Dissociation of increases in cyclic GMP from relaxation of arterial smooth muscle

Glyceryl trinitrate increased the cyclic GMP content of epinephrine-contracted aortic strips from glyceryl trinitrate-tolerant rats in a dose-dependent manner. Glyceryl trinitrate (.05 millimicron/ml) at a concentration that caused to relaxation of aortas from tolerant animals but a 42.4 3.7% relaxation of control aortas, elevated cyclic GMP levels in both groups approximately 3-fold. The results are not consistent with the hypothesis that cyclic GMP is involved in the relaxation of arterial smooth muscle.     

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     

Modulation of relaxation to levcromakalim by S-nitroso-N-acetylpenicillamine (SNAP) and 8-bromo cyclic GMP in the rat isolated mesenteric artery

1. Levcromakalim caused concentration-dependent relaxations of methoxamine-induced tone in both endothelium-denuded and intact vessels. Its potency was reduced by the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP; 0.1 μM or 1 μM) in both denuded and intact vessels. The maximal relaxation (R_max) was reduced only in denuded vessels.
2. SNAP was more potent in endothelium-denuded than intact vessels but there were no differences in R_max. Glibenclamide (10 μM) did not affect relaxation to SNAP in endothelium-denuded or intact vessels.
3. The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) increased the potency and R_max of levcromakalim in endothelium-intact vessels. ODQ had no effect in denuded vessels.
4. ODQ (10 μM) reduced the vasorelaxant potency of SNAP in both intact and endothelium-denuded vessels by 190-fold and 620-fold, respectively.
5. 8-bromo cyclic GMP (10 or 30 μM) reduced both the potency and R_max of levcromakalim in de-endothelialized vessels, but had no effect in intact vessels although it reduced both the potency and R_max of levcromakalim in intact vessels incubated with ODQ (10 μM).
6. In the presence of ODQ (10 μM), SNAP (0.1 μM or 1 μM) reduced the potency of levcromakalim in intact vessels, without altering R_max, but had no effect in denuded vessels. SNAP (50 μM) reduced both the potency and R_max of levcromakalim in intact and endothelium-denuded vessels.
7. Therefore, although SNAP causes relaxation principally through generation of cyclic GMP, it can modulate the actions of levcromakalim through mechanisms both dependent on, and independent of, cyclic GMP; the former predominate in endothelium-denuded vessels and the latter in intact vessels.

     

Negative functional effects of cyclic GMP are altered by cyclic AMP phosphodiesterases in rabbit cardiac myocytes

In this study, we tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes would be affected by the actions of cyclic GMP on cyclic AMP phosphodiesterases. Ventricular myocytes from eight rabbits were used to determine the functional and cyclic AMP changes caused by 10(-7), 10(-6), 10(-5) M 8-Bromo-cGMP alone and after the administration of 10(-6) M milrinone (cyclic GMP-inhibited cyclic AMP phosphodiesterase inhibitor) or 10(-6) M erythro-9-(2-Hydroxy-3-3-nonyl)adenine (EHNA, cyclic GMP-stimulated cyclic AMP phosphodiesterase inhibitor). 8-Br-cGMP dose-dependently reduced %shortening by 35+/-4% of baseline at 10(-5) M. This effect was significantly blunted by EHNA at all doses. The maximum rate of shortening was reduced by 31+/-3% by 10(-5) M 8-Br-cGMP. This effect of 8-Br-cGMP was significantly enhanced (42+/-4%) in the milrinone group. A similar pattern was observed in the maximum rate of relaxation data. Cyclic AMP levels were significantly increased from a baseline level of 4.0+/-0.8 pmol/10(5) myocytes by milrinone (+60%), EHNA (+61%) and 8-Br-cGMP (+47%). The combination of EHNA plus 8-Br-cGMP increased cyclic AMP levels significantly more that the combination of milrinone plus 8-Br-cGMP. Exogenous cyclic GMP reduces myocyte function, while raising cyclic AMP possibly through cyclic GMP-inhibited cyclic AMP phosphodiesterase effects. Blocking cyclic GMP-inhibited cyclic AMP phosphodiesterase enhances the functional effects cyclic GMP, while blocking cyclic GMP-stimulated cyclic AMP phosphodiesterase reduced these effects. The study demonstrated a functional interaction between cyclic GMP and cyclic AMP related to the cyclic GMP affected cyclic AMP phosphodiesterases.     

