Effects of 8-bromo cyclic GMP and verapamil on depolarization-evoked Ca2+ signal and contraction in rat aorta.

1. The pharmacological action of NO donors is usually attributed to a cellular rise in guanosine 3':5'-cyclic monophosphate (cyclic GMP), but this hypothesis is based only on indirect evidence. Therefore, we have studied the effects of cyclic GMP on Ca2+ movements and contraction in rat isolated endothelium-denuded aorta stimulated by KCl depolarizing solution using the permeant analogue 8-bromo cyclic GMP (BrcGMP). Isometric contraction and fura-2 Ca2+ signals were measured simultaneously in preparations treated with BrcGMP and with verapamil. The activation of calcium channels was estimated by measuring the quenching rate of the intracellular fura-2 signal by Mn2+ and by the depolarization-dependent influx of 45Ca2+. 2. Stimulation with 67 mM KCl-solution evoked an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and a contractile response which were inhibited by pretreatment with verapamil (0.1 microM) or BrcGMP (0.1-1 mM). However, the inhibition of the fura-2 Ca2+ signal was significantly higher with verapamil than with BrcGMP, whereas the contraction was inhibited to a similar extent. 3. When preparations were exposed to K(+)-depolarizing solution in which the calcium concentration was cumulatively increased, the related increase in fura-2 Ca2+ signal was barely affected by BrcGMP, whereas the contractile tension was strongly and significantly inhibited. 4. Cellular Ca2+ changes were also estimated with 45Ca2+. 45Ca2+ influx in resting preparations was significantly reduced by BrcGMP (0.1 mM) but not by verapamil (0.1 microM); 45Ca2+ influx in KCl-depolarized preparations was reduced by verapamil but was unaffected by BrcGMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells

Summary The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca²⁺]_i) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 M) enhanced the increase in [Ca²⁺]_i elicited by bradykinin (3 nM) and also caused an increase in resting [Ca²⁺]_i when given alone. This increase in resting [Ca²⁺]_i was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N^G-nitro-l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca²⁺]_i and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B₂-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B₂-receptors with a subsequent increase in [Ca²⁺]_i and nitric oxide formation. Send offprint requests to R. Busse at the above address     

CGS 9343B and W7 (calmodulin antagonists) inhibit KCl-induced increase in cytosolic free calcium and insulin secretion of RINm5F cells

Summary CGS 9343B:1,3-Dihydro-l-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin-4-yl)methyl)-4-piperidinyl]-2 H-benzimidazol-2-one maleate and W7: N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide) are calmodulin antagonists with different specificities. The effects of CGS 9343B and W7 on cytosolic free calcium concentration ([Ca²⁺]_i) and insulin release were investigated in rat insulinoma cells (RINm5F). As measured with the Quin-2 technique, preincubation with CGS 9343B (0.3–10 M) and W7 (5–50 M) concentration dependently decreased KCl (25 mM)-mediated accumulation of cytosolic calcium. Both, CGS 9343B (10 M) and W7 (50–100 M) almost abolished the alanine- and KCl-induced increase in [Ca²⁺]_i and significantly inhibited KCl (25 mM)- and alanine (10 mM)-mediated insulin release. W5 (100 LM), the chlorine-deficient analogue of W7 with decreased affinity for calmodulin, did not inhibit the KCl-induced increase in [Ca²⁺]_i and enhanced basal and KCl-mediated insulin release by 56% and 189%, respectively. Our data suggest that CGS 9343B and W7 inhibit the depolarization-induced calcium uptake and subsequent increase in [Ca²⁺]_i. Send offprint requests to H. P. T. Ammon at the above address     

Inhibitory Effects of Calcium Channel Antagonists on Motor Dysfunction Induced by Intracerebroventricular Administration of Paraquat

