The effect of histamine on cyclic AMP levels in guinea-pig tracheal smooth muscle

Histamine (10(-5)--3 x 10(-4) M) increased the cylic AMP content of guinea-pig tracheal smooth muscle, the maximal effect being a 3-fold increase after 2-min incubation with 10(-4) histamine. Histamine-induced accumulation of cyclic AMP was not affected by propranolol or atropine, but was reduced by mepyramine. Aspirin and indomethacin abolished the cyclic AMP response to histamine and potentiated histamine-induced contractions of the smooth muscle. These results suggest that the elevation of cyclic AMP levels in response to histamine is mediated by prostaglandins, and represents an important negative feedback regulatory mechanism which modulates the contractile response of guinea-pig tracheal smooth muscle to histamine.     

Inhibition of histamine-stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle.

1. The effect on histamine-stimulated [3H]-inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2. Salbutamol (1 microM), forskolin (1 microM) and vasoactive intestinal peptide (VIP, 1 microM) inhibited the inositol phosphate response to 0.1 mM histamine and increased the accumulation of [3H]-cyclic AMP in [3H]-adenine-labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutrylcyclic AMP (1 mM) and 8-bromo-cyclic AMP (1 mM). 3. In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]-cyclic AMP accumulation (EC50 = 1.2 microM) at much higher concentrations than those required for inhibition of histamine-stimulated [3H]-inositol phosphate accumulation (EC50 = 0.09 microM). However, significant increases in [3H]-cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4. In the presence of histamine (0.1 mM), isobutylmethylxanthine (IBMX, 1 mM) and rolipram (0.1 mM) both significantly (P less than 0.05) elevated tissue [3H]-cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P less than 0.05) reduced histamine-stimulated [3H]-inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]-cyclic AMP or histamine-induced [3H]-inositol phosphate accumulation. 5. Both rolipram and forskolin reduced the increase in incorporation of [3H]-inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]-inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]-inositol incorporation.     

The identification of apparently novel cyclic AMP and cyclic GMP phosphodiesterase activities in guinea-pig tracheal smooth muscle.

Phosphodiesterase (PDE) activities that were capable of hydrolysing cyclic AMP (Km = 6.8 +/- 2 microM) and cyclic GMP (Km = 6.7 +/- 1.6 microM) were isolated from tracheal smooth muscle. These enzyme(s) activities were insensitive to stimulation by calcium/calmodulin and to inhibition by cyclic GMP, rolipram (type IV inhibitor) and siguazodan (type III inhibitor). Zaprinast was a relatively poor inhibitor of both cyclic AMP and cyclic GMP hydrolysis (IC50 = 46 +/- 9 microM and 45 +/- 14 microM respectively). These results suggest that tracheal smooth muscle may contain an apparently novel PDE. However, KCl (30 mM) which facilitates calcium entry in cells, depressed bradykinin-stimulated intracellular cyclic AMP formation, suggesting that the type I PDE may be functionally present. We suggest that considerable caution be exercised in identifying apparently novel PDE isoforms.     

P2 purinoceptor-mediated cyclic AMP accumulation in bovine vascular smooth muscle cells.

Extracellular ATP has been shown to induce intracellular Ca2+ mobilization and adenylate cyclase inhibition via P2 purinoceptors in several species of cells. Now we found that in calf vascular smooth muscle cells the addition of ATP to the medium did not induce inhibition but stimulation of cyclic AMP accumulation, in addition to stimulation of inositol phosphate production. Adenosine and AMP also induced cyclic AMP accumulation but their efficacy was much less than that of ATP. The ATP action was not influenced by the presence of either adenosine deaminase or of an ATP regenerating system, whereas the AMP action was increased by the regenerating system. The results indicate that the cyclic AMP accumulation by ATP is due to ATP itself but neither to adenosine nor to AMP, both of which are produced from ATP. ATP receptor coupled to the cyclic AMP generation was shown to be different from that coupled to phospholipase C based on the difference in the potency order of the receptor agonists and in the sensitivity of P2 receptor agonists to 8-cyclopentyl-1,3-dipropylxanthine (CPX)- and suramin-induced antagonism. We conclude that in the aortic smooth muscle cells a novel P2-type receptor directly coupled to adenylate cyclase activation exists in addition to the previously known P2 receptor linked to phospholipase C activation.     

