induced contraction in both normal and sensitized tissues. Incubation (20 min) in a Ca²⁺ -free solution inhibited cooling-induced contraction in normal but not in sensitized trachea. Exposure to a low Na⁺ (25 mmol/l) or a K⁺-free medium abolished contraction to cooling in normal and sensitized trachea. Ouabain (0.1–10 mol/l) and vanadate (0.01–5 mmol/l) inhibited cooling-induced-contraction to a greater extent in normal than in sensitized trachea. Polymyxin B (0.5 mmol/l) selectively depressed responses to cooling in sensitized trachea. In a separate series of experiments, it was shown that sensitized trachea was hyperresponsive to ouabain and vanadate. Previous cooling to 20°C abolished responses to ouabain but only attenuated those to vanadate. These results are compatible with an enhancement of Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities in sensitized trachea and further support the notion that intracellularly stored Ca²⁺ plays a decisive role in the activation of sensitized tracheal muscle. Send offprint requests to J. L. Ortiz at the above address     

Levcromakalim-induced modulation of membrane potassium currents,intracellular calcium and mechanical activity in rat mesenteric artery

In freshly-dispersed cells from rat mesenteric artery, levcromakalim (1 and 10 M) induced a non-inactivating potassium current (I_KCO), an event which was associated with increased current noise. I_KCO was fully inhibited in the presence of 10 M glibenclamide. Stationary fluctuation analysis of the current noise associated with I_KCO induced by levcromakalim at a holding potential of –10 mV indicated that the unitary conductance of the underlying K-channels was 10.2 pS at 0 mV under the quasi-physiological conditions of the experiment.In isolated arterioles both levcromakalim (10 nM - 10 M) and nifedipine (10 nM - 10 M) each elicted full, concentration-dependent, parallel reductions of the increases in [Ca²⁺]_i (assessed using fura-2) and tension induced by 10 M noradrenaline. However, the effects of both drugs on KCl-induced increases in tension and in [Ca²⁺]_i, did not follow a simple relationship. Levcromakalim relaxed KCl- and noradrenaline-induced sustained contractions with a similar potency. This was in contrast to nifedipine which was approximately 20 times more potent against KCl-induced contractions.It is concluded that levcromakalim relaxes rat mesenteric arterioles primarily by the opening of a small conductance, glibenclamide-sensitive K-channel. An additional action of levcromakalim is suggested by its relative inability to suppress the increase in [Ca²⁺]_i produced by 30 mM K⁺-PSS. Correspondence to: A. H. Weston at the above address     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.     

Caffeine depression of spontaneous activity in rabbit sino-atrial node cells

• 1.1. Effects of caffeine on the action potentials and the membrane currents in spontaneously beating rabbit sino-atrial (SA) node cells were examined using a two-microelectrode technique.
• 2.2. Cumulative administrations of caffeine (1–10 mM) caused a negative chronotropic effect in a concentration-dependent manner, which was not modified by atropine (0.1 μM). At 10 mM, caffeine increased the amplitude and prolonged the duration of action potentials significantly; the other parameters were unaffected.
• 3.3. In 3 of 16 preparations, caffeine (5 mM) elicited arrhythmia. At high Ca²⁺ (8.1 mM), caffeine (5 mM) increased the incidence of arrhythmia.
• 4.4. Caffeine (0.5–10 mM) enhanced the slow inward current, but at 10 mM decreased the enhanced peak current by 5 mM. The hyperpolarization-activated inward current was also enhanced by caffeine, but 10 mM caffeine decreased the current peak as compared with that at 5 mM. In addition, caffeine inhibited the delayed rectifying outward current in a concentration-dependent manner, accompanied by a depressed activation curve without any shift in the half-maximum activation voltage.
• 5.5. Caffeine elevated the cytoplasmic Ca²⁺ level in the SA node cells loaded with Ca²⁺-sensitive fluorescent dye (fura-2).
• 6.6. These results suggest that caffeine enhances and/or inhibits the ionic currents and elicits arrhythmia due to the induction of cellular calcium overload.