Abstract: This study reports the effects of Ca²⁺ channel blockers (Ca antagonists) on intraneuronal Ca²⁺ ([Ca²⁺]_i) movements and on the disturbance of rotarod performance produced in rats by intracerebroventricular administration of paraquat. Paraquat (50 nmol) produced a decrement in rotarod performance which was present at 30 min. and maximal at 60 min. and was not associated with overt behavioural changes; larger doses of paraquat (100-400 nmol intracerebroventricularly) produced paresis and convulsions which severely disrupted rotarod behaviour. The disruption of rotarod performance after paraquat (50 nmol intracerebroventricularly) was significantly reduced by giving Ca antagonists (flunarizine, verapamil and nicardipine) not only intraperitoneally 15 min. after paraquat but also intracerebroventricularly immediately before paraquat. The order of pharmacological potency was flunarizine ≥ verapamil > nicardipine. In contrast, intracerebroventricular administration of Bay K 8644, a Ca agonist, enhanced the disruption of rotarod performance caused by paraquat (50 nmol). In in vitro studies, paraquat markedly potentiated the rapid increase in [Ca²⁺]_i levels evoked by 50 mM KCl in rat brain synaptosomal fraction, although paraquat alone produced a small prolonged rise in [Ca²⁺]_i levels which had a slow onset. The above results suggest that paraquat induced neurotoxicity is associated with increased [Ca²⁺]_i levels in brain neuronal cells, and that paraquat might effect on membrane activity instability.     

Influence of oligopeptide aldehydes on intracellular Ca2+ concentration in rat pituitary cells

We investigated the effects of some synthetic tripeptide aldehydes, earlier shown to influence pituitary hormone secretion and ⁴⁵Ca²⁺ uptake, on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of rat anterior pituitary cells in suspension. Boc-D-Phen- Leu-Phenylalaninal or Boc-D-Phe-Leu-Prolinal in the tested range of 1–100 or 200 μM, respectively, were ineffective in influencing basal [Ca²⁺]_i but caused a concentration-dependent inhibition in K⁺ (25 mM)-induced [Ca²⁺]_i elevation. The IC₅₀ of both effects was about 50 μM. In contrast, they did not interfere with the stimulation caused by the calcium channel agonist BAY K 8644 and were also ineffective in influencing the receptor-mediated stimulus of thyrotropin-releasing hormone on [Ca²⁺]_i. On the basis of the present and foregoing results the possible involvement of calcium channels is discussed, but different mechanisms mediating the tripeptide aldehyde inhibition are also considered. A third tripeptide aldehyde. Boc-Gln-Leu-Lysinal (Boc-GLL), showed ionophore-like properties. This nontoxic substance caused a dose-dependent rise up to 400% (at 100 μM) in [Ca²⁺]_i. Its effect is not mediated by voltage-dependent calcium channels, as it cannot be inhibited either by the classicalp calcium channel antagonists verapamil and nifedipine, or by the above-mentioned inhibitory tripeptide aldehydes. When we decreased the extracellular Ca²⁺ concentration by the addition of 4 mM EGTA, the effect was inverted and Boc-GLL caused a large fall in [Ca²⁺]_i. We suggest that Boc-GLL may open cell membrane pores through which Ca²⁺ moves along the concentration gradient. The calcium flux can be inhibited by 20 mM Mg²⁺ and 100 μM Co²⁺ but not by 500 μM La³⁺. Thus, tripeptide aldehydes. depending on their structure, may decrease or increase [Ca²⁺]_i via uncoventional mechanisms and may serve as tools for dissecting details of cell calcium homeostasis.     