Modulation of carbachol-induced inositol phosphate formation in bovine tracheal smooth muscle by cyclic AMP phosphodiesterase inhibitors.

An investigation was made of a range of agents capable of elevating tissue cyclic AMP levels, or acting as a stable analogue of cyclic AMP, upon carbachol induced inositol phosphate responses in bovine tracheal smooth muscle slices. Whereas the beta 2 adrenoceptor agonist salbutamol (1 microM) and the membrane permeable analogue of cyclic AMP, 8-bromo-cyclic AMP (1 mM) were without effect upon total [3H]inositol phosphate formation induced by carbachol, 3-iso-butyl-1-methylaxanthine (IBMX) (EC50 140 microM), the high Km, cyclic AMP selective phosphodiesterase inhibitor rolipram (EC50 41 microM) and theophylline (EC50 76 microM) all inhibited the inositol phosphate response to low (1 microM) concentrations of carbachol. IBMX (IC50 13 microM), rolipram (IC50 4.6 microM) and theophylline (IC50 180 microM) all relaxed bovine tracheal muscle strips precontracted with methacholine (1 microM). The adenylate cyclase activator forskolin (1 microM), produced a much smaller (10% inhibition) effect upon inositol phosphate formation induced by carbachol. Carbachol (1 microM-1 mM) did not inhibit forskolin induced [3H]cyclic AMP formation. An inhibitor of the cyclic GMP preferring phosphodiesterase isozyme, M&B 22948 (1-100 microM), was without effect upon either carbachol induced inositol phosphate formation or trachealis tone. It is concluded that IBMX, rolipram and theophylline inhibit carbachol stimulated inositol phosphate formation, possibly through a cyclic AMP independent mechanism.     

Beta-adrenoceptor stimulation and cyclic AMP levels in bovine tracheal muscle of old and young animals.

The relationship between cyclic AMP levels and mechanical activity after exposure to isoprenaline was studied in tracheal smooth muscle. It was found that the basal cyclic AMP content decreased with age. The relaxing effects of isoprenaline, theophylline and papaverine were tested on muscles contracted by histamine, acetylcholine or carbacholine. Isoprenaline completely relaxed histamine contracted tracheas, but not those contracted by acetylcholine or carbacholine. Theophylline and papaverine completely relaxed the tracheas irrespective of whether the contracting agent was histamine, acetylcholine or carbacholine. Isoprenaline increased the cyclic AMP content of bovine trachea; this effect was stronger in muscles with spontaneous tension than in histamine contracted muscles. In muscles contracted by carbachol, isoprenaline increased the cyclic AMP level after 5 min. The correlations between the changes in the cyclic AMP levels and the tension in tracheal smooth muscle support the hypothesis that this nucleotide plays a role in the relaxation process.     

The effect of cyclic AMP elevating agents on bradykinin- and carbachol-induced signal transduction in canine cultured tracheal smooth muscle cells.

