cAMP and cGMP do not mediate the vasorelaxation induced by iodinated radiographic contrast media in isolated swine renal arteries.

PURPOSE: Vasodilatation is a frequent side effect of radiographic contrast media, partially due to a direct effect on vascular smooth muscles. Our purpose was therefore to examine any possible implication of the cyclic adenosine monophospate (cAMP) and cyclic guanosine monophosphate (cGMP) pathways in contrast media induced vasorelaxation. MATERIAL AND METHODS: Isolated segments of swine renal arteries were precontracted with 10 microM phenylephrine and relaxed with iomeprol before and after blockade of the cAMP and cGMP pathways. RESULTS: 80 mM and 160 mM of iomeprol significantly relaxed about 52% and 68% of the precontracted arterial rings, respectively. 10 microM of IBMX, a phosphodiesterase inhibitor, did not increase the relaxant effect of 80 mM iomeprol but increased the relaxations induced by 400 nM forskolin by about 1.9 times (which stimulates the production of cAMP), and by 1 microM sodium nitroprusside (which stimulates the production of cGMP). 1 microM of H89 (an inhibitor of the cAMP-dependent protein kinase), was able to reduce the relaxation induced by 4 microM forskolin by about 2.5 times but had no significant effect on the relaxation induced by 160 mM iomeprol. 10 microM of ODQ (an inhibitor of the soluble guanylate cyclase), could reduce the relaxation induced by 10 microM of SNP but not the one induced by 160 mM iomeprol. Moreover, the absence of endothelial cells did not alter the relaxation induced by iomeprol. CONCLUSION: The activation of the cAMP and cGMP pathways are not involved in the in vitro relaxation induced by iomeprol in swine renal arteries.

Iodinated radiographic contrast media inhibit the capacitative calcium entry into smooth muscle cells of the swine renal artery.

RATIONALE AND OBJECTIVES: To investigate whether nonionic, iodinated, radiographic contrast media (RCM) could modulate calcium release from or calcium entry into smooth muscle cells of the swine renal artery. METHODS: Intracellular calcium concentrations of isolated myocytes loaded with the calcium-sensitive dye fluo-3 were analyzed using a fluorescence imaging system. Calcium signals were compared with isometric contractions of vascular segments in an organ bath. The effects of the triiodinated monomer iomeprol were compared with those of a mannitol solution. RESULTS: Stimulation of alpha-receptors by phenylephrine caused a biphasic calcium signal. Transient liberation of calcium from intracellular stores triggered the sustained entry of extracellular calcium (capacitative entry). Iomeprol and mannitol slightly inhibited the initial transient spike to the same extent. The calcium influx was reversibly inhibited by RCM by about 50%. Up to a concentration of 40 mmol/L, the inhibition induced by iomeprol was significantly higher than that induced by mannitol (P < 0.05-0.01). In isolated arterial segments, relaxation of the contraction phase depending on the calcium influx was significantly higher with the RCM than with mannitol (P < 0.01). CONCLUSIONS: Vasodilatation by CM is associated with a reduction in the capacitative entry of calcium. The mechanism of this effect is not clear, but it can be ruled out that it is mainly due to the high osmolarity of these compounds.

Changes of intracellular calcium concentrations by phenylephrine in renal arterial smooth muscle cells.

In smooth muscle cells isolated from swine renal interlobar arteries, phenylephrine (PE) at concentrations of 1-10 microM produced biphasic increases of the intracellular calcium concentration. An early transient rise was followed by a maintained plateau. The maintained component was sensitive to extracellular calcium, in contrast to the early transient, which was still observed in nominally calcium-free solution. Nifedipine (1 microM) and NiCl2 (100 microM) only weakly affected the calcium signal, suggesting that voltage-sensitive calcium channels play only a minor role in the PE-induced changes in intracellular calcium. Thapsigargin (0.5 microM) elevated the intracellular calcium concentration and depressed both the early transient and the maintained component of the PE response. In calciumfree medium PE induced a transient rise of the intracellular calcium concentration with a depressed plateau. Readmission of calcium elevated the intracellular calcium concentration above the baseline. Both components of the PE-induced calcium signal were completely abolished when the cells were pretreated with the phospholipase C (PLC) inhibitor U73122 (2 microM). LaCl3 (100 microM, 1 mM), an inhibitor of calcium-release-activated current (ICRAC), had no effect on the PE-induced calcium signal. GdCl3 (50 microM), SKF 96365 (10 microM) and flufenamic acid (100 microM), reported to inhibit nonselective cation channels, blocked or transiently reduced the maintained calcium signal. Several protein kinase inhibitors such as genistein (10 microM), H7 (50 microM), H89 (1 microM) and bisindolylmaleimide (0.2 microM) reduced the maintained calcium signal. We conclude that the initial transient spike of the PE-induced calcium signal is due to release of calcium from inositol 1,4,5-trisphosphate-sensitive calcium stores evoked by alpha 1-adrenoceptor-coupled stimulation of PLC and that the maintained component is due to capacitative calcium entry, which is modulated by protein kinases.