Nitric oxide synthesis in endothelial cytosol: Evidence for a calcium-dependent and a calcium-independent mechanism

Summary Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 M free calcium; EC₅₀ 0.3 M). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 M) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation N^G-nitro-L-arginine (1 mM) and by LY 83583 (1 M), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells. Send of fprint requests to A. Mülsch, at the above address     

The phosphatase inhibitor okadaic acid blocks KCl-depolarization-induced rise of cytosolic calcium of rat insulinoma cells (RINm5F)

It has been shown that okadaic acid (OA) diminishes insulin secretion of rat pancreatic islets in response to glucose, glyceraldehyde and KCl. Glucose, glyceraldehyde and KCl cause release of insulin by depolarization and subsequent opening of L-type calcium channels. Calcium entry into cells is thought to be related to protein phosphorylation. To evaluate whether or not OA mediated inhibition of insulin secretion in response to depolarization might be due to an interference with calcium uptake, we studied its effect on KCl (30 mM)-induced increases of cytosolic calcium and discharge of insulin in the insulin secreting clonal tumor cell line RINm5F. OA inhibited KCl-stimulated insulin release in concentrations 1 M. In intact RINm5F cells similar concentrations of OA decreased the activity of protein phosphatases PP-1/PP-2A and inhibited the depolarization-induced rise of cytosolic calcium ([Ca²⁺]_i). The latter action could also be achieved with the protein phosphatase inhibitor calyculin A, whereas the OA analogue 1-nor-okadaone, which is without effect on phosphatases, did not affect [Ca²⁺]_i or insulin release. It is concluded that depression of depolarization-induced insulin secretion by OA is due to inhibition of calcium entry along voltage dependent calcium channels. The data also suggest that in RINm5F cells protein phosphatases PP-1/PP-2A are related to the function of voltage-dependent calcium channels.     

Effects of the Chinese herb component phellopterin on the increase in cytosolic free calcium in PC12 cells

The present study investigated the effects of the Chinese Herb component, phellopterin on high K⁺ and glutamate‐induced extracellular calcium influx and caffeine or cyclopiazonic acid (CPA)‐induced calcium release from internal stores in attached PC12 cells. Attached cells were loaded with the calcium fluorescent indicator Fluo‐3/AM with the final concentration of 5 µM for 50 min at 37°C and cytosolic free Ca²⁺ measured as fluorescent intensity (FI) (excitation: 488 nm; emission: 535 nm). When PC12 cells were exposed to extracellular Ca²⁺([Ca²⁺]₀) 2.0 mM, the FI for resting [Ca²⁺]_i was 1,188±163, high K⁺ (75 mM) and glutamate (10 mM) induced an increase in [Ca²⁺]_i with peak values of 4,270±982 and 3,096±402, respectively. Phellopterin (0.1–100 µM) had no apparent effect on resting [Ca²⁺]_i, but inhibited high K⁺ and glutamate induced the increase in [Ca²⁺]_i in a dose‐dependent manner. When PC12 cells were exposed to Ca²⁺‐free solution, the FI for resting [Ca²⁺]_i was 804±77. Caffeine (40 mM) and CPA (30 µM) stimulated Ca²⁺ release from caffeine‐ryanodine and inositol 1,4,5‐tris‐phosphate (InsP₃₎‐sensitive internal calcium stores, inducing an increase in [Ca²⁺]_i to 2,938±362 and 1,816±291, respectively. Phellopterin (0.1–100 µmol/L) inhibited caffeine and CPA stimulated intracellular calcium release in a dose‐dependent manner. In summary, phellopterin, a novel component isolated from Changii radix, inhibited Ca²⁺ influx induced by stimulation of voltage‐gated and receptor‐dependent calcium channels with a greater inhibition of receptor‐dependent calcium channels. It also inhibited Ca²⁺ release from caffeine‐ryanodine and InsP₃‐sensitive internal stores, being more potent for caffeine stimulation. Phellopterin may be a promising candidate for the development of new classes of calcium antagonists. Drug Dev Res 68:79–83, 2007. © 2007 Wiley‐Liss, Inc.     