     

Inhibitory effects of caffeine on contractions and calcium movement in vascular and intestinal smooth muscle.

1. The mechanism of the inhibitory effect of caffeine was investigated using vascular smooth muscle of rabbit aorta and intestinal smooth muscle of taenia isolated from guinea-pig caecum. 2. Caffeine, 0.5-10 mM, relaxed the sustained contraction induced by 65.4 mM KCl or 10(-6) M noradrenaline in aorta, and by 45.4 mM KCl or 10(-6) M carbachol in taenia. The inhibitory effect of caffeine on the high K+-induced contraction was antagonized by external Ca2+ but not by the Ca2 channel activators, Bay K 8644 (10(-7) M) or CGP 28,392 (10(-7) M). Forskolin (2 x 10(-7) M) potentiated the inhibitory effect of caffeine on the noradrenaline-induced contraction but not on the high K+- or carbachol-induced contraction. Caffeine induced a time- and concentration-dependent increase in the cyclic AMP content of aorta and forskolin caused a further augmentation. 3. 45Ca2+ uptake was increased by high K+ or noradrenaline in aorta and by high K+ or carbachol in taenia. The increments were inhibited by caffeine at concentrations needed to inhibit muscle contractions. 4. 45Ca2+ in the cellular releasable site in aorta was decreased either by noradrenaline or by caffeine. Simultaneous application of noradrenaline and caffeine did not induce an additive decrease. 5. In aorta treated with a Ca2+-free solution, caffeine induced only a small contraction. Noradrenaline induced a greater contraction which was inhibited by caffeine. After washout of caffeine and noradrenaline, the second application of noradrenaline induced a transient contraction suggesting that caffeine does not deplete the noradrenaline-sensitive store.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium dependent 3H-noradrenaline release from rat neocortical slices in the absence of extracellular calcium presynaptic modulation by μ-opioid receptor and adenylate cyclase activation

Summary In Ca²⁺-free EGTA (1 mmol/l)-containing medium veratrine (3 mol/l) and ouabain (100 mol/l) strongly enhanced the efflux of ³H-noradrenaline from superfused rat brain neocortical slices prelabelled with the radioactive amine. In both cases ³H-noradrenaline release was prevented by tetrodotoxin (1 mol/l). These effects of veratrine and ouabain were virtually additive and independent of whether the noradrenaline uptake carrier was blocked with 1 mol/l desipramine or not. The adenylate cyclase activator forskolin (10 nmol/l–10 mol/l) strongly enhanced veratrine- and ouabain-induced ³H-noradrenaline release, without affecting spontaneous tritium efflux. The release induced by both stimuli was profoundly inhibited by the selective -opioid receptor agonist [d-Ala, MePhe⁴, Gly-ol⁵]enkaphalin (DAGO, 3 nmol/l–1 mol/l) in a concentration-dependent manner. The inhibitory effects of 1 mol/l DAGO were abolished by 1 mol/l naloxone. On the other hand, preincubation of the slices for 1 h with the -opioid receptor-selective irreversible ligand fentanyl isothiocyanate (1 pmol/l) did not change the inhibitory effects of DAGO.These data show that veratrine- and ouabain-induced ³H-noradrenaline release from central noradrenergic nerve terminals is facilitated by increasing intracellular cyclic AMP levels and reduced by activation of presynaptic -opioid receptors, indicating the involvement of exocytotic neurotransmitter release. The results provide further evidence for the hypothesis that under these conditions neurotransmitter release from central noradrenergic neurons is triggerred by a Na⁺-induced efflux of Ca²⁺ ions from intracellular stores.Abbreviations DAGO [d-Ala², McPhe⁴, Gly-ol⁵]enkephalin Send offprint requests to A. N. M. Schoffelmeer at the above address     

The mechanism of honokiol-induced and magnolol-induced inhibition on muscle contraction and Ca2+ mobilization in rat uterus