1. The effects of the beta 2-adrenoceptor agonist, procaterol, on sympathetic neuroeffector transmission were studied in the pithed adrenal demedullated rat to determine if generation of angiotensin II was involved in its effect. Pressor responses were elicited by either electrical stimulation (20 V, 2 Hz) of the entire spinal sympathetic outflow or methoxamine (0.1 mg kg-1, i.v.). 2. Sodium nitroprusside (3 and 5 micrograms kg-1 min-1, i.v.) produced hypotension and the pressor responses to both sympathetic nerve stimulation and methoxamine were reduced. This indicates that decreasing blood pressure in pithed rats reduces pressor responses. Procaterol (10 and 30 ng kg-1 min-1, i.v.) also produced hypotension but did not alter pressor responses to sympathetic nerve stimulation. Nevertheless, procaterol (10 and 30 ng kg-1 min-1, i.v.) did reduce pressor responses to to methoxamine. Together these results suggest that procaterol may have enhanced sympathetic neurotransmitter release. This was confirmed in another series of experiments where procaterol (30 ng kg-1 min-1, i.v.) increased plasma noradrenaline levels during sympathetic nerve stimulation. 3. Captopril (5 mg kg-1, i.v.) produced hypotension and as expected reduced pressor responses to sympathetic nerve stimulation. When the hypotensive effect of captopril was abolished by concomitant vasopressin infusion (1.5-4.5 i mu kg-1 min-1, i.v.), pressor responses to sympathetic nerve stimulation were restored to pre-captopril levels. (ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of cyclic AMP accumulation and relaxant actions of salmeterol and salbutamol in bovine tracheal smooth muscle.

1. The ability of salmeterol to stimulate cyclic AMP accumulation and relaxation has been compared with that of salbutamol in bovine tracheal smooth muscle. In addition, the anti-spasmogenic effects of these agents and their abilities to modulate histamine-stimulated [3H]-inositol phosphate accumulation have also been investigated. 2. In tissue strips, a close temporal correlation was found to exist between salmeterol (0.1 microM)-induced relaxation of methacholine (500 nM)-induced tone and cyclic AMP accumulation, both maximal reversal of induced tone (26.2 +/- 6.0%) and maximal levels of cyclic AMP accumulation being achieved after 30-40 min. In contrast to salmeterol, salbutamol exerted greater and more rapid effects on both parameters. Maximal reversal of methacholine-induced tone (79.3 +/- 14.0%) and maximal levels of cyclic AMP accumulation were produced within 5 min. 3. Salmeterol-induced cyclic AMP accumulation (EC50 = 5.3 [1.8 - 15.2] nM) and inhibition of histamine (0.1 mM)-stimulated [3H]-inositol phosphate accumulation (IC50 = 1.4 [0.3-6.3] nM) were both more potent than those induced by salbutamol (EC50 = 169 [99 - 290] nM; IC50 = 13.8 [7.0 - 27.4] nM). However, maximal effects exerted by each of these agents were similar in magnitude. 4. Anti-spasmogenic effects were examined by beta-adrenoceptor agonist application to tissue strips prior to construction of spasmogen concentration-effect curves. Both salmeterol and salbutamol exerted more marked inhibition of the contractile response induced by histamine than that induced by methacholine, salmeterol being the more potent agent, while salbutamol produced a greater maximal inhibitory effect. 5. The results demonstrate that salmeterol is a more potent agent than salbutamol and have highlighted a close temporal correlation between promotion of cyclic AMP accumulation and tissue relaxation stimulated by each agent when both parameters are measured under identical conditions.     

Inhibition of a high affinity cyclic AMP phosphodiesterase and relaxation of canine tracheal smooth muscle

The cyclic nucleotide phosphodiesterase (PDE) activity of canine tracheal smooth muscle (CTS,) was examined. Column chromatography of soluble CTSM-PDE revealed five peaks of activity. One of these peaks (V) was examined further in this study and showed a high affinity for adenosine 3',5'-cyclic monophosphate (Km = 0.63 microM). Seven pharmacological PDE inhibitors were tested for their abilities to inhibit the peak V enzyme and also for their abilities to cause mechanical relaxation of CTSM strips in isolated tissue baths. A strong correlation (P greater than 0.001) between peak V PDE inhibition (-log Ki) and airway muscle relaxation (-log ED50) was found.     

Modulation of fluoroaluminate-induced inositol phosphate formation by increases in tissue cyclic AMP content in bovine tracheal smooth muscle.