Effect of papaverine on cyclic nucleotide levels in the isolated rat aorta.

An increase in cAMP but no significant modification in cGMP content could be demonstrated in rat aorta strips after applying papaverine in concentrations which reduced contractile responses. Accumulation of cAMP was induced in noradrenaline-stimulated on K+-depolarized strips, under omission of external Ca2+. Thus the elevation of cAMP level preceded the reduction of contraction subsequently elicited by readdition of Ca2+. The effects could not be dissociated under the experimental conditions used here.     

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.

     

The effects of theophylline and cyclic AMP on intestinal smooth muscle contractions

The effects of 10^?4-10^?3 M theopphylline, cyclic 3'5'-adenosine monophosphate (cyclic AMP), dibutyryl cyclic AMP (DBC) and theophylline plus DBC were observed on the spontaneous and potassium (K)-induced contractions of the guinea-pig taenia coli. All of the compounds inhibited spontaneous contractions as well as the phasic and tonic components of the K-contracture. The effects of cyclic AMP, a compound which will not readily penetrate the cell membrane, were similar to those of the other compounds in inhibiting the spontaneous and phasic contractions, but it was a less effective inhibitor of the tonic contracture. Inhibition of spontaneous and phasic activity may occur at the cell membrane while the inhibitory actions on the tonic contracture may depend on cellular penetration by the inhibitor. There was no evidence that theophylline and DBC have different sites of action.     

Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.
2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity.
3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.
4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

     

Stimulation of calcium uptake into aortic microsomes by cyclic AMP and cyclic AMP-dependent protein kinase

Summary Enhancement of calcium uptake into rabbit aortic microsomes was seen at a cyclic AMP concentration of 10^–6M in the presence of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37). Protein kinase alone also increased calcium uptake, but cyclic AMP alone was without effect. The results suggest that stimulation of calcium sequestration may be the mechanism of cyclic AMP involvement in vascular smooth muscle relaxation.     

Effects of phthalazinol on smooth muscle cells of the porcine coronary artery and on neuromuscular junction on the guinea-pig vas deferens

Summary In smooth muscle cells of the porcine coronary artery, phthalazinol (10^–5 M) did not modify the membrane potential and the membrane resistance. At a concentration of 10^–4 M or higher, it hyperpolarized the membrane, reduced the membrane resistance and enhanced the rectifying property of the membrane. At the concentration of 10^–5 M, phthalazinol raised the threshold for the induction of a contraction and suppressed nonselectively the amplitude of the contraction evoked by application of high [K]₀, acetylcholine or electrical depolarization of the membrane. Phthalazinol (10^–5–10^–4 M) did not modify the membrane properties of smooth muscle cells from the guinea-pig vas deferens. However, it suppressed the amplitude of the excitatory junction potentials and the facilitation phenomenon produced by repetitive stimulation at various rates. The action potential recorded from the adrenergic nerves was not affected in the presence of phthalazinol (10^–5 and 10^–4 M). The mean amplitude of the miniature excitatory junction potentials (m.e.j.p.s.) was not affected by treatment with phthalazinol (10^–5–10^–4 M), but the rate of which of m.e.j.p.s. appeared was reduced by phthalazinol (>5×10^–5 M). These results indicate that the vasodilator property of phthalazinol may result from a suppression of Ca-mobilization in both the smooth muscle cells and the adrenergic nerve terminals. The former affects the mechanical response directly and the latter leads to an inhibition of noradrenaline release.     

Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: Differential subcellular localization and regulated expression by cyclic AMP

1. A combination of pharmacological, molecular biological and biochemical approaches were used to investigate the differential expression of two cyclic GMP-inhibited cyclic nucleotide phosphodiesterase genes (PDE3A and PDE3B) in the rat.
2. RT–PCR using PDE3A- or PDE3B-specific oligonucleotide primers allowed amplification of products encoding PDE3A (508 bp) or PDE3B (499 bp) sequences from several rat tissues (heart, aorta, liver, kidney and epididymal fat), from primary cultures of aortic vascular smooth muscle cells (VSMC) as well as from an SV40 large T-antigen immortalized aortic VSMC line.
3. Immunoblotting experiments with PDE3-selective antisera allowed the detection of both PDE3A and PDE3B immunoreactive proteins in several rat tissues, including tissues of the cardiovascular system, in primary cultures of aortic VSMC and in an SV40 large T-antigen immortalized aortic VSMC line. In all cases, PDE3A was expressed as a 120 kDa protein which was only detected in the cytosolic fraction. PDE3B was expressed as a 135 kDa protein and its expression was limited to the particulate fraction of all tissues and cells studied.
4. Prolonged incubation of cultured aortic VSMC with agents that increase VSMC cyclic AMP (forskolin or 8-bromo-cyclic AMP) produced marked time-dependent increases in PDE3 activity which correlated with increases in PDE3A and PDE3B RT–PCR signals and a marked increase in particulate PDE3 activity and PDE3B protein.
5. The physiological, pharmacological and biochemical implications of these findings are discussed based on previous reports of the effects of PDE3 inhibitors in the cardiovascular system and the relevance of our findings are presented in the context of the development of PDE3A and/or PDE3B-selective pharmacological agents.

     

Effects of 8-S-benzyl cyclic AMP on mechanical characteristics and cyclic AMP levels of the canine ventricular myocardium

Effects of 8-S-benzyl cyclic AMP on the mechanical characteristics of cardiac contradiction and on teh intracellular cyclic AMP level were studied on the isolated canine right ventricular myocardium. For comparison, inotropic effects of dibutyryl and 8-bromo cyclic AMP were also investigated. 8-S-Benzyl cyclic AMP caused a concentration-dependent positive intropic action. The action of 8-S-benzyl cyclic AMP developed and reached a steady level much faster thant hat of dibutyryl cyclic AMP (dbcAMP), while the time courses of the disappearance of the actions following washout of the compounds did not differ. The dose-response curve for 8-S-benzyl cyclic AMP lay substantially in the same concentration range as did that for dbcAMP. The duration of a single contraction was decreased by 8-S-benzyl cyclic AMP in a concentration-dependent manner, chiefly because of shortening of the relaxation time. The cyclic AMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) enhanced the positive inotropic action of 8-S-benzyl cyclic AMP; 8-bromo cyclic AMP caused a definite positive inotropic action only in the presence of IBMX. The intracellular cyclic AMP levels determined 5, 15 and 30 min after the administration of 1 mM 8-S-benzyl cyclic AMP were not significantly different from the corresponding control values determined in the paired muscle. The present results indicte that the changes in mechanical characteristics caused by 8-S-benzyl cyclic AMP were very similar to those induced by β-aderenoceptor stimulation. Since the intracellular cyclic AMP level was not changed after administration of 8-S-benzyl cyclic AMP, this compound is probably not metabolized intracellularly to cyclic AMP but acts directly as the original compound on the protein kinase. This compound provides an excellent pharmacological tool to mimic the effect of β-adrenoceptor stimulation on myocardial contractility.     

Papaverine, cyclic AMP and the dependence of the rat aorta on extracellular calcium.

The spasmolytic effects of papaverine and dibutyryl cyclic AMP (db-cAMP) were compared on isometric contractile responses induced by addition of increasing amounts of external calcium to K+-depolarized or noradrenaline-stimulated rat aorta strips. Papaverine at a concentration active on depolarized strips (3 times 10(-5) moles/1) reduced the maximal contraction (Emax) elicited by Ca2+ in these preparations, while db-cAMP did not. Contrary to what was observed on depolarized aortae, the degree of inhibition of noradrenaline-stimulated strips did not decrease with increasing extracellular calcium concentration (Ca)e. Both db-cAMP and papaverine at a concentration which did not depress Emax (5 times 10(-6) moles/1) potentiated the relaxing effect of high (Ca)e on contractions elicited by noradrenaline. In conclusion, cyclic AMP is probably implicated in the mode of action of papaverine on the noradrenaline-stimulated rat aorta. At a concentration active on depolarized strips, papaverine is also able to impair contractility directly.     