Inhibition of L-type calcium currents in guinea pig ventricular myocytes by the kappa-opioid agonist U50488H does not involve binding to opiate receptors.

In guinea pig ventricular myocytes the kappa-opioid agonist trans(+/-)-3,3-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate salt (U50488H) inhibited L-type calcium currents (ICa) as measured by the patch clamp technique in a dose-dependent fashion. The basal, as well as the isoprenaline and 8-Br-cAMP-stimulated, ICa were found to be reduced by U50488H. The inhibition was reversible and was not prevented on preincubation with pertussis toxin or the receptor antagonists naloxone and naltrexone. Naltrexone alone also caused an inhibition of ICa. Leucine enkephaline, a peptide opiate agonist, had no effect on ICa. U50488H did not alter the current/voltage relationship of the calcium current. The inhibition was independent of the cytosolic calcium concentration because it was also observed in the presence of 10 mM BAPTA in the pipette. If the compound was applied intracellularly via a perfused patch pipette there was no inhibition of ICa. The calcium current stimulated by the dihydropyridine calcium agonist Bay K 8644 was also reduced by U50488H. Conversely the inhibition of ICa by U50488H could be antagonized by Bay K 8644. In conclusion, these results demonstrate that binding to specific membrane receptors is not involved in the inhibition of L-type calcium current by U50488H and other nonpeptide opioid agonists. A direct interaction with the channel molecule at the exterior of the cell is probably involved.

Muscular cavernous single cell analysis in patients with venoocclusive dysfunction.

Enzymatically isolated smooth muscle cells of the corpora cavernosa obtained from open biopsies of 15 patients, clinically nonresponding to papaverine/phentolamine and prostaglandin E1 (PGE1) and classified by cavernosometry, were examined using the patch-clamp technique in the whole-cell configuration mode simultaneously monitoring the intracellular calcium concentration by means of the Ca(2+)-sensitive fluorescence dye FURA-II. It could be demonstrated that extracellularly applied PGE1 induces smooth muscle relaxation by inhibition of voltage-dependent L-type Ca2+ currents (58 +/- 8%). Compared to intact cavenous tissue (n = 5), the smooth muscle cells of 14/15 PGE1 nonresponders had no evidence of functional disturbance. Due to intact smooth muscle cells in most cases, etiology of venoocclusive dysfunction remains unclear.

Involvement of intracellular Ca2+ release mechanism in adenosine-induced cardiac Ca2+ current inhibition.

BACKGROUND: Myocardial contractility is regulated by changes in the free intracellular Ca2+ concentration [Ca2+]i, which is determined by concerted interactions of Ca2+ influx through voltage-dependent Ca2+ channels, release of Ca2+ from intracellular pools, and Ca2+ extrusion systems. Although adenosine has been reported to attenuate postischemic stunning, the mechanism is unknown. METHODS: The patch clamp technique and the Ca(2+)-sensitive fluorescence dye FURA II were used in isolated ventricular cardiomyocytes to study the effects of adenosine on the L-type Ca2+ channel current (ICa) and on [Ca2+]i. RESULTS: Adenosine decreased the basal ICa by 76% +/- 4% (100 nmol/L, n = 48) and simultaneously elevated [Ca2+]i up to 1.8 mumol/L (n = 37) by adenosine-A1-receptor activation. The adenosine-evoked responses were not sensitive to pertussis toxin (n = 12), but intracellular application of guanosine diphosphate-beta sulfate (10 mumol/L) reduced the effects of adenosine by 86.7% +/- 7% (n = 9). Block of intracellular Ca2+ release by ryanodine and thapsigargin attenuated, whereas intracellularly applied inositol 1,4,5-trisphosphate (n = 18) mimicked the adenosine-evoked responses. CONCLUSIONS: These findings suggest that coupling of adenosine to A1-receptors induced G protein-mediated activation of phospholipase C followed by generation of inositol 1,4,5-trisphosphate, which releases Ca2+ from intracellular Ca2+ stores. The inhibition of ICa by depletion of intracellular Ca2+ stores could play a major role in preventing ischemia-induced Ca2+ overload.