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca²⁺]_i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10⁻⁷ M), NO (10⁻¹⁰–10⁻⁶ M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca²⁺]_i and cation influx as indicated by fura-2 fluorimetry.
2. Zaprinast (10⁻⁴ M), increased NO-stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca²⁺]_i reached after administration of NO, the initial decreases in [Ca²⁺]_i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast.
3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10⁻⁵ M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10⁻¹⁰–10⁻⁸ M, but the decrease in [Ca²⁺]_i or cation influx caused by higher concentrations of NO (10⁻⁷–10⁻⁶ M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10⁻⁷–10⁻⁵ M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO.
4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca²⁺]_i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca²⁺]_i and smooth muscle relaxation to NO.

     

Effects of Apigenin on Glutamate-induced [Ca2+]i Increases in Cultured Rat Hippocampal Neurons

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin (1 µM to 100 µM) for 5 min inhibited glutamate (100 µM, 1 min) induced [Ca²⁺]_i increase, concentration-dependently. Pretreatment with apigenin (30 µM) for 5 min significantly decreased the [Ca²⁺]_i responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 µM, 1 min) and N-methyl-D-aspartate (NMDA, 100 µM, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca²⁺]_i response induced by 50 mM KCl solution, decreased the [Ca²⁺]_i responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100 µM, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca²⁺]_i responses. Furthermore, treatment with apigenin (30 µM) significantly inhibited the amplitude and frequency of 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.     

Effects of Calcium Channel Modulators on the Regulation of Cytoplasmic Ca2+ and Hormone Secretion of Parathyroid Cells

Abstract: Effects of Ca²⁺-channel modulators were examined in human, bovine and murine parathyroid tissue. In 0.5 mM external Ca²⁺, 100 μM verapamil inhibited parathyroid hormone release, stimulated uptake and efflux of ⁴⁵Ca and raised cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). However, in 3.0 mM Ca²⁺ the drug only affected efflux. Methoxyverapamil (50 μM) inhibited parathyroid hormone release in 0.5 mM but stimulated secretion in 3.0 mM Ca²⁺. BAY K. 8644 (10 μM) had similar actions as verapamil on parathyroid hormone release and [Ca²⁺]_i, whereas nifedipine (1μM) and diltiazem (50-100 μM) lacked effects. Despite the lack of voltage-dependent Ca²⁺ channels in parathyroid cells, drugs with established actions on such channels affect [Ca²⁺]_i and parathyroid hormone release. However, these actions are not sufficiently pronounced and tissue specific to allow their use for correcting hyperparathyroidism.     

Effects of manganese on cyclic GMP levels in the rat ductus deferens

Summary Mn²⁺ and other divalent cations (Me²⁺) are capable of preventing contractile responses of smooth muscle to various stimuli. The effects of Mn²⁺, of other inhibitory Me²⁺ and of Ca²⁺ on basal and hormone-stimulated cyclic nucleotide levels were studied in the ductus deferens of the rat.In tissue segments that had been preincubated with no Me²⁺ added, Mg²⁺ (2 or 10 mM), Co²⁺, Ni²⁺ and Cd²⁺ (2 mM each) caused 2-fold elevations of the cyclic GMP level within 5 min, whereas Mn²⁺ and Ca²⁺ caused 7-and 5-fold elevations, respectively. Cyclic AMP levels were not significantly changed except for a 10–20% increase by Mn²⁺. Whereas Mn²⁺ caused a continuous rise of the cyclic GMP level over at least 10 min, the stimulatory effect of Ca²⁺ was most pronounced during the first minute. The effects of Ca²⁺ and Mn²⁺ were not additive. When Ca²⁺ was added with or after Mn²⁺ (2 mM each), the stimulatory effect of Mn²⁺ was depressed. Similarly, Mn²⁺ caused only a small increase in the cyclic GMP level of tissue incubated with Ca²⁺.With the hormonal stimuli, acetylcholine and noradrenaline (0.1 mM each), a dissociation of their effects on tissue tone and on the cyclic GMP level occurred. Whereas either stimulus caused about 2-fold elevation of the cyclic GMP in the presence of Mn²⁺ with or without Ca²⁺ present, contractile responses were prevented by Mn²⁺. In contrast, K⁺ at a high, depolarizing concentration, which increased the cyclic GMP level in the presence of Ca²⁺, had no effect on the cyclic nucleotide with Mn²⁺ added instead of Ca²⁺. Whereas hormone-induced elevations of the cyclic GMP level depended on the presence of Me²⁺, the effect of hydroxylamine (0.1 mM) to increase the level of this nucleotide in the absence of Me²⁺ was not augmented by Ca²⁺ or Mn²⁺.The present data indicate that hormone-induced elevations of the cyclic GMP level in the absence of extracellular Ca²⁺ do not promote contraction. The findings also suggest that hormones are capable of stimulating cyclic GMP formation by mechanisms other than hormone-induced increase in cytoplasmic Ca²⁺ concentration.Abbreviations cGMP cyclic - GMP guanosine 3:5-monophosphate - cAMP cyclic AMP, adenosine 3:5-monophosphate - Me²⁺ divalent cation(s) - EDTA ethylenediaminetetraacetic acid This work was supported by the Deutsche Forschungsgemeinschaft. A preliminary report was presented (Schultz and Schultz, 1976). The data have partially been taken from a dissertation submitted by K.D.S. to the University of Heidelberg in partial fulfillment of the requirements for the Ph. D. degree     