The kappa-opioid agonist (+/-)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway

In view of the scarce information about the analgesic mechanism of kappa-opioid receptor agonists, the objective of the present study was to determine whether nitric oxide (NO) is involved in the peripheral antinociception of bremazocine, a kappa-opioid receptor agonist. Three drugs all interfering with the L-arginine/NO/cyclic GMP pathway were tested using the rat paw model of carrageenan-induced (250 microg) hyperalgesia: (a) N(G)-nitro-L-arginine (a nonselective NO-synthase inhibitor), (b) methylene blue (a guanylate cyclase inhibitor), and (c) zaprinast (a cyclic GMP phosphodiesterase inhibitor). Intraplantar administration of bremazocine (20, 40 and 50 microg) caused a dose-dependent peripheral antihyperalgesia against carrageenan-induced hyperalgesia. The possibility of the higher dose of bremazocine (50 microg) having central or systemic effect was excluded since administration of the drug into the left paw did not elicit antinociception in the contralateral paw. However, when the dose of bremazocine was increased to 100 microg, a significant increase in the nociceptive threshold was observed, as measured in the hyperalgesic contralateral paw. Peripheral antihyperalgesia induced by bremazocine (50 microg) was significantly reduced in a dose-dependent manner when N(G)-nitro-L-arginine (6, 9, 12 and 25 microg) or methylene blue (250, 375 and 500 microg) was injected before. Previous treatment with 50 microg of zaprinast (which had no effect when administered alone) potentiated the antihyperalgesic effect of bremazocine (20 microg).Our data suggest that bremazocine elicits peripheral antinociception by activation of the L-arginine/NO/cyclic GMP pathway and that nitric oxide is an intermediary in this mechanism, forming cyclic GMP.     

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide-cyclic GMP pathway

Fencamfamine (FCF) is an indirect dopamine agent with effects similar to amphetamine and cocaine. In the present study, we investigate changes in Na,K-ATPase, cyclic AMP-dependent protein kinase (PKA) and nitric oxide synthase (NOS) activity and cyclic GMP levels in the nucleus accumbens (NAc) and striatum (ST) of animals acutely or repeatedly treated with FCF (3.5 mg/kg). Na,K-ATPase had a similar activity in control and repeatedly treated animals, but was reduced in the NAc of the acute group. This enzyme was reduced in the ST in acute and repeatedly treated animals, compared to the control group. Expression of the _1,2,3-Na,K-ATPase isoforms in the NAc and the ST was not altered in all groups studied. Acute FCF induced a significant increase in PKA activity in both the ST and the NAc. Repeatedly treated animals showed a higher increase in PKA activity in the NAc, but not in the ST, when compared to the acute group. There was also an increase in both NOS activity and cyclic GMP levels only in the NAc of FCF repeatedly treated animals compared to the acute and control groups. We suggest that chronic FCF treatment is linked to a modification in Na,K-ATPase activity through the PKA and NO–cyclic GMP pathway.     

Effect of a novel inhibitor of cyclic AMP phosphodiesterase,E-1020, on cytosolic Ca++ level and contraction in vascular smooth muscle

Summary 1. The effects of a novel cyclic AMP phosphodiesterase inhibitor, E-1020 (1,2-dihydro-6-methyl-2-oxo5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on cytosolic Ca⁺⁺ level ([Ca⁺⁺]_cyt) and muscle tension were examined in rat aorta using a fluorescent Ca⁺⁺ indicator, fura-2. 2. The sustained contraction induced by norepinephrine was more strongly inhibited by E-1020 than that induced by high K⁺. The contraction induced by a higher concentration of the stimulant was less sensitive to E-1020 than that due to a lower concentration. 3. Contractions induced by high K⁺ and norepinephrine followed the increase in [Ca⁺⁺]_cyt. E-1020 inhibited the increments in [Ca⁺⁺]_cyt and muscle tension. A Ca⁺⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca⁺⁺]_cyt more strongly than the contraction. E-1020 inhibited the verapamil-insensitive portion of the norepinephrine-stimulated [Ca⁺⁺]_cyt and contraction. 4. Norepinephrine transiently increased [Ca⁺⁺]_cyt and muscle tension in Ca⁺⁺-free solution. E-1020 inhibited the transient contraction but not the stimulated [Ca⁺⁺]_cyt. 5. E-1020 increased the cyclic AMP content of the muscle. The effects of E-1020 on cyclic AMP content and contraction were potentiated by an activator of adenylate cyclase, forskolin. 6. These results suggest that E-1020 inhibits the vascular contractility by the decrease in [Ca⁺⁺]_cyt and decrease in Ca⁺⁺ sensitivity of contractile elements. These effects may be mediated by the increase in cyclic AMP content of the muscle. Send offprint requests to M. Täjimi at the above address     