Role of intracellular Ca(2+)-stores in the 5-hydroxytryptamine-induced Ca(2+)-current inhibition in NG108-15 hybrid cells.

Mouse neuroblastoma x rat glioma hybrid cells (N x G, NG108-15) were used to study the mechanism of Ca(2+)-current (ICa) inhibition by 5-hydroxytryptamine (5-HT). 5-HT caused a dose-dependent decrease of ICa which was abolished by ICS 205-930 (10)(-8) M) while 2-methyl-5-HT was an agonist. Intracellular infusion of GDP beta S (50 microM) prevented the 5-HT-induced inhibition of ICa whereas pertussis toxin (PTX) pretreatment did not alter the 5-HT response. The 5-HT-induced inhibition depended on the free Ca(2+)-concentration in the pipette solution. Pretreating N x G cells with low molecular weight (LMW) heparin (160 micrograms/ml), 200 microM ryanodine or 2-10 mM caffeine attenuated the 5-HT-induced inhibition of ICa. From these results we suggest that the 5-HT-induced ICa inhibition requires release of Ca2+ from intracellular stores.

Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels.

In the heart, the guanosine 5'-triphosphate (GTP)-binding protein Gs is activated by hormone binding to beta-adrenergic receptors and stimulates the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A. Additionally, Gs can modulate cardiac Ca channels directly in cell-free systems. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (ICa) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the ICa density increased from 12 to 40 microA/cm2 following application of 1 microM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1 microM ISP enhanced ICa only from 9 to 17 microA/cm2 after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPs (the Rp-isomer of adenosine 3',5'-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on ICa. Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of ICa, which probably reflects the direct stimulation of Ca channels by Gs under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 microM cAMP, bath application of 20-40 microM 3-isobutyl-1-methylxanthine (IBMX) enhanced the ICa density to about 41 microA/cm2 indicating saturation of the cAMP pathway. Under this condition, 1 microM ISP was without significant effect on ICa. This result may suggest that direct Gs stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the Gs pathway in intact myocytes. Third, simultaneous application of 1 microM ISP and 40 microM IBMX enhanced ICa up to densities of around 75 microA/cm2 during cell dialysis with 100 microM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that Gs is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of Gs may act to prime L-type Ca channels for cAMP-dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes.

Potentiation by cyclic GMP of beta-adrenergic effect on Ca2+ current in guinea-pig ventricular cells.

1. Effects of cyclic GMP on L-type Ca2+ current (ICa) were investigated in myocytes isolated from guinea-pig ventricles using the patch clamp method in the whole-cell configuration combined with intracellular perfusion. 2. When ICa was increased by bath application of isoprenaline (0.001-0.1 microM) or forskolin (0.5-1 microM), or by intracellular dialysis with cyclic AMP (50-100 microM), dialysis with 10 microM-cyclic GMP resulted in an additional stimulation of ICa. Without these pre-treatments, cyclic GMP (1-100 microM) had no effect on the basal ICa. 5'-GMP was without effect. 3. The stimulatory effect of cyclic GMP was observed at concentrations higher than 0.1 microM with a maximum at around 10 microM in the pipette. The dose-response relation between isoprenaline and ICa was shifted to the left by (10 microM) cyclic GMP; the half-maximum isoprenaline concentration shifted from 16 to 4.6 nM. 4. The increase of ICa on dialysing 50 microM-cyclic AMP varied from cell to cell, probably due to a difference in phosphodiesterase activity. The cells responding weakly to cyclic AMP showed a greater response to cyclic GMP, and vice versa. In cells dialysed with hydrolysis-resistant derivatives (10-50 microM-8-(4-chlorophenylthio)-cyclic AMP or 50 microM-8-bromo-cyclic AMP), additional dialysis with cyclic GMP failed to modify ICa. Dialysis with cyclic GMP abolished the stimulatory effect of milrinone, a specific inhibitor of cyclic GMP-inhibited phosphodiesterase. These findings suggested that inhibition of cyclic GMP-sensitive phosphodiesterase was responsible for the stimulatory effect of cyclic GMP. 5. In the presence of isoprenaline, direct application of an active fragment of cyclic GMP-dependent protein kinase (PKG) failed to modify ICa in most cells. Activation of native PKG by intracellular dialysis with 8-bromo-cyclic GMP, or higher concentrations of cyclic GMP (100-1000 microM), depressed ICa in about 25% of the cells. Furthermore, dialysis of cyclic GMP reversed the increase of ICa by the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX). These findings suggested the presence of antagonistic mechanisms of cyclic GMP, which are independent from the above synergistic action. PKG may be involved in this antagonistic effect.