1. The effect of fluoroaluminate complexes (AlCl3 plus NaF) upon smooth muscle tone, [3H]-inositol phosphate accumulation and [3H]-cyclic AMP accumulation has been investigated in slices of bovine tracheal smooth muscle. 2. Fluoroaluminate (10 microM AlCl3 + various concentrations of NaF) elicited concentration-dependent contractions of bovine tracheal smooth muscle strips at concentrations of NaF in the range 1-10 mM. The resultant contractile response was reversed by isoprenaline (50 nM) and was preserved in calcium-free medium. 3. Fluoroaluminate stimulated [3H]-inositol phosphate formation at concentrations of NaF over 1 mM. The response to 20 mM NaF + 10 microM AlCl3 was 164 +/- 29% of the response to 1 mM histamine. Fluoroaluminate also increased the incorporation of [3H]-myo-inositol into membrane phospholipids. 4. Fluoroaluminate produced a small rise in [3H]-cyclic AMP levels (2.1 fold increase over basal with 20 mM NaF). The response to forskolin (1 microM, 8.6 fold over basal) was reduced by fluoroaluminate in a concentration-dependent manner, but still remained significantly (P less than 0.05) elevated over the response to fluoroaluminate alone. 5. The [3H]-inositol phosphate response to fluoroaluminate was inhibited by salbutamol (maximum inhibition 60%, IC50 = 0.08 microM), forskolin (1 microM, 46% inhibition) and isobutylmethylxanthine (1 mM, 73% inhibition). 6. These data suggest that inhibition of agonist-induced inositol phospholipid turnover by cyclic AMP in this tissue can occur at the post-receptor level.     

Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle.

The hypotheses were tested that the relaxant effect of adenosine and related compounds in the longitudinal muscle of the rabbit small intestine involves interaction with adenylate cyclase and/or the elevation of tissue cAMP levels. Adenylate cyclase was prepared by gentle homogenization of an isolated smooth muscle cell fraction obtained after collagenase digestion of longitudinal muscle strips. A number of analogs and derivatives of adenosine possessing a primary or secondary 6-amino group were found to inhibit the enzyme similarly to adenosine; however, there was no correlation between compounds known to relax the intact tissue and the existence, or the degree of, cyclase inhibition. Isolated muscle strips were exposed to adrenaline, isoprenaline, adenosine or ATP, at doses causing 30-60% relaxation, for 60 sec prior to sampling and analysis of cAMP content. While small increments in cAMP levels were found after administering adrenaline or isoprenaline, no change was found with adenosine in the absence or presence of theophylline of 1-methyl-3-isobutylxanthine. Neither adenylate cyclase inhibition nor changes in cAMP levels appear to be part of the mechanism of the smooth muscle relaxant action of adenosine or ATP.     

Intracellular cyclic AMP level and intestinal smooth muscle relaxation

The intracellular level of cyclic AMP in the taenia from the guinea pig caecum was significantly increased by isoprenaline in a dose-dependent manner. Papaverine also increased the intracellular cyclic AMP level, but a synthetic antispasmodic agent, Aspaminol (1,1-diphenyl-3-piperidinobutanol hydrochloride) did not modify it. The results suggest that there are two mechanisms for the so-called papaverine-like actions. The increase in the intracellular cyclic AMP concentration produced by isoprenaline was prevented by propranolol. In taenia depolarized by KCl, papaverine caused relaxation but isoprenaline did not. This difference can be explained by the observation that the intracellular cyclic AMP level in the KCl-depolarized taenia is increased by papaverine but not by isoprenaline.     

Nitric oxide donors enhance calcitonin gene-related peptide-induced elevations of cyclic AMP in vascular smooth muscle cells.