Low concentrations of caffeine raise intracellular calcium concentration only in the presence of extracellular calcium in cultured molluscan neurons

• 1.1. The effects of low concentrations of caffeine (100 and 300 μM) on the intracellular calcium concentration [Ca²⁺]_i in four cultured, identified neurons of the pond snail Lymnaea stagnalis (L) were investigated.
• 2.2. Intracellular CA²⁺ levels in these neurons were measured with the cell-permeable Ca²⁺ indicator Fura-2/AM, both in the presence and absence of extracellular Ca²(O-Ca²⁺/EGTA).
• 3.3. In the presence of Ca²⁺ in the external medium, caffeine was found to induce a substantial elevation in the free [Ca²⁺]_i in all cell types.
• 4.4. In some cases, the rise in [Ca²⁺]_i was found to be both time- and concentration-dependent.
• 5.5. Low doses of caffeine did not produce any appreciable rise in [Ca²⁺]_i in the absence of Ca²⁺ in the external medium, but calcium was still available from stores, as clinical concentrations of halothane rose [Ca²⁺]_i in the absence of extracellular calcium.
• 6.6. These results indicate that the actions of caffeine, when applied at low concentrations, are dependent on extracellular calcium.

     

Caffeine inhibits cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5′-O-(3-thiotriphosphate) in hyperpolarized pancreatic ß-cells

The effects of caffeine on cytoplasmic Ca²⁺ oscillations induced by carbachol and guanosine 5-O-(3-thiotriphosphate) (GTP--S) were studied in individual mouse pancreatic ß-cells clamped at a hyperpolarized potential. Addition of 10 mM caffeine did not affect the cytoplasmic Ca²⁺ concentration ([Ca²⁺]₁) in ß-cells exposed to 20 mM glucose and hyperpolarized with diazoxide. Under similar conditions 100 M carbachol induced a typical response with a marked [Ca²⁺]_i peak followed by a lower sustained elevation. Irrespective of whether 10 mM caffeine was present, there were [Ca²⁺]_i transients with frequencies of 1–5/min superimposed on the sustained phase in 50–60% of the cells. In previously non-exposed cells the introduction of 10 mM caffeine caused temporary lowering of the sustained phase with disappearance of the transients. Subsequent omission of caffeine in the continued presence of carbachol caused a marked [Ca²⁺]_i peak followed by reappearance of the [Ca²⁺]_i, transients. However, in cells oscillating in the presence of caffeine its omission caused disappearance of the transients. In this case reintroduction of caffeine restored the transients.In cells kept at –70 mV by a patch pipette containing 100 M GTP--S and 3 mM Mg-ATP there were [Ca²⁺]_i transients with frequencies of 0.5–2.5/min. These transients were sufficiently pronounced to activate repetitively a K⁺ current. Addition of 10 mM caffeine caused disappearance of the [Ca²⁺]_i transients or reduction of their amplitudes and frequencies.The results indicate that caffeine does not activate Ca²⁺-induced Ca²⁺ release in hyperpolarized ß-cells but inhibits the Ca²⁺-mobilizing effect of inositol 1,4,5-trisphosphate. Correspondence to: E. Gylfe at the above address     