Inhibitory modulation of fast and slow Ca(2+)-currents in neuroblastoma x glioma cells during differentiation.

Mouse neuroblastoma x rat glioma hybrid cells (N x G, 108CC15) were used to study the inhibitory effects of the synthetic opioid D-Ala2-D-Leu5-enkephalin (DADLE), somatostatin, adrenaline-alpha 2 and angiotensin II on voltage-dependent Ca(2+)-currents (ICa) using the patch-clamp technique in the whole-cell configuration mode. The inhibitory effects could be abolished by pretreatment of N x G cells with pertussis toxin or intracellular infusion of GDP beta S indicating an involvement of a pertussis toxin sensitive GTP-binding protein (G-protein), presumably Go. The effect of DADLE, the strongest inhibitor of ICa, was studied during dibutyryl cyclic AMP (dBcAMP) induced differentiation. Using omega-conotoxin GVIA (omega-CTX) and methoxyverapamil (D600) as specific Ca(2+)-channel blockers of the N- and L-type Ca(2+)-channels, it was found that in N x G cells DADLE predominantly induces inhibition of T- and N-type Ca(2+)-channels.

Calcium currents of neuroblastoma x glioma hybrid cells after cultivation with dibutyryl cyclic AMP and nickel.

The long-term modulation of calcium (Ca2+) currents (ICa) was studied in 108CC15 neuroblastoma x glioma hybrid (NxG) cells grown under various culture conditions. The following results were obtained: 1. Addition of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP) or 0.1 microM forskolin to the culture medium increased a transient component of ICa two-fold within 3 days, from 21.0 +/- 1.6 pA/pF (n = 22) to a maximum of 40.0 +/- 2.6 pA/pF (n = 28). Under these conditions, cells also expressed a slowly inactivating ICa component (maximum after 3 days, 20.5 +/- 1.6 pA/pF, n = 28). 2. The fast inactivating ICa as well as the db-cAMP-induced slowly inactivating ICa were completely down-regulated during incubation of NxG cells with the inorganic Ca2+ channel blocker, nickel (Ni2+, 100 microM). The suppressing effect was reversed within 3 days of incubation in db-cAMP-containing medium lacking Ni2+. 3. Binding studies on membrane preparations of control and Ni2(+)-pretreated NxG cells revealed a marked difference in the maximal (+)3H-PN200-110 binding. The difference was seen in undifferentiated as well as in db-cAMP-incubated cells. 4. The protein synthesis blocker, cycloheximide, suppressed both the db-cAMP-induced increase and the reappearance of ICa following Ni2+ pretreatment. It is suggested that chronic application of db-cAMP or Ni2+ to NxG cells increases and decreases the number of Ca2+ channel proteins, respectively.

Ionic currents contributing to the action potential in single ventricular myocytes of the guinea pig studied with action potential clamp.

With the action potential clamp procedure we studied the contribution of various ionic currents to the action potential in single ventricular myocytes. Action potentials were elicited by a current pulse through the suction pipette and recorded by a computer. A representative action potential was then repetitively replayed to the same cell under voltage-clamp conditions. Successive pharmacological blocks of ionic currents allowed for the first time the measurement of the contribution of the L-type calcium current (ICa) and the [Ca2+]i-activated currents as well as the potassium current to the action potential. Experiments using caffeine as a tool to increase calcium release from the sarcoplasmic reticulum supported the idea that INaCa contributes to the plateau during the second half of the action potential and even lasts into diastole, whereas strong elevation of the intracellular [Ca]i during the action potential additionally activated the non-specific cation channel.

Whole-cell calcium current in guinea-pig ventricular myocytes dialysed with guanine nucleotides.