Vasorelaxant effects of calcitonin gene-related peptide (CGRP) are dependent on endothelium-derived nitric oxide (NO) in some arteries. The mechanism involved is still not clear. In the present study, we used NO donors (sodium nitroprusside (SNP) and 6-(2-hydroxy-1-methyl-2-nitrisohydrazino)-N-methyl-1-hyxanamine (NOC-9)), cyclic GMP elevator (brain natriuretic peptide (BNP)) and a selective type III (cyclic GMP-inhibited) phosphodiesterase (PDE) inhibitor 5-(4-acetamidophenyl)pyrazin-2(1H)-one (SK&F94120) to investigate involvement of NO, cyclic GMP and type III PDE in CGRP-induced accumulation of cyclic AMP in cultured rat aortic smooth muscle cells. SNP (10 microM), NOC-9 (10 microM) and BNP (1 microM) all increased intracellular cyclic GMP to similar levels (2- to 2.5-fold above basal) and caused significant enhancement of CGRP (10 nM)-induced cyclic AMP accumulation similar to that caused by 10 microM SK&F 94120. The data are therefore consistent with our hypothesis that the mechanism of endothelium-dependent vasorelaxation effect of CGRP involves cyclic GMP-mediated inhibition of type III PDE and subsequent accumulation of cyclic AMP in smooth muscle cells.     

Effects of a range of beta2 adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells.

1. The effects of the selective beta2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non-selective beta adrenoceptor agonist isoprenaline on [3H]-cyclic AMP formation and cyclic AMP response element (CRE) driven luciferase expression, assessed using the construct p6CRE/luc, were studied in primary cultures of human airway smooth muscle (HASM) cells. 2. Optimal transfection conditions for transient expression of pGL3 Control were 4 microg DNA/well71 in a 6 well plate and 1.8 microl Transfectam/microg DNA. Expression was maximal at 48 - 72 h. 3. Salbutamol (maximum response 19%, EC50 0.6 microM), terbutaline (maximum response 38%, EC50 2.3 microM) and salmeterol (maximum response 18%, EC50 0.0012 microM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC50 0.08 microM). However, all of the beta2 adrenoceptor agonists produced increases in CRE-driven luciferase activity, in cultured HASM transfected with the vector p6CRE/luc, which were equivalent or greater (salmeterol) than those seen with isoprenaline. 4. Both salbutamol and salmeterol were more potent at increasing luciferase expression than in elevating cyclic AMP levels in these cells. The potency ratios (EC50 (cyclic AMP)/EC50 (LUC)) for the agents studied were isoprenaline: 0. 2 fold, terbutaline: 3 fold, salbutamol: 24 fold, salmeterol: 38 fold. 5. These data suggest that important quantitative differences exist in the ability of beta2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE-driven mechanism.     

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides in guinea pig tracheal smooth muscle

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides were investigated in epithelium-denuded guinea pig tracheal rings, treated with indomethacin (5 μM) and phosphoramidon (1 μM) and contracted with histamine (3 μM). Atrial natriuretic peptide (ANP) was a more potent relaxant than C-type natriuretic peptide whereas ANP-(4–23) was inactive suggesting the involvement of ANP_A receptors in the relaxant effect of ANP. ODQ (1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one, 10 μM), a selective inhibitor of soluble guanylyl cyclase, markedly inhibited the relaxant response to sodium nitroprusside. The relaxant response to ANP was not altered by ODQ demonstrating the involvement of particulate guanylyl cyclase. ANP-induced relaxations, as well as sodium nitroprusside-induced relaxations, were similarly potentiated by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, 3 μM), a type IV phosphodiesterase inhibitor, and by zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one, 10 μM), a type V phosphodiesterase inhibitor. ANP-mediated response was unaffected by glibenclamide (10 μM), a selective blocker of ATP-sensitive K⁺ channels, and by apamin (1 μM), a selective blocker of small-conductance Ca²⁺-activated K⁺ channels. Iberiotoxin (100 nM) extensively prevented the relaxant effect of ANP suggesting the activation of large-conductance Ca²⁺-activated K⁺ channels. In addition, ANP (10 nM) and ANP-(4–23) (100 nM) significantly reduced forskolin (1 μM)-stimulated cAMP accumulation suggesting, for the first time, the presence of functional ANP_C receptors in guinea pig airway smooth muscle. However, relaxations to forskolin and to isoproterenol were not altered in the presence of ANP-(4–23) or ANP demonstrating that the inhibitory effect of ANP-(4–23) and ANP on adenylyl cyclase was not sufficient to alter the functional response induced by these two activators of adenylyl cyclase.     

Procaterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells

β₂ agonists reduce the frequency of exacerbations in patients with bronchial asthma and chronic obstructive pulmonary disease caused by respiratory virus infection. β₂ agonists reduce the production of pro-inflammatory cytokines. However, the inhibitory effects of β₂ agonists on the infection of rhinovirus, the major cause of exacerbations, have not been well studied. To examine the effects of a β₂ agonist, procaterol, on rhinovirus infection and rhinovirus infection-induced airway inflammation, human tracheal epithelial cells were infected with a major group rhinovirus, type 14 rhinovirus. Rhinovirus infection increased viral titers and the content of pro-inflammatory cytokines, including interleukin-1β and interlukin-6, in supernatant fluids and rhinovirus RNA in the cells. Procaterol reduced rhinovirus titers and RNA, cytokine concentrations, and susceptibility to rhinovirus infection. Procaterol reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for type 14 rhinovirus, and the number of acidic endosomes in the cells from which rhinovirus RNA enters into the cytoplasm. Procaterol inhibited the activation of nuclear factor kappa-B (NF-κB) proteins including p50 and p65 in the nuclear extracts, while it increased the cytosolic amount of the inhibitory kappa B-α and intracellular cyclic AMP (cAMP) levels. A selective β₂-adrenergic receptor antagonist ICI 118551 [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] reversed the inhibitory effects of procaterol on rhinovirus titers and RNA, susceptibility to rhinovirus infection, pro-inflammatory cytokines production, ICAM-1 expression, acidic endosomes, and NF-κB. ICI 118551 also reversed the effects of procaterol on cAMP levels. Procaterol may inhibit rhinovirus infection by reducing ICAM-1 and acidic endosomes as well as modulate airway inflammation in rhinovirus infection.     

Mechanisms underlying the inhibitory effect of dibutyryl cyclic AMP in vascular smooth muscle.

The mechanism by which dibutyryl cyclic AMP (db-cAMP) induces vasodilatation was examined in isolated rat aorta. The contraction induced by norepinephrine (NE) was more sensitive to the inhibitory effect of db-cAMP than that induced by high K+, and the contraction induced by lower concentrations of each stimulant was more sensitive to db-cAMP than that induced by higher concentrations. Db-cAMP at 10 microM inhibited the increases in muscle tension and cytosolic Ca2+ level ([Ca2+]i) without changing the [Ca2+]i-tension relationship, suggesting that the inhibitory effect is mainly due to a decrease in [Ca2+]i. A higher concentration (300 microM) of db-cAMP inhibited muscle tension more strongly than [Ca2+]i suggesting that db-cAMP decreases Ca2+ sensitivity of contractile elements. In contrast, 10 microM verapamil inhibited the NE-stimulated [Ca2+]i more strongly than the NE-induced contraction. The verapamil-insensitive portion of the NE-stimulated [Ca2+]i and contraction was inhibited by db-cAMP, suggesting that db-cAMP and verapamil act by different mechanisms. In Ca(2+)-free solution, 1 microM NE induced transient increases in muscle tension and [Ca2+]i. The transient contraction was inhibited by 1 mM db-cAMP more strongly than [Ca2+]i. An activator of adenylate cyclase, forskolin, showed inhibitory effects similar to those of db-cAMP. The inhibitory effects of db-cAMP and forskolin were inversely proportional to [Ca2+]i before the addition of these inhibitors. These results suggest that db-cAMP inhibits smooth muscle contraction by decreasing [Ca2+]i and the Ca2+ sensitivity of contractile elements, and that both of these effects are stronger when [Ca2+]i is lower.