Vasorelaxant effect of formononetin in the rat thoracic aorta and its mechanisms

The purpose of the present study was to investigate the effect of formononetin and the related mechanisms on isolated rat thoracic aorta. Formononetin concentration dependently relaxed aortic rings precontracted with norepinephrine (NE, 1 μM) or KCl (80 mM). Pretreatment with formononetin noncompetitively inhibited contractile responses of aortas to NE and KCl. The vasorelaxant effect of formononetin partially relied on intact endothelia, which was significantly attenuated by incubation with N^ω-nitro-l-arginine methyl ester (100 μM). In endothelium-denuded rings, glibenclamide (10 μM) and tetraethylammonium (5 mM) showed slight reduction in the vasorelaxant effect of formononetin. Moreover, formononetin reduced NE-induced transient contraction in Ca²⁺-free solution and inhibited the vasocontraction induced by increasing external calcium in medium plus 80 mM KCl. Our results suggested that formononetin induced relaxation in rat aortic rings through an endothelium-dependent manner via nitric oxide synthesis pathway, and also involving an endothelium-independent vasodilatation by the blockade of Ca²⁺ channels. The opening of K⁺ channels might also be one of the mechanisms of formononetin-induced vasorelaxation.     

Arterial contractions induced by cumulative addition of calcium in hypertensive and normotensive rats: influence of endothelium

Responses to cumulative addition of Ca²⁺ (0.2–2.5 mM) after precontraction with potassium chloride (KCl) and noradrenaline in Ca²⁺-free medium were studied in isolated mesenteric arterial rings from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The Ca²⁺ contractions in 125 mM KCl-stimulated endothelium-denuded rings in the presence of atenolol (10 M) and phentolamine (10 M) were less marked in SHR than WKY, although the contractions to high concentrations of KCl in normal organ bath Ca²⁺ (1.6 mM) were similar in these strains. The difference in Ca²⁺ contractions between SHR and WKY during KCl stimulation was also present after 10-min pretreatment with 1 mM ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) in Ca²⁺-free medium. However, when noradrenaline (1 M) was used as the agonist the Ca²⁺ contractions of endothelium-denuded rings in the two strains were comparable, while exposure to EGTA reduced these responses more effectively in SHR than WKY. Nifedipine (0.5 nM and 10 nM in KCl- and noradrenaline-stimulated rings, respectively) more efficiently inhibited the Ca²⁺ contractions in hypertensive than in normotensive rats.The presence of intact vascular endothelium attenuated the contractions to Ca²⁺ addition comparably (during KCl stimulation) or even more (during noradrenaline) in SHR when compared with WKY NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) counteracted this attenuation correspondingly in WKY and SHR, and L-arginine (1 mM) restored it in both strains, whereas indomethacin (10 mM) was without effect on the response. However, mesenteric arterial relaxations induced by the endothelium-dependent agonists acetylcholine and ADP in noradrenaline-precontracted (1 M) rings were clearly impaired in SHR, and also L-NAME (0.1 mM) reduced the responses to acetylcholine more efficiently in SHR. In contrast, the relaxations to acetylcholine and ADP in KCl-precontracted (60 mM) rings in the absence and presence of L-NAME were comparable between the two strains.In conclusion, attenuated contractile response to cumulative Ca²⁺ addition during stimulation with KCl clearly differentiated arterial smooth muscle of hypertensive and normotensive rats, suggesting altered function of cell membrane in SHR. The more pronounced effect of nifedipine on the response indicates abnormal function of voltage-dependent Ca²⁺ channels, and higher diminishing effect of EGTA on the contraction during noradrenaline suggests exaggerated action of the chelator on membrane-bound Ca²⁺ in SHR. Interestingly, the depressant effect of intact endothelium on the Ca²⁺ contraction response, mediated largely via nitric oxide, was not attenuated in SHR. Furthermore, impaired endothelium-dependent agonist-induced relaxations can be attributed to reduced release of endothelium-derived hyperpolarizing factor in this type of genetic hypertension. Correspondence to: M. Kähönen at the above address     

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.     