The stimulation of human neutrophil migration by angiotensin II: its dependence on Ca2+ and the involvement of cyclic GMP

1. Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10⁻¹⁰ M it caused a maximal increase of migration; migration increased from 47.2±2.1 μm in the absence of angiotensin II, to 73.1±2.2 μm with 10⁻¹⁰ M angiotensin II present in the lower compartment of the Boyden chamber (n=5, P<0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10⁻⁸ M and higher inhibited chemotactic peptide-stimulated chemotaxis.
2. The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca²⁺. In the presence of 1 mM Ca²⁺, angiotensin II stimulated migration to 76.1±1.7 μm, while migration in the absence of Ca²⁺ was 42.2±1.9 μm (n=4, P<0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II-activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca²⁺ than in electroporated cells. This supports the view that there is an influx of Ca²⁺ through the plasma membrane, and a requirement of calcium for an intracellular target.
3. Angiotensin II-stimulated migration was inhibited by pertussis toxin; from 71.6±2.0 μm in the absence, to 43.6±1.5 μm in the presence of pertussis toxin (n=4, P<0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTPγS. This suggests that one or more G-proteins are involved in the activating effect of angiotensin II.
4. Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP-dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP-dependent kinase.

     

Blockade by haloperidol of the increase in tryptophan hydroxylase activity induced by incubation of slices of brain stem with dibutyryl cyclic AMP

Pretreatment of rat brain stem slices with dibutyryl cyclic AMP, caffeine, theophylline and 3-isobutyl-1-methylxanthine increased the activity of tryptophan hydroxylase in supernatant preparations of enzyme made from the slices. This effect does not appear to be mediated by a cyclic AMP sensitive mechanism since it was not reproduced by exposure of the slices to 8-bromo cyclic AMP, to papaverine, a nonxanthine phosphodiesterase inhibitor, or to other treatments known to raise tissue cyclic AMP levels. The ability of haloperidol to block this increase in enzyme activity is consistent with a role for calmodulin and calcium as mediators of the enzyme activation, particularly when this observation is considered in conjunction with the evidence that supernatant preparations of this enzyme was activated under phosphorylating conditions by a calcium-calmodulin dependent process [5]. Nevertheless, in view of the high concentrations of haloperidol employed in the present experiments, the possibility that this drug may produce its effects in the brain stem slices through some other action, unrelated to its ability to bind to calmodulin, should be kept in mind.     

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.     

Importance of intracellular angiotensin II in vascular smooth muscle cell apoptosis: inhibition by the angiotensin AT1 receptor antagonist irbesartan

The intracellular uptake of Angiotensin II has been described, although its physiological role is not yet understood. We aimed to study the role of Angiotensin II internalization in Angiotensin II-induced apoptosis. Vascular smooth muscle cells were cultured from male Wistar-Kyoto rats and treated with Angiotensin II (1 microM, 48 h). Apoptosis was assessed by DNA fragmentation, cell cytometry and caspase-3 activity. The Angiotensin AT(1) receptor antagonist irbesartan (0.1-10 microM) and the inhibitors of Angiotensin II internalization phenylarsine oxide (PAO, 20 microM), but not the AT(2) receptor antagonist PD123319 (S-(+)-1-[(4-(Dimethylamino)-3-methylphenyl)methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid di(trifluoroacetate) salt), decreased Angiotensin II-mediated apoptosis. Pre-treatment with irbesartan, but not with PD123319, blocked Angiotensin II internalization. We found a strong correlation between intracellular Angiotensin II staining and Angiotensin II-induced apoptosis for all compared groups. We therefore conclude that internalization of Angiotensin II is involved in apoptosis of vascular smooth muscle cells induced by this peptide.