1. Whole-cell calcium current (ICa) was recorded in guinea-pig ventricular myocytes superfused with Na+,K(+)-free solution and dialysed with a substrate-free solution (minimum intracellular solution, MICS). A dual tight-seal pipette method was often used to permit pressure-enhanced dialysis of a test solution after a given pre-dialysis. 2. In dual-pipette experiments, test dialysates contained 100 mM-GTP-gamma-S (guanosine 5'-O-(3-thiotriphosphate] or 100 microM-GMP-PNP (guanyl-5'-imidodiphosphate). These non-hydrolysable analogues of guanosine triphosphate (GTP) enhanced ICa amplitude (+ 10 mV) by 20-40%. Dialysates containing 100 microM-GTP or GDP-beta-S (guanosine 5'-O-(2-thiodiphosphate] were ineffective, and pre-dialysis with GDP-beta-S blocked stimulation by GTP-gamma-S. 3. Non-hydrolysable GTP analogues slowed the inactivation of ICa and shifted the voltage eliciting maximum ICa by 5-10 mV in the negative direction. 4. ICa enhancement by GTP analogues was attributed to the activation of three GTP-binding regulatory (G) proteins (Gi, Gp and Gs). In single-pipette experiments, the inactivation of Gi by pre-treatment with pertussis toxin did not block enhancement, and a Gp-activating regimen (external acetylcholine-internal GTP) was without effect. Thus, it is probable that the effects of GTP analogues on ICa were primarily mediated by Gs activation. 5. PI-MICS dialysates contained phosphorylation-pathway inhibitors and were used to inhibit Ca2+ channel phosphorylation via the adenyl cyclase pathway. These were deemed effective since forskolin (1-5 microM) doubled ICa during control dialysis but was without effect after 8 min PI-MICS dialysis. However, 0.1 microM-isoprenaline increased ICa by 35% in myocytes totally unresponsive to forskolin, suggesting that beta-adrenergic receptor occupation can stimulate ICa even when the phosphorylation pathway is blocked. 6. After prolonged dialysis of myocytes with PI-MICS, ICa was still enhanced by pressure-assisted dialysis of 100 microM-GTP-gamma-S or GMP-PNP. We conclude that activated Gs has a direct effect on cardiac Ca2+ channels.

On the mechanism of the "specific bradycardic action" of the verapamil derivative UL-FS 49.

Membrane currents were measured in single sino-atrial node cells of guinea pig and rabbit hearts as well as in guinea pig ventricular myocytes using the patch-clamp technique. UL-FS 49 blocked the L-type calcium current (ICa) in sino-atrial node cells at drug concentrations which had little or no effect on the amplitude of the hyperpolarization-activated current ih(f). In guinea pig ventricular myocytes UL-FS 49 also blocked ICa but not as strongly as in sino-atrial node cells. In a computer simulation of the sino-atrial node action potential the extent of rate reduction by block of either ih(f) or ICa was estimated. From the data obtained by single cell measurements and the computations we concluded that rate reduction in primary pacemaker cells by application of UL-FS 49 is mainly due to a use dependent block of the L-type calcium current. Voltage dependent unblock of iCa at potentials more negative than -50 mV together with the lower drug sensitivity of ventricular cells can explain the "specific bradycardic action" of UL-FS 49.

A dual-pipette technique that permits rapid internal dialysis and membrane potential measurement in voltage-clamped cardiomyocytes.

Guinea pig ventricular myocytes were voltage-clamped and dialysed using two glass patch pipettes (P1, P2) with tip openings of around 2 microns. A substantial improvement in the efficacy of dialysis from P2 was achieved by the application of positive pressure (15-30 cm H2O) to P2, and similar negative pressure to P1. Evidence of enhanced dialysis was obtained by measuring the effect on Ca channel current of P2 dialysates containing Ca, cAMP, GTP [gamma-S], trypsin, or the catalytic subunit of protein kinase A. Times to maximum response were 3-5 times shorter than those calculated or observed by others using a single-pipette method. The speeding-up was verified in comparative experiments with 100 microM GTP [gamma-S] dialysates; maximum stimulation of ICa occurred after 1.3-1.8 min with the dual-pipette method, versus 8.2 min with a single pipette. Other advantages of the dual-pipette method include the option of following a control dialysis from P1 with a test dialysis from P2, and the measurement of actual membrane potential. The disadvantages are that the rate of success is lower than with single-pipette experiments, and that smaller cardiomyocytes are difficult subjects.