Vasorelaxant effect of the analgesic clonixin on rat aorta

• 1.1. A novel vasorelaxant effect of clonixinate of l-lysine (Clx), analgesic and anti-inflammatory, was studied in rat aortic rings.
• 2.2. Clx completely relaxed aortic rings contracted by KCl 70 mM and together with its analog flunixin exhibited lesser potency but equal efficacy than verapamil. In comparison, indomethacin, which is a more potent cyclo-oxygenase inhibitor relaxed only about 40% of the maximal contraction of aortic rings.
• 3.3. Furthermore, Clx antagonized Ca²⁺ dependent aortic contraction and BAY K-8644 induced aortic contraction suggesting its calcium antagonist character.
• 4.4. From these results it can be concluded that the hypotensive effect seen in rats in vivo after Clx i.v. injection arises because of vasodilatory effect of Clx and gives further support to the proposal that the pharmacological mechanism of action of Clx should be calcium antagonism.

     

Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells--ionic dependence and effects of local anesthetics.

A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [³H]guanine and isolating [³H]cyclic GMP with a cation exchange resin (Dowex-50⁺) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [³H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [³H]cyclic GMP. In the absence of external Na⁺ ([Na⁺]_e, with cesium chloride as osmotic filler) or external ([Ca²⁺]_e), the carbamylcholine-stimulated formation of [³H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM?[Na⁺]_e, whereas the optimal [Ca²⁺], was around 5 mM. Ca²⁺ increased [³H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (K_B) in the range of 6–250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.     

Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores

The results of binding studies suggest the presence of histamine H₁-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and superoxide anion (O₂ ^– ) formation in HL-60 cells differentiated towards monocytes with 1,25-dihydroxychole-calciferol. In HL-60 monocytes, HA increased [Ca²⁺]_i with a half-maximal effect at 8 M and a maximum at 30–100 M. Pertussis toxin (PTX) partially inhibited the stimulatory effects of HA on [Ca²⁺]_i. Betahistine, a weak partial H₁-receptor agonist, also increased [Ca²⁺]_i, whereas H₂- and H₃-receptor agonists were ineffective. H₁- but not H₂- and H₃-receptor antagonists inhibited HA induced rises in [Ca²⁺]_i. HA-induced rises in [Ca²⁺]_i were desensitized in a homologous manner and were also inhibited by the activator of protein kinase C, 4\-phorbol 12-myristate 13-acetate. Various protein kinase C inhibitors did not interfere with homologous desensitization. The stimulatory effects of HA on [Ca²⁺]_i were completely dependent on the presence of extracellular Ca²⁺ and were inhibited by the blocker of non-selective cation (NSC) channels, 1-{\-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1 H-imidazole hydrochloride (SK & F 96365). HA was much less effective than the chemotactic peptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), to induce rises in [Ca²⁺]_i. Unlike fMLP, HA did not activate O₂ ^– formation. Our data indicate that HL-60 monocytes possess H₁-receptors coupled to heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) of the G_i-family and PTX-insensitive G-proteins which mediate activation of NSC channels without concomitant activation of Ca²⁺ mobilization from intracellular stores, that homologous desensitization of HA-induced Ca²⁺ influx is independent of protein kinase C and that the stimulatory effect of HA on Ca²⁺ influx is too small to result in activation of O_{2 –} formation. Correspondence to: R. Seifert at the above address