The effects of honokiol and magnolol extracted from the Magnolia officinalis on muscular contractile responses and intracellular Ca²⁺ mobilization were investigated in the non-pregnant rat uterus. Honokiol and magnolol (1–100 mol/l) were observed to inhibit spontaneous and uterotonic agonists (carbachol, PGF₂, and oxytocin)-, high K⁺-, and Ca²⁺ channel activator (Bay K 8644)-induced uterine contractions in a concentration-dependent manner. The inhibition rate of honokiol on spontaneous contractions appeared to be slower than that of magnolol-induced response. The time periods that were required for honokiol and magnolol, at 100 mol/l, to abolish 50% spontaneous contractions were approximately 6 min. Furthermore, honokiol and magnolol at 10 mol/l also blocked the Ca²⁺-dependent oscillatory contractions. Consistently, the increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by PGF₂ and high K⁺ were suppressed by both honokiol and magnolol at 10 mol/l. After washout of these treatments, the rise in [Ca²⁺]_i induced by PGF₂ and high K⁺ was still partially abolished. In conclusion, the inhibitory effects of honokiol and magnolol on uterine contraction may be mediated by blockade of external Ca²⁺ influx, leading to a decrease in [Ca²⁺]_i. Honokiol and magnolol may be considered as putative Ca²⁺ channel blockers and be of potential value in the treatment of gynecological dysfunctions associated with uterine muscular spasm and dysmenorrhea.     

In vitro vasodilator mechanisms of the indole alkaloids rhynchophylline and isorhynchophylline,isolated from the hook of<Emphasis Type="Italic"> Uncaria rhynchophylla</Emphasis> (Miquel)

Rhynchophylline (Rhy) and isorhynchophylline (Isorhy), indole alkaloids from Uncaria hooks, reportedly exert hypotensive and vasodilatory effects, but the mechanism of action is unclear. We therefore investigated the relaxant effects of these two isomeric alkaloids in rat arteries in vitro, in particular in respect of the various functional Ca²⁺ pathways. Both Rhy and Isorhy relaxed aortic rings precontracted with phenylephrine (PE, 1 µM) in a dose-dependent manner (3–300 µM). Removal of endothelium and preincubation with L-NAME (300 µM) slightly inhibited but did not prevent the relaxant response. These results indicate that Rhy and Isorhy act largely in an endothelium-independent manner. Unlike nicardipine, both alkaloids not only inhibited the contraction induced by 60 mM KCl (IC₅₀ 20–30 µM), but also that induced by PE and U46619, albeit to a lesser extent (IC₅₀ 100 and 200 µM, respectively). These results suggest that Rhy and Isorhy may act via multiple Ca²⁺ pathways. In contrast to their inhibitory effects on KCl-induced and receptor-mediated contractions, where both isomers were comparably potent, Rhy was more potent than Isorhy at higher concentrations (>100 µM) in inhibiting both caffeine (25 mM)- and cyclopiazonic acid (CPA, 30 µM)-induced contractions. Similar results observed with caffeine in Ca²⁺-containing medium were also observed in Ca²⁺-free medium. However, 0.1–0.3 µM nicardipine (which completely inhibited KCl-induced contraction) had no significant inhibitory effect on CPA-induced contractions. Taken together, these results indicate discrimination between these two isomers with respect to Ca²⁺-induced Ca²⁺ release and non-L-type Ca²⁺ channel, but not for IP₃-induced Ca²⁺ release and L-type Ca²⁺ channels. Similar relaxant responses to KCl- and caffeine-induced contractions were seen when these two alkaloids were tested on the smaller mesenteric and renal arteries. In conclusion, the vasodilatory effects of Rhy and Isorhy are largely endothelium independent and are mediated by L-type Ca²⁺ channels. At higher concentrations, they also affect other Ca²⁺-handling pathways, although to a lesser extent. While there is no discrimination between the two isomers with respect to the contraction induced by KCl or agonists (PE and U46619), differential effects between Rhy and Isorhy were seen on caffeine- and CPA-induced